Lewandowski JP, Dumbović G, Watson AR, Hwang T, Jacobs-Palmer E, Chang N, Much C, Turner K, Kirby C, Schulz JF, Muller C-L, Rubenstein ND, Groff AF, Liapis SC, Gerhardinger C, Hubner N, van Heesch S, Hoekstra HE, Sauvageau M, Rinn JL. The Tug1 locus is essential for male fertility. BioRxiv Forthcoming;Abstract
Several long noncoding RNAs (lncRNAs) have been shown to function as central components of molecular machines that play fundamental roles in biology. While the number of annotated lncRNAs in mammalian genomes has greatly expanded, their functions remain largely uncharacterized. This is compounded by the fact that identifying lncRNA loci that have robust and reproducible phenotypes when mutated has been a challenge. We previously generated a cohort of 20 lncRNA loci knockout mice. Here, we extend our initial study and provide a more detailed analysis of the highly conserved lncRNA locus, Taurine Upregulated Gene 1 (Tug1). We report that Tug1 knockout male mice are sterile with complete penetrance due to a low sperm count and abnormal sperm morphology. Having identified a lncRNA loci with a robust phenotype, we wanted to determine which, if any, potential elements contained in the Tug1 genomic region (DNA, RNA, protein, or the act of transcription) have activity. Using engineered mouse models and cell-based assays, we provide evidence that the Tug1 locus harbors three distinct regulatory activities - two noncoding and one coding: (i) a cis DNA repressor that regulates many neighboring genes, (ii) a lncRNA that can regulate genes by a trans-based function, and finally (iii) Tug1 encodes an evolutionary conserved peptide that when overexpressed impacts mitochondrial membrane potential. Our results reveal an essential role for the Tug1 locus in male fertility and uncover three distinct regulatory activities in the Tug1 locus, thus highlighting the complexity present at lncRNA loci.
Preprint PDF
Barrett RDH, Laurent S, Mallarino R, Pfeifer SP, Xu CCY, Foll M, Wakamatsu K, Duke-Cohan JS, Jensen JD, Hoekstra HE. Linking a mutation to survival in wild mice. Science 2019;363:499-504.Abstract
Adaptive evolution in novel or changing environments can be difficult to predict because the functional connections between genotype, phenotype, and fitness are complex. Here, we make these explicit connections by combining field and laboratory experiments in wild mice. We first directly estimate natural selection on pigmentation traits and an underlying pigment locus, Agouti, using experimental enclosures of mice on different soil colors. Next, we show how a mutation in Agouti associated with survival causes lighter coat color via changes in its protein binding properties. Together, our findings demonstrate how a sequence variant alters phenotype and then reveal the ensuing ecological consequences that drive changes in population allele frequency, thereby illuminating the process of evolution by natural selection.
Baier F, Hoekstra HE. The genetics of morphological and behavioral island traits in deer mice. Proceedings of the Royal Society B 2019;286:20191697.Abstract
Animals on islands often exhibit dramatic differences in morphology and behavior compared to mainland individuals, a phenomenon known as the "island syndrome". These differences, such as changes in body size and aggression, are thought to be adaptations to island environments, where there are high resource levels, low predation, limited dispersal, and thus high population densities. However, the extent to which island traits have a genetic basis or instead represent plastic responses to environmental extremes is often unknown. Here, we revisit a classic case of island syndrome in deer mice (Peromyscus maniculatus) from British Columbia. Previous field studies suggested that Saturna Island mice evolved several island traits, including higher body weight and reduced aggression relative to mainland populations. Using historical collections, we show that Saturna Island mice and those from neighboring islands are approximately 35% (~5g) heavier than mainland mice. We then collected mice from two focal populations: Saturna Island and a nearby mainland population. First, using molecular data, we find that these populations are genetically distinct, having diverged approximately 10 thousand years ago. Second, we established laboratory colonies and find that Saturna Island mice are heavier both because they are longer and have disproportionately more lean mass. These trait differences are maintained in second-generation captive-born mice raised in a common environment, implying a strong heritable component. In addition, island-mainland hybrids are heavier when born to island mothers than to mainland mothers, revealing a maternal genetic effect on body weight. Next, using behavioral testing in the lab, we also find that wild-caught island mice are less aggressive than mainland mice. However, lab-raised mice born to these founders do not differ in aggression, regardless of whether they are tested in conditions that induce low or high aggression, suggesting the large behavioral difference observed between wild-caught island and mainland individuals is likely a plastic response. Together, our results reveal that these mice respond differently to environmental conditions on islands, evolving both heritable changes in a morphological trait and also expressing a plastic phenotypic response in a behavioral trait.
Delaney EK, Hoekstra HE. Diet-based assortative mating through sexual imprinting. Ecology & Evolution 2019;00:1-6.Abstract

