#### Research Questions

Here, the two behavioral processes we sought to investigate were explored in a series of questions. A fundamental question concerned the true unit of analysis—that is, should the level of analysis be the individual or the dyad? We first considered if, when treated as independent actors, male and female parents demonstrate unique patterns of change across four sequentially reared litters. Further, we considered if change can be best characterized with a model of no growth, linear growth, or nonlinear growth. A model of no growth could be indicative of either constant (e.g., invariant) parenting or an inability to identify a mean trajectory followed by all parents. Support for a linear growth model would indicate an approximately equal growth or decline of the mean parental behavior across litters. A non-linear growth model would indicate growth or decline of the mean parental behavior in an unequal way across litters. We made the same considerations of change at the level of the dyad, using a model that considers the dyad as two covarying individuals that is, we considered an alternative model in which the level of analysis is the individual (either male or female parent), but in which some interaction between partner parents is permitted through time-varying covariances.

#### Subjects and Laboratory Conditions

All data were collected from the prairie vole colony at the University of California, Davis over a period of approximately 6 years. One-hundred forty-one parenting dyads were selected from archival data on the basis that no experimental manipulation had been applied to the dyad or their pups and that observations of parenting behavior had been made upon the dyad during post-natal day (PND) one through three. All subjects were descendants of wild prairie voles that were originally caught near Champaign, Illinois and continually outbred to maintain genetic diversity, and included new genetic stock from another captive colony in 2015. Animals were maintained on a 14:10 light-dark cycle with lights on at 06:00. Water and food (high-fiber Purina rabbit chow) were provided ad libitum. Breeder pairs and their offspring were maintained and observed in large, polycarbonate cages (44 × 22 × 16 cm). Each cage had aspen wood bedding (i.e., Sani-Chips), and cotton was provided for nesting material. Humidity was controlled and room temperature maintained near 70◦F. All pups were weaned from the home cage on PND20, and subsequent litters of pups were not exposed to older siblings. Home cages were left undisturbed beyond weekly cage changes and three daily checks of food and water.

Litter size for each cage was recorded at PND1 and corrected at weaning in the event of a miscount. Corresponding information on litter size in each observed litter and the age of each parent at the time of birth of their first litter was also found in laboratory archives and recorded when available. The mean number of pups per litter across all measured litters (1–4) was 4.75 (SD = 1.47); the number of pups per litter ranged from 1 to 8. At the time of birth of their first litter, mothers' mean age was 99.56 days (SD = 26.56); the range of maternal age at first litter was 64 to 180 days. At the same time point, the fathers had a mean age of 145 days (SD = 73.00); the range of paternal age at first litter was 51–422 days.

When measures of parental behavior were (in some cases) available for many litters, litters one through four were used in this study. This decision was both practical and theoretical. Practically, the availability of observational data beyond litter four was increasingly sparse. Under natural conditions, prairie vole dyads are unlikely to produce more than four litters of pups before the death of one or both parents, as the average life expectancy of both male and female prairie voles is short (approximately 50–80 days), with most breeding pairs surviving only 1–2 months (Getz and Hofmann, 1986; Getz et al., 1997, 2000; Thomas and Wolff, 2004). In laboratory conditions, animals may live over 2 years and breeding pairs may produce well in excess of 10 successive litters.

#### Behavioral Measures

Parental care observations were collected through live focal sampling by graduate students and undergraduate research assistants who were trained and validated according to the ethogram presented in Perkeybile et al. (2013). Observers sat approximately 1–2 feet from the home cage, and observations were recorded on laptop computers using behavioral software (www.behaviortracker.com). For each litter, both mother and father were simultaneously observed for 20 min focal samples during PND1-3. Mothers were distinguished from fathers using individual characteristics (e.g., size, color, etc.) in addition to observations of pup attachment to nipples or evidence that nursing had occurred (e.g., swollen nipples or milk-wetted fur). Variation in parental behavior in this perinatal period has been predictive of pup bio-behavioral outcomes (Perkeybile et al., 2013). The perinatal period is also the period in which the most extensive parental care is shown, and it is a period in which, prior to fur growth, pups have little ability to thermoregulate (Gebczynski, 1981; Blake, 1992). The observed home cage was left undisturbed on a metal cage rack with overhead coverage from the rack above. Observations were not conducted on days on which cage changes occurred.

The primary outcome measure was a composite score of pup-directed behaviors (PDBs), which was calculated for each respective litter from the focal sampling output. The number of seconds spent in a variety of pup-directed behaviors (i.e., nursing, licking and grooming, physical contact, etc.) were summed across each focal sample, added across all focal samples for each respective litter, and then divided by the total number of focal samples collected (between two and four) for a mean value of pup-directed behavior observed for each parent in the perinatal period of each respective litter. In all, 93.2% of composite scores were generated from four focal samples, 4.1% from three focal samples, and 2.6% from two focal samples with a mean of 3.91 (median = 4) focal samples per composite score of PDBs. Some behaviors were not considered mutually exclusive (e.g., nursing and licking), thus allowing for summed totals of PDB to exceed 1,200 s; and maternal and paternal care were often contemporaneous.

#### Missing Data

The mean number of litters observed per breeder pair was 2.13 (median = 2 litters). Across all 141 breeding pairs and across all four time points, 303 litters were observed; Thus, of the 564 potential opportunities for observation (i.e., across all 141 breeder pairs at four time points), there were 261 missing observations, due to attrition across the 4 litters. A complete summary of the distribution of missing observations (by litter) is provided in **Table 1**. The number of missing observations in the data increased as the litter number increased from 19 in litter 1 to

TABLE 1 | Descriptive statistics for pup-directed behaviors, number of litters observed, and number of litters left unobserved (of the 141 total observed pairs) across litters and by litter.

108 in litter 4. Data were not missing because of active researcher selection for any particular behavioral trait(s). This behavioral paradigm was designed originally as a behavioral diagnostic to classify new parents as low-, medium, or high-contact parents, and the diagnostic was originally designed such that generally only the first two litters of a pair were observed. However, in some circumstances, later litters (i.e., litters 3 and 4) had been opportunistically observed. The choice to halt observation after two litters or to continue observation in later litters was not determined by any behavioral characteristic of the pairs observed. For some pairs, these observations were not necessarily in consecutive litters (due to inconvenience, logistic difficulty, etc. of observation timing). For example, for some pairs there are observations for litters 1 and 3, rather than litters 1 and 2; and, for others there were observations for litters 1–4. Thus, we believe it is reasonable to assume data to be missing completely at random (MCAR), and we do not expect missing observations to introduce bias into the estimated model parameters.

#### Model Specification

In this study, we explored three separate clusters of models. The first cluster contains two identical sets of models for mothers and fathers. The second cluster adds to the cluster 1 models both a time-varying covariate for age and a timevarying covariate for litter size. The third cluster allows for time-varying covariance between parenting partners—that is, if a significant covariation exists between maternal and paternal care, and how that covariation exists across successive litters. The level of analysis was primarily exploratory, with the primary interest being in model selection based on comparative indices of model fit, i.e., what pattern of change best describes the observed data on parental care across successive litters. Within each cluster, models of no growth, linear growth, and non-linear growth were compared in a structural equation modeling framework (see Kline, 2016 for further reading) to determine which growth pattern best fit observed data from mothers and fathers independently (i.e., independent maternal and paternal models), and mothers and fathers within the context of one-another (i.e., a maternal-paternal bivariate model). The models run for mothers and fathers could not be compared directly to each other given different data were used for the generation of their respective models. However, model parameters for each sample can be interpreted and compared (yet informally) across samples. Path diagrams representing the respective models are found in (**Figures 3A–C**) along with detail for their interpretation. For further reading on these techniques, we recommend Bollen and Curran (2006).

#### Model Cluster 1: Independent Maternal and Paternal Models

There were three models in this cluster: a No-Growth Model, a Linear Growth Model, and a Non-linear Growth Model (see **Figure 1A**). All three models include an observed outcome measure of a composite score of pup-directed behaviors for each litter (1–4).

The No-Growth Model for the maternal parent is therefore expressed as:

$$\begin{aligned} \mathcal{X}\_{[t]n} &= \mathcal{x}\_{[0]n} + \mathcal{e}\_{[t]n} \\ \mathcal{x}\_{[0]n} &= \mu\_{\mathfrak{x}[0]} + d\_{\mathfrak{x}[0]n} \end{aligned}$$

where X[t]n is the observed score for the outcome variable (i.e., PDBs) at any given point, t (i.e., litter 1–4), x[0]n is an individual's intercept (i.e., PDB for litter 1), and e[t]n is the residual variance; µ is a fixed group mean, and d represents the variation in such mean across individuals. Whereas the no-growth model predicts only a starting value (i.e., the intercept) and no further change, this model is expanded upon in the Linear Growth Model and the Non-linear Growth Model to include an individual's slope (i.e., pattern of change in PDBs from litter 1 onward). Thus, the general model is:

$$\begin{aligned} X\_{[t]n} &= \varkappa\_{[0]n} + B\_{[t]} \cdot \varkappa\_{\mathfrak{N}n} + \varrho\_{[t]n} \\ \varkappa\_{[0]n} &= \mu\_{\mathfrak{x}[0]} + d\_{\mathfrak{x}[0]n} \\ \varkappa\_{[s]n} &= \mu\_{\mathfrak{x}[s]} + d\_{\mathfrak{x}[s]n} \end{aligned}$$

where B[t] represents a set of coefficients expressing the shape of the curve. These coefficients can be fixed to test various hypotheses of growth. For example, for a linear growth model, the coefficients can be fixed as = 0, 0.33, 0.67, 1, such that the effect of parity on the outcome of PDBs is constrained to a linear pattern. Alternatively, some of the B[t] can be estimated from the data to detect non-linear changes, thus to test the Non-linear Growth Model, which is also often referred to as a latent basis model. For this, the first and last loading (leading from xs to the PDBs for litters 1 and 4, respectively) can be set to 0 and 1, respectively, and the loadings for litters 2 and 3 are left to be freely estimated from the data.

#### Model Cluster 2: Independent Maternal and Paternal Models With Covariates

Cluster two expanded on the models in cluster one with the addition of covariates for parental age at the birth of their first litter and litter size for each litter (see **Figure 1B**). There were similarly three models in this cluster: a No-Growth Model, a Linear Model, and a Non-linear Growth Model. All models

FIGURE 1 | (A–C) path diagrams for Clusters 1–3. Path diagrams are visual representations of model parameters and their relationships, in which each type of model parameter is provided a graphical symbol, i.e., squares or rectangles are used for observed variables (i.e., indicators), circles are used for latent variables and error terms, straight arrows for hypothetical causal or direct effects, and curved arrows for covariances (Kline, 2016). In all path diagrams presented here (A–C), composite scores of pub-directed behaviors (PDBs) for each litter (1–4) are represented as sequential squares (from left to right) around indicators by Xt or Yt , where t = litter. Each represented composite score (e.g., Xt or Yt ) has an error term, ex or ey, which represents variance not explained by the composite scores, including variance due to measurement error. Elements of regression, intercept and slope, are represented by x0 and xs, respectively, and circumscribed to represent their status as latent variables, each with their own covariance and a covariance between the two. Triangles containing the number 1 represent the inclusion of a term for the analysis of means. As the intercept for each model does not change across time, straight arrows from the intercept factor to each time point are labeled with the number 1, indicating a constraint on estimation of the regression between the intercept and each time point. Straight arrows from the slope factor (xs or ys) are variably constrained, according to the growth pattern in consideration. Thus, for the no growth model, regression paths from the slope factor to indicators at all time points are constrained to 0; for the linear growth model, regression paths from the slope factor to indicators are constrained to 0, .33, .67, and 1 for litters 1, 2, 3, and 4, respectively; and for the non-linear growth model, only the regression path from the slope factor to litter 1 is constrained (to 0) and all others are freely estimated, as represented by the term λ*t* . Path diagram (A) represents models tested in Cluster 1, the Independent Maternal and Paternal Models, which includes only the common parameters outlined above. Path diagram (B) represents models tested in Cluster 2, the Independent Maternal and Paternal Models with Covariates, which expands upon the model given for Cluster 1 with the addition of a time-invariant covariate for parental age (Xage) and time-varying covariates for litter size (Pt ), both given in squares. Path diagram (C) represents models tested in Cluster 3, the Maternal-Paternal Bivariate Model, which expands upon the model given for Cluster 1 by presenting models for change in maternal and paternal care in parallel with double headed arrows representing covariance between the two models, and therefore covarying trajectories of change between mothers and fathers.

included an observed measure for each litter (1–4), each with its own residual and time-varying covariate (Pt) to account for litter size. Both models also accounted for parental age with the inclusion of an exogenous, time-invariant covariate for age (Xage, Yage, respectively). In the models generated for the combined dyad, both exogenous covariates for age are included. The model for which time does not contribute to a pattern of growth for the maternal parent is expressed as:

$$\begin{aligned} X\_{[t]n} &= \varkappa\_{[0]n} + P\_{[t]n} + \mathsf{Xage}\_n + e\_{[t]n} \\ \varkappa\_{[0]n} &= \mu\_{\mathfrak{x}[0]} + d\_{\mathfrak{x}[0]n} \end{aligned}$$

#### Model Cluster 3: Maternal-Paternal Bivariate Model

Cluster 3 took the same elements from the first cluster to form a bivariate growth model, which estimates two individual models (maternal and paternal) simultaneously. This third cluster of models allows for covariation between the female and male parent such that the intercepts and slopes of the two covary. Here, again, factor loadings could be fixed to = 0, .33, .67, 1, or left open and allowed to vary freely in order to form the Non-linear Growth Model (see **Figure 1C**); specifically, maternal factor loadings were fixed for a linear model while paternal factor loadings were fixed for a non-linear growth model, according to the best fitting models in Cluster 1. Either could be modified, as in cluster 2, to include either or both time-invariant and/or timevariant covariates. This model affords new parameter estimates, including the covariance between slopes of each parent (i.e., the extent to which changes are related), as well as the covariance between the parents' intercepts.

