Hull JM, Mindell DP, Talbot SL, Kay EH, Hoekstra HE, Ernest HB. Population structure and plumage polymorphism: The intraspecific evolutionary relationships of a polymorphic raptor, Buteo jamaicensis harlani. BMC Evolutionary Biology 2010;10:224.Abstract

BACKGROUND: Phenotypic and molecular genetic data often provide conflicting patterns of intraspecific relationships confounding phylogenetic inference, particularly among birds where a variety of environmental factors may influence plumage characters. Among diurnal raptors, the taxonomic relationship of Buteo jamaicensis harlani to other B. jamaicensis subspecies has been long debated because of the polytypic nature of the plumage characteristics used in subspecies or species designations. RESULTS: To address the evolutionary relationships within this group, we used data from 17 nuclear microsatellite loci, 430 base pairs of the mitochondrial control region, and 829 base pairs of the melanocortin 1 receptor (Mc1r) to investigate molecular genetic differentiation among three B. jamaicensis subspecies (B. j. borealis, B. j. calurus, B. j. harlani). Bayesian clustering analyses of nuclear microsatellite loci showed no significant differences between B. j. harlani and B. j. borealis. Differences observed between B. j. harlani and B. j. borealis in mitochondrial and microsatellite data were equivalent to those found between morphologically similar subspecies, B. j. borealis and B. j. calurus, and estimates of migration rates among all three subspecies were high. No consistent differences were observed in Mc1r data between B. j. harlani and other B. jamaicensis subspecies or between light and dark color morphs within B. j. calurus, suggesting that Mc1r does not play a significant role in B. jamaicensis melanism. CONCLUSIONS: These data suggest recent interbreeding and gene flow between B. j. harlani and the other B. jamaicensis subspecies examined, providing no support for the historical designation of B. j. harlani as a distinct species.

Vignieri SN, Larson JG, Hoekstra HE. The selective advantage of crypsis in mice. Evolution 2010;64:2153-8.Abstract

The light color of mice that inhabit the sandy dunes of Florida's coast have served as a textbook example of adaptation for nearly a century, despite the fact that the selective advantage of crypsis has never been directly tested or quantified in nature. Using plasticine mouse models of light and dark color, we demonstrate a strong selective advantage for mice that match their local background substrate. Further our data suggest that stabilizing selection maintains color matching within a single habitat, as models that are both lighter and darker than their local environment are selected against. These results provide empirical evidence in support of the hypothesis that visual hunting predators shape color patterning in Peromyscus mice and suggest a mechanism by which selection drives the pronounced color variation among populations.

Metz HC, Manceau M, Hoekstra HE. Turing patterns: How the fish got its spots. Pigment Cell & Melanoma Research 2010;24:12-4. PDF
Hubbard JK, Uy JA, Hauber ME, Hoekstra HE, Safran RJ. Vertebrate pigmentation: From underlying genes to adaptive function. Trends in Genetics 2010;26:231-9.Abstract

Animal coloration is a powerful model for studying the genetic mechanisms that determine phenotype. Genetic crosses of laboratory mice have provided extensive information about the patterns of inheritance and pleiotropic effects of loci involved in pigmentation. Recently, the study of pigmentation genes and their functions has extended into wild populations, providing additional evidence that pigment gene function is largely conserved across disparate vertebrate taxa and can influence adaptive coloration, often in predictable ways. These new and integrative studies, along with those using a genetic approach to understand color perception, raise some important questions. Most notably, how does selection shape both phenotypic and genetic variation, and how can we use this information to further understand the phenotypic diversity generated by evolutionary processes?

