Development of a Partner Preference Test That Differentiates Between Established Pair Bonds and Other Relationships in Socially

American Journal of Primatology

RESEARCH ARTICLE Development of a Partner Preference Test that Differentiates Between Established Pair Bonds and Other Relationships in Socially Monogamous Titi Monkeys (Callicebus cupreus)

SARAH B. CARP1#, EMILY S. ROTHWELL1,2*,#, ALEXIS BOURDON3, SARA M. FREEMAN1, EMILIO FERRER4, 1,2,4 AND KAREN L. BALES 1California National Primate Research Center, University of California—Davis, Davis, California 2Animal Behavior Graduate Group, University of California—Davis, Davis, California 3AgroParisTech, Paris, France 4Department of Psychology, University of California—Davis, Davis, California

Partner preference, or the selective social preference for a pair mate, is a key behavioral indicator of social monogamy. Standardized partner preference testing has been used extensively in rodents but a single test has not been standardized for primates. The goal of this study was to develop a partner preference test with socially monogamous titi monkeys (Callicebus cupreus) adapted from the widely used rodent test. In Experiment 1, we evaluated the test with pairs of titi monkeys (N ¼ 12) in a three-chambered apparatus for 3 hr. The subject was placed in the middle chamber, with grated windows separating it from its partner on one side and an opposite sex stranger on the other side. Subjects spent a greater proportion of time in proximity to their partners’ windows than the strangers’, indicating a consistent preference for the partner over the stranger. Touching either window did not differ between partners and strangers, suggesting it was not a reliable measure of partner preference. Subjects chose their partner more than the stranger during catch and release sessions at the end of the test. In Experiment 2, we compared responses of females with current partners (N ¼ 12) in the preference test with other relationship types representing former attachment bonds (N ¼ 13) and no attachment bond (N ¼ 8). Only females from established pair bonds spent signiﬁcantly more time near their partner’s window compared to the stranger’s indicating that this measure of preference was unique to current partners. Other measures of preference did not differentiate behavior toward a current partner and other relationship types. This test reproduces behavioral patterns found in previous studies in titi monkeys highlighting the accuracy of this new partner preference test. This test can be used as a standardized measure of partner preference in titi monkeys to quantitatively study pair bonding and evaluate factors inﬂuencing partner preference. Am. J. Primatol. © 2015 Wiley Periodicals, Inc.

Key words: nonhuman primate; behavior; monogamy; New World monkey; social attachment

INTRODUCTION Only 3–5% of all mammals are classiﬁed as socially Contract grant sponsor: National Science Foundation Graduate monogamous [Kleiman, 1977]. However, social monog- Research Fellowship; contract grant sponsor: HD053555; amy is more prevalent among non-human primates and contract grant sponsor: Office of Research Infrastructure is observed in approximately 10% of primate species Programs; contract grant number: P51OD01107; contract grant [Fuentes, 1998]. Studies of social monogamy have sponsor: Good Nature Institute focused on a variety of species, including rodents (e.g., Conflicts of interest: None. prairie voles and California mice) [Martin et al., 2006; # Williams et al., 1992], New World primates (e.g., titi Sarah B. Carp and Emily S. Rothwell are co-first authors. monkeys, marmosets, and owl monkeys) [Anzenberger, Correspondence to: Emily S. Rothwell, Animal Behavior 1992; Fernandez-Duque & Huck, 2013; Smith et al., Graduate Group, University of California—Davis, One Shields 2010], lesser apes (e.g., gibbons and siamangs) Avenue, Davis, CA 95616. E-mail: [email protected] [Fuentes, 2000; Lappan, 2008], and humans [Hazan Received 3 July 2014; revised 21 June 2015; revision accepted 4 & Shaver, 1987]. By studying the behavior and biology July 2015 of species that are capable of forming the adult male– DOI: 10.1002/ajp.22450 female bonds that characterize a socially monogamous Published online XX Month Year in Wiley Online Library mating system, we can better understand the biological (wileyonlinelibrary.com).