#### Statistical Analyses

All models were fit with the package lavaan (Rosseel, 2012) in R (R Core Team, 2017) using full information maximum likelihood (FIML). All behavioral scores were divided by 100 to put all measures in the same metric and aid convergence. Standard measures of model fit (chi-square difference test, CFI, TLI, RMSEA) and information criteria [AICi , 1i(AIC), and wi(AIC)] were used to evaluate the models.

"Good fit" is first characterized, here, by a p-value above 0.05, an indication that the hypothesis of exact model fit is not rejected. Ideal Comparative Fit Index (CFI; Bentler, 1990) and Tucker-Lewis Index (TLI; Tucker and Lewis, 1973) scores are >0.95 (Hu and Bentler, 1999). Better Root Mean Square Error of Approximation (RMSEA; Steiger, 1990) scores are below 0.080. A chi-square difference test can be used to determine significant differences (improvement) in fit between two models that are nested, that is, when one model can be represented as a more constrained sub-model of the other. Thus, our no growth model is nested within linear growth model, which is in turn nested within the non-linear growth model.

The additional measure of fit, Akaike's Information Criterion (AICi), was generated as well as transformations of AICi , including 1i(AIC), the change in AIC between each model and the best candidate model, and Akaike Weights [wi(AIC)], which can be interpreted as a probability that a model is the best in its set (Wagenmakers and Farrell, 2004). All fit indices for Cluster 1 and Cluster 3 can be found in **Table 3**.

In some cases, there was an occurrence of a Heywood Case, an instance in which there are parameter estimates of illogical value (Chen et al., 2001; Kolenikov and Bollen, 2012). Here, such cases were restricted to the production of negative slope variances; thus, where the model with negative variances demonstrated significant improvement of model fit over alternative models, the variance and all covariances for the slope parameter were fixed to zero and treated as a "corrected" model. Corrected latent base models are comparable to no-growth models, as the nogrowth models are nested within the latent base model; model comparison between corrected latent base models and linear models is, however, impossible for lack of nesting.

### RESULTS

#### Descriptive Statistics

Maternal pup-directed behaviors have a mean value of 1,055 (SD = 251.8) with a range of 250–1,522. Maternal PDBs appear close to normally distributed with a slight negative skew and kurtosis. Paternal PDBs have a mean value of 628.5 (SD = 307.9) with a range of 0–1,366. Paternal PDBs appear to be normally distributed with positive skewness and kurtosis. Combined dyad PDBs (i.e., the sum of maternal and paternal PDBs) have a mean value of 1,684 (SD = 335.9) with a range of 585.8–2606.0. Dyad PDBs are normally distributed. The slope of plotted observed means across the four measured litters for maternal PDB, paternal PDB, and combined dyad PDB appear flat-to-negative, flat-to-positive, and flat-to-positive, respectively (see **Figure 2**).

FIGURE 2 | Parental care observed data. Pup directed behaviors are tracked for fathers (blue), mothers (red), and as a dyadic composite (black) across the first four litters. Mean values for each group at each time are respectively indicated by the Mars symbol (♂), Venus symbol (♀), or a "D". The shading in blue, red, and gray respectively indicates standard error intervals around the means.

Correlation coefficients between maternal and paternal care care at each time point are presented in **Table 2**.

### Independent Maternal and Paternal Models

For analyses of maternal care, the exact-fit hypothesis was not rejected for no-growth [χ 2 M (11) = 18.521, p = 0.070], linear [χ 2 M (8) = 9.923, p = 0.318], and latent base [χ 2 M (6) = 10.194, p = 0.117] models, giving first evidence that all three models could fit the observed data. A chi-square difference test shows that the addition of a linear growth parameter significantly improves model fit compared to the no-growth model [χ 2 D (3) = 9.229, p = 0.026] (see **Table 3**, **Figure 3A**). The non-linear growth model, however, does not significantly improve model fit over the nogrowth model [χ 2 D (5) = 8.328, p = 0.139]. CFI, TLI, RMSEA, and AICi show better fit for the linear model over the no-growth model, and wi(AIC) sets the conditional probability that the linear model is the best fitting model of the set at 0.778.

For analyses of paternal care, the exact-fit hypothesis is not rejected for no-growth [χ 2 M (11) = 17.246, p = 0.101], linear [χ 2 M (8) = 15.021, p = 0.059, and latent base [χ 2 M (6) = 6.537, p = 0.366] models, giving first evidence that all three models could fit the observed data; however, the non-linear growth model demonstrated negative variance while also displaying better

TABLE 2 | Correlation coefficients for measures of pup-directed behaviors across litters (1–4) by individual (Female, X; Male, Y).

#### TABLE 3 | Indices of model fit.

relative fit (as assessed by CFI, TLI, RMSEA, and AICi) than either the no-growth and linear models. For a corrected nonlinear growth model, the exact-fit hypothesis is also not rejected [χ 2 M (8) = 5.565, p = 0.585] and the corrected latent base model shows improvement of model fit compared to the no-growth model [χ 2 D (3) = 10.681, p = 0.014] (see **Table 3**, **Figure 3B**). The conditional probability that the corrected non-linear growth model is the best fitting model of the set is 0.912.

### Independent Maternal and Paternal Models With Covariates

For both instances (maternal care and paternal care), the addition of covariates controlling for age and litter size yielded models with exceptionally poor fit. Cluster 2 models yield estimates of intercept and slope that are comparable, yet further from the observed data, to those from models in Cluster 1.

#### Maternal-Paternal Bivariate Model

For analyses of parental care in a corrected bivariate process, in which the trajectory for maternal care is fixed as linear and the trajectory for paternal care is fixed as non-linear, the exact-fit hypothesis is not rejected [χ 2 M (24) = 30.616, p = 0.165]. The result of negative slope variance for paternal care and subsequent correction for the Heywood Case means results for slope covariance between maternal and paternal care are uninterpretable. The covariance of intercepts for maternal and paternal care is −0.057 (p = 0.93) (see **Table 3**, **Figure 3C**).

### DISCUSSION

#### Parenting Changes Over Time

We hypothesized that prairie vole parental care does not vary from one litter to the next. Our results depart from this hypothesis of consistency. Contrary to previous observations that indicated increased parental care in multiparous mothers (Wang and Novak, 1994b), our findings suggest that maternal care is best described as having a negative trajectory, with a decline across litters (see **Figures 3A,C**). Differences in methodology

*Rows in bold indicate the model with best comparative fit (as determined through comparative fit indices) for each observed group [Mothers, Fathers, or the Dyad (Bivariate)]. Models include patterns of no growth, linear growth, non-linear growth, corrected non-linear (CNL) growth and/or a Linear/CNL Mix. Analyses of Cluster 2 are not given; for the mother, father, and dyad, the addition of covariates of litter size and parental age yielded models with exceptionally poor fit/non-converging models. In order from left to right, model fit indices presented are chi-square statistic (X*2 *), degrees of freedom (DF), p-value (P), Comparative Fit Index (CFI), Tucker-Lewis Index (TLI), Root Mean Square Error of Approximation (RMSEA), Akaike's Information Criterion (AICi ),* 1*I (AIC), and Akaike Weights [wi (AIC)].*

FIGURE 3 | (A–C) (A) maternal, (B) paternal, and (C) bivariate dyadic models of pup-directed behavior. In all sub-figures: black lines indicate the plotted means of the sample data; red lines indicated the plotted means of the calculated no-growth model; green lines indicate the plotted means of the calculated linear model; blue and purple lines indicate the plotted means of the uncorrected and corrected latent base models, respectively. In Figure 1C, the black line in the lower half of the figure indicates the means of sample data for fathers, and the black line in the upper half of the figure indicates the means of sample data for mothers. Black error bars represent standard error.

may explain these differences, particularly the use of crosssectional vs. mixed-longitudinal analysis in the former and current studies, respectively, as well as significant differences in sample size. Previous study of parental change and parity in male and female prairie vole parents from litter 1 and 2 (using longitudinal methods) also find a decline in parental care from litters 1 and 2 (Stone and Bales, 2010). Moreover, our findings of decline in maternal care with parental experience aligns more closely with the human literature (Boukydis and Burgess, 1982). The mechanism behind a decline in care for neonates across subsequent litters is not particularly clear. We hypothesize that mothers may become more efficient in their investment with parenting experience; alternatively, reduced sensitivity to pup cues may play some role, as could the effect of aging. Posthoc investigation of the individual behaviors that compose the composite score for PDBs show that decline in maternal care is reflected in time nursing (Supplementary Table 1, Supplementary Figure 3); a similar decline is observed in time spent in the nest (Supplementary Table 1, Supplementary Figure 1), which is not a component of the composite score for PDBs.

Paternal care is best described as having an initial decline from the first to second litter and a subsequent uptake in paternal activity from litter 2 through 4 (see **Figures 3B,C**). The comparison of mothers' and fathers' respective patterns of parental care highlights their opposing trajectories (**Figures 2**, **3C**). A pattern of initial decline followed by an increase in paternal behavior could be explained by competing factors; that is, one responsible for the decline, and another which counteracts the decline. Similar mechanisms behind maternal care decline could be responsible for paternal care decline, including an improved paternal efficiency, reduced sensitivity, or a broad effect of aging. We would then hypothesize that paternal care increases following an initial decline in order to compensate for maternal decline in investment in of their offspring. Post-hoc investigation of paternal care shows that the paternal pattern of decline and subsequent incline is reflected in paternal time in Rogers et al. Trajectories of Biparental Care

nest (Supplementary Table 1, Supplementary Figure 2), which is not a component of the composite score for PDBs. The increase of PDBs appears to be driven largely by an increase in huddling and non-huddling contact (Supplementary Table 1, Supplementary Figures 5, 7, respectively). Other individual parental behaviors are displayed in Supplementary Figures 4, 6, 8–11.

#### Compensatory Fathers

The models of individual change for mothers and fathers fit quite well and suggest opposing trends in parental care between mothers and fathers from litters 2 to 4. The general stability seen in the composite of maternal and paternal behavior (**Figure 2**) may be a consequence of opposing trends, where when mothers decline in maternal care across time, fathers display compensatory behavior to fill an apparent void. We hypothesize that the increase of paternal care could be a response intended to decrease the amount of time pups are left unattended on the nest, or more simply that fathers are aware of unattended pups and act to ameliorate the pups' condition. A potential mechanism by which fathers become aware of unattended pups is through pup ultrasonic vocalization, to which male prairie voles are responsive (Rabon et al., 2001). The mechanism behind paternal motivation to respond to ultrasonic vocalization may be mediated through stress, as pup-vocalizations may be stress inducing and male prairie voles have been demonstrated to increase parental care when stressed (Bales et al., 2006). Moreover, the initial decline and subsequent increase of paternal care parallels previous trends of weight loss and subsequent recovery in fathers across their first 3 l of pups (Campbell et al., 2009). This suggests energetics may play an important role in the patterns of paternal behavior.

