Publications

In Preparation
Harringmeyer OS, Hu CK, Metz HC, Mihelic EL, Rosher C, Sanguinetti-Scheck J, Hoekstra HE. A single genetic locus lengthens deer mouse burrows via motor pattern evolution. bioRxiv 2023.07.03.547545 In Preparation; PDF
Baier F, Reinhard K, Tong V, Murmann J, Farrow K, Hoekstra HE. The neural basis of defensive behaviour evolution in Peromyscus mice. bioRxiv 2023.07.04.547734 In Preparation; PDF
Forthcoming
Kingsley EP, Hager ER, Lassance J-M, Tuner KM, Harringmeyer OS, Kirby C, Neugebroen BI, Hoekstra HE. Adaptive tail-length evolution in deer mice is associated with differential Hoxd13 expression in early development. bioRxiv Forthcoming;Abstract
SUMMARY
Variation in the size and number of axial segments underlies much of the diversity in
animal body plans. Here, we investigate the evolutionary, genetic, and developmental
mechanisms driving tail-length differences between forest and prairie ecotypes of deer mice
(Peromyscus maniculatus). We first show that long-tailed forest mice perform better in an
arboreal locomotion assay, consistent with tails being important for balance during climbing. The
long tails of these forest mice consist of both longer and more caudal vertebrae than prairie mice.
Using quantitative genetics, we identify six genomic regions that contribute to differences in
total tail length, three of which associate with vertebra length and the other three with vertebra
number. For all six loci, the forest allele increases tail length, consistent with the cumulative
effect of natural selection. Two of the genomic regions associated with variation in vertebra
number contain Hox gene clusters. Of those, we find an allele-specific decrease in Hoxd13
expression in the embryonic tail bud of long-tailed forest mice, consistent with its role in axial
elongation. Additionally, we find that forest embryos have more presomitic mesoderm than
prairie embryos, and that this correlates with an increase in the number of neuromesodermal
progenitors (NMPs), which are modulated by Hox13 paralogs. Together, these results suggest a
role for Hoxd13 in the development of natural variation in adaptive morphology on a
microevolutionary timescale.
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Bedford NL, Gable JT, Hu CK, Wooldridge TB, N.A. Sokolov, Lassance JM, Hoekstra HE. Automated tracking reveals the social network of beach mice and their burrows. bioRxiv Forthcoming; PDF
2024
Kautt AF, Chen J, Lewarch CL, Hu C, Turner K, Lassance J-M, Baier F, Bedford NL, Bendesky A, Hoekstra HE. Evolution of gene expression across brain regions in behaviourally divergent deer mice. Molecular Ecology 2024;(e17270)Abstract
The evolution of innate behaviours is ultimately due to genetic variation likely acting in the nervous system. Gene regulation may be particularly important because it can evolve in a modular brain-region specific fashion through the concerted action of cis- and trans-regulatory changes. Here, to investigate transcriptional variation and its regulatory basis across the brain, we perform RNA sequencing (RNA-Seq) on ten brain subregions in two sister species of deer mice (Peromyscus maniculatus and P. polionotus)—which differ in a range of innate behaviours, including their social system—and their F1 hybrids. We find that most of the variation in gene expression distinguishes subregions, followed by species. Interspecific differential expression (DE) is pervasive (52–59% of expressed genes), whereas the number of DE genes between sexes is modest overall (~3%). Interestingly, the identity of DE genes varies considerably across brain regions. Much of this modularity is due to cis-regulatory divergence, and while 43% of genes were consistently assigned to the same gene regulatory class across subregions (e.g. conserved, cis- or trans-regulatory divergence), a similar number were assigned to two or more different gene regulatory classes. Together, these results highlight the modularity of gene expression differences and divergence in the brain, which may be key to explain how the evolution of brain gene expression can contribute to the astonishing diversity of animal behaviours.