underpinnings involved in the formation and mainte- studies of titi monkeys (Callicebus spp.), which were nance of these social bonds. tested in the current study, several different methods Male–female dyads in some socially monogamous have been used (Table I). Furthermore, partner prefer- species form stable adult attachments to one another, ence tests previously utilized with primates differ called pair bonds. Pair bonds are characterized considerably from the standardized rodent test. As primarilybyspendingtimeinextendedphysicalcontact can be seen from Table I, primate partner preference and maintaining physical proximity with one’spair tests are variable in both duration (e.g., minutes mate [Carter et al., 1995]. Pair bonded animals often compared to hours) and apparatus size (e.g., t-maze show aggression toward same-sex strangers and compared to home-cage). Furthermore, they have been occasionally to opposite-sex strangers, who might utilized to study changes in partner preference under pose a threat to the stability of the bond [Cubicciotti biological manipulation where baseline partner prefer- & Mason, 1978; Winslow et al., 1993]. Perhaps the best ence was not initially validated. studied behavioral indicator of social monogamy is The first goal of the current study was to develop a partner preference, or a specific social preference partner preference test for socially monogamous displayed for the pair mate over an unfamiliar primates that was adapted from the standardized opposite-sex conspecific [Mason & Mendoza, 1998]. rodent partner preference test in order to facilitate In order to study these pair bond related behaviors comparisons across mammals on this behavioral in a standardized way, laboratory assessments use a indicator of social monogamy. The second goal was to validated behavioral paradigm called the partner evaluate this new partner preference test by utilizing preference test. In rodents, this test typically involves titi monkeys (Callicebus cupreus)inestablishedpair a three-chambered testing arena in which the familiar bonds, which display characteristically similar adult pair mate (“partner”) and an unfamiliar opposite-sex attachment bonds to those of prairie voles (“Experi- conspecific(“stranger”) are restricted to the two outside ment 1” of the current study). The final goal was to chambers. The test subject is placed into the central validate this test by applying it to different relationship “neutral” chamber and then allowed to move to spend types. Here, we capitalized on the fact that infant titi time in, or adjacent to, the two outside chambers monkeys form an attachment only to their fathers, with containing each stimulus animal or to remain in the a distress response only observed when infants are neutral portion of the apparatus [Williams et al., 1992]. separated from their father and not when separated The time that the test animal spends in proximity to (or from their mother [Mendoza & Mason, 1986]. There- in physical contact with) either of the two stimulus fore, we specifically tested relationship types in adult animals is measured, as well as any affiliative or female titi monkeys that represent former attachment aggressive behaviors exhibited by the test animal. The bonds (i.e., former partner, father) and a relationship partner preference test (and its derivatives from that presumably never constituted an attachment studies of non-monogamous species, the “social prefer- bond (i.e., mother) [Mendoza & Mason, 1986], to see if ence test”) has been most widely used in rodents [Millan this preference test can differentiate between a partner & Bales, 2013]. The widespread use of this behavioral preference and a more general social preference paradigm across rodent laboratories and the conse- (“Experiment 2” of the current study). quent high consistency in test parameters across Validating this partner preference test for titi studies have arguably allowed this behavioral task to monkeys will provide a standardized way to assess become as reliable and reproducible as other consis- pair bonding in future studies across socially monog- tently used rodent behavior tasks, such as the Morris amous primate and mammalian species. For example, water maze or elevated plus maze. As a result of this a partner preference paradigm can be used to reproducibility, the partner preference test has been determine the effect of different pharmacological used to establish the roles of the oxytocin, vasopressin, manipulations on the formation or maintenance of dopamine, and opioid systems in mediating pair bond social bonds, and to learn more about the changing formationinmonogamousprairievoles[Burkettetal., dynamics of pair bonds under different social contexts 2011; Cho et al., 1999; Gingrich et al., 2000; Resendez or over time. Furthermore, establishing the factors et al., 2012; Williams et al., 1994; Young et al., 2001], that influence adult social bonds in nonhuman and it has also been used to study social preferences in primates will have important implications for under- non-monogamous rodents like meadow voles [Beery & standing human adult attachment relationships. Zucker, 2010; Parker et al., 2001; Ross et al., 2009]. In contrast with rodents, the field of primatology has not yet adopted a standardized test to evaluate METHODS partner preference across species. Although partner preference has been studied in several primate species, a Subjects wide variety of methodological approaches have been Experiment 1: Evaluate the partner preference test utilized (Table I). These varied approaches likely stem with mates in established pair bonds from accommodating for behavioral or housing differ- In Experiment 1, 24 titi monkeys (C. cupreus)were ences across species and laboratories. But even among tested between February 2013 and February 2014.

Am. J. Primatol. TABLE I. Previous Partner Preference Tests in Socially Monogamous Rodents and Primates

Stimulus animal Preference Length of Test access and measure (partner vs. partner Species length Apparatus presentation type1 stranger)2 relationship

Rodents Prairie voles Exp. 1: 8 hr Three chambers Full physical; Contact Exp. 1: >24 hr (Microtus ochrogaster)a simultaneous cohabitation; Exp. 2: 3 hr Exp. 2: 6 hr cohabitation Primates Titi monkeys 2.5 hr; choice sessions Three chambers Grated; simultaneous Proximity, touching >6mo (Callicebus cupreus) grated window, (present study) choice sessions Titi monkeys Repeated 15 min Three chambers Full physical; both Proximity, social and >7mo (Callicebus cupreus)b trials separate and sexual behavior simultaneous Titi monkeys Repeated 30 min One chamber Full physical; Proximity, social and >17 mo (Callicebus cupreus)c trials separate sexual behavior Titi monkeys Repeated 15 min Two chambers Grated; tested in Proximity, social and >2yr (Callicebus cupreus)d trials pairs sexual behavior Titi monkeys Repeated 5 min trials Three chambers Grated; both Proximity >2wk 3,4 Test Preference Partner Primate Nonhuman (Callicebus cupreus)e, separate and simultaneous Titi monkeys 90 min trials One chamber Full physical; Proximity, social >8wk 3,4 (Callicebus cupreus)f, separate behavior, heart rate Titi monkeys Repeated 15 min Two chambers Visual only; tested Proximity, social >8mo 3,4 (Callicebus cupreus)g, trials with pair mate in behavior, heart rate cage Titi monkeys Repeated 5 min trials V shaped Grated; both Proximity, agonistic >1yr 3,4 (Callicebus cupreus)h, separate and behavior simultaneous Black-Tufted Marmosets 20 min T shaped Grated; simultaneous Proximity, social and 24 hr or 3 wk (Callithrix penicillata)i sexual behavior Common Marmosets 20 min T shaped Grated; simultaneous Proximity, social and >8wk j m .Primatol. J. Am. (Callithrix jacchus) sexual behavior White-Lipped Tamarins Repeated 30 min Three chambers Grated; both Proximity, social and >6mo (Saguinus labiatus)k trials separate and sexual behavior simultaneous /3 4/Carp et al.

