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Insectes Sociaux (2019) 66:153–163 https://doi.org/10.1007/s00040-018-00679-4

RESEARCH ARTICLE

The influence of sociality, caste, and size on behavior in a facultatively eusocial bee

A. Smith1 · M. Simons1,3 · V. Bazarko4 · M. Seid2

Received: 2 July 2018 / Revised: 5 October 2018 / Accepted: 17 November 2018 / Published online: 28 November 2018 © International Union for the Study of Social Insects (IUSSI) 2018

AbstractSocial cooperation requires increased tolerance of other individuals. We used social and solitary individuals of the faculta-tively eusocial bee Megalopta genalis to compare interactions with non-nestmate individuals in a standardized behavioral assay, a circle tube. We set up interactions between bees from different nests matched for caste (solitary, social: queen or worker). We found more tolerance in social than solitary pairs, but found no difference in aggression. We also found that workers continued expressing caste-typical behavior even when matched against other workers from different nests. However, there was no difference in expression of queen-typical behaviors between the three groups. Our data on social caste show that outside of the queen–worker social context, both queens and workers express similar levels of queen-like behavior. However, workers still express higher levels of worker-like behavior than do queens. We found no effect of variation in ovary size on behavior. We found that body size correlated positively with queen-like behaviors, and negatively with worker-like behav-iors. Our body size data suggest that the worker phenotype may result from naturally occurring size-correlated variation in behavior, combined with maternal manipulation of both body size through nutrition and behavior and ovary development through social aggression.

Keywords Circle tube · Maternal manipulation · Social evolution · Caste · Aggression

Introduction

A basic prerequisite of social cooperation is tolerating the presence of other individuals without aggression (Wilson 1971, Lin and Michener 1972, Brockmann and Dawkins 1979). Among the nesting Hymenoptera, solitary species are typically intolerant of other individuals in their nests, while social species, whether eusocial or communal, must tolerate the presence of nestmates (Michener 1974, West-Eberhard 1987). However, the influence of social or solitary behavior on aggression has never been directly measured in the same

species because most species are either solitary or social. A major challenge for social species is nestmate recognition: how to keep out intruders but let in nestmates (Reeve 1989, Sherman et al. 2009, Breed 2014). Theory predicts that if the criteria for entry are strict enough to bar all non-nestmates they will also exclude some nestmates. But if criteria are lenient enough to admit all nestmates, they will also permit some intruders to enter (Reeve 1989, Sherman et al. 2009). However, solitary insects do not have this dilemma: they can treat any other individual in the nest as an intruder. Thus, we hypothesize that solitary individuals will generally show less tolerance of non-nestmates than will social ones. To test this hypothesis we compare the behavior of social and solitary individuals of the same species.

Facultatively eusocial bees can nest solitarily or in a social group with a queen and worker(s). Comparing social and solitary nests of the same species offers the opportunity to study the behavioral transitions involved in expressing the two phenotypes (Schwarz et al. 2007, Kocher and Pax-ton 2014, Shell and Rehan 2017). In this case, we hypoth-esize that as part of the social phenotype, socially nesting individuals are more tolerant and less aggressive towards

Insectes Sociaux

* A. Smith adam_smith@gwu.edu

1 Department of Biological Sciences, George Washington University, Washington, USA

2 Biology Department, University of Scranton, Scranton, USA3 Department of Ecology and Evolutionary Biology, University

of Michigan, Ann Arbor, USA4 Department of Ecology and Evolutionary Biology, Princeton

University, Princeton, USA

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non-nestmates than solitary conspecifics are towards non-nestmates. In these nests, social individuals must balance the probability of excluding a nestmate with the probability of admitting an intruder (e.g., Bell 1974). For this study, we use the facultatively eusocial or solitary halictid bee Mega-lopta genalis, which can nest either solitarily or eusocially (in small groups with one queen and one–three workers) in the same population in central Panama (Wcislo et al. 2004, Kapheim et al. 2012).

Circle tubes are a standardized behavioral assay to com-pare levels of aggression. A circle tube is particularly well suited to measure non-nestmate agonistic behaviors because it is a neutral arena with no effects of nest ownership. Pre-vious studies of bees using circle tubes have shown that primitively social species tend to discriminate between nestmates and non-nestmates, while communal species are less discriminating and often tolerate unfamiliar individu-als (reviewed by McConnell-Garner and Kukuk 1997, Peso and Richards 2010, Rehan and Richards 2013, Dew et al. 2014). The behavior of larger colony eusocial species appar-ently depends on the genetic structure of the colony and caste (foundress queen vs. worker) tested. Eusocial bees may be more aggressive towards non-nestmates, especially as reproductive nest foundresses (Smith and Weller 1989, Pabalan et al. 2000). However, species in which nestmates are less genetically related tend to be more tolerant of non-nestmates (Polidori and Borruso 2012, Gonzalez et  al. 2018). Solitary nesting species are generally characterized by high levels of avoidance in circle tubes, and less aggres-sion and tolerance (Packer 2006, Richards and Packer 2010). However, Wcislo (1997) showed that in the sweat bee Lasi-oglossum figueresi, which was usually solitary (~ 80% of nests), the few nests with multiple females recognized nest-mates. Flores-Prado et al. (2008) showed that even strictly solitary bees had the ability to recognize nestmates using dead individuals from previous generations of the same or different nests. Pentane washes of these dummy “intruders” erased any differences, which suggested a chemical basis for the template to recognize “self” vs. “intruder” (Flores-Prado et al. 2008). However, no study has ever compared social and solitary individuals of the same species to test whether the expression of social cooperation entails a generalized reduction in aggression and increase in tolerance relative to solitary nesters.

