Plymouth Marine Laboratory, Plymouth, England
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Abstract |
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Introduction |
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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 1970
; Kirby, Berry, and Powers 1997
). 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 1988
). 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 1992
; Amano, Vermeij, and Narita 1993
).
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 1990
; Kirby, Berry, and Powers 1997
); (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 1990
; Kirby, Berry, and Powers 1997
), suggesting that mMDH may characterize different genetic compositions (chromosome rearrangements and allozymes) (Day 1992
); (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 1977
; Powers and Place 1978
; Powers et al. 1991
; Nielsen, Page, and Crossland 1994
; Harrison, Nielsen, and Page 1996
), suggesting that in some cases variation in MDHs may represent the outcome of natural selection (Nielsen, Page, and Crossland 1994
; Harrison, Nielsen, and Page 1996
).
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Materials and Methods |
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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 2000
). 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-Itreated mRNA (extracted from N. lapillus foot muscle tissue) as template and broad taxon degenerate PCR primers (Kirby 2000
). Rapid amplification of cDNA ends (RACE) (Frohman, Dush, and Martin 1988
) 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)
. 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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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 P1P4 (fig. 1
) and methods similar to those used to amplify internal fragments of mMDH from N. lapillus (Kirby 2000
). 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 1994
). Unrooted phylogenetic trees relating N. lapillus mMDH and cMDH amino acid sequences to other MDHs were constructed using PHYLIP (Felsenstein 1993
) by neighbor-joining on pairwise amino acid distances calculated according to the formula of Kimura (1983)
. Support for the branching order of the trees was provided by bootstrap analysis performed with 500 replications.
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Results |
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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 1994
) 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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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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Discussion |
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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 1997
) and date to the Albian of the mid-late Cretaceous (Taylor, Morris, and Taylor 1980
). The fossil record of the genus Nucella spans an interval of about 25 Myr from the early Miocene to the recent (Cambridge and Kitching 1982
), and according to this fossil record and estimates based on rates of evolution of mtDNA in Nucella (Collins et al. 1996
), 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 (1937
, pp. 307308) 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, 1992
) and associations between the mMDH genotype and the shell phenotype (Kirby, Bayne, and Berry 1994a
), 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 1975
]) provides a convenient mechanism by which genetic variation could now be maintained and evolve separately. Robertsonian translocations reduce recombination (White 1978
, pp. 45105), causing genetic isolation between alleles (Gregorius and Herzog 1989
). 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 1939
, pp. 8792; Wallace 1955
) conferring an important selective advantage on N. lapillus (Wright 1931
; Lewontin and Kojima 1960
). 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 1994a
) 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 1978
; Nielsen, Page, and Crossland 1994
; Harrison, Nielsen, and Page 1996
). 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 1997
). 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 1993
).
Since the discovery of considerable protein polymorphism in natural populations, there has been debate as to how this variation may be maintained (Lewontin 1974
, pp. 189271; Nei 1975
, pp. 154174; Wright 1978
, pp. 242322; Kimura 1983
, pp. 253299). 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 1997
; Filatov and Charlesworth 1999
; Flajnik et al. 1999
; Su and Nei 1999
). 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 1982
; Collins et al. 1996
), may predate Nucella. Transpecific polymorphisms, like that seen in mMDH in Nucella, are macroevolutionary phenomena (Mayr 1963
, pp. 586620) (as opposed to microevolutionary intraspecific polymorphisms), so other macroevolutioary phenomena such as coadaptation (Dobzhansky 1937
, pp. 307308) and epigenetic interactions (Waddington 1957
, pp. 188) are likely to be associated with them. The physical stresses associated with life in the intertidal environment (Lewis 1964
, pp. 217221) 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 1931
) 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 1994a
); 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 1973
; Newkirk and Doyle 1975, 1979
; Janson and Sundberg 1983
; Grahame and Mill 1992
). The study of the origin and molecular evolution of variation in mMDH in Nucella and related taxa consequently comprises ongoing research.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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1 Keywords: cline
coadaptation
macroevolution
malate dehydrogenase
Nucella lapillus,
transpecific
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
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