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2023
Jourjine N, Woolfolk ML, Sanguinetti-Scheck JI, Sabatini JE, McFadden S, Lindholm AK, Hoekstra HE. Two pup vocalization types are genetically and functionally separable in deer mice. Current Biology 2023;33(7):1237-1248.e4. PDF
Gozashti L, Feschotte C, Hoekstra HE. Transposable element competition shapes the deer mouse genome. Molecular Biology and Evolution 2023;40(4):msad069. PDF
2022
Khadraoui M, Merritt JR, Hoekstra HE, Bendesky A. Post-mating parental behavior trajectories differ across four species of deer mice. PLoS ONE 2022;17(10):e0276052.Abstract
Among species, parental behaviors vary in their magnitude, onset relative to reproduction, and sexual dimorphism. In deer mice (genus Peromyscus), while most species are promiscuous with low paternal care, monogamy and biparental care have evolved at least twice under different ecological conditions. Here, in a common laboratory setting, we monitored parental behaviors of males and females of two promiscuous (eastern deer mouse P. maniculatus and white-footed mouse P. leucopus) and two monogamous (oldfield mouse P. polionotus and California mouse P. californicus) species from before mating to after giving birth. In the promiscuous species, females showed parental behaviors largely after parturition, while males showed little parental care. In contrast, both sexes of monogamous species performed parental behaviors. However, while oldfield mice began to display parental behaviors before mating, California mice showed robust parental care behaviors only postpartum. These different parental-care trajectories in the two monogamous species align with their socioecology. Oldfield mice have overlapping home ranges with relatives, so infants they encounter, even if not their own, are likely to be closely related. By contrast, California mice disperse longer distances into exclusive territories with possibly unrelated neighbors, decreasing the inclusive fitness benefits of caring for unfamiliar pups before parenthood. Together, we find that patterns of parental behaviors in Peromyscus are consistent with predictions from inclusive fitness theory.
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Harringmeyer OS, Hoekstra HE. Massive inversion polymorphisms shape the genomic landscape of deer mice. Nature Ecology & Evolution 2022;6:1965–1979.Abstract
Chromosomal inversions are an important form of structural variation that can affect recombination, chromosome structure and fitness. However, because inversions can be challenging to detect, the prevalence and hence the significance of inversions segregating within species remains largely unknown, especially in natural populations of mammals. Here, by combining population-genomic and long-read sequencing analyses in a single, widespread species of deer mouse (Peromyscus maniculatus), we identified 21 polymorphic inversions that are large (1.5–43.8 Mb) and cause near-complete suppression of recombination when heterozygous (0–0.03 cM Mb−1). We found that inversion breakpoints frequently occur in centromeric and telomeric regions and are often flanked by long inverted repeats (0.5–50 kb), suggesting that they probably arose via ectopic recombination. By genotyping inversions in populations across the species’ range, we found that the inversions are often widespread and do not harbour deleterious mutational loads, and many are likely to be maintained as polymorphisms by divergent selection. Comparisons of forest and prairie ecotypes of deer mice revealed 13 inversions that contribute to differentiation between populations, of which five exhibit significant associations with traits implicated in local adaptation. Taken together, these results show that inversion polymorphisms have a significant impact on recombination, genome structure and genetic diversity in deer mice and likely facilitate local adaptation across the widespread range of this species.
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Stenseth NC, Andersson L, Hoekstra HE. Gregor Johann Mendel and the development of modern evolutionary biology. PNAS 2022;119(30):e2201327119. PDF
Hoekstra HE, Robinson GE. Behavioral genetics and genomics: Mendel’s peas, mice,and bees. PNAS 2022;119(30):e21221541119.Abstract
The question of the heritability of behavior has been of long fascination to scientists and the broader public. It is now widely accepted that most behavioral variation has a genetic component, although the degree of genetic in fl u- ence differs widely across behaviors. Starting with Men- del ’ s remarkable discovery of “ inheritance factors, ” it has become increasingly clear that speci fi c genetic variants that in fl uence behavior can be identi fi ed. This goal is not without its challenges: Unlike pea morphology, most natu- ral behavioral variation has a complex genetic architec- ture. However, we can now apply powerful genome-wide approaches to connect variation in DNA to variation in behavior as well as analyses of behaviorally related varia- tion in brain gene expression, which together have pro- vided insights into both the genetic mechanisms underlying behavior and the dynamic relationship between genes and behavior, respectively, in a wide range of species and for a diversity of behaviors. Here, we focus on two systems to illustrate both of these approaches: the genetic basis of bur- rowing in deer mice and transcriptomic analyses of division of labor in honey bees. Finally, we discuss the troubled rela- tionship between the fi eld of behavioral genetics and eugenics, which reminds us that we must be cautious about how we discuss and contextualize the connections between genes and behavior, especially in humans.