One challenge to the hypothesis of independent actors is the observation of "forced baby sitting"—an behavior in which mothers or fathers may push one another back into the nest—, which, while anecdotal, we and others have observed in prairie voles under laboratory conditions (although, not seen in McGuire et al., 2007) and empirically observed in other vole species (e.g., M. socialis guentheri; Libhaber and Eilam, 2002); thus, it is possible that mothers increasingly encourage paternal behavior through forced baby sitting with each successive litter.

### Consequences for Offspring

Responsive fathers may play an important role in stabilizing early life experiences for offspring, particularly offspring of later litters which experience less maternal care. Without the stabilizing influence of fathers, pups in later litters might experience significantly less direct parental care than those of earlier litters. Despite shared genetics, we could then expect differential adult behavioral phenotypes in siblings of different litters if fathers cannot or do not stabilize the parental environment.

In prairie voles, increased parental investment may result in higher survival rate in the laboratory (Wang and Novak, 1994a), although other studies would suggest no such effect in the laboratory setting (Mcguire et al., 1992; McGuire et al., 2007; Wang and Novak, 1992). Under more challenging circumstances, the conferred benefit of paternal care on pup survival may be more pronounced in some biparental species, such as under conditions of cold temperature (in California mice, Gubernick et al., 1993), presence of predators (in prairie voles, Getz et al., 1992), or increased effort to access food (in California mice, Gubernick et al., 1993; Cantoni and Brown, 1997). High contact parenting is also associated with slower development, as indicated through the achievement of developmental milestones of eye opening, leaving the nest, and eating solid food (Perkeybile et al., 2013). Contrasts between high- and low-contact parents also indicate variation in neuroendocrine (Perkeybile and Bales, 2016) and cortical development (Seelke et al., 2016a); for example, pups receiving high tactile contact develop greater intrinsic and more localized connections in the primary somatosensory area than their low-contact counterparts (Seelke et al., 2016b).

Of particular relevance here is the expression of monogamy in adulthood, which has been linked to early life experience of parental care. Experimental manipulations of the family unit during the neonatal period induce increased parental behavior, an effect that resulted in facilitated partner preference formation in manipulated (higher contact) female offspring, but inhibited partner preference formation in non-manipulated (lower-contact) female offspring (Bales et al., 2007). High contact parenting as a result of natural variation also promotes social behavior (e.g., physical contact) when compared to mediumand low-contact parenting (Arias Del Razo and Bales, 2016). These effects are demonstrated to have intergenerational effects on parenting and alloparenting behaviors (Stone and Bales, 2010; Arias Del Razo and Bales, 2016). After paternal deprivation, male and female offspring reared with their mother only show deficits in pair-bond formation, thus indicating that paternal care facilitates partner preference formation (Ahern and Young, 2009).

Another potential stabilizing factor not considered here is the presence of alloparental care from non-dispersed siblings. The presence of fathers and alloparental siblings is associated with decline in maternal care and time spent in the natal nest as well as increased maternal locomotion, eating, and drinking (Wang and Novak, 1992). Pups reared in the presence of a mother, father and alloparents are left unattended less frequently than those reared with only a mother (Solomon, 1991). While well reviewed by Kenkel et al. (2017), more research on the effects of alloparental care on early life development is needed.

### Limitations and Future Directions

A major limitation of this study is the relatively small sample size of 141 dyads (282 individuals), which is compounded by a high rate of missing data. As discussed earlier, the missing data are likely to be missing completely at random, which means that they have little potential to introduce systematic bias into the results. However, missing data translate directly into loss of power and loss of precision in the estimation. Continued data collection, particularly in later litters (e.g., litters 3 and 4) would be particularly beneficial to future analyses. Another benefit of an increased sample size would be the ability to utilize multiple groups models to consider the classification of "high contact" and "low contact" parents—that is, parents who show stability in the extremes of parenting over time. It should also be noted that our analyses do not provide information as to whether observed change from one litter to the next is statistically significant, but rather that one pattern of change fits the observed data significantly better than another. Whether or not these changes in parental behavior translate into significant differences in pup bio-behavioral development is not studied here; however, the hypothesis that parity affects pup bio-behavioral development is testable.

Another major limitation of this study is its having been done with individuals exclusively from a single laboratory colony that had been outbred several generations prior from an ancestral population in natural conditions. The removal of the study population from natural environmental cues could result in patterns specific to laboratory conditions, just as could any laboratory study of this species. However, the amount of control on external conditions (e.g., ad libitum provisioning of food and water, no threat of predation or natural disaster, etc.) aids in our identification of this latent process of change in parental behavior that might otherwise be masked by confounding environmental factors. Finally, it should be noted that the age of the parents observed for this study is significantly older than what would be seen in the wild. It is unlikely that in the wild, pairs would live to produce more than 2 l or to begin parenting at such an advanced age. Nevertheless, given the longevity of voles in the laboratory, these findings are particularly useful for those who study the species in the laboratory setting.

#### CONCLUSIONS

This study provides valuable information for our understanding of the process of parenting in a monogamous species. We find evidence, here, that prairie vole parenting is indeed a process of change, rather than a constant. Our findings support previous findings of sexual dimorphism in the parental behaviors of prairie voles, and our findings extend the conceptualization of sexual dimorphism to patterns of change in parental behaviors across litters. Moreover, we find evidence for a potentially important role of fathers in the maintenance of monogamous behavior in offspring. These results generate new hypotheses that can and should be tested with further research. Thus, we conclude that prairie vole parenting is a dynamic process of two individuals, each with their own potential to dramatically alter trajectories of bio-behavioral development in offspring. Understanding longitudinal and inter-individual dynamics of

#### REFERENCES

complex social behaviors and their development is critical for the informed investigation of both proximate and ultimate mechanisms that may subserve these very behaviors.

#### ETHICS STATEMENT

Protocols for general husbandry and any studies generative of data used here and collected from the standing vole breeding colony were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California, Davis, and complied with National Institutes of Health ethical guidelines as set forth in the Guide for Lab Animal Care.

#### AUTHOR CONTRIBUTIONS

FR designed and executed all aspects of this work under the advisement of MR, EF, and KB. All authors participated in writing and editing of this article.

### FUNDING

This research was supported by funding from NIH HD060117 and NIH HD071998. FR was supported by an Affective Science Training Fellowship from NIH 5T32MH020006-19 at the time this manuscript was written.

#### ACKNOWLEDGMENTS

We would like to thank Anurupa Kar for her contributions in compiling data used in this study, and thanks is due to those individuals who originally collected the data, especially Dr. Allison Perkeybile and Trenton Simmons. Thank you to Dr. Siwei Liu for commentary. We would also like to thank Dr. Cindy Clayton and Dr. Rhonda Oates-O'Brien for veterinary care of the prairie voles, as well as Dr. Adele M. H. Seelke, Jessica Bond, and all undergraduate volunteers in our lab who performed animal husbandry duties.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fevo. 2018.00073/full#supplementary-material

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Rogers, Rhemtulla, Ferrer and Bales. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Dopamine Modulation of Reunion Behavior in Short and Long Term Marmoset Pairs

Sarah B. Carp1 \*, Jack H. Taylor 1 , Stephanie L. Womack 1,2 and Jeffrey A. French1,2

*1 Department of Psychology, Callitrichid Research Center, University of Nebraska at Omaha, Omaha, NE, United States, 2 Department of Biology, Callitrichid Research Center, University of Nebraska at Omaha, Omaha, NE, United States*

One major neurobiological substrate regulating social processes is dopamine (DA). DA is implicated in social behavior in species as diverse as fish and birds, and has an established role in regulating relationships between mates in socially monogamous rodents. Marmoset monkeys display traits associated with social monogamy including high rates of affiliation, biparental care, distress upon separation, and aggression toward strangers; several of these behavioral patterns change throughout the development of relationships. This temporal change may represent changing demands, as pairs are likely to jointly face new experiences (e.g., parenthood) throughout pairing. We investigated the role of DA and pairing length on social behavior during reunion after separation from the mate. Marmosets were removed from their home environment and treated with agonists and antagonists for the D1 and D2 receptor subtypes. They were exposed to a novel environment containing an opposite-sex stranger and their pair mate, and then reunited with their mate in the home enclosure. Marmosets in long term pairs exhibited higher levels of food sharing during reunion than marmosets in short term pairs, with females in long term pairs sharing food more than males; no sex difference was observed in short term pairs. Subjects in short term pairs spent more time grooming their mate than receiving grooming during reunion, while marmosets in long term pairs displayed similar amounts of both initiated and received grooming. DA treatment altered pair-level behavior. When females received either a D2 agonist or antagonist, short term pairs spent less time in proximity, compared to when males received the same treatments. In long term pairs, treatment of females with either a D1 agonist or antagonist resulted in pairs spending less time in social proximity than when males were treated. These findings suggest that the function of the DA system in mate behavior may be similar between rodents and primates, with the D1 system modulating the expression of behavior in long term pairs and the D2 system regulating behavior in short term pairs. Furthermore, these results supplement a large body of work suggestive of deep evolutionary roots of the DA system in regulating social behavior.

Keywords: social monogamy, pairing length, social relationships, formation, mating system

#### Edited by:

*Nancy G. Solomon, Miami University, United States*

#### Reviewed by:

*Zachary V. Johnson, Georgia Institute of Technology, United States Joseph Lonstein, Michigan State University, United States*

> \*Correspondence: *Sarah B. Carp<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Behavioral and Evolutionary Ecology, a section of the journal Frontiers in Ecology and Evolution*

> Received: *15 January 2018* Accepted: *04 April 2018* Published: *09 May 2018*

#### Citation:

*Carp SB, Taylor JH, Womack SL and French JA (2018) Dopamine Modulation of Reunion Behavior in Short and Long Term Marmoset Pairs. Front. Ecol. Evol. 6:46. doi: 10.3389/fevo.2018.00046*

## INTRODUCTION

A close, selective, though not necessarily exclusive, social and sexual relationship with a single partner is a hallmark of social monogamy. There are several behavioral components of social monogamy, all of which reference this relationship between partners. Within the construct of social monogamy there is variability both within, and especially among, species in the specific social and behavioral components that are displayed (Díaz-Muñoz and Bales, 2015; Tecot et al., 2016). In nonhuman primates, social monogamy is associated with a host of behavioral traits, including a selective social preference for the mate over other conspecifics, biparental care, and aggression toward sameor opposite-sex conspecifics (French et al., 2017). Of particular interest are several phenomena associated with separation of the mating pair, which include distress upon separation from the mate, social buffering of stressful experiences while in the presence of the mate, and high rates of affiliative behavior upon reunion with the mate after a period of social separation (French et al., 2017).

Marmoset monkeys (Callithrix spp.) display many characteristics associated with social monogamy. These features include social buffering of stress responses by the presence of the pair mate (Smith et al., 1998; Rukstalis and French, 2005; Cavanaugh et al., 2015), affiliation toward the mate (Ågmo et al., 2012), biparental care (Snowdon, 1996; Ziegler et al., 2009), and aggression toward same-sex conspecifics (Evans, 1983; Ross et al., 2004; Ross and French, 2011). However, marmosets do not display a strong selective social preference for their mate in standard partner preference tests (Smith et al., 2010; Cavanaugh et al., 2014), and they also demonstrate high levels of social and sexual interest in opposite-sex strangers (Cavanaugh et al., 2014; Mustoe et al., 2015). In addition, there is variability in group demography in wild marmosets, ranging from single pair and offspring to multimale/multifemale groups (Digby, 1999; Sousa et al., 2005). Thus, marmosets demonstrate traits that are associated with social monogamy, as well as flexible and conditional social responses in ways that resemble the complexities of human "social monogamy" (Chapais, 2013).

One behavioral trait that may be particularly important to the stability of a mating pair is the degree to which pairs re-establish their relationship during reunion after a period of separation, stress, or disruption. Separation from social partners is often accompanied by behavioral signs of distress, and increased glucocorticoid concentrations (Smith et al., 1998; DeVries et al., 2003; Hennessy et al., 2009; French et al., 2012; Ziegler and Crockford, 2017). Monogamous male titi monkeys demonstrate differential brain activation in regions associated with the regulation of social behavior (limbic and striatal) during separation from a mate, and release of the neuropeptide oxytocin upon reunion with the mate (Hinde et al., 2016), indicating physiological responses to both separation from, and reunion with, a mate. In marmosets, separation from a mating partner and short- or long-term exposure to a novel environment is associated with elevated hormonal stress responses (cortisol increases) (Smith et al., 1998; Cavanaugh et al., 2016). Furthermore, levels of affiliation are higher between mates after a separation, compared to pre-separation levels, suggesting that elevated rates of affiliative behavior upon reunion in marmosets is an important mechanism for down-regulating the negative affect and stress response associated with partner separation (Shepherd and French, 1999; Cavanaugh et al., 2018). High rates of affiliative behavior upon reunion among other classes of social relationships in marmosets is also associated with regulation of the arousal associated with separation from close social contacts. Juvenile marmosets that engage in high levels of affiliation during reunion after social isolation demonstrate a faster return to baseline glucocorticoid levels (Taylor et al., 2015), highlighting the importance of reunion behavior across social contexts in marmosets.

