Evolution of Boldness and Exploratory Behavior in Giant Mice from Gough Island

bioRxiv preprint doi: https://doi.org/10.1101/2020.09.10.292185; this version posted September 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

6 Evolution of Boldness and Exploratory Behavior in Giant Mice from Gough Island

9 Jered A. Stratton, Mark J. Nolte, and Bret A. Payseur

12 Laboratory of Genetics, University of Wisconsin – Madison, Madison, WI 53706

14 Corresponding Author: Jered A. Stratton

15 Email for Correspondence: [email protected]

17 Abstract

18 Island populations are hallmarks of extreme phenotypic evolution. Radical changes

19 in resource availability and predation risk accompanying island colonization drive changes

20 in behavior, which Darwin likened to tameness in domesticated animals. Although many

21 examples of animal boldness are found on islands, the heritability of observed behaviors, a

22 requirement for evolution, remains largely unknown. To fill this gap, we profiled anxiety

23 and exploration in island and mainland inbred strains of house mice raised in a common

24 laboratory environment. The island strain was descended from mice on Gough Island, the

25 largest wild house mice on record. Experiments utilizing open environments across two

26 ages showed that Gough Island mice are bolder and more exploratory, even when a shelter

27 is provided. Concurrently, Gough Island mice retain an avoidance response to predator

28 urine. F1 offspring from crosses between these two strains behave more similarly to the

29 mainland strain for most traits, suggesting recessive mutations contributed to behavioral

30 evolution on the island. Our results provide a rare example of novel, inherited behaviors in

31 an island population and demonstrate that behavioral evolution can be specific to different

32 forms of perceived danger. Our discoveries pave the way for a genetic understanding of

33 how island populations evolve unusual behaviors.

35 Keywords

36 Island syndrome, house mice, anxiety, island evolution, island tameness, behavior

40 Introduction

41 Organisms that colonize islands experience unique environmental challenges that

42 require novel solutions [1]. New kinds and distributions of resources on islands can select

43 for new foraging strategies [2-4]. For many island colonizers, a loss of predators alleviates

44 the need for behavioral or morphological defenses [2, 5-6]. If resources are difficult to

45 acquire and predatory risk is absent, the optimal foraging-risk ratio is highly skewed,

46 favoring exploration and boldness [7-9]. Island populations, therefore, provide

47 opportunities to test hypotheses about the evolution of behavior in novel environments.

48 Populations that spread to islands often display behavioral changes. Many

49 populations lose anti-predator behaviors [6]. Some island inhabitants are more easily

50 approached by humans, a phenomenon Darwin called “island tameness” [10-11]. In some

51 island rodents, territoriality and conspecific aggression are reduced compared to mainland

52 populations [12-13]. Behavioral responses to island environments sometimes occur in the

53 context of other phenotypic shifts. New behaviors are often accompanied by transitions in

54 life history and morphology [12,14-15] in a pattern referred to as the “island syndrome”

55 [16].

56 Although behavior is suspected to be an important component of adaptation to

57 island conditions, the question of whether island populations harbor heritable changes in

58 behavior has rarely been answered [13,17]. Demonstrating that behavior has evolved on

59 islands requires evidence of genetic change in behavioral traits.

60 A promising organismal system for testing hypotheses about behavioral evolution in

61 response to island environments is found on Gough Island, a remote volcanic island in the

62 middle of the South Atlantic Ocean. Despite the remote location of Gough Island, over

63 2,700 miles from the nearest mainland, house mice (Mus musculus domesticus) colonized

64 the island, probably via sealing ships from western Europe a few hundred generations ago

65 [18-20]. In a remarkable phenotypic transformation, these mice evolved a body size twice

66 that of their mainland counterparts [21-22]. Laboratory-born offspring of Gough Island

67 mice (hereafter “GI mice”) maintain their unusual size, confirming that this morphological

68 distinction has a genetic basis [23-24]. On Gough Island, mice live without the predators

69 and without the human commensals they commonly exploit for food and shelter on the

70 mainland [21,25]. The diet of GI mice is highly variable and seasonal, with invertebrates

71 (mainly earthworms) and seeds being the most stable food source [22]. During the winter

72 season, GI mice predate on endangered seabird populations that use the island as a nesting

73 ground, leading to the deaths of an estimated 2 million chicks and/or eggs per year [26].

