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1 Repeated evolution of circadian dysregulation in cavefish populations 2 3 Katya L. Mack1, James B. Jaggard2, Jenna L. Persons3, Courtney N. Passow4, Bethany A. 4 Stanhope2, Estephany Ferrufino2, Dai Tsuchiya3, Sarah E. Smith3, Brian D. Slaughter3, Johanna 5 Kowalko5, Nicolas Rohner3,6, Alex C. Keene2, Suzanne E. McGaugh4 6 7 1Biology, Stanford University, Stanford, CA, USA 8 2Department of Biological Sciences, Florida Atlantic University, Jupiter, FL, USA 9 3Stowers Institute for Medical Research, Kansas City, MO, USA 10 4Ecology, Evolution, and Behavior, University of Minnesota, Saint Paul, MN, USA 11 5Wilkes Honors College, Florida Atlantic University, Jupiter FL, USA 12 6Department of Molecular and Integrative Physiology, The University of Kansas Medical 13 Center, Kansas City, KS, USA 14 15

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16 Abstract 17 Circadian rhythms are nearly ubiquitous throughout nature, suggesting they are critical for 18 survival in diverse environments. Organisms inhabiting environments with arrhythmic days, such 19 as caves, offer a unique opportunity to study the evolution of circadian rhythms in response to 20 changing ecological pressures. Here we demonstrate that the cave environment has led to the 21 repeated disruption of the biological clock across multiple populations of Mexican cavefish, with 22 the circadian transcriptome showing widespread reductions in rhythmicity and changes to the 23 timing of the activation/repression of in the core pacemaker. Then, we investigate the 24 function of two genes with decreased rhythmic expression in cavefish. Mutants of these genes 25 phenocopy reductions in sleep seen in multiple cave populations, suggesting a link between 26 circadian dysregulation and sleep reduction. Altogether, our results reveal that evolution in an 27 arrhythmic environment has resulted in dysregulation to the biological clock across multiple 28 populations by diverse molecular mechanisms. 29 30

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31 Introduction 32 Circadian rhythms that maintain 24-hour oscillations in physiology and behavior are nearly 33 ubiquitous in nature1,2. Considered an adaptive mechanism for organisms to anticipate 34 predictable changes in their environment3,4, the biological clock coordinates diverse biological 35 processes, from the sleep-wake cycle and metabolism in animals to growth and photosynthesis in 36 plants5–7. In the majority of organisms, the biological clock is endogenous, but environmental 37 time-cues (“zeitgebers”) including light and temperature play a key role in synchronizing the 38 clock with an organism’s external environment8–11. Subjecting animals to light-dark cycles that 39 differ from that of a 24-hour day has profound impacts on organismal health, including reduced 40 performance, increased illness, and decreased longevity12–14. Consequently, systems where these 41 environmental zeitgebers have been lost provide a unique opportunity to examine how ecology 42 drives evolution in the biological clock, and characterize how clock evolution affects 43 downstream physiology and behavior15. Here, we examine daily expression changes across the 44 transcriptomes of cavefish populations that evolved under darkness and relatively low daily 45 temperature variation16,17. 46 47 The Mexican tetra, Astyanax mexicanus, exists as surface populations that live in rivers with 48 robust light and temperature rhythms, and at least 30 cave populations that live in perpetual 49 darkness with limited fluctuations in temperature or other environmental cues18. The existence of 50 multiple cave populations of independent origin and conspecific surface populations provides a 51 powerful comparative framework to study phenotypic evolution. Cave populations of A. 52 mexicanus have repeatedly evolved a suite of traits related to a cave-dwelling lifestyle, including 53 degenerate eyes19–21, reduced pigmentation22–25, and changes in metabolism and behavior26–35. 54 Circadian rhythms and sleep behavior are also substantially altered in cavefish27,36–38. While 55 cavefish largely maintain locomotor and physiological rhythms in light-dark conditions, many 56 populations show loss of these rhythms under constant darkness36–41. Cavefish populations have 57 also convergently evolved a drastic reduction in sleep compared to surface fish27. Alterations can 58 also be seen at the molecular level; an examination of clock genes (, , cry1a) in cave 59 and surface fish found changes in the expression level and timing of activation of these genes in 60 multiple cave populations.36 Consequently, the Mexican tetra provides a unique opportunity to

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61 study the evolutionary response of the biological clock and circadian rhythms to the arrhythmic 62 cave environment across multiple cave populations. 63 64 In vertebrates, circadian rhythms are regulated by transcriptional feedback loops, where clock 65 directly or indirectly regulate the expression of the genes from which they are 66 transcribed. The feedback loops of the circadian clock result in oscillations of expression of 67 ~24 hours42. These oscillating transcripts make up the “circadian transcriptome” and are a 68 substantial source of rhythmic physiology and behavior43–45. The conserved nature of the 69 biological clock in vertebrates make comparative studies extremely powerful. Even across 70 evolutionary timescales, many clock genes and downstream regulators of circadian behavior 71 maintain similar functions.45 While many species of cave-dwelling organisms show evidence of 72 weakened or absent locomotor rhythms46–52, no global analysis of transcriptional changes 73 associated with clock evolution has been performed in any cave species, including A. mexicanus. 74 Comparing global transcriptional shifts over circadian time between cave and surface 75 populations can provide insight into naturally occurring dysregulation of the biological clock 76 across multiple cave populations. 77 78 Here we characterize and compare the circadian transcriptome of A. mexicanus surface fish with 79 that of three cave populations. We find widespread changes in the circadian transcriptome, with 80 cave populations showing convergent reductions and losses of transcriptional oscillations and 81 alterations in the phase of key circadian genes. Visualization of clock genes mRNAs of in the 82 midbrain and liver of surface and cave fish support the dysregulation observed in the RNAseq 83 data, and identify tissue-specific patterns unique to cave individuals. To functionally assess the 84 contribution of dysregulated genes to sleep and behavior, we investigate the function of two 85 genes linked to circadian regulation by creating CRISPR/cas9 mutants, and find that these genes 86 are involved in regulating sleep behavior in A. mexicanus. Our results suggest that circadian 87 rhythm, a highly conserved mechanism across most metazoans, is repeatedly disrupted at the 88 molecular level in cavefish, with potential consequences for organismal physiology and 89 behavior. 90 91 Results

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92 Fewer genes show evidence of daily cycling in cavefish populations 93 To identify changes in rhythmic expression between cave and surface populations, we performed 94 RNAseq with total RNA from whole fish from three cave populations (Molino, Pachón, and 95 Tinaja) and one surface population (Río Choy) collected every 4-hours for one daily cycle under 96 constant darkness at 30 days post-fertilization (dpf)(see Methods). Fry were raised in a light-dark 97 cycle (14:10) and then transferred into total darkness 24 hours prior to the start of the sampling 98 period. An average of 14,197,772 reads were mapped per sample (File S1, Fig. S1-4). Filtering 99 of genes with low expression (< 100 total counts across all samples) resulted in 21,048 annotated 100 genes used for downstream analysis. Although the cave populations are not monophyletic53, 101 principle component analysis showed that the primary axis of differentiation among samples is 102 habitat (i.e., cave or surface; Fig. S5). 103 104 We used JTK_cycle54 to detect 24-hour oscillations in transcript abundance. We found that the 105 surface population had the greatest number of rhythmic transcripts (539), followed by Tinaja 106 (327), Pachón (88), and Molino (83), respectively (FDR < 0.05, see Table S1). Surprisingly few 107 genes (19) showed significant cycling across all populations (Fig. 1A,B)(File S1). In all 108 populations except Molino, we found significant overlap between rhythmic transcripts in A. 109 mexicanus and those identified in zebrafish55,56 (See Methods; at p < 0.05: surface, Pachón and 110 Tinaja, all p < 1 x 10-4; Molino p = 1, hypergeometric tests). Rhythmic transcripts in both the 111 surface and cave populations were enriched for the GO terms related to circadian rhythm, 112 including “regulation of circadian rhythm” (GO:0042752)(surface, q = 4.85 x 10-10; Tinaja, q = 113 4.16 x 10-8; Pachón, q = 2.33 x 10-6; Molino, q = 4.98 x 10-6). Rhythmic transcripts in the surface 114 population were also strongly enriched for “visual perception” (GO:0007601)(q = 4.80 x 10-12), 115 “sensory perception of light stimulus” (GO:0007602) (q = 9.28 x 10-12), and “phototransduction” 116 (GO:0007602)(q = 1.14 x 10-7), unlike cave populations (lists of enriched terms in File S1). 117 118 Nearly 22% (117) of genes found to be rhythmic in the surface population were arrhythmic (p- 119 value > 0.5) in all three cave populations and 77% (416) were arrhythmic in at least one cave 120 population (Table S2, File S1)(see Methods), highlighting that the loss of rhythmicity has 121 evolved independently among independent origins of the cave phenotype. Conversely, no genes 122 that were rhythmic across all caves were arrhythmic in the surface population. Genes that were

