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.

Coyne JA, Hoekstra HE. Evolution of protein expression: New genes for a new diet. Current Biology 2007;17:R1014-6.Abstract

A new study identifies gene duplication of a salivary enzyme as a recent adaptation to changes in diet among human populations, highlighting the diverse ways that gene regulation can evolve.

Hoekstra HE, Coyne JA. The locus of evolution: Evo devo and the genetics of adaptation. Evolution 2007;61:995-1016.Abstract

An important tenet of evolutionary developmental biology ("evo devo") is that adaptive mutations affecting morphology are more likely to occur in the cis-regulatory regions than in the protein-coding regions of genes. This argument rests on two claims: (1) the modular nature of cis-regulatory elements largely frees them from deleterious pleiotropic effects, and (2) a growing body of empirical evidence appears to support the predominant role of gene regulatory change in adaptation, especially morphological adaptation. Here we discuss and critique these assertions. We first show that there is no theoretical or empirical basis for the evo devo contention that adaptations involving morphology evolve by genetic mechanisms different from those involving physiology and other traits. In addition, some forms of protein evolution can avoid the negative consequences of pleiotropy, most notably via gene duplication. In light of evo devo claims, we then examine the substantial data on the genetic basis of adaptation from both genome-wide surveys and single-locus studies. Genomic studies lend little support to the cis-regulatory theory: many of these have detected adaptation in protein-coding regions, including transcription factors, whereas few have examined regulatory regions. Turning to single-locus studies, we note that the most widely cited examples of adaptive cis-regulatory mutations focus on trait loss rather than gain, and none have yet pinpointed an evolved regulatory site. In contrast, there are many studies that have both identified structural mutations and functionally verified their contribution to adaptation and speciation. Neither the theoretical arguments nor the data from nature, then, support the claim for a predominance of cis-regulatory mutations in evolution. Although this claim may be true, it is at best premature. Adaptation and speciation probably proceed through a combination of cis-regulatory and structural mutations, with a substantial contribution of the latter.

Mullen LM, Hirschmann RJ, Prince KL, Glenn TC, Dewey MJ, Hoekstra HE. Sixty polymorphic microsatellite markers for the oldfield mouse developed in Peromyscus polionotus and Peromyscus maniculatus. Molecular Ecology 2006;6:36-40.Abstract

We isolated and characterized 60 novel microsatellite markers from the closely related oldfield mouse (Peromyscus polionotus) and deer mouse (Peromyscus maniculatus) for studies of conservation, ecological, quantitative and population genetics. We assessed all 60 markers in a wild population of Peromyscus polionotus rhoadsi (N = 20) from central Florida and found an average of nine alleles per marker and an observed heterozygosity (HO) of 0.66 (range = 0.00–1.00). These polymorphic markers contribute to the growing number of genomic resources for Peromyscus, an emerging model system for ecological and evolutionary research.

Turner LM, Hoekstra HE. Adaptive evolution of fertilization proteins within a genus: Variation in ZP2 and ZP3 in deer mice (Peromyscus). Molecular Biology and Evolution 2006;23:1656-69.Abstract

Rapid evolution of reproductive proteins has been documented in a wide variety of taxa. In internally fertilized species, knowledge about the evolutionary dynamics of these proteins between closely related taxa is primarily limited to accessory gland proteins in the semen of Drosophila. Investigation of additional taxa and functional classes of proteins is necessary in order to determine if there is a general pattern of adaptive evolution of reproductive proteins between recently diverged species. We performed an evolutionary analysis of 2 egg coat proteins, ZP2 and ZP3, in 15 species of deer mice (genus Peromyscus). Both of these proteins are involved in egg-sperm binding, a critical step in maintaining species-specific fertilization. Here, we show that Zp2 and Zp3 gene trees are not consistent with trees based on nonreproductive genes, Mc1r and Lcat, where species formed monophyletic clades. In fact, for both of the reproductive genes, intraspecific amino acid variation was extensive and alleles were sometimes shared across species. We document positive selection acting on ZP2 and ZP3 and identify specific amino acid sites that are likely targets of selection using both maximum likelihood approaches and patterns of parallel amino acid change. In ZP3, positively selected sites are clustered in and around the region implicated in sperm binding in Mus, suggesting changes may impact egg-sperm binding and fertilization potential. Finally, we identify lineages with significantly elevated rates of amino acid substitution using a Bayesian mapping approach. These findings demonstrate that the pattern of adaptive reproductive protein evolution found at higher taxonomic levels can be documented between closely related mammalian species, where reproductive isolation has evolved recently.

