Dean and Thornton (2007) reviewed a number of case studies that employed manipulative biochemical experiments of specific gene products to gain insight into the mechanistic basis of adaptive evolution. In some cases, allelic differences in protein function or protein expression may have measurable phenotypic effects at the level of whole-organism performance. This opens up the possibility of measuring fitness-related trait variation among individuals with known genotypes under natural conditions. Although such studies face the challenge of controlling for genetic background to isolate the effects of specific genes, the potential pay-off is that it may be possible to establish direct, mechanistic connections between whole-organism performance and fitness in an ecologically relevant context.
Below we highlight several case studies that have integrated population genetic analyses of DNA sequence polymorphism with functional studies of specific gene products and/or whole-organism phenotypes to gain insight into the mechanistic basis of adaptation. There are a number of relevant case studies that could be reviewed in this context (for additional examples, see Eanes 1999; Watt and Dean 2000; Dean and Thornton 2007; Dalziel et al. 2009). We have chosen to highlight a few select examples in which evidence for selection on naturally occurring variation can be interpreted within a clear ecological context. In each of these case studies, the specific genes under investigation were chosen for study based on their known or predicted effects on fitness-related measures of organismal performance. Each of these studies succeeds in illuminating different pairwise links in the adaptive recursion, and each provides reasonably detailed (but still incomplete) descriptions of potential mechanisms of adaptation.
PGI POLYMORPHISM, FLIGHT PERFORMANCE, AND DISPERSAL ABILITY IN BUTTERFLIES
Metabolic performance can affect organismal fitness through the allocation of metabolic currencies to competing bioenergetic demands such as growth and reproduction (Watt 1986). Measures of metabolic performance that may have fitness consequences include the rate of pathway flux and the efficiency of flux (i.e., productivity of the pathway relative to the energy demands of maintenance metabolism; Clark and Koehn 1992; Watt and Dean 2000). The study of phosphoglucose isomerase (PGI) polymorphism in alfalfa butterflies (Colias eurytheme) provides a classic example of adaptive genetic variation in metabolic performance, and provides a mechanistic description of how differences in metabolic performance translate into fitness differences under natural conditions (Watt 1977, 1983; Watt et al. 1983).
In Colias butterflies, genetic variation at PGI affects the transient-state resupply of ATP to flight muscle via glycolysis (Watt 1977, 1983; Watt and Dean 2000). Natural populations of C. eurytheme segregate multiple electophoretically distinguishable PGI alleles, and the various diploid genotypes vary several-fold in measures of enzyme function such as Vmax/Km, where Vmax is the maximum velocity of the enzyme-catalyzed reaction under substrate-saturating conditions, and Km is the Michaelis rate constant for the reaction. The three most common PGI genotypes (3/3, 3/4, and 4/4) exhibit significant differences in enzyme kinetics at temperatures that fall below the thermal optimum for butterfly flight (35–39°C; Watt 1983). At suboptimal temperatures, 3/4 heterozygotes exhibit elevated Vmax/Km relative to the 3/3 and 4/4 homozygotes. Importantly, PGI enzymes of the two alternative homozygotes are not equal due to a trade-off between catalytic activity and thermal stability. As predicted by the observed genotypic differences in biochemical performance, PGI 3/4 heterozygotes are capable of maintaining active flight during a larger fraction of the daily thermal cycle, followed closely by 3/3 and then 4/4 (Watt et al. 1983, 1996). These effects of PGI genotype on flight capacity are expected to have important consequences for both female fecundity (because females lay eggs one at a time and are actively flying between each successive egg-laying) and male mating success (by determining opportunities for mating with flight-active females). Consistent with predictions based on metabolic performance, measures of female fecundity in the field revealed a rank-order of PGI genotypes that was concordant with the rank-order of Vmax/Km values: 3/4 > 3/3 ≫ 4/4 (Watt 1992). Field studies of male mating success exploited the phenomenon of sperm precedence, where the last male to mate with a given female fertilizes all the eggs (Boggs and Watt 1981; Carter and Watt 1988). Similar to the documented rank-order of PGI genotypes with respect to female fecundity, the distribution of PGI genotypes in progeny arrays revealed an overrepresentation of kinetically effective genotypes among contributing sires relative to all males in the general population (Watt et al. 1985, 1986). In summary, there is a striking concordance between the rank-order of biochemical performance among PGI genotypes and field-based measurements of survival and reproductive success in both males and females (Watt 2003).