74 The combination of increased body size, novel consumption of birds, loss of predatory

75 danger, and removal of human commensals predicts the evolution of increased exploration

76 and boldness in GI mice. Because GI mice are western European house mice (Mus musculus

77 domesticus), the same subspecies as the laboratory mouse [20], established methods in

78 biomedical research can be applied to profile behavioral evolution.

79 In this article, we use GI mice to examine recent behavioral evolution on an island.

80 By exposing juvenile and adult mice to novel environments with different levels of

81 perceived risk, we uncovered multiple lines of evidence that GI mice are bolder (i.e. less

82 anxious) and more exploratory than mice from a mainland reference strain. The detection

83 of these differences in inbred strains raised in a common environment demonstrates that

84 they are inherited. Our findings indicate that GI mice evolved enhanced boldness and

85 exploration over a short timescale and in concert with other substantial phenotypic

86 changes. This work lays the foundation for identifying the genetic changes responsible for

87 behavioral evolution in organisms that colonize islands.

89 Materials and Methods

90 Mouse Strains and Husbandry

91 Inbred strains of GI mice and mainland mice were used throughout this study. In

92 2009, GI mice were live-caught and shipped to the University of Wisconsin School of

93 Veterinary Medicine Charmany Instructional Facility, where a breeding colony was

94 established [23]. GI mice for this study belonged to a strain maintained for 21-23

95 generations of brother-sister mating. At this stage of inbreeding, we expect most of the

96 genome to be homozygous, allowing us to treat individual mice as replicates of the same

97 genetic background. Mainland mice belonged to the WSB/EiJ inbred strain founded from

98 breeding pairs caught in Maryland (purchased from the Jackson Laboratory, Bar Harbor,

99 ME) and were maintained in the same colony as GI mice for the same time period. F1s

100 were generated by crossing mice from the GI strain and the mainland strain in both

101 maternal directions. All mice were housed in micro-isolator cages with corn cob substrate

102 (1/8th inch; The Andersons Lab Bedding), with ad libitum access to food (Envigo 2020X

103 Teklad Global Diet) and water. Breeders were provided with a higher fat chow (Envigo

104 2019 Teklad Global Diet) and a red mouse igloo (Bio Serv). All cages were provided with

105 nesting material and irradiated sunflower seeds (Envigo). Cage changes occurred every 6-

106 8 days or, in the case of a new litter, 10 days after parturition. The colony was kept in a

107 temperature-controlled room (68-72°F) under a 12-hour light/dark cycle.

108 Mice used for behavioral testing were weaned 20-21 days after parturition and

109 housed with one littermate of the same sex to reduce behavioral effects of within-cage

110 hierarchies [27]. All mice were weighed to the nearest tenth of a gram during cage

111 changeouts and after the last behavioral assay (i.e. weekly from ages 4-10 weeks old +/- 1

112 day).

113

114 Behavioral Assays

115 All behavioral assays were conducted in a room separate from the main colony.

116 Each subject was tested four times with the following regimen (see Figure 1A): open field

117 (4 and 8 weeks old +/- 1 day), light/dark (9 weeks old +/- 1 day), and predator cue (10

118 weeks old +/- 1 day). All tests were conducted during the light phase of the light/dark

119 cycle. Females were scored for stage of estrous cycle according to Caligioni [28] on the

120 same day as testing, beginning with the second open field test. Twelve females (all from the

121 mainland strain) were in estrous the same day as testing. These animals fell within the

122 range of values for each trait measured in that strain and were included in the final

123 analysis. The order of subjects tested within litters each day was randomized. Each test

124 began by bringing the subjects’ cage into the room for a 30-minute acclimation period. The

125 experimenter remained in the room out of sight of the subjects throughout the acclimation

126 and testing periods except for transfer to and from the arena. The relevant arena for each

127 test (see Figure 1B for examples) was placed in the center of the room next to a movable

128 cart where a computer and video recording hardware were located. The arena was cleaned

129 with 70% ethanol after each test and at least five minutes was allowed for the ethanol to

130 evaporate before testing a new subject. All tests were video recorded using Debut Video

131 Capture Software v 5.33 at default settings with Focus set to 0. A Logitech HD Pro Webcam

132 C920 was positioned directly above the center of the arena. Two videos were taken before

133 each test to assist in video analysis: an “empty” video containing a short recording of the

134 empty arena, and a calibration video containing a short recording of the arena with specific

135 positions marked. The “empty” video was used in aligning all images of the test recording.

136 The calibration video was used in defining coordinates of regions of interest and converted

137 pixels to millimeters. Subsequent subsections provide additional details of experimental

138 design for each test.