The notion that reunion behavior modulates stress responses to separation is supported by the observation that marmosets experiencing a stressor with their partner exhibit no difference in affiliative behavior upon reunion from pre-stressor levels (Smith et al., 1998). This suggests that in marmosets when a mate is present during a stressor, behavioral and physiological responses during the stressor are reduced (e.g., social buffering), and the need to downregulate stress upon reunion in the home enclosure is hence less important (Cavanaugh et al., 2016). The duration of separation also shapes the nature of affiliative interactions upon reunion. Marmoset pairs separated for an extended period of time (7 days) showed elevated pair-directed affiliation upon reunion, while short separations (5–15 min) did not increase rates of affiliative behavior (Duarte et al., 2017), suggesting that longer separations require larger behavioral responses during reunion, presumably to aid in re-establishment of the social relationship. Together, these data suggest that high rates of affiliative interactions with significant social partners after a period of separation has important consequences for regulating the biobehavioral responses associated with social separation. Thus, reunion after separation represents a time at which individuals in the pair are engaging in behavior to reduce behavioral and endocrine components of the stress response and to re-establish social relationships.

The expression of reunion behavior with a pair mate may be dependent on the phase of the relationship. The underlying relationship between mating partners in socially monogamous rodents has been delineated into two phases: formation and maintenance. Each of these phases is associated with distinct behavioral traits, as well as neurobiology, in rodent models of social monogamy (i.e., prairie voles) (Young and Wang, 2004; Curtis et al., 2006; Lim and Young, 2006; Young et al., 2008, 2011; Aragona and Wang, 2009). Social preference for the mate is a key marker of pair bond formation, and in voles is observed after 24 h of cohabitation (Williams et al., 1992), while selective aggression toward conspecifics is considered a marker of pair bond maintenance and is tied to onset of mating with a mate (Carter et al., 1995). While the phases of pair formation and maintenance in prairie voles appear to follow a strict and short timeline, primates tend to show longer and more variable transitions in relationships (Maninger et al., 2017). Patterns of behavior between mates in marmosets shift across time, including levels of sexual behavior (typically, high in the beginning and lower with increased length of pairing) and affiliative social behavior across pairing (typically, lower in the beginning and increasing with pairing length) (Schaffner et al., 1995; Ågmo et al., 2012; c.f. Evans and Poole, 1984). Thus, while there are differences in affiliative behavior in marmosets between short and long term pairs, there is no clear behavioral marker of an "established" pair. Furthermore, social monogamy is theorized to have evolved from an environment in which females were highly dispersed and males experienced fitness benefits from guarding and maintaining a relationship with a single female (Lukas and Clutton-Brock, 2013). Thus, male and female mates experience a suite of different evolutionary pressures in terms of mating success. While males may benefit from engaging in as many sexual encounters as possible given the minimal cost associated with sperm production, they also benefit from continuous presence near their female mates (e.g., mate guarding, proximity maintenance) to reduce the risk of her engaging in extra pair copulations. Especially in species that exhibit paternal care, it is critically important, from an energetic perspective, that males raise offspring genetically related to them. It is therefore likely that males and females will display different behavioral profiles from one another at both initial and later stages of a relationship.

The neurotransmitter dopamine (DA) has been identified as a key player in decision making and social behavior in species as diverse as invertebrate leeches, fish, birds, and mammals (O'Connell and Hofmann, 2011). Of particular note is the role that DA plays in the formation and maintenance of bonds between mating partners in rodents. DA is a neurobiological regulator of the reward system, and its role in associative learning may facilitate its involvement in social behavior (Aragona et al., 2003; Curtis et al., 2006; Brom et al., 2014). The DA system has five receptor types that can be divided into two subfamilies of receptors: D1-like (D1 and D5), and D2-like (D2, D3, and D4). These subtypes differentially regulate bond formation and maintenance in prairie vole pairs (Curtis et al., 2006; Aragona and Wang, 2009): activation of the D2 system facilitates selective social preferences and thereby formation (Wang et al., 1999; Gingrich et al., 2000; Aragona et al., 2003, 2006; Edwards and Self, 2006) and activation of the D1 system regulates selective social aggression associated with bond maintenance (Aragona et al., 2006). In prairie voles, bond formation is accompanied by an upregulation of D1 receptors in the nucleus accumbens, a brain region central to the reward system (Aragona et al., 2006). To date, there has not been a systematic assessment of the role of the D1 and D2 system in regulating the formation and maintenance of attachments in socially monogamous nonhuman primates.

There is, however, some evidence for the importance of the DA system in mediating social relationships in primates. D1 receptor binding in monogamous male titi monkeys is increased after pairing with a female in the lateral septum, a brain region associated with motivation, reward, and reinforcement (Hostetler et al., 2016), suggesting that pairing with a social partner may alter the expression of D1 receptors. Genetic variation in a DA receptor in humans is associated with variability in measures of fidelity and sexual promiscuity (Garcia et al., 2010), and brain regions associated with DA show increased activation in human males treated with oxytocin in response to viewing images of a romantic partner (Scheele et al., 2013), indicating that other neural systems may be working with, or through, the DA system to induce social effects. General cooperative behaviors even outside of a pairing context in humans appears to be mediated through activation of brain regions rich in DA and involved in reward processing (Rilling et al., 2002). Furthermore, blocking either D1 or D2 receptor types in macaques, a polygynous primate, reduced attention toward a social stimulus (Yamaguchi et al., 2017), indicating that both the D1 and D2 systems function in assessing social stimuli in nonhuman primates. While there is evidence indicating that the DA system influences social behavior in primates, it is unclear to what extent the DA system has a conserved role in regulating relationships in socially monogamous primates in relatively new vs. well-established pairs.

The current study assessed the ways in which reunion behavior after separation in marmosets was influenced by manipulation of the D1 and D2 signaling systems, and whether the effects of DA manipulation differed as a function of the length of the social relationship between pair mates. Marmoset pairs cohabiting for 8 weeks (short-term) or 3 years (longterm) were physically separated from pair mates and housed in a novel environment for 60–75 min, and affiliative behavior upon reunion was quantified. D1 and D2 receptor activation was pharmacologically manipulated with selective receptor agonists and antagonists. If the role of the DA system in relationship dynamics in marmosets is similar to voles, then we expected that manipulation of the D2 system would alter reunion behavior in short term, but not long term pairs, while manipulation of the D1 system would alter reunion behavior in long term, but not short term pairs.

#### MATERIALS AND METHODS

#### Subjects

A total of 20 marmosets (Callithrix jacchus) were used in this study. Subjects included animals in short term (n = 7 pairs, average pair length = 8.76 weeks, SD = 3.07 weeks), and long term (n = 5 pairs, average pairing length = 3.07 years, SD = 1.25 years) pairs. None of the pairs had parental experience with their current mate, and one female in the study had previous parental experience. Four animals were repaired and studied both in a short term and a long term pair context, but the remaining subjects were studied in only one pairing context. Both members of a pair served as subjects on different days of testing, with a minimum of 3 days between tests as a treated focal animal. Marmosets were housed at the Callitrichid Research Center at the University of Nebraska at Omaha. Animals were housed in enclosures with minimum dimensions of 101 × 76 × 160 cm.

Marmosets received a daily diet of a prepared commercial marmoset food (Zupreem R ) supplemented by fresh fruits, vegetables, yogurt, apple sauce, eggs, and mealworms. The production of offspring was prevented by either surgical vasectomy of the male or monthly treatment of the female with the luteolytic agent Estrumate R (Merck). Hormonal states of the subjects are not anticipated to be highly impacted by these procedures and treatments. Surgical vasectomy is not known to reduce testosterone levels, and estrumate produces a normative nonconceptive ovarian cycle, with post-ovulatory progesterone levels equivalent to those of untreated females (Hodges et al., 1988; Mustoe et al., 2012). Additional information regarding animal care can be found in Schaffner et al. (1995). This study was carried out in accordance with the PHS Policy on the Humane Care and Use of Laboratory Animals. The protocol was approved by the University of Nebraska Medical Center/University of Nebraska at Omaha Institutional Animal Care and Use Committee (protocol # 15-033-05-FC).

#### Drug Treatments

Subjects were treated with one of five treatments: D1 agonist (SKF 38393, 0.05 mg/kg), D1 antagonist (SCH 23390, 0.01 mg/kg), D2 agonist (Quinpirole, 0.05 mg/kg), D2 antagonist (Raclopride 0.03 mg/kg), or saline vehicle. Treatment order was counterbalanced among subjects. Thus, each marmoset received five treatments and served as an untreated pair mate five times for a total of 10 exposures to the testing paradigm. Treatment doses were selected based on a systematic dose-response study conducted in our lab that identified doses at which motoric side effects were not observed (Carp, unpublished data). Injections were given intramuscularly in a volume of 0.5 mL/kg.  Both raclopride (Farde et al., 1986) and SCH 23390 (Hostetler et al., 2016) are regularly utilized in PET imaging studies. Quinpirole is able to cross the blood brain barrier (Kostrzewa et al., 1993). It is unclear how well SKF 38393 penetrates the blood brain barrier (Kamien and Woolverton, 1985), however, intramuscular administration does alter neuronal activity (Boraud et al., 2001). Therefore, peripheral administration of all compounds are anticipated to produce effects through central activation. All treatments were purchased from Sigma Aldrich and prepared in sterile saline and kept frozen at −20◦C until day of treatment. As per recommendations from the manufacturer, D1 and D2 antagonist treatments were reconstituted from stock every 30 days, while the D1 and D2 agonist treatment solutions were reconstituted every 90 days.

### Reunion Observations

Subjects were removed from their home enclosure and administered a treatment, housed alone in a small transport cage in an isolated room for 30 min during the drug uptake period, and then placed in a novel T-shaped enclosure in a separate room for 30–45 min. During the time in the T-enclosure, the marmoset had simultaneous visual, auditory, olfactory, and limited tactile access to their untreated mate and to an untreated unfamiliar opposite-sex marmoset in stimulus cages at each end of the T-portion of the cage [for more details on the preference testing apparatus, see (Cavanaugh et al., 2014) and (Smith et al., 2010)]. The T maze is a novel environment for subjects, and simultaneous separation from a mate, and exposure to a novel environment results in a reliable stress response in marmosets (Smith et al., 1998). After this procedure, the treated marmoset and mate were reunited in their home enclosure. Interactions between males and females were recorded for 10 min (longterm pairs) or 15 min (short-term pairs), and included rates of food sharing, approaches to partner, instances of initiating TABLE 1 | Ethogram of marmoset (*Callithrix jacchus*) behavior recorded during reunion observations.

*a Indicates the behavior was recorded for number of occurrences.*

*b Indicates the duration of the behavior was recorded.*

and receiving grooming, and durations of grooming and time spent in social proximity. Definitions for the behavioral patterns are found in **Table 1**. Duration of separation and reunion were different because data were collected for two projects, one with short term and one with long term pairs. To correct for the different observation lengths, all behaviors were converted to duration or count per hour of observation for data analysis purposes. This correction assumes that the rates of recorded behavior did not vary across the observation period.

### Data Analysis

To assess the effects of treatment, sex, and pair type (short term or long term) we used a Linear Mixed Model analysis that nested marmoset ID within Pair ID. This nesting allowed us to account for the non-independence of marmosets being tested in both the short term and long term pairing context. Because age at testing varied between social conditions, this measure was included as a covariate. For behavior that could be exhibited by either the treated marmoset or the untreated mate, factors were added to include behavior initiated and received. As such, our template model is as follows: Behavior = **DA Treatment** × **Sex** × **Pair Type** × Initiate/Receive + Age + error(Pair ID) + error(Subject ID) + error(residual), with those factors in bold as the tests of our hypotheses, and those in italics added when grooming behavior was analyzed. We calculated a Hinde index for approach and leave behavior of subjects using the following equation: [Number of Subject Approaches/(Number of Subject Approaches + Number of Pair Mate Approaches)]–[Number of Subject Leaves/(Number of Subject Leaves + Number of Pair Mate Leaves)]. This index allows for a simultaneous measure of responsibility of both initiation (approach) of proximity and breaking proximity (leave) by the subject. Post hoc probing was conducted only if significant main effects or interactions were obtained. Statistical tests were conducted with Fisher's tests and a Satterthwaite approximation for degrees of freedom. Cohen's d effect sizes for post hoc Fisher's t-tests were calculated using model estimated marginal means and standard errors, and

standard deviations were calculated from standard errors using Satterthwaite approximated degrees of freedom +1 to estimate n. ANOVA tables for all reported analyses can be found in Supplementary Tables S1–S4.