A complication with comparing social and solitary indi-viduals is that social individuals have castes (queen and worker) while solitary nesters do not (Smith et al. 2009, Kapheim et al. 2012). A previous study on M. genalis using circle tubes showed that queens and workers exhibited caste-specific behaviors in nestmate interactions (that is, inter-actions between the queen and worker of the same nest in a circle tube; Smith et al. submitted). To control for this, we used caste-matched non-nestmate pairings: two solitary

females from different nests in a circle tube, two queens from different nests in a circle tube, and two workers from different nests in a circle tube.

Even in circle tubes, when bees are removed from their in-nest social context, the effects of social caste may persist. In M. genalis specifically, and primitively eusocial sweat bees more generally, the worker caste phenotype develops from social aggression directed from the queen to the worker and results in suppression of ovarian development and other associated physiological effects (e.g., Michener et al. 1990, Kapheim et al. 2012, 2016, Smith et al. 2013). We previ-ously showed that worker sterility in M. genalis is reversible over a period of weeks when the queen is removed from the nest (Smith et al. 2009). However, in circle tube tests performed the same day as nest collection, we predict that queens will express queen-like behavior, and workers will express worker-like behavior even when matched against non-nestmates of the same caste. The alternative hypothesis is that these caste-typical behaviors disappear outside of the queen–worker social context.

Lastly, previous studies of bees in circle tubes have found effects of body size and ovary development on behavior, with larger bees and bees with larger ovaries sometimes (but not always) more aggressive and dominant (Wcislo 2004, McConnell-Garner and Kukuk 1997, Pabalan et al. 2000, Arneson and Wcislo 2003, Richards and Packer 2010, Rehan and Richards 2013, Lawson et al. 2017). Our previous stud-ies of M. genalis nesting biology showed that queens and solitary reproductives are larger bodied and have larger ova-ries than workers (Smith et al. 2009, Kapheim et al. 2012, 2013). We predict that body size and ovary size will be posi-tively correlated with aggression and queen-like behavior, and negatively correlated with worker-like behavior.

Here we test predictions from three hypotheses. First, we predict that solitary females will show less tolerance, more aggression, and more avoidance behaviors in circle tubes with non-nestmates than social females will. Second, we predict that caste-typical behaviors of queens and workers will persist even when paired against non-nestmates of the same caste, outside of their normal social dominance hierar-chy. Third, we predict that body size and ovary development will influence behavior in non-nestmate interactions similar to nestmate interactions. This is the first paper to compare interactions with non-nestmates in social and solitary indi-viduals from the same species while controlling for caste.

Methods

Research site and study organism

We collected all bees for this study from the forest of Barro Colorado Island, Panama (BCI; 9°09′N, 79°51′W). We

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have previously described the biology of Megalopta genalis (Augochlorini, Halictidae) (Smith et al. 2003, Wcislo et al. 2004, Kapheim et al. 2012). In summary, a foundress female constructs a tunnel nest in a dead stick suspended in the vegetation. Bees forage during the 60–90 min before sunrise and after sunset; they are inside their nest during the day (Kelber et al. 2006, Smith et al. 2017). M. genalis is faculta-tively solitary or eusocial; in some nests, the first daughter remains in the nest as sterile, subordinate worker, and the original foundress now becomes a social queen. Social nests typically have 1–3 workers (mode = 1). In other nests, all offspring disperse about 1 week after adult emergence, and the foundress remains a solitary reproductive. Thus, solitary females do have interactions with their offspring (subsocial, sensu Plateaux-Quénu 2008). In social nests, the workers perform all foraging trips, and the queen rarely leaves the nest. Reproductive division of labor is maintained by social dominance; workers can enlarge their ovaries and reproduce if the queen is removed (Smith et al. 2009). Workers are typ-ically smaller than queens and solitary reproductives, reflect-ing reduced pollen provisioning by the foundress (Kapheim et al. 2011, 2012).