139

140 Open Field Test

141 The open field (58cm W x 58cm D x 58cm H) was constructed from expanded, white

142 PVC (Grainger Industrial Supply). Lighting in the room was set so that the center of the

143 open field measured 300 +/- 5 lux. A calibration video was taken using a poster board on

144 the bottom of the arena with the center and each corner (1 inch from both edges) marked

145 by circles drawn with black marker. Subjects were initially placed in the center of the

146 arena facing away from the movable cart. The video recording software was started and

147 the subject was allowed to freely explore the arena. After 30 minutes of uninterrupted

148 exploration, the subject was returned to its home cage and the number of fecal boli in the

149 arena was counted. The open field was cleaned with 70% ethanol and five minutes were

150 allotted for the ethanol to evaporate before testing the cagemate.

151

152 Light/Dark Test

153 A mouse-sized place-preference chamber (27” W x 8” D x 15” H, San Diego

154 Instruments) was used for a light/dark test. One half of the arena had a black floor and was

155 exteriorly covered with black static cling film. The opposite chamber had a white floor and

156 was interiorly covered with white static cling film (except for the lid to allow video

157 observation). The chamber divider was positioned 4 cm above the floor to allow free

158 access between both chambers. Lighting was set so that the center of the light chamber

159 measured 300 +/- 5 lux. A calibration video was taken using a separate floor panel placed

160 on top of the floor in the white chamber with the center and each corner (0.5 inch from

161 both edges) marked by circles drawn with black marker. Subjects were initially placed in

162 the center of the light chamber facing the entrance to the dark chamber. The video

163 recording software was started and the subject was allowed to freely explore the arena.

164 After 30 minutes of uninterrupted exploration, the subject was returned to its home cage

165 and the number of fecal boli in each chamber was counted. Both chambers were cleaned

166 with 70% ethanol and the chamber floors were removed and shaken to evaporate the

167 ethanol before testing the cagemate.

168

169 Predator Cue Test

170 A rat-sized y-maze (San Diego Instruments) was used for a predator cue test. Walls

171 of the y-maze were increased to 20 inches to prevent escape during testing. Lighting was

172 set so that the center of the y-maze measured 300 +/- 5 lux. Two arms were designated as

173 cue arms where either a predator or control cue would be placed. The third arm was

174 designated as the “blank arm”. Due to the lighting arrangement of the room, the blank arm

175 was darker (~150 lux) than the center or cue-containing arms. A calibration video was

176 taken using tightly balled black garbage bags inside the ports of each cue arm and a poster

177 board in the blank arm marking the center and edge of the blank arm with black marker.

178 Subjects were initially placed in the center of the empty y-maze facing the blank arm. The

179 video recording software was started and the subject was allowed to freely explore the

180 arena. After 10 minutes, the subject was corralled to the end of the blank arm and a barrier

181 was inserted isolating the subject. Fecal boli were then counted and removed. A cotton

182 ball soaked in 4 ml of red fox urine (Minnesota Traplines) was placed in the port of a

183 randomly chosen cue arm as a predator-associated cue. A cotton ball soaked in 4 ml of

184 white doe urine (Code Blue) was placed in the port of the other cue arm as a non-predator-

185 associated cue. The barrier was then removed and the video recording software was

186 started again. After 20 minutes, the subject was corralled to the end of the blank arm and

187 contained using an insertable barrier. The subject was then anaesthetized using a cassette

188 stuffed with isoflurane-soaked gauze and transferred to a glass jar along with the cassette

189 for blood collection. The cotton balls were removed from the y-maze and fecal boli were

190 counted. The y-maze was cleaned with 70% ethanol and five minutes were allotted for the

191 ethanol to evaporate. The cagemate was then tested using the same procedure and cotton

192 balls.

193

194 Analysis of Videos

195 Videos were translated into x and y coordinates of the subject for each frame of the

196 video using the following pipeline. First, the raw videos were converted using ffmpeg via

197 the following command:

198

199 ffmpeg -i inputFileName.avi -pix_fmt nv12 -f avi -vcodec rawvideo convertedFileName.avi

200

201 The converted video file was run through an ImageJ script associated with Mousemove

202 [29], which we modified to label frames deleted due to inability to maintain the framerate

203 or frames when no mouse was detected. Deleted frames and frames when multiple objects

204 were detected were reincorporated using the position of flanking frames. Calibration

205 videos were run through the same process to obtain positions of known locations in the

206 arena. A small subset of videos had a large number of frames (>1%) deleted by

207 erroneously tracking multiple objects. The trajectory files for these videos were manually

208 edited to remove objects that never moved (i.e. not the mouse).

