Play behavior occurs during the juvenile period in a broad range of mammalian species, is inherently rewarding, and can importantly influence development (Bekoff and Byers, 1998; Burghardt, 2005; Trezza et al., 2010; Pellis et al., 2014). We evaluated the relationship between social play in juvenile Belding’s ground squirrels (Urocitellus beldingi) and the development of temperament along the caution/boldness continuum. Young animals are often vulnerable to predation and in some cases infanticide by conspecifics, and caution should thus generally be favored among young animals (Hrdy, 1979; Sherman, 1981; Lima and Dill, 1990; Nunes and Monroy Montemayor, 2023).

Temperament includes behavioral traits of an individual that are consistent over time and across situations. Temperaments are expressed along continua that can include caution-boldness, exploration-avoidance, and ranges of docility (Réale et al., 2007; Petelle et al., 2013). The caution-boldness continuum specifically encompasses responses to danger or threats, whereas exploration-avoidance refers to responses to novel objects or unfamiliar situations, and docility refers to the degree to which responses are passive or active. Elements of temperament can exhibit plasticity in young animals during periods of behavioral development, and social, physiological, and ecological factors in early life can have important and long-lasting effects on temperament (Sinn et al., 2008; Rödel and von Hulst, 2009; Rödel and Monclús, 2011; Petelle et al., 2017; DiRienzo et al., 2018; Cabrera et al., 2021). Temperament in turn can affect a range of ecologically important variables including physiological responses to stress, reproductive success, and survival (Carere et al., 2001; Dingemanse et al., 2004; Both et al., 2005; Boon et al., 2008; Réale et al., 2010; Baugh et al., 2013; Colchester and Harrison, 2016; Rasmussen and Belk, 2017; Vetter et al., 2016). Temperament during the juvenile period can provide a foundation for development of adult behavioral and personality traits. Factors that influence juvenile temperament might thus have an important impact of the expression of behavior in adulthood (Rothbart and Ahadi, 1994; Eisenberg, 2012; Wright and Jackson, 2022).

Temperament can be an element of coping styles, which include correlated behavioral and physiological responses that help animals navigate ecological and social challenges. For example, proactive coping styles tend to be characterized by active responses and quick action, greater agression, lower behavioral flexibility, and lower responsiveness along the hypothalamus-pituitary-adrenal (HPA) axis involving release of glucocorticoids after exposure to a physical or psychological stressor. By contrast, reactive coping styles tend to be characterized by passive responses, information gathering, lower aggression, greater behavioral flexibility, and greater responsiveness along the HPA axis (Koolhaas et al., 1999; Coppens et al., 2010). Coping styles can vary according to ecological and social variables, with different coping styles being adaptive under different conditions (Duckworth et al., 2023).

Play behavior can appear frivolous with no immediate or obvious benefits for animals, creating challenges for defining or quantifying play. Nevertheless, various adaptive functions have been linked to play behavior (Palagi, 2018). The expression of play behavior can vary greatly across species; however, within a species play is typically characterized by specific behavior patterns that are exhibited in varied sequences (Bekoff, 1974; Burghardt, 2005). Moreover, Burghardt (2005) identified five elements common to play across species. Play behaviors are 1) voluntary and spontaneous, 2) characteristic of a species and performed repeatedly but not invariantly, 3) functionless in the context in which they occur but may resemble functional behaviors, 4) distinct in their expression from functional behaviors, and 5) expressed in healthy, unstressed individuals. Play behavior can be categorized as non-social, involving manipulation of objects or locomotor activity, or as social, involving interaction with other individuals (Burghardt, 2005; Pellis et al., 2014; Pellis and Pellis, 2017). Play most commonly occurs among juveniles, but in some cases may continue beyond the juvenile period into adulthood (Bekoff and Byers, 1998; Palagi, 2018, 2023).

A diverse range of adaptive benefits have been suggested to result from juvenile play behavior across different species (Nunes and Monroy Montemayor, 2023). In some cases, these benefits are experienced early in the life of individuals. For example, juvenile play can contribute to motor development in young animals. Play behavior boosts acquisition of motor skill and coordination in Assamese macaques (Macaca assamensis; Berghänel et al., 2015) and U. beldingi (Nunes et al., 2004a). Juvenile play can also contribute to social development. Play facilitates establishment and maintenance of dominance relationships in juvenile red foxes (Vulpes vulpes; Meyer and Weber, 1996), helps young seals (Phoca vitulina) integrate into their social groups (Wilson, 1974). Moreover, play enhances survival in young brown bears (Ursus arctos) by reducing stress-related mortality (Fagen and Fagen, 2004).

