Enriched sleep environments lengthen lemur sleep duration.

Characteristics of the sleep-site are thought to influence the quality and duration of primate sleep, yet only a handful of studies have investigated these links experimentally. Using actigraphy and infrared videography, we quantified sleep in four lemur species (Eulemur coronatus, Lemur catta, Propithecus coquereli, and Varecia rubra) under two different experimental conditions at the Duke Lemur Center (DLC) in Durham, NC, USA. Individuals from each species underwent three weeks of simultaneous testing to investigate the hypothesis that comfort level of the sleep-site influences sleep. We obtained baseline data on normal sleep, and then, in a pair-wise study design, we compared the daily sleep times, inter-daily activity stability, and intra-daily activity variability of individuals in simultaneous experiments of sleep-site enrichment and sleep-site impoverishment. Over 164 24-hour periods from 8 individuals (2 of each species), we found evidence that enriched sleep-sites increased daily sleep times of lemurs, with an average increase of thirty-two minutes. The effect of sleep-site impoverishment was small and not statistically significant. Though our experimental manipulations altered inter-daily stability and intra-daily variability in activity patterns relative to baseline, the changes did not differ significantly between enriched and impoverished conditions. We conclude that properties of a sleep-site enhancing softness or insulation, more than the factors of surface area or stability, influence lemur sleep, with implications regarding the importance of nest building in primate evolution and the welfare and management of captive lemurs.

Introduction

Sleep is a period of behavioral quiescence and reduced responsiveness to external stimuli, thus making it a vulnerable and dangerous state [1]. Consistent with this observation, comparative studies have revealed that risk of predation at the sleep-site covaries negatively with sleep duration across mammals [2-4]. Sleep-sites likely also vary in quality and security along dimensions that involve the physical comfort of the substrate, level of concealment from conspecifics and predators, and thermal properties [5-7]. A major question concerns how characteristics of the sleep site influence sleep quality, and how that in turn influences organismal function and fitness.

Considerable effort has been put into studying the role of sleep-site characteristics for apes. The sleep quality hypothesis, for example, proposes that apes construct beds to permit less fragmented, undisturbed sleep that promotes sleep quality through either greater sleep intensity [deeper slow-wave sleep (SWS) and/or REM sleep] or longer individual sleep stages [8-11]. This hypothesis has been supported by increased effort chimpazees put into building more complex nests [12] and observations of orang-utans (Pongo pygmaeus wurmbii) in Southern Borneo selecting sleep sites for comfort and stability rather than predator defence [13]. More recently, comparative analysis has shown that the sleep of captive orang-utans (Pongo spp.) is less frequently interrupted by movements of the head and body than the sleep of baboons (Papio papio.) [14]. Moreover, experimental work has demonstrated that orang-utans exhibit higher quality sleep (defined by less gross-motor movement and greater overall sleep times) when using complex sleeping platforms [15]. For wild, individually sleeping apes, sleep-site modifications may improve sleep through several mechanisms. For one, the removal or covering of protruding substrate reduces stress on tissues [12]. The relaxed skeletal muscle tone of REM sleep also puts branch sleeping animals at risk of falling; enlarged surface area and functional concavity of nests likely reduce this risk and enable greater quantities of REM sleep.

Other primates exhibit a wide range of sleep-site selection behaviors, but do not build daily nests as do great apes [16]. Guinea baboons (Papio papio), for example, will sleep on both cliffs and tree branches, often on terminal branches of emergent trees. Many New World monkeys such as the golden lion tamarin (Leontopithecus rosalia) often sleep in tree cavities, while one population of tufted capuchin monkeys (Cebus apella) has shown a proclivity for sleeping in the leaves of Jessenia palms. Among lemurs, variation is sleep-site behavior is as broad as in other primates. Some lemurs, like great apes, make nests (Mirza coquereli, Microcebus myoxinus, Galagoides demidoff), many sleep in tree holes, and yet more are branch sleepers [6].

Investigation of how sleep-sites affect lemur sleep is interesting for at least three reasons: 1) the use of nests by some lemurs and by great apes, but not Haplorrhine monkeys, begs the question of why (and when) this behavior has appeared and disappeared in the primate lineage, 2) variation amongst lemur species creates opportunities to study the ecological factors that select for different sleep phenotypes, and 3) reconstructing the sleep phenotype of ancestral lemurs will help us understand the starting point, and subsequent constraints, of evolution in primate sleep.

The first question has been partly answered by observing differences in the nesting behavior of lemurs and apes: lemurs do not conduct nightly sleep site modifications as great apes do–making their nests functionally more akin to those of birds than apes [6]. Cladistic differences in sleep-site modification behavior are also likely explained in part by advanced cognition and social learning in apes [5, 17]. An important aspect of understanding how and why sleep phenotypes vary across primates is determining the role that sleep-site comfort has on the sleep quality of non-nest sleeping primates. If aspects of nests, such as comfort, shelter, and stability improve sleep quality, it would suggest non-nesting species face greater costs of nest building, perhaps from the need to defend nests or the inability to change sleeping locations.

In previous work, we found that disrupted sleep influences aspects of lemur behavior and cognition [18]. In wild grey mouse lemurs, similar cognitive tests predicted body condition and survival [19], suggesting that sleep-related changes have functional consequences. Here, we investigate the effect of sleep-site characteristics on sleep-wake activity and duration in four species of lemur (Eulemur coronatus, Lemur catta, Propithecus coquereli, and Varecia rubra) by experimentally enriching and impoverishing sleep-sites in pairs of captive lemurs simultaneously. We experimentally tested the hypothesis that the comfort and stability of the sleep-site influences lemur sleep quality. Based on this hypothesis, we predicted that 1) enriching a sleep-site with soft, insulating materials would increase sleep duration and decrease sleep fragmentation (measured through intra-daily variability in activity patterns) and 2) impoverishing a sleep-site by removing flat, stable, above-ground surfaces would decrease total sleep time and increase sleep fragmentation. Thus, in addition to implications for animal welfare, understanding the links between sleep sites and sleep may also inform understanding of primate behavior, ecology, and evolution.

Methods

Study subjects

Research was performed at the Duke Lemur Center (DLC), in Durham, North Carolina, USA, where subjects were housed in dyadic, sex-balanced groups (i.e., with one male and one female). We generated actigraphic data from eight individuals, with one male and one female from each species. For detailed biographical information on the study subjects, see Bray et al. [20]. Animals received unlimited access to water and fresh fruit, vegetables, and Purina monkey chow on a daily basis. Animal use and methods were approved by the Duke University Institutional Animal Care and Use committee (Protocol #: A236-13-09) and the DLC Research Committee.

Data collection

The study was conducted over four months from April to July in 2016. Given each experimental protocol involved the work of multiple DLC staff and researchers, the research needed to be distributed on a per species basis as follows: Varecia: April 6 –April 26, Lemur: May 26 –June 15, Propithecus: Tuesday June 7 –Monday June 27, Eulemur: July 14 –August 3. This effort generated a dataset of 40–42 nights per species, totaling 164 twenty-four-hour periods, with circadian activity continuously recorded using MotionWatch 8 (CamNtech) tri-axial accelerometers. These actigraphic sensors are lightweight (7 g) and attached to standard nylon pet collars. Animals were monitored by DLC caretaking staff closely for two hours after collaring and at regular intervals throughout the study to ensure no adverse reactions to the collar; there were no reports of abnormal behavior beyond some scratching and head-shaking immediately following the collaring. The study took place in indoor housing to control for temperature and light conditions.