209 Combined trajectory files were then analyzed to measure a variety of different traits

210 depending on the test (see below). Pixels were translated to mm using known distances

211 between objects in the calibration trajectory file. A mobile threshold was set for each

212 subject using the method of Shoji [30]. Output for each trait was recorded for every minute

213 of the test to observe temporal patterns during the test.

214 The center of the open field was defined as a circle with diameter half the length of a

215 side (29cm). Distance traveled was the sum of all positional changes above the mobile

216 threshold in the combined trajectory file.

217 Times spent past three different thresholds in the light chamber were recorded: 1)

218 0.5 inches from the dark chamber, 2) midpoint of the light chamber, and 3) 0.5 inches from

219 the edge of the wall of the light chamber.

220 For the predator cue test, entrance into each arm was recorded once a mouse was

221 12 inches from the end of the arm. Distance traveled was computed as the sum of all

222 positional changes above the mobile threshold in the combined trajectory file.

223

224 Corticosterone Quantification

225 Immediately following the predator cue test, each mouse was anaesthetized using

226 an isoflurane-soaked gauze and transferred to a glass jar. Once unresponsive, the mouse

227 was decapitated, and blood was collected and stored on ice until all tests for the day were

228 completed. Blood was centrifuged at 1200 rpm for 10 minutes at 4°C to collect plasma.

229 Samples were stored at -80°C until assay submission. Plasma corticosterone concentration

230 was quantified by ELISA at the Wisconsin National Primate Research Center.

231

232 Statistical Analyses

233 All statistical analyses were conducted in R (v 3.4.1) [31]. Linear models were built

234 separately for each behavior using the lm function {stats}. Behaviors were treated as

235 dependent variables. Strain, sex, age (for the open field test), and cross direction (for F1s)

236 were treated as fixed, independent variables. Interactions suggested by graphical patterns

237 were also evaluated. The significance of an independent variable was evaluated using

238 additional sum-of-squares tests comparing models that included or excluded the variable.

239 Behaviors in F1s and parental strains were compared using t-tests. F1s from mothers of

240 different strains were tested separately when the cross direction term in the linear model

241 was significant. F1s were inferred to be similar to a parental strain when they were

242 statistically indistinguishable from that strain but distinct from the other parental strain.

243 F1s were inferred to be similar to the mid-parent value when they were indistinguishable

244 from that value but distinct from both parental strains. When F1s were statistically distinct

245 from both parental distributions and the mid-parent value they were inferred to be similar

246 to midpoint values between the mid-parent value and the parental mean to which they

247 were closest.

248

249 Data Availability

250 All computational scripts used in video analysis are available in Supplementary Files

251 1-9. Raw measurements and associated metadata for each mouse are included in

252 Supplementary Table 1.

253

254 Results

255 Open Field Test

256 We conducted open field tests at two life stages (juvenile and adult) to investigate

257 how GI mice and mainland mice explore a brightly lit, open environment with high risk of

258 detection. We observed extensive behavioral differences between GI mice and mainland

259 mice at both juvenile and adult ages (Figure 2; Table 1). Overall, GI mice travel more,

260 spend more time in the center of the arena, and deposit fewer fecal boli than mainland mice

261 (Figure 2; Table 1). Strain differences for time spent in the center of the arena and the

262 number of fecal boli deposited are similar between ages. Alternatively, distance traveled

263 shows a strong strain-by-age interaction, with the disparity between strains expanding

264 after puberty.

265 F1 offspring from crosses between GI mice and mainland mice show behavioral

266 patterns consistent with a nonadditive genetic architecture with occasional parental effects

267 (Figure 2; Table 2). For most behaviors, F1 averages are closer to the mainland strain.

268 Only the number of fecal boli deposited by F1 adults is statistically indistinguishable from

269 the mid-parent value (Table 2). Cross direction influences distance traveled in both

270 juveniles and adults. F1s with a GI mother travel less than F1s with a mainland mother

271 (Supplementary Table 2).

272 Collectively, these results indicate that GI mice evolved an increased willingness to

273 explore novel, risky environments. Whereas boldness-related behaviors are consistent

274 across life stages and cross directions, exploratory behaviors are influenced by age and

275 parental effects.