Collections and experimental trials

We collected nests the morning of the circle tube trial, dur-ing daylight when all bees were inside. We plugged the nest entrance with cotton wool, wrapped the stick containing the nest in a plastic bag, and brought it back to the BCI lab facilities. We refrigerated collected stick nests for ~ 15 min and then opened the nests. Nests were designated solitary or social based on the number of adult females found inside. For social nests, we assumed that the largest female was the queen. We later confirmed worker and queen status by ovar-ian dissection (see below). Following ovary dissection, if the putative queen did not have the largest ovaries or if one of the workers appeared to be a newly emerged female rather than a mature worker, that pair was excluded from the study.

To determine if some behaviors were typically “queen-like” and others “worker-like”, we performed circle tube tri-als between the queen and one worker from the same nest (N = 21 nests). Individuals were not marked because they were distinguishable by size difference.

For the non-nestmate trials, we marked both individuals on the wing with Decocolor paint pens to distinguish them. We marked both individuals in case marking influenced behavior (Packer 2005). All trials were run the same day as the nests were collected. Queen–queen, solitary–solitary, and worker–worker trials were between non-nestmates, each from a different nest collected the day of the trial. All bees were used only once.

Queen–worker trials were conducted from June to July 2015 and February to May of 2016. All other trials were

conducted from February to May 2016. After opening the nests and marking each bee (bees were not marked in the queen–worker trials), we placed each bee in a 1.5-ml cen-trifuge tube with the end cut off and plugged with cotton wool for 15 min at ambient temperature. To begin the trial, we removed the cotton from each tube and placed the open end into the end of a 30-cm tube of 8-mm-inner diameter clear flexible PVC tubing. One bee was inserted at each end, and the ends of the tube were joined with a length of wider tubing to secure the tube in a circle. We recorded each tube for 15 min using a Logitech c920 camera. Tri-als were run in ambient temperature under natural light between 14:00 and 17:00 local time.

We freeze killed bees after each trial. We measured head width as a measure of body size, and dissected the abdomen to measure ovary size by photographing the ovaries dorsally at 10x magnification through a dissecting microscope and measuring their total area using ImageJ following Smith et al. (2008, 2009). We calculated rela-tive values for head and ovary size by dividing the value for each bee by that of the bee to which it was matched in the circle tube.

We scored videos using the ethogram of behaviors modified from Kapheim et al. (2016) and Dew et al. (2014; Table 1). Videos were scored blind to experimental group (queens, workers, or solitary) and individual identity; queen–worker trials could not be scored blind due to the obvious size difference between the castes. We designated queen-like and worker-like behaviors based on the results of the queen–worker trials (see “Results”). We used pre-vious studies of bees in circle tubes to designate tolerant, aggressive, and avoidance behaviors (Dew et al. 2014, Law-son et al. 2017, Gonzalez et al. 2018). ‘Pass’, ‘antennate’, ‘mandible touch’, and ‘head–head’ were designated tolerant behaviors. ‘Back up’ and ‘reverse’ were designated avoid-ance behaviors. ‘Push’, ‘nudge’, ‘bite’, and ‘back into’ were designated as aggressive behaviors. Most previous studies included ‘C-posture’ as an aggressive behavior; however, our work with M. genalis in both circle tubes and natural nests suggests that it is a defensive behavior in this species, so here we analyze it separately (Smith et al. 2003, Kapheim et al. 2016; see below in “Results” for evidence that C-pos-ture is not associated with the other behaviors we designate as aggressive). ‘Follow’ behavior has been described incon-sistently in previous studies as well, so here we analyze it separately (Dew et al. 2014). Our behavior ‘chase’ may have been included in ‘follow’ by previous authors, so we also analyze the two together for consistency with the literature. Note that the queen- and worker-like designations are not exclusive of the tolerance, avoidance, and aggression des-ignations; for instance, reverse is part of both ‘worker-like behaviors’ and ‘avoidance’ behaviors, and all of the ‘aggres-sive’ behaviors are also ‘queen-like’ (see “Results”).

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Ovary area and head width were normally distributed continuous variables, and analyzed with standard paramet-ric statistics. Even though we used body size and ovary size to define queens and workers within a social nest, we still tested queen–worker body size differences across the study as between-nest variation might overwhelm within-nest variation. All of the recorded behavioral vari-ables were count variables with non-normal distribution and many zeros. Thus, we used Wilcoxon paired rank tests to compare queens and workers from the same nest, Mann–Whitney U tests to compare solitary and social groups, and Kruskal–Wallis tests followed by Dunn–Bon-ferroni post hoc pairwise comparisons when comparing queen, worker, and solitary results. Because we were measuring social interactions between two individuals, the behaviors of each bee are not independent of the other bee that it was in a circle tube with. Thus, for the non-nestmate trials we used each circle tube pair, rather than each bee, as the unit of analysis. However, when analyzing correla-tions between behavior and body size or ovary size, we used the individual as the unit of analysis. When analyz-ing queen–worker trial results, we used the individual as the unit of analysis. We used Pearson correlations when comparing ovary size and head width, and Spearman cor-relations for all comparisons involving circle tube behav-iors. Because activity levels may vary between groups, we also analyzed behaviors as a proportion of total behaviors. To calculate this proportion, we divided the number of observations of the focal behavior for each bee by the total number of all observed behaviors for that individual. We then summed the proportions of the two bees in a pair. All statistics were performed in SPSS v. 21.