Surveys of DNA sequence variation at the Pgi gene revealed the specific amino acid substitutions that distinguish the kinetically distinct electromorphs, and homology-based modeling of the PGI protein structure provided insights into the structural mechanism that may be responsible for the overdominance of enzyme kinetics (Wheat et al. 2006). The primary candidate sites for molecular adaptation are a pair of charge-changing amino acid substitutions that are located in an exterior, interhelical loop that is positioned at the interface between subunits of the dimeric enzyme (Fig. 2A). These amino acid changes may alter subunit interactions and/or stereochemistry of the catalytic center (Wheat et al. 2006). Consistent with a causal role for these two substitutions, a sliding window analysis revealed a significant excess of intermediate-frequency silent-site polymorphisms in the specific region of exon 9 that harbors both nonsynonymous changes (Fig. 2B), a pattern suggestive of long-term balancing selection.
Figure 2. Polymorphic amino acid residues in the PGI enzyme of Colias eurytheme, and variation in site-frequency spectra across the coding region of the underlying gene. (A) Homology-based model of a single PGI monomer (green) showing segregating amino acid sites (369 and 375) located in an exterior interhelical loop that is positioned at the intersubuint interface of the dimeric enzyme. This region is part of a peptide chain (yellow) that connects active site residues Glu 361 and His 392. (B) A sliding window analysis of Tajima's D based on synonymous site polymorphism across exons of the Pgi gene in C. eurytheme. Codons 369 and 375 are located in exon 9 (step size = 25bp and window length = 70bp, which is half the average exon length). Modified from Wheat et al. (2006).
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In addition to evolutionary and functional evidence for the adaptive maintenance of PGI polymorphism in C. eurytheme and other Colias species (Watt et al. 1996; Watt 2003), recent field-based studies of the Glanville fritillary butterfly, Melitaea cinxia, have also revealed evidence for balancing selection on the Pgi gene. Melitaea cinxia is common throughout Eurasia, and it has been intensively studied in the Åland archipelago in Finland where it persists as a classic metapopulation. Dispersal among habitat patches plays a key role in maintaining the viability of the metapopulation as a whole, as extinction–recolonization dynamics result in a rapid turnover of local demes (Hanski 1999; Hanski and Ovaskainen 2000; Nieminen et al. 2004; Ovaskainen and Hanski 2004).
The study of PGI polymorphism in the M. cinxia metapopulation has provided a number of important insights into the genetic basis of fitness variation under natural conditions. First, PGI variation is associated with flight capacity, female fecundity, and survivorship (Haag et al. 2005; Saastamoinen 2007; Klemme and Hanski 2009; Niitepõld et al. 2009; Orsini et al. 2009; Saastamoinen et al. 2009). Second, PGI genotypes associated with increased flight capacity and increased fecundity are also found at a higher frequency in isolated, newly established demes relative to older demes. Third, variation in PGI allele frequencies among demes is also correlated with variation in deme growth rates, which is a measure of realized fitness (Hanski and Saccheri 2006). Thus, variation in flight performance and fitness variation among PGI genotypes has ramifying effects on metapopulation dynamics, illustrating how studies of evolutionary mechanism can enrich our understanding of ecological process (Saccheri and Hanski 2006).
Consistent with evidence for overdominant selection on enzyme function in contemporary populations of M. cinxia (Haag et al. 2005; Hanski and Saccheri 2006; Niitepõld et al. 2009; Orsini et al. 2009), population genetic analysis of DNA sequence polymorphism at the Pgi gene revealed strong evidence for long-term balancing selection. The inferred age of the most recent common ancestor of the two main Pgi alleles within the Åland population predates the divergence times of five extant Melitaea species (Wheat et al. 2010). In both Colias and Melitaea, overdominant selection on PGI enzyme function appears to stem from the ability of heterozygotes to maintain flight activity over a broader range of ambient air temperatures (Watt et al. 1983, 1985, 1996; Watt 1992; Saastamoinen and Hanski 2008; Niitepõld et al. 2009). The documented genotype × temperature interaction on flight capacity of M. cinxia suggests that the PGI polymorphism of this species may be characterized by the same trade-off between catalytic activity and thermal stability that has been documented for the PGI polymorphism in Colias (Saastamoinen and Hanski 2008; Niitepõld et al. 2009). In addition, comparative analysis of Pgi structures in M. cinxia and C. eurytheme suggests that a similar structural mechanism may underlie overdominance of enzyme kinetics and metabolic performance in both species (Wheat et al. 2010). The survey of nucleotide variation in the Pgi gene of M. cinxia revealed that the two most common allele classes were distinguished by a pair of charge-changing amino acid substitutions in the same interhelical loop domain identified in C. eurytheme butterflies, although the specific sequence changes were different in each species. A detailed investigation of PGI enzyme kinetics of M. cinxia will be necessary to determine whether a similar mechanism underlies the apparent overdominance of metabolic performance in both Colias and Melitaea.