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123 rhythmic in the surface population but arrhythmic in cavefish populations include genes known 124 to be involved in the regulation of circadian rhythm (Table S3). For example, is a gene 125 that encodes a transcriptional activator that forms a core component of the circadian clock and 126 shows conserved rhythmic activity in zebrafish55 and mice57. Despite exhibiting a conserved 127 function across vertebrates, this gene is arrhythmic in all three cave populations (surface, p = 128 0.0004, q=0.02; all caves, p > 0.5). Further, in comparing the p-values of genes with known roles 129 in circadian rhythm (61 genes, see Methods, File S1), we found that these genes were 130 significantly more rhythmic in their expression in the surface population than in cave populations 131 (Wilcoxon signed rank test, p < 0.002 in all cave-surface pairwise comparisons). 132 133 Cave and surface populations show differences in periodicity and amplitude of rhythmic 134 transcripts 135 To identify genes with changes in rhythmicity between cave and surface populations, we 58 136 calculated a differential rhythmicity score (SDR) for each gene for each population pair . This 137 metric accounts for both changes in how rhythmic a transcript is, as defined by differences in the 138 JTK_cycle p-value for each gene between surface and cave populations, as well as differences in 139 the robustness of a transcript’s oscillation, as defined by differences in amplitude of gene 140 expression between the cave and surface populations. We found that 103 genes showed greater 141 rhythmicity in the surface fish for all surface-cave comparisons (e.g., surface-Pachón, surface- 142 Molino, surface-Tinaja; File S1)(Fig. 1C). This set of genes was highly enriched for the pathway 143 “Circadian clock system,” with a 21-fold enrichment compared to the background set of genes 144 used in this analysis (q = 9.96 x 10-11), and was the only pathway significantly enriched after 145 false-discovery rate correction. Comparatively, relatively few genes showed significantly 146 improved rhythmicity in cave populations (Fig. 1C, genes below zero for differential periodicity 147 and differential amplitude z-score), and no genes showed increases in rhythmicity across 148 multiple caves. 149 150 This unbiased assessment revealed that genes with reductions in rhythmicity in cave populations 151 include several primary and accessory components of the core transcriptional clock (Fig. 152 2A)(Table 1). In the circadian clock’s primary feedback loop, members of the (bHLH)-PAS 153 family (e.g., CLOCKs, ARNTLs) heterodimerize and bind to E-box DNA response elements to

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154 transcriptionally activate key clock proteins (e.g., PERs, CRYs). Negative feedback is then 155 achieved by CRY:PER heterodimers which inhibit the CLOCK:ARNTL complex. Another 156 regulatory loop is induced by the CLOCK:ARNTL complex activating the of nr1d1 157 and rorc genes (e.g., rorca and rorcb), which in turn both positively and negatively regulate the 158 transcription of (see Fig. 2B). Many of the genes that transcribe these activators and 159 repressors show reductions or loss of cycling in one or more cave populations, and a few show 160 reductions in rhythmicity in all three caves (e.g., rorca, rorcb, arntl2)(Table 1, Fig. 2). 161 Reductions in rhythmicity at core clock genes suggest an overall dampening of the core circadian 162 mechanism in cave populations compared to surface forms. 163 164 Genes with reduced rhythmicity in cavefish populations also include genes involved in 165 downstream regulation of oscillations in physiology and behavior (Table 1). Among the genes 166 which show reductions in rhythmicity across all three caves is arylalkylamine N- 167 acetyltransferase 2 (aanat2), which is involved in melatonin synthesis in the pineal gland59–61. 168 Aanat2 is required for the circadian regulation of sleep in zebrafish, and mutant zebrafish sleep 169 dramatically less at night due to decreased bout length, but not decreased bout number61. 170 Interestingly, cave populations show a dramatic reduction in sleep bout length compared to 171 surface fish27. Like in zebrafish59,61, the expression of aanat2 in surface A. mexicanus shows 172 robust rhythmic behavior (q = 6.6 x 10-4) with peak expression during subjective night. In 173 contrast, cavefish do not show evidence of rhythmic transcription of aanat2 (Molino, p = 1.0; 174 Tinaja, p = 0.82; Pachón, p = 0.33) and expression does not increase in these populations during 175 subjective night (Fig. 2). 176 177 Another gene that shows reductions in rhythmicity across all three caves is camk1gb, a gene 178 involved in linking the pineal master clock with downstream physiology of the pineal gland. 179 Knock-downs of this gene in zebrafish significantly disrupt circadian rhythms of locomotor 180 activity62, similar to what is observed in Pachón cavefish39,40. Camk1gb also plays a role in 181 regulating aanat2; knockdowns of camk1gb reduce the amplitude of aanat2 expression rhythm 182 by half in zebrafish62. 183

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184 Rhythmic transcripts in cave populations show a delay in phase compared to the surface 185 population 186 Next, we investigated whether the timing of the circadian clock differs between cave and surface 187 populations by comparing the peak expression time (i.e., phase) of cycling transcripts. Most 188 circadian genes show peak expression immediately before dawn or dusk63, and consistent with 189 this, surface and cave populations of A. mexicanus all showed a bimodal distribution of phases 190 (Fig. 3A). However, this distribution was shifted towards later in the day in fish from all three 191 cave populations compared to the surface population. Our results are consistent with the 192 individual gene analysis of Beale et al.36, which showed that the clock genes per1 and cry1a 193 display phase delays in fish from the Pachón and Chica caves relative to surface fish (Fig. S6). 194 We then compared the phase of A. mexicanus genes with phase estimates of orthologous core 195 clock genes in zebrafish sampled under dark conditions55. While zebrafish and A. mexicanus 196 diverged ~146 MYA64, the timing of peak expression of core clock genes was highly similar in 197 zebrafish and surface fish (median difference of 0.8 hours between orthologs, Table S4). 198 Comparatively, the phase of clock genes in the core and accessory loops are often more shifted 199 in cave populations relative to zebrafish phase than the surface population (median shifts of 4.7, 200 2.2, and 3.5 hours for Tinaja, Molino, and Pachón, respectively)(Table S4). 201 202 To further characterize differences in phase between surface and cave populations, we compared 203 the phase of all genes with evidence for rhythmic expression across cave-surface population 204 pairs (where p < 0.05 for a gene in the surface and the cave being compared). Consistent with the 205 phase shifts observed in core clock genes and accessory loops, for each cave-surface comparison, 206 we found that rhythmic transcripts showed significantly later peak expression in cave 207 populations (Wilcoxon signed rank test, all pairwise comparisons p < 2.2 x 10-16, Table S5)(Fig. 208 3B, phase of all genes in File S1). Rhythmic transcripts in Pachón showed the greatest average 209 shift, with a delay of 2.03 hours compared to surface fish (average shift, surface-Tinaja: 1.3 210 hours, surface-Molino: 0.48 hours). 211 212 Genes with circadian cis- elements show loss of cycling and changes in phase in cavefish 213 In light of the alterations we see in the expression of genes in the circadian transcriptome, we 214 next investigated circadian cis- elements in cavefish and surface fish. In vertebrates, circadian

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215 oscillations in are a consequence of interlocking transcriptional feedback loops 216 of core clock proteins binding to a combination of known cis-elements (E-box, RRE, D-box) in 217 the promoters and enhancers of target genes55,65. The timing of activator and repressor binding to 218 these cis- elements results in the phase of oscillating transcripts65. 219 220 To better understand what drives oscillation patterns in A. mexicanus, we extracted promoter 221 sequences of genes with rhythmic transcription in the surface population and identified 222 (TF) binding motifs in each sequence (Table S6). We consider rhythmic 223 genes with significant circadian binding motifs (i.e., E-box, RRE, D-box sequences) putative 224 targets of clock proteins in the circadian feedback loop. We then used a sliding window approach 225 to identify the specific time window when the phases of circadian TF targets are most enriched in 226 surface fish (see Methods). Consistent with observations in zebrafish55, the E-box, which is 227 bound by CLOCK-ARNTL complexes as part of the core feedback loop66, showed phase- 228 specific enrichment within the early peak (CT 2), and RRE elements, which are bound by RORA 229 proteins and NR1D1/NR1D2 of the accessory loop67, were enriched within the later peak (CT 230 14-16)(Fig. S7)(see Methods). The D-box, which binds the repressor NFIL3, was also enriched 231 within the early peak (CT 0-6), as in zebrafish55. The correspondence between surface A. 232 mexicanus surface fish and zebrafish suggests that the timing of key regulatory cascades 233 mediating circadian oscillations in gene expression is largely conserved between these species. 234 235 We then searched for circadian cis- elements in cavefish sequences upstream of genes that have 236 lost rhythmicity in cave populations. A large proportion of the putative targets of core clock 237 elements identified in the surface fish (e.g., genes identified as having an E-box, RRE or D-box 238 motifs in their promoter sequence as described above) do not cycle in one or more cave 239 populations: >77% are arrhythmic in at least one cave population. However, for nearly all gene 240 for which we identified a proximal circadian motif in the surface fish, we also identified a 241 binding site in cavefish (see Table S7 and Supplemental Methods). The maintenance of clock 242 binding sites in cavefish populations suggests that the reduction in the number of rhythmic 243 transcripts in cavefish is likely to be a consequence of changes in the transcription of core clock 244 genes, rather than divergence at the target cis- binding sites at downstream genes. 245