These subjects represented 12 current pairs (N ¼ 12 male, N ¼ 12 female), housed at the California National Primate Research Center in Davis, California. Titi

ete citation in monkeys are small-bodied (approximately 1 kg) New 4mo 6mo

> > World primates. The average age of the subjects was partner Length of

Cubicciotti & Mason – relationship 8.34 1.01 years (range: 2.15 23.08 years). All individ- h uals were housed with their partner and, if applicable, offspring during the course of the study. Twenty-three of the subjects were born in captivity at the California National Primate Research Center and one was wild caught in 1990 and imported to the colony. Seventeen additional monkeys were used as stranger stimulus 2 animals during the partner preference test sessions. Anzenberger et al. [1986],

g Stranger animals were unfamiliar to the test animal before the study and were approximately matched with r others) the most liberal access is listed for the table. behavior stranger) Preference the test animal’s partner on sex, age, and weight. sexual behavior measure (partner vs. Proximity, social and Experiment 2: Validate the partner preference test

. by applying it to other relationship types In Experiment 2, 13 adult female titi monkeys

1 were tested between November 2013 and February 2014, all of which were born in captivity. Due to Cubicciotti & Mason [1975], f limited availability of animals in the colony for which these relationship types were available, only females Callicebus cupreus were tested in Experiment 2. There was no overlap

access and with the test subjects in Experiment 1. The average Inglett et al. [1990]. Mason [1975],

m Stimulus animal e age of these subjects was 6.09 4.5 years (range: separate and simultaneous presentation type 2.42–15.83 years). All females lived with their pair mate and, if applicable, offspring during the course of the study. All females were tested with a stranger stimulus animal and a familiar stimulus animal. Familiar animals constituted the following Relation- Epple [1990], and l ship Types: (i) former mate (N ¼ 5) representing a former adult attachment, (ii) father (N ¼ 8) repre- but has since been updated to be senting a former infant attachment, or (iii) mother ¼ Fernandez-Duque et al., 2000,

Three chambersT shaped Grated; simultaneous Proximity, sexual Grated; both (N 8) representing no attachment at any life stage. d The average length of time that the test animal had been separated from the familiar stimulus animals was 1.7 1.4 years for former mates and 1.51 1.17

Callicebus moloch years for parents. Sixteen additional titi monkeys were used as the stranger stimulus animals during ed as

ﬁ these test sessions. As in Experiment 1, stranger Buchanan-Smith & Jordan [1992],

k animals were unfamiliar to the test animal and were Test length Apparatus ). approximately matched with the familiar stimulus animal on sex, age, and weight. trials trials Fernandez-Duque et al. [1997], c Saimiri Repeated 30 min Repeated 30 min Housing and Care ed into broad categories to make comparison between methodologies simpler. For more detailed information about exact behaviors measured, see compl ﬁ Animals in both Experiments 1 and 2 were l

) housed indoors, in two types of cages, based on where Cavanaugh et al. [2014], j they were located in the facility. One type of home

Anzenberger [1988],

b cage measured 1.2 m 1.2 m 2.1 m with four horizontal perches running the width of the cage in a staggered fashion. The second type of home cage m ) measured 1.2 m 1.2 m 1.8 m with four horizontal perches running the width of the cage in a staggered Smith et al. [2010], i fashion. In both rooms, animals were kept on a 12-hr rosalia Leontopithecus Saguinus fuscicollis ( ( light: 12-hr dark cycle (lights on at 0600 hr and off at Golden Lion Tamarins Brown-Mantled Tamarins Williams et al. [1992], In cases where trials involvedPreference multiple measure types has of been access classi to stimulus animals (e.g., removable barriersStudy were also in conducted place for on someAt squirrel conditions the monkeys and time ( removed of fo testing and publication, species was identi TABLE I. Continued Species a 1 2 3 4 the References. [1978], 1,800 hr). Temperature in both housing facilities was

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kept at approximately 21°C. All animals were Test Conditions provided the same diet consisting of monkey chow, Experiment 1 fruits, and rice cereal provided twice per day (at 0800 All tests were conducted with mates from and 1,300 hr), as well as additional availability of established pair bonds with two test sessions for enrichment with water available ad libitum. All each pair, one with the male as the test animal and housing conditions and experimental procedures one with the female as the test animal. Pair mates’ were approved by the University of California Davis tests took place within approximately 1 week of each Institutional Animal Care and Use Committee and other in order to minimize the potential effect of pair adhered to the American Society of Primatologists bond dynamics over time on results. principles for the ethical treatment of primates. Experiment 2 Test Apparatus In Experiment 2, animals were tested with one of three relationship types with familiar stimulus Figure 1 depicts the partner preference test animals: (i) former partner, (ii) father, and (iii) apparatus used in both Experiments 1 and 2. The test mother. These test conditions facilitated our ability apparatus consisted of three adjacent cages with to see whether the partner preference test could grated windows measuring 0.3 m 0.3 m connecting discriminate between a partner preference and a each of the side cages to the center cage. These grated more general social preference. The same females windows were made of a mesh wirework with were tested in conditions (ii) and (iii), with approxi- openings of 1.3 cm 1.3 cm and allowed full visual, mately 1 week separating each test session. The olfactory and minimal physical contact between the female test subjects in condition (i) were distinct from test animal in the center and that in either of the side those tested in conditions (ii) and (iii). cages which held the stimulus animals. Two perches 1.2 m long ran the width of the cage and were measured into three equal sections, each 0.4 m, and Procedure classiﬁed as either a preference zone (the one-third of At the beginning of the test sessions in both the perch closest to either grate) or a neutral zone Experiments 1 and 2, a visual block was put in front of (the middle third). All other areas of the cage were the three-cage apparatus to prevent visual access to considered no preference, or neutral, zones. The two other animals housed in the same room. Trained stimulus animals had visual access to one another personnel caught animals in their home cages and when both animals were located near their respective placed them into transport cages measuring 0.6 m grated windows at the same time. Likewise, when 0.3 m 0.3 m to be brought to the testing apparatus. stimulus animals were near their grated windows The two stimulus animals were released into the they could observe the test animal interacting with stimulus animal cages on each side of the test animal the other stimulus animal. While no other animals cage. The location of the two stimulus animals was were within the visual ﬁeld of the testing animals counterbalanced within each experiment, so that half there was auditory and olfactory contact with non- of the preference test sessions had the familiar (i.e., testing animals housed in the same room. partner, former partner, father, mother) stimulus

Fig. 1. Front view of the test apparatus for the partner preference test.