Results

We designated the following behaviors as ‘queen-like’ because they were expressed significantly more by queens than workers in the queen–worker trials: ‘push’, ‘back into’, ‘bite’, back up’, ‘nudge’ and ‘chase’ (see Table 2 for means and statistics). We include ‘nudge’ with the queen-like behaviors even though the queen–worker dif-ference was marginally non-significant (p = 0.06) because a larger sample of queen–worker trials used for a separate study on aggression (Smith et al., submitted) showed sig-nificantly more nudges in queens (N = 42 queen–worker pairs, p < 0.001). ‘Worker-like’ behaviors consist of ‘C-posture’ and ‘reverse’; these behaviors were performed more often by workers than queens. All further results presented below refer to the non-nestmate circle tube tri-als: queen–queen, worker–worker, and solitary–solitary.

We recorded circle tube trials from seven pairs of queens, seven pairs of workers, and seven pairs of solitary females. In one of the worker–worker trials, the two bees performed no behavior of any type; these were excluded from analysis, leaving six pairs of workers.

Hypothesis 1: differences between social and solitary nests

Solitary bees showed significantly less tolerance than the social bees (U = 4.60, p = 0.03; Fig. 1a. Median soli-tary ± IQR = 14 ± 16, social = 42 ± 58). When all three groups were compared separately there were no significant

Table 1 Ethogram of behaviors included in the study (Modified from Dew et al. (2014), Kapheim et al. (2016) Statistics)

Behavior Definition

C-posture Abdomen curled anteriorly under head to present sting and mandibles to the other beeNudge Quick contact with the other bee; forward movement toward the other bee and backward again without pause in betweenPush One bee applies force to another with its headBack into One bee backs up, pushing the other with its abdomenNip One bee closes mandibles < 1 cm from the other beeBite One bee closes mandibles around a body part of another beeBack up One bee walks backwards from an interaction without turning aroundReverse One bee turns around so that its abdomen is facing the other bee before walking away while facing forwardsFollow One bee follows another at a walking pace after they either back or reverse out of an interactionChase One bee quickly pursues another that is moving away. Distinguished from follow by moving at faster than normal walking

speedAntennate One bee touches its antennae to the head of the other beeHead–head Both bees have heads touching each other without pushingPass Bees walk past each other in the tubeMandible touch Bees touch opened mandibles without biting or offering trophallaxis

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differences (Kruskal–Wallis H = 5.67, p = 0.06, Fig. 1a). The difference in tolerance behaviors was driven largely by significant differences in the behaviors pass (U = 72.0, p = 0.03; median social = 16 ± 34, solitary = 2 ± 4) and antennate (U = 70.5, p = 0.046; median social = 18 median social = 18 ± 16, solitary = 10 ± 15 16, solitary = 10 ± 15). There were no differences between social and solitary bees in the frequencies of avoidance behavior (U = 49.5, p = 0.76; Fig.  1b) or aggressive behavior (U = 32.5, p = 0.31; Fig. 1c). There was no difference in the behavior ‘follow’ between social (including both queens and work-ers) and solitary bees (U = 57.5.5, p = 0.35) nor was there a difference with ‘follow’ and ‘chase’ combined together (U = 56.5, p = 0.39). The behavior ‘C-posture’ negatively correlated with aggressive behavior (rho = − 0.46, N = 20, p = 0.04).

Overall activity level (the sum of all recorded behaviors) was affected by caste (Kruskal–Wallis H = 6.34, p = 0.04; median queen = 188 ± 81, median solitary = 126 ± 54, median worker = 191 ± 157). We thus also analyzed behav-iors as the relative proportions of all behaviors to account for variation in activity levels. When analyzed as relative proportions, differences between social and solitary bees approached but did not reach significance in tolerance (U = 2.90, p = 0.09), avoidance (U = 3.47, p = 0.06) and aggression (U = 3.47, p = 0.06; Fig. 1d–f). When all three

groups were compared separately, aggression significantly differed because workers showed proportionally less aggres-sion than either queens or solitary females (Kruskal–Wallis H = 10.48, p = 0.005; worker–queen pairwise Bonferroni-corrected p = 0.02, worker–solitary p = 0.01, queen–solitary p = 1.0; Fig. 1f).