### RESULTS

### Effects of Length of Relationship on Reunion Behavior

#### Social Proximity

After a social stressor involving partner separation and exposure to an opposite-sex stranger, marmosets in short and long term pairs behaved differentially upon reunion. Marmosets in both short and long term pairs spent similar amounts of time in proximity during reunion, **Figure 1**; main effect: F(1, 14.11) = 2.69, p = 0.123, but differed in the specific behavior patterns associated with close spatial proximity.

#### Food Sharing

Marmosets in long term pairs engaged in higher levels of food sharing upon reunion than did marmosets in short term pairs, **Figure 2A**; main effect: F(1, 14.90) = 6.17, p = 0.025. There was also a sex difference observed in long term, but not short term, pairs of marmosets in frequency of food sharing, interaction effect: F(1, 11.98) = 5.84, p = 0.032. In long term pairs of marmosets, females engaged in higher levels of food sharing during reunion than did male marmosets, **Figure 2B**; post hoc: t(12.1) = 2.59, p = 0.024, 95% CI [0.009, 0.103], d = 0.695. However, in short term pairs of marmosets females and males displayed no difference in their rate of food sharing, **Figure 2B**; post hoc: t(12.2) = −0.66, p = 0.519, 95% CI [−0.052, 0.028], d = −0.181.

#### Grooming

Although overall duration of grooming did not differ in reunion between short term and long term pairs, **Figure 3A**; main effect:

FIGURE 2 | Food sharing (model estimates ± SEM) among short and long term marmoset pairs. (A) Long term marmoset pairs engaged in more food sharing during reunion than did short term marmoset pairs. (B) Females in long term pairs engaged in more food sharing during reunion than did males in long term pairs. There was no significant difference in food sharing between males and females in short term pairs. \*Indicates significant difference at *p* < 0.05.

F(1, 14.798) = 0.33, p = 0.576, the duration of grooming initiated by subjects or mates differed by pair length, interaction effect: F(1, 215.99) = 4.87, p = 0.028.  However, treated subjects and their mates in long term pairs displayed no difference in the duration of grooming during reunion after separation, **Figure 3B**; post hoc: t(216) = −0.59, p = 0.556, 95% CI [−0.625, 0.337], d = −0.095.

### Effects of Dopamine Treatment on Reunion Behavior

#### Dopamine Effects in Short Term Pairs

During reunion, marmoset pairs spent similar amounts of time in proximity regardless of DA treatment, main effect: F(4, 96.00) = 1.91, p = 0.114. However, time in proximity differed between short and long term pairs based on DA treatment and whether the male or female received the treatment, interaction effect: F(4, 96.00) = 2.50, p = 0.047. During reunion, male and female marmosets in short term pairs spent similar amounts of time in proximity when they received a saline treatment, **Figure 4A**;

post hoc: t(70.70) = −0.29, p = 0.772, 95% CI [−11.471, 8.556], d = −0.047. However, marmosets in short term pairs spent less time in proximity during reunion when females were treated with either a D2 receptor agonist, **Figure 4A**; post hoc: t(70.70) = −2.04, p = 0.045, 95% CI [−20.237, −0.213], d = −0.333, or a D2 receptor antagonist, **Figure 4A**; post hoc: t(70.70) = −2.18, p = 0.032, 95% CI [−20.973, −0.946], d = −0.356, compared to when males received the same treatment. Additionally, pairs spent less time in proximity when females were treated with a D2 receptor agonist compared to when females were treated with saline, post hoc: t(96.0) = −2.29, p = 0.024, 95% CI [−19.530, −1.399], d = −0.336. No other treatments were significantly different from saline. Short term pairs spent similar amounts of time in proximity when either males or females were treated with a D1 agonist, **Figure 4A**; post hoc: t(70.8) = 0.48, p = 0.634, 95% CI [−7.607, 12.407], d = 0.078, or D1 antagonist, **Figure 4A**; post hoc: t(70.8) = −0.37, p = 0.716, 95% CI [−11.842, 8.175], d = −0.060.

#### Dopamine Effects in Long Term Pairs

A different pattern of treatment effects was observed in long term pairs. There was no sex difference in time spent in proximity in long term pairs when males and females were treated with saline,

FIGURE 4 | Duration in proximity (model estimates ± SEM) to the opposite sex partner during reunion. (A) Short term marmoset pairs spent less time in proximity during reunion when the female was treated with a dopamine receptor subtype 2 agonist or antagonist compared to when the male was treated with the same compound. Duration in proximity during reunion did not differ when males or females in short term pairs were treated with a dopamine receptor subtype 1 agonist or antagonist or saline. Bars marked with N denote significant within sex differences from saline. Pairs spent less time in proximity during reunion when females were treated with a D2 agonist compared to when females received saline. There were no significant effects within males compared to saline. (B) Long term marmoset pairs spent less time in proximity during reunion when the female was treated with a dopamine receptor subtype 1 agonist or antagonist compared to when the male was treated with the same compound. Duration in proximity during reunion did not differ when males or females in long term pairs were treated with a dopamine receptor subtype 2 agonist or antagonist or saline (significant sex × pair type × treatment interaction). There were no significant within sex treatment effects for either males or females. \*Indicates significant difference at *p* < 0.05. D1 Ant = D1 antagonist, D1 Ag = D1 agonist, D2 Ant = D2 antagonist, D2 Ag = D2 agonist.

**Figure 4B**; post hoc: t(71.1) = −0.83, p = 0.412, 95% CI [−16.704, 6.925], d = −0.134. However, male and female marmosets in long term pairs spent less time in proximity when females received treatment with either a D1 agonist, **Figure 4B**; post hoc: t(71.00) = −2.23, p = 0.029, 95% CI [−25.061, −1.415], d = −0.365, or D1 antagonist, **Figure 4B**; t(71.00) = −2.03, p = 0.046, 95% CI [−23.880, −0.238], d = −0.329, compared to when males received the same treatment. Long term marmoset pairs spent similar amounts of time in proximity upon reunion when males and females were treated with a D2 agonist, **Figure 4B**; post hoc: t(71.00) = 0.19, p = 0.846, 95% CI [−10.663, 12.971], d = 0.031, or D2 antagonist, **Figure 4B**; post hoc: t(71.00) = −0.81, p = 0.421, 95% CI [−16.615, 7.028], d = −0.131.

#### Responsibility for Proximity Maintenance

The Hinde index revealed no effect of DA treatment on the rate at which treated subjects were initiating and breaking proximity with their mates, main effect: F(4, 96.00) = 0.88, p = 0.477, suggesting that the observed differences in proximity behavior were not attributable to changes in approach or leave behavior by the subject.

### DISCUSSION

Reunion in marmoset pairs constitutes an important time for assessing affiliative behavior. We noted several differences in the ways in which pairs housed together for differing lengths of time interact upon reunion. Marmosets in long term pairs engaged in higher levels of food sharing than did marmosets in short term pairs. Females in long term pairs demonstrated higher rates of food sharing than their mates, while males and females shared food at equal rates in short term pairs. It has been hypothesized that in social settings in which females have multiple mating partner options, males should share food with females at high rates in order to enhance the chance for mating opportunities (Jaeggi and Van Schaik, 2011). However, in our study females in long term pairs were observed to share food more often than males. Though the social context in the current study differs from that in Jaeggi and Van Schaik (2011), females were reunited with their mate after exposure to a strange male. Presumably, males are cognizant of the odors/sounds of the other marmoset in the testing room and therefore may be more likely to behave during reunion as if their female mate had additional mating opportunities. In a comparative analysis of food sharing in primate species whose groups are composed of a single adult male, high rates of male-to-female food sharing predominantly occurs in socially monogamous species (Jaeggi and Van Schaik, 2011), however, that study did not measure female-to-male instances of food sharing. Food sharing is a behavior that is sensitive to the context of the pair (i.e., pregnancy). Monogamous male and female owl monkeys share food at equal rates, however, male-to-female food sharing increases when females are lactating compared to when they are cycling or pregnant, while female-tomale food sharing occurs at equal rates regardless of reproductive status (Wolovich et al., 2010). Though male-to-female food sharing may facilitate reduced energy expenditure by females and shortened interbirth interval (Wolovich et al., 2010), underlying proximate and ultimate causes of female-to-male food sharing are less clear.

Marmosets in short term pairs displayed a different pattern of grooming compared to that observed in long term pairs. Grooming behavior was altered such that rather than initiating and receiving comparable levels of grooming, as in long term pairs, treated marmosets in short term pairs spent more time grooming their partners upon reunion than their partner spent grooming them. This difference in behavior may indicate that while in long term pairs the context of the separation (e.g., experience as the treated subject vs experience as the untreated mate) does not change the expression of grooming, members of short term pairs are sensitive to the experimental context. Thus, in short term pairs, treated subjects engaged in longer duration of grooming than did untreated mates during reunion, thereby displaying a behavioral difference dependent on experimental experience. In prairie voles, the opposite effect is found: separated voles receive, rather than initiate, higher rates of grooming from their partners (Burkett et al., 2016). These contrasts point to potentially important species differences in the roles of initiated vs. received sociality upon reunion in regulating and reestablishing relationships after separation.

There was no difference in the total amount of time spent in proximity between short term and long term pairs. Though there are documented differences in normative levels of affiliation as measured by sexual behavior, time in proximity, and overall time spent grooming in marmoset pairs dependent on length of pairing (Evans and Poole, 1984; Schaffner et al., 1995; Ågmo et al., 2012), we did not find these differences expressed during reunion. Thus, while marmosets in short and long term pairs may use slightly different strategies to reestablish their relationship upon reunion (e.g., initiation of grooming and rates of food sharing), they spent similar amounts of time in proximity. The pattern of change in behavior dependent on the actions of both members of the pair is largely consistent across findings in the current study. Both food sharing and grooming (behaviors that differed between short and long term pairs in reunion) can only occur when both members of the pair engage in the appropriate dyadic interaction. This dependence on both partners highlights the complexities of studying pair-level interactions and the behavioral richness of dyads.

The DA system appears to be involved in the way that pairs behave during reunion, with DA treatment affecting pair-level interactions. Marmoset pairs, of both short and long term, spent similar amounts of time in proximity upon reunion under saline conditions regardless of whether the male or female was treated. However, alteration of the D2 system, either through receptor agonism or antagonism, altered this pattern in short term pairs, with pairs spending less time in proximity when females received treatment compared to when males received the same treatment. D1 treatments produced different effects in long term pairs, such that pairs spent less time in proximity when the female was treated with either a D1 receptor agonist or antagonist compared to when males received the same treatment. Furthermore, short term pairs spent less time in proximity when females were treated with a D2 agonist compared to when females were treated with saline, indicating that the sex difference observed between males and females was likely due to a decrease in proximity when females were treated. Though other treatments did not produce significant differences from saline, this suggests that at least in short term pairs, treatment of females may yield differences in the way in which pairs regulate social proximity. Male marmosets increase proximity regulation as their mate progresses through pregnancy (Evans and Poole, 1984), suggesting that pairs may be primed to be sensitive to alterations in female physiology, and our data suggest that DA signaling in females may be included in changes in female physiology that alter pair social dynamics. DA, particularly D2 agonists, also reduce prolactin release in marmoset monkeys (Almond et al., 2006), indicating the potential for DA treatments to have off-target effects on other hormonal systems. Given the known role of prolactin (PRL) in facilitating parental behavior (Ziegler et al., 2009) and parent-infant bonding, it is likely that DA-PRL interactions may have meaningful implications for behavior between mates. This may provide another mechanism through which marmoset females are more sensitive to manipulation of the DA system than males. While overall proximity duration was affected by both DA treatment and sex, other measures, such as initiating and breaking proximity did not differ among DA treatment conditions. Future research should evaluate aspects of individual behavior that may underlie the observed differences in pairlevel behavior. In the current study individual initiation and breaking of proximity (the Hinde index) was not able to explain observed differences of time spent in proximity. However, other measures of social interest, such as social gaze, a measure known to be important in directing human social behavior (Frischen et al., 2007), may help to illuminate the behavioral mechanisms through which pairs are altering interactions in response to DA treatment.