Play can also have longer-term benefits that extend beyond the juvenile period in a wide array of animals. Long-term benefits of play can be related to motor skill. For example, locomotor play in juvenile mule deer (Odocoileus hemionus) and white-tailed deer (O. virginianus) promotes development of motor skills important in evading predators (Carter et al., 2019). In some cases, juvenile play appears to enhance the competence of adult behavior. For example, rough and tumble play during the juvenile period in American minks (Neovison vison) is associated with increased proficiency of copulatory behavior in adulthood (Ahloy Dallaire and Mason, 2017). In female U. beldingi juvenile play is associated with greater intensity of maternal territorial behavior and increased likelihood of weaning a litter as a yearling (Nunes, 2014). In many animals, long-term benefits of juvenile play are related to social behavior and social organization. In horses (Equus caballus; Rho et al., 2007), domestic dogs (Canis lupus familiaris; Ward et al., 2008), female African elephants (Loxodonta africana; Lee and Moss, 2014), and geladas (Theropithcus gelada; Gallo et al., 2021), play interactions help young animals forge long-term social relationships. Play behavior in yellow-bellied marmots (Marmota flaviventris) helps to establish hierarchical relationships that persist into adulthood (Blumstein et al., 2013), and in baboons (Papio papio, P. hamadryas) and mandrills (Mandrillus leucophaeus, M. sphinx) juvenile play fighting is related to the structural rigidity of long-term dominance relationships (Kraus et al., 2019). In spotted hyenas (Crocuta crocuta) and male African elephants play facilitates social assessment by providing young animals with information about conspecifics with whom they will interact throughout their lives (Lee and Moss, 2014; Nolfo et al., 2021). In rats and an array of primate species play behavior is critical for development of socially competent adult behavior (Himmler et al., 2016; Pellis et al., 2017; Palagi, 2018; Stark and Pellis, 2020; Stark et al., 2021; Bijlsma et al., 2022, Bijlsma et al., 2024).

We evaluated the relationship between juvenile social play and development of temperament in U. beldingi, a species that is a well-established model system for studies of play (Holmes, 1994; Nunes and Monroy Montemayor, 2023). We used an experimental approach to build on prior studies reporting correlations between social play and development of temperament in U. beldingi (Marks et al., 2017; Hurst-Hopf et al., 2023; Shehan et al., 2023). Because U. beldingi is diurnal, it is well-suited for studies involving observation of behavior. The habitat of U. beldingi extends across high elevation meadows in the western United States. Squirrels live together in groups but do not have a complex social system. The active period of U. beldingi occurs during a 3–4 month period in late spring and summer coinciding with the local growing season. Squirrels hibernate in underground burrows between active seasons, subsisting on stored fat. Mating begins soon after squirrels emerge from hibernation. Because of the short active period, females bear at most one litter each year (Michener, 1983; Jenkins and Eshelman, 1984; Sherman and Morton, 1984). Young squirrels remain underground in natal burrows during lactation and first appear above ground between 25–28 days of age near the time of weaning (Holekamp et al., 1984). Juvenile U. beldingi engage in play behavior primarily during their first two weeks above ground. Most play interactions (> 97%) occur between littermates (Nunes et al., 1999). The short, well-defined play interval for U. beldingi makes the species especially amenable to studies involving experimental manipulation of play behavior.

Prior studies of juvenile U. beldingi suggested juvenile social play may contribute to development of temperament in this species. Social play in young squirrels was associated with increased exploration in an unfamiliar environment, a shift from docile to more active behavioral responses, and increased caution in response to a potential threat (Marks et al., 2017; Hurst-Hopf et al., 2023: Shehan et al., 2023). These prior studies were correlational and provided preliminary evidence supporting a relationship between social play and development of temperament in young animals. Here we expanded on this prior work and used an experimental approach to evaluate the hypothesis that social play behavior promotes development of cautious responses in juvenile U. beldingi. We disrupted the play behavior of juvenile U. beldingi engaged in social play by walking toward them until the play interactions were halted, and used undisrupted juveniles and juveniles disrupted while not engaged in play as controls. To evaluate caution, we conducted behavioral tests in which a human intruder approached a squirrel and noted the distances at which the squirrel first noticed and then fled from the intruder. We reasoned that if social play promotes development of cautious responses in U. beldingi, then play-disrupted juveniles should have smaller increases in caution across the developmental period in which play primarily occurs than undisrupted and nonplay-disrupted juveniles.

