Understanding the evolutionary factors that affect genetic variation in natural populations remains the primary focus of population genetics. Early surveys of allozyme variation using protein electrophoresis revealed abundant polymorphism, but only about one third of the possible amino acid changes, and none of the variation at silent and non-coding sites, could be detected with this technique . Analyses of genetic variation now involve nucleotide data, which can be used in a variety of statistical tests to discriminate between patterns of variation due to neutral processes (drift, population expansion, bottlenecks) and various types of selection (positive, purifying, balancing). Most studies have shown that levels of variation at silent and non-coding sites are substantially higher than levels of protein variation, suggesting that the majority of non-synonymous mutations are deleterious . Even so, patterns of allozyme (protein) variation often appear to conform to neutral or nearly neutral expectations . Conversely, detailed studies showing associations with different habitats, or clinal variation across wide geographic ranges have provided evidence that functional differences among allozyme variants can affect fitness (e.g. Alcohol dehydrogenase and Glucose-6-phosphate dehydrogenase in Drosophila melanogaster and Phosphoglucose isomerase in Colias butterflies; reviewed in ). Thus, a major challenge has been to determine the adaptive significance, if any, of polymorphism and interspecific divergence at allozyme loci .
L-Lactate dehydrogenase (L-LDH, EC 220.127.116.11) is involved in the interconversion of pyruvate and L(-)-lactate, which allows the aerobic metabolism of lactate accumulated by anaerobic glycolysis following periods of exposure to lowered environmental oxygen tension, or hypoxia . There is evidence that selection is acting on LDH variation in well-studied species such as the killifish, Fundulus heteroclitus [6, 7]. For example, an amino acid substitution between two alleles that vary along a cline in temperature affects thermostability , and experimental studies have shown that this variation is involved in the adaptation of killifish to hypoxia [9, 10].
A strong association between Ldh genotype and habitat has been also observed in members of the Daphnia pulex species complex. Based on relative electrophoretic mobility, the pond species, D. pulex is characterized by homozygosity for the slow (S) Ldh allele and the closely related lake species, D. pulicaria by homozygosity for the fast (F) allele in North America [11–13]. In addition, the two species hybridize and hybrids are generally found in ponds or disturbed, intermediate habitats . The genetic and habitat segregation is also associated with differences in physiological and life history traits [14, 15]. Surveys of allozyme variation in most Daphnia species are consistent with a one-locus model, but a recent analysis of the D. pulex genome sequence has identified two Ldh genes, LdhA and LdhB, that are approximately 26 cM apart [16, 17]. Preliminary gene expression work suggests that LdhA is expressed at a significantly higher level than LdhB , but the link between the allozyme locus and the genome sequences has not yet been made. Nevertheless, the fixation of different LDH variants in D. pulex and D. pulicaria in different aquatic habitats, despite the fact that they share polymorphisms at other allozyme loci [19–21], suggests the possibility that Ldh variation is adaptive. However, the hypothesis that selection has played a role in Ldh divergence in Daphnia has yet to be tested.
Genetic studies suggest that D. pulex and D. pulicaria are members of a relatively young species complex and appear to be undergoing ecological speciation . Both species reproduce by cyclical parthenogenesis in which females produce offspring by apomictic parthenogenesis during favorable conditions and have a sexual phase that results in the production of diapausing eggs prior to the onset of unfavorable conditions. Natural hybrids between the two species reproduce by obligate parthenogenesis [23, 24] in which females produce broods of parthenogenetic offspring (like cyclic females), but also produce their diapausing eggs parthenogenetically. This results in a completely ameiotic life history and allows clonal genotypes to persist for many years.
The objectives of the present study are to analyze both LdhA and LdhB sequence variation to (1) determine which locus is detected in allozyme surveys, (2) elucidate the evolutionary history of the two genes in the context of speciation and hybridization, and (3) determine if there is evidence that selection is acting on the variation at each locus. To achieve these objectives, isolates of D. pulex and D. pulicaria from North America were chosen to maximize geographic coverage of the three Ldh genotypes (SS, SF and FF) and the two breeding systems.