Colony Size and Initial Conditions Combine to Shape Colony Reunification Dynamics

Behavioural Processes 170 (2020) 103994

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Behavioural Processes

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Colony size and initial conditions combine to shape colony reuniﬁcation dynamics T

Grant Navid Doeringa,*, Kirsten A. Sheehyb, James B. Barnetta, Jonathan N. Pruitta a Department of Psychology, Neuroscience & Behaviour, McMaster University, Hamilton, Ontario, L8S 4K1, Canada b Department of Ecology, Evolution & Marine Biology, University of California – Santa Barbara, Santa Barbara, CA, 93106, USA

ARTICLE INFO ABSTRACT

Keywords: Group cohesion and collective decision-making are important for many social animals, like social insects, whose Consensus societies depend on the coordinated action of individuals to complete collective tasks. A useful model for un- Polydomy derstanding collective, consensus-driven decision-making is the fluid nest selection dynamics of ant colonies. Quorum sensing Certain ant species oscillate between occupