Diversity in rest-activity patterns among Lake Malawi cichlid fishes suggests novel axis of habitat partitioning

Animals display remarkable diversity in rest and activity patterns that are regulated by endogenous foraging strategies, social behaviors, and predator avoidance. Alteration in the circadian timing of activity or the duration of rest-wake cycles provide a central mechanism for animals to exploit novel niches. The diversity of the 3000+ cichlid species throughout the world provides a unique opportunity to examine variation in locomotor activity and rest. Lake Malawi alone is home to over 500 species of cichlids that display divergent behaviors and inhabit well-defined niches throughout the lake. These species are presumed to be diurnal, though this has never been tested systematically. Here, we measure locomotor activity across the circadian cycle in 12 cichlid species from divergent lineages and distinct habitats. We document surprising variability in the circadian time of locomotor activity and the duration of rest. In particular, we identify a single species, Tropheops sp. “red cheek” that is nocturnal. Nocturnal behavior was maintained when fish were provided shelter, but not under constant darkness, suggesting it results from acute response to light rather than an endogenous circadian rhythm. Finally, we show that nocturnality is associated with increased eye size, suggesting a link between visual processing and nighttime activity. Together, these findings identify diversity of locomotor behavior in Lake Malawi cichlids and provide a system for investigating the molecular and neural basis underlying the evolution of nocturnal activity.


Introduction 32
Animals display remarkable diversity in rest and activity patterns. Circadian differences in 33 locomotor activity and rest can differ dramatically between closely related species, or 34 even between individuals of the same species, raising the possibility that it can be 35 adaptive and subject to selection[1-3]. Indeed, circadian regulation of locomotor activity 36 is strongly associated with foraging strategies, social behaviors, and predator avoidance 37 that are critical factors in organismal fitness [4,5]. Alteration in the circadian timing of 38 activity or the duration of rest-wake cycles provide a central mechanism for animals to 39 exploit novel niches. 40 41 Across phyla the timing of rest and activity is regulated by a circadian clock that persists 42 under constant conditions, as well as acute response to environmental cues that include 43 light and food availability [6]. For example, many teleost species display robust diurnal 44 locomotor rhythms including the goldfish (Carassius Auratus), the Mexican tetra 45 (Astyanax mexicanus), and the zebrafish (Danio rerio) [1,7,8]. Conversely, limited 46 examples of nocturnal teleosts have been identified including the plainfin midshipman, 47 the Senegalese sole, and the doctor fish, Tinca tinca [9][10][11]. Despite these conspicuous 48 differences, variation in rest and activity patterns have not been well described within a 49 lineage. Moreover, the ecological basis of such variation, and its relationship to niche 50 exploitation has not been studied systematically. 51

52
Cichlids represent a leading model for investigating the evolution of development, 53 morphology, and complex behavior. In Lake Malawi alone, there are over 500 species of 54 cichlids, inhabiting a diversity of environmental and feeding niches. Cichlid species exhibit 55 a high degree of habitat fidelity and partition their environment along discrete ecological 56 axes, including distinct biotic (food availability, predation, and parasites) and abiotic (light, 57 water chemistry) environments that play a critical role in the origins and maintenance of 58 cichlid biodiversity [12][13][14][15][16][17]. Predation on Malawi cichlids is considered to be relatively low, 59 which is thought to have contributed to their evolutionary and ecological success [18]. 60 However, the lake is home to predators, including the Cornish jack Mormyrops 61 anguilloides, that feed on cichlids in the intermediate and near-shore rocky habitat. M. 62 anguilloides are weakly electric fish that hunt at night using electrical pulses thought to 63 be undetectable by cichlids [19]. Field studies on this predatory behavior have suggested 64 that cichlids are largely diurnal [19], in agreement with the notion that rest represents a 65 form of adaptive inactivity that allows for predator avoidance [20]. Deviations from diurnal 66 activity have been noted for new world cichlids, which exhibit nocturnal parental care of 67 eggs [21,22], and the ability of some Malawi cichlids to forage in low-light conditions, via 68 widened lateral line canals, suggests the potential for nocturnal behaviors to evolve in this 69 group [23]. Given that Malawi cichlids exhibit an impressive magnitude of diversity in an 70 array of anatomical and behavioral traits, we reasoned that they may also exhibit variation 71 in rest-activity patterns. Indeed, this could represent an important, but underappreciated, 72 dimension of habitat partitioning. 73