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246 We then compared the phase of putative clock targets in surface fish and cavefish. Consistent 247 with the phase shifts seen in the core clock (Table S4), putative clock targets also show shifts in 248 their phase in cavefish compared to surface fish (Table S8, Fig. S7). In the case of the E-box, in 249 cave populations the phase intervals with the highest proportion of these motifs occur later (CT 250 4, 6, 6-10 in Molino, Pachón, and Tinaja, respectively) than what is seen in the surface 251 population (CT 2) (Fig. S7). In contrast, for the RRE and D-box motif, there is no phase with 252 enrichment in cave populations. 253 254 Visualization of key circadian mRNAs reveals tissue-specific expression patterns in cave and 255 surface populations 256 Our whole-fry RNAseq data suggest that rhythms in the core clock are dampened, and that 257 rhythmic transcripts arephase shifted. Teleost circadian systems are highly decentralized and 258 autonomous core clock gene expression may be observed in many tissues68–70, and tissue-specific 259 differences may be obscured in whole organisms preparations71. 260 261 To address potential tissue-specific contributions to differences between cave and surface 262 populations, we used RNAscope® fluorescence in situ hybridization (FISH)72. We collected 263 brains and livers from individual 30dpf fry at 3 timepoints (CT0, CT8, and CT16)70 and 264 performed RNA FISH for key circadian mRNAs in the primary loop (per1 and arntl1a) and 265 regulatory loop (rorca and rorcb) (Advanced Cell Diagnostics, Hayward, CA, USA, see 266 Methods for details and controls). Brains were sectioned to allow visualization of the optic 267 tectum and periglomerular grey zone (Fig. S8). These are both sites showing significant clock 268 gene expression in zebrafish and these brain structures were consistently identifiable despite the 269 small size of the 30dpf midbrain73. 270 271 We find that temporal expression patterns of per1a and arntl1a mRNA in the midbrains of 272 surface fish is consistent with our RNAseq results and expression patterns in zebrafish55 (Fig. 4, 273 ‘B’). Per1a expression peaks strongly at dawn (CT0) and rapidly diminishes. Arntl1a cycles in 274 anti-phase and shows high expression at dusk (CT16). In contrast, cavefish show a number of 275 population specific alterations to the temporal expression of per1a and arntl1a over the day (Fig. 276 4, S10, S11, S12). Per1a expression in cavefish shows broader temporal expression, consistent

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277 with phase shift at this gene seen in whole fish (Fig. S6). Where per1a expression rapidly 278 diminishes in surface midbrain after CT0, expression of per1a persists through CT8 in cave 279 populations. Surprising, Tinaja per1a expression is at its lowest at CT0, when the surface 280 population per1a expression is greatest. Similarly, we see the persistence of arntl1a expression 281 in Tinaja and Molino outside of CT16, the primary window of expression in the surface 282 populations. Molino midbrains in particular showed high basal expression compared to the 283 surface, specifically at CT8 when per1a and arntl1a expression is nearly absent in surface fish 284 (Figs. S10, S12). 285 286 Temporal expression patterns for per1a and arntl1a in surface fish is also similar to zebrafish 287 (Fig. 4, ‘L’). Surface fish liver expression of per1a and arntl1a are largely in synchrony with the 288 expression of these genes in surface midbrain, however, expression of per1a and arntl1a in the 289 liver is also seen at the intermediate timepoint, CT8. Compared to surface, Pachón and Tinaja 290 livers show similar arntl1a expression but peak expression of per1a is delayed (occurring at CT8 291 instead of CT0). Per1a expression persists in Pachón and Tinaja at CT16, when per1a transcripts 292 are low in surface livers. Intriguingly, peak and trough expression in Pachón and Tinaja livers 293 appear to be out of sync with those in the midbrains (Fig. 4, ‘L’ vs. ‘B’). Per1a expression in 294 Tinaja liver is greatest at CT8, but is expressed highest in brains at CT16. In Pachón midbrains, 295 per1a expression is highest at CT0, but expression is highest in the livers at CT8 and CT16. In 296 contrast, Molino livers exhibit temporal expression of per1a that is synchronous with brains. 297 However, as with the midbrain, per1a has high basal expression in Molino compared to surface, 298 Pachón, and Tinaja. 299 300 Next, we examined the expression pattern and timing of rorca and rorcb, two gene members of 301 the regulatory loop that regulate the expression of arntl paralogs and circadian transcription67. 302 Our RNAseq results indicate that rorca and rorcb show peak expression in whole surface fish at 303 CT12 (Fig. 5, S13), similar to what has been observed in zebrafish (Table S4). Consistent with 304 this, in RNA FISH of surface fish livers and midbrains, rorca and rorcb expression was highest 305 midday (Fig. 5, S13). Temporal expression of rorca and rorcb was similar between livers and the 306 midbrain in cave populations. When compared to surface tissues, the expression of rorca and 307 rorcb is delayed in Pachón and much broader in Tinaja and Molino populations. Molino

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308 midbrains have high expression of rorca and rorcb compared to all other populations. In 309 addition, rorcb expression is seen across timepoints in all caves, particularly in Molino, whereas 310 it is nearly absent in surface fish at CT0 and CT8 (Fig. 5). 311 312 Thus, as observed in the whole organism RNAseq analysis, tissue-specific expression of these 313 primary loop and regulatory loop genes confirm the phase shifts and dysregulation of cavefish 314 transcripts compared to surface fish, though, our RNAscope analysis suggests that disruptions to 315 the temporal regulation per1a and arntl1a expression may not manifest uniformly across tissues 316 in Pachón and Tinaja cavefish (Fig. 4). 317 318 Aanat2 and rorca regulate sleep behavior in surface fish 319 The circadian clock plays an important role in regulating the sleep-wake cycle70. We have found 320 that expression of the genes in the core pacemaker, as well as a circadian sleep regulator 321 (aanat2), are differentiated between cave and surface populations (see Fig. 2). Cavefish 322 populations have repeatedly evolved towards a reduction in sleep at night and overall, raising the 323 question of whether dysregulation of the biological clock is contributing to changes in sleep 324 behavior between cave and surface populations. However, the function of circadian genes in A. 325 mexicanus sleep regulation has not been investigated. 326 327 To explore the role of clock genes in sleep regulation in A. mexicanus, we created crispant 328 surface fish mosaic for mutations in aanat2 and rorca. Rorca and aanat2 were selected because 329 they show differences in expression between the surface and all cave populations, and because 330 aanat2 plays an important role sleep regulation in zebrafish61. Mutagenized 30 dpf fish and 331 wildtype (WT) sibling controls were phenotyped for sleep behavior. Raw locomotor behavior 332 was processed to quantify sleep duration and behavioral architecture (locomotor distance, 333 waking activity, sleep bout duration, and sleep bout number) as previously described38,74. For 334 each injected construct, three separate biological replicates were carried out to ensure the 335 phenotype was consistently reproducible. Genotyping confirmed mutagenesis (Figs. S14, S15). 336 337 Aanat2 crispants demonstrate the role of this gene in the regulation of sleep in A. mexicanus. 338 While there was no difference in total sleep duration between WT surface and aanat2 surface

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339 fish crispants (unpaired t-test, p = 0.98, Fig. 6A), night sleep duration (minutes/hour) was 340 significantly reduced in aanat2 crispants (unpaired t-test, p = 0.0108, Fig. 6B,C). These results 341 are consistent with the function of this gene in zebrafish, where increased expression of this gene 342 during subjective night increases melatonin production, regulating nighttime sleep61. Zebrafish 343 aanat2 knockout mutants produce little or no melatonin and sleep almost half as much during the 344 night as control fish61. 345 346 Rorca crispants also show differences in sleep behavior compared to WT surface fish. Like 347 aanat2 crispants, rorca mutants sleep significantly less minutes per hour at night compared to 348 WT sibling fish (unpaired t-test, p < 0.0001, Fig. 6E,F); however, rorca crispants also sleep less 349 overall during a 24-hr period (unpaired t-test, p < 0.0001, Fig. 6D). While WT surface fish sleep 350 an average of 5.16 hours per 24-hr period, rorca surface mutants sleep just 2.26 hours per 24-hr 351 period. Rorca expression has not been previously associated with sleep regulation, but it is a 352 transcriptional regulator of the core clock gene arntl1a/b. The mammalian ortholog of arntl1a/b, 353 arntl1, has been implicated as a regulator in the sleep-wake cycle and deletions alter sleep 354 behavior in mammals75–77. Altogether, these results demonstrate that alterations to the biological 355 clock in surface fish can alter sleep behavior. Future investigations into the specific genetic 356 variants between surface and cavefish will help delimit the role of the biological clock in 357 cavefish sleep phenotypes. 358 359 Discussion 360 Circadian rhythms are nearly ubiquitous in eukaryotes, directing aspects of behavior, physiology, 361 and metabolism2. The biological clock is thought to provide a mechanism for organisms to 362 synchronize their physiology and behavior to predictable daily cycles3,4. However, we have a 363 limited understanding of how the biological clock is altered in the face of arrhythmic 364 environments, where organisms are isolated from regular environmental cues15,36,42,78,79. Caves 365 provide a natural laboratory for perturbations to circadian biology: isolated from light-dark 366 cycles and with little fluctuation in temperature, cave-dwelling organisms lack many of the 367 environmental stimuli found outside caves15,16,36. 368