276

277 Light/Dark Test

278 To understand how exploration of a novel environment changes when a sheltered

279 area is available, we conducted a light/dark test using a place preference chamber with

280 equally sized light and dark chambers. We found that GI mice spend nearly twice as much

281 time in the light chamber as mainland mice (Figure 3; Table 1). GI mice also enter the light

282 chamber nearly twice as many times. Strain differences are maintained when considering

283 only exploratory bouts past the center of the light chamber. As with the open field test, GI

284 mice deposited less fecal boli, suggesting that baseline anxiety is similar in the two tests

285 (Table 1). These patterns indicate that GI mice evolved increased willingness to leave

286 sheltered areas and to venture farther from shelters compared to mainland mice.

287

288 Predator Cue Test

289 To reveal how exploration is impacted by the presence of a predator cue, we

290 conducted tests in a y-maze with one arm containing a cotton ball soaked in fox urine. In

291 the first 10 minutes of the test with no cues present, both strains show a slight preference

292 for the blank arm where no cue would be placed (Figure 4A). This pattern could reflect the

293 slight shading of this arm due to lighting constraints in the room. GI mice travel farther

294 overall and deposit fewer fecal boli than mainland mice during these first ten minutes,

295 echoing results from the open field test (Table 1). After both cues are presented, GI mice

296 and mainland mice spend less time in the arm containing the fox urine than in the arm

297 containing the deer urine (Figure 4A; Table 1). We find no evidence for strain differences

298 in this avoidance of the predator cue.

299 Plasma corticosterone concentrations collected immediately following the predator

300 cue test do not differ between strains (Figure 4B, t-test; P-value = 0.447). This result

301 indicates that GI mice are as physiologically stressed as mainland mice by the presence of a

302 predator cue though they continue to deposit fewer fecal boli (t-test; P-value < 1.2E-7).

303 Despite the apparent similarity in anxiety among the two strains, GI mice travel more than

304 mainland mice after cues are presented (Table 1). These findings suggest that GI mice

305 retain avoidance and stress responses associated with exposure to threats from terrestrial

306 predators even while evolving a greater willingness to explore.

307

308 Discussion

309 Gough Island mice are more exploratory and less fearful than their mainland

310 counterparts in novel, open environments without cues from terrestrial predators. Data

311 from across our experiments demonstrate that GI mice show constant motion throughout

312 each test with little preference for location except when direct cues of terrestrial predators

313 are present. In contrast, mainland mice have high innate anxiety and prefer to remain in

314 regions of each arena where a predator would have the smallest chance of detecting and/or

315 grasping them (e.g. periphery of the open field, dark chamber of the light/dark box). Since

316 all mice in this study were raised in a common setting, these behavioral differences among

317 inbred strains have a genetic basis.

318 Although understanding which aspects of the environment on Gough Island

319 stimulated behavioral evolution will require ecological studies, characteristics of GI mice

320 and the island suggest potential causes. GI mice are the largest wild house mice in the

321 world [23], offering an extreme case of the gigantism commonly observed among island

322 rodents [15-16], Larger bodies demand greater energetic requirements, particularly during

323 winter [32]. The willingness of GI mice to explore could have been driven by expanded

324 caloric demand, especially with a diet that is highly varied and likely opportunistic [22,33].

325 Additionally, the evolution of boldness may have facilitated the transition to eating seabird

326 chicks, which are a rich source of nutrients during winter when food is scarce and mortality

327 is high [33]. In contrast to mice inhabiting typical mainland environments, less anxious

328 mice on Gough Island can forage in open areas without danger from predators. For these

329 reasons, exploration and boldness likely provide significant advantages to mice on Gough

330 Island.

331 Comparisons across juvenile and adult life stages suggest that enhanced exploration

332 and reduced anxiety have evolved along distinct developmental trajectories in GI mice.

333 Although both juvenile and adult GI mice spend more time in the center of an open field

334 and deposit fewer fecal boli, GI mice travel farther than mainland mice only after puberty.

335 Results from other studies support the idea that exploration and boldness can be

336 uncoupled. Pumpkinseed fish approach a novel food source and a potential threat

337 differently [34]. Wild-caught starlings show greater escape motivation than hand-reared

338 starlings when placed in a new cage, but the two groups of birds respond similarly to novel

339 objects [35]. Organisms living in a complex environment are expected to benefit from

340 context-specific evolution of behavior [34,36]. Exploration, which is tied to the need for

341 resources, including food and shelter, takes on greater importance when mice leave the

342 nest and care of their mother. Alternatively, reducing unnecessary anxiety may increase

343 both juvenile and adult fitness by reducing negative health consequences from continuous

344 stress [9,37]. Therefore, natural selection could uncouple the developmental timing of

345 exploration and anxiety.

