New Results

Evolution and genetics of precocious burrowing behavior in Peromyscus mice

Hillery C. Metz, Nicole L. Bedford, Linda Pan, Hopi E. Hoekstra
doi: https://doi.org/10.1101/150243

Summary

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.

  • Juvenile P. polionotus construct burrows precociously compared to its sister species P. maniculatus

  • Cross-fostering does not alter species-specific burrowing behavior

  • A QTL linked to adult tunnel length predicts developmental onset of burrow construction in hybrids

  • Pleiotropic genetic variant(s) may affect behavioral drive across life stages

eTOC

Metz et al. find that oldfield mice, a species that digs long, complex burrows, also digs burrows earlier in development compared to its sister species. In F2 hybrids, precocious burrowing is co-inherited with long adult tunnels, and an allele linked to adult tunnel length also predicts timing of first burrow construction, suggesting that a single genetic region controls different aspects of the same behavior across distinct life stages.

Results

P. polionotusconstruct burrows earlier in life thanP. maniculatus

To examine the developmental onset of burrow construction in Peromyscus mice, we assayed burrowing behavior in juveniles starting at 17 days of age (these mice are typically weaned at postnatal day [P] 24). We found striking interspecific differences in both the timing and progression of burrow construction (Figure 1; Table S1). Notably, P. polionotus were precocious diggers, constructing complete burrows—defined as excavations with at least two components: an entrance tunnel plus a nest chamber—on average 10 days earlier than P. maniculatus. The first appearance of a complete burrow was at P17 in P. polionotus (1 of 5 mice; Figure 1b), but not until P27 in P. maniculatus (3 of 14 mice; Figure 1b), a considerable difference in developmental stage (see Figure S1 for timeline of development). Moreover, P. polionotus burrowed at adult-like frequencies from P19 onward, a developmental benchmark P. maniculatus did not reach until P27 (Figure 1b; Table S1).

Figure 1.
Figure 1.

The ontogeny of burrow construction in sister species P. maniculatus (yellow) and P. polionotus (blue). All animals were naïve and tested only once. (A) The ancestral burrow architecture, built by P. maniculatus, is short (<15cm) and simple. In contrast, adult P. polionotus dig stereotyped burrows with a long entrance tunnel, nest chamber and escape tunnel (total excavation length ~50 cm). (B) Proportion of tested mice constructing a complete burrow (i.e., entrance tunnel and nest chamber). Curves and shaded areas represent binary generalized linear smoothers with 95% confidence intervals. Species differences evaluated by Fisher’s exact test (juveniles: see text for details, adults: P. maniculatus n=17; P. polionotus n=9). (C) Length of total excavation. Juvenile differences evaluated by ANCOVA (see text for details). Adult differences between species evaluated by t-test (P. maniculatus n=17; P. polionotus n=9). Error bars at +/- 1 SE of the mean. (D) Proportion of tested mice constructing an escape tunnel. Statistical tests as in (B). (E) Distance run on a wheel during a 90-minute trial by P. maniculatus and P. polionotus juveniles (P17-P31, see text for details) and adults (>P60, P. maniculatus n=10; P. polionotus n=10). Statistical tests as in (C). (F) Cartoon depiction of data shown in panels B-D highlighting variation in burrow shape over development. Significance levels: p ≥ 0.05 = ns (not significant), p ≤ 0.05 = *, p ≤ 0.01 = **, p ≤ 0.001 = ***.

Whereas tunnel length increased with age in both species, reflecting a progression in burrowing ability with growth and development (Figure 1c; ANCOVA, p < 0.0001), tunnel length varied considerably between species. P. polionotus consistently produced significantly longer burrows than P. maniculatus (Figure 1c; ANCOVA, p < 0.0001), consistent with the known differences in adult tunnel length [3-5]. Furthermore, the rate of increase in tunnel length across ontogeny was significantly greater for P. polionotus (Figure 1c; ANCOVA, age x species interaction, p = 0.030). Thus, both the expression of adult-like burrowing frequency and an increase in excavation length develops more rapidly in P. polionotus than in P. maniculatus.