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animal located on the right of the test animal and the In Experiment 1, we recorded the proximity of reverse for the remaining test sessions. Half of test both stimulus animals (i.e., partner, stranger) to the sessions within each experiment took place in the test animal in order to assess whether their social morning and half in the afternoon. All animals were availability during the partner preference test provided food and water throughout the duration of affected the behavior of the test animal. This the test session. behavioral data, consisting of the stimulus animals’ Each test session consisted of 2.5 hr of focal proximity to the grated window, was collected from animal observations, which were split into five video recordings of the stimulus animal cages taken consecutive 30-min observations. Splitting observa- during Experiment 1 test sessions. This measure was tions into these 30-min blocks allowed us to evaluate similar to the preference zone designated for the test whether the animals’ preferences changed over the animal. These videos were scored using the DVRe- length of the test. The beginning of the first corder module of Behavior Tracker 1.5. Multiple observation was signaled by the release of the test trained observers were validated for video scoring animal into the center cage of the apparatus. The with greater than 85% observer reliability prior to final observation was followed by five catch and independent scoring, with approximately 10% of release preference sessions in which the test animal videos co-scored in order to ensure that reliability was caught from the center cage in a transport cage was maintained. and immediately released again into the center cage. Following release, the test animal’s preference was recorded as the first preference zone that the test Measuring Preference animal entered into and remained in for at least We evaluated three measures of preference in 3 sec. If the test animal did not enter a preference order to see which measures differentiated between a zone within 5 min, it was recorded as no preference partner preference for the pair mate (Experiment 1) choice. This measure of preference was included at and other types of relationships (Experiment 2). the end of the test session when the subjects would be First, preference was measured by proportion of total familiar with the location of the stimulus animals in time the test animal spent in the preference zone of relation to the center test cage. While all animals his or her familiar stimulus animal (Experiment 1: were familiar with transport and capture, this partner; Experiment 2: former partner, father, procedure provided a potential stressor to which mother) compared to the proportion of total time in the animals could respond by choosing to immedi- the preference zone of the stranger stimulus animal. ately seek proximity with one of the two stimulus Second, preference was measured as the proportion animals. Finally, we included this measure to of total time spent by the test animal touching the evaluate a potential simplification of the partner grated window that separates the center test cage preference test, whereby detailed behavioral data from the familiar stimulus animal cage (Experiment collection would be foregone, and instead, the catch 1: partner; Experiment 2: former partner, father, and release preference measure would be performed mother) compared to the proportion of total time the at the conclusion of a 2.5 hr habituation session in the test animal spent touching the grated window that test apparatus. In total, each test session lasted separates the center test cage from the stranger approximately 3 hr and 15 min. stimulus animal’s cage. Third, preference was measured as the test animal choosing the familiar stimulus animal (Experiment 1: partner; Experi- Behavioral Data Collection ment 2: former partner, father, mother) more often Behavioral data were collected during test than the stranger animal during the five catch and sessions via focal animal sampling with a defined release preference sessions at the end of each test ethogram of behaviors. The recorded behaviors session. focused on the physical location of the test animal including presence in the preference zone of either the familiar stimulus animal (i.e., current partner, Data Analysis former partner, father, mother) or stranger and Experiment 1 directly touching the grate of either stimulus animal. We utilized a t-test and Wilcoxon Rank Sum test Behavioral data were collected using Windows in R Statistical Software [version 2.15.3, R Core tablets (Dell Windows 8 Tablet) running the Record- Development Team, 2013] for the first two measures er module of Behavior Tracker 1.5 (www. of preference, respectively. For the first measure of behaviortracker.com). Multiple trained observers preference, we compared the proportion of total time were validated for live focal animal sampling with the test animal spent in the partner’s preference zone greater than 90% observer reliability prior to the to the proportion of total time the test animal spent in start of the study. Approximately 20% of observa- the stranger stimulus animal’s preference zone. For tions were co-scored between two observers to ensure the second measure of preference, we compared the that observer reliability was maintained. proportion of time spent touching the grate of the