Weber JN, Hoekstra HE. The evolution of burrowing behavior in deer mice. Animal Behavior 2009;77:603-09.Abstract

The evolutionary history of most behaviours remains unknown. Here, we assay burrowing behaviour of seven species of deer mice in standardized environments to determine how burrowing evolved in this genus (Peromyscus). We found that several, but not all, species burrow even after many generations of captive breeding. Specifically, there were significant and repeatable differences in both the frequency of burrowing and burrow shape between species. Moreover, these observed species-specific behaviours resemble those reported in wild mice. These results suggest that there is probably a strong genetic component to burrowing in deer mice. We also generated a phylogeny for these seven species using characters from four mtDNA and two autosomal loci. Mapping burrowing behaviour onto this phylogeny suggests a sequence for how complex burrowing evolves: from small, simple burrows to long, multitunnel burrows with defined entrance and escape tunnels. In particular, the most ‘complex’ burrows of P. polionotus appear to be derived. These behavioural data, when examined in a phylogenetic context, show that even closely related species differ in their burrowing behaviours and that the most complex burrows probably evolved by the gradual accumulation of genetic change over time. 2008 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Hoekstra HE. The Evolution Ringmaster: Review of The Greatest Show on Earth: The Evidence for Evolution by Richard Dawkins. Cell 2009;139:454-55. PDF
Berry AJ, Hoekstra HE. (Re)Reading The Origin. Current Biology 2009;19(3):R9. PDF
Mullen LM, Vignieri SN, Gore JA, Hoekstra HE. Adaptive basis of geographic variation: Genetic, phenotypic and environmental differences among beach mouse populations. Proceedings of the Royal Society B: Biological Sciences 2009;276:3809-18.Abstract

A major goal in evolutionary biology is to understand how and why populations differentiate, both genetically and phenotypically, as they invade a novel habitat. A classical example of adaptation is the pale colour of beach mice, relative to their dark mainland ancestors, which colonized the isolated sandy dunes and barrier islands on Florida's Gulf Coast. However, much less is known about differentiation among the Gulf Coast beach mice, which comprise five subspecies linearly arrayed on Florida's shoreline. Here, we test the role of selection in maintaining variation among these beach mouse subspecies at multiple levels-phenotype, genotype and the environments they inhabit. While all beach subspecies have light pelage, they differ significantly in colour pattern. These subspecies are also genetically distinct: pair-wise F(st)-values range from 0.23 to 0.63 and levels of gene flow are low. However, we did not find a correlation between phenotypic and genetic distance. Instead, we find a significant association between the average 'lightness' of each subspecies and the brightness of the substrate it inhabits: the two most genetically divergent subspecies occupy the most similar habitats and have converged on phenotype, whereas the most genetically similar subspecies occupy the most different environments and have divergent phenotypes. Moreover, allelic variation at the pigmentation gene, Mc1r, is statistically correlated with these colour differences but not with variation at other genetic loci. Together, these results suggest that natural selection for camouflage-via changes in Mc1r allele frequency-contributes to pigment differentiation among beach mouse subspecies.

Aminetzach YT, Srouji JR, Kong CY, Hoekstra HE. Convergent evolution of novel protein function in shrew and lizard venom. Current Biology 2009;19:1925-31.Abstract

How do proteins evolve novel functions? To address this question, we are studying the evolution of a mammalian toxin, the serine protease BLTX [1], from the salivary glands of the North American shrew Blarina brevicauda. Here, we examine the molecular changes responsible for promoting BLTX toxicity. First, we show that regulatory loops surrounding the BLTX active site have evolved adaptively via acquisition of small insertions and subsequent accelerated sequence evolution. Second, these mutations introduce a novel chemical environment into the catalytic cleft of BLTX. Third, molecular-dynamic simulations show that the observed changes create a novel chemical and physical topology consistent with increased enzyme catalysis. Finally, we show that a toxic serine protease from the Mexican beaded lizard (GTX) [2] has evolved convergently through almost identical functional changes. Together, these results suggest that the evolution of toxicity might be predictable-arising via adaptive structural modification of analogous labile regulatory loops of an ancestral serine protease-and thus might aid in the identification of other toxic proteins.