In trials when mice did not construct full burrows, individuals of both species usually excavated shallow cup-shaped cavities (divots) instead. Only three of 97 mice (two P17 P. polionotus and one P27 P. maniculatus) failed to leave any signs of digging activity. These data suggest that the motor patterns for digging were partly, if not completely, developed in both species by at least P17.

Juveniles construct burrows with miniaturized adult architecture

Juveniles from both species produced burrows with architecture typical of adults of their respective species. Starting at P19, nearly the earliest age tested, the burrows of P. polionotus included escape tunnels at a frequency not significantly different from conspecific adults (Figure 1d; Fisher’s exact test, p = 0.523). Likewise, P. maniculatus juvenile burrows invariably featured only a single tunnel leading to the nest chamber, always lacking an escape tunnel (Figure 1d). Although complete with regard to architectural components, juvenile excavations were significantly shorter than those of adults (Figure 1c; t-tests, p < 0.0001 for both species), thus representing miniature versions of adult burrows.

Precociousness is specific to burrowing behavior

To evaluate whether precocious burrow construction in P. polionotus might be due to advantages in physical rather than behavioral development (e.g. [6]), we examined general measures of morphological and motor development in both species. Two lines of evidence refute this hypothesis. First, P. polionotus did not perform better in a second motor activity task: P. polionotus juveniles travelled less distance in a 90- minute wheel-running assay than P. maniculatus. While total distance run increased with age at a comparable rate in both species (Figure 1e; age × species interaction term, p = 0.5993), P. maniculatus ran significantly greater distances than age-matched P. polionotus (ANCOVA, p < 0.001). Second, P. polionotus are smaller than P. maniculatus in both body mass (ANCOVA, p < 0.0001) and hindfoot length (ANCOVA, p < 0.0001) across development (Figure S1). Likewise, we did not observe heterochrony favoring P. polionotus with respect to additional developmental milestones, as P. maniculatus reached them earlier in life (Figure S1). Thus, precocious burrowing in P. polionotus juveniles reflects a behavioral difference, likely specific to burrowing, not an advantage in overall activity level, motor ability, or morphological development.

Species-specific burrowing behavior unaltered by interspecific cross-fostering

To disentangle the effects of genetics from environment, pups were reciprocally cross-fostered between the two sister species (Figure 2a). We reasoned that any effects on burrowing behavior resulting from parental environment were likely to be greatest during post-natal development.

Figure 2.
Figure 2.

Reciprocal interspecific cross-fostering between P. maniculatus (yellow) and P. polionotus (blue). (A) Schematic of cross-fostering design with cross-fostered pups highlighted in red. (B,E) Proportion of mice constructing complete burrows, (C,F) proportion of mice building an escape tunnel, and (D,G) length of excavations. Sample sizes for each age and foster group are shown. For B, C, E and F, differences between foster treatments were evaluated by Fisher’s exact test; for D and G, by ANCOVA (see text for details). Significance levels: p ≥ 0.05 = ns (not significant).

In P. maniculatus, the developmental onset of burrow building did not differ between cross-fostered and non-fostered animals. Prior to P27, P. maniculatus juveniles did not build complete burrows regardless of foster treatment (Figure 2b). Following the onset of burrowing, fostered animals constructed burrows no more frequently than pups reared by their biological parents (Figure 2b; Fisher’s exact test, one-tailed, p = 0.57). Cross-fostered P. maniculatus also did not build escape tunnels (Figure 2c), and the excavations of cross-fostered animals closely matched those of mice raised by their biological parents with regard to length (Figure 2d; ANCOVA, p = 0.63).