Using actigraphy data, response variables were generated from processed activity logs recorded at one-minute epochs. Recent advances in scoring algorithms have increased accuracy in detecting wake-sleep states and total sleep times [21]. We generated twenty-four-hour total sleep times (TST) for individuals in each species. We followed protocols used in previous primate sleep studies [22-26] and in prior work by our group performed at the DLC [20, 27]. The sensor sampled movement once per second at 50 Hz and assigned an activity value, referred to as “counts”, per one-minute epoch. Previous studies have used the operational definition of behavioral sleep measured via actigraphy as the absence of any force in any direction during the measuring period [28]. We similarly determined that animals were consistently at rest, and thus inferred sleep states (i.e., sustained quiescence in a species-specific posture), when actigraphy count values were equal to zero. We selected this criteria as the most conservative measure of sleep available to us because CamNtech does not make available its algorithm for determining count values from acceleration data (but see Van Hees et al. [29] for an approximation of a similar proprietary actigraphy algorithm). Following recommendations for validating actigraphy-based inferences of sleep state [30], a previous study compared infrared videography of sleeping lemurs to actigraphy counts from the same model of collar used in the current study. Lemurs could be clearly seen in the videos to make minor movements such as looking around or adjusting body position during 1 minute epochs with four or more activity counts, but such motions were not detected during epochs with fewer than four activity counts [27]. Nonetheless, using motion as a proxy for sleep leaves open the possibility that we may detect periods of relaxed wakefulness as sleep, and thus requires appropriate care when interpreting the results.

Experimental procedure

In a pair-wise experimental design, individuals from each species underwent two weeks of simultaneous testing after being measured in a baseline condition. During week one (baseline), each pair experienced normal sleep conditions. These conditions were the same as normal indoor operating protocol for the DLC, where individuals had access to two housing cells with mounted, raised shelves and basic amenities (e.g., raised square crates attached to the walls). To reduce the confounds of outdoor lighting and temperature, the baseline conditions only differed from normal conditions in that individuals were restricted to sleeping indoors, where temperatures were maintained within a few degrees of 78°F and the most substantial source of light came from dim emergency lighting at the ends of enclosure hallways. Baseline conditions differed from the experimental conditions in the options for sleep, with animals allowed to sleep socially (in pairs) during baseline but separated at night in the experiments. Though this presents a potential confound (with regard to the effect of sleep substrates) in comparisons of baseline conditions to experimental conditions, this decision reduced the overall impact of our study on the lemurs and we control for this confound by contrasting sleep times in our two experimental conditions, in which social conditions were the same.

During week two–the beginning of the pair-wise experiment–an individual was randomly chosen for one of two experimental conditions: sleep enrichment or sleep impoverishment. The other individual of the pair underwent the opposite treatment. One treatment was applied to each of the two housing cells the lemur pair had access to during baseline, and subjects restricted to their respective housing cell following evening caretaking. The third week was a reversal of the previous week’s condition, with individuals switching sleep-sites and thus experiencing the opposite condition. During experimental nights, individuals were isolated from the pair-mate, but they were in vocal communication and could touch one another between the enclosures, thus providing some of the typical social conditions they experienced in the baseline period. This was done to reduce the influence of separation on individual level sleep-wake expression.

Housing cells were enriched by providing lemurs with a high-quality plastic sleeping box (30 cm x 60 cm) with open slats on the side to permit ventilation; additionally, a 2.5 cm slab memory foam mattress was embedded in the base of the box with a small nylon blanket placed on the top of the mattress (Fig 1). Housing cells were impoverished by removing all enrichments items (including sleeping crates available in baseline conditions) and removing normally available wall shelves, leaving only narrow ledges for above ground perching during sleep.

Fig 1

The sleep enrichment experimental condition.

A subject Propithecus coquereli perched in the enriched sleep-site provided in the sleep enrichment experimental condition.

We used infrared videography to determine whether the subject in the sleep enrichment condition was using the sleeping box. On only one night did an individual lemur (Beatrice, P. coquereli) fail to use the enriched sleeping crate when available. In all conditions, the nighttime period was considered to start at 18:00 and end at 06:00. During the day, enclosures were returned to baseline conditions and the animals were allowed to move freely between cells.

Data analysis

We generated descriptive statistics characterizing the distribution of inferred sleep time throughout the 24-hour period by individuals and across the different experimental conditions. To test whether our experimental manipulations of sleep-site condition had a meaningful effect on lemur sleep times, we followed the approach of Pinhiero & Bates [31], building and testing a series of nested linear mixed-effects models. We began by modeling only the random effect of individual (nested within species), thus establishing a reference model that acknowledges sleep times are individually variable but assumes our experimental manipulations had no effect (Model 0). Because sleep is a biorhythm and likely to contain temporal dependencies, we expanded the reference model to include within-subject temporal autocorrelation in TST across 24-hour periods. Based on the autocorrelation function of our sleep data, we compared Model 0 to a first order autoregressive model of TST within subjects (Model 1). Finally, we tested the null hypothesis that our experimental manipulations had no effect on TST by adding to Model 1 parameters describing our experimental structure. These parameters were the coefficients for the three levels of experimental condition, the two orders in which the conditions were presented (used to control for potential order effects [32]), and their interactions (Model 2), resulting in the equation where TST is the TST of individual j of species s on day t, μ is the intercept (the baseline TST for subjects presented with the enrichment condition prior to the impoverishment condition), ϕ1 is the magnitude of the first order temporal auto-regression, β is the regression coefficient for the experimental condition given by Condition, β is the regression coefficient for the order of experimental conditions (enrichment first versus impoverishment first) given by Order, β is the regression coefficient for the interaction of the condition-order pair given by Condition*Order, U is a random intercept for species s, V is a random intercept for individual j, E is an error term, and the latter three terms are assumed to follow normal distributions with mean 0 and variances , and , respectively.

We used delta AICs at each step to assess whether the more complex model provided a sufficiently improved fit to the data. Before making inferences about the effects of our experimental structure, we plotted the normalized residuals of Model 2 against 1) predicted values and 2) the quantiles of a standard normal distribution to conduct diagnostic checks of our model assumptions. Concluding that Model 2 met all necessary assumptions, we calculated the contrasts of each level of experimental condition (including baseline and marginal to order) and tested for significant differences in TST between the baseline condition and the two experimental conditions using ANOVA. Finally, we calculated the intra-class correlation coefficients (ICCs) to compare the proportion of unstructured variance in our data and used this comparison to determine when our limited sample size prevented us from adding additional complexity to our statistical model. We fit all models to our data using the function lme of the library nlme v3.1 [33] in R version 4.0.4 [34]. Our analysis can be fully reproduced using code and documentation reported in the S1 File.

Inter-daily stabililty and intra-daily variability

In addition to testing for changes in total sleep time, we also calculated from the raw actigraphy data two metrics of sleep disruption first described in Van Someren et al. [35]: inter-daily stability (IS) and intra-daily variability (IV). Inter-daily stability is defined as the ratio of 1) the variance of averaged actigraphy counts across epochs around the grand mean to 2) the overall variance. In other words, it measures how consistent actigraphy measures are for each minute in a day across days, and thus gives an indication of how well activity levels are entrained to dial rhythms. Intra-daily variability is the ratio of 1) the squares of the difference in counts between all successive minutes and 2) the mean square differences in counts relative to the grand mean; it gives an indication of how fragmented activity patterns are within a given experimental period.

We calculated both metrics within each experimental condition for all subjects. We then analyzed these descriptive measures much like TST, fitting a linear model to each using fixed effects for experimental condition and order as well as their interaction and random effects for individual and species (nested). As with TST, we contrasted the effects of each experimental condition after marginalizing over the effects of order and its interaction with experimental condition.