The distances at which juvenile U. beldingi in the study noticed and fled from intruders during behavioral tests increased across the primary play period. Increases in distances to flee from an intruder were significantly greater in juveniles whose play behavior was not disrupted compared to play-disrupted juveniles. We further observed a trend approaching significance for increases in distances to first notice an intruder to be greater in juveniles whose play behavior was not disrupted. These results are consistent with social play behavior in juvenile U. beldingi contributing to development of cautious responses. Shehan et al. (2023) similarly observed that higher rates of social play in juvenile U. beldingi were correlated with greater increases in cautious responses across the play interval. Together with the results of Shehan et al. (2023), our findings here provide strong support for the hypothesis that juvenile social play in U. beldingi influences the development of temperament along the caution-boldness continuum, promoting shifts toward more cautious behavior in response to a potential threat.

Marks et al. (2017) observed that juvenile U. beldingi that engaged in social play at higher rates tended to have greater increases in exploratory behavior in a novel environment. Hurst-Hopf et al. (2023) further observed that greater social play among juvenile U. beldingi was associated with greater shifts from passive to active behavioral responses across the play interval. Thus, temperament appears to be plastic in U. beldingi during the juvenile period, and experiences associated with social play may promote refinement of various temperament traits. These refinements might better equip young squirrels to express adaptive responses across a diverse array of situations they may encounter as they interact with the environment. For example, as young squirrels begin to venture farther from the natal area, they may shift to a more proactive coping style accompanied by greater caution to better equip them to explore unfamiliar surroundings and interact with unfamiliar conspecifics.

A range of variables are associated with development of temperament in young animals (Cabrera et al., 2021). For example, in European rabbits (Oryctolagus cuniculus) greater body mass among juvenile males is associated with greater aggressiveness in agonistic interactions, as well as greater exploration in an unfamiliar environment (Rödel and von Hulst, 2009; Rödel and Monclús, 2011). Moreover, maternal effects on the development of temperament and other variables have been suggested to prepare offspring to be successful under prevailing environmental conditions (Storm and Lima, 2010; Kapheim et al., 2011; Dantzer et al., 2013). Environmental stresses experienced by mothers can directly affect the way they raise their young, which in turn can affect the behavior and other characteristics of offspring (Mousseau and Fox, 1998; Weinstock, 2001; Hayward and Wingfield, 2004). For example, environmental stressors and prior experience raising young have been observed to influence glucocorticoid concentrations in mothers, and glucocorticoids passed to offspring through milk during lactation can influence development of behavior and elements of temperament such as boldness and docility (Hinde et al., 2015; Petelle et al., 2017).

Possible influences of social play on the development of temperament in U. beldingi might be mediated by effects of play on the developing brain. Pellis et al. (2023) noted that juvenile play behavior in rats promotes increased behavioral skill and social competency during adulthood, and suggested that a general benefit of play may be to refine or improve skills and behaviors that can be applied adaptively across a variety of social and non-social contexts. Various aspects of brain development exhibit plasticity during neonatal and juvenile periods, and a range of experiences after birth can direct elements of neural and behavioral development (Johnson, 2001; Stiles and Jernigan, 2010; Kolb and Gibb, 2011; Sakai and Sugiyama, 2018). Various brain regions help to modulate the expression of play behavior in mammals (Pellis and Iwaniuk, 2004; Siviy, 2016; Achterberg and Vanderschuren, 2023). Moreover, juvenile play in rats and hamsters (Mesocricetus auratus) has been shown to modify development of areas in the frontal cortex associated with motor and social behavior as well as behavioral flexibility. These modifications have been suggested to facilitate more nuanced neural responses and more adaptable behavior later in life as well as context-appropriate behavioral responses (Bell et al., 2010; Pellis et al., 2010; Himmler et al., 2013; Burleson et al., 2016; Himmler et al., 2017; Bijlsma et al., 2022; Cooper et al., 2023; Pellis et al., 2023; Bijlsma et al., 2024).