Likewise, P. polionotus raised by heterospecific parents began burrowing at the earliest age tested (P19; Figure 2e), and from P21 onward, nearly all cross-fostered P. polionotus excavated burrows (12 of 14 mice; Figure 2e). Burrow structure also did not change with cross-fostering treatment. Cross-fostered P. polionotus dug escape tunnels as early in ontogeny (from P19), and as frequently (50%, 8 of 16 mice), as non-fostered juveniles (41%, 22 of 53 mice; Figure 2f, Fisher’s exact test, p = 0.34) and as frequently as conspecific adults (67%, 6 of 9 mice; Fisher’s exact test, p = 0.58). Finally, excavation lengths did not differ between cross-fostered and non-fostered animals (Figure 2g; ANCOVA, p = 0.06). In summary, we found no differences in burrowing behavior following cross-fostering, consistent with there being a strong genetic component to the development of burrowing behavior.

Ontogeny of burrow construction isP. polionotus-dominant

We next tested the hypothesis that differences in the developmental onset of burrowing in juveniles share a common genetic basis with the well-characterized differences in adult burrow architecture [3-5] using a P. polionotus x P. maniculatus experimental cross (Figure 3a).

Figure 3.
Figure 3.

Genetic dissection of precocious burrowing in P. polionotus X P. maniculatus hybrids. (A) Schematic of breeding design showing P. maniculatus (yellow), P. polionotus (blue), first-generation F1 hybrids (green) and second-generation F2 hybrids (grey). (B) Proportion of juvenile animals digging complete burrows (upper panel) and escape tunnels (lower panel); groups compared using Fisher’s exact tests (see text for details). (C) Length of excavations in F1 hybrids compared to P. maniculatus and P. polionotus; species differences were evaluated by ANCOVA (see text for details). Sample sizes for each group are shown below. (D) Timeline of the four behavioral assays completed for each F2 hybrid. (E) Average adult excavation length of backcross F2 hybrids that, as juvenile burrowers, were either delayed (i.e., no burrows dug at P21 or P24) or precocious (i.e., at least one complete burrow dug at P21 or P24). Black lines indicate means for each group, which were compared by Welch’s two-sample t-test. (F) Least squares regression between juvenile and adult excavation lengths in backcross F2 hybrids (R2 = 0.1681, p = 0.0013). Shaded area is a Loess smoother (locally weighted smoother). Significance levels: p ≥ 0.05 = ns (not significant), p ≤ 0.05 = *, p ≤ 0.01 = **, p ≤ 0.001 = ***.

The development of burrowing behavior in first generation (F1) hybrids closely matches P. polionotus in each parameter examined, including the proportion of mice constructing burrows (Figure 3b; Fisher’s exact test, p = 0.378), the proportion of mice constructing escape tunnels (Figure 3b; Fisher’s exact test, p = 1.00), and the length of excavations (Figure 3c; ANCOVA, p = 0.086). Moreover, F1 hybrids differ significantly from P. maniculatus in all of these measures of burrowing behavior: proportion of mice constructing burrows (Figure 3b; Fisher’s exact test, p < 0.0001), proportion of mice constructing escape tunnels (Figure 3b; Fisher’s exact test, p = 0.019), and length of excavations (Figure 3c; ANCOVA, p < 0.0001). This inheritance pattern indicates that the genetic underpinnings of precocious burrowing, a developmental trait, are P. polionotus-dominant, consistent with the pattern of inheritance observed for adult burrowing behavior (F1 hybrid adults build P. polionotus-like burrows with regard to both length and shape [3,5]).