74
The development of automated tracking of locomotor activity in fish species has been 75 applied for the study of sleep and locomotor activity in zebrafish and Mexican 76 cavefish [24]. These methodologies provide the opportunity for comparative approaches 77 that examine differences in activity between populations, and across contexts. Here, we 78 extend this methodology to study sleep across 12 species of cichlids, from diverse 79 habitats. Our choice of species focuses on the near-shore rock-dwelling clade of Malawi 80 cichlids (i.e., mbuna), but we also include representative species from other major 81 lineages. Our goal is not to characterize the evolution of rest-activity patterns per se, but 82 rather to better understand the degree and type of variation exhibited by this group. We 83 identify robust variation in the quantity, as well as the circadian timing, of rest and activity. 84 In addition, this analysis reveals, for the first time, a nocturnal species of Malawi cichlid, 85 suggesting that circadian regulation of activity may provide a mechanism for niche 86 exploitation in African cichlids. Together, these findings suggest cichlids can be used as 87 in length x 1" outer diameter, was added to each chamber at the beginning of the 123 acclimation period. For experiments testing the effect of light, fish were acclimated to 124 their tanks on a normal 14:10 LD cycle, and then recorded in 24 hours of darkness. 125 126 Following acquisition, recordings were processed in Ethovision XT 15 (Noldus) to 127 extract positional data for individual fish throughout the 24 hour period, and this data 128 was used to calculate velocity and locomotor activity, as previously described [25]. Fish were imaged using a digital camera (Olympus E520) mounted to a camera stand. 141 All images included a ruler. Using the program Image J[26], measures of standard 142 length, head length, and eye area were obtained for each fish. Eye size was measured 143 in fish used in the behavioral analysis. In addition, when possible, we augmented these 144 samples with wild-caught animals from the Albertson lab collections. In particular, we 145 added wild-caught samples to the L. fuelleborni, M. zebra, T. sp. "red cheek", and T. sp. 146 "red fin" populations. Two measures of eye-size were obtained: total area, and area 147 relative to head length. Because relative eye-size exhibits strong allometric 148 effects[27],residuals were obtained via a linear regression of eye-size on standard 149 length in R[28]. All statistical analyses were based on residuals. Results were the same 150 whether we used absolute eye-size or eye-size relative to head length. We therefore 151 only present data using absolute eye-size. 152

(e) Statistics and analysis 154
One-Way ANOVAs were carried to identify inter-species differences in overall locomotor 155 activity, average waking velocity, rest duration, and total time in shelter between 156 species. To identify differences between multiple conditions, such as activity in the light 157 vs. dark, or shelter vs no-shelter conditions, a Two-Way ANOVA was carried out, and 158 followed by Sidak's multiple comparisons post-hoc test. To identify significant rhythms in 159 activity across the day-night cycle, an "activity change ratio" was calculated as follows: 160 First, average hourly day-and night-time activity were calculated for each fish. Night-161 time activity was subtracted from day-time activity, and the result was divided by their 162 sum, providing a normalized day/night preference score. To identify significant 163 rhythmicity, one-sample t-tests were performed. To identify differences between the 164 mbuna and non-mbuna groups, nested ANOVAs were performed. All statistical 165 analyses were carried out using InStat software (GraphPad Prism 8). 166