Results

Individual sleep times are presented in Fig 2. TST, inter-daily stability, and intra-daily variability are summarized by condition in Table 1. AIC revealed that Model 2 is the preferred model for TST (Model 2 AIC = 1742; Model 1 ΔAIC = 12.4; Model 0 ΔAIC = 16.6). The first order autoregressive effect in this model is small (, 95% CI [-0.08, 0.26]). Estimated contrasts of TST between experimental conditions are presented in Fig 3. The estimated contrast of TST between the enriched condition and the baseline condition (marginal to order) is 32.0 minutes with a standard error of 8.72 and the hypothesis of no difference between enriched and baseline can be ruled out (Enriched—Baseline: df = 152, F-value = 13.0, p < 0.001). The contrast between the impoverished and baseline condition is not significantly different from 0 (Impoverished—Baseline: mean = -0.18 minutes, SE = 8.72, df = 152, F-value = <0.001 p = 0.984). The majority of unstructured variance in TST given Model 2 is attributable to individual differences (ICCsubject = 0.592), most of the remaining variance to the error term (ICCError = 0.301), and only a small proportion to species level differences (ICCSpecies = 0.107).

Fig 2

Daily lemur sleep patterns.

Total sleep time in minutes for each observed 24-hour period, plotted by individual across days. Lemurs exposed to the impoverished condition first are presented on the left, those exposed to the enriched condition first on the right.

Table 1

Summarized sleep times by individual and experimental condition.

ID	Sex	Species	Baseline			Enrichment			Impoverishment
			Mean TST	IS	IV	Mean TST	IS	IV	Mean TST	IS	IV
Aria	F	E. coronatus	560	0.36	0.77	563	0.45	0.67	550	0.41	0.67
Geb	M	E. coronatus	643	0.39	0.74	642	0.42	0.64	565	0.51	0.56
Bertha	F	P. coquereli	729	0.32	0.85	759	0.40	0.76	763	0.40	0.74
Beatrice	M	P. coquereli	654	0.32	0.99	726	0.38	0.85	670	0.39	0.87
Avior	M	V. rubra	626	0.38	0.67	666	0.31	0.72	631	0.32	0.55
Josephine	F	V. rubra	601	0.31	0.83	636	0.27	0.90	583	0.29	0.94
Persephone	F	L. catta	568	0.34	0.80	611	0.31	0.71	613	0.29	0.79
Teres	M	L. catta	731	0.28	0.97	769	0.28	0.82	745	0.27	0.71

Mean sleep times (TST) per 24-hour period (minutes), inter-daily stability, and intra-daily variability for each study individual across conditions. All experimental periods were seven days, except Aria, Geb, Persophone, and Teres in the Baseline condition, which were six days.

Fig 3

Estimated effects of experimental condition on total sleep time.

The estimated differences in total sleep time (in minutes and marginalized over the estimated effects of condition order) for each pair of experimental conditions. Error bars represent two standard errors of the estimated population mean difference for the given contrast.

Linear mixed-effect models of Inter-Daily Stability and Intra-Daily variability both produced normally distributed residuals; the estimated effects of experimental conditions on both metrics are presented in Fig 4. Both the enriched condition and the impoverished condition yielded higher inter-daily stability in sleep than the baseline condition, though the effects were small and non-significant (Enriched v. Baseline: mean difference = 0.013, se = 0.021, df = 12, F-Value = 0.38, p = 0.55; Impoverished v. Baseline: mean difference = 0.021, se = 0.021, df = 12, F-Value = 1.02, p = 0.33). Both the enriched and impoverished conditions yielded decreased intra-daily variability relative to baseline conditions, where the effect was noteworthy for the enrichment condition and statistically significant in the impoverishment condition (Enriched v. Baseline: mean difference = -0.069, se = 0.035, df = 12, F-Value = 3.75, p = 0.08; Impoverished v. Baseline: mean difference = -0.095, se = 0.035, df = 12, F-Value = 7.24, p = 0.02).

Fig 4

Marginalized differences in activity patterns between experimental conditions.

The estimated mean difference in Inter-Daily Stability (IS) and Intra-Daily Variability (IV) of lemur activity patterns between experimental condition, marginalized over estimated effects of experimental order. Error bars represent two standard errors of the estimated population mean difference for the given contrast.

Discussion

The results of our study provide evidence that, for a limited subset of lemur species that do not nest outside the context of infant care, provisioning sleep sites with soft and insulated materials lengthens daily sleep durations. Among the models that we compared, the one that explicitly accounted for experimental manipulations was the most likely given our data. More importantly, we can conclude with confidence from this model that sleep site enrichment adds, on average, between 23 and 41 minutes of daily sleep relative to the baseline conditions (these values are the marginal contrasts, which account for any effects of the order experimental conditions were presented in). These conclusions are robust to sample size, but the small number of individuals in our study prevents us from making further conclusions about whether the results were driven by particularly strong effects in any subset of age, sex, or species classifications. An additional caveat is that we measure body-motion, rather than brain activity, as a proxy for sleep; it is possible we are measuring changes in wakeful resting states and we make no conclusions regarding specific sleep states such as REM or slow-wave sleep.

Interestingly, the manipulations of our impoverishment condition had no discernable impact on daily sleep times of our lemur subjects. Individuals in this condition were typically observed sleeping on narrow window ledges in their enclosures rather than on the ground. Though we cannot say for certain that the surface area–and hence stability–of lemur sleep sites do not affect sleep (we simply fail to reject this hypothesis), the differing impact of our impoverishment and enrichment conditions allows for some important inferences. First, we infer that the presence of soft, insulating materials is more important to our subjects’ sleep duration than the size of the sleeping substrate. Second, it allows us to treat the impoverishment condition as a control, indicating that incidental effects common to both manipulations, such as the separation of lemur pairs at night or the disruption of typical caretaking routines, are not sufficient to explain the differences between the enriched and impoverished conditions. Such confounds, however, may alter the differences of both conditions from baseline; thus, caution is warranted in determining whether it was the enriched or impoverished condition that had the greater effect on sleep time.

Measures of inter-daily stability and intra-daily variability did not differ between experimental conditions. Intra-daily variability in activity patterns did, however, decrease substantially in both experimental conditions relative to the baseline. This suggests a confounding aspect of our experimental manipulations resulted in less fragmented sleep; the change in social sleep conditions (from paired to isolated sleeping) offers a logical explanation. Some caution should be taken in interpreting the reduction in intra-daily variability as a consequence of less fragmented sleep per se, as this is a measure of general activity patterns and could also be affected by fragmentation in other activities such as play or alert rest. Regardless of what caused changes in activity between baseline and experimental conditions, we failed to find any significant difference in intra-daily variability or inter-daily stability between the enriched and impoverished conditions.

Given that lemurs in our study experienced overall greater sleep duration in the enriched sleeping condition, it may have been that the sleep architecture of ancestral fixed-point sleeping primates was deep and high quality, whereas later larger bodied primates that were branch sleepers traded longer sleep periods for other advantages. There are many potential trade-offs that could result in the use of less comfortable sleep sites, despite the evidence we found that such sites could reduce total sleep times. These include predator defense, thermoregulation, parasite avoidance, group cohesion, and resource distribution [5, 36]. The role of predation may be particularly important for lemurs, all of which are highly predated by raptors, boas, and fossa [37]. The high number of cathemeral (active both at night and during the day) lemur species is hypothesized to result from the similarly 24-hour threat of these predator species [38]. High predation threat may also help explain why lemurs, but not other Malagasy mammals, show marked increases in female to male body size ratios relative to their mainland sister clades [39]. Whereas male-male competition has resulted in stark sexual-size dimorphism (larger males) in most primate species, the prevalence of medium sized arboreal predators, particularly the fossa, in Madagascar may have pushed body size selection in both lemur sexes toward the threshold of what the canopy can support. Thus, the anti-predator benefits that larger body size and/or social living provide throughout daily activity cycles may have outweighed the benefits of secure, comfortable sleeping sites such as tree-cavities.