346 Rodents living on islands without terrestrial predators are expected to lose the

347 avoidance response to predator cues [38]. It is interesting, therefore, that GI mice and

348 mainland mice respond similarly to fox urine. There are several explanations for this

349 finding, none of which are mutually exclusive. First, GI mice may not have been on the

350 island long enough for new genetic variants that relax the response to predator cues to

351 arise and spread. Perhaps this phenotype has a smaller mutational target size than general

352 anxiety, a trait for which we observed a heritable reduction in GI mice. Secondly, the

353 source population(s) of GI mice may have experienced frequent terrestrial predation but

354 not avian predation, leaving intact the response to cues from terrestrial predators while

355 allowing the loss of anxiety about aerial threats. This idea is consistent with the “predator

356 archetype” hypothesis [39], which postulates disparate defense responses to avian and

357 terrestrial predators. A third possibility is that selection on general anxiety may be more

358 direct than selection to remove predator cue responses. The presence or absence of a

359 predator cue response makes no difference when predators are absent, whereas reducing

360 general anxiety can facilitate the optimization of foraging strategies and reduce energy

361 expenditure from unnecessary stress [9]. A final explanation is that the pathway for

362 detecting terrestrial predators could play additional functional roles in GI mice. Sulfur-

363 containing byproducts of meat-eating vertebrates are known to be aversive stimuli for

364 rodents [40], but there may be other sulfur-containing compounds on Gough Island that

365 mice need to detect. Since fish-eating seabirds are a major source of nutrition for GI mice

366 during the winter season [26,33], the ability to locate nests emitting sulfur signals could be

367 beneficial.

368 The phenotypic patterns we documented in F1s provide insights into the genetic

369 architecture that underlies the evolution of new behaviors in GI mice. We found that F1s

370 resemble mainland mice more than GI mice in open field tests, implying that GI mouse

371 alleles are recessive to mainland mouse alleles for several behaviors connected to boldness

372 and exploration. Recessive mutations tend to reduce function, suggesting that the variants

373 of interest could have decreased the expression of genes or pathways that are typically

374 active in mainland mice. The evolutionary trajectory of recessive mutations in GI mice

375 would have depended on their initial frequencies. Whereas new recessive variants would

376 have required stronger selection to become established and spread to high frequency than

377 new dominant variants [41], the probability of fixation of standing variants that contribute

378 to adaptation is independent of dominance [42]. The inference of recessive gene action

379 also raises the prospect that behavioral evolution was accomplished through a small

380 number of mutations with large phenotypic effects. Although loci that affect behavior in

381 laboratory strains of mice have been mapped to every chromosome [43], alleles with

382 substantial effects exist for some behaviors, including boldness and exploration [44-45].

383 Our characterization of F1s uncovered additional factors that shape behavior in GI

384 mice. An apparent maternal effect on distance traveled in the open field acts in the

385 opposite direction of the strain effect (i.e. GI mouse mothers reduce distance traveled by F1

386 offspring). This result implies that the genetic increase in distance traveled in GI mice is

387 greater than it appears based on comparisons to mainland mice since this maternal effect

388 must be overcome. Antagonism between maternal and direct genetic effects provides a

389 barrier to selection and could draw populations away from optimal trait values, depending

390 on the degree of covariance between these effects [46].

391 Several caveats accompany our interpretations. The mainland strain we profiled

392 was chosen based on its availability and common usage in mouse genetics. If the behavior

393 of this wild-derived inbred strain departs significantly from the mainland mice from which

394 GI mice are descended, our conclusions about the evolution of new behaviors in GI mice

395 could be incorrect. Western Europe is the most likely ancestral origin of M. m. domesticus

396 in North America and on Gough Island [20]. Detailed reconstruction of behavioral

397 evolution in GI mice will ultimately require behavioral studies in specific source

398 populations, which remain to be identified. Our study also assumes behavior in the

399 laboratory is representative of behavior in the wild. While the simplicity of our

400 experimental design facilitates the identification of causal factors in behavioral differences,

401 those interpretations are limited to the environment in which the tests were conducted.

402 Mice in the wild experience complex environments without ad libitum access to food.

403 Incorporating more realistic scenarios (e.g. fasting, outside enclosures) in future behavioral

404 studies will help broaden our interpretations of the behavioral changes in GI mice.