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369 Here we have utilized comparisons between conspecific Astyanax mexicanus cavefish and 370 surface fish to explore changes to the biological clock following adaptation to the cave 371 environment. Using RNAseq to survey gene expression changes over a 24-hr period, we 372 identified several repeated alterations in the biological clock in cavefish populations, including: 373 1) a reduction in the number of transcripts with evidence of daily cycling, 374 2) the changes in the oscillation patterns of genes in the primary circadian feedback loop (e.g., 375 arntl2, arntl1a/b, cry1ba, cry1bb) and accessory loops (rorca/b, bhlhe40/1), and 3) an overall 376 delay in phase of the genes in the circadian transcriptome, including core clock genes and their 377 putative targets. Population genetic analyses indicate that a number of genes in the circadian 378 transcriptome are also genetically differentiated between the surface and one or more cave 379 populations (see Supplemental Material and Methods). Visualization of mRNAs of core (arntl1a, 380 per1a) and accessory loop (rorca, rorcb) in the midbrain and the liver of cave and surface fish 381 support changes in the regulation of key clock genes and highlight tissue-specific changes in the 382 expression of core clock genes in two of the cavefish populations relative to surface fish. The 383 decoupling of rhythms between tissues in caves is an interesting biological feature that warrants 384 further investigation across more tissues in these populations. 385 386 In addition to changes in the core and accessory loops, we also found that an important regulator 387 of circadian physiology, aanat2, shows reductions in rhythmicity across all three cave 388 populations. Aanat2 encodes a that regulates melatonin production in the pineal gland, 389 and robust expression of this gene is considered a marker for circadian clock function59,61,80,81. 390 We found that cavefish populations, unlike surface fish and other teleosts82, did not show 391 increased aanat2 expression during the subjective night (Fig. 2), making dysregulation of the 392 circadian clock an interesting candidate for the repeated changes in sleep regulation in seen 393 across cavefish populations27. Investigating the function of aanat2 and rorca through the 394 creation of aanat2 and rorca crispants revealed that these genes play a role in sleep regulation in 395 A. mexicanus. Consequently, we speculate that dysregulation of the biological clock may have 396 behavioral consequences for cavefish, and in particular, may contribute to the reductions in sleep 397 seen across cavefish populations. 398

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399 Beyond the regulation of circadian physiology, environmental light plays an essential role in cell 400 cycle control and activating DNA repair in teleosts83–86. Previous work in this system has found 401 that cave populations of A. mexicanus show higher levels of DNA repair activity and increased 402 expression of DNA repair genes in the dark36. As a common light signaling pathway controls 403 both DNA repair and clock entrainment, one purposed mechanism for this is that cavefish have 404 sustained upregulation of clock genes normally driven by light, akin to ‘perceiving’ sustained 405 light exposure36. While we found that DNA repair genes are more often upregulated in cave 406 populations (Table S10, see Supplemental Material and Methods), we did not find that genes 407 associated with light induction were more likely to be upregulated in cavefish compared to 408 surface fish under dark-dark conditions (Table S11). Nor did we find that light-activated genes in 409 the core circadian feedback loop are consistently upregulated in cave populations (see 410 Supplemental Material and Methods, Figure S16). Thus, more work is necessary to understand 411 the relationship between clock evolution and DNA repair in this system. 412 413 Altogether, these results suggest that highly conserved features of circadian biology have been 414 repeatedly disrupted across populations of cavefish. Further research on the molecular basis of 415 clock dysregulation will help delineate if clock dysfunction is a consequence of molecular 416 alterations to (1) the pathways that perceive, or synchronize the biological clock to, external 417 stimuli, or (2) alterations to the core transcriptional feedback loop, and whether this 418 dysregulation is a consequence of selection or drift after the movement into the cave 419 environment. 420 421 Methods 422 Sampling 423 Samples from each population were derived from the same mating clutch and raised in 14:10 424 light-dark cycle. All fish were exactly 30 days post fertilization at the start of the experiment. 425 Fish were kept in total darkness for 24 hours prior to sampling and throughout the duration of the 426 experiment. Six replicates were first sampled at 6am (Circadian Time 0) and then every four 427 hours throughout until 2am (corresponding to CT 20). During the experiment, fish were fed ad 428 libitum twice daily, in the morning and evening (exactly at 8am and 8pm). Individuals were 429 immediately flash frozen in liquid nitrogen. RNA was extracted from the whole organism with

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430 extraction batch randomized for time of sampling and population. Samples were sequenced on 431 the Illumina HiSeq 2500 platform to produce 125-bp paired-end reads (File S1) using strand- 432 specific library preparations. Samples were randomized across lanes relative to population and 433 time of sampling. Previous work showed no extraction batch or lane effect on these samples87. 434 435 Read mapping 436 Reads were cleaned of adapter contamination and low-quality bases with Trimmomatic 437 (v0.33)88. Cleaned reads were mapped to the A. mexicanus draft genome assembly v1.02 438 (GCA_000372685.1) with STAR89 and reads overlapping exonic regions were counted with 439 Stringtie (v1.3.3d)90 based on the A. mexicanus Ensembl v91 annotation. Genes with fewer than 440 100 reads across all samples were removed from the analysis. Reads were subsequently 441 normalized for library size and transformed with a variance stabilizing transformation with 442 DESeq291 for principle component analysis. 443 444 Identification of rhythmic transcripts 445 Rhythmic transcripts with 24-h periodicity were identified using the program JTK_cycle54, using 446 one 24-h cycle, with a spacing of 4 hours and 6 replicates per time point. JTK_cycle was also 447 used to estimate the amplitude and phase of each rhythmic transcript. We retained genes as 448 rhythmic at an False Discovery Rate (FDR) of 5%. Our overall result, that more genes are 449 cycling in the surface population that in cave populations (Table S1), was insensitive to specific 450 value of the FDR cut-off. We defined arrhythmic transcripts at p-value > 0.5, a conservative cut- 451 off for arrhythmic expression that has been used in other studies58,92. 452 453 Differential rhythmicity analysis 454 To identify differential rhythmicity between pairs of populations, we calculated a differential

455 rhythmicity score (SDR) for genes with a JTK_cycle p-value < 1 in either population. As in 58 456 Kuintzle et al. , SDR was defined as:

&' + &# 457 !"# = √2 458

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459 Where Zp is the Z-score for changes in periodicity between populations, where changes in

460 periodicity are defined as log(p -value of population 1) – log(p -value of population 2). ZR is the 461 Z-score computed for changes in amplitude between populations, where changes in amplitude

462 are defined as: log2(Amplitude of population 2 / Amplitude of population 1). Amplitude for each 463 gene was estimated with JTK_cycle. 464

465 We then computed a p-value for SDR values using a Gaussian distribution based on the fit to the 466 empirical distribution. We used R’s p.adjust to perform a Benjamini & Hochberg correction for 467 multiple testing (Table S9). 468 469 Promoter analysis of rhythmic genes 470 We defined putative promoters as the region 1-kb upstream to 200bp downstream from the 471 transcription start site (TSS). Population genetic data53 was used to identify variants with 472 putative promoter regions segregating between populations and create alternative reference 473 genomes for each population. Genes with a JTK_cycle FDR < 0.1 in the surface were used to 474 identify phase enrichment of circadian cis- elements in the surface population. FIMO of the 475 MEME suite93 was used to identify motifs in each promoter. FIMO was used to estimate p- 476 values for motifs in each sequence; motifs with p-values < 0.0001 (the default cut-off) were 477 considered for downstream analyses. To identify phase-specific enrichment of binding motifs, 478 we tested sliding windows of time with Fisher’s exact tests for motifs in phase versus motifs out 479 of phase compared to the total set of in- and out- of phase genes. To compute changes in phase of 480 genes that are putative targets of circadian transcription factors between surface and cave 481 populations, we calculated the minimum distance between the two phases based on a 24-h 482 clock. Further details of this analysis are available in the Supplemental Methods. 483 484 Tissue preparation for RNA fluorescence in situ hybridization (FISH) 485 Pachón, Molino, Tinaja, and surface fish derived from the Río Choy population were raised in 486 densities of 20-25 individuals/3L tank in 14:10LD cycle at 23°C and 750-800μS/cm 487 conductivity, with feeding and water quality control as previously described94. 488

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489 29dpf fish were placed in constant darkness in the dark phase of the day preceding tissue 490 collection. 30dpf fish were fed ad libitum at 8am and 8pm on the day of tissue collection. 491 Individual fish were euthanized in MS-222 at CT0, CT8, and CT16. Experiments were 492 performed in duplicate: two fish were euthanized and dissected at each time point from each 493 population. All efforts were made to reduce light exposure prior to fixation. Dissected brains and 494 livers were placed into a freshly prepared solution of 4% PFA in DEPC 1X PBS on ice. Samples 495 were transferred to room temperature after 3-5 minutes on ice and allowed to fix for 1 hour. 496 Samples were then placed at 4°C for 36 hours. After 36 hours, samples were rinsed briefly in 497 DEPC water and washed in DEPC 1X PBS three times for 15 minutes with constant shaking. A 498 graded EtOH wash was performed (30%- 50%- 70%- 100%) in DEPC treated water with 5 499 minutes between steps. 500 501 Tissue processing and paraffin embedding were performed with a PATHOS Delta hybrid tissue 502 processor (Milestone Medical Technologies, Inc, MI, USA)95. Paraffin sections were cut with 503 12μm thickness using a Leica RM2255 microtome (Leica Biosystems Inc. Buffalo Grove, IL, 504 USA) under RNase free conditions. Brains were sectioned coronally through the mesencephalon- 505 diencephalon to allow visualization of the optic tectum and periventricular grey zone, sites 506 showing significant clock gene expression in zebrafish73 (Fig. S8). We were unable to 507 consistently section through the same region of the brain in order to compare expression in 508 hypothalamic nuclei due to the size of 30dpf brains73. Livers were sectioned longitudinally. 509 Sections were mounted on SureBond charged microscope slides (cat#SL6332-1, Avantik, 510 Springfield, NJ, USA). To allow for comparison between populations, all brains or livers from a 511 single probe set were processed at once (e.g., rorca-rorcb brains from all populations, and all 512 time points were processed at the same time). In addition, brains or livers from each time point 513 (CT0, CT8, CT16) were processed on a single slide to allow for a direct comparison between 514 time points within populations. 515 516 RNA FISH, imaging and analysis 517 RNAscope® probes were designed by ACDBio to target all known mRNA transcripts of genes 518 per1a (ENSAMXG00000019909), arntl1a (ENSAMXG00000011758), rorca 519 (ENSAMXG00000009363), and rorcb (ENSAMXG00000015029) (Advanced Cell Diagnostics,

