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The genetics of adaptation is a key problem in evolutionary biology. Pocket gophers of the species Thomomys bottae provide one of the most striking examples of coat color variation in mammals. Dorsal pelage color is strongly correlated with soil color across the range of the species, presumably reflecting the selective pressure exerted by predation. To investigate the genetic basis of coat color variation in T. bottae, we cloned and sequenced the melanocortin-1 receptor locus (Mc1r), a candidate pigmentation gene, in 5 dark and 5 light populations of the species. Our results show that, in contrast to many other species of mammals and other vertebrates, coding variation at Mc1r is not the main determinant of coat color variation in T. bottae. These results demonstrate that similar phenotypic variation may have a different genetic basis among different mammalian species.
A basic goal in evolutionary genetics is to find the genes underlying adaptive traits. Finding the genes underlying adaptations might allow us to answer a number of questions. For example, do adaptive phenotypes derive mainly from changes in gene structure or gene regulation? Does adaptation usually involve one or a few mutations of major effect or many mutations of small effect? What kinds of genes are involved? Are similar phenotypes produced by similar genetic changes? Finding genes involved in adaptation has been difficult and many of the best examples come from response to human disturbance (e.g., antibacterial drug resistance: Walsh 2000; insecticide resistance: Daborn et al. 2002). One promising approach is to choose ecologically important phenotypes for which a clear set of candidate genes exists (Palopoli and Patel 1996).
Thomomys bottae is a species of pocket gopher in the southwestern United States and northwestern Mexico that exhibits a high degree of geographic variation, both morphologically and genetically (Patton and Smith 1990). In particular, these gophers exhibit dramatic variation in the color of the dorsal pelage, and this color variation is strongly correlated with soil color across the range of the species (e.g., Ingles 1950; Patton and Smith 1990), presumably due to selection from predation. Avian predators, including owls, are known to prey upon Thomomys (Fassler and Leavitt 1975; Janes and Barss 1985; Cutler and Hays 1991; Young et al. 1997), and several species of owls discriminate between light and dark mice on light and dark backgrounds in experimental tests, even under very low light intensities (Dice 1947).
A large amount of information exists on the genetic, developmental, and biochemical details underlying pigmentation (reviewed in Bennett and Lamoreux 2003), making this an excellent phenotype to explore using a candidate gene approach. In mammals, pigment is produced in specialized cells known as melanocytes, and there are 2 basic kinds of pigment: eumelanin (dark brown or black) and phaeomelanin (light cream, yellow, or red). A key switch between the production of these 2 kinds of pigment involves the interaction of 3 proteins: the agouti signalling protein (ASP), alpha melanocyte stimulating hormone (α-MSH), and the melanocortin-1 receptor (MC1R). When α-MSH binds to MC1R, the latter is activated and eumelanin is produced. ASP, on the other hand, is an antagonist of MC1R and when expressed phaeomelanin is produced. In a surprisingly large number of vertebrate species, mutations in Mc1r have been shown to cause changes from light to dark color over much of the body (e.g., cattle: Klungland et al. 1995; mouse: Barsh 1996; horse: Marklund et al. 1996; chicken: Takeuchi et al. 1996; fox: Vage et al. 1997; pig: Kijas et al. 1998; sheep: Vage et al. 1999; dog: Newton et al. 2000; black bear: Ritland et al. 2001; bananaquit: Theron et al. 2001; jaguar and jaguarundi: Eizirik et al. 2003; pocket mouse: Nachman et al. 2003; and lesser snow geese and arctic skuas: Mundy et al. 2004). The common finding of a role for Mc1r in color variation is probably due in part to publication bias. Mc1r is a single-exon gene that is easily studied and has been investigated far more than other pigmentation genes. There are some cases, however, in which Mc1r does not seem to be involved in color differences (pocket mice: Hoekstra and Nachman 2003; old-world leaf warblers: MacDougall-Shackleton et al. 2003; and some primates: Mundy and Kelly 2003), although at least in the case of warblers the phenotypic differences investigated were the presence or absence of unmelanized pattern elements rather than overall body coloration. In spite of these negative results, the widespread role of Mc1r in coat color evolution in many species, and the fact that some of the phenotypic changes described in these species resemble T. bottae color phenotypes, make Mc1r a reasonable candidate gene for the variation in pelage color in T. bottae. We note that other genes, particularly Agouti, may also be good candidates.