Speciation is facilitated by “magic traits,” where divergent natural selection on such traits also results in assortative mating. In animal populations, diet has the potential to act as a magic trait if populations diverge in consumed food that incidentally affects mating and therefore sexual isolation. While diet‐based assortative mating has been observed in the laboratory and in natural populations, the mechanisms causing positive diet‐based assortment remain largely unknown. Here, we experimentally created divergent diets in a sexually imprinting species of mouse, Peromyscus gossypinus (the cotton mouse), to test the hypothesis that sexual imprinting on diet could be a mechanism that generates rapid and significant sexual isolation. We provided breeding pairs with novel garlic‐ or orange‐flavored water and assessed whether their offspring, exposed to these flavors in utero and in the nest before weaning, later preferred mates that consumed the same flavored water as their parents. While males showed no preference, females preferred males of their parental diet, which is predicted to yield moderate sexual isolation. Thus, our experiment demonstrates the potential for sexual imprinting on dietary cues learned in utero and/or postnatally to facilitate reproductive isolation and potentially speciation.

Kocher SD, Mallarino R, Rubin BER, Yu DW, Hoekstra HE, Pierce NE. The genetic basis of a social polymorphism in halictid bees. Nature Communications 2018;9:4338.Abstract
The emergence of eusociality represents a major evolutionary transition from solitary to group reproduction. The most commonly studied eusocial species, honey bees and ants, represent the behavioral extremes of social evolution but lack close relatives that are non-social. Unlike these species, the halictid bee Lasioglossum albipes produces both solitary and eusocial nests and this intraspecific variation has a genetic basis. Here, we identify genetic variants associated with this polymorphism, including one located in the intron of syntaxin 1a (syx1a), a gene that mediates synaptic vesicle release. We show that this variant can alter gene expression in a pattern consistent with differences between social and solitary bees. Surprisingly, syx1a and several other genes associated with sociality in Lalbipes have also been implicated in autism spectrum disorder in humans. Thus, genes underlying behavioral variation in Lalbipes may also shape social behaviors across a wide range of taxa, including humans.
Fisher HS, Hook KA, Weber WD, Hoekstra HE. Sibling rivalry: Males with more brothers develop larger testes. Ecology and Evolution 2018;8(16):8197-8203.Abstract
When females mate with multiple partners in a reproductive cycle, the relative number of competing sperm from rival males is often the most critical factor in determining paternity. Gamete production is directly related to testis size in most species, and is associated with both mating behavior within a system and perceived risk of competition. Peromyscus maniculatus is naturally promiscuous and males invest significantly more in sperm production than males of P. polionotus, their monogamous sister-species. Here we show that the relatively larger testes in P. maniculatus are retained, even after decades of enforced monogamy in captivity. While these results suggest that differences in sperm production between species with divergent evolutionary histories can be maintained, we also show that the early rearing environment of males can strongly influence their testis size as adults. Using a second-generation hybrid population to increase variation in testis size, we show that males reared in litters with more brothers develop larger testes as adults. Importantly, this difference in testis size is also associated with increased fertility. Together, our findings suggest that sperm production may be both broadly shaped by natural selection over evolutionary timescales and also finely tuned during early development.
Mallarino R, Pillay N, Hoekstra HE, Schradin C. Quick Guide: African striped mice. Current Biology 2018;28:R293–R305. PDF
Goncalves GL, Masestri R, Moreira GRP, Jacobi MAM, Freitas TRO, Hoekstra HE. Divergent genetic mechanisms lead to spiny hair in mammals. PLoS ONE 2018;13(8):e0202219.Abstract
In humans, a single amino acid change (V370A) in the Ecdysoplasin A receptor (Edar) gene is associated with a unique hair phenotype in East Asian populations. Transgenic experiments in mouse show that this mutation enhances Edar signaling in vitro, which in turn alters multiple aspects of hair morphology. Here we tested whether this substitution contributes to the spiny hair observed in six families of rodents. We first measured hair traits, focusing on guard hairs and their physical properties, such as tension and deformation, and compared the morphology between spiny and non-spiny sister lineages. Two distinct hair morphologies were repeatedly observed in spiny rodent lineages: hairs with a grooved cross-section and a second near cylindrical form, which differ in their cross-section shape as well as their tensiometric properties. Next, we sequenced a portion of the Edar locus in these same species. Most species surveyed have the common amino acid valine at position 370, but the kangaroo rat and spiny pocket mouse have an isoleucine. We also found one additional amino acid variant: tree rats have a Leu422Val polymorphism. However, none of these variants are associated with changes in hair morphology. Together these data suggest that the specific Edar mutation associated with variation in human hair morphology does not play a role in modifying hairs in wild rodents, highlighting that different evolutionary pathways can produce similar spiny hair morphology.
Lewarch CL, Hoekstra HE. The evolution of nesting behaviour in Peromyscus mice. Animal Behaviour 2018;139:103-115.Abstract