Results 168 (a) Variation in activity and rest behaviors 169
To measure variation in activity across Lake Malawi cichlids, we compared the locomotor 170 activity in twelve different species, across eight genera, of cichlids that were selected for 171 diversity in habitat, behavior, and lineage representation. We sampled more deeply in the 172 rock-frequenting mbuna clade (n=7 species; n=4 genera), which occupy a complex, three-173 dimensional habitat characterized by a high density of cichlid individuals ( Fig 1A). In 174 addition, we analyzed activity patterns in four non-mbuna species, which occupy the 175 intermediate to open-water habitat ( Fig 1B). Following an initial 24 hour period of 176 acclimation, activity was recorded in individually housed juvenile fish across 24 hours in 177 standard light-dark conditions, with infrared lighting used to monitor locomotor activity 178 during the night as previously described in A. mexicanus [25]. Quantification of total 179 locomotor activity over 24 hours identified marked variation across species, with certain 180 species (i.e., S. fryeri) exhibiting significantly lower activity than all other species tested, 181 while the activity of others (i.e., T. sp. "elongatus Boadzulu") was significantly greater than 182 all other species (Fig 1C). Notably, variation in mean activity was continuously distributed 183 between these two extremes. In addition, there was a division between mbuna and non-184 mbuna species, with mbuna species trending towards increased locomotion relative to 185 non-mbuna species (p = 0.052). 186

187
To determine whether these differences were due to hyperactivity or differences in rest, 188 we measured the average waking velocity for each population. Among all species tested, 189 only one (T. sp. "elongatus Boadzulu"), displayed significantly higher swimming velocity, 190 suggesting the bulk of the variation among species is due to differences in rest/activity 191 regulation ( Fig 1D). In agreement with this notion, there were significant inter-species 192 differences in the duration of rest bouts lasting greater than one minute (Fig 1E). The 193 majority of species displayed very little rest, averaging less than 3 hours/day, while three 194 species, C. trewavasae, D. compressiceps, and S. fryeri (all non-mbuna) spent 195 significantly longer resting than all other species tested. The average rest duration of S. 196 fryeri was over 10-fold different than other species tested. Together, these findings 197 suggest that differences in total locomotor activity between cichlid species are largely 198 attributable to differences in rest. Notably, mbuna species together rested significantly 199 less than non-mbuna species (Fig 1E), possibly reflecting adaptation to the near-shore 200 rocky habitat. Support for this possibility, as opposed to historical contingency, is the 201 observation that A. stuartgranti, a non-mbuna species that co-occurs with mbuna, rests 202 less than other non-mbuna species (Fig 1E). 203

(b) Variation in patterns and magnitudes of rhythmic activity 205
To determine whether there are differences in circadian modulation of activity, we 206 compared activity over the light-dark cycle (Fig 2A). We found evidence for strong diurnal 207 activity in three mbuna species (L. fuelleborni, T. sp. "elongatus Boadzulu", and I. 208 sprengerae), while activity did not significantly differ based on light or dark phases in 209 seven species tested (Fig 2B). A single species, Tropheops sp. "red cheek", was 210 significantly more active in the night, providing the first evidence for nocturnality in a Lake 211 Malawi cichlid (Fig 2B). To account for variation in total locomotion between fish of 212 different species, we quantified preference for light and dark activity for each individual 213 tested. In agreement with quantification of average locomotor activity, T. sp. "red cheek" 214 had significantly greater preference for nighttime activity while L. fuelleborni, T. sp. 215 "elongatus Boadzulu", and I. sprengerae, had significantly greater preference for daytime 216 activity (Fig 2C). This analysis also suggests a preference for diurnal activity in L. 217 trewavasae, and for nocturnal activity in two additional non-mbuna species (C. dark periods (Fig S1). This analysis is largely in agreement with analysis of locomotor 224 activity, with day-active species consolidating rest during the dark period, and vice versa. 225 226 Since T. sp. "red cheek" is a highly territorial and aggressive species [29,30], it is possible 227 that the nighttime activity of this species represents a search strategy for locations that 228 provide shelter from predators, as opposed to a natural reflection of activity patterns. To 229 differentiate between these possibilities, we provided each animal with a 3 inch cylindrical 230 shelter (PVC piping), and measured behavior across light and dark conditions (Fig 3A). 231 We analyzed the total activity across the circadian cycle, as well as time spent in the 232 shelter in T. sp. "red cheek", as well as in L. trewavasae and M. zebra, closely related 233 mbuna species that co-occur with T. sp. "red cheek". These two species also exhibited 234 lower and indistinguishable activity levels during the day and night, and we were 235 interested to see if the addition of shelter would alter this pattern. When provided a hiding 236 spot, T. sp. "red cheek" remained robustly nocturnal, while M. zebra and L. trewavasae 237 did not show light dark preference, which is consistent with their activity patterns in the 238 absence of shelter (Fig 3B). We quantified the total time animals spent within the shelter 239 and found that L. trewavasae spent significantly more time in the shelter than M. zebra 240 and T. sp. "red cheek" (Fig 3C), which is consistent with this species' behavior in the wild. avoid nocturnal predators ( Fig 3D). Conversely, there were no differences in shelter 245 preference between light or dark periods for M. zebra and T. sp. "red cheek". Together, 246 these findings suggest that the presence of a shelter does not significantly impact the 247 activity pattern of the cichlid species tested, and that the nocturnal locomotor activity of 248 T. sp. "red cheek" does not represent a search for shelter. 249 250 It is possible that the nocturnal locomotor behavior of T. sp. "red cheek" is due to an 251 endogenous circadian rhythm or a differential response to light. To distinguish between 252 these possibilities, we measured locomotor activity under constant dark conditions. 253 Briefly, fish were acclimated under standard 14:10 light dark conditions, then activity was 254 recorded for 24 hours under constant darkness (Fig 4A). While T. sp. "red cheek" are 255 significantly more active during the dark period under light:dark conditions, there was no 256 difference between light and dark activity under constant darkness. (Fig 4A). A 257 comparison of total activity between the day (with light present) and the subjective day 258 (darkness) reveals that activity is significantly lower in the presence of light (Fig 4B). 259 These findings are consistent with a role for light in suppressing activity, thereby inducing 260 nocturnal behavior. 261