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2 partner to the proportion of time spent touching the s2k ) and could co-vary among themselves (s01k, s02k, grate of the stranger. For the final measure of and s12k). In addition to these within-unit random preference, choosing the partner more than the effects, covariances between intercepts and slopes stranger during the catch and release sessions, we could also be allowed between individuals. In utilized a Friedman’s x2 test. practice, often due to data constraints (e.g., small As a measure of reliability of the partner sample size), restrictions can be imposed as needed in preference test, we evaluated how consistently the the covariance matrix of individual residual varian- test measured an individual animal’s preference. To ces T ¼ cov(Ent) and in the between-individual do this, we correlated the proportion of time animals covariance matrix S ¼ cov(Ut). spent in their partners’ preference zones when they This dyadic model allowed us to account for served as the test animals with the proportion of time statistical interdependence between pair mates in animals spent in the partners’ preferences zone when behavioral expressions of preference. We accounted they served as the stimulus animals. for interdependence by nesting male and female pair We utilized a dyadic mixed model in SAS PROC mates’ data within dyad-level dummy variables and MIXED (version 9.2, SAS Institute, Cary, NC) to regressing the dependent variables on these dummy assess whether or not the proportions of time the test variables [Laurenceau & Bolger, 2005; Raudenbush animal spent in the preference zone of the partner et al., 1995]. Using a mixed model allowed us to and stranger were consistent over the course of the account for the hierarchical structure in our data (i. 2.5 hr test by looking at each of the five 30-min e., repeated observations within individuals and the observations (Observation Number). With this mod- individuals within dyads) and incorporate random el, we also evaluated whether the proximity of the effects [Bryk & Raudenbush, 1987]. With the dyadic stimulus animals to the test animal (Stimulus Mate mixed model, we evaluated fixed effects, as described Proximity to Test Animal; Stimulus Stranger Prox- above (Observation Number, Stimulus Mate Prox- imity to Test Animal) influenced the proportions of imity to Test Animal, Stimulus Stranger Proximity time the test animal spent in the respective prefer- to Test Animal, Observation NumberStimulus Mate ence zone of these animals. Finally, with this model, Proximity to Test Animal, Observation Number- we also assessed the interactions between Observa- Stimulus Stranger Proximity to Test Animal). The tion NumberStimulus Mate Proximity to Test features of this model make it appropriate for Animal and Observation NumberStimulus Strang- evaluating preference as a behavioral characteristic er Proximity to Test Animal to see if the influence of that likely incorporates (but is rarely evaluated as the proximity of the stimulus animals on the test incorporating) a dyad interdependent component. animals’ behavior was consistent through the partner preference test. We used the following model in Experiment 2 these analyses: In Experiment 2, we compared across the different relationship types utilizing a General m ¼ S ðb þb þb þ Þ; Linear Mixed Model (GLMM) [Littell et al., 1996] Yntk dk 0nk 1nk Timentk 2nk Xntk entk fi k¼1 in SAS PROC GLM with xed effects for Relationship Type (partner, former partner, father, mother), Stimulus Animal (familiar stimulus, stranger stim- where Y is the outcome variable for dyad n,at ntk ulus) and the interaction of Relationship Type- occasion t, for each of the two units k in the dyad. Stimulus Animal with planned comparisons for Thus, d is a dummy variable d to d (0 or 1, in our k 1 m familiar stimulus versus stranger stimulus at each case), to indicate each unit in the dyad, b is the 0nk level of Relationship Type. We also included a intercept for unit k on dyad n, b is the slope 1nk random effect for individual animal in order to associated with time for each unit k on dyad n, b is 2nk account for some animals being tested for different the slope associated with the set of predictors X, for relationship types. We applied this GLMM to the first each unit k on dyad n, and e is the residual. In ntk two measures of preference, specifically the propor- theory, the intercepts and slopes for each of the units tion of time that test animals spent in the preference in the dyads can be decomposed as zones of the stimulus animals and the proportion of b ¼ b þ b ; time that the test animals spent touching the grates 0nk 0k 0nk of the stimulus animals. b ¼ b þ m ; fi 1nk 1k 1nk and For the nal measure of preference, we utilized a b ¼ b þ m ; GLMM with a fixed effect for Relationship Type 2nk 2k 2nk (partner, former partner, father, mother) and a random effect for individual animal. We applied which indicates that, for each unit k, the intercept this GLMM to the proportion of choices for the and slopes have fixed group means (b0k, b1k, and b2k) familiar stimulus animal (partner, former partner, and random residuals (u0k, u1k, and u2k), and these father, mother), the proportion of choices for the 2 2 residuals have variance components (s0k , s1k , and stranger, and the proportion of no preference choices

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during the catch and release sessions. For all partner to that of the stranger (Wilcoxon rank sum test: statistical tests in Experiments 1 and 2, a was set W ¼ 332, P ¼ 0.37; Fig. 2B). Subjects chose their at 0.05 and all tests were two-tailed. partner more than the stranger during the five catch 2 2 and release sessions (Friedman’s x test: x 23 ¼ 23.4, P < 0.001, Fig. 2C). RESULTS We found a significant positive correlation Experiment 1 between the proportion of time that individuals Current pair mates displayed a partner preference spent in the preference zone of their partner when on two of our three measures of preference. First, they were the test animal and the proportion of time subjects spent a significantly greater proportion of time the same individual spent in the stimulus animal in the preference zone of their partner than of the preference zone when their partner was the test ¼ ¼ stranger (t-test: t ¼ 4.84, df ¼ 46, P < 0.001; Fig. 2A). In animal (Pearson correlation: r 0.64, df 22, < contrast, there was no significant difference in the time P 0.001). This result indicates within-individual subjects spent touching the grated window of their consistency in partner preference regardless of the animal’s position in the test apparatus (Fig. 3).

Predictive parameters of spending time in the partner’s preference zone Table II presents the unstandardized linear regression coefficients of the fixed effects for our dyadic mixed model. These coefficients are presented separately for males and females, as they were not compared directly within the dyadic structure of the model. For females (Fig. 4A) and males (Fig. 4B), the proportion of time spent in the partner’s preference zone was not predicted by Observation Number, indicating consistency throughout the partner preference test. Likewise, the proportion of time male and female test animals spent in the partner’s preference zone was not predicted by the Stimulus Mate Proximity to Test Animal, indicating that the test animal’s location in the partner’s preference zone was not influenced by the location of their pair mate in the stimulus animal cage (Table II). Simi- larly, Stimulus Stranger Proximity to Test Animal did not predict the proportion of time that test animals spent in their partners’ preference zones (Table II). Finally, there were no significant

Fig. 2. Measures of preference when applying the partner preference test to current pair bonds in Experiment 1. A: Test animals spent a greater proportion of time in the preference zone of their mate compared to the stranger. B: Test animals did not Fig. 3. There was a positive association between the proportion differentiate between their mate and the stranger with touching of time that test animals (Experiment 1) spent in their partner’s the grated windows separating them. C: Test animals chose their preference zone and the proportion of time the same individual partner more than the stranger in the catch and release sessions spent in the preference zone of their partner when they served as at the end of the test. P < 0.001. Error bars indicate SEM. a stimulus animal for their pair mate.