Steiner CC, Rompler H, Boettger LM, Schoneberg T, Hoekstra HE. The genetic basis of phenotypic convergence in beach mice: Similar pigment patterns but different genes. Molecular Biology and Evolution 2009;26:35-45.Abstract

Convergent evolution is a widespread phenomenon seen in diverse organisms inhabiting similar selective environments. However, it is unclear if similar phenotypes are produced by the same or different genes and mutations. Here we analyze the molecular mechanisms underlying convergent pigment pattern among subspecies of the beach mouse (Peromyscus polionotus) inhabiting the Gulf and Atlantic coasts of Florida. In these two geographic regions, separated by more than 300 km, "beach mice" have lighter colored coats than do their mainland counterparts, produced by natural selection for camouflage against the pale coastal sand dunes. We measured color pattern in eight beach mouse subspecies and showed that three of the Gulf Coast subspecies are more phenotypically similar to an Atlantic coast subspecies than to their Gulf Coast neighbors. However, light-colored beach mice do not form a monophyletic group. Previous results implicated a single derived amino acid change in the melanocortin-1 receptor (Mc1r) as a major contributor to pigment pattern in the Gulf Coast beach mice; despite phenotypic similarities, the derived Mc1r allele was not found in the Atlantic coast beach mouse populations. Here we show that Atlantic coast beach mice have high levels of Mc1r polymorphism but they lack unique alleles. Functional assays revealed that single amino acid mutations segregating in Atlantic coast beach mice do not cause any change in Mc1r activity compared with the activity of Mc1r from dark-colored mice. These joint results show that convergent pigment patterns in recently diverged beach mouse subspecies--whose developmental constraints are presumably similar--have evolved through a diversity of genetic mechanisms.

Kingsley EP, Manceau M, Wiley CD, Hoekstra HE. Melanism in Peromyscus is caused by independent mutations in Agouti. PLoS One 2009;4:e6435.Abstract

Identifying the molecular basis of phenotypes that have evolved independently can provide insight into the ways genetic and developmental constraints influence the maintenance of phenotypic diversity. Melanic (darkly pigmented) phenotypes in mammals provide a potent system in which to study the genetic basis of naturally occurring mutant phenotypes because melanism occurs in many mammals, and the mammalian pigmentation pathway is well understood. Spontaneous alleles of a few key pigmentation loci are known to cause melanism in domestic or laboratory populations of mammals, but in natural populations, mutations at one gene, the melanocortin-1 receptor (Mc1r), have been implicated in the vast majority of cases, possibly due to its minimal pleiotropic effects. To investigate whether mutations in this or other genes cause melanism in the wild, we investigated the genetic basis of melanism in the rodent genus Peromyscus, in which melanic mice have been reported in several populations. We focused on two genes known to cause melanism in other taxa, Mc1r and its antagonist, the agouti signaling protein (Agouti). While variation in the Mc1r coding region does not correlate with melanism in any population, in a New Hampshire population, we find that a 125-kb deletion, which includes the upstream regulatory region and exons 1 and 2 of Agouti, results in a loss of Agouti expression and is perfectly associated with melanic color. In a second population from Alaska, we find that a premature stop codon in exon 3 of Agouti is associated with a similar melanic phenotype. These results show that melanism has evolved independently in these populations through mutations in the same gene, and suggest that melanism produced by mutations in genes other than Mc1r may be more common than previously thought.

Linnen CR, Kingsley EP, Jensen JD, Hoekstra HE. On the origin and spread of an adaptive allele in deer mice. Science 2009;325:1095-8.Abstract

Adaptation is a central focus of biology, although it can be difficult to identify both the strength and agent of selection and the underlying molecular mechanisms causing change. We studied cryptically colored deer mice living on the Nebraska Sand Hills and show that their light coloration stems from a novel banding pattern on individual hairs produced by an increase in Agouti expression caused by a cis-acting mutation (or mutations), which either is or is closely linked to a single amino acid deletion in Agouti that appears to be under selection. Furthermore, our data suggest that this derived Agouti allele arose de novo after the formation of the Sand Hills. These findings reveal one means by which genetic, developmental, and evolutionary mechanisms can drive rapid adaptation under ecological pressure.

Abzhanov A, Extavour CG, Groover A, Hodges SA, Hoekstra HE, Kramer EM, Monteiro A. Are we there yet? Tracking the development of new model systems. Trends in Genetics 2008;24:353-60.Abstract

It is increasingly clear that additional 'model' systems are needed to elucidate the genetic and developmental basis of organismal diversity. Whereas model system development previously required enormous investment, recent advances including the decreasing cost of DNA sequencing and the power of reverse genetics to study gene function are greatly facilitating the process. In this review, we consider two aspects of the development of new genetic model systems: first, the types of questions being advanced using these new models; and second, the essential characteristics and molecular tools for new models, depending on the research focus. We hope that researchers will be inspired to explore this array of emerging models and even consider developing new molecular tools for their own favorite organism.