Here, we have cloned and sequenced the entire Mc1r gene in pocket gophers from 10 populations, representing 5 paired light and dark localities across the range of the species. We found no association between Mc1r variation and coat color, in contrast to many other vertebrates, suggesting that similar phenotypes may evolve through changes at different genes in different species.
Fifty-two individuals were studied (Appendix 1). Specimens were obtained from the Museum of Vertebrate Zoology at the University of California Berkeley and the Mammal Collection of the Department of Ecology and Evolutionary Biology at the University of Arizona, Tucson. The sampling was designed to simultaneously maximize dorsal color differences and minimize genetic distances between populations for each comparison. Five pairs of populations were chosen (Figure 1). Four of these pairs belong to different major intraspecific genetic units, defined previously by Patton and Smith (1990) based on allozyme allele frequencies. The fifth comparison (T.bottae. connectens and T.b. ruidosidae) involves populations from different genetic units (Patton and Smith 1990) that are very similar with respect to their mtDNA (Smith 1998). The maximum geographic distance between populations within pairs (T.b. awanhee and T.b. perpallidus) is 517 km. The rest of the populations (within pairs) are 85–424 km apart.
Genomic DNA was extracted from tissue samples preserved in ethanol using Qiagen (Valencia, CA) extraction kits. The entire Mc1r gene consists of a single exon and was isolated in gophers by polymerase chain reaction (PCR) amplifying and cloning a conserved central region and then using genome walking to capture the 5′ and 3′ ends. First, primers GWMc1rF: 5′-CTCTTYCTCDGCYTGGGGCT-3′ and GWMc1rR: 5′-ACCABRAGMAYDKYAGCACCT-3′ were designed in conserved regions of Mc1r across mammals to PCR amplify and sequence the middle portion of the coding sequence in T. bottae. This sequence was then used to design 2 pairs of species-specific nested primers (MC1RTB1R: 5′-AGTAGTACATGGGCGAGTGCAGGTTT-3′, MC1 RTB2R: 5′-AATCACTACCAGCACATTCTCCACCA-3′, MC1RTB1F: 5′-CTCCTGGGCATTTTCTTCTTATGCTG-3′, MC1RTB2F: 5′-TAACTCCATTGTTGACCCCCTCAT CT-3′) that were used to capture the 3′ and 5′ ends of Mc1r by genome walking (Universal Genome Walking Kit; Clonetech, Palo Alto, CA). A third set of primers (described below) flanking the coding region were then developed from the sequences in the previous step to amplify a fragment of 1123 bp.
The entire coding sequence of Mc1r was PCR amplified using the following primers: Mc1r3F: 5′-TGACACCAT-GAAATGAGCAG-3′ and Mc1r8R: 5′-CATAGGGAT-CAGGACACTGG-3′, under the following conditions: 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 65 °C for 1 min, preceded by a denaturizing step of 2 min at 94 °C, and followed by an extension step of 8 min at 65 °C. The diploid PCR products were sequenced with the same primers. Potential heterozygous sites were identified by visual inspection of the chromatograms and in most cases confirmed by sequencing the complementary strand.
Sequence data for the mitochondrial cytochrome b (Cyt b) gene were obtained for the 10 specimens of T.b. awahnee and T.b. perpallidus because in that comparison a statistically significant association was found between a replacement polymorphism in Mc1r and coat color. Primers MVZ69 and MVZ14 (Smith 1998) were used to PCR amplify the entire coding region (1140 bp), according to the conditions described by Smith and Patton (1991). MVZ69 and TBcytb14b (5′-GGTCTTCATCTYHGGYTTAC-3′) were used as sequencing primers.
In all cases, PCR products were sequenced on an ABI377 or ABI3700 automated sequencer (Applied Bio-systems, Foster City, CA). Sequences were edited and aligned using SEQUENCHER (Gene Codes, Ann Harbor, MI). Sequences were deposited in GenBank under the following accession numbers: Mc1r (EF488832–488875) and Cyt b (EF488876–488885).