While large body size and social living may relax the need for secure sleep sites, additional factors are necessary to explain the benefits of flexible sleep-site selection over fixed-point nesting; we consider four such factors most important to consider. 1) Proximity to food resources has been shown to influence primate nest-construction and sleep-sites [36, 40, 41]. In landscapes with dynamic resource distributions, flexible sleep-sites could optimize foraging efficiency by reducing daily commutes to resource hotspots that last more than a day. 2) Similarly, sleep-site flexibility may allow adaptive responses to changes in predation risk; why should an animal invest in sleep-site modification if it is likely to be chased off by a predator at any time? 3) Parasite risk may play multiple roles in shaping sleep site selection. Sleeping areas can become contaminated with parasites, suggesting that exposure to parasites might be a cost of reusing sleep-sites [42]. Conversely, certain sleeping sites can provide parasite-related benefits to primates. In the context of vector-borne diseases, for example, an enclosed site may help to obscure cues that mosquitoes use to locate hosts. Supporting this hypothesis, malaria prevalence decreases in species of New World monkeys that sleep in enclosed microhabitats [43]. 4) Finally, factors such as group size and body mass likely limit the ability of some primate species to use fixed-point nesting habitats and, in general, to obtain consolidated sleep [44].

Only with the emergence of frequent, secure platform construction in ancestral apes has deep, REM heavy sleep architecture (re)-emerged. This sleep pattern is most strong expressed in humans [45], perhaps due to a more recent transition to sleeping on the ground in sentinalized groups permitting even higher quality sleep along the human lineage [46]. In general, mammals with larger encephalization quotients exhibit more REM sleep [4]; cognitive ability likely explains some differences in nesting behavior between apes and other primates. While black and white ruffed lemurs (Varecia variagata), for example, will build nests regardless of their ability to observe con-specifics doing so [47], whether and how chimpanzees build nests depends on the nest-building behavior of their social group [48]. Accordingly, site-flexible nest construction is thought to reflect great apes’ capacity for social learning and environmental problem solving [49], and is considered by some to be the most pervasive form of material culture in great apes [5, 15, 17]. Thus, our results provide further evidence that an integration of the sleep quality hypothesis, sleep-site flexibility, and environmental cognition are necessary to explain early hominid evolution [44]; great apes are not the only primates that would benefit from flexibly and regularly building secure sleep sites, but they are the only ones that do so. One remaining question is whether inter-species cognitive differences also mediate the fitness benefits of sleep quality. For example, primate brain size and diet are correlated, with larger brained species being more reliant on resources that are predictable, but sparse and ephemeral [50, 51]; does high quality sleep aid species that rely on sophisticated cognitive maps to keep track of temporal patterns of resource availability at multiple locations?

The precise nature of the tradeoffs in sleep-site selection–and the ecological conditions that favor the evolution of one strategy over another–would be further elucidated by expanding the comparisons in sleep among lemur species and between other primates. Though it was our hope that this study might help differentiate the ecological factors mediating the impact of sleep quality, the nearly negligible amount of variance in our data that can be attributed to subject species tells us there is little to be gained from attempting to explicitly model species level effects with the data at hand. This is perhaps unsurprising given the limited number of individuals per species in our study, and speaks more to a lack of statistical power than the absence of species level variation. Our small sample size also prevents us from conducting additional tests to explore other individual-level predictors of TST, such as age or sex. Here, we were limited by the constraints of working with globally threatened species and the logistical difficulty of maintaining controlled sleep conditions in a large, multipurpose facility.

None-the-less, we can offer some insights to researchers who wish to answer these questions through the collection of additional data. We did not, for example, consider in our experimental design the important interaction of experimental condition with the order of presentation. The potential of carryover effects (the impact of one condition differentially affecting sleep during the next condition) resulting from this interaction required us to include additional parameters in our statistical model. Allowing a recovery period between experimental conditions would remove the need to statistically control for this effect, increasing the statistical power per observation without creating additional work or disturbance to the lemurs [32]. We also take a fairly simple approach to modeling the temporal dynamics of lemur sleep due to the limited duration of our observation periods. Study of sleep patterns during extended baseline conditions would allow for more sophisticated models of baseline sleep behavior (e.g. by adding additional moving average or autoregressive parameters to account for long term homeostatic trends). These additional data would not require the time intensive and disruptive efforts of experimental manipulations, but would facilitate inference about the effects of such experiments when they are conducted.

Future work that employs these ideas may also identify other ecological co-variates of increased sleep duration in enriched sleep sites. The inclusion of smaller, fixed-point nesting species in a similar study, for example, could reveal trade-offs between more flexible sleep locations and periods of intense, high-quality sleep in primates. Comparison of lemurs to lorisiforms may also prove fruitful; a comparative analysis of lorisiform sleep behavior revealed predation pressure strongly influenced sleep-site selection, but neither the effect of predation risk nor of specific sleep sites on sleep quality has been investigated [52]. Many lemurs, including those in our study, also display a high degree of cathemerality [38]; with sufficient data, it may be possible to link flexible sleep site locations with flexible sleep timing. As affordable and mobile sleep monitoring technology improves upon current methods, future research can increase the number of subjects per species used in such a study. Though we are limited in the inferences we can make about species level differences in the relationship between sleep site and sleep duration, our experimental approach has clearly demonstrated that there is a relationship in at least some lemurs. With further replication and refinement, this approach can be used to understand not just where and how primates sleep, but why they do so.

In conclusion, we found that enriched sleep-sites increase sleep duration in at least some lemurs. This highlights the importance of sleep-site conditions for lemurs, as is also known for hominoids [12, 53–55] and cercopithecoids [56]. With respect to captive primate welfare, previous work has illustrated the importance of enriching sleep-sites for large brained and large bodied great apes [15, 57]. In light of our findings, we suggest that managing institutions for any primate species should take care to allot resources to ensure that primates that are primed to sleep in species-specific ways by way of environmentally modified sleep-sites. Finally, we conclude that behaviors that influence sleep-site selection, thereby augmenting sleep quality, are evolutionarily conserved in primates and may be critically important for not only apes and monkeys, but certain strepsirrhines as well.

Statistical analysis.

Code and diagnostic figures from which the statistical analysis in this paper can be fully replicated.

(DOCX)

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18 Jun 2021

PONE-D-21-17696

Enriched sleep environments lengthen lemur sleep duration

PLOS ONE

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Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This study addresses the question how sleep environment influence sleep duration in primates. Effects of an enriched or impoverished sleep-site on sleep time were measured in a total of 8 individuals of 4 different lemur species by means of actigraphy and video recordings. The results suggest that an enriched sleep-site, particularly an increased softness and insulation, increases daily sleep time by about half an hour. The study is interesting and well introduced, but I do have a number of questions and concerns regarding the experimental design and data analysis that need to be addressed.

1. In general, there is a lack of detail on the processing and analysis of the data, which in the end makes it hard to appreciate and interpret the value of the finding. It may be a cliché, but recordings of rest-activity patterns are only a correlate of true sleep-wake patterns. How strong the correlation is depends on many variables and may differ between species, sexes, and experimental conditions. More details are needed on how the actigraphy and video data were processed. Particularly, it needs to be explained in detail how these data were then transformed and used as an indicator of sleep time. I also don’t quite understand the operational definition for sleep being “the absence of any force in any direction during the measuring period” (line 119-120).

2. It is somewhat surprising that the manuscript only presents data on total daily resting time whereas the actigraphy and video data surely contain a lot more interesting and relevant information. For example, what about the duration of sleep/rest episodes in the different conditions as a possible measure of sleep continuity? And what about the distribution of sleep/rest and activity across the 24h cycle?

3. The experimental design is complicated. The study aimed to compare actigraphy-based total daily sleep time under baseline conditions with sleep time in an enriched or impoverished nest-site condition. However, the methods section mentions that the Lemurs had access to all the cage enrichments during the day (line 163). This is where the results become difficult to interpret. For example, is sleep in the impoverished condition determined by the impoverished sleep-site at night or by the enriched environment during the day?

4. Another complication in the design is that animals were sleeping in pairs during baseline but not during the enriched and impoverished nest-site conditions (line 135-136 and 143-144). So, how much of the effect that is observed is due to the difference in cage enrichment or simply the fact that the animals where individually housed?