405 Our study is among the first to report inherited differences in behavior between

406 island and mainland populations [17,47]. Island deer mice that evolved larger bodies are

407 less aggressive than their mainland relatives, but this behavioral difference is not heritable

408 [13]. Perhaps the extreme ecological conditions on Gough Island (e.g. lack of predators,

409 lack of human commensals, presence of seabird chicks as a source of food in winter) have

410 created particularly strong selective pressure for behavioral evolution. Regardless of the

411 environmental causes, our demonstration of heritable differences between island and

412 mainland populations in a genetic model organism sets the stage for identifying genes

413 responsible for behavioral evolution associated with island colonization. GI mice could also

414 serve as a useful experimental model for exploration and anxiety in humans [48-49] where

415 the list of candidate genes for these behaviors continues to grow [50].

416

417 Authors’ Contributions

418 JAS helped design the study, carried out the behavioral testing, performed the video

419 analysis, conducted the statistical testing, and drafted the manuscript; MJN conceived of

420 the study, helped design the study, and critically revised the manuscript; BAP helped

421 design the study, and critically revised the manuscript. All authors gave final approval for

422 publication and agree to be held accountable for the work performed therein.

423

424 Acknowledgments

425 We thank Megan Latsch and Dr. Michelle Parmenter for conducting the pilot study

426 that inspired this project. We thank Dr. Andrew Bakken for helping construct the open

427 field and Dr. John Stratton for assistance in developing scripts.

428

429 Funding

430 This research was funded by National Institutes of Health (NIH) grant R01

431 GM100426. JAS was partially supported by the NIH Graduate Training Grant in Genetics at

432 the University of Wisconsin – Madison (T32 GM007133). MJN was partially supported by

433 an NHGRI training grant (5T32HG002760) to the UW-Madison Genomic Sciences Training

434 Program.

435

436 Competing Interests

437 The authors have no competing interests to declare.

438

439 Ethics

440 All experiments were approved by the University of Wisconsin-Madison

441 Institutional Animal Care and Use Committee (protocol no. V005209).

442

443 Figure and Table Captions

444 Figure 1: Schematic of testing design and arenas. A) Timeline of a subject mouse’s life. B)

445 Schematic of the open field arena. The center defined during video analysis is outlined by

446 the dashed circle. C) Schematic of the light/dark box. The subject has free access to both

447 equally-sized chambers during the test. Ditance ot the center threshold is noted by the

448 dashed line. D) Schematic of the y-maze use din the predator cue test. Each arm is equally

449 sized and freely accessible. Threshold for each arm defined during video analysis is

450 indicated in the right arm by the dashed line. Locations of the predator and control cues

451 are noted with black dots.

452

453 Figure 2: Open field comparisons between strain and ages. Means across sexes are

454 designated by horizontal black bars. F1s are separated based on strain of the mother. A)

455 Time spent in the center of the open field. B) Distance traveled in meters.

456

457 Figure 3: Results from light/dark test. Means across sexes are designated by horizontal

458 black bars. A) Left: amount of time spent in light chamber of light/dark box. Right: number

459 of entrances to the light chamber. B) Left: amount of time spent past the center of the light

460 chamber. Right: number of crosses past the center of the light chamber.

461

462 Figure 4: Results from Predator Cue Test. A) Mean ratio and standard error of time spent

463 in arm with a given cue to time spent in blank arm. Ratios on the left are for the ten

464 minutes of exploration before cues were added. B) Corticosterone concentration of mice

465 immediately after the predator cue test. Means across sexes are designated by horizontal

466 bars.

467

468 Table 1: Summaries of linear model results for each trait across experiments. The third

469 column shows transformations performed on the dependent variable. The fourth column

470 shows significant independent variables among Sex, Strain, and Age (for traits from the

471 open field test). When none of these predictors were significant, the overall mean is

472 presented as the Intercept Estimate.

473

474 Table 2: Summary of effects influencing behaviors in the open field. A "+" indicates

475 significance of that variable for the given trait. F1 groupings are based on which values

476 (mainland mean, GI mean, or midparent) the F1 distribution is significantly different from

477 using a t-test. For traits with a significant Cross Direction effect the two groups were

478 treated separately.