Hoekstra HE. Genetics, development and evolution of adaptive pigmentation in vertebrates. Heredity 2006;97:222-34.Abstract

The study of pigmentation has played an important role in the intersection of evolution, genetics, and developmental biology. Pigmentation's utility as a visible phenotypic marker has resulted in over 100 years of intense study of coat color mutations in laboratory mice, thereby creating an impressive list of candidate genes and an understanding of the developmental mechanisms responsible for the phenotypic effects. Variation in color and pigment patterning has also served as the focus of many classic studies of naturally occurring phenotypic variation in a wide variety of vertebrates, providing some of the most compelling cases for parallel and convergent evolution. Thus, the pigmentation model system holds much promise for understanding the nature of adaptation by linking genetic changes to variation in fitness-related traits. Here, I first discuss the historical role of pigmentation in genetics, development and evolutionary biology. I then discuss recent empirically based studies in vertebrates, which rely on these historical foundations to make connections between genotype and phenotype for ecologically important pigmentation traits. These studies provide insight into the evolutionary process by uncovering the genetic basis of adaptive traits and addressing such long-standing questions in evolutionary biology as (1) are adaptive changes predominantly caused by mutations in regulatory regions or coding regions? (2) is adaptation driven by the fixation of dominant mutations? and (3) to what extent are parallel phenotypic changes caused by similar genetic changes? It is clear that coloration has much to teach us about the molecular basis of organismal diversity, adaptation and the evolutionary process.

Hoekstra HE, Hirschmann RJ, Bundey RA, Insel PA, Crossland JP. A single amino acid mutation contributes to adaptive beach mouse color pattern. Science 2006;313:101-4.Abstract

Natural populations of beach mice exhibit a characteristic color pattern, relative to their mainland conspecifics, driven by natural selection for crypsis. We identified a derived, charge-changing amino acid mutation in the melanocortin-1 receptor (Mc1r) in beach mice, which decreases receptor function. In genetic crosses, allelic variation at Mc1r explains 9.8% to 36.4% of the variation in seven pigmentation traits determining color pattern. The derived Mc1r allele is present in Florida's Gulf Coast beach mice but not in Atlantic coast mice with similar light coloration, suggesting that different molecular mechanisms are responsible for convergent phenotypic evolution. Here, we link a single mutation in the coding region of a pigmentation gene to adaptive quantitative variation in the wild.

Hoekstra HE, Nachman MW. Coat-color variation in rock pocket mice (Chaetopidus intermedius): from phenotype to genotype. Zoology 2005;133:79-99.Abstract


In a series of classic studies in mammalian evolutionary biology, Sumner (1921), Benson (1933), and Dice and Blossom (1937) described striking coat color variation in the rock pocket mouse, Chaetodipus intermedius, in the deserts of Arizona and New Mexico. These authors showed that C. intermedius coat color typically matches the color of the rocks on which the mice live; the dorsal pelage varies from a light, sandy color for populations found on some granites to a dark, nearly black color for populations found on basalt lava flows. Dice and Blossom (1937) suggested that this crypsis is an adaptation to avoid predation. Motivated by the wealth of data on the genetics, biochemistry, and molecular biology of the pigmentation process, we have used a candidate-gene approach to identify the genetic basis of adaptive coat color variation in C. intermedius. We review our recent studies on this topic with emphasis on the following key results: the identification of a single gene (the melanocortin-1-receptor, Mc1r) in one population that appears to be largely responsible for color differences, the balance between selection and migration among neighboring melanic and light races, and the finding that melanism has evolved independently on different lava flows through changes at different genes. 



Hoekstra HE, Krenz JG, Nachman MW. Local adaptation in the rock pocket mouse (Chaetodipus intermedius): Natural selection and phylogenetic history of populations. Heredity 2005;94:217-28.Abstract

Elucidating the causes of population divergence is a central goal of evolutionary biology. Rock pocket mice, Chaeotdipus intermedius, are an ideal system in which to study intraspecific phenotypic divergence because of the extensive color variation observed within this species. Here, we investigate whether phenotypic variation in color is correlated with local environmental conditions or with phylogenetic history. First, we quantified variation in pelage color (n=107 mice) and habitat color (n=51 rocks) using a spectrophotometer, and showed that there was a correlation between pelage color and habitat color across 14 sampled populations (R2=0.43). Analyses of mtDNA sequences from these same individuals revealed strong population structure in this species across its range, where most variation (63%) was partitioned between five geographic regions. Using Mantel tests, we show that there is no correlation between color variation and mtDNA phylogeny, suggesting that pelage coloration has evolved rapidly. At a finer geographical scale, high levels of gene flow between neighboring melanic and light populations suggest the selection acting on color must be quite strong to maintain habitat-specific phenotypic distributions. Finally, we raise the possibility that, in some cases, migration between populations of pocket mice inhabiting different lava flows may be responsible for similar melanic phenotypes in different populations. Together, the results suggest that color variation can evolve very rapidly over small geographic scales and that gene flow can both hinder and promote local adaptation.