Hypothesis 2: caste‑typical behaviors in the absence of social hierarchy

Workers expressed more worker-like behaviors in worker–worker pairs than did either queens in queen–queen pairs or solitary reproductives in solitary–solitary pairs (Kruskal–Wallis H = 13.46, p < 0.001. Queen–worker pair-wise p = 0.001, solitary–worker pairwise p = 0.07, soli-tary–queen pairwise p = 0.48; Fig. 2a). Both worker-like behaviors, ‘C-posture’ and ‘reverse’, were expressed more by workers (C-posture: H = 13.85, p = 0.001; queen–worker pairwise p = 0.001, solitary–worker pairwise p = 0.12; reverse: H = 6.77, p = 0.03; queen–worker pairwise = 0.03, solitary–worker pairwise p = 0.31). There was no differ-ence between queens, workers, or solitary females in the expression of queen-like behavior (H = 0.62, p = 0.73; Fig. 2b). Results were similar when analyzed as propor-tions. Proportion of worker-like behavior significantly dif-fered between groups (H = 10.82, p = 0.004. Queen–worker

Table 2 Summary statistics of behaviors from the queen–worker trials

The test statistic (W) and p value for Wilcoxon paired rank tests (N = 21 queen–worker pairs for each behavior) are presented. Behaviors in bold type significantly differed between queens and workers. Means and standard deviations for the occurrence of each behavior among queens and workers are presented, but note that the Wilcoxon test compares paired rank rather than overall means. For behaviors in which many pairs showed a tied rank, no test statistic could be calculated (NA in table)

Wilcoxon paired rank test results

Queen Worker

W p mean ± SD mean ± SD

Queen-like behaviors Push 14 0.03 1.48 2.49 0.31 0.81 Back into 6 0.004 3.60 4.52 1.26 2.56 Bite 17.5 0.009 3.72 3.82 1.22 1.80 Back up 20 0.01 6.62 10.10 4.20 6.23 Chase 2.5 0.03 0.91 1.47 0.23 0.55 Nudge 22 0.06 4.03 4.51 1.11 1.92

Worker-like behaviors C-posture 28 0.002 2.06 3.46 9.55 8.04 Reverse 8.5 0.02 1.55 2.74 2.46 3.68

Other behaviors Antennate NA 2.85 3.94 2.20 2.56 Follow 22 0.60 2.08 3.65 1.68 3.03 Head–head NA 2.54 2.54 1.89 1.72 Mandible touch NA 0.77 1.25 0.78 1.27 Nip 89.5 0.84 11.88 13.31 9.54 12.87 Pass NA 5.91 5.51 5.74 5.55

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pairwise p = 0.004, solitary–worker pairwise p = 0.07, solitary–queen pairwise p = 0.93; Fig. 2c). Proportion of queen-like behavior did not differ between groups (H = 4.38, p = 0.11; Fig. 2d).

Hypothesis 3: effects of ovary size and body size on behavior

Workers had smaller ovaries than the other two groups; there were no differences between solitary and queen ovaries (mean ± SD queen = 1.69 ± 0.91  mm2, soli-tary = 1.97 ± 0.69 mm2, worker = 0.64 ± 0.60 mm2, ANOVA F2,37 = 11.18, p < 0.001; solitary–worker Tukey p < 0.001, solitary–queen p = 0.60, worker–queen p = 0.003). Workers were also smaller bodied than queens and solitary females; there were no differences in body size between queens and solitary females (queen mean head width = 4.24 ± 0.44 mm,

solitary = 4.11 ± 0.43 mm, worker = 3.66 ± 0.19, ANOVA F2,37 = 7.69, p = 0.002; queen–worker p = 0.002, soli-tary–worker p = 0.10, queen–solitary p = 0.81). Because body size, ovary development, and worker caste are con-founded, we analyze the effects of body size and ovary size variation in workers separately from the two reproductive groups below.

Among workers, there was no correlation between body size and ovary size (r = 0.18, N = 12, p = 0.57). Ovary size was not significantly correlated with queen-like, worker-like, tolerance, avoidance, or aggressive behavior. There was a significant correlation between ovary size and the proportion of avoidance behavior (rho = 0.59, N = 12, p = 0.04), but no significant correlation between ovary size and proportion of queen-like, worker-like, tolerance, or aggressive behavior. There was no correlation between relative ovary size and queen-like, worker-like, avoidance, tolerance, or aggressive

A B C

D E F

Fig. 1 Number of tolerant a, avoidance b and aggressive c behaviors for queens, workers, and solitary reproductives. Proportion of tolerant d, avoidance e and aggressive f behaviors. Both numbers and propor-tions represent sums of both bees in a circle tube pair, so proportions can range from zero to two. Boxes that do not share a letter are sta-tistically different from each other in f. The bracket a indicates a sig-

nificant difference between solitary reproductives and the social bees (workers and queens combined) for tolerant behaviors. There were no statistically significant differences between groups for other behav-iors. For all boxplots, horizontal lines show the median, boxes the interquartile range (IQR), and whiskers up to 1.5*(IQR)