Turner LM, Hoekstra HE. Causes and consequences of the evolution of reproductive proteins. The International Journal of Developmental Biology 2008;52:769-80.Abstract

Proteins involved in reproduction often evolve rapidly, raising the possibility that changes in these proteins contribute to reproductive isolation between species. We review the evidence for rapid and adaptive change in reproductive proteins in animals, focusing on studies in recently diverged vertebrates. We identify common patterns and point out promising directions for future research. In particular, we highlight the ways that integrating the different but complementary approaches of evolutionary and developmental biology will provide new insights into fertilization processes.

Stinchcombe JR, Hoekstra HE. Combining population genomics and quantitative genetics: Finding the genes underlying ecologically important traits. Heredity 2008;100:158-70.Abstract

A central challenge in evolutionary biology is to identify genes underlying ecologically important traits and describe the fitness consequences of naturally occurring variation at these loci. To address this goal, several novel approaches have been developed, including 'population genomics,' where a large number of molecular markers are scored in individuals from different environments with the goal of identifying markers showing unusual patterns of variation, potentially due to selection at linked sites. Such approaches are appealing because of (1) the increasing ease of generating large numbers of genetic markers, (2) the ability to scan the genome without measuring phenotypes and (3) the simplicity of sampling individuals without knowledge of their breeding history. Although such approaches are inherently applicable to non-model systems, to date these studies have been limited in their ability to uncover functionally relevant genes. By contrast, quantitative genetics has a rich history, and more recently, quantitative trait locus (QTL) mapping has had some success in identifying genes underlying ecologically relevant variation even in novel systems. QTL mapping, however, requires (1) genetic markers that specifically differentiate parental forms, (2) a focus on a particular measurable phenotype and (3) controlled breeding and maintenance of large numbers of progeny. Here we present current advances and suggest future directions that take advantage of population genomics and quantitative genetic approaches - in both model and non-model systems. Specifically, we discuss advantages and limitations of each method and argue that a combination of the two provides a powerful approach to uncovering the molecular mechanisms responsible for adaptation.

Turner LM, Chuong EB, Hoekstra HE. Comparative analysis of testis protein evolution in rodents. Genetics 2008;179:2075-89.Abstract

Genes expressed in testes are critical to male reproductive success, affecting spermatogenesis, sperm competition, and sperm-egg interaction. Comparing the evolution of testis proteins at different taxonomic levels can reveal which genes and functional classes are targets of natural and sexual selection and whether the same genes are targets among taxa. Here we examine the evolution of testis-expressed proteins at different levels of divergence among three rodents, mouse (Mus musculus), rat (Rattus norvegicus), and deer mouse (Peromyscus maniculatus), to identify rapidly evolving genes. Comparison of expressed sequence tags (ESTs) from testes suggests that proteins with testis-specific expression evolve more rapidly on average than proteins with maximal expression in other tissues. Genes with the highest rates of evolution have a variety of functional roles including signal transduction, DNA binding, and egg-sperm interaction. Most of these rapidly evolving genes have not been identified previously as targets of selection in comparisons among more divergent mammals. To determine if these genes are evolving rapidly among closely related species, we sequenced 11 of these genes in six Peromyscus species and found evidence for positive selection in five of them. Together, these results demonstrate rapid evolution of functionally diverse testis-expressed proteins in rodents, including the identification of amino acids under lineage-specific selection in Peromyscus. Evidence for positive selection among closely related species suggests that changes in these proteins may have consequences for reproductive isolation.