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TABLE II. Partner Preference Test Parameters Predicting Proportion of Time in Preference Zone

Coefficient Coefficient (SE) t-value (SE) t-value

Partner’s preference zone Females Males

Intercept 0.37 (0.09) 3.96 0.36 (0.08) 4.44 Observation number 0.01 (0.03) 0.42 0.02 (0.02) 0.84 Stimulus mate proximity to test animal 0.06 (0.14) 0.44 0.32 (0.24) 1.35 Stimulus stranger proximity to test animal 0.15 (0.21) 0.72 0.13 (0.28) 0.49 Observation number stimulus mate proximity to test animal 0.003 (0.06) 0.06 0.07 (0.08) 0.94 Observation number stimulus stranger proximity to test animal 0.10 (0.09) 1.05 0.01 (0.12) 0.11

Stranger’s preference zone Females Males

Intercept 0.06 (0.05) 1.37 0.17 (0.05) 3.47 Observation number 0.05 (0.02) 2.43 0.003 (0.01) 0.24 Stimulus mate proximity to test animal 0.14 (0.09) 1.57 0.12 (0.14) 0.84 Stimulus stranger proximity to test animal 0.007 (0.13) 0.05 0.22 (0.17) 1.29 Observation number stimulus mate proximity to test animal 0.07 (0.04) 1.66 0.05 (0.05) 1.11 Observation number stimulus stranger proximity to test animal 0.08 (0.06) 1.42 0.07 (0.07) 0.92

P < 0.05. P < 0.001.

interactions between Observation NumberStimulus Mate Proximity to Test Animal or Observation NumberStimulus Stranger Proximity to Test Ani- mal, suggesting there was a consistent lack of inﬂuence of the stimulus animals on all test animals’ proportion of time in the partner’s preference zone throughout the test (Table II).

Predictive parameters of spending time in the stranger’s preference zone The proportion of time that female, but not male, test animals spent in the preference zone of the stranger stimulus animal was predicted by Observa- tion Number (Table II). Specifically, females spent a significantly greater proportion of time in the strangers’ preference zones through the course of the partner preference test (Fig. 4A). In contrast, Observation Number did not predict male test animals’ proportion of time spent in the preference zones of the strangers, indicating consistency throughout the partner preference test (Fig. 4B). The proportion of time that male and female test animals spent in the preference zone of the stranger was not predicted by Stimulus Mate Proximity to Test Animal nor by Stimulus Stranger Proximity to Test Animal, suggesting that the test animals’ time spent in the preference zone of the strangers was not influenced by the location of the partner or stranger in the stimulus animal cages (Table II). Similarly Fig. 4. Consistency of spending time in the partner and stranger’s preference zone throughout the partner preference there were no significant interactions for Observa- test for males (A) and females (B) in Experiment 1. Males and tion NumberStimulus Mate Proximity to Test females were consistent across the test in the proportion of time spent in the partners’ preference zone. Males were consistent in Animal or Observation Number Stimulus Stranger the proportion of time spent in the strangers’ preference zones Proximity to Test Animal, meaning that this lack of throughout the test. Females spent a significantly greater influence on the location of the partner or stranger in proportion of time in the strangers’ preference zones as the partner preference test progressed. P < 0.05. Error bars indicate the stimulus animal cages was consistent through- SEM. out the partner preference test (Fig. 4A and B).

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Inter-individual variation in partner preference The dyadic model reports significant intercepts (Table II) for both males and females along with significant variance around these intercepts (Dyadic mixed model: female variance: B ¼ 0.04, P < 0.05; male variance: B ¼ 0.04, P < 0.05) on the proportion of time spent in the partner’s preference zone. This result indicates significant individual differences in the proportion of time subjects spent in their partners’ preference zones during the first 30 min of the partner preference test. Combined with the non-significant effect of Observation Number for females and males, these individual differences in partner preference were consistent over time through the course of the partner preference test. Similarly, males displayed significant inter- individual differences in the proportion of time they spent in the strangers’ preference zones during the first 30 min of the partner preference test, as indicated by a significant intercept (Table II) and variation around that intercept (Dyadic mixed model: B ¼ 0.01, P < 0.05). Again, combined with a non-significant effect of Observation Number, this indicates that individual differences among males were consistent throughout the test. In contrast, there was no significant intercept for females, indicating similarity across females in the proportion of time spent in the strangers’ preference zone during the first 30 min of the partner preference test. This result, combined with the significant effect for Observation Number, suggests that females became less similar to one another in their proportion of time in the stranger’s preference zone throughout the partner preference test.

Experiment 2 For the proportion of time spent in the preference zones, there was no significant effect for Relationship Fig. 5. Measures of preference when applying the partner Type (GLMM: F(3, 65) ¼ 1.03, P ¼ 0.39) or the preference test across different types of relationships in Experi- ments 1 and 2. A: Only in the case of a current partner did test interaction of Relationship Type Stimulus Animal animals spend a significantly greater proportion of time in the (GLMM: F(3, 65) ¼ 1.02, P ¼ 0.39). However, there preference zone of the familiar stimulus animal compared to the was a significant effect of Stimulus Animal on the stranger. B: In the case of a former infant attachment to the father, test animals displayed a trend to spend a greater proportion of time spent in the preference zones proportion of time touching the grated window of the familiar (GLMM: F(1, 65) ¼ 5.26, P < 0.05). Planned compar- compared to the stranger stimulus animals. C: Test animals chose the familiar stimulus animal more frequently when tested isons of the Relationship Type Stimulus Animal with their current partner compared to when they were tested interaction for familiar stimulus versus stranger with their mother or father. Test animals also chose the stranger stimulus at each level of Relationship Type found less frequently when tested with their current partner compared to when they were tested with their mother or father. P < 0.01; that only in the partner condition subjects spent a P < 0.05. #P ¼ 0.07. Error bars indicate SEM. significantly greater proportion of time in the preference zone of the familiar stimulus (i.e., partner) compared to the stranger (t ¼ 2.95, planned comparisons between familiar versus P < 0.01; Fig. 5A). stranger for the Relationship TypeStimulus Animal For the proportion of time spent touching the interaction at each level of Relationship Type grated windows of the stimulus animals, there were revealed a trend for females to spend a greater no significant fixed effects observed for Stimulus proportion of time touching the grate of their father Animal (GLMM: F(1, 65) ¼ 1.84, P ¼ 0.18) or the compared to the stranger stimulus male (t ¼ 1.85, interaction between Relationship TypeStimulus P ¼ 0.07; Fig. 5B). The fixed effect of Relationship Animal (GLMM: F(3, 65) ¼ 0.68, P ¼ 0.57). However, Type was not estimated.