Possible effects of play on the development of temperament might also be mediated in some capacity by play-related modulation of glucocorticoid secretion. Social play behavior in dogs is associated with decreased concentrations of salivary cortisol and increased performance on learning-related memory tests (Horváth et al., 2008; Affenzeller et al., 2017). Moreover, glucocorticoids early in life can have important effects on the course of brain development (Gunnar and Barr, 1998; Davis et al., 2013). Thus, one possibility is that social play alters glucocorticoid secretion in ways that influence behavioral learning or brain development during the juvenile period, resulting in refinement in the development of temperament.

Experimental disruption of play in this study decreased the rates of play and duration of play bouts among juvenile U. beldingi. Thus, the effects of play that we observed on the cautious responses of young squirrels might have been related to the amount of play in which squirrels engaged. Various studies have suggested that reduced play can influence development of the brain in young animals and have long-term consequences on the expression of behavior (e.g., Stark and Pellis, 2020; Stark et al., 2021; Bijlsma et al., 2023; Pellis et al., 2023). Leca (2023) and Pellis et al. (2024) noted that the structure of play behavior is related to its adaptive functions, and Bijlsma et al. (2024) observed that the environment in which play occurs can affect the way in which it influences development of the brain and behavior. Thus, results in our study might also be related to the disruptions that occurred while juveniles were engaged in play. Disruption of play behavior under natural conditions might provide information about the density of conspecifics or predators, and might alter play in ways that promote development of coping styles that are adaptive under prevailing environmental conditions. For example, disruption of play interactions might have shaped the ways in which juvenile U. beldingi in our study assessed their play partners or surroundings, or gauged potential risk or danger during play interactions. Ecological conditions can influence temperament in young animals (Sinn et al., 2008; DiRienzo et al., 2018; Cabrera et al., 2021). In yellow-bellied marmots, for example, environmental conditions influence the glucocorticoid content of milk in mothers, which in turn influences the temperament of their offspring (Petelle et al., 2017). We note that currently little is known about how the specific structure of play behavior or context in which play occurs might influence the development of behavior in free-living animals.

Human disturbance can influence the behavior of animals. For example, elk (Cervus elaphus) have been observed to exhibit greater vigilance at the cost of foraging in response to human activity (Ciuti et al., 2012). Similarly, in yellow-eyed penguins increased vigilance at the cost of preening is associated with human activity (French et al., 2019). Moreover, increased aggressive behavior has been observed in little penguins (Eudyptula minor) living on islands that have greater human activity (Colombelli-Négrel and Katsis, 2021), and increased avoidance behavior associated with human activity has been observed in zoo populations of E. minor (Sherwen et al., 2015). In this study, disruption of juvenile U. beldingi when they were not engaged in play interactions did not influence their cautious responses relative to undisrupted juveniles. We note that disruption of the nonplay behavior of juvenile U. beldingi during observation sessions in our study was relatively infrequent and low intensity, suggesting that low-levels of human activity may not have a notable impact on development of cautious responses in juvenile U. beldingi.

Field studies of play behavior allow for evaluation of play in the context in which it evolved and under conditions in which it is naturally expressed, but tend to be observational or correlational (Palagi, 2018; Nunes, 2023). Here we conducted a simple manipulation of play behavior in the field that did not appear to be overly disruptive for the squirrels. Our results suggest that play behavior in young U. beldingi contributes to development of caution across the play interval. Play in young U. beldingi promotes development of motor skill and coordination (Nunes et al., 2004a; Nunes et al., 2004b), and our results further suggest that play behavior might also promote or refine development of nonplay elements of behavior.

The dataset evaluated in this study is available through Zenodo: https://doi.org/10.5281/zenodo.10667044.

Ethical approval was not required for the study involving animals in accordance with the local legislation and institutional requirements because the University of San Francisco does not have an Animal Care and Use Committee and does not require review of research using animals. We followed guidelines for the use of mammals in field studies published by the American Society of Mammalogists.

WR: Conceptualization, Data curation, Formal Analysis, Investigation, Writing – original draft, Writing – review & editing. MK: Data curation, Investigation, Writing – original draft, Writing – review & editing. SN: Conceptualization, Data curation, Formal Analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Writing – original draft, Writing – review & editing.