Reflectance spectrophotometry was used to characterize dorsal coloration. Reflectance, calibrated to a white standard, was measured over the UV (200–400 nm) and visible (400–850 nm) spectra with a USB2000 spectrophotometer (Ocean Optics) coupled to a MiniDT1000 light source (Analytical Instrument Systems, Inc.). Five measurements were taken per individual on the dorsum and then combined into an average individual spectrum, as in Hoekstra et al. (2005). Total reflectance intensity (brightness) was obtained summing the total area (UV + visible) under the average reflectance curve.
Associations between Mc1r genotypes at each polymorphic site and phenotype (dark versus light) were tested in 3 × 2 contingency tables using Monte Carlo simulations. The 3 rows thus correspond to the 3 possible genotypes, and the 2 columns correspond to light versus dark animals.
To assess population structure, the statistic FST (Hudson et al. 1992) was calculated from the mitochondrial sequences using the software DNAsp (Rozas et al. 2003). This was done with the pair of populations showing strong association between variation at Mc1r and coat color.
The amino acid alignment of a consensus sequence of Mc1r and several other "wild-type" Mc1r mammalian sequences is shown in Appendix 2. The consensus length of Mc1r in vertebrates is 954 bp. In T. bottae, we observed a deletion of 21 bp (in frame) in the first extracellular region, resulting in a gene length of 933 bp (including the stop codon). The first extracellular region constitutes the most variable portion of the protein among vertebrates, probably indicating low functional constraints.
The distribution of nucleotide variation within and among different pairs of populations is presented in Figure 2. We observed 18 single-nucleotide polymorphisms, of which 6 resulted in nonsynonymous changes. The overall level of nucleotide variability (π = 0.25%) was slightly higher than seen in other mammals at Mc1r (e.g., humans, π = 0.1%: Harding et al. 2000; pocket mice, π = 0.21%: Nachman et al. 2003).
Despite this variability, there was no consistent association between replacement polymorphisms (Figure 2) and coat color (Figure 1 and Table 1), suggesting that Mc1r is not a major determinant of coat color variation in this species.
In one pair of populations (T.b. perpallidus-T.b. awahnee), however, we did observe an association between Arg208Cys and dorsal color (Monte Carlo simulation P < 0.004). In spite of this significant association between Arg208Cys and dorsal color among these 10 animals, several observations suggest that this mutation is not causing the observed color differences. First, the amino acid associated with the light phenotype in T.b. perpallidus (e.g., specimen MVZ166253) is associated with the dark phenotype in another population (T.b. ruidosidae; e.g., specimen MVZ147056). Second, there are no obvious color differences between Arg/Cys and Cys/Cys among T.b. perpallidus. Individuals of both genotypes exhibit similar total reflectance intensities and reflectance peaks at the same wavelengths in the UV and visible spectra. This pattern would, in principle, be compatible with a dominant light mutation. However, all known dominance relationships among Mc1r alleles in other species show the reverse: dark alleles are dominant over light ones (e.g., Klungland et al. 1995; Vage et al. 1997, 1999; Kijas et al. 1998; Newton et al. 2000; Nachman et al. 2003). Third, T.b. perpallidus and T.b. awahnee represent the most distant comparison in our sample, in terms of both genetic and geographic distances. A spurious correlation between genotype and phenotype can be generated due to population structure. To test for population structure, we used mitochondrial cyt b sequences to calculate FST. The observed FST (0.988) confirms an extreme level of genetic differentiation between these populations. Therefore, the observed differences at Mc1r might simply reflect population divergence. Fourth, the Arg208Cys change lies at a site that is variable across mammals (Appendix 2) and is not associated with coat color polymorphisms in other species (Mundy 2005), suggesting that it may not be functionally important. Together these observations suggest that population structure may account for the association between the Arg208Cys genotype and color phenotype.
Thus, coding sequence variation in Mc1r does not appear to be the principal determinant of coat color differences in T. bottae. Our reduced sample sizes, however, only allow the identification of strong associations between phenotypic and genotypic variation. The possibility that weak associations might exist remains open. Similarly, despite the fact that so far associations between regulatory variation at Mc1r and coat color differences have not been described in other species, a role for variation in the regulatory region of Mc1r cannot be ruled out with this data.