It is also worth noting that the same pattern of change in social proximity to the partner during reunion is observed when marmosets are treated with either the agonist or antagonist, suggesting that pairs may be responding to an alteration of DA signaling rather than enhancement or inhibition of the system. The similarity in agonist and antagonist effects stands in contrast to research in other species indicating differential effects of agonist and antagonist treatment. One potential explanation for the similarity in effect is differential efficacy of some of the compounds. The D1 receptor agonist (SKF 38393) has been documented to have a lower efficacy in primate than in rodent brain tissue, and in both rodents and primates the efficacy of the agonist is lower than that of DA itself (Arnt et al., 1988; Pifl et al., 1991). Additionally, there is evidence of a U shaped dose-response curve for agonists of the D1 system (Cai and Arnsten, 1997), indicating the potential that observed effects are dose-dependent. Therefore, at least within the D1 system, it is possible that the effects of the agonist and antagonist are both producing effects less potent than if DA were acting alone.

The consistency of findings across pair types (D2 manipulation altering behavior in short term pairs, and D1 manipulation altering behavior in long term pairs) indicates that there may be dynamic shifts in behavior that affect the dyadic nature of pair interactions dependent on pairing length and DA subsystem. Research in prairie voles has indicated a role for the D2 system in measures of relationship formation (Gingrich et al., 2000; Aragona et al., 2003, 2006), and the D1 system in measures of relationship maintenance (Aragona et al., 2006). Thus, it is interesting to note that social proximity in short term pairs of marmosets is changed by manipulation of the D2 system, and social proximity in long term pairs of marmosets is changed by manipulation of the D1 system, as would be predicted if the DA system has a conserved role in pair behavior during reunion.

DA itself is an evolutionarily conserved neurotransmitter with widespread effects on regulating behavior. Though commonly recognized as having a role in motoric function, DA also has known roles in decision making and social behavior (O'Connell and Hofmann, 2011). DA receptor distribution has been characterized in cichlid fish and D1 and D2 receptors are found in regions homologous to those associated with social behavior in other vertebrate species (O'Connell et al., 2011). The effect of DA on mate behavior is not limited to mammals. DA has been associated with male courtship and pairing behavior in monogamous zebra finch birds (Huang and Hessler, 2008; Goodson et al., 2009), and higher levels of DA and its metabolites are found in paired than unpaired zebra finches (Banerjee et al., 2013). Furthermore, immediately early gene markers indicated increased neuronal activity of dopamine rich brain regions after pairing compared to unpaired finches (Banerjee et al., 2013). Together, these studies highlight the conserved nature of DA in facilitating both social behavior generally, and regulating interactions between mates.

There is further evidence for the role of DA in primate pair interactions from studies on humans. Subjects looking at pictures of a partner with whom they considered themselves to be in love showed increased activation of brain regions rich in DA, compared to looking at pictures of an acquaintance (Fisher et al., 2005). This indicates that the DA system may be selectively important in romantic attachments rather than overall sociality. Additionally, genetic variability in the DA system has been linked to differences in human sexual behavior.  Though DA has a role in modulating sociosexual relationships, it is not the only regulatory neurotransmitter that impacts sociality in mammals. Other neural systems, especially oxytocin and vasopressin, interact with dopaminergic signaling to facilitate social behavior (Johnson and Young, 2015). Thus, research on the specific roles of the D1 and D2 systems in primate pair behavior is necessary in order to inform not only the independent role of the dopaminergic system, but the potential co-modulatory effects with other neural systems. Specifically, the current study indicates that DA subsystems do maintain a role in regulating reunion behavior in a primate species displaying social monogamy. These findings fit into the larger emerging evolutionary notion that there are neurobiological systems, including those involving dopamine signaling, conserved across vertebrate species that may play crucial roles in regulating sociality.

### AUTHOR CONTRIBUTIONS

SC: designed the study, carried out experiments, organized the dataset, and wrote the first draft of the manuscript; JF and SC: obtained funding for the project; JT and SC: performed statistical analyses and interpreted analyses; JT, SW, and JF: contributed to and revised the manuscript. All authors read, revised and approved the final version of the submitted manuscript.

#### FUNDING

The research in this manuscript was supported by the National Institutes of Health (HD089147 awarded to JF) and the University of Nebraska at Omaha's Graduate Research and Creative Activity Award (awarded to SC, title: The Role of Social Context and Dopamine in Partner Fidelity in Marmoset Monkeys).

#### ACKNOWLEDGMENTS

We would like to thank members of the French lab for their comments on study design and feedback during manuscript

#### REFERENCES

preparation. We would also like to thank Heather Jensen, Haley Hassenstab, Jacqueline Muellner, and Drs. Liz Gunkelman and Noel Johnson for providing excellent care of the marmoset colony. We greatly appreciate research assistance by the following volunteers: Kathy Barnett, Haley Classe, Brianna Martinie, Amber Park, Mariah Wulf, and Colton Wulfekuhl.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fevo. 2018.00046/full#supplementary-material

Ziegler, T. E., Prudom, S. L., Zahed, S. R., Parlow, A. F., and Wegner, F. (2009). Prolactin's mediative role in male parenting in parentally experienced marmosets (Callithrix jacchus). Horm. Behav. 56, 436–443. doi: 10.1016/j.yhbeh.2009.07.012

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Carp, Taylor, Womack and French. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Why Monogamy? A Review of Potential Ultimate Drivers

#### Hope Klug\*

Department of Biology, Geology, and Environmental Science, University of Tennessee at Chattanooga, Chattanooga, TN, United States

The existence of monogamy in animals is perplexing from an evolutionary perspective. If individuals: (1) have the opportunity to mate with more than one individual and (2) doing so provides fitness benefits (e.g., indirect benefits, increased mating success or fecundity), why does monogamy ever occur in animals? To address this question, we must examine how the potential benefits and costs of monogamy differ between the sexes and how such costs and benefits interact with factors including resource availability, offspring need, parental care, and mating dynamics (i.e., the costs and benefits associated with acquiring mates and mate availability). In this review, I examine the interplay between parental and offspring dynamics, resource availability and mate distribution, and mating dynamics. In doing so, I highlight the life history and ecological conditions under which monogamy is expected vs. not. I then discuss areas of research that are needed to enhance our evolutionary understanding of monogamy. In particular, enhanced understanding of monogamy will come from: (1) more explicit consideration that the factors that lead to the origin of monogamy vs. the maintenance of monogamy might differ and (2) identifying how potential interactions among factors influence the origin and/or maintenance of monogamy.

#### Edited by:

Alexander G. Ophir, Cornell University, United States

#### Reviewed by:

Ivar Folstad, UiT The Arctic University of Norway, Norway Andreas Walzer, Universität für Bodenkultur Wien, Austria

> \*Correspondence: Hope Klug<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Behavioral and Evolutionary Ecology, a section of the journal Frontiers in Ecology and Evolution

> Received: 11 January 2018 Accepted: 13 March 2018 Published: 29 March 2018

#### Citation:

Klug H (2018) Why Monogamy? A Review of Potential Ultimate Drivers. Front. Ecol.  For example, Bateman (1948) found that male reproductive success is often limited by the number of female mates that a male acquires; as such, males are predicted to increase their lifetime reproductive success by mating with multiple females (Bateman, 1948; Jones and Ratterman, 2009). The expectation that male reproductive success is highly dependent on male mating success has been used to explain male multiple mating in a range of taxonomic groups (Jones and Ratterman, 2009). Likewise, females frequently benefit from mating with multiple males. For instance, a meta-analysis in insects revealed that multiple mating provides direct benefits to females in terms of increased egg production and fertility (Arnqvist and Nilsson, 2000). Similarly, extra-pair paternity, which is a proxy for multiple mating, was associated with higher hatching success, and hence greater female reproductive success, across 113 bird species (Reding, 2015). Female multiple mating can also in some, but not all, cases be associated with infanticide avoidance (Hrdy, 1979) or indirect benefits (Blomqvist et al., 2002; García-González and Simmons, 2005).

Given that multiple mating is often associated with fitness benefits, the existence of monogamy is perplexing from an evolutionary perspective. Indeed, if individuals: (1) have the opportunity to mate multiply and (2) doing so provides fitness benefits (e.g., increased reproductive success; see also Thonhauser et al., 2014; for review of relevant hypotheses), why does monogamy ever occur in animals? To address this question from an ultimate perspective, we must consider whether multiple mating is always possible and explore the costs and benefits of multiple mating. If multiple mating is possible, monogamy would most generally be expected to occur when there are no benefits of multiple mating or when there are costs of multiple mating. Understanding how monogamy can be beneficial to individuals, and hence, persist in populations requires that we consider: (1) how the benefits and costs of monogamy differ between the sexes and (2) how such costs and benefits interact with factors such as resource availability, offspring need, parental care, and mating dynamics.

In this review, I focus on the ultimate causes of monogamy in animals. I first discuss operational definitions and classic hypotheses of monogamy; I then discuss more recent work that has focused on the evolutionary interplay between parental and offspring dynamics, resource availability, and mating dynamics.

### MONOGAMY: WHAT'S IN A NAME AND WHAT DO WE SEE IN NATURE?

Monogamy occurs when individuals have one mate for their entire lifetime or some specified period of time, and monogamy has been classified as a mating system in which neither sex is able to monopolize additional members of the opposite sex (Emlen and Oring, 1977). Historically, monogamy was thought to occur primarily in birds, and Lack (1968) initially estimated that more than 90% of birds were monogamous. However, as molecular tools became more accessible, researchers began to discover that many species that were once thought to be monogamous mate multiply. For instance, in one of the first studies on this topic, Gowaty and Karlin (1984) found that at least 5% of male and 15% of female eastern bluebirds (Sialia sialis) were caring for at least one offspring that was not genetically theirs; such research was surprising at the time because eastern bluebirds were thought to be monogamous. Such studies led to a distinction between genetic and social monogamy (Gowaty, 1996). Social monogamy occurs when a male-female pair cohabit and/or form a pair bond for some specified period of time; social monogamy does not assume that the male-female pair is exclusive with regard to mating. In contrast, genetic monogamy assumes that a malefemale pair is exclusive for some specified period of time with regard to mating and fertilization, although the pair doesn't necessarily pairbond.

The distinction between social and genetic monogamy has led to a range of studies that have revealed unique combinations of genetic and social monogamy. For instance, the largemouth bass (Micropterus salmoides) is socially monogamous and exhibits near-genetic monogamy (DeWoody et al., 2000). Likewise, long-eared owls (Asio otus) are socially and genetically monogamous (Marks et al., 1999). Similarly, Kentish plovers (Charadrius alexandrines) tend to primarily, but not always, exhibit social and genetic monogamy (Küpper et al., 2004). In contrast, in the prairie vole (Microtus ochrogaster), most males form pairbonds and cohabit with a single female but also engage in extra-pair mating; thus, most, but not all, individuals engage in social but not genetic monogamy (Ophir et al., 2007, 2008a,b). In the pot-bellied seahorse (Hippocampus abdominalis) the opposite trend has been observed: despite social promiscuity (e.g., promiscuous courtship) and a lack of pairbonding, genetic monogamy has been observed (Wilson and Martin-Smith, 2007). Mating systems have also been found to be dynamic (e.g., Forsgren et al., 2004; Wilson and Martin-Smith, 2007), suggesting that in some systems, individuals that are socially and/or genetically monogamous at one point in time might adopt a different strategy under different ecological conditions. Indeed, genetic monogamy occurs across a range of animals, but there is often inter-specific variation in the extent to which genetic monogamy occurs (**Table 1**).

When we focus on understanding the occurrence of monogamy in nature, it is important to recognize that a mating system is a descriptor of population- or species-level dynamics. Individuals themselves can exhibit strategies that are consistent with a given mating system, and these individual-level strategies that are associated with a given mating system (e.g., a particular propensity to engage in monogamous courtship, monogamous mating, multiple mating) are traits that can experience and respond to selection. That is, such traits can evolve. In contrast, the mating system of a population is not a trait and can therefore not evolve in a biological sense; a mating system can change on short and long time scales and across spatial contexts, and a mating system can create selection pressures, but ultimately traits are what evolve biologically. In considering the factors that are hypothesized to influence monogamy (discussed below), it is important to keep in mind that mating systems are influenced by traits, which can evolve, and that a mating system can create selective pressures that lead to future evolution of relevant traits. This evolutionary interplay and feedback between mating systems and traits is likely key to understanding the variation in mating dynamics that exist in nature, and hence, key to understanding the occurrence of monogamy.