5. Also, if I read the methods section correctly, the baseline condition always preceded the enriched and impoverished conditions. How do the authors exclude the possibility of an order effect or time effect? Do they have additional baseline recordings after the experimental treatment weeks to assess whether sleep time normalized to baseline values?

6. It is not clear why the authors chose to study 4 different species of lemur (with only 2 individuals for each species) rather than 8 individual of a single species. Also, it seems like species was not included as a variable in the analysis.

7. For each of the 4 species, 2 individuals were included, one male and one female. Were there any sex differences in rest-activity patterns?

8. The Figure shows the data in its most basic form; that is, daily resting time separately for each individual on each of the 6-7day in each of the 3 conditions. However, this makes it quite hard to read the figure and get an overall picture. Perhaps the authors should include additional figures or panels that simply shows the average resting times per condition. After all, they claim that the claim is that there is a statistical significant effect of experimental condition.

9. Based on the current figure, the reader may have the impression that the differences in daily resting time between the conditions is rather small compared to even the variation within an individual and within a certain condition. Moreover, the differences between the conditions is certainly small compared to the differences between the individuals. This raises questions as to how relevant these differences are and what other variables might explain variation in resting time. Perhaps this should be discussed.

10. The discussion speaks about the preference of the subjects for enriched sleeping-sited (line 283). However, in this study the lemurs were not offered a choice between the different conditions, so how can we know what their preference was.

11. The discussion mentions that the lemurs chose to sleep on narrow, elevated ledges rather than the ground during the impoverishment condition (line 286). If this is a meaningful observation worthy of discussion, the authors should consider showing the data to support this. In a way this goes back to point 2, the feeling that there is much more in the actigraphy data and video recordings than only the total daily resting time now presented.

Minor comments:

- Methods, line 111: animals were housed indoors to control for temperature and light. Please add what the indoor temperature and light conditions were during the study

- Results, Figure 2: Specify the unit of the y-axis (minutes).

Reviewer #2: Dear Dr Vyazovskiy:

It was my pleasure to read the paper by Vining et al. entitled, “Enriched sleep environments lengthen lemur sleep duration” for the journal PLOS ONE. The paper reports original findings on the effect of sleep site comfort on sleep duration in four species of primate. Although the low size is very low (two animals per species), the data collected is unique and hard to replicate. Moreover, owing to strict regulations on primate research (even behavioural studies), it is best to take whatever data can be had. I recommend the data be published in PLOS ONE subject to minor revision of the paper, as per below.

Title page: Please give City, Country for all affiliations.

Introduction, lines 64, 65: Please define “deep” and “efficient” and “higher quality” sleep. These are never actually explained in the paper, yet much seems to rest on their definition. For instance, sleep depth can be measured directly, with arousal thresholds, or inferred using slow wave activity during slow wave sleep. Slow wave activity cannot be compared across species (for a variety of physiological and non-physiological reasons) and so some explanation would be warranted.

Introduction, line 70. Consider this: The reduced skeletal muscle tone that accompanies REM sleep might make small surface areas particularly dangerous and thus animals sleeping in such locations might selectively reduce REM sleep (either in duration or %sleep).

Methods, line 110: How do you know the animals acclimated to the collars within 2 hours?

Methods: line 115: Total sleep time (TST) is a standard acronym for 24-h sleep amount. Otherwise it is TRT - total recording time. I would suggest you use TST and not TTST as the latter is not familiar to most sleep readers.

Methods, line 123: Are you certain you validated sleep states? First, no data on sleep states is presented. Second, this seems rather a big deal to gloss over. Please explain, clarify or correct.

Methods: the low sample size needs to be addressed. I can guess as to the reasons and am empathetic but it will be eye-catching to many. I’d be proactive and defend against criticism straight-up.

Methods, line 170: Should species not be a fixed factor in the model?

Results, line 223-225: I would not say “sample size of 7”. This is misleading. I would replace with “All values were an average over 7 days, except….”

Discussion, line 255: The issue of sleep depth and quality re-emerges here too. Sleep depth is difficult to compare across species. You cannot compare SWA across species. Moreover, some data challenges comparing arousal thresholds across species. For instance, sleeping emperor penguins arouse quickly with lightly touched on their feet, but no where else on their body. Does this make an emperor penguin a light or deep sleeper compared to a starling? I have strong doubts over the ability to make such statements.

Discussion, line 295-297: This result was also found by Lesku et al. 2006 in that mammalian species with greater encephalization had a higher %REM sleep. Thus, not only do humans have more REM sleep (%) than non-human primates but so too do mammals, in general.

References: There are heaps of sloppy errors in the reference section. Endnote appears to have been used, but unchecked. Please check each reference individually for consistent formatting and accuracy.

Reviewer #3: First, I would like to say that the ethics statement in this study was excellent and reassuring - when I read "sleep impoverishment" alarm bells rang for me, although I know that at DLC, all care would be given to the animals, and the excellent and detailed ethics statement made that clear.

Overall, this is a "neat" study with an excellent design. It is novel and indeed, for me, not only has implications for primate evolution and nest building, but my first thought went to conservation, where habitats are destroyed and nesting materials may not be available, long loss of even half an hours sleep per night could have long term negative effects. I think the authors should add a few lines about this to the discussion and perhaps one phrase in the abstract.

Introduction

Line 72 - not all apes build nests - I guess you mean great apes - it would be nice to have a such as after sleep site selection behaviours with a few examples as this paragraph reads very thin

72-75 - unpack this sentence - need a better flow into lemurs - maybe that many strepsirrhines build nests, and a more detailed review of the main functions (also comfort, parasite control etc)

77 - I would be more broad since most galagos also use nests - and many lemurs don't - I feel this work has broader impact if you are looking at strepsirrhine evolution - there are a few good reviews on sleep site use by galagos (Bearder earlier on, Svensson more recently) - then justify the choice of the lemur species selected...I actually had no idea that sifakas use nests...nor ring-tailed lemurs - this makes me realise that the previous paragraph is confusing - are you testing just sleep sites? or nests? clarify and justify the examples

Line 86 - I feel that this section should be either be higher or in the discussion - you can simply state that you discuss sleep site comfort in relation to welfare and evolution (and I suggest add conservation)

Methods - really cool experimental design! I am just curious about battery life and if you had to change the collars or recharge the battery (or remove the sensor regularly for download of data)

Discussion - AH now you say these are species that do NOT nest - this needs to be more clear in the intro

Line 241 - can you not compare with any other studies? You mentioned a mouse lemur study in the wild...you mentioned several bird studies - and surely some human studies - no references in this paragraph

Line 261 - as this is not a primate journal, you need to explain what cathemeral is

Line 263 - turn this around to explain to the reader about sex dimorphism then you can interpret the findings

Lie 268 - the lemurs in your sample certainly do not fit the definition of large body size in a mammal - but medium bodied - Maybe you want to include a threshold in grams related to security in branches to support a size of that nature - this is why virtually no large bodied mammals are arboreal (the exception mainly being some great apes!)

Paragraph 268 - a lot to unpack in this paragraph - follow the hypotheses paragraph by paragraph rather than mixing

Line 297 - this implies that human evolved to sleep in tree platforms - do you think this is the case? provide more evidence

Line 300 - this feels a bit 1950s and very primatological - most animals that make nests have some form of learning, at least in choice of materials etc

Line 305 - do you mean hominid?? or do you mean hominin? are you including the ancestors of chimps etc?