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Fig. 2 Number of worker-like a and queen-like b behaviors for queens, workers, and solitary reproductives. Proportion of all behaviors that were worker-like c and queen-like d. Both numbers and proportions represent sums of both bees in a circle tube pair, so proportions can range from zero to two. Boxes that do not share a letter are statistically different from each other in a. There were no differences between groups for queen-like behaviors b. For all boxplots, horizontal lines show the median, boxes the interquar-tile range (IQR), and whiskers up to 1.5*(IQR). Dots represent data points > 1.5*(IQR) from the median. Queen-like behav-iors are ‘push’, ‘back into’, ‘bite’, back up’, ‘nudge’ and ‘chase’. Worker-like behaviors are ‘C-posture’ and ‘reverse’

A B

C D

Fig. 3 Number of queen-like behaviors correlated with head width for reproductive bees (queens, dark filled circles, and solitary females, light-filled circles), but not for workers (open circles). The trend line is a linear regression for queens and solitary females combined

Fig. 4 Number of worker-like behaviors correlated negatively with head width for reproductive bees (queens, dark filled circles, and solitary females, light-filled circles) and workers (open circles). The dashed trend line is a linear regression for workers. The solid trend line is a linear regression for queens and solitary females combined

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behaviors, either as counts or proportions. Head size nega-tively correlated with worker-like behavior (rho = − 0.58, N = 12, p = 0.046); no other correlations were significant (Figs. 3, 4). Relative head size showed a nearly significant correlation with aggressive behavior (rho = 0.522, N = 12, p = 0.08) and a significant correlation with proportion of aggressive behavior (rho = 0.71, N = 12, p = 0.009). There were no other significant correlations with relative head size.

Among queens and solitary females, body size and ovary size were not significantly correlated (r = 0.39, N = 28, p = 0.13). Neither ovary size nor relative ovary size signifi-cantly correlated with queen-like, worker-like, tolerance, avoidance or aggressive behavior, either as counts or propor-tions. Body size, however, correlated positively with queen-like behavior (rho = 0.68, N = 28, p < 0.001), negatively with worker-like behavior (rho = − 0.48, p = 0.01), positively with avoidance (rho = 0.48, p = 0.01) and positively with aggres-sion (rho = 0.43, p = 0.02); it did not significantly correlate with tolerance. Body size also correlated with proportion queen-like behavior (rho = 0.64, N = 28, p < 0.001) and nega-tively with proportion worker-like behavior (rho = − 0.70, N = 28, p < 0.001). Body size did not significantly correlate with the proportion of tolerance, avoidance, or aggressive behaviors. Relative head size also significantly correlated with queen-like behavior (rho = 0.45, N = 28, p = 0.02) and negatively with proportion of worker-like behavior (rho = − 0.43, N = 28, p = 0.02).

The correlation of body size and queen-like behavior was significant among the solitary females when analyzed alone, but was not significant when queens were analyzed alone (solitary: N = 14, rho = 0.83, p < 0.001; queens: N = 14, rho = 0.43, p = 0.12). Results were similar for proportions of queen-like behaviors (solitary: rho = 0.68, N = 14, p = 0.007; queens: rho = 0.47, N = 14, p = 0.09). Relative head size did not correlate with either measure of queen-like behavior in either group.

The correlation with worker-like behavior was not sig-nificant among the solitary females when analyzed alone, but was significant among the queens when analyzed alone (solitary: N = 14, rho = − 0.49, p = 0.07; queens: N = 14, rho = − 0.55, p = 0.04). The correlations with proportion of worker-like behavior were significant for each group alone (solitary: rho = − 0.80, N = 14, p = 0.001; queens: rho = − 0. 68, N = 14, p = 0.007). Relative head size significantly corre-lated with proportion worker-like behavior among solitaries (rho = − 0.60, N = 14, p = 0.03). Relative head size did not significantly correlate with worker-like behavior analyzed as a count among solitaries, or either measure of worker-like behavior among queens.

While ovary size showed no significant correlations with behavior among queens or solitary females, relative ovary size negatively correlated with aggression (rho = − 0.58, N = 14, p = 0.03) and proportion aggression (rho = − 0.63,

N = 14, p = 0.02) among solitary females. There were no other significant correlations with relative ovary size among solitary females or queens.

Discussion

Our results showed mixed support for each of our hypoth-eses. First, tolerance, but not aggression, differed between social and solitary bees. Second, worker-like, but not queen-like, behaviors persisted even when bees were removed from their natural social context. Third, body size, but not ovary size, influenced behavior in our circle tube assays. Below we discuss each set of predictions in depth.