HEMOGLOBIN POLYMORPHISM AND AEROBIC METABOLISM OF HIGH-ALTITUDE DEER MICE
The deer mouse (Peromyscus maniculatus) has the broadest altitudinal distribution of any North American mammal, and therefore represents an ideal study organism for investigating mechanisms of physiological adaptation to different elevational zones. Deer mice are remarkably abundant in alpine environments at elevations up to 4300 m, where the partial pressure of O2 (PO2) is ∼60% of the sea level value. At such altitudes, the reduced PO2 of inspired air results in a reduced O2 saturation of arterial blood (hypoxemia), which in turn leads to a reduced supply of O2 to the cells of aerobically metabolizing tissues. In the absence of other compensatory physiological adjustments, this hypoxia-induced hypoxemia can impose severe constraints on aerobic metabolism and may therefore influence an animal's food and water requirements, the capacity for sustained locomotor activity, and the capacity for internal heat production.
Evidence from a number of vertebrate species indicates that physiological adaptation to high-altitude hypoxia often involves fine-tuned adjustments in blood-O2 affinity (Storz and Moriyama 2008). Under conditions of extreme hypoxia when pulmonary O2 loading is at a premium, an increased blood-O2 affinity helps maximize the level of tissue oxygenation for a given difference in PO2 between the sites of O2 loading in the pulmonary capillaries and the sites of O2 unloading in the tissue capillaries. Studies of deer mice have demonstrated that the divergent fine-tuning of blood-O2 affinity plays an important role in adaptation to different elevational zones (Storz 2007). Surveys of natural variation revealed that blood-O2 affinity is positively correlated with the native altitude of different deer mouse subspecies (Snyder 1981, 1985; Snyder et al. 1982, 1988), and physiological studies of wild-derived strains of deer mice revealed that this variation in blood biochemistry is strongly associated with allelic variation at two tandemly duplicated genes that encode the α-chain subunits of adult hemoglobin (Hb; Chappell and Snyder 1984; Chappell et al. 1988).
Remarkably, phenotypic effects of this two-locus α-globin polymorphism are also manifest at the level of whole-animal physiological performance, as measured by maximal rates of O2 consumption (VO2max) elicited by aerobic exercise or cold exposure. Both measures of aerobic power output exhibited consistent variation among strains of mice with different α-globin genotypes: mice with the high-affinity α-globin genotype exhibited a higher VO2max when tested under hypoxic conditions, whereas mice with the low-affinity genotype exhibited a higher VO2max when tested under normoxic conditions at sea level, and double heterozygotes were characterized by intermediate measures of aerobic performance under both treatments (Chappell and Snyder 1984; Chappell et al. 1988). This genetically based variation in VO2max can be expected to have important fitness consequences in alpine environments because mice that are capable of attaining a higher VO2max under hypoxia can maintain a constant body temperature by means of aerobic thermogenesis at lower ambient temperatures. Physiological studies have revealed that high-altitude deer mice are often operating close to their aerobic performance limits (Hayes 1989a, b), and even during summer months, thermogenic demands associated with low nighttime temperatures are sufficient to outstrip VO2max (Hayes and O’Connor 1999). Because VO2max is impaired under hypoxic conditions (Rosenmann and Morrison 1975; Chappell et al. 2007), small endotherms such as deer mice face a double bind as their thermogenic capacity is compromised under conditions in which thermoregulatory demands are especially severe.
Consistent with these expected effects on fitness, a survivorship study of high-altitude deer mice in the White Mountains of eastern California revealed strong directional selection on thermogenic capacity (Hayes and O’Connor 1999). In one season that was characterized by especially high spring snow-melt, the magnitude of the standardized, directional selection gradient on thermogenic capacity (0.523) represents one of the largest linear selection gradients ever measured in a free-ranging vertebrate species (Kingsolver et al. 2001). The results of Hayes and O’Connor (1999) indicate that, during periods of extreme cold, the average survivor would have been able to stave off hypothermia at air temperatures 1–2°C lower than the average nonsurvivor.