Structures built by animals, such as nests, often can be considered extended phenotypes that facilitate the study of animal behaviour. For rodents, nest building is both an important form of behavioural thermoregulation and a critical component of parental care. Changes in nest structure or the prioritization of nesting behaviour are therefore likely to have consequences for survival and reproduction, and both biotic and abiotic environmental factors are likely to influence the adaptive value of such differences. Here we first develop a novel assay to investigate interspecific variation in the nesting behaviour of deer mice (genus Peromyscus). Using this assay, we find that, while there is some variation in the complexity of the nests built by Peromyscus mice, differences in the latency to begin nest construction are more striking. Four of the seven taxa examined here build nests within an hour of being given nesting material, but this latency to nest is not related to ultimate differences in nest structure, suggesting that the ability to nest is relatively conserved within the genus, but species differ in their prioritization of nesting behaviour. We also find that latency to nest is not correlated with body size, climate or the construction of burrows that create microclimates. However, the four taxa with short nesting latencies all have monogamous mating systems, suggesting that differences in nesting latency may be related to social environment. This detailed characterization of nesting behaviour within the genus provides an important foundation for future studies of the genetic and neurobiological mechanisms that contribute to the evolution of behaviour.

Pfeifer SP, Laurent S, Sousa VC, Linnen CR, Foll M, Excoffier L, Hoekstra HE, Jensen JD. The evolutionary history of Nebraska deer mice: local adaptation in the face of strong gene flow. Molecular Biology and Evolution 2018;35:792-806.Abstract
The interplay of gene flow, genetic drift, and local selective pressure is a dynamic process that has been well studied from a theoretical perspective over the last century. Wright and Haldane laid the foundation for expectations under an island-continent model, demonstrating that an island specific beneficial allele may be maintained locally if the selection coefficient is larger than the rate of migration of the ancestral allele from the continent. Subsequent extensions of this model have provided considerably more insight into the conditions under which such a beneficial allele may be maintained, lost, or fixed. Yet, connecting theoretical results with empirical data has proven challenging, owing to a lack of information on the relationship between genotype, phenotype, and fitness. Here, we examine the demographic and selective history of deer mice in and around the Nebraska Sand Hills, a system in which variation at the Agouti locus affects cryptic coloration that in turn affects the survival of mice in their local habitat. We first genotyped 250 individuals from eleven sites along a transect spanning the Sand Hills at 670,000 SNPs across the genome. Using these genomic data, we found that deer mice first colonized the Sand Hills following the last glacial period. Subsequent high rates of gene flow have served to homogenize the majority of the genome between populations on and off the Sand Hills, with the