479

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30 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.10.292185; this version posted September 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Table 1: Summaries of linear model results for each trait across experiments. The third column shows transformations performed on the dependent variable. The fourth column shows significant independent variables among Sex, Strain, and Age (for traits from the open field test). When none of these predictors were significant, the overall mean is presented as the Intercept Estimate. Experiment Trait Transformation Independent Variables Intercept Estimate Strain (Mainland) Effect Distance Traveled (meters) None Sex, Strain, Age, Strain*Age 201.686 -17.416 Time Spent Mobile (seconds) None Sex, Strain, Age, Strain*Age 648.324 -6.506 Open Field Time in Center (seconds) Natural Log Strain 5.16728 -0.91655 Number of Fecal Boli None Strain 6.833 2.7137

Time in Light Chamber (seconds) None Strain 774.53 -271.62 Light Chamber Entrances Natural Log Strain 5.52242 -0.678 Light/Dark Box Time Past Center (seconds) None Strain 409.12 -155.35 Center Crosses Natural Log Strain 4.63887 -1.10467 Number of Fecal Boli None Strain 6.9697 2.5829

Ratio of Time in Cue Arm/Blank Arm Pre-exposure None None 0.7235549 - Ratio of Time in Control Arm/Blank Arm Pre-exposure None None 0.7340024 - Distance Traveled (meters) Pre-exposure None Strain 80.37 -28.213 Time Spent Mobile (seconds) Pre-exposure None Strain 186.497 -40.137 Predator Cue Ratio of Time in Cue Arm/Blank Arm Post-exposure None None 0.5019955 - Ratio of Time in Control Arm/Blank Arm Post-exposure None None 0.6987754 - Distance Traveled (meters) Post-exposure None Strain, Sex 131.017 -35.684 Time Spent Mobile (seconds) Post-exposure Natural Log Strain, Sex 5.86979 -0.14411 Corticosterone Concentration (ng/mL) None Sex 715.63 - bioRxiv preprint doi: https://doi.org/10.1101/2020.09.10.292185; this version posted September 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Table 1: Summaries of linear model results for each trait across experiments. The third column shows transformations performed on the dependent variable. The fourth column shows significant independent variables among Sex, Strain, and Age (for traits from the open field test). When none of these predictors were significant, the overall mean is presented as the Intercept Estimate. Strain p-value Sex (Male) Effect Sex p-value Age (Adult) Effect Age p-value Strain:Age Effect Strain:Age p-value 0.0204 -17.896 0.00135 62.007 1.80E-14 -116.698 2.00E-16 0.74908 -44.306 0.00351 99.081 2.82E-06 -245.43 6.66E-15 2.00E-16 ------1.79E-14 ------

6.25E-10 ------9.76E-10 ------2.54E-07 ------2.00E-16 ------9.97E-05 ------

------1.21E-13 ------2.57E-09 ------4.91E-08 -17.732 0.00374 - - - - 0.000247 -0.10018 0.010094 ------100.01 0.0481 - - - - bioRxiv preprint doi: https://doi.org/10.1101/2020.09.10.292185; this version posted September 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Table 2: Summary of effects influencing behaviors in the open field. A "+" indicates significance of that variable for the given trait. F1 groupings are based on which values (mainland mean, GI mean, or midparent) the F1 distribution is significantly different from using a t-test. For traits with a significant Cross Direction effect the two groups were treated separately. Experiment Trait Sex Effect Cross Direction Effect F1 Grouping Distance Traveled + + Underdominant (GI Mother), Mainland (Mainland Mother) Open Field Juvenile Time in Center - - Midparent/Mainland midpoint Number of Fecal Boli - - Gough Island

Distance Traveled + + Midparent/Mainland midpoint Open Field Adult Time in Center - - Midparent/Mainland midpoint Number of Fecal Boli - - Midparent bioRxiv preprint doi: https://doi.org/10.1101/2020.09.10.292185; this version posted September 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 1 A Predator Cue Light/Dark and Blood Open Field Open Field Collection Birth Wean

Puberty 0 1 2 3 4 5 6 7 8 9 10 Weeks B C D

58 cm 20 20 cm 29 cm 58 cm 17 cm bioRxiv preprint doi: https://doi.org/10.1101/2020.09.10.292185; this version posted September 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 2 A

F1 F1 F1 F1 Gough Mainland Gough Mainland (Gough mother) (Mainland mother) (Gough mother) (Mainland mother) Juvenile Adult bioRxiv preprint doi: https://doi.org/10.1101/2020.09.10.292185; this version posted September 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 3 A

Gough Mainland Gough Mainland bioRxiv preprint doi: https://doi.org/10.1101/2020.09.10.292185; this version posted September 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 4 A B

Gough Mainland