This system represents a rare case in which it has been possible to establish a mechanistic connection between allelic variation in protein function and fitness-related variation in whole-animal physiological performance. More recently, physiological studies of highland and lowland deer mice documented that variation in Hb-O2 affinity is also strongly associated with allelic variation at two tandemly duplicated genes that encode the β-chain subunits of the Hb tetramer (Storz et al. 2009, 2010). These causal connections between genotype, phenotype, and fitness have helped to illuminate the role of natural selection in shaping altitudinal variation in allele frequencies. Patterns of nucleotide diversity and LD at the two duplicated α-globin genes (HBA-T1 and HBA-T2) and the two duplicated β-globin genes (HBB-T1 and HBB-T2) are clearly indicative of local adaptation to different elevational zones (Storz et al. 2007, 2009; Storz and Kelly 2008). The divergent fine-tuning of Hb-O2 affinity appears to be attributable to the combined effects of eight amino acid mutations in the α-chains and four amino acid mutations in the β-chains, yielding a total of 12 candidate sites for molecular adaptation (Fig. 3; Storz et al. 2010).
Figure 3. Homology-based structural model of deer mouse hemoglobin (Hb) showing the location of 12 polymorphic amino acid sites that exhibit allele frequency differences between high- and low-altitude populations. Mutations in the α- and β-chain subunits are shown in panels A and B, respectively. These represent candidate sites for the adaptive fine-tuning of Hb-O2 affinity between highland and lowland populations. Based on data reported in Storz et al. (2009, 2010).
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Measurements of O2-equilibrium curves of purified Hbs revealed that allelic differences in Hb-O2 affinity were attributable to a suppressed sensitivity to the inhibitory effects of 2,3-diphosphoglycerate (DPG) and Cl− ions, allosteric cofactors that preferentially bind to sites in deoxyHb (Storz et al. 2009, 2010). Because the binding of DPG and Cl− ions helps stabilize the low-affinity deoxyHb quaternary structure, a suppressed sensitivity to both cofactors results in an increased O2 affinity by shifting the allosteric equilibrium in favor of the high-affinity oxyHb conformation. Comparisons between matched pairs of high- and low-altitude mice with Hbs containing the same α-chains but different β-chains revealed that the suppressed DPG sensitivity was associated with the two-locus β-globin haplotype that predominates in the high altitude population (Fig. 4). This important physiological property is therefore attributable to the independent or joint effects of four amino acid mutations that distinguish the alternative β-globin alleles.
Figure 4. O2-equilibrium curves of deer mouse hemoglobins showing allelic differences in Hb-O2 affinity. (A) Curves for high-altitude mice that express the βII Hb isoform (product of the d1β-globin allele) in the presence and absence of allosteric cofactors (2,3-DPG and Cl− ions); (B) Curves for low-altitude mice that express the βI Hb isoform (product of the d0β-globin allele) under the same experimental treatments; (C) Summary of allelic differences in O2 affinity and cofactor sensitivity between the β-chain Hb isoforms of high- and low-altitude mice (P50 is the PO2 at which Hb is 50% saturated). Based on data reported in Storz et al. (2009).
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The discovery that allelic differences in anion sensitivity contribute to the adaptive fine-tuning of Hb-O2 affinity illustrates the value of integrating evolutionary analyses of sequence variation with mechanistic appraisals of protein function. The population genetic analysis revealed evidence that the observed patterns of β-globin polymorphism have been shaped by a history of divergent selection between elevational zones, and this result motivated experimental investigations into the functional significance of the allelic variation. Experimental measures of O2-binding properties corroborated the tests of selection by demonstrating a functional difference between the products of alternative alleles.