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Bedford NL, Weber JN, Tong W, Baier F, Kam A, Greenberg RA, Hoekstra HE. Interspecific variation in cooperative burrowing behavior by Peromyscus mice. Evolution Letters 2022;https://doi.org/10.1002/evl3.293Abstract
Animals often adjust their behavior according to social context, but the capacity for such behavioral flexibility can vary among species. Here, we test for interspecific variation in behavioral flexibility by comparing burrowing behavior across three species of deer mice (genus Peromyscus) with divergent social systems, ranging from promiscuous (Peromyscus leucopus and Peromyscus maniculatus) to monogamous (Peromyscus polionotus). First, we compared the burrows built by individual mice to those built by pairs of mice in all three species. Although burrow length did not differ in P. leucopus or P. maniculatus, we found that P. polionotus pairs cooperatively constructed burrows that were nearly twice as long as those built by individuals and that opposite-sex pairs dug longer burrows than same-sex pairs. Second, to directly observe cooperative digging behavior in P. polionotus, we designed a burrowing assay in which we could video-record active digging in narrow, transparent enclosures. Using this novel assay, we found, unexpectedly, that neither males nor females spent more time digging with an opposite-sex partner. Rather, we demonstrate that opposite-sex pairs are more socially cohesive and thus more efficient digging partners than same-sex pairs. Together, our study demonstrates how social context can modulate innate behavior and offers insight into how differences in behavioral flexibility may evolve among closely related species.
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Wooldridge TB, Kautt AF, Lassance JM, McFadden S, Domingues VS, Mallarino R, Hoekstra HE. A novel enhancer of Agouti contributes to parallel evolution of cryptically colored beach mice. PNAS 2022;119(27):e220286119.Abstract
Identifying the genetic basis of repeatedly evolved traits provides a way to reconstruct their evolutionary history and ultimately investigate the predictability of evolution. Here, we focus on the old fi eld mouse ( Peromyscus polionotus ), which occurs in the southeastern United States, where it exhibits considerable color variation. Dorsal coats range from dark brown in mainland mice to near white in mice inhabiting sandy beaches; this light pelage has evolved independently on Florida ’ s Gulf and Atlantic coasts as camou fl age from predators. To facilitate genomic analyses, we fi rst generated a chromosome-level genome assembly of Peromyscus polionotus subgriseus . Next, in a uniquely variable mainland population ( Peromyscus polionotus albifrons ), we scored 23 pigment traits and performed targeted resequencing in 168 mice. We fi nd that pigment variation is strongly associated with an ∼ 2-kb region ∼ 5 kb upstream of the Agouti sig- naling protein coding region. Using a reporter-gene assay, we demonstrate that this reg- ulatory region contains an enhancer that drives expression in the dermis of mouse embryos during the establishment of pigment prepatterns. Moreover, extended tracts of homozygosity in this Agouti region indicate that the light allele experienced recent and strong positive selection. Notably, this same light allele appears fi xed in both Gulf and Atlantic coast beach mice, despite these populations being separated by > 1,000 km. Together, our results suggest that this identi fi ed Agouti enhancer allele has been main- tained in mainland populations as standing genetic variation and from there, has spread to and been selected in two independent beach mouse lineages, thereby facilitating their rapid and parallel evolution.
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Hu CK, York RA, Metz HC, Bedford NL, Fraser HB, Hoekstra HE. Cis-regulatory changes in locomotor genes are associated with the evolution of burrowing behavior. Cell Reports 2022;38:110360.Abstract
How evolution modifies complex, innate behaviors is largely unknown. Divergence in many morphological
traits, and some behaviors, is linked to cis-regulatory changes in gene expression. Given this, we compare
brain gene expression of two interfertile sister species of Peromyscus mice that show large and heritable differences
in burrowing behavior. Species-level differential expression and allele-specific expression in F1 hybrids
indicate a preponderance of cis-regulatory divergence, including many genes whose cis-regulation is
affected by burrowing behavior. Genes related to locomotor coordination show the strongest signals of
lineage-specific selection on burrowing-induced cis-regulatory changes. Furthermore, genetic markers
closest to these candidate genes associate with variation in burrow shape in a genetic cross, suggesting
an enrichment for loci affecting burrowing behavior near these candidate locomotor genes. Our results provide
insight into how cis-regulated gene expression can depend on behavioral context and how this dynamic
regulatory divergence between species may contribute to behavioral evolution.