Developmental and adult traits share a common genetic basis

To test if developmental traits (namely, precocious burrow construction) and adult traits (long entrance tunnels, presence of an escape tunnel) are genetically linked, we generated 60 backcross F2 hybrids. If traits have an independent genetic basis, they are expected to become uncoupled in the F2 generation. We assessed burrowing performance for each F2 hybrid at four time points: two juvenile (P21 and P24) and two adult trials (P61 and P64) (Figure 3d). Half of the F2 hybrids (31 of 60) dug at least one juvenile burrow (at the P21 or P24 time point) and thus were scored as precocious burrowers, while the remaining half (29 of 60) completed no juvenile burrows and were scored as delayed burrowers. Consistent with there being a shared genetic basis for burrowing traits across life stages, we found that precocious burrowers went on to dig significantly longer burrows as adults (Figure 3e; t-test, p = 0.024). Furthermore, juvenile excavation length was significantly associated with adult tunnel length (Figure 3f; least-squares linear regression, R2 = 0.1681, p = 0.001). Neither cross direction (i.e. whether the F1 parent was the sire or dam) nor sex of the F2 had an effect on juvenile or adult burrow length (t-tests, cross direction, juveniles: p = 0.371; adults: p = 0.673; sex, juveniles: p = 0.682; adults: p = 0.431). Together, these data suggest that for burrowing, juvenile (precocious onset of burrowing) and adult (tunnel length) behavior share some pleiotropic genetic basis, are influenced by closely linked genes, or both.

To determine which regions of the genome affect both juvenile and adult behaviors, we genotyped backcross F2 mice at four unlinked markers, each representing a genetic locus previously linked to differences in adult burrow architecture [5]. We found that inheritance of a P. polionotus allele was predictive of juvenile phenotype at one of the four markers (Figure 4). Specifically, a marker on linkage group 2 was associated with earlier onset of burrowing (Figure 4a; Fisher’s exact test, one-tailed, p = 0.044), juvenile excavation length (Figure 4b; t-test, p = 0.014), and, as expected, adult excavation length (Figure 4c; t-test p = 0.033). For each of the other markers examined, no significant relationships between genotype and phenotype were detected (Figure S2; Fisher’s exact tests and t-tests, p > 0.05), possibly due, in part, to the limited number of F2 hybrids examined. These data suggest that a gene, or closely linked genes, on linkage group 2 affects variation in burrowing behavior at different life stages.

Figure 4.
Figure 4.

Effect of a single P. polionotus allele on juvenile burrowing behavior in F2 hybrids at a QTL previously linked to adult tunnel length [17]. Three traits are shown: (A) precociousness score across two juvenile trials (1 = mouse dug at least one discrete burrow at P21 and/or P24 behavior trials; 0 = mouse dug no burrow at P21 or P24); (B) average juvenile excavation length; and (C) average adult excavation length. Genotypes for 60 backcross F2 hybrids are either MM (homozygous for the P. maniculatus allele) or PM (heterozygous). For each genotype, trait means are plotted with error bars at +/- 1 SE of the mean. The mean trait values for each parental species are plotted as horizontal bars: P. polionotus (blue) and P. maniculatus (yellow). Parental species trait values are based on one trial per individual, aged P21-P24 (juveniles, P. maniculatus n=16; P. polionotus n=22), or >P60 (adults, P. maniculatus n=17; P. polionotus n=9). F2 hybrid trait values are the average of two juvenile (A,B) or two adult (C) trials. Significance levels, determined by Fisher’s exact test (A) or t-test (B,C), are: p ≤ 0.05 = *.

Discussion

Huxley likes to speak of ‘the three major problems of biology’: that of causation, that of survival value and that of evolution—to which I should like to add a fourth, that of ontogeny.

—Tinberg en (1963)

Striking behavioral differences between closely-related species can be a powerful resource for understanding the evolution of behavior and its mechanistic underpinnings—both major goals of biology. Behaviors are among the most complex phenotypes, and to successfully tease apart how species-specific differences evolve requires an integrative approach, as championed by Tinbergen [7]. More specifically, Tinbergen’s 1963 landmark paper advocates for the addition of ontogeny to Huxley’s existing framework for behavioral research [8].

Ontogeny, the study of how behavior changes across the life of an individual, can provide understanding that is not discernible using other approaches; for example, it can uncover unexpected ancestral state reconstructions and generate novel hypotheses (e.g. [9-12]), or expose underlying proximate mechanisms driving changes in behavior (e.g. [13-16]). In short, ontogeny informs and edifies each of Tinbergen’s four questions and can provide novel insights into how behavior evolves.