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520 Hayward, CA, USA) based on Astyanax_mexicanus-2.0, INSDC Assembly (GCA_000372685). 521 Probes were ordered for two-plexing (per1a-arntl1a and rorca-rorcb) to provide an internal 522 control in each sample for mRNA phase and quantity. Probes may be ordered at the ACD Online 523 Store using the following catalog numbers: per1a (590801), arntl1a (590831-C2), rorca 524 (590811), and rorcb (590821-C2). In situ hybridization of per1a, arntl1a, rorca, and rorcb 525 mRNA was performed on paraffin sections using RNAscope Multiplex Fluorescent Detection 526 Kit v2 (Advanced Cell Diagnostics, Hayward, CA, USA) according to the manufacturer’s 527 protocol. 528 529 Slides were stored in the dark at 4°C before imaging. Fluorescent images of sections were taken 530 with a Nikon Eclipse TI equipped with a Yokogawa CSU W1 spinning disk head and 531 Hamamatsu ORCA-Flash 4.O camera for high-resolution imaging using a Nikon 20x/0.75 Plan 532 Apo objective. Probes rorca and per1a were imaged with 561nm laser and collected with an 533 ET605/70m emission filter, and arntl1a and rorcb with 633nm laser and collected with an 534 ET700/75m emission filter. DAPI was excited at 405nm and collected with an ET455/50m 535 emission filter. Samples were identified automatically for imaging using a custom script as in 536 Guo et al.96, and each sample was imaged as a tile scan with 10% overlap and z-stack of depth of 537 18µm with 0.9 µm optical slice thickness. Microscope and camera settings during acquisition 538 were identical across all samples to allow for a direct comparison between timepoints and 539 populations. Image processing in Fiji97 was identical between populations and time points within 540 a tissue and probe set. Briefly, tiles were stitched into a complete image using Grid/Collection 541 Stitching98, maximum projected and contrast adjusted. To properly compare cave populations to 542 surface populations, we set the intensity ranges for each image to that of the surface sample 543 images (Fig. 4). The intensities of Molino and Tinaja brain images in Fig. S11 are adjusted for 544 oversaturation. Figs. 4 and 5 exclude the DAPI channel. Corresponding DAPI staining for these 545 figures can be found in Fig. S9. Images are representative of two fish collected from each 546 timepoint per population. For Figs. 4 and 5, maximum projected images are shown. 547 548 For quantification in Fig. S10, tiles were stitched into a complete image using Grid/Collection 549 Stitching98. Stitched images were sum projected and background subtracted. 400x400 anatomical 550 regions of interest were identified by visual inspection and average intensities were measured for

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551 each channel. To allow for a comparison of relative expression per cell from multiple samples, 552 DAPI intensity was measured as a proxy for cell density, and FISH signal intensity was 553 normalized against DAPI intensity for each region. Technical replicates (two to three sections 554 from the same liver or brain) were averaged to make biological replicates represented as points 555 in Fig. S10. All groups have two biological replicates with the exception of rorca-rorcb Pachón 556 brains which have only one due to sample loss. Graphing and statistical analysis were performed 557 using GraphPad Prism software (GraphPad, Prism version 8.3.0, GraphPad Software, San Diego, 558 California USA). For each mRNA probe, cave population means were compared with the control 559 mean (Surface) within timepoints using 2-way ANOVA. Dunnett's test was used to correct for 560 multiple comparisons across populations and timepoints. Original data underlying this part of the 561 manuscript can be accessed from the Stowers Original Data Repository at 562 http://www.stowers.org/research/publications/libpb-1485. 563 564 CRISPR/Cas9 design and genotyping 565 Functional experiments were performed by generating crispant fish mosaic for mutations in 566 aanat2 and rorca. CRISPR gRNAs were designed using ChopChop v3 software99. gRNAs were 567 designed to target an exon in each of the genes and to produce a double stranded break close to a 568 restriction enzyme site for genotyping purposes. For aanat2, the gRNA was 5’- 569 GGTGTGCCGCCGCTGCCGGA-3’ and for rorca the gRNA was 5’- 570 gGAGAACGGTAACGGCGGGCA-3’ where the lower case g at the 5’ end was added to the 571 sequence for T7 transcription (restriction enzyme target sites are underlined). gRNAs were 572 synthesized as previously described100 with modifications101,102. Briefly, gRNA specific oligos 573 were synthesized (IDT) that contained the gRNA target site, T7 promoter, and an overlap 574 sequence: 575 576 aanat2 oligo A:

5’- TAATACGACTCACTATAGGTGTGCCGCCGCTGCCGGAGTTTTAGAGCTAGAAATAGC-5773’

578 rorca oligo A:

5’-TAATACGACTCACTATAgGAGAACGGTAACGGCGGGCAGTTTTAGAGCTAGAAATAGC-3’

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579 Each oligo A was annealed to the oligo B:

5’- GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATT TCTAGCTCTAAAAC-3’ 580 581 and these annealed oligos were amplified. gRNAs were transcribed using the T7 Megascript kit 582 (Ambion), as in Klaassen et al.103 and Stahl et al.102, and purified using a miRNeasy mini kit 583 (Qiagen). Nls-Cas9-nls104 mRNA was transcribed using the mMessage mMachine T3 kit (Life 584 Technologies) following the manufacturer’s instructions and purified using the RNeasy MinElute 585 kit (Qiagen) following manufacturer’s instructions. 150 pg Cas9 mRNA and 25 pg aanat2 gRNA 586 or 150 pg Cas9 mRNA and 25 pg rorca gRNA were injected into single-cell surface fish 587 embryos (2 nL/embryo were injected). 588 589 Genomic DNA from injected embryos and wild-type (uninjected) controls was extracted at 48 590 hours post-fertilization101,102 and used for genotyping by PCR to determine if mutagenesis was 591 achieved at the locus through gel electrophoresis (Figs. S14, S15). Briefly, gRNAs were 592 designed to disrupt specific restriction enzyme sites; samples were incubated with that restriction 593 enzyme to determine if the Cas9 cut. The undigested fragment acts as a control while the 594 digested fragment is used to genotype. Cases where we see a band where the undigested 595 fragment is indicates that the restriction enzyme was not able to cut due to the site being 596 disrupted because of the CRISPR/Cas9 + gRNA. Gene specific primers are given in File S1. 597 PCRs were performed with a 56 °C annealing temperature and a 1-minute extension time for 35 598 cycles. The resulting PCR product was split in half, and one half was restriction enzyme 599 digested. A DNA fragment containing the aanat2 target fragment was digested with BbvI (New 600 England Biolabs Inc.). The rorca DNA fragment was digested with Cac8I (New England 601 Biolabs). Both DNA fragments were digested at 37 °C for 1 hour. 602 603 For wildtype individuals, the PCR fragment was used for cloning. For crispant fish, restriction 604 enzyme digests were performed (as described above) and the uncleaved bands were gel purified 605 using a gel extraction kit (Qiagen). PCR products were cloned into the pGEM®-T Easy Vector 606 (Promega), and three clones per individual were picked, grown, and purified using QIAprep Spin

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607 Miniprep Kit (Qiagen) and then sequenced by Sanger sequencing to confirm mutagenesis 608 (Eurofins Genomics). 609 610 Quantifying sleep behavior 611 30 dpf injected crispant fish and WT (uninjected) sibling controls were used for all behavioral 612 experiments. Fish were maintained on a 10-14 light-dark cycle throughout development as well 613 as for behavioral experiments. Fish were placed in 12-well tissue culture plates (Cellstar) 18-24 614 hours before behavioral recording to acclimate to the recording chamber. Fish were fed normal 615 brine shrimp meals before the start of the recording, which began at ZT0 (zeitgeber time) and 616 lasted 24 hours. Video recordings were processed in Ethovision XT (v13). Raw locomotor data 617 was processed with custom-written scripts to quantify sleep duration and behavioral architecture 618 such as locomotor distance, waking activity, sleep bout duration, and sleep bout number38,74. For 619 each injected construct, three separate biological replicates were carried out to ensure the 620 phenotype was consistently reproducible. Each biological replicate represented different clutches 621 of fish, injected on different days. Both wildtype and crispant individuals were assessed from 622 each clutch. 623 624 Data accessibility 625 All sequence data were deposited in the short-read archive (SRA) under the accession 626 PRJNA421208. Data underlying RNA FISH analysis can be found at 627 http://www.stowers.org/research/publications/libpb-1485. 628 629 Acknowledgements 630 We thank the University of Minnesota Genomics Center for their guidance and performing the 631 cDNA library preparations and Illumina HiSeq 2500 sequencing. The Minnesota 632 Supercomputing Institute (MSI) at the University of Minnesota provided resources that 633 contributed to the research results reported within this paper. Funding was supported by NIH 634 (1R01GM127872-01 to SEM and ACK) and NSF award IOS 165674 to ACK, and a US-Israel 635 BSF award to ACK. KLM was supported by a Ruth Kirschstein National Research Service 636 Award from NIH and a Stanford Center for Computational, Evolutionary and Human Genomics 637 (CEHG) postdoctoral fellowship. CNP was supported by Grand Challenges in Biology