Mullen LM, Hoekstra HE. Natural selection along an environmental gradient: A classic cline in mouse pigmentation. Evolution 2008;62:1555-70.Abstract

We revisited a classic study of morphological variation in the oldfield mouse (Peromyscus polionotus) to estimate the strength of selection acting on pigmentation patterns and to identify the underlying genes. We measured 215 specimens collected by Francis Sumner in the 1920s from eight populations across a 155-km, environmentally variable transect from the white sands of Florida's Gulf coast to the dark, loamy soil of southeastern Alabama. Like Sumner, we found significant variation among populations: mice inhabiting coastal sand dunes had larger feet, longer tails, and lighter pigmentation than inland populations. Most striking, all seven pigmentation traits examined showed a sharp decrease in reflectance about 55 km from the coast, with most of the phenotypic change occurring over less than 10 km. The largest change in soil reflectance occurred just south of this break in pigmentation. Geographic analysis of microsatellite markers shows little interpopulation differentiation, so the abrupt change in pigmentation is not associated with recent secondary contact or reduced gene flow between adjacent populations. Using these genetic data, we estimated that the strength of selection needed to maintain the observed distribution of pigment traits ranged from 0.0004 to 21%, depending on the trait and model used. We also examined changes in allele frequency of SNPs in two pigmentation genes, Mc1r and Agouti, and show that mutations in the cis-regulatory region of Agouti may contribute to this cline in pigmentation. The concordance between environmental variation and pigmentation in the face of high levels of interpopulation gene flow strongly implies that natural selection is maintaining a steep cline in pigmentation and the genes underlying it.

Turner LM, Hoekstra HE. Reproductive protein evolution within and between species: Maintenance of divergent ZP3 alleles in Peromyscus. Molecular Ecology 2008;17:2616-28.Abstract

In a variety of animal taxa, proteins involved in reproduction evolve more rapidly than nonreproductive proteins. Most studies of reproductive protein evolution, however, focus on divergence between species, and little is known about differentiation among populations within a species. Here we investigate the molecular population genetics of the protein ZP3 within two Peromyscus species. ZP3 is an egg coat protein involved in primary binding of egg and sperm and is essential for fertilization. We find that amino acid polymorphism in the sperm-combining region of ZP3 is high relative to silent polymorphism in both species of Peromyscus. In addition, while there is geographical structure at a mitochondrial gene (Cytb), a nuclear gene (Lcat) and eight microsatellite loci, we find no evidence for geographical structure at Zp3 in Peromyscus truei. These patterns are consistent with the maintenance of ZP3 alleles by balancing selection, possibly due to sexual conflict or pathogen resistance. However, we do not find evidence that reinforcement promotes ZP3 diversification; allelic variation in P. truei is similar among populations, including populations allopatric and sympatric with sibling species. In fact, most alleles are present in all populations sampled across P. truei's range. While additional data are needed to identify the precise evolutionary forces responsible for sequence variation in ZP3, our results suggest that in Peromyscus, selection to maintain divergent alleles within species contributes to the pattern of rapid amino acid substitution observed among species.

Kay EH, Hoekstra HE. Rodents. Current Biology 2008;18:R406-10. PDF
Steiner CC, Weber JN, Hoekstra HE. Adaptive variation in beach mice produced by two interacting pigmentation genes. PLoS Biology 2007;5:e219.Abstract

Little is known about the genetic basis of ecologically important morphological variation such as the diverse color patterns of mammals. Here we identify genetic changes contributing to an adaptive difference in color pattern between two subspecies of oldfield mice (Peromyscus polionotus). One mainland subspecies has a cryptic dark brown dorsal coat, while a younger beach-dwelling subspecies has a lighter coat produced by natural selection for camouflage on pale coastal sand dunes. Using genome-wide linkage mapping, we identified three chromosomal regions (two of major and one of minor effect) associated with differences in pigmentation traits. Two candidate genes, the melanocortin-1 receptor (Mc1r) and its antagonist, the Agouti signaling protein (Agouti), map to independent regions that together are responsible for most of the difference in pigmentation between subspecies. A derived mutation in the coding region of Mc1r, rather than change in its expression level, contributes to light pigmentation. Conversely, beach mice have a derived increase in Agouti mRNA expression but no changes in protein sequence. These two genes also interact epistatically: the phenotypic effects of Mc1r are visible only in genetic backgrounds containing the derived Agouti allele. These results demonstrate that cryptic coloration can be based largely on a few interacting genes of major effect.