EDA POLYMORPHISM AND ARMOR PLATING IN FRESHWATER STICKLEBACKS
The parallel loss of armor plating in multiple, independently derived freshwater populations of threespine stickleback fish (Gasterosteus aculeatus) has been the focus of a highly integrative, multiteam research enterprise that has yielded important insights into the genetic basis of morphological evolution (Colosimo et al. 2004, 2005; Cresko et al. 2004; Hohenlohe et al. 2010), as well as the complex chain of causal relationships between genotype, phenotype, and fitness (Barrett et al. 2008). Since the end of the last glacial maximum, threespine sticklebacks have independently colonized multiple postglacial, freshwater lake and stream systems in the Northern Hemisphere. These freshwater populations have consistently evolved a reduction in the number of external bony lateral plates relative to their marine counterparts. Comparative mapping experiments implicated the same major-effect QTL in the parallel loss of armor plating in multiple populations (Colosimo et al. 2004; Cresko et al. 2004) and subsequent fine-mapping and transgenic experiments suggested a causal role for the derived “low” allele of the Ectodysplasin-A (Eda) gene (Colosimo et al. 2005). Population surveys of nucleotide polymorphism in the Eda gene revealed that the low allele is present at low frequency in the ancestral marine population of sticklebacks, phylogenetic analysis suggested that the age of the low allele vastly predates the postglacial colonization of freshwater habitats, and a high-density SNP-based genome scan revealed that the Eda gene region has been subject to positive directional selection in multiple, independently derived freshwater populations (Colosimo et al. 2005; Hohenlohe et al. 2010). These lines of evidence indicate that the parallel evolution of reduced armor plating in different freshwater populations has been driven by repeated selection on standing genetic variation.
Armed with information about the phenotypic effects of alternative Eda alleles, Barrett et al. (2008) conducted a transplantation experiment to obtain direct measurements of fitness variation among alternative Eda genotypes under natural conditions. The experiment was designed to test the hypothesis that reduced armor plating confers a fitness advantage in freshwater sticklebacks by permitting a reallocation of energy toward juvenile growth. An increased rate of juvenile growth appears to enhance a stickleback's prospects for overwinter survival and early reproduction in the following spring. Barrett et al. (2008) phenotyped thousands of marine sticklebacks from coastal British Columbia to identify partially plated individuals that were likely to be heterozygous for the low allele and the wild-type “complete” allele. After using DNA markers to determine the Eda genotypes of wild-caught fish, a total of 182 low/complete heterozygotes were then transplanted to replicate ponds and Eda genotype frequencies were monitored over the course of a full annual cycle (=1 stickleback generation). As predicted, the low Eda allele was associated with higher rates of juvenile growth and overwinter survival, and over the course of the annual cycle the low allele underwent a parallel net increase in frequency across each of the replicate ponds. Surprisingly, however, the increase in net frequency of the low allele was primarily attributable to overdominance of fitness at the Eda gene after the fish had attained the final adult number of lateral plates, roughly midway through the annual cycle (Fig. 5). During the earlier stage of ontogenetic development when plate number was not yet finalized, low/complete Eda heterozygotes actually had lower fitness than either of the alternative homozygotes.
Figure 5. Temporal changes in allele and genotype frequencies at the Eda gene in four replicate freshwater populations of threespined stickleback. (A) Changes in frequency of the “low plated”Eda allele in four replicate ponds (different colored lines). All samples are from the first (F1) cohort of offspring, except the June and July 2007 samples, which are from the second (F2) pond generation. (B). Approximate life-history stages through the course of the experiment. Fish were stained with Alizarin red to highlight bone. (C). Genotype frequencies averaged across all four ponds. Purple, homozygous complete genotype (CC); orange, heterozygote genotype (CL); green, homozygote low genotype (LL). Vertical bars show standard errors on the basis of n= 4 ponds. From Barrett et al. (2008).
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This ontogenetic shift in the dominance of fitness was not expected under the “burden of plates” hypothesis, because Eda heterozygotes are characterized by an intermediate level of armor plating relative to the low/low and complete/complete homozygotes. This shift in the dominance of fitness during ontogeny—coupled with the reversal of fitness ranks between the two alternative homozygotes—produced parallel oscillations in allele frequency across all four replicate ponds. These parallel oscillations suggest the possibility of antagonistic pleiotropy, where Eda alleles have opposing fitness effects on different traits at different stages of development. Alternatively, the seasonal changes in frequency of the low allele may reflect a correlated response to selection on other loci that are in LD with the Eda gene. It seems clear that the overall fitness effects of Eda are not solely determined by differences in the level of armor plating. This study demonstrates the power of ecological experiments to reveal causes and mechanisms of fitness variation under natural conditions.
The study also provides a rare example of a case in which it was actually possible to measure fitness variation among alternative genotypes in real time. However, in this particular case the striking patterns of parallel evolution that had been documented in stream systems throughout the northern hemisphere left little doubt that the armor-plating phenotype was subject to strong directional selection in freshwater environments. It is not clear whether this same experimental approach can generally be expected to reveal detectable levels of fitness variation in cases where the adaptive significance of a trait is not obvious from the start.