Am. J. Primatol. Nonhuman Primate Partner Preference Test /11

The proportion of choices for the familiar stimu- subject spent touching the grated window of their lus animal compared to choices for the stranger partner compared to that of the stranger indicating stimulus animal during the catch and release this measure was not a reliable measure of partner sessions was significantly predicted by Relationship preference within the test. Finally, we found that test Type (GLMM: F(3, 23) ¼ 5.53, P < 0.05; Fig. 5C). subjects chose the preference zone of their partner Post-hoc tests revealed that the proportion of choices significantly more than the preference zone of the for the current partner was significantly higher than stranger during the catch and release preference choosing the father (t ¼3.65, P < 0.01) or mother sessions at the end of the test. This result indicates (t ¼3.91, P < 0.01), but not different from the that these sessions could be used as a simplified former partner (t ¼0.31, P ¼ 0.76). version of the partner preference test, but may be Relationship Type did not predict the proportion superfluous when combined with measuring the of choosing the stranger stimulus animal during the proportion of time spent in the preference zones. catch and release sessions (GLMM: F(3, 23) ¼ 2.41, Based on these results, Experiment 1 suggested that P ¼ 0.13; Fig. 5C). However, post-hoc tests revealed spending time in the partner’s preference zone and that the proportion of choices for the stranger were choosing the partner during the catch and release significantly lower when animals were tested with sessions serve as candidate behaviors representing their current partner compared to their father partner preference within this new paradigm. (t ¼ 2.55, P < 0.05) or mother (t ¼ 2.21, P < 0.05) but Results from Experiment 1 indicated that did not differ from the proportion of choices for the spending time in the partner’s preference zone stranger when they were tested with their former reliably measured individual animal’s preference partner (t ¼7.33, P < 0.48). Relationship Type did for his or her partner. Specifically, we found that the not predict the proportion of no preference choices proportion of time spent in the partner’s preference during the catch and release sessions (GLMM: F(3, zone was highly correlated between trials when an 23) ¼ 0.87, P < 0.49; Fig. 5C). individual served as the test subject and when the same individual served as the stimulus partner for his or her pair mate. We interpret this finding as an DISCUSSION indication of high within-individual consistency in Titi monkeys and other socially monogamous preference to remain in proximity to one’s partner, primates establish pair bonds that are largely irrespective of the position of the monkey as the test maintained by a preference for their partners over subject or stimulus animal. strangers, which is expressed by maintaining prox- As a behavioral construct, one individual’s imity to their partner. Our goals in the current study preference for his or her mate is likely influenced were to develop a primate partner preference test by the reverse preference from that mate to the first that was adapted from the standardized paradigm partner. Despite the bidirectionality of this con- used in rodents and to assess whether it would be a struct, partner preference is typically measured as a reliable way to measure partner preference via time behavioral characteristic of a single individual. We spent in the preference zone of one’s partner in titi consider it a strength of the current study that we monkeys. Our intent in developing this new partner accounted for the bidirectionality of partner prefer- preference test was to further our understanding of ence in Experiment 1 by testing both pair mates in the role of partner preference in social monogamy the partner preference test and incorporating part- and to specifically facilitate more direct comparison ner interdependence into our statistical model. This within primates and across other socially monoga- approach allowed us to account for pair level effects mous mammals. when evaluating which parameters of the partner Our goal in Experiment 1 was to evaluate this preference test were predictive of spending time in new partner preference test using established pairs the partner and strangers’ preference zones. Within of titi monkeys. We measured partner preference via this dyadic statistical approach, we found that the the test animal’s behavior in three ways. First, we availability of the partner or stranger at the grated found that titi monkeys spent significantly more time interaction window did not predict the proportion of in the preference zone of their partner than that of time test animals spent in the preference zones of the stranger. We observed individual differences either stimulus animal, which indicates that the within both males and females in the proportion of tendency of the test animals to spend more time near time they spent in their partners’ preference zones. their partners is a valid measure of preference However, all individuals maintained a preference for outside variable behavior of the stimulus animals. their partner throughout the course of the test. We Taken together, these findings indicate that individ- interpret this as an advantage of this new test to ual titi monkeys behave in a consistent manner detect differences across individuals and relation- regardless of whether they are the test subject or ships in partner preference, which we intend to stimulus animal and that this consistent behavior is describe in future studies. Second, we found no not affected by the physical location of either difference in the proportion of time that the test stimulus animal (partner or stranger).