Line 309 - bold statement - reference needed

Line 319- globally threatened - not endangered (that is an IUCN category)

310-335 - a lot of unreferenced reflection - can be dramatically shortened or made stronger with referencing

Suggest here to include something about conservation implications

Line 355 - substandard is an odd choice- indeed, captive facilities should consider more the ecology of the species and try to closely match it

Line 357 - larger bodied apes and monkeys (again monkeys are not large for the most part)...strepsirrhines unless you mean tarsiers but you never mentioned tarsiers anywhere else and you also did not mention anything other than lemurs so this comes out of the blue anyway! Indeed, there are some very nice new studies of sleep in slow lorises using similar methods as to here, as well as the aforementioned studies on galagos that would really complement the work and make the discussion broader

Some minor comments

Line 65 etc - the accepted spelling of orang-utan is generally with a hyphen and at the very least this is how Indonesians would spell it in English

Figure 1 text a subject (Propithecus coquereli)

Table 1 - random change of font and the table heading needs to be more detailed and explain the parameters

line 235 - among the models THAT we compared

Line 274 - delete you - replace with alternative - an animal, a lemur, a primate

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24 Aug 2021

Reviewer 1

1. In general, there is a lack of detail on the processing and analysis of the data, which in the end makes it hard to appreciate and interpret the value of the finding. It may be a cliché, but recordings of rest-activity patterns are only a correlate of true sleep-wake patterns. How strong the correlation is depends on many variables and may differ between species, sexes, and experimental conditions. More details are needed on how the actigraphy and video data were processed. Particularly, it needs to be explained in detail how these data were then transformed and used as an indicator of sleep time. I also don’t quite understand the operational definition for sleep being “the absence of any force in any direction during the measuring period” (line 119-120).

We are thankful to the reviewer for this input. We added the raw actigraphy data collected from our collars to the public repository for this publication (https://github.com/aqvining/Lemur_Sleep_Site_Enrichment) and the code to calculate TST and all other secondary metrics to Supplementary Material 1. Additinally, upon reviewing this process, the initial steps of our data processing were not fully replicable, and we found an error with the TST calculations for one individual. We corrected this, which led to some minor changes to the quantitative results and no changes to conclusions.

Unfortunately, CamNtech has been unwilling to share the algorithm by which their actigraphy counts (our raw data) are derived from underlying acceleration measurements. However, we added citation to a paper that attempts to recreate this algorithm for a similar collar and also clarified the relationship between actigraphy counts and our inference of sleep state (lines 158-163).

Data from infrared videography collected in the course of the current study were not collected in a reproducible manner, so we have removed reference to those data. Instead, we discuss how infrared videos were used to more rigorously validate our protocol for inferring sleep state from CamNtech MotionWatch8 collars in a previous study at the Duke Lemur Center (lines 165-170)

2. It is somewhat surprising that the manuscript only presents data on total daily resting time whereas the actigraphy and video data surely contain a lot more interesting and relevant information. For example, what about the duration of sleep/rest episodes in the different conditions as a possible measure of sleep continuity? And what about the distribution of sleep/rest and activity across the 24h cycle?

Thank you for these suggestions. We made the raw actigraphy data available, as described in our response to the previous comment. We also added analyses to the paper to investigate the inter-daily stability and intra-daily variation of actigraphy data. New predictions regarding these metrics can be found in lines 114-116, the calculation and analysis of these metrics are now provided in lines 268-283, results from these analyses, including a new figure, are in lines 319-336, and we discuss these new results in lines 365-375. We have also modified Table 2 to present these data, removing standard deviations of TST to keep the data presentation compact and readable.

3. The experimental design is complicated. The study aimed to compare actigraphy-based total daily sleep time under baseline conditions with sleep time in an enriched or impoverished nest-site condition. However, the methods section mentions that the

Lemurs had access to all the cage enrichments during the day (line 163). This is where the results become difficult to interpret. For example, is sleep in the impoverished condition determined by the impoverished sleep-site at night or by the enriched environment during the day?

Thank you for this comment. In the referenced line, we confused the “enrichment condition” specific to our study with the assortment of items added to lemur enclosures to create a more dynamic, habitat-relevant environment, often also referred to as enrichment outside the context of this paper. We rephrased to remove this ambiguity.

4. Another complication in the design is that animals were sleeping in pairs during baseline but not during the enriched and impoverished nest-site conditions (line 135-136 and 143-144). So, how much of the effect that is observed is due to the difference in cage enrichment or simply the fact that the animals where individually housed?

We appreciate the reviewer’s attention to possible confounds in our experimental design. Because the social aspect of sleep conditions is controlled for in the contrast of our enriched vs. impoverished conditions, we can safely conclude isolated sleep does not explain the observed differences in total sleep time between these two conditions. This is noted in lines 345-346 and we have now further clarified our rationale for the decision in lines 186-190 so that it is clear to the reader when we introduce our methods. Though we successfully control for the confound of social sleeping in the contrast between enriched and impoverished conditions, we agree that this confound would affect the absolute measures of difference from baseline. This raises the possibility that our results could potentially be explained by an increase of TST from sleeping alone PLUS a decrease in TST from the impoverished condition, rather than simply an increase in TST during the enriched condition. We have added this caveat to our conclusions (line 361).

5. Also, if I read the methods section correctly, the baseline condition always preceded the enriched and impoverished conditions. How do the authors exclude the possibility of an order effect or time effect? Do they have additional baseline recordings after the experimental treatment weeks to assess whether sleep time normalized to baseline values?

The reviewer is correct that the baseline condition always precedes the enriched and impoverished conditions, and is astute to note the possibility of an order effect. We explicitly account for the possibility that the order of experimental conditions may affect observed TST by including the parameter Order in our statistical model. Additionally, we account for the possibility that the effect of Enrichment preceding Impoverishment may differ from the effect of Impoverishment preceding Enrichment by including an interaction term between Order and Condition in our statistical model. When calculating our observed differences in TST between conditions, we do so marginal to Order (258, 291), meaning we used the estimated coefficients for Order and its interaction with Condition to calculate the differences in TST between conditions attributable to order and crossover effects. We then subtract these estimates from our final, reported values. The details of these calculations (which use matrix multiplication to propagate estimated error in our coefficients through to our final confidence intervals) are included in the Statistical Inference section of S1: Analysis.

To clarify the use of these methods in our manuscript without becoming overly technical, we added new language and additional citations (238). We have also rephrased line 345 to explicitly link the technical term “marginal contrasts” to the way we controlled for order effects.

As the reviewer notes, experimental controls such as additional baseline periods would be helpful, potentially adding statistical power to future analyses; we clarify the role the order and carryover effects would have on such decisions in our discussion of future research (lines 460-470).

6. It is not clear why the authors chose to study 4 different species of lemur (with only 2 individuals for each species) rather than 8 individual of a single species. Also, it seems like species was not included as a variable in the analysis.

We agree with the reviewer that a study including 8 individuals of the same species and sex would allow for more precise conclusions. Unfortunately, we did not have access to any single species at the DLC for which that many pair-housed individuals were available for this type of study. Additionally, a motivating factor for this study was the development and use of standardized methods with which we could compare the results across species. Unfortunately, our limited sample size did not allow us to directly test hypotheses of species level differences. We do, however, include species in our model as a random effect (U). From the Interclass Correlation Coefficients reported in line 297 we concluded that only a small proportion of the unstructured variance in our modeled data were attributable to species level differences; including species as a fixed effect in our model would be unlikely to reveal significant effects while creating a major risk of overfitting the model. We discuss the rationale behind these decisions in detail in lines 447-459. To address the reviewer’s concerns, we have also added clarifying language at the top of our discussion section (lines 346-349)

7. For each of the 4 species, 2 individuals were included, one male and one female. Were there any sex differences in rest activity patterns?

Thank you for the question, it would indeed be interesting to know if there are any sex differences in the sleep of these species. Unfortunately, due to the small number of individuals in our study, we are limited in our ability to test additional hypotheses. Attempting to simultaneously model species, sex, and individual level effects on TST leads to overparameterization and an unidentifiable model. Because we have no specific hypotheses about the role of sex on total sleep time, and attempting separate tests for any such effect would constitute questionable use of multiple testing, we have chosen to refrain from reporting and speculating on sex-biased total sleep time. We added a sentence at line 455 to help explain our rationale.