(c) Activity is higher in territorial species 263
General information regarding each species', habitat, behavior, prey-preference, and 264 phylogenetic relationship is provided in Table 1. To determine whether any variables of 265 rest or activity associate with these ecological factors, we compared locomotor data with 266 known ecological variables. Unsurprisingly, species described as territorial exhibited, on 267 average, greater overall activity compared to those characterized as weakly or non-268 territorial. We note, however, that any conclusion about the relationship between 269 locomotor activity and ecology/phylogeny may be premature, as significant differences in 270 rest-activity behavior exist between closely related and ecologically similar species (e.g., 271 within Tropheops and Labeotropheops). The more general conclusion to be drawn from 272 these data is that Lake Malawi cichlids exhibit substantial and continuous variation in 273 activity levels and patterns. 274

(d) Eye size is associated with night-time activity 276
Across fish species, nocturnality or adaptation to low-light conditions is associated with 277 larger eye size. In addition, species that rely on visual modes of foraging generally 278 develop larger eyes [32][33][34]. On the other hand, species adapted to forage on attached 279 algae generally possess smaller eyes, consistent with a functional tradeoff for the 280 production of power during jaw closure [35]. Specifically, algal scrapers tend to exhibit 281 smaller and dorsally shifted eyes to accommodate larger adductor muscles that are 282 situated below the eyes [36]. To understand how eye size relates to these variables, we 283 measured eye size in cichlid individuals in all species tested (Fig 5A, 5B), and tested for 284 significant correlations. Notably, we did not observe an obvious association between eye 285 size and lineage or foraging mode (Fig 5B). While the visual hunting species C. 286 trewavasae and S. fryeri possess larger eyes on average, D. compressiceps, an ambush-287 hunter, has the smallest eyes of the species measured. Likewise, while the algal scraping 288 species within the genus Labeotropheus has relatively small eyes, the attached algae 289 specialists, T. sp. "red cheek", has the largest relative eye size of the species measured. 290 The other species with large eyes was A. stuartgranti, which is a sonar hunter with 291 enlarged lateral line canals capable of foraging in low-light conditions [23]. Neither did we 292 identify a correlation between rest amount and eye size (Fig 5C). However, there was a 293 strong correlation between eye size and preference for night-time activity (Fig 5D). 294 Whether the large eye size in these species represents an adaptation to nocturnality 295 remains to be tested, but it is a notable morphological correlate worthy of further 296 investigation. 297 298