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908 Table 1. Known circadian regulators with losses or reductions in rhythmicity in cave 909 populations1 Gene name Populations Predicted functions of transcribed proteins

Core loop

arntl1a Tinaja, Pachón Activator in core circadian feedback loop105

arntl1b Tinaja, Pachón Activator in core circadian feedback loop105

arntl2 Tinaja, Molino, Pachón Activator in core circadian feedback loop105

cry1aa Pachón Repressor in core circadian feedback loop106

cry1ba Tinaja, Pachón Repressor in core circadian feedback loop106

cry1bb Tinaja, Pachón Repressor in core circadian feedback loop106

cry4 Pachón Repressor in core circadian feedback loop106

Accessory pathways

rorca Molino, Tinaja, Pachón Regulator of core feedback loop107

rorcb Molino, Tinaja, Pachón Regulator of core feedback loop107

bhlhe40 Molino Regulator of core feedback loop108

Tinaja, Pachón Regulator of core feedback loop108

fbxl3a Molino, Tinaja, Pachón Regulator of core feedback loop109,110

nfil3 Tinaja, Pachón Regulator of core feedback loop111

nfil3-5 Tinaja, Pachón Regulator of core feedback loop55

cipcb Pachón Regulator of core feedback loop112

Mediators of downstream physiology and behavior

aanat2 Molino, Tinaja, Pachón Controls daily changes in melatonin production61

camk1gb Molino, Tinaja, Pachón Involved in linking pineal master clock with peripheral circadian activity62

nptx2b Pachón, Molino Modulates circadian synaptic changes113 910 1Exhaustive list of genes with changes in rhythmicity can be found in File S1. 911

35 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.14.906628; this version posted January 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