8. The Figure shows the data in its most basic form; that is, daily resting time separately for each individual on each of the 6- 7 day in each of the 3 conditions. However, this makes it quite hard to read the figure and get an overall picture. Perhaps the authors should include additional figures or panels that simply shows the average resting times per condition. After all, they claim that the claim is that there is a statistical significant effect of experimental condition

Thank you for the suggestion. We have added an additional figure (Figure 3) that shows the estimated mean and standard error for the difference (in minutes) of total sleep time between conditions.

9. Based on the current figure, the reader may have the impression that the differences in daily resting time between the conditions is rather small compared to even the variation within an individual and within a certain condition. Moreover, the differences between the conditions is certainly small compared to the differences between the individuals. This raises questions as to how relevant these differences are and what other variables might explain variation in resting time. Perhaps this should be discussed.

Thank you for the comment. We hope that the figure added in response to the previous comment, along with our discussion of the sources of variance in these data in lines 450-453 will be sufficient to address these concerns.

10. The discussion speaks about the preference of the subjects for enriched sleeping-sited (line 283). However, in this study the lemurs were not offered a choice between the different conditions, so how can we know what their preference was.

The discussion mentions that the lemurs chose to sleep on narrow, elevated ledges rather than the ground during the impoverishment condition (line 286). If this is a meaningful observation worthy of discussion, the authors should consider showing the data to support this. In a way this goes back to point 2, the feeling that there is much more in the actigraphy data and video recordings than only the total daily resting time now presented.

We appreciate the reviewer’s concern. At question are the observations that lemurs prefer to sleep in enriched sleep-sites crates (relative to the rest of their enclosure) when available, and on narrow ledges (relative to the rest of their enclosure) when no enrichment was available. These observations were made through a mix of the authors’ experiences, reports by DLC caretaking staff, and a limited sub-sample of the data for which infrared video was available. However, as this evidence is neither un-biased nor fully reproducible, we removed the relevant paragraph.

11. Minor Comments: Thank you, we have made changes to address these.

Reviewer 2

1. Introduction, lines 64, 65: Please define “deep” and “efficient” and “higher quality” sleep. These are never actually explained in the paper, yet much seems to rest on their definition. For instance, sleep depth can be measured directly, with arousal thresholds, or inferred using slow wave activity during slow wave sleep. Slow wave activity cannot be compared across species (for a variety of physiological and non-physiological reasons) and so some explanation would be warranted.

Thank you for the comment, we agree that some additional clarity was needed. We revised lines 67-70 to more accurately define the measures reported in the studies being described.

2. Introduction, line 70. Consider this: The reduced skeletal muscle tone that accompanies REM sleep might make small surface areas particularly dangerous and thus animals sleeping in such locations might selectively reduce REM sleep (either in duration or %sleep).

A useful note, thank you. We used it to enrich the language at line 73.

3. Methods, line 110: How do you know the animals acclimated to the collars within 2 hours?

Thank you for your question. We have rephrased to clarify the protocol by which DLC caretaking staff monitored the lemurs to ensure lemurs were not responding adversely to the collars.

4. Methods: line 115: Total sleep time (TST) is a standard acronym for 24-h sleep amount. Otherwise it is TRT - total recording time. I would suggest you use TST and not TTST as the latter is not familiar to most sleep readers.

Thank you for this useful suggestion. We have adopted the use of TST throughout our materials.

5. Methods, line 123: Are you certain you validated sleep states? First, no data on sleep states is presented. Second, this seems rather a big deal to gloss over. Please explain, clarify or correct

Thank you pointing out this ambiguity. We have described our process for inferring sleep state in greater detail in lines 160-170.

6. Methods: the low sample size needs to be addressed. I can guess as to the reasons and am empathetic but it will be eyecatching to many. I’d be proactive and defend against criticism straight-up.

We understand the reviewer’s concern and appreciate the input. To more proactively address the issue of sample size, we added a sentence to the beginning of the Discussion (lines 346-349).

7. Methods, line 170: Should species not be a fixed factor in the model?

Ideally, yes. However, one of the limitations of our sample size is in our ability to add additional parameters to the model. We compared inter-class correlation coefficients to determine when our statistical model had reached a level of parameterization that approached the limits of our ability to make sound inferences – a process we have now clarified in lines 261-262. Unfortunately, given the large ratio of individual-level variance to species-level variance when both were included as random variables, we lack the ability to detect species level effects even should they exist, and thus refrain from further complexifying our model by including species fixed-effect parameters. This is discussed in lines 455-458.

8. Results, line 223-225: I would not say “sample size of 7”. This is misleading. I would replace with “All values were an average over 7 days, except….”

Thank you for the suggestion, we have adopted it.

9. Discussion, line 255: The issue of sleep depth and quality re-emerges here too. Sleep depth is difficult to compare across species. You cannot compare SWA across species. Moreover, some data challenges comparing arousal thresholds across species. For instance, sleeping emperor penguins arouse quickly with lightly touched on their feet, but no where else on their body. Does this make an emperor penguin a light or deep sleeper compared to a starling? I have strong doubts over the ability to make such statements.

Thank you, we agree that is important to be clear about what exactly has been measured and to draw justifiable conclusions. We rephrased this paragraph to focus on sleep duration instead of sleep quality. Additionally, we added analyses to explore stability and fragmentation of sleep patterns during our experiment, detail of which are provided in response to Reviewer 1’s second question.

10. Discussion, line 295-297: This result was also found by Lesku et al. 2006 in that mammalian species with greater encephalization had a higher %REM sleep. Thus, not only do humans have more REM sleep (%) than non-human primates but so too do mammals, in general.

Thank you for highlighting these results here; we have used an additional citation to the study mentioned to strengthen our argument in this paragraph (line 428).

11. References: There are heaps of sloppy errors in the reference section. Endnote appears to have been used, but unchecked. Please check each reference individually for consistent formatting and accuracy.

We thank the review for their attention to detail, and apologize for the mistakes – we failed to realize our reference manager was overwriting manual fixes with automatic updates. We have carefully checked the references upon final submission and made corrections where necessary.

Reviewer 3

1. Overall, this is a "neat" study with an excellent design. It is novel and indeed, for me, not only has implications for primate evolution and nest building, but my first thought went to conservation, where habitats are destroyed and nesting materials may not be available, long loss of even half an hours sleep per night could have long term negative effects. I think the authors should add a few lines about this to the discussion and perhaps one phrase in the abstract.

Thank you for the encouraging words! We share your enthusiasm for conservation and likewise hope that our research might facilitate conservation efforts. However, we have struggled to come up with direct links between our findings and currently employed strategies for conservation management. We would feel uncomfortable promoting the conservation benefits of our study without a better understanding of what these might be, especially given that the subjects in our study are not primarily nest builders.

2. Introduction

Line 72 - not all apes build nests - I guess you mean great apes - it would be nice to have a such as after sleep site selection behaviours with a few examples as this paragraph reads very thin

72-75 - unpack this sentence - need a better flow into lemurs - maybe that many strepsirrhines build nests, and a more detailed review of the main functions (also comfort, parasite control etc)

77 - I would be more broad since most galagos also use nests - and many lemurs don't - I feel this work has broader impact if you are looking at strepsirrhine evolution - there are a few good reviews on sleep site use by galagos (Bearder earlier on,

Svensson more recently) - then justify the choice of the lemur species selected...I actually had no idea that sifakas use nests...nor ring-tailed lemurs - this makes me realise that the previous paragraph is confusing - are you testing just sleep sites? or nests? clarify and justify the examples

Line 86 - I feel that this section should be either be higher or in the discussion - you can simply state that you discuss sleep site comfort in relation to welfare and evolution (and I suggest add conservation)

. . .

Discussion - AH now you say these are species that do NOT nest - this needs to be more clear in the intro

Thank you for the detailed suggestions for how we can improve our introduction and discussion of sleep phenotype variation in primates. We have approached this set of comments together, substantially expanding and re-organizing the final two paragraphs (lines 78-121) of our introduction to include additional examples and clearer justification for the choice of species in our study.