Hypothesis 1: differences between social and solitary nests

Social females (the queens and workers) were more tolerant of non-nestmates than were solitary females. This supports the prediction that in addition to cooperation with nestmates, living in a social group requires a generalized increase in tolerance to other individuals. Other studies that made inter-specific comparisons of social and solitary species (Packer 2006, Richards and Packer 2010) found increased avoid-ance behavior in the solitary bees relative to social bees. Our data did not show this pattern when analyzed as counts, but solitary bees did show more avoidance behavior when analyzed as a proportion of all behaviors, although this dif-ference was not significant (p = 0.06). However, given our small sample size (n = 13 social pairs and 7 solitary pairs), further study may reveal that in addition to being less toler-ant of other bees than social females, solitary females also exhibit increased avoidance.

Our prediction of increased tolerance among social rela-tive to solitary females was supported by our results. How-ever, the literature on inter-specific comparisons shows the opposite trend: solitary species generally exhibit more pass-ing (a tolerant behavior) than do social ones (Richards and Packer 2010). Our intra-specific comparison found the oppo-site: consistent with tolerant behaviors in general, solitary females passed less than social females.

Previous inter-specific studies found less aggression in solitary than social females (reviewed in Richards and Packer 2010). Our study found no difference in aggression between social and solitary females. However, when ana-lyzed as a proportion of all behaviors, workers were sig-nificantly less aggressive than solitary females and social queens. The aggressive behaviors are also queen-like behaviors. This may explain why workers show low lev-els of aggression, but we found no suggestion of systematic social–solitary differences in aggression independent of the queen–worker differences.

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We excluded the behavior ‘C-posture’ from our aggre-gate aggression measure because a previous observation nest study of M. genalis and our results suggest that it is expressed most by subordinate workers (Kapheim et al. 2016). The other behaviors that we chose for calculating aggression following the literature (‘push’, ‘bite’, ‘nudge’, and ‘back into’) were among those expressed most by queens in the queen–worker circle tube trials. In our study, ‘C-pos-ture’ negatively correlated with aggression. This suggests that including ‘C-posture’ in the larger combined aggression variable would have been a mistake for M. genalis. Perhaps if other species use C-posture for apparent self-defense like M. genalis (e.g., Buckle 1982), relationships with aggression may have been obscured by including C-posture with the other aggressive behaviors into a combined variable. Dew et al. (2014) caution against assigning behaviors a priori to behavioral classes, as they may be used for different pur-poses in different species. ‘C-posture’ appears to be a case where a behavior may be aggressive in some species, but not in M. genalis.

Hypothesis 2: caste‑typical behaviors in the absence of social hierarchy

Our study showed that caste-typical expression of worker-like behaviors, but not queen-like behaviors, persisted even when queens and workers were removed from their social context (queen and worker(s) of the same nest) and placed in a circle tube with a non-nestmate of the same caste. Workers showed significantly more worker-like behaviors in interac-tions with other workers than queens showed in interactions with other queens. Our data suggest that caste-typical behav-ior patterns of worker-like behavior are robust to short-term removal from their normal queen–worker social context.

The pattern we observed with queen-like behavior was the opposite of that seen for worker-like behavior: no differ-ence between groups. This suggests that in the absence of an established social dominance hierarchy, all individuals express similar levels of queen-like behavior. Our data sug-gest that the expression of worker-like behavior is at least somewhat long lasting, whereas queen–worker differences in queen-like behavior disappear outside of the queen–worker social context.

In primitively social sweat bees, the worker phenotype (foraging to nourish the queen’s offspring, reproductively sterile, and submissive to the queen) emerges from queen aggression directed toward her worker daughter (Michener and Brothers 1974, Michener 1990, Kapheim et al. 2016). In M. genalis, this maternal manipulation includes manipula-tion of pollen provisions to create smaller worker daughters, and adult dominance interactions to suppress worker ovar-ian development, juvenile hormone levels, and vitellogenin, the egg precursor protein, titers (Smith et al. 2009, 2013,

Kapheim et al. 2011, 2012). These physiological effects, including the influence of size (see below), may explain why workers continue expressing worker-like behavior outside of their normal social context.

Solitary individuals were behaviorally similar to queens in both queen-like and worker-like behaviors. However, the interpretation of worker-like behavior requires some caution: while the queen–solitary pairwise comparison was not sig-nificant (p = 0.16, n = 7 pairs of solitaries and seven pairs of queens, Fig. 2a), a larger sample size may show that solitar-ies are actually intermediate between queens and workers; this awaits further study. Our study used field-collected nests with unknown history, which may also have obscured dif-ferences between queens and solitary reproductives. Thus, some of our solitary females may have been social queens whose workers recently died or new nest foundresses whose future workers have not yet developed into adults. Neverthe-less, our data suggest that workers are the ‘modified’ caste, while queens are relatively similar to solitaries.