912 Figure 1. A. Overlap of genes with rhythmic expression between populations. B. Heatmap of 913 genes with rhythmic patterns in surface fish (Río Choy), ordered by gene phase, compared to 914 expression in cave populations. Each column represents gene expression at a single-time-point, 915 sampled every four hours from 0-20 hours. Redder boxes correspond to higher expression. C. 916 Identifying genes with changes in rhythmicity between cave and surface populations. Genes with 917 greater amplitude values have larger differences between their expression peak and trough, 918 where genes with greater periodicity show stronger cyclical oscillation patterns (see Methods). 919 Genes are colored based on their SDR q-values. Genes with positive values for both show 920 increased rhythmicity in the surface population, where genes with negative values show 921 increased rhythmicity in cave populations. 922 Surface Molino Pachon Tinaja ENSAMXG00000028873 ENSAMXG00000021795ENSAMXG00000024728ENSAMXG00000025648ENSAMXG00000028873 ENSAMXG00000025648ENSAMXG00000028873 ENSAMXG00000021795ENSAMXG00000024728ENSAMXG00000025648 ENSAMXG00000024728ENSAMXG00000025648ENSAMXG00000028873 A B ENSAMXG00000018025ENSAMXG00000019111ENSAMXG00000019307 ENSAMXG00000019111ENSAMXG00000019307ENSAMXG00000021795ENSAMXG00000024728 ENSAMXG00000017870ENSAMXG00000018025ENSAMXG00000019111ENSAMXG00000019307 ENSAMXG00000019111ENSAMXG00000019307ENSAMXG00000021795 ENSAMXG00000017378ENSAMXG00000017665ENSAMXG00000017870 ENSAMXG00000017870ENSAMXG00000018025 ENSAMXG00000017378ENSAMXG00000017665 ENSAMXG00000017665ENSAMXG00000017870ENSAMXG00000018025 ENSAMXG00000013705ENSAMXG00000016091ENSAMXG00000017328 ENSAMXG00000016091ENSAMXG00000017328ENSAMXG00000017378ENSAMXG00000017665 ENSAMXG00000013103ENSAMXG00000013705ENSAMXG00000016091ENSAMXG00000017328 ENSAMXG00000016091ENSAMXG00000017328ENSAMXG00000017378 ENSAMXG00000011798ENSAMXG00000011870ENSAMXG00000012953ENSAMXG00000013103 ENSAMXG00000012953ENSAMXG00000013103ENSAMXG00000013705 ENSAMXG00000011798ENSAMXG00000011870ENSAMXG00000012953 ENSAMXG00000011870ENSAMXG00000012953ENSAMXG00000013103ENSAMXG00000013705 ENSAMXG00000009800ENSAMXG00000010828ENSAMXG00000011797 ENSAMXG00000010828ENSAMXG00000011797ENSAMXG00000011798ENSAMXG00000011870 ENSAMXG00000009418ENSAMXG00000009800ENSAMXG00000010828ENSAMXG00000011797 ENSAMXG00000010828ENSAMXG00000011797ENSAMXG00000011798 ENSAMXG00000008112ENSAMXG00000008834ENSAMXG00000009250ENSAMXG00000009418 ENSAMXG00000009250ENSAMXG00000009418ENSAMXG00000009800 ENSAMXG00000008112ENSAMXG00000008834ENSAMXG00000009250 ENSAMXG00000008834ENSAMXG00000009250ENSAMXG00000009418ENSAMXG00000009800 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ENSAMXG00000002780ENSAMXG00000003145ENSAMXG00000004327 ENSAMXG00000003145ENSAMXG00000004327ENSAMXG00000004730 ENSAMXG00000002337ENSAMXG00000002780ENSAMXG00000003145ENSAMXG00000004327 ENSAMXG00000002780ENSAMXG00000003145ENSAMXG00000004327 ENSAMXG00000001036ENSAMXG00000002059ENSAMXG00000002337 ENSAMXG00000001036ENSAMXG00000002059ENSAMXG00000002337ENSAMXG00000002780 ENSAMXG00000025749ENSAMXG00000001036ENSAMXG00000002059 ENSAMXG00000025749ENSAMXG00000001036ENSAMXG00000002059ENSAMXG00000002337 6 3 ENSAMXG00000021563ENSAMXG00000025204ENSAMXG00000025525ENSAMXG00000025749 ENSAMXG00000025204ENSAMXG00000025525ENSAMXG00000025749 ENSAMXG00000021563ENSAMXG00000025204ENSAMXG00000025525 ENSAMXG00000021563ENSAMXG00000025204ENSAMXG00000025525 ENSAMXG00000019070ENSAMXG00000019972ENSAMXG00000020411 ENSAMXG00000019972ENSAMXG00000020411ENSAMXG00000021563 ENSAMXG00000018992ENSAMXG00000019070ENSAMXG00000019972ENSAMXG00000020411 ENSAMXG00000019070ENSAMXG00000019972ENSAMXG00000020411 ENSAMXG00000018466ENSAMXG00000018625ENSAMXG00000018992 ENSAMXG00000018466ENSAMXG00000018625ENSAMXG00000018992ENSAMXG00000019070 ENSAMXG00000018220ENSAMXG00000018466ENSAMXG00000018625 ENSAMXG00000018220ENSAMXG00000018466ENSAMXG00000018625ENSAMXG00000018992 ENSAMXG00000015171ENSAMXG00000016188ENSAMXG00000016317ENSAMXG00000018220 ENSAMXG00000016188ENSAMXG00000016317ENSAMXG00000018220 ENSAMXG00000015171ENSAMXG00000016188ENSAMXG00000016317 ENSAMXG00000015171ENSAMXG00000016188ENSAMXG00000016317 ENSAMXG00000012543ENSAMXG00000012837ENSAMXG00000013857 ENSAMXG00000012837ENSAMXG00000013857ENSAMXG00000015171 ENSAMXG00000009922ENSAMXG00000012543ENSAMXG00000012837ENSAMXG00000013857 ENSAMXG00000012543ENSAMXG00000012837ENSAMXG00000013857 ENSAMXG00000009483ENSAMXG00000009702ENSAMXG00000009922 ENSAMXG00000009483ENSAMXG00000009702ENSAMXG00000009922ENSAMXG00000012543 ENSAMXG00000009136ENSAMXG00000009483ENSAMXG00000009702 ENSAMXG00000009136ENSAMXG00000009483ENSAMXG00000009702ENSAMXG00000009922 ENSAMXG00000008450ENSAMXG00000008452ENSAMXG00000008745ENSAMXG00000009136 ENSAMXG00000008452ENSAMXG00000008745ENSAMXG00000009136 ENSAMXG00000008450ENSAMXG00000008452ENSAMXG00000008745 ENSAMXG00000008450ENSAMXG00000008452ENSAMXG00000008745 ENSAMXG00000007169ENSAMXG00000007243ENSAMXG00000008353 ENSAMXG00000007243ENSAMXG00000008353ENSAMXG00000008450 ENSAMXG00000007020ENSAMXG00000007169ENSAMXG00000007243ENSAMXG00000008353 ENSAMXG00000007169ENSAMXG00000007243ENSAMXG00000008353 ENSAMXG00000005717ENSAMXG00000006283ENSAMXG00000007020 ENSAMXG00000005717ENSAMXG00000006283ENSAMXG00000007020ENSAMXG00000007169 ENSAMXG00000004926ENSAMXG00000005717ENSAMXG00000006283 ENSAMXG00000004926ENSAMXG00000005717ENSAMXG00000006283ENSAMXG00000007020 ENSAMXG00000004462ENSAMXG00000004650ENSAMXG00000004754ENSAMXG00000004926 ENSAMXG00000004650ENSAMXG00000004754ENSAMXG00000004926 ENSAMXG00000004462ENSAMXG00000004650ENSAMXG00000004754 ENSAMXG00000004462ENSAMXG00000004650ENSAMXG00000004754 ENSAMXG00000003558ENSAMXG00000003954ENSAMXG00000003989 ENSAMXG00000003954ENSAMXG00000003989ENSAMXG00000004462 ENSAMXG00000003097ENSAMXG00000003558ENSAMXG00000003954ENSAMXG00000003989 ENSAMXG00000003558ENSAMXG00000003954ENSAMXG00000003989 ENSAMXG00000001724ENSAMXG00000001885ENSAMXG00000003097 ENSAMXG00000001724ENSAMXG00000001885ENSAMXG00000003097ENSAMXG00000003558 ENSAMXG00000001684ENSAMXG00000001724ENSAMXG00000001885 ENSAMXG00000001684ENSAMXG00000001724ENSAMXG00000001885ENSAMXG00000003097 ENSAMXG00000000845ENSAMXG00000001184ENSAMXG00000001240ENSAMXG00000001684 ENSAMXG00000001184ENSAMXG00000001240ENSAMXG00000001684 ENSAMXG00000000845ENSAMXG00000001184ENSAMXG00000001240 ENSAMXG00000000845ENSAMXG00000001184ENSAMXG00000001240 ENSAMXG00000027396ENSAMXG00000028641ENSAMXG00000000101 ENSAMXG00000028641ENSAMXG00000000101ENSAMXG00000000845 ENSAMXG00000026328ENSAMXG00000027396ENSAMXG00000028641ENSAMXG00000000101 ENSAMXG00000027396ENSAMXG00000028641ENSAMXG00000000101 ENSAMXG00000025016ENSAMXG00000025388ENSAMXG00000026328 ENSAMXG00000025016ENSAMXG00000025388ENSAMXG00000026328ENSAMXG00000027396 ENSAMXG00000024783ENSAMXG00000025016ENSAMXG00000025388 ENSAMXG00000024783ENSAMXG00000025016ENSAMXG00000025388ENSAMXG00000026328 ENSAMXG00000020921ENSAMXG00000021145ENSAMXG00000021489ENSAMXG00000024783 ENSAMXG00000021145ENSAMXG00000021489ENSAMXG00000024783 ENSAMXG00000020921ENSAMXG00000021145ENSAMXG00000021489 ENSAMXG00000020921ENSAMXG00000021145ENSAMXG00000021489 25 ENSAMXG00000020090ENSAMXG00000020220ENSAMXG00000020349 ENSAMXG00000020220ENSAMXG00000020349ENSAMXG00000020921 ENSAMXG00000019909ENSAMXG00000020090ENSAMXG00000020220ENSAMXG00000020349 ENSAMXG00000019909ENSAMXG00000020090ENSAMXG00000020220ENSAMXG00000020349 ENSAMXG00000019480ENSAMXG00000019909 ENSAMXG00000019909ENSAMXG00000020090 ENSAMXG00000019273ENSAMXG00000019480 ENSAMXG00000019273ENSAMXG00000019480 ENSAMXG00000019162ENSAMXG00000019170ENSAMXG00000019273 ENSAMXG00000019162ENSAMXG00000019170ENSAMXG00000019273ENSAMXG00000019480 ENSAMXG00000018435ENSAMXG00000019162ENSAMXG00000019170 ENSAMXG00000018435ENSAMXG00000019162ENSAMXG00000019170 ENSAMXG00000016729ENSAMXG00000016859ENSAMXG00000017293ENSAMXG00000018435 ENSAMXG00000016859ENSAMXG00000017293ENSAMXG00000018435 ENSAMXG00000016729ENSAMXG00000016859ENSAMXG00000017293 ENSAMXG00000016729ENSAMXG00000016859ENSAMXG00000017293 ENSAMXG00000015752ENSAMXG00000016033ENSAMXG00000016153 ENSAMXG00000016033ENSAMXG00000016153ENSAMXG00000016729 ENSAMXG00000015752ENSAMXG00000016033ENSAMXG00000016153 ENSAMXG00000015531ENSAMXG00000015752ENSAMXG00000016033ENSAMXG00000016153 ENSAMXG00000014794ENSAMXG00000015442ENSAMXG00000015531 ENSAMXG00000014794ENSAMXG00000015442ENSAMXG00000015531ENSAMXG00000015752 ENSAMXG00000014519ENSAMXG00000014794ENSAMXG00000015442ENSAMXG00000015531 ENSAMXG00000014519ENSAMXG00000014794ENSAMXG00000015442 ENSAMXG00000012371ENSAMXG00000012973ENSAMXG00000013099ENSAMXG00000014519 ENSAMXG00000012973ENSAMXG00000013099ENSAMXG00000014519 ENSAMXG00000012371ENSAMXG00000012973ENSAMXG00000013099 ENSAMXG00000011973ENSAMXG00000012371ENSAMXG00000012973ENSAMXG00000013099 Genes rhythmic rhythmic Genes ENSAMXG00000011595ENSAMXG00000011973 ENSAMXG00000011973ENSAMXG00000012371 ENSAMXG00000011595ENSAMXG00000011973 ENSAMXG00000011280ENSAMXG00000011595 ENSAMXG00000009949ENSAMXG00000010054ENSAMXG00000011280 ENSAMXG00000009949ENSAMXG00000010054ENSAMXG00000011280ENSAMXG00000011595 ENSAMXG00000009547ENSAMXG00000009949ENSAMXG00000010054ENSAMXG00000011280 ENSAMXG00000009547ENSAMXG00000009949ENSAMXG00000010054 ENSAMXG00000006476ENSAMXG00000007679ENSAMXG00000009339ENSAMXG00000009547 ENSAMXG00000007679ENSAMXG00000009339ENSAMXG00000009547 ENSAMXG00000006476ENSAMXG00000007679ENSAMXG00000009339 ENSAMXG00000006168ENSAMXG00000006476ENSAMXG00000007679ENSAMXG00000009339 ENSAMXG00000005029ENSAMXG00000006152ENSAMXG00000006168 ENSAMXG00000005029ENSAMXG00000006152ENSAMXG00000006168ENSAMXG00000006476 ENSAMXG00000004932ENSAMXG00000005029ENSAMXG00000006152ENSAMXG00000006168 ENSAMXG00000004932ENSAMXG00000005029ENSAMXG00000006152 ENSAMXG00000004098ENSAMXG00000004118ENSAMXG00000004486ENSAMXG00000004932 ENSAMXG00000004118ENSAMXG00000004486ENSAMXG00000004932 ENSAMXG00000004098ENSAMXG00000004118ENSAMXG00000004486 ENSAMXG00000003688ENSAMXG00000004098ENSAMXG00000004118ENSAMXG00000004486 ENSAMXG00000002346ENSAMXG00000002962ENSAMXG00000003688 ENSAMXG00000002346ENSAMXG00000002962ENSAMXG00000003688ENSAMXG00000004098 ENSAMXG00000001940ENSAMXG00000002346ENSAMXG00000002962ENSAMXG00000003688 ENSAMXG00000001940ENSAMXG00000002346ENSAMXG00000002962 ENSAMXG00000000505ENSAMXG00000000784ENSAMXG00000001420ENSAMXG00000001940 ENSAMXG00000000784ENSAMXG00000001420ENSAMXG00000001940 ENSAMXG00000000505ENSAMXG00000000784ENSAMXG00000001420 ENSAMXG00000000056ENSAMXG00000000505ENSAMXG00000000784ENSAMXG00000001420 ENSAMXG00000025891ENSAMXG00000028482ENSAMXG00000000056 ENSAMXG00000025891ENSAMXG00000028482ENSAMXG00000000056ENSAMXG00000000505 ENSAMXG00000025811ENSAMXG00000025891ENSAMXG00000028482ENSAMXG00000000056 ENSAMXG00000025811ENSAMXG00000025891ENSAMXG00000028482 ENSAMXG00000020572ENSAMXG00000024788ENSAMXG00000025303ENSAMXG00000025811 ENSAMXG00000024788ENSAMXG00000025303ENSAMXG00000025811 ENSAMXG00000020572ENSAMXG00000024788ENSAMXG00000025303 ENSAMXG00000020483ENSAMXG00000020572ENSAMXG00000024788ENSAMXG00000025303 ENSAMXG00000018783ENSAMXG00000020366ENSAMXG00000020483 ENSAMXG00000018783ENSAMXG00000020366ENSAMXG00000020483ENSAMXG00000020572 ENSAMXG00000018696ENSAMXG00000018783ENSAMXG00000020366ENSAMXG00000020483 ENSAMXG00000018696ENSAMXG00000018783ENSAMXG00000020366 ENSAMXG00000016053ENSAMXG00000016382ENSAMXG00000017909ENSAMXG00000018696 ENSAMXG00000016382ENSAMXG00000017909ENSAMXG00000018696 ENSAMXG00000016053ENSAMXG00000016382ENSAMXG00000017909 ENSAMXG00000015855ENSAMXG00000016053ENSAMXG00000016382ENSAMXG00000017909 ENSAMXG00000013348ENSAMXG00000014846ENSAMXG00000015855 ENSAMXG00000013348ENSAMXG00000014846ENSAMXG00000015855ENSAMXG00000016053 ENSAMXG00000012889ENSAMXG00000013348ENSAMXG00000014846ENSAMXG00000015855 ENSAMXG00000012889ENSAMXG00000013348ENSAMXG00000014846 ENSAMXG00000011114ENSAMXG00000012052ENSAMXG00000012408ENSAMXG00000012889 ENSAMXG00000012052ENSAMXG00000012408ENSAMXG00000012889 ENSAMXG00000011114ENSAMXG00000012052ENSAMXG00000012408 ENSAMXG00000010495ENSAMXG00000011114ENSAMXG00000012052ENSAMXG00000012408 ENSAMXG00000009065ENSAMXG00000009417ENSAMXG00000010495 ENSAMXG00000009065ENSAMXG00000009417ENSAMXG00000010495ENSAMXG00000011114 ENSAMXG00000008981ENSAMXG00000009065ENSAMXG00000009417ENSAMXG00000010495 ENSAMXG00000008981ENSAMXG00000009065ENSAMXG00000009417 ENSAMXG00000008486ENSAMXG00000008740ENSAMXG00000008918ENSAMXG00000008981 ENSAMXG00000008740ENSAMXG00000008918ENSAMXG00000008981 ENSAMXG00000008486ENSAMXG00000008740ENSAMXG00000008918 ENSAMXG00000007301ENSAMXG00000008486ENSAMXG00000008740ENSAMXG00000008918 ENSAMXG00000004636ENSAMXG00000004726ENSAMXG00000007301 ENSAMXG00000004636ENSAMXG00000004726ENSAMXG00000007301ENSAMXG00000008486 ENSAMXG00000004334ENSAMXG00000004636ENSAMXG00000004726ENSAMXG00000007301 ENSAMXG00000004334ENSAMXG00000004636ENSAMXG00000004726 ENSAMXG00000003446ENSAMXG00000004224ENSAMXG00000004238ENSAMXG00000004334 ENSAMXG00000004224ENSAMXG00000004238ENSAMXG00000004334 ENSAMXG00000003446ENSAMXG00000004224ENSAMXG00000004238 ENSAMXG00000002597ENSAMXG00000003446ENSAMXG00000004224ENSAMXG00000004238 ENSAMXG00000001309ENSAMXG00000002414ENSAMXG00000002597 ENSAMXG00000001309ENSAMXG00000002414ENSAMXG00000002597ENSAMXG00000003446 ENSAMXG00000001309ENSAMXG00000002414ENSAMXG00000002597 ENSAMXG00000001309ENSAMXG00000002414 V2 V3 V4 V5 V6 V7 V2 V3 V4 V5 V6 V7 V2 V3 V4 V5 V6 V7 0 20 0 V2 V3 V4 V5 V6 20V7 0 20 0 20 C CT CT CT CT