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Hager ER, Harringmeyer OS, Wooldridge TB, Theingi S, Gable JT, McFadden S, Neugeboren B, Turner KM, Hoekstra HE. A chromosomal inversion contributes to divergence in multiple traits between deer mouse ecotypes. Science 2022;377 (6604):399-405.Abstract
How locally adapted ecotypes are established and maintained within a species is a long-standing question in evolutionary biology. Using forest and prairie ecotypes of deer mice (Peromyscus maniculatus), we characterized the genetic basis of variation in two defining traits—tail length and coat color—and discovered a 41-megabase chromosomal inversion linked to both. The inversion frequency is 90% in the dark, long-tailed forest ecotype; decreases across a habitat transition; and is absent from the light, short-tailed prairie ecotype. We implicate divergent selection in maintaining the inversion at frequencies observed in the wild, despite high levels of gene flow, and explore fitness benefits that arise from suppressed recombination within the inversion. We uncover a key role for a large, previously uncharacterized inversion in the evolution and maintenance of classic mammalian ecotypes.
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2021
Hager ER, Hoekstra HE. Tail length evolution in deer mice: linking morphology, behavior and function. Integrative and Comparative Zoology 2021;61(2)Abstract

Determining how variation in morphology affects animal performance (and ultimately fitness) is key to understanding the complete process of evolutionary adaptation. Long tails have evolved many times in arboreal and semi-arboreal rodents; in deer mice, long tails have evolved repeatedly in populations occupying forested habit even within a single species (Peromyscus maniculatus). Here we use a combination of functional modeling, laboratory studies, and museum records to test hypotheses about the function of tail-length variation in deer mice. First, we use computational models, informed by museum records documenting natural variation in tail length, to test whether differences in tail morphology between forest and prairie subspecies can influence performance in behavioral contexts relevant for tail use. We find that the deer mouse tail plays little role in statically adjusting center of mass or in correcting body pitch and yaw, but rather it can affect body roll during arboreal locomotion. In this context, we find that even intraspecific tail-length variation could result in substantial differences in how much body rotation results from equivalent tail motions (i.e., tail effectiveness), but the relationship between commonly-used metrics of tail-length variation and effectiveness is non-linear. We further test whether caudal vertebra length, number, and shape are associated with differences in how much the tail can bend to curve around narrow substrates (i.e., tail curvature) and find that, as predicted, the shape of the caudal vertebrae is associated with intervertebral bending angle across taxa. However, although forest and prairie mice typically differ in both the length and number of caudal vertebrae, we do not find evidence that this pattern is the result of a functional trade-off related to tail curvature. Together, these results highlight how even simple models can both generate and exclude hypotheses about the functional consequences of trait variation for organismal-level performance.

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Jourjine N, Hoekstra HE. Expanding evolutionary neuroscience: insights from comparing variation in behavior. Neuron 2021;2(11):1084-1099.Abstract
Neuroscientists have long studied species with convenient biological features to discover how behavior emerges from conserved molecular, neural, and circuit level processes. With the advent of new tools, from viral vectors and gene editing to automated behavioral analyses, there has been a recent wave of interest in developing new, “nontraditional” model species. Here, we advocate for a complementary approach to model species development, that is, model clade development, as a way to integrate an evolutionary comparative approach with neurobiological and behavioral experiments. Capitalizing on natural behavioral variation in and investing in experimental tools for model clades will be a valuable strategy for the next generation of neuroscience discovery.
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Harringmeyer OS, Woolfolk ML, Hoekstra HE. Fishing for the genetic basis of migratory behavior. Cell 2021;184(2):303-305.Abstract
For many species, migrating at just the right time is essential for both survival and reproduction. A new study in salmon localizes a small genomic region associated with migration timing, which in turn affects other physiological traits, suggesting that a seemingly complex suite of migration traits is linked by one “simple” phenotype.
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2020
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. Genome Biol. 2020;21(1):237.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.
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2019
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.
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