Here, we focused on the ontogeny of burrow construction, an ecologically important behavior that varies dramatically between closely related species of North American Peromyscus rodents. Most species in this genus build small (<20cm), simple burrows as adults, but one species, P. polionotus, has recently evolved a stereotyped burrowing behavior that results in a considerably longer burrow (>100cm in the wild) comprised of an elongated entrance tunnel, a nest chamber, and a secondary tunnel that extends upward from the nest toward the soil surface. This second tunnel does not penetrate the soil surface except during emergency evacuation, and thus is often referred to as an escape tunnel ([3-5, 17-20]; Figure 1a). The burrows of P. polionotus have inspired studies of phylogenetic history [4], genetic mechanisms of behavior [3,5], and speculations of adaptive function—namely that P. polionotus burrows may provide refuge from the elevated rates of predation that occur in open, exposed habitats (e.g. [21,22]). However, the ontogeny of the behavior—the last of Tinbergen’s four questions—remained unexamined until now.

We report on how the final product of digging behavior—the extended phenotype [23], or burrow—originates and progresses during the post-natal development of two sister species of Peromyscus with dramatically different adult burrow architectures. We first find that P. polionotus are precocious with respect to burrow construction, building their first burrows 10 days earlier in development than P. maniculatus. This is surprising given that P. maniculatus is larger, tends to reach developmental milestones earlier, and outperforms age-matched P. polionotus in a wheel-running assay. These results suggest that P. polionotus has evolved a life history change—a precocious expression of behavior—that is likely specific to burrow construction.

We also examined the shape of burrows produced by juvenile Peromyscus mice. We found that each species’ characteristic burrow architecture is intact in juveniles. This result suggests that in pure species, the neurobiological control of each component of the complete burrow architecture (frequency of burrow construction, entrance tunnel, and escape tunnel) is expressed together throughout life. This result is especially surprising in light of previous work showing that the genetic control of adult burrow construction in P. polionotus is modular [5]. Although the shape of juvenile burrows is similar to adult burrows, they are smaller in overall size, likely due to the energetic cost of burrowing.

Using a cross-fostering experiment, we next tested if these juvenile burrowing traits were primarily learned postnatally or were driven by interspecific genetic differences. It is important to note, however, that our experiments cannot rule out prenatal maternal effects (e.g. [24]). We found that cross-fostering results do not differ if single or multiple pups are transferred to heterospecific parents, suggesting there is no measurable effect of sibling’s genotype on juvenile behavior. We report that all aspects of species-specific burrowing behavior are preserved in cross-fostered individuals of both species, demonstrating that juvenile expression of burrowing behavior is likely to have a strong genetic basis.

Finally, we examined the genetic underpinnings of behavioral ontogeny in hybrids of P. polionotus and P. maniculatus using a genetic cross. We found that a developmental trait (precocious onset of burrowing) and an adult trait (long tunnels characteristic of adult P. polionotus burrows) are genetically dominant and co-inherited, both at the level of phenotypic co-variation and with respect to a specific genetic marker. These data point to a shared—likely pleiotropic—genetic basis influencing behavior across life stages.

These results have implications for the evolution of burrowing behavior. First, pleiotropy (or tight linkage of multiple causal mutations) can facilitate or inhibit evolution. On one hand, pleiotropy can produce effects that are not directly selected for (and potentially even harmful), but that are nevertheless secondarily “dragged along” by evolution [25,26]. On the other hand, because changes in several traits are often involved during adaptation to a new environment [27-29], co-inheritance of groups of phenotypes (e.g. by pleiotropy or linkage) can expedite adaptation [30-32]. Indeed, a common experimental outcome is to map multiple traits to a shared genomic region [33-38], and this genetic architecture can affect how evolution proceeds.