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Finally, in Experiment 1, we found a different this social partner is the one with which titi monkeys temporal pattern across sexes in the test animal’s arguably share the longest period of physical contact. interaction with the opposite sex strangers. First, we Finally, we found that the catch and release found that males display consistent individual sessions at the end of the partner preference test differences throughout the partner preference test differentiated between adult and former infant in the time they spend in proximity with the female attachment bonds. Specifically, we found that female strangers. Despite these differences, all males still titi monkeys chose their partners (both current and spent a greater proportion of time in the test near former) over strangers more frequently than choos- their partners compared to strangers. In contrast, ing their mother or father over strangers. However, female proximity to the male stranger increased over there was no difference between the test animal time in the partner preference test. Although these choosing the current partner over a stranger and sex differences may be viewed as a limitation to the choosing their former partner over a stranger. validity of the partner preference test, we interpret Moreover, titi monkeys tested with their partner this finding as a presentation of species typical sex (either current or former) and a stranger chose the differences consistent with previous research. It has stranger less frequently than titi monkeys tested been demonstrated in titi monkeys that male and with their mother and a stranger or their father and a female pair mates respond differently to the presen- stranger. Because this potential measure of prefer- tation of a stranger animal of the opposite sex. Male ence does not differentiate between current and titi monkeys tend to be more proactive in approach- former adult attachment bonds, we do not recom- ing unfamiliar females, both alone and in the mend utilizing the catch and release session alone presence of their mates, while females are more within this new paradigm. likely to rebuff stranger male attempts at contact in In summary, across Experiments 1 and 2, a both settings [Anzenberger, 1988; Fernandez-Duque single measure of partner preference emerged for et al., 1997]. Thus, we interpret the temporal change established pair bonds that did not generalize to for females as titi monkey females becoming less other relationship types. Specifically, this measure inhibited over time during the course of the test. was time spent in the partner’s preference zone, In Experiment 2, we applied the partner prefer- which broadly translates to individuals attempting ence test to different relationship types with the goal to maintain proximity with their pair mates. to see if the preference test could differentiate Therefore, we recommend using this measure in between a partner preference and a more general future uses of this partner preference test. These social preference. Specifically, we evaluated relation- results are supported by a large body of literature in ships that represent former attachment bonds for socially monogamous primates, including titi mon- adult female titi monkeys (i.e., former partner, keys, that has demonstrated consistent results of a father) and a relationship that presumably never proximity preference for the partner thereby lending constituted an attachment bond (i.e., mother) and credibility to the accuracy of this new partner compared responses to the preference test setting to preference test [e.g., Anzenberger, 1988; Fernan- those displayed when tested with a current adult dez-Duque et al., 1997; Mason, 1975; Smith et al., attachment (i.e., partner) from Experiment 1. We 2010]. We propose this new partner preference test found that female titi monkeys differentiated be- has several strengths in that it provides consistent tween their familiar social partner (i.e., partner, patterns of partner preference across individuals of former partner, father, mother) and the stranger in both sexes while still being able to detect consistent time spent in preference zone—a proxy measure of individual differences. Further, this test also detects proximity maintenance—only in the case of current species specific behavior patterns expected for titi adult attachment partner. Therefore, we interpret monkeys based on previous research. One caveat of time spent in the preference zones of the stimulus the current study was that we were only able to test animals in the partner preference test as a valid female titi monkeys in Experiment 2. However, measure by which to differentiate a preference for a based on the fact that we found similar patterns of current attachment partner compared to other partner-related behaviors in both sexes in Experi- relationship partners for female titi monkeys. ment 1, we expect we would find similar validation of In contrast, we found that touching the grated the partner preference test when applied to adult window of the stimulus animals tended to differenti- male titi monkeys. ate between a former infant attachment and other Future studies in titi monkeys, and other socially social preferences. Specifically, only in the case of monogamous primates, can use this paradigm to tests with the father did titi monkeys tend to touch evaluate a variety of research questions. First, it the grated window of the familiar stimulus more would be interesting to use this paradigm to than the stranger stimulus animal. Because titi determine the effect of pair bond length on partner monkey fathers primarily carry infants [Mendoza & preference. There is some evidence that behavioral Mason, 1986], we suggest that this measure of indicators of pair bond formation, such as mating, preference may be specific to the father because time in proximity, and time in contact with a new

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mate, are highly variable between pairs in the first necessary for adult social attachment in monogamous 48 hr of pair introduction [Bales et al., 2007]. prairie voles. Neuropsychopharmacology 36:2200–2210. However, no studies to date have characterized Carter CS, DeVries AC, Getz LL. 1995. Physiological substrates of mammalian monogamy: the prairie vole how these behavioral indicators of pair bond forma- model. Neuroscience and Biobehavioral Reviews tion change over the course of the bond (i.e., 19:303–314. measures of pair bond maintenance) or when Cavanaugh J, Mustoe AC, Taylor JH, French JA. 2014. precisely the preference for one’s partner emerges Oxytocin facilitates fidelity in well-established marmoset pairs by reducing sociosexual behavior toward opposite-sex after pairing. In addition, future studies using strangers. Psychoneuroendocrinology 49:1–10. behavioral pharmacology in New World monkeys Cho MM, DeVries AC, Williams JR, Carter CS. 1999. The are now possible using this validated paradigm. For effects of oxytocin and vasopressin on partner preferences in example, it is possible to design experiments analo- male and female prairie voles (Microtus ochrogaster). – gous to those performed in prairie voles and other Behavioral Neuroscience 113:1071 1079. Cubicciotti DD III, Mason WA. 1975. Comparative studies of monogamous or highly social rodents to determine social behavior in Callicebus and Saimiri: male-female the contribution of various neuropeptide (oxytocin, emotional attachments. Behavioral Biology 16:185–197. vasopressin) and neurotransmitter (dopamine, sero- Cubicciotti DD III, Mason WA. 1978. Comparative studies of tonin, opiate) systems to pair bond formation and the social behavior in Callicebus and Saimiri: heterosexual jealousy behavior. Behavioral Ecology and Sociobiology expression of partner preference in nonhuman 3:311–322. primate species. Epple G. 1990. Sex differences in partner preference in mated pairs of saddle-back tamarins (Saguinus fuscicollis). Be- havioral Ecology and Sociobiology 27:455–459. ACKNOWLEDGMENTS Fernandez-Duque E, Huck M. 2013. Till death (or an intruder) do us part: intrasexual-competition in a monogamous This work was supported by a National Science primate. PloS ONE 8:e53724. Foundation Graduate Research Fellowship to ESR, Fernandez-Duque E, Mason WA, Mendoza SP. 1997. Effects of HD053555, Office of Research Infrastructure Pro- duration of separation on responses to mates and strangers grams P51OD01107, and the Good Nature Institute. in the monogamous titi monkey (Callicebus moloch). American Journal of Primatology 43:225–237. Thanks to members of the Bales lab who assisted Fernandez-Duque E, Valeggia CR, Mason WA. 2000. 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