Surface 10 10 1010 10 Surface 10 Surface score score - - 5 score 5

- 5

p.adj.SDRFDR p.adj.SDR p.adj.SDR 1.00 1.00 1.00 0.75 0.75 0.75 0 0.50

0.50 Z_AMP 0 0.50 0 Z_AMP Z_AMP 0 0.25 0.25 0 0 0.25

−5 -5−5 -5−5 Differential periodicity z Differential Differential periodicity z Differential

Differential periodicity z Differential Pachon -10−10 Molino Tinaja

−10 −5 0 5 10 −10 −5 0 5 10 15 −10 −5 0 5 10 -10 Z_Per0 10 -10 0Z_Per 15 -10 Z_Per0 10 Differential amplitude z-score Differential amplitude z-score Differential amplitude z-score 923 924

36 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.14.906628; this version posted January 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

925 Figure 2. A. Key circadian genes with changes in rhythmicity in cave populations (see Fig. S17 926 for all core circadian genes with changes in rhythmicity). Colored lines represent a loess 927 regression of gene expression through time for each population. B. Simplified schematic of the 928 circadian feedback loops based on proposed interactions in zebrafish43,45,55. Grey boxes indicate 929 genes that are either arrhythmic or show significantly reduced rhythmicity between cave and 930 surface. White boxed genes do not show significant differences between cave and surface. 931 Highlighted in yellow is the core loop. Bright yellow circles represent regulating protein 932 complexes. Red lines indicate negative regulation, black lines indicate positive regulation. Genes 933 that are not boxed did not show evidence of rhythmic expression in any cave or surface 934 population. Dotted lines are for visual clarity. Genes without an annotated ortholog in cavefish 935 were not included in the schematic. A arntl1a arntl2 nfil3 500 cipcb Surface 30 600 400 Molino 90 Tinaja pop pop pop pop 300 CHOY 20CHOY 400CHOY CHOY Pachón MOLINO 60 MOLINO MOLINO MOLINO nfil3 PACHON PACHON PACHON PACHON TINAJA TINAJA 200TINAJA TINAJA

30 10 200 100

0 0 Normalized expression Normalized 0 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20

500 80 rorca rorcb cry1bb aanat2

400 75 60 60

pop pop pop300 pop 50CHOY 40CHOY CHOY CHOY 40 MOLINO MOLINO MOLINO MOLINO PACHON PACHON 200PACHON PACHON TINAJA TINAJA TINAJA TINAJA

20 25 20 100 Normalized expression Normalized 0 0 0 0 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 Time B rorca rorcb

dbpb

nfil3 tefa cipca cry1ab nfil3-5 tefb cipcb cry1aa nfil3-6 cry1ba per1a cry1bb per1b cry4 per2 CLOCK BMAL cry5

arntl1a CRY PER clock1a arntl1b Arrhythmic or differentiated in all clock1b arntl2 caves Arrhythmic or differentiated in 2 cave nr1d1 populations Arrhythmic or differentiated in 1 cave bhlhe40 population bhlhe41 936 937

37 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.14.906628; this version posted January 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

938 Figure 3. A. Phase (e.g., peak expression time) distribution of rhythmic transcripts in each 939 population. B. Cycling genes on average show a delay in phase in cave populations relative to 940 their phase in the surface population. 941

● A B 20 ● ● 100100 ● 15 Surface 15 Molino Pachón Tinaja ● ● ●

● 150 150 1010 7575 4040

1010 Delay in cave

0 100100 V1 0 50 count 50 count count count

2020 ● 5 5 -10−10 ● ● Number of Number genes 50 50 ● 2525 ● ●

● ● Delay in surface

● ●

−20 ● ●

0 ● 00 00 0 00 0 5 10 15 20 Molino−choy Pachon−choy Tina−choy 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 10V2 10 V2 0 10V2 20 0 10V2 20 0 20 0 V2 20 Surface Phase time -Surface - -Surface Tinaja Molino Pachón 942

38 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.14.906628; this version posted January 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

943 Figure 4. Temporal expression patterns of per1a and arntl1a in brain and liver tissue in 944 Astyanax mexicanus populations. In-situ staining of per1a (cyan) and arntl1a (magenta) using 945 RNAscope® in the midbrain (‘B’, top panels for each timepoint) and liver (‘L’, bottom panels 946 for each timepoint) of surface fish and cavefish (Pachón, Tinaja, Molino) at CT0, CT8, and 947 CT16. Each time point is a single fish sample with probes separated into two channels. Images 948 are representative sections of two fish collected per time point, per population. Scale bar is 949 25μM.

950

951 952 953

39 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.14.906628; this version posted January 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

954 Figure 5. Temporal expression patterns of rorca and rorcb in midbrain and liver tissue in 955 Astyanax mexicanus populations. In-situ staining of rorca (cyan) and rorcb (magenta) using 956 RNAscope® in midbrain (‘B’, top panels for each timepoint) and liver (‘L’, bottom panels for 957 each timepoint) of Surface fish and cavefish (Pachón, Tinaja, Molino) at CT0, CT8, and CT16. 958 Each time point is a single fish sample. Images are representative sections of two fish collected 959 per time point, per population. Scale bar is 25μM. 960

961 962

40 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.14.906628; this version posted January 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

963 Figure 6. Mutant aanat2 and rorca fish reveal a role for these genes in sleep behavior in A. 964 mexicanus. A. Total sleep is not significantly altered between control and aanat2 crispant surface 965 fish (unpaired t-test, p=0.99). B-C. Day sleep is not significantly altered between WT and 966 crispant aanat2 fish (unpaired t-test, p=0.55). Night sleep is significantly reduced in aanat2 967 crispants compared to WT controls (unpaired t-test, p<0.0001). C. Total sleep is significantly 968 reduced in crispant rorca fish compared to WT controls (unpaired t-test, p<0.0001). E-F. Day 969 sleep was not significantly altered between WT and rorca crispants (unpaired t-test, p=0.056). 970 Night sleep is significantly reduced in rorca crispants compared to WT controls (unpaired t-test, 971 p<0.0001). 972 973 974 A B C N.S. /24hrs) hrs Min. sleep/hour Min. sleep/hour Sleep duration (

D E F Surface WT Surface rorca /24hrs) hrs Min. sleep/hour Min. sleep/hour Sleep duration ( Surface WT Surface rorca 975

41