Discussion 299
The diversity of the ~3000 cichlid species throughout the world provides a unique 300 opportunity to examine the effects of ecological niche and evolutionary history on the 301 regulation of locomotor activity and rest. Cichlid species have undergone adaptive 302 radiations, resulting in morphologies and behaviors that can be highly specialized to 303 specific ecological niches. The ecology inhabited by cichlids includes species with habitat 304 fidelity to shorelines, deep water, and the intermediate zone between rocky and sandy 305 regions. In addition, many species are generalists and inhabit multiple different niches. 306 Here we focused our analysis on Lake Malawi cichlids, that alone likely contains over 500 307 species of fish, many of which share overlapping ecological niches [37]. The well 308 characterized ecosystem within the lake, as well as the taxonomic diversity uniquely 309 positions cichlids for investigating the role of ecology in shaping behavioral evolution. 310 Indeed, an important outstanding question is how can so many species with dietary 311 overlap co-exist in this lake? Many factors have been proposed to contribute including 312 the multitude of ecological resources available in this large tropical lake, low predation, 313 as well as the ability of cichlid species to evolve highly specific courtship and feeding 314 behaviors [18,38]. Circadian regulation of activity and rest may provide an additional 315 contributor to niche partitioning, reproductive isolation and even speciation, yet these 316 behaviors have not been studied systematically. The finding that the timing and duration 317 of rest and activity varies dramatically, and continuously, between populations of Lake 318 Malawi cichlids suggest this is a fruitful line of inquiry. conditions that ranges from diurnal to nocturnal, yet the majority of animals maintain 324 rhythms of ~24hrs under constant dark conditions [39]. Feeding is likely a critical mediator 325 of activity rhythms, though in some species, the daily timing of feeding differs from 326 locomotor activity. For example, zebrafish are highly diurnal and maintain 24 hour 327 rhythms, yet feeding occurs primarily during the night [40]. A similar trend has been noted 328 in cichlids, where diurnal species exhibit mating and brooding behaviors primarily at 329 night [21,41]. These findings suggest a high degree of flexibility in the circadian regulation 330 of behavior, and that the circadian timing of many behaviors may differ from locomotor 331 behavior that is typically used as a primary readout of the circadian clock [42]. Here, we 332 focused specifically on locomotor activity and did not provide social conspecifics or food 333 that could influence the timing of activity. Fully understanding the evolution circadian 334 behavior of each species and its relationship to its natural environment will require 335 examining additional behaviors that may be under circadian regulation. 336

337
A notable finding from this study is a species that appears to be nocturnal. Tropheops sp. 338 "red cheek" is a member of a highly speciose and ecologically diverse lineage [13,30,43]. 339 It is a vigorously territorial species that occupies the near shore rocky habitat, where 340 males defend large patches of rocks, cultivating algae gardens that they only allow 341 potential mates to feed from. This species exhibits significant habitat and dietary overlap 342 with L. fuelleborni, another algae foraging species from the rocky shallows. L. fuelleborni 343 is arguably one of the most ecologically successful species in the lake, with numerous 344 anatomical adaptations that enable it to dominate this niche[31, [44][45][46]. How then might 345 another species coexist with such a well-adapted forager? Based on the results presented 346 here, it is tempting to speculate that L. fuelleborni and T. sp. "red cheek" are partitioning 347 their habitat by rest-activity patterns. Consistently, these two species (1) are among the 348 most active of any measured, (2) are both strongly rhythmic, and (3) their rhythmicity is 349 opposite of one another. 350

351
Our findings raise the possibility that T. sp. "red cheek" is nocturnal in the wild, and the 352 limited amount of night filming that has been performed in Lake Malawi supports this 353 notion. Specifically, Arnegard and Carlson (2005) documented the nocturnal predatory 354 behavior of the weakly electric species, M. anguilloides, on cichlids in the rocky habitat. 355 The footage (available at https://malawicichlids.com/mw19000.htm), is impressive and 356 shows the success of the "pack" hunting strategy employed by M. anguilloides. Two 357 cichlid species (based on male breeding color) are readily apparent in the footage, A. 358 stuartgranti and T. sp. "red cheek". Indeed, the very first fish seen in the night footage is 359 a male T. sp. "red cheek" (@ 1:15). This fish is not resting within a rocky cave, crack or 360 crevice, but rather it is actively swimming well above the rocks. In fact, in the ~6 minutes 361 of night footage, no fewer than 5 T. sp. "red cheek" individuals can be observed, many A. 362 stuartgranti are observed as well. As a point of comparison, no Labeotropheus or 363 Maylandia species are readily observed at night, though they are common in the day 364 footage at the beginning and end of the ~8 minute film. This filming was not intended to 365 address questions related to rest-activity patterns in cichlids, and so we are cautious 366 about drawing firm conclusions; however, the trends are conspicuously consistent with 367 our laboratory results. 368