3. Methods - really cool experimental design! I am just curious about battery life and if you had to change the collars or recharge the battery (or remove the sensor regularly for download of data)

Thank you! The collars stayed on the lemurs for the duration of study with no need to recharge or download data. The battery life of the collars was one of the determining factors in the length of each study period – adding additional baseline periods, for example, would likely have required us to catch the lemurs and replace their collars.

4. Line 241 - can you not compare with any other studies? You mentioned a mouse lemur study in the wild...you mentioned several bird studies - and surely some human studies - no references in this paragraph

The paragraph referenced discusses the specific results of our study and the statistical support behind our conclusions; we do not have any relevant references to add. As the reviewer notes, we discuss these results in the larger context of other studies at different points in our Introduction and Discussion.

5. Line 261 - as this is not a primate journal, you need to explain what cathemeral is

Thank you for the observation. We have added clarity to our definition at line 384.

6. Line 263 - turn this around to explain to the reader about sex dimorphism then you can interpret the findings

Lie 268 - the lemurs in your sample certainly do not fit the definition of large body size in a mammal - but medium bodied - Maybe you want to include a threshold in grams related to security in branches to support a size of that nature - this is why virtually no large bodied mammals are arboreal (the exception mainly being some great apes!)

We appreciate the opportunity to improve clarity here, and have added a sentence at line 388 to address both of these points.

7. Paragraph 268 - a lot to unpack in this paragraph - follow the hypotheses paragraph by paragraph rather than mixing.

Thank you for pointing out the density of this paragraph. We have added some organizational phrasing and numbering to help distinguish the hypotheses and make this paragraph easier to digest.

8. Line 297 - this implies that human evolved to sleep in tree platforms - do you think this is the case? provide more evidence

Thank you for this observation. Out intent was to imply that common ancestors of apes and humans evolved to sleep in tree platforms, but that early hominins likely transitioned to different sleep patterns sometime after their divergence from other Great Apes. We rewrote these two sentences (lines 425-427) to clarify.

9. Line 300 - this feels a bit 1950s and very primatological - most animals that make nests have some form of learning, at least in choice of materials etc.

We appreciate the note, and have rephrased to clarify the results of studies being cited and add nuance to distinguishing features of learning being discussed.

10. Line 305 - do you mean hominid?? or do you mean hominin? are you including the ancestors of chimps etc?

Correct, we mean to use hominid. In this paragraph, we discuss the emergence of secure, flexible platform building in ancestral apes, and how our research helps us understand the context of this evolutionary shift. We have elaborated on the sentence at line 439 to help reinforce this point.

11. Line 309 - bold statement - reference needed

We have rephrased to make it clearer this was meant as a question, not a statement, and added reference to some of the underlying literature that motivated the question (line 442).

12. Line 319- globally threatened - not endangered (that is an IUCN category)

Thank you, we corrected this.

13. 310-335 - a lot of unreferenced reflection - can be dramatically shortened or made stronger with referencing

We added a reference to back up some statistical suggestions made in this section. Largely, however, we are leaning on our own expertise and experiences conducting this study to make suggestions for improving similar, future efforts. We believe that a thorough discussion regarding the strengths and weaknesses of a study such as the one presented here belongs in any manuscript. As the subject of this discussion is our own study and what we learned in the process of conducting it, we do not have additional references to add.

14. Line 355 - substandard is an odd choice- indeed, captive facilities should consider more the ecology of the species and try to closely match it

Thank you. We agree that the ending clause containing “substandard” distracts from this point and we removed it.

15. Line 357 - larger bodied apes and monkeys (again monkeys are not large for the most part)...strepsirrhines unless you mean tarsiers but you never mentioned tarsiers anywhere else and you also did not mention anything other than lemurs so this comes out of the blue anyway! Indeed, there are some very nice new studies of sleep in slow lorises using similar methods as to here, as well as the aforementioned studies on galagos that would really complement the work and make the discussion broader

Thank you for helping us to add clarity here. We have removed “large bodied” to avoid ambiguity, changed “prosimian” to strepsirrhine (line 500), and added reference to Svensson et al.’s comparative study of lorisiform sleep at line 478.

Some minor comments

Line 65 etc - the accepted spelling of orang-utan is generally with a hyphen and at the very least this is how Indonesians would spell it in English

Figure 1 text a subject (Propithecus coquereli)

Table 1 - random change of font and the table heading needs to be more detailed and explain the parameters

line 235 - among the models THAT we compared

Line 274 - delete you - replace with alternative - an animal, a lemur, a primate

Thank you, we have made the suggested changes.

Submitted filename: Response to Reviewers_PlosONE.docx

Click here for additional data file.

27 Sep 2021

PONE-D-21-17696R1Enriched sleep environments lengthen lemur sleep durationPLOS ONE

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Reviewer #1: The authors did an admirable job in addressing the comments and the revision is certainly a major improvement. While some aspects of the experimental design remain a potential confound (e.g., previous points 4 and 5), at least now the authors acknowledge and address these issues in the discussion.

One issue that has not been fully addressed yet is that recordings of rest-activity patterns are only a correlate of true sleep-wake patterns (mentioned under my previous point 1). In other words, the authors equate a lack of movement as measured with motion watches with sleep. However, it is not excluded that differences in the the amount of movement between conditions reflect, perhaps partly, differences in relaxed wakefulness rather than differences in true sleep. The lemurs in the enriched condition with the high quality sleeping-box, lined with comfortable soft bedding may just be more inclined to stay in their nest a bit longer, even when they are awake. I feel this is not a trivial point, given that the differences in rest and/or sleep between the enriched and impoverished condition amounts to not much more than 30 min on average.

Also, with the previous points in mind, I have mild concerns about the use of the wording 'strong evidence' in both the abstract (line 37) and discussion (line 316). They may want to tone down a little bit.

Reviewer #2: I am satisfied that the authors have effectively addressed my comments and queries and thank them for their thoughtful responses.

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4 Oct 2021

1. “One issue that has not been fully addressed yet is that recordings of rest-activity patterns are only a correlate of true sleep-wake patterns (mentioned under my previous point 1). In other words, the authors equate a lack of movement as measured with motion watches with sleep. However, it is not excluded that differences in the the amount of movement between conditions reflect, perhaps partly, differences in relaxed wakefulness rather than differences in true sleep. The lemurs in the enriched condition with the high quality sleeping-box, lined with comfortable soft bedding may just be more inclined to stay in their nest a bit longer, even when they are awake. I feel this is not a trivial point, given that the differences in rest and/or sleep between the enriched and impoverished condition amounts to not much more than 30 min on average.”

We would like to thank the reviewer for their concern, and agree that we should be as transparent as possible about what we measured. Though we think that our inferential criteria for categorizing sleep are quite conservative and unlikely to capture much restful wakefulness, we have added a few sentences to make clear to our readers that it is a possibility. First, we added a sentence to the methods (line 157) explaining the possibility that we may be capturing wakefulness in our quantitative definition of sleep. Additionally, we now highlight in the first paragraph of our discussion (line 228) that using body-motion as a proxy for sleep limits our ability to make conclusions about specific sleep states such as REM of SWS.

2. Also, with the previous points in mind, I have mild concerns about the use of the wording 'strong evidence' in both the abstract (line 37) and discussion (line 316). They may want to tone down a little bit.

Thank you for the note. We removed “strong” from both of these sentences.

Submitted filename: Response to Reviewers_Lemur_Sleep2.docx

Click here for additional data file.

5 Oct 2021

Enriched sleep environments lengthen lemur sleep duration

PONE-D-21-17696R2

Dear Dr. Vining,

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11 Oct 2021

PONE-D-21-17696R2

Enriched sleep environments lengthen lemur sleep duration

Dear Dr. Vining:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

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PLOS ONE