Hypothesis 3: effects of ovary size and body size on behavior

Our data showed little influence of ovary development on behavior, but a strong influence of body size on behavior. One problem with our analysis is that workers are smaller than the two reproductive groups, likely because their mother (the queen) provisions them with less pollen (Kapheim et al. 2011). Workers also have suppressed ovarian development due to social dominance from their mother, the queen (Smith et al. 2009, Kapheim et al. 2012, 2016). Thus, size, ovary development, and caste are inherently confounded. For this reason, we analyzed workers separately from the two repro-ductive groups. A previous study (Kapheim et al. 2012) found that queens were slightly larger than solitary reproduc-tives, although other studies have not found this difference (Smith et al. 2008, 2009). All previous studies found that queens had larger ovaries than solitary reproductives (Smith et al. 2008, 2009, Kapheim et al. 2012), but in the current study, we found no size difference or difference in ovary development between queens and solitary reproductives.

Here we show that ovary development generally does not influence behavior in non-nestmate interactions. The only exception was a negative correlation (not the predicted direction) between relative (but not absolute) ovary size and aggression among solitary females. A previous study in this species showed a positive correlation between ovary size and aggression in non-nestmates, but it did not control for caste (Arneson and Wcislo 2003). West-Eberhard (1987, 1996) suggested that changes in aggression and social tolerance associated with ovarian development cycles may have been coopted in the evolution of social cooperation. However, our data on solitary females show no obvious effect of ovary

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size on aggression or tolerance. The results from other non-nestmate circle tube studies are mixed. Ovary size influenced behavior in some (Wcislo et al. 1997) but not other sweat bee species studied (McConnell-Garner and Kukuk 1997, Richards and Packer 2010).

Body size had a strong effect on behavior in our study. In the two reproductive groups, queens and solitary females, body size correlated with queen-like behavior, and corre-lated negatively with worker-like behavior: larger females acted more like queens and less like workers (Figs. 3, 4). However, among workers, there was no correlation between size and queen-like behavior, but a strong negative correla-tion between size and worker-like behavior: smaller workers were more worker-like, even in interactions with other work-ers (Fig. 4). In our previous work with this species, we inter-preted queen–worker body size differences in the context of social dominance: smaller daughters could be more easily dominated by the queen, and queen dominance suppressed reproduction (Smith et al. 2008, 2009, Kapheim et al. 2012, 2013, 2016). Because the queens control the provisioning of their daughters, they may manipulate pollen provisions to create smaller daughters (Kapheim et al. 2011, 2015). How-ever, our results suggest that in addition to social dominance, body size per se may also influence the expression of worker behavior. This is consistent with broader developmental nutrition effects on adult social behavior (Kapheim 2017). Another interpretation is that the smaller daughters may be more strongly dominated by their queens, and thus continue expressing worker-like behavior more intensely than larger workers in our circle tube trials.

It is not clear how to interpret the correlation between body size and avoidance, especially since one of the avoid-ance behaviors is worker-like (‘reverse’) and the other is queen-like (‘back up’). One explanation may be that it is simply more difficult for a large bee than a small bee to pass in the confined space of a circle tube, so larger bees are more likely to avoid each other. The correlation with aggres-sive behavior and body size makes sense, given the high degree of overlap between the behaviors used to calculate the aggregate variables ‘aggression’ and ‘queen-like’ (see “Methods”). The relationship between size and aggressive/queen-like behavior among the solitary, as well as social, bees suggests that body size may be a source of behavioral variation among solitary females from which social coop-eration and division of labor could emerge (Jeanson et al. 2005). Unfortunately, in the only ancestrally solitary halictid species tested to date (Xeralictus bicuspidariae), there was no effect of body size on behavior (Richards and Packer 2010; other solitary halictid species studied are derived from social ancestors; Gibbs et al. 2012).

Given that size and caste are inherently confounded in M. genalis, the persistence of caste-typical expression of worker-like behaviors even in caste-matched non-nestmate

circle tubes may be a result of size effects on behavior rather than caste. However, given that worker body size is influ-enced by maternal manipulation of pollen provisions, body size can be seen as one of the caste-typical traits resulting from nestmate social interactions (Kapheim et al. 2011, 2012). In the bee Ceratina calcarata (Apidae), Lawson et al. (2017) showed that experimental reduction of larval pro-visions created small-bodied, less aggressive bees, similar to natural workers. M. genalis awaits similar experimental manipulations.

Conclusions

Social cooperation requires increased tolerance of other individuals relative to solitary living. Our data suggest that this increased tolerance is at least partially generalized to all interactions, as social bees were more tolerant of non-nestmates than solitary bees were. Our data on social caste suggest that queen-like behavior is relatively constant in the absence of social dominance hierarchies, but that worker-like behaviors persist. Our body size data suggest that the worker phenotype may result from naturally occurring size-correlated variation in behavior combined with mater-nal manipulation of both body size through nutrition and behavior and ovary development through social aggression.

Acknowledgements This work was supported by NSF Grant #17-1028536545 to ARS and MAS. Yi Ling and Callum Kingwell helped collect nests.

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