Related, these findings make it difficult to identify the precise phenotypic targets of selection, if any. While variation in adult burrows can affect fitness [22,39], juvenile burrowing behavior may also be a target of selection. For example, natural selection for burrowing earlier in P. polionotus may reflect (i) its open habitat [17], which may expose young mice to predation and thus increase the survival value of burrowing, or (ii) a form of “play” during a critical period of motor development [40-42]. Our results, which implicate a broadly-acting pleiotropic genetic mechanism, highlight the challenge in identifying which specific trait or traits have been selected—in this case, precocious juvenile burrowing, long adult burrows, or both.

Finally, the shared genetic control of the timing of behavior onset and adult behavior also sheds light on the underlying neural mechanism. One parsimonious explanation for the co-inheritance of precocious burrowing with long entrance tunnels is that species-specific genetic differences produce heritable internal states that persist in individuals across life stages. Specifically, genetic variation shaping internal states may affect innate, species-specific behavioral drives or time budgets (i.e. apportionment of time spent performing different behaviors) such that P. polionotus more frequently engages in burrowing rather than alternative behaviors, compared to P. maniculatus, whose innate drives are tuned differently). Divergent neural tuning often has been linked to variation in neuromodulators or their receptors, rather than to variation in the underlying circuitry (e.g. [43-46]). Our results are thus consistent with a role for neuromodulators (and behavioral drives) in the evolution of burrowing in Peromyscus rodents, adding to the accumulating evidence that neuromodulatory systems are a frequent substrate for behavioral diversity and evolution [47].

Experimental Procedures

Animal care and breeding

We used captive strains of Peromyscus originally acquired from the Peromyscus Genetic Stock Center (University of South Carolina, Columbia SC, USA). We used only offspring of experienced parents (≥1 previous litter weaned) for experiments. Mice were housed in standard conditions. Due to genomic imprinting in these species, production of F1 hybrids was limited to crosses of P. polionotus sires to P. maniculatus dams [48,49]. Thus, this cross design excludes any P. polionotus maternal effects acting in favor of P. polionotus-like burrowing behavior. All procedures were approved by the Harvard University Institutional Animal Care and Use Committee.

Burrowing behavior trials: parental species and F1 hybrids

We tested burrowing behavior in a total of 131 juvenile and 26 adult mice in in large, indoor sand-filled arenas as previously described [4,5], except duration was reduced from 48 hours to 14-17 hours for juveniles. Briefly, we released animals into 1.2 × 1.5 × 1.1 m enclosures filled with approximately 700 kg of moistened, hard-packed premium playground sand (Pharma-Serv Corp.), under otherwise normal housing conditions. We tested juveniles once, without previous experience, and thus our experiment targeted innate behavior and not learned ability. Each mouse was tested once, singly.

Burrowing behavior trials: backcross F2 hybrids

We generated 60 backcross F2 hybrids by crossing F1 hybrids to P. maniculatus mates (Figure 3a). Both male and female F1 hybrids were backcrossed to P. maniculatus (reciprocal pairings): 22 animals were produced from an F1 dam and 38 from an F1 sire. We then characterized the juvenile and adult burrowing behavior of these backcross mice, collecting developmental and adult phenotypes in the same individuals: each F2 was tested four times in total, at juvenile ages 21 and 24 days, and adult ages 61 and 64 days. Enclosure area was reduced by half (i.e. to 0.6 × 1.5 × 1.1 m) for testing both juvenile and adult backcross individuals to accommodate the large number of animals being tested.

Burrow Measurements

To quantify burrow construction, at the conclusion of each trial, we inspected enclosures for any excavations, which were qualitatively characterized as either burrows (comprised of ≥1 tunnel plus a nest area) or divots (broad cup-shaped vertical diggings <10 cm; see Figure 1f). Next, we injected burrows with polyurethane insulation foam (Hilti Corp., Schaan, Liechtenstein) as previously described [4,5] and measured the lengths of burrow components (entrance tunnel, nest chamber, and escape tunnel if present) from dried polyurethane casts. We measured the length of divots directly in the enclosures.