## CLASSIC PERSPECTIVES ON MONOGAMY

#### Resources, Parental Care, and Sexual Selection Matter

Emlen and Oring (1977) were not the first to study mating systems (Darwin, 1888; Verner and Willson, 1966; Orians, 1969; Bartholomew, 1970), but the synthetic conceptual framework of mating systems that they developed was remarkably influential. Emlen and Oring (1977) hypothesized that there is a link between mating system and sexual selection and suggested that sexual selection will be relatively slight in monogamous systems (**Figure 1**). They emphasized that understanding diversity in mating systems requires that we understand the factors that influence sexual selection. Specifically, they suggested that sexual selection will be influenced by the "ability of a portion of the population to control the access of others to potential mates" and that monogamy will occur when there is "economic defendability of a mate" (Emlen and Oring, 1977; **Figure 1**). Emlen and

#### TABLE 1 | Examples of monogamy in nature.

Oring (1977) noted that critical non-mate resources will vary spatially and temporally and that this can affect the spatial and temporal distribution—and hence the defendability—of potential mates (**Figure 1**). They additionally predicted a link between biparental care and monogamy and noted that monogamy is most likely to occur when the potential for or the benefit of multiple mating is low (**Figure 1**). They hypothesized that under such conditions, individuals can increase their fitness by remaining with their current mate and increasing offspring survival.

More recent work (discussed below) has demonstrated that co-evolutionary feedback can occur and lead to patterns that are more complex than those initially predicted (e.g., we now know that sexual selection isn't always slight in monogamous systems; Jones and Hunter, 1993). Indeed, as noted by Wittenberger and Tilson (1980), no single hypothesis alone is sufficient to explain monogamy; instead a series of hypotheses and an understanding of when each should apply is required to understand monogamy (Wittenberger and Tilson, 1980). Nonetheless, the relatively simple verbal arguments of Emlen and Oring (1977) highlighted the key role that sexual selection, resources, and parental care can play in influencing monogamy.

### The Role of Female Benefits in Monogamous Mating Systems

Wittenberger and Tilson (1980) expanded the conditions under which monogamy is expected. They focused more explicitly on the role that female fitness benefits have in maintaining monogamy and hypothesized that monogamous mating systems require: (1) female benefits of monogamous pair bonds that cannot be obtained in the absence of monogamy; (2) the ability of females to assess the mated status of males; and (3) a lack of male desertion. They additionally hypothesized that monogamy is most likely to occur when: (1) male parental care is essential and non-shareable among offspring; (2) the benefits of mating with a superior polygynous male do not outweigh the costs of polygyny that is associated with reduced parental care; (3) males achieve the greatest fitness benefits by mating with and defending a single female; (4) aggression by females occurs; and (5) increased competition for resources and/or increased predation associated with multiple female mates occurs (Wittenberger and Tilson, 1980). Importantly, these hypotheses are inter-related and not mutually exclusive.

In the years following the work of Emlen and Oring (1977) and Wittenberger and Tilson (1980), numerous authors refined the conditions under which monogamy is expected (e.g., Greenlaw and Post, 1985; Björklund and Westman, 1986; Mock and Fujioka, 1990). Many of these early studies that focused explicitly on monogamy utilized verbal arguments that were largely based on our knowledge of mating systems at the time (but see, e.g., mathematical models by Parker and Macnair, 1978 and McLean and Manning, 1985). Many of these early hypotheses have now been tested or expanded upon empirically and/or by using mathematical modeling. Below, I review the insights that such work has provided.

## RECENT PERSPECTIVES ON THE ORIGIN AND MAINTENANCE OF MONOGAMY

### Parental Care Can Matter

A frequent prediction of early work is that paternal care is associated with monogamy (Emlen and Oring, 1977; Wittenberger and Tilson, 1980; Gowaty, 1996; **Figure 1**). Indeed, some theoretical work has found that paternal care influences the occurrence of monogamy. Iwasa and Harada (1998) found that monogamy can occur when parental ability is the same between parents if females choose their mates and males invest in paternal care. If females and males vary in quality (i.e., fecundity and parental ability), this can lead to assortative mating and monogamy, although the mating dynamics will depend on the costs of care to males and variation in female fecundity (Iwasa and Harada, 1998). In animals, monogamy is often associated with bi-parental care, but whether bi-parental care precedes or follows the evolution of monogamy is debatable (reviewed in Brotherton and Komers, 2003 and discussed in section "But, Is Bi-parental Care Really Necessary for Monogamy"? below).

## Co-evolutionary Dynamics Matter

More recent work has suggested that understanding the origin and persistence of any mating system, including monogamy, requires that we develop a framework that accounts for interactions among mating dynamics, parental investment, and costs of care, and mating (**Figure 1**). The likelihood of paternal care, which as discussed above is in some cases predicted to influence monogamy, is affected by a range of factors, including the costs and benefits of caring vs. competing for mates (**Figure 1**). Kokko and Jennions (2008) developed a model focused on sex roles that accounted for feedback associated with the costs and benefits of caring vs. competing for mates. Their work revealed that when providing parental care is associated with higher mortality than competing for mates, individuals of the deserting sex (i.e., the sex that provides no care) will become more common in the population and in turn have difficulty

finding a mate; under such a scenario, across evolutionary time, we would expect (1) bi-parental care to be more likely and (2) for males and females to differ relatively little in the amount of care provided (Kokko and Jennions, 2008). While monogamy was not explicitly focused on in the modeling work of Kokko and Jennions (2008), if bi-parental care is associated with monogamy (see above), we might expect monogamy to be more likely in systems in which parental care is associated with high adult mortality.

### But, Is Bi-parental Care Really Necessary for Monogamy?

While bi-parental care and monogamy commonly co-occur (reviewed in Brotherton and Komers, 2003), some researchers have questioned whether bi-parental care is necessary for, or rather a consequence of, monogamy. In humans, Schacht and Bell (2016) found that mate guarding rather than paternal care leads to monogamy, as monogamy allows males to maintain high paternity. Likewise, Lukas and Clutton-Brock (2013) suggested that in non-human mammals, male care is a consequence rather than a cause of monogamy. They instead suggested that monogamy is caused by low female density and the inability of males to defend multiple females. However, Dobson et al. (2010) found that socially monogamous and nonmonogamous mammals have similar local densities and home ranges, suggesting that local density and home range area might not be a primary driver of monogamy in all mammals. Instead, Dobson et al. (2010) suggested that the origin of monogamy in mammals is caused by a range of factors. In primates, Opie et al. (2013) found a correlation between social monogamy, female home range, and bi-parental care; however, their research suggests that infanticide is likely the key factor that leads to a shift toward social monogamy in primates.

In some cases, paternal care is decoupled from monogamy. In extreme cases, males die after mating and are thus unable to provide paternal care. Fromhage et al. (2005) found that monogyny can be favored in the absence of male care when monogyny increases paternity if the sex ratio is male biased. Thus, while bi-parental care and monogamy frequently co-occur, it does not appear that bi-parental care is always a pre-requisite for the origin or maintenance of monogamy. Indeed, in some cases mate guarding and paternity assurance can directly favor monogamy.

In summary, bi-parental care commonly co-occurs with monogamy, but the role that bi-parental care plays in driving the origin of monogamy is unclear, particularly in mammals.

### Interactions Between Ecology and Parental Care Can Lead to Monogamy

While bi-parental care is not always essential for monogamy, parental care and offspring need can in some cases interact with ecological factors to drive the origin of monogamy. For instance, Brown et al. (2010) found that in frogs, a single ecological factor (breeding pool size) is related to the origin of care. In poison frogs, they found that feeding of offspring co-evolved with the use of small pools and that feeding behavior was associated with the origin of bi-parental care, as bi-parental care is essential to offspring survival in small (but not large) pools. Molecular and field analyzes revealed that social and genetic monogamy occur in Ranitomeya imitator, a species that utilizes small pools, but not Ranitomeya variabilis, a species that uses large pools, suggesting that an ecological factor (pool size) led to the co-evolution of bi-parental care and monogamy in R. imitator (Brown et al., 2010). These results highlight the important interactions that can occur between ecological factors, parental care, offspring need, and monogamy.

Decades of empirical and theoretical research suggest that there is no single factor that drives monogamy across animals. This is perhaps not surprising given the immense variation in life history, evolutionary history, and ecological factors in animals. Numerous studies, however, have demonstrated that the following likely influence monogamy: (1) spatial and temporal distribution of females, (2) parental care costs and benefits, (3) offspring need, (4) infanticide, (5) costs and benefits of multiple mating, (7) mate competition, (8) paternity assurance, (9) the potential for mate guarding, and (10) resource use (**Figure 1**). This is a broad list, and the relative importance of each factor likely varies across systems. Additionally, the factors above are likely to interact. For example, ecology can affect mate distribution and offspring need; infanticide can affect offspring need; costs of parental care will influence mate availability, which will in turn affect benefits of mate searching. Additional research on such interactions warrants further attention (discussed below).

#### WHAT'S NEXT?

I suggest that there are two primary areas of research that are needed to more fully understand monogamy from an ultimate

#### REFERENCES

perspective. First, we need to recognize that the factors that promote the origin vs. the maintenance of monogamy might differ (see discussion in Gowaty, 1996). Within animals, it will be important to better understand if the factors that promote the origin of monogamy are the same (or different) than those that promote the maintenance of monogamy.

Second, it will be critical to better understand how life-history, ecological, and mating factors interact to influence monogamy. As mentioned earlier, monogamous mating dynamics can create selective pressures that influence evolutionary trajectories; for example, West (2014) found that the evolution of large brain size is associated with social but not genetic monogamy in birds. Likewise, Jašarevic et al. (2013) ´ found that monogamy can influence the evolution of female life histories in mammals. Further, previous work has found that mate guarding, male attractiveness, and paternity can interact in complicated ways to influence social monogamy (Kokko and Morrell, 2005). Recent studies have begun to focus on the interplay between such factors, but there is still more work to do in identifying the interactions (rather than the individual factors) that lead to the origin and maintenance of monogamy. Focusing on such interactions will likely also be necessary to better understand how monogamy varies within a population and through time. Indeed, understanding variation in the propensity to be monogamous within and across individuals of a population has received relatively little attention from an ultimate perspective and warrants future attention.

#### AUTHOR CONTRIBUTIONS

The author confirms being the sole contributor of this work and approved it for publication.

#### ACKNOWLEDGMENTS

This material is based upon work supported by the National Science Foundation under Grant No. 1552721. I am grateful to Dylan Hackett for his discussion and research on the ideas discussed herein.

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Klug. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Socio-Ecological Disruptions at Critical Periods During Development Alter Stress Responses and Hippocampal Dendritic Morphology of Prairie Voles: Implications for Social Monogamy

Omar A. S. Al-Naimi, Jose R. Delvalle, Sophia S. Carryl, Natalia A. Rodriguez, Fayeza Aliou, Monica Cambi and Maryam Bamshad\*

*Department of Biological Sciences, Lehman College-City University of New York, Bronx, NY, United States*

#### Edited by:

*Nancy G. Solomon, Miami University, United States*

#### Reviewed by:

*Karen Lisa Bales, University of California, Davis, United States Angela J. Grippo, Northern Illinois University, United States*

#### \*Correspondence:

*Maryam Bamshad maryam.bamshad-alavi@ lehman.cuny.edu*

#### Specialty section:

*This article was submitted to Behavioral and Evolutionary Ecology, a section of the journal Frontiers in Ecology and Evolution*

> Received: *28 December 2017* Accepted: *29 May 2018* Published: *19 June 2018*

#### Citation:

*Al-Naimi OAS, Delvalle JR, Carryl SS, Rodriguez NA, Aliou F, Cambi M and Bamshad M (2018) Socio-Ecological Disruptions at Critical Periods During Development Alter Stress Responses and Hippocampal Dendritic Morphology of Prairie Voles: Implications for Social Monogamy. Front. Ecol. Evol. 6:84. doi: 10.3389/fevo.2018.00084* Emotions are often associated with the evolution of monogamy. For example, fear of cuckoldry has been recently proposed as the driving force for human monogamy. We used prairie voles (*Microtus ochrogaster*) as a model of human behavior to study how stress reactivity is shaped by socio-ecological disruptions experienced as neonates and as subadults. We hypothesized that social disruptions would have a greater impact on the vole's stress levels than ecological disruptions, and that females would be more susceptible to the experience than males. At 6 days postpartum, the housing conditions were manipulated to have offspring raised by: (1) both parents under a protective cover (NoDisrupt); (2) Both parents uncovered (EcoDisrupt); (3) Mother alone covered (SocDisrupt); (4) Mother alone uncovered (SocEcoDisrupt). To experience disruptions as subadults, offspring were weaned then housed either alone (Isolate) or with a same-sex sibling (Social). As adults, each offspring was placed in an open-field arena and tested over 3 consecutive days to measure its behavior in response to an empty space (Day1), a same-sex vole in a container (Day2), and an empty container (Day3). The brain of a subgroup of subjects was processed for Golgi staining to assess the impact of disruptions on hippocampal dendritic morphology in adulthood. Males that experienced social disruption in early life displayed lower stress levels on Day2 of testing than males and females in other groups. This effect was only evident in males that did not experience social disruptions as subadults. Socio-ecological disruptions at postpartum had an unanticipated impact on the hippocampus of the voles. The apical dendrites of the CA3 neurons in male and female voles that experienced either social or ecological disruptions in early life and remained socially isolated as subadults were longer than those in undisturbed voles. Our results suggest that social disruptions experienced in early life modulate the male's stress-related behaviors and may thus influence his monogamous tendencies. Exposure to disruptions may also impact the memory circuits of the brain that monogamous animals use to make mating decisions.