Although there are a few documented cases in which Mc1r is not implicated in pigmentation differences (e.g., some dog breeds: Kerns et al. 2003; mustelids: Hosoda et al. 2005; and some populations of pocket mice: Hoekstra and Nachman 2003), for a wide range of taxa, Mc1r has been shown to be responsible for coat color differences both in domestic and wild species.
Why is Mc1r not involved in color variation in gophers? In many of the species in which Mc1r has been implicated in color variation, the color differences are relatively discrete rather than continuous. For example, in black bears, Mc1r mutations are associated with a light race, and intermediates have not been observed. Similarly, of 200 pocket mice captured on and adjacent to a lava flow in Arizona, all were easily categorized as light or dark, although some minor variation within the classes is also evident (Hoekstra et al. 2004). In cases such as these, Mc1r alleles seem to have large effects, and phenotypic variants presumably segregate roughly as Mendelian traits. In several situations, however, Mc1r mutations explain a smaller amount of phenotypic variation, and color variation is more quantitative. For example, in beach mice, Mc1r mutations account for 10–36% of the variation in pigmentation phenotypes (Hoekstra et al. 2006). Pocket gophers show nearly continuous variation from very dark to very light (Patton and Smith 1990). Mc1r mutations of large effect may not be tolerated in environments where selection is favoring incremental differences. A long-standing question in evolutionary biology is whether genes identified originally from laboratory mutations (such as Mc1r), most of which are of large effect, will also contribute to adaptive evolution in nature (e.g., Palopoli and Patel 1996). The results presented here suggest that Mc1r mutations of large effect have not contributed to adaptive differences among gopher populations. However, the continuous variation in coat color in pocket gophers suggests that this trait might have a polygenic basis. Finding the genes underlying this variation will likely be a more daunting task, requiring mapping and association studies involving many more markers and individuals.
We especially thank J.L. Patton for suggestions. We thank the Museum of Vertebrate Zoology, University of California, Berkeley, and the Mammal Collection at the University of Arizona, Tucson for providing the tissue samples and skin samples used in this study.
This work was funded by National Science Foundation (DEB 9981810) and National Institutes of Health (R01 GM074245-01 A1) grants to MWN.
Individuals are listed by population. Collection number is followed by sex: m, male; f, female.
T.b. albatus: MVZ 154176 (m), MVZ 154177 (f), MVZ 154178 (m), MVZ 156116 (f), MVZ 156117 (f), UA25203.
T.b. angularis: MVZ 156221 (f), MVZ 162159 (m), MVZ 162162 (f), MVZ 164595 (m), MVZ 164601 (f).
T.b. awahnee: MVZ 158712 (f), MVZ 158713 (f), MVZ 158718 (f), MVZ 158722 (f), MVZ 158726 (f).
T.b. catalinae: MVZ 146822 (m), MVZ 146823 (f), MVZ 146824 (m), MVZ 146825 (m), MVZ 146826 (f).
T.b. connectens: MVZ 150251 (m), MVZ 150260 (f), MVZ 150261 (m), MVZ 150262 (m), MVZ 150263 (m).
T.b. laticeps: MVZ 160608 (f), MVZ 160614 (f), MVZ 160618 (m), MVZ 160674 (m), MVZ 160682 (f).
T.b. navus: MVZ 162073 (m), MVZ 162075 (f), MVZ 163174 (f), MVZ 163176 (m), MVZ 163194 (f).
T.b. pascalis: MVZ 156161 (f), MVZ 156179 (f), MVZ 156180 (m), MVZ 162166 (f), MVZ 162863 (m).
T.b. perpallidus: MVZ 166250 (f), MVZ 166252 (m), MVZ 166253 (m), MVZ 166256 (m), MVZ 166257 (f), UA26596.
T.b. ruidosidae: MVZ 147023 (f), MVZ 147026 (f), MVZ 147027 (f), MVZ 147055 (f), MVZ 147056 (f).
Protein alignment of the consensus T. bottae Mc1r with several species of mammals (pig, AF326520; cow, U39469; dog, AF064455; horse, AF288357; human, AF326275; and pocket mouse, AY258992). Putative transmembrane portions are shaded. Amino acid position corresponding to the Arg208Cys replacement change is indicated with an arrow (position 208 in the T. bottae sequence corresponds to position 218 in the consensus sequence).