Cross-fostering

Age-matched (≤ 48 hrs age difference) P. maniculatus (n=18) and P. polionotus (n=16) pups were traded between experienced (≥1 previous litter) heterospecific breeding pairs 24-48 hours after birth. Then, we measured the burrowing behavior of each resultant juvenile at a single time point (during 19-31 days).

Wheel-running Behavioral Trials

To compare the ontogeny of a second motor behavior (and general activity level) between species, we performed a standardized wheel running assay [50]. We tested naïve, juvenile P. maniculatus (n=43) and P. polionotus (n=40) at P17 - P31. After 4 hours of home cage habituation to the wheel (Ware Manufacturing Inc., Phoenix, AZ), we recorded 90 minutes of wheel running activity with a CC-COM10W wireless bike computer (Cateye Co. Ltd., Osaka, Japan). We performed all tests between 16:00 and 22:00h during the dark cycle.

Statistical Analyses

To disentangle effects of age on burrowing behavior, we employed several statistical tests. We first tested for effects of age and species on burrowing behavior as well as for effects of sex, postnatal litter size, enclosure, and foster status at the intraspecific level using ANCOVA. Because we did not detect statistical differences between treatments, singly cross-fostered individuals and litter-fostered animals were pooled (fostering details above). We used Fisher’s exact test to evaluate the frequencies of burrow and escape tunnel digging between different genetic groups. We used t-tests to compare means in F2 hybrids. To evaluate phenotypic correlations in our F2 cross, we used least squares linear regression. To detect associations between quantitative trait loci (QTLs) linked to adult behavior and precocious burrowing, we used Fisher’s exact test, and for continuous traits in juveniles (i.e. excavation length), we used t-tests. Two P. polionotus individuals that appeared in poor health (age >23 days) were excluded from all analyses. All statistical analyses were performed using the R language.

Genotyping

We genotyped the backcross F2 population (n=60) at four loci corresponding to known QTL underlying adult burrowing behavior (identified in [5]) using species-specific restriction fragment length polymorphism (RFLP) differences. We verified that the selected RFLPs were fixed between species by sequencing PCR amplicons of 4 unrelated individuals of each species as well as the P. maniculatus and F1 parents of the cross (BigDye® Terminator v3.1 Cycle Sequencing Kit, Life Technologies). PCR products were digested with restriction enzymes (New England Biolabs, Ipswich MA), separated by gel electrophoresis, and genotypes were called based on the resultant banding pattern.

Author Contributions

H.C.M., N.L.B. and H.E.H. conceived and designed experiments. H.C.M., N.L.B. and L.P. performed experiments, H.C.M. and N.L.B. analyzed the data. H.C.M. and H.E.H wrote the paper.

Acknowledgements

We thank S. Scalia, J. Mason and the Hoekstra Lab for assistance with behavioral assays and animal husbandry; A. Bendesky, B. de Bivort, C. Dulac, J. Losos, B. Ölveczky, H. Fisher, B. Peterson, M. Munoz, and J. Weber for helpful discussions and comments on the manuscript; Harvard’s Office of Animal Resources, particularly J. Rocca for animal husbandry; and S. Worthington for statistical consultation. This research was funded by a Chapman Memorial Scholarship, a National Science Foundation Graduate Research Fellowship, a Doctoral Dissertation Improvement Grant (NSF 1209753), and an American Fellowship from the American Association of University Women to H.C.M.; a Natural Sciences and Engineering Research Council of Canada Graduate Fellowship to N.L.B.; Harvard College Research Program award and a Harvard Museum of Comparative Zoology Grant for Undergraduate Research to L.P., and a Beckman Young Investigator Award to H.E.H. H.E.H. is an Investigator in the Howard Hughes Medical Institute.

Footnotes

  • ↵1 Co-first author

  • ↵2 Lead Contact

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Posted June 14, 2017.
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Evolution and genetics of precocious burrowing behavior in Peromyscus mice
Hillery C. Metz, Nicole L. Bedford, Linda Pan, Hopi E. Hoekstra
bioRxiv 150243; doi: https://doi.org/10.1101/150243
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