exception of the Agouti pigmentation locus. Furthermore, we observe strong haplotype structure around putatively beneficial mutations within the Agouti locus, and these mutations are strongly associated with the pigment traits that are strongly correlated with local soil coloration and thus responsible for cryptic coloration. We discuss these empirical results in light of theoretical expectations, thereby providing a complete example of the dynamics between ancestral gene flow and local adaptation in a classic mammalian system.
Delaney EK, Hoekstra HE. Sexual imprinting and speciation in two Peromyscus species. Evolution 2018;72(2):274–287.Abstract
Sexual isolation, a reproductive barrier, can prevent interbreeding between diverging populations or species. Sexual isolation can have a clear genetic basis; however, it may also result from learned mate preferences that form via sexual imprinting. Here, we demonstrate that two sympatric sister species of mice -- the white-footed mouse (Peromyscus leucopus) and its closest relative, the cotton mouse (P. gossypinus) -- hybridize only rarely in the wild despite co-occurring in the same habitat and lack of any measurable intrinsic postzygotic barriers in laboratory crosses. We present evidence that strong conspecific mate preferences in each species form significant sexual isolation. We find that these mating preferences are learned in one species but may be genetic in the other: P. gossypinus sexually imprints on its parents, but innate biases or social learning affects mating preferences in P. leucopus. Our study demonstrates that sexually imprinting contributes to reproductive isolation that reduces hybridization between otherwise inter-fertile species, supporting a previously underappreciated role for learning in mammalian speciation.
Kingsley EP, Kozak KM, Pfeifer SP, Yang D-S, Hoekstra HE. The ultimate and proximate mechanisms driving the evolution of long tails in forest deer mice. Evolution 2017;71(2):261-273.Abstract

Understanding both the role of selection in driving phenotypic change and its underlying genetic basis remain major challenges in evolutionary biology. Here, we use modern tools to revisit a classic system of local adaptation in the North American deer mouse, Peromyscus maniculatus, which occupies two main habitat types: prairie and forest. Using historical collections, we find that forest-dwelling mice have longer tails than those from nonforested habitat, even when we account for individual and population relatedness. Using genome-wide SNP data, we show that mice from forested habitats in the eastern and western parts of their range form separate clades, suggesting that increased tail length evolved independently. We find that forest mice in the east and west have both more and longer caudal vertebrae, but not trunk vertebrae, than nearby prairie forms. By intercrossing prairie and forest mice, we show that the number and length of caudal vertebrae are not correlated in this recombinant population, indicating that variation in these traits is controlled by separate genetic loci. Together, these results demonstrate convergent evolution of the long-tailed forest phenotype through two distinct genetic mechanisms, affecting number and length of vertebrae, and suggest that these morphological changes — either independently or together — are adaptive.