Keywords: paternal deprivation, early-life environmental disruptions, socio-spatial memory circuit, social monogamy, adolescence, social isolation, HPA-axis

## INTRODUCTION

The evolutionary pressure that led humans and other mammals to form monogamous relationships is an enigma (Wittenberger and Tilson, 1980; Henrich et al., 2012). Given the low cost of reproduction for male mammals, they should maximize their fitness by pursuing as many females as possible (Clutton-Brock and Parker, 1992; Kappeler, 2013), but not all of them do (Lukas and Clutton-Brock, 2012). Males of a few species across a broad range of taxa display pair-living and copulate with their partner rather than with novel females (Lukas and Clutton-Brock, 2013). Although some of these males occasionally come in contact with other females and engage in extra-pair copulations (Solomon and Jacquot, 2002; Cohas and Allaine, 2009; Streatfeild et al., 2011), most share a home range with one female for an extended period of time and some even participate in parental care (Reichard, 2003). Studies from a variety of species that have attempted to explain why some males forgo their mating opportunities to invest in their family have produced contradictory results (Dobson et al., 2010). While the conflicting data continues to puzzle the evolutionary biologists (Kappeler, 2013), the research has provided neurobiologists with multiple avenues to explore the underlying physiological mechanisms that may maintain social monogamy within a population.

The neurobiology of social monogamy is studied from multiple angles. Some have focused on the biology of attachment in monogamous species as a rewarding emotion that keeps sexual partners in close proximity of each other and fosters paternal care (Lim et al., 2004; Young and Wang, 2004; Young, 2009; Young et al., 2011; Burkett and Young, 2012; Johnson and Young, 2015). This line of research, which has been immensely fruitful in discovering the neurochemistry of selective partner preference and social bonding, has been based on the theory that the evolution of monogamy was driven by the need for males to invest in paternal care, thus making bonding an adaptive behavior (Geary, 2000; Fraley et al., 2005; Young et al., 2011). However, others have viewed the problem of monogamy as a male tactic to maximize his fitness in environments where access to mates is limited and where he could be cuckolded into caring for another male's progeny (Phelps and Ophir, 2009; Ophir, 2017). This approach that is focused on cognition is more in line with the theory that the pressure driving the evolution of monogamy was mate guarding or resource scarcity instead of paternal investment (Schacht and Bell, 2016). Accordingly, individuals choose a mating strategy to increase their fitness and lower their chances of being cuckolded by processing social and spatial information within a given space. That information enables them to identify and remember the location of potential mates and their competitors. Additionally, a unified model of emotions and cognition controlled by the so called "social decision-making network" has been proposed to show how animals could evaluate their social environment when deciding whether to remain attached to a single mating partner, mate outside the relationship or seek many partners (Ophir, 2017). A key brain region in this model is the hippocampus, which along with the retrosplenial cortex, anterior thalamus, and septal nuclei is part of a network for forming spatial and social memories, and is also a main accessory area of the mesolimbic reward circuitry (Ikemoto, 2010; O'Connell and Hofmann, 2011; Ophir, 2017).

If social monogamy is a cost-benefit assessment evolved to enhance fitness, then it is likely to be entangled not only with the positive emotion of attachment, but also with stress that can affect decisions making. In real life, when individuals are challenged with physical threats or face uncertainty, neither humans nor other animals weigh all alternatives to choose the best strategy and their cognitive abilities can be affected by their stress responses (Giora, 1987; Lima, 1998; Porcelli and Delgado, 2017; Summers et al., 2017). Although several studies have examined the role of stress in monogamy, most have focused on how stress influences or is impacted by partner preference and paternal care (DeVries et al., 1996; Bosch et al., 2009; Hostetler and Ryabinin, 2013; Hyer and Glasper, 2017; Kowalczyk et al., 2018). Our aim in the current study was to examine the development of stress reactivity in a socially monogamous rodent that displays alternative reproductive tactics in nature to understand how stress might shape an individual's decision-making processes associated with reproductive strategies.

To investigate the development of stress reactivity in a monogamous species, we looked at studies in other rodents. Research in rats has shown that the rearing environment in early life impacts the developing brain to shape the adult stress responses, cognition, and reproductive behavior (Liu et al., 2000; Cameron et al., 2008; Kundakovic and Champagne, 2015). However, the process is complex and depends on multiple factors including the offspring genotype, sex, and social interactions experienced later in life (Daskalakis et al., 2013). We used prairie voles as an animal model of social monogamy to examine the impact of early rearing environment and later social experience on stress reactivity of male vs. female offspring. Prairie voles are one of the rare mammalian species that display socially monogamous relationships and biparental care in captivity and in nature (Getz et al., 1981; Oliveras and Novak, 1986; Terleph et al., 2004). Like humans, prairie voles are designated as a socially monogamous species because the adult male and female pairs show a preference to live together but do not mate exclusively (Carter et al., 1995; Strassmann, 2003; Solomon et al., 2004; Streatfeild et al., 2011). Laboratory studies have shown that when male prairie voles are given the opportunity to access multiple mates, they show a preference to mate with only one of them (Blocker and Ophir, 2016). Although pair-bonded males are attentive to the sensory stimuli of other sexually-receptive females (Parker et al., 2011; Rodriguez et al., 2013), when given a choice, they prefer to remain in contact with their mate rather than a novel female (Blocker and Ophir, 2016). This suggests that males may be predisposed to forgo a desire to seek other mates once they form a pair bond.

Nevertheless, observations of prairie voles under natural and semi-natural settings have shown that the behavior of both males and females is dynamic and can shift from exclusive mating with a single partner to occasional mating with a novel conspecific (Solomon and Jacquot, 2002; Ophir et al., 2008; McGuire and Getz, 2010). For example, wild male prairie voles are reported to switch their mating tactics from being residents that share a home range with a single female to being wanderers that visit multiple nests (Solomon and Jacquot, 2002; Ophir et al., 2008). Given that the reproductive advantage of monogamy vs. polygamy for prairie voles is equivocal (Mabry et al., 2011; Okhovat et al., 2015), it is unclear why some male voles choose a pair-living tactic while others do not and why some males switch their tactic. An insight into this conundrum might be found by examining the stress responses of voles when they encounter conspecifics and must decide to form a monogamous bond or to copulate outside the relationship. It is possible that stress reactivity of prairie voles affects their decision-making processes when exploring their mating options in unpredictable environments.

By varying the social and ecological conditions of the natal nest, we attempted to identify the early-life factors that might contribute to the predisposition of male and female voles for responding to stress. We hypothesized that the anxiety level shown by adult voles in presence of a conspecific would differ based on their sex, whether they were exposed to either social or ecological disruptions in their early-life environment, and if they experienced additional disruptions by being socially isolated as subadults later in life. Given that the hippocampus can be reshaped by stress throughout life (McEwen and Magarinos, 2001) and is a key brain region within the so called "social decision-making network" (Ophir, 2017), we examined the dendritic morphology of its neurons to determine the impact of disruptions experienced in early life superimposed on disruptions experienced in later life. As voles are social animals that sometimes live in groups and are raised by both parents (McGuire and Getz, 1995), we predicted that social disruptions would increase the anxiety levels of adult offspring more than ecological disruptions. Based on previous research in prairie and mandarin voles showing that females are more sensitive than males to manipulations of their social environment such as paternal deprivation and social isolation (Ruscio et al., 2009; Yu et al., 2012), we also predicted that our female subjects would be more susceptible to the impact of postpartum disruptions than males. Given the adverse effects of social isolation in voles (Scotti et al., 2015), we expected the early-life disruptions to have a greater negative effect on the stress reactivity and neuronal development of voles that were isolated as subadults than the socially-housed voles that remained with their same-sex sibling.

## MATERIALS AND METHODS

### Subjects

The subjects were the F2 generation of prairie-vole breeding pairs that are maintained in the animal facility at Lehman College. The breeding colony was formed from descendants of wildcaught voles that were originally captured in east-central Illinois, U.S.A. We minimize inbreeding in our colony by monitoring the relatedness of males and females that we select for breeding. All voles in our facility are housed in standard clear plastic cages (48 × 27 × 20 cm) and supplied with nesting material to simulate their natural habitat. Voles are underground burrowers that occupy a variety of habitats including ungrazed pastures, hay fields, and alfalfa fields (Cole and Batzli, 1979; Mankin and Getz, 1994). They prefer to live in areas of thick rather than sparse vegetation; they use vegetation as both food and cover (Lin and Batzli, 2001). They are more likely to disperse from areas of no vegetative cover (Lin et al., 2006). Given that voles are considered as pests, a way to control their population is to mow their vegetative cover (Carter and Getz, 1993; O'Brien, 1994). To create a protective and familiar environment for them in the lab, we first place a layer of ∼5 cm of moistened peat moss at the bottom of their cage then fill the cage to the top with straw. Water and food, consisting of a mixture of sunflower seeds, rabbit chow, and cracked corn are available at libitum. The colony animals are kept in rooms with fluorescent lighting and at temperatures around 20–25◦C. The light:dark of the room is set at 14:10 with lights on at 6:00 a.m. The offspring of our breeding colony serve as experimental subjects. They are weaned at 20 days then transferred to a separate room where they are housed with their same-sex siblings under a protective cover of straw until adulthood. All experimental and stimulus animals used in the study were at least 60-days-old, were sexually inexperienced at the beginning of the experiment, were raised by both parents. They were removed from their natal nest before their mother gave birth to another litter.

All applicable national and institutional guidelines for the care and use of animals were followed. Animal care and all procedures performed were in accordance with the ethical standards of Lehman College and were approved by the Lehman College Institutional Animal Care and Use Committee.

### Housing Manipulations

We used a total of 64 adult males and 64 adult females that we separated from their same-sex siblings to create four sets of breeding pairs (N = 16 pairs for each of four conditions), whose offspring served as our experimental subjects. The animals selected for breeding were between 60 and 120 days old. They were unrelated and unfamiliar to each other. Each male was held in a wire mesh cylindrical container (10 × 7.7 cm) with some food. The container was capped and placed in the cage of a female for 24 h, so the partners would become accustomed to one another's sensory stimuli. The male was then released into the female's cage and the pair was left undisturbed, except for cage cleaning, until the birth of their first offspring. At 6 days postpartum, the cages of the breeding pairs were manipulated to expose offspring to early-life disruptions that they may experience in nature such as loss of their father, protective cover or both their father and their protective cover. A total of 16 family units were left undisturbed and served as control (NoDisrupt). The NoDisrupt offspring were reared by both parents under a protective cover of straw. In the 16 family units whose ecological condition was disrupted (EcoDisrupt) the protective cover was permanently removed leaving the mother and the father to raise the young with only the peat moss at the bottom of the cage and a handful of loose straws. In the 16 family units whose social condition was disrupted (SocDisrupt), the father was permanently removed leaving the mother to raise the young under the protective cover of straw. In the 16 family units whose social and ecological conditions were disrupted (SocEcoDisrupt), the father and the protective cover were permanently removed leaving the mother to raise the young alone with only the peat moss at the bottom of the cage and a handful of loose straws. All

family units were then left undisturbed until the offspring were 20 days of age. To expose offspring to later-life disruptions, the subadults at weaning were sexed and housed either with a same sex-same sibling (Social) or housed alone (Isolate). The Social and Isolate groups were housed under a protective cover of straw until adulthood.

#### Anxiety Testing