369
It is important to note that our analyses are limited to rest, and we did not examine sleep 370 per se. Across phyla, ranging from jellyfish to humans, sleep can be defined by shared 371 behavioral characteristics that include consolidated periods of behavioral quiescence, 372 homeostasis following deprivation and increased arousal threshold, and species-specific 373 posture [47]. In teleosts, the duration of inactivity associated with sleep has been defined 374 as one minute of immobility in larval A. mexicanus and zebrafish, and the same duration 375 for adult A. mexicanus [48,49]. The duration of sleep and rest is highly variable across 376 many other teleost species, and even between individuals of the same species. For 377 example, different populations of A. mexicanus display extreme differences in sleep and 378 activity, with cave dwelling populations of A. mexicanus sleeping less than river-dwelling 379 surface fish counterparts. These differences presumably evolved, at least in part, due to 380 increased foraging needs in a nutrient-poor cave environment [50]. Based on previous 381 work in fishes, we defined rest as the total duration of inactivity bouts longer than one 382 minute, and therefore these phenotypes may reflect differences in sleep duration across 383 cichlid species. While specifically examining sleep in cichlids will require defining the 384 period of immobility associated with changes in arousal threshold, posture, poster, and 385 other behavioral characteristics of sleep, we submit that it represents a fruitful line of 386 inquiry as it offers an ideal system in which to delve further into the evolution of sleep and 387 its molecular underpinnings. and R.C.A interpreted the results and wrote the manuscript. All authors were involved 396 with rounds of editing and proofing of the figures and manuscript text. 397 Table 1 563 General information about the Lake Malawi species under study. Information based on [30,31]. Abbreviations: G = 564 generalist, S = specialist, N = non-territorial, T = territorial, W = weakly territorial, SL = standard length. 565 566 567 568 1 These species have been observed to penetrate much deeper waters (e.g., ~40m). 569 2 This distribution pattern suggests a lake-wide historical distribution. varies significantly across 11 cichlid species (one-way ANOVA: F10, 91 = 11.10, 577 p<0.0001). Mbuna species trend towards higher activity than non-mbuna species, 578 although this relationship does not reach significance (nested ANOVA, F1,9 = 5.009). (D) 579 Waking velocity over 24 hours is significantly elevated in only one species of cichlid, 580 Tropheops sp. "elongatus Boadzulu" (one-way ANOVA, F10,89 = 5.398). (E) 581 Consolidated periods of rest (>60 seconds) vary significantly across mbuna and non-582 mbuna groups. (nested ANOVA, F1,9 = 7.808, p = 0.0209). 583 significantly increased activity during the subjective day (L. fuelleborni, T. sp. "elongatus 587 Boadzulu", I. sprengerae) while a single species exhibits increased locomotor activity 588 during the subjective night (T. sp. "red cheek") (two-way ANOVA, F10,91 = 13.27). (C) 589 Activity change scores, calculated as the difference between daytime and nighttime 590 activity, divided by their sum, reveal differences in day/night preference across cichlid 591 species (one-sample t-test; L. trewavasae (t12 = 3.419, p = 0.0051), L. fuelleborni (t7 = 592 12.93, p < 0.0001), T. sp. "red cheek" (t13 = 2.930, p < 0.0117), T. sp. "elongatus 593 Boadzulu" (t9 = 7.182, p < 0.0001), I. sprengerae (t6 = 5.374, p = 0.0017), C. trewavasae 594 (t6 = 3.555, p = 0.012), S. fryeri (t7 = 4.693, p = 0.0022). 595