Metz HC, Bedford NL, Pan L, Hoekstra HE. Evolution and genetics of precocious burrowing behavior in Peromyscus mice. Current Biology 2017;27:3837-3845.Abstract
A central challenge in biology is to understand how innate behaviors evolve between closely related species. One way to elucidate how differences arise is to compare the development of behavior in species with distinct adult traits. Here, we report that Peromyscus polionotus is strikingly precocious with regard to burrowing behavior, but not other behaviors, compared to its sister species P. maniculatus. In P. polionotus, burrows were excavated as early as 17 days of age, while P. maniculatus did not build burrows until 10 days later. Moreover, the well-known differences in burrow architecture between adults of these species -- P. polionotus adults excavate long burrows with an escape tunnel, while P. maniculatus dig short, single-tunnel burrows -- were intact in juvenile burrowers. To test whether this juvenile behavior is influenced by early-life environment, pups of both species were reciprocally cross-fostered. Fostering did not alter the characteristic burrowing behavior of either species, suggesting these differences are genetic. In backcross F2 hybrids, we show that precocious burrowing and adult tunnel length are genetically correlated, and that a single P. polionotus allele in a genomic region linked to adult tunnel length is predictive of precocious burrow construction. The co-inheritance of developmental and adult traits indicates the same genetic region -- either a single gene with pleiotropic effects, or closely linked genes -- acts on distinct aspects of the same behavior across life stages. Such genetic variants likely affect behavioral drive (i.e. motivation) to burrow, and thereby affect both the development and adult expression of burrowing behavior.
Bendesky A, Kwon Y-M, Lassance J-M, Lewarch CL, Yao S, Peterson BK, He MX, Dulac C, Hoekstra HE. The genetic basis of parental care evolution in monogamous mice. Nature 2017;554:434-439.Abstract


Parental care is essential for the survival of mammals, yet the mechanisms underlying its evolution remain largely unknown. Here we show that two sister species of mice, Peromyscus polionotus and Peromyscus maniculatus, have large and heritable differences in parental behaviour. Using quantitative genetics, we identify 12 genomic regions that affect parental care, 8 of which have sex-specific effects, suggesting that parental care can evolve independently in males and females. Furthermore, some regions affect parental care broadly, whereas others affect specific behaviours, such as nest building. Of the genes linked to differences in nest-building behaviour, vasopressin is differentially expressed in the hypothalamus of the two species, with increased levels associated with less nest building. Using pharmacology in Peromyscus and chemogenetics in Mus, we show that vasopressin inhibits nest building but not other parental behaviours. Together, our results indicate that variation in an ancient neuropeptide contributes to interspecific differences in parental care. 


Hu CK, Hoekstra HE. Peromyscus burrowing: A model system for behavioral evolution. Seminars in Cell and Developmental Biology 2017;61:107-114.Abstract

A major challenge to understanding the genetic basis of complex behavioral evolution is the quantification of complex behaviors themselves. Deer mice of the genus Peromyscusvary in their burrowing behavior, which leaves behind a physical trace that is easily preserved and measured. Moreover, natural burrowing behaviors are recapitulated in the lab, and there is a strong heritable component. Here we discuss potential mechanisms driving variation in burrows with an emphasis on two sister species: P. maniculatus, which digs a simple, short burrow, and P. polionotus, which digs a long burrow with a complex architecture. A forward-genetic cross between these two species identified several genomic regions associated with burrow traits, suggesting this complex behavior has evolved in a modular fashion. Because burrow differences are most likely due to differences in behavior circuits, Peromyscus burrowing offers an exciting opportunity to link genetic variation between natural populations to evolutionary changes in neural circuits.

Mallarino R, Linden TA, Linnen CR, Hoekstra HE. The role of isoforms in the evolution of cryptic coloration in Peromyscus mice. Molecular Ecology 2017;26:245-258.Abstract

A central goal of evolutionary biology is to understand the molecular mechanisms underlying phenotypic adaptation. While the contribution of protein-coding and cis-regulatory mutations to adaptive traits have been well documented, additional sources of variation—such as the production of alternative RNA transcripts from a single gene, or isoforms—have been understudied. Here, we focus on the pigmentation gene Agouti, known to express multiple alternative transcripts, to investigate the role of isoform usage in the evolution of cryptic color phenotypes in deer mice (genus Peromyscus). We first characterize the Agouti isoforms expressed in the Peromyscus skin and find two novel isoforms not previously identified in Mus. Next, we show that a locally adapted light-colored population of P. maniculatus living on the Nebraska Sand Hills shows an up-regulation of a single Agouti isoform, termed 1C, compared to their ancestral dark-colored conspecifics. Using in vitro assays, we show that this preference for isoform 1C may be driven by isoform-specific differences in translation. In addition, using an admixed population of wild-caught mice, we find that variation in overall Agouti expression maps to a region near exon 1C, which also has patterns of nucleotide variation consistent with strong positive selection. Finally, we show that the independent evolution of cryptic light pigmentation in a different species, P. polionotus, has been driven by a preference for the same Agouti isoform. Together, these findings present an example of the role of alternative transcript processing in adaptation and demonstrate molecular convergence at the level of isoform regulation.

Mallarino R, Hoekstra HE, Manceau M. Developmental genetics in emerging rodent models: Case studies and perspectives. Current Opinion in Genetics & Development 2016;39:182-186.Abstract


For decades, mammalian developmental genetic studies have focused almost entirely on two laboratory models: Mus and Rattus, species that breed readily in the laboratory and for which a wealth of molecular and genetic resources exist. These species alone, however, do not capture the remarkable diversity of morphological, behavioural and physiological traits seen across rodents, a group that represents >40% of all mammal species. Due to new advances in molecular tools and genomic technologies, studying the developmental events underlying natural variation in a wide range of species for a wide range of traits has become increasingly feasible. Here we review several recent studies and discuss how they not only provided technical resources for newly emerging rodent models in developmental genetics but also are instrumental in further encouraging scientists, from a wide range of research fields, to capitalize on the great diversity in development that has evolved among rodents. 


Mallarino R, Henegar C, Mirasierra M, Manceau MC, Shradin C, Vallejo M, Beronja S, Barsh GS, Hoekstra HE. Developmental mechanisms of stripe patterns in rodents. Nature 2016;539:518-523.Abstract


Mammalian colour patterns are among the most recognizable characteristics found in nature and can have a profound impact on fitness. However, little is known about the mechanisms underlying the formation and subsequent evolution of these patterns. Here we show that, in the African striped mouse (Rhabdomys pumilio), periodic dorsal stripes result from underlying differences in melanocyte maturation, which give rise to spatial variation in hair colour. We identify the transcription factor ALX3 as a regulator of this process. In embryonic dorsal skin, patterned expression of Alx3 precedes pigment stripes and acts to directly repress Mitf, a master regulator of melanocyte differentiation, thereby giving rise to light-coloured hair. Moreover, Alx3 is upregulated in the light stripes of chipmunks, which have independently evolved a similar dorsal pattern. Our results show a previously undescribed mechanism for modulating spatial variation in hair colour and provide insights into how phenotypic novelty evolves.


Mallet J, Hoekstra HE. Ecological genetics: A key gene for mimicry and melanism. Current Biology 2016;26:R802-804.Abstract


 Mimicry and melanism in Lepidoptera provided the first convincing examples of natural selection in action. Genetic analysis has now shown that, surprisingly, mimicry in Heliconius butterflies and melanism in peppered moths are switched at precisely the same gene: cortex .


Bear DM, Lassance JM, Hoekstra HE, Datta SR. The evolving neural and genetic architecture of vertebrate olfaction. Current Biology 2016;26:R1039-R1049.Abstract


Evolution sculpts the olfactory nervous system in response to the unique sensory challenges facing each species. In vertebrates, dramatic and diverse adaptations to the chemical environment are possible because of the hierarchical structure of the olfactory receptor (OR) gene superfamily: expansion or contraction of OR subfamilies accompanies major changes in habitat and lifestyle; independent selection on OR subfamilies can permit local adaptation or conserved chemical communication; and genetic variation in single OR genes can alter odor percepts and behaviors driven by precise chemical cues. However, this genetic flexibility con- trasts with the relatively fixed neural architecture of the vertebrate olfactory system, which requires that new olfactory receptors integrate into segregated and functionally distinct neural pathways. This organization allows evolution to couple critical chemical signals with selectively advantageous responses, but also con- strains relationships between olfactory receptors and behavior. The coevolution of the OR repertoire and the olfactory system therefore reveals general principles of how the brain solves specific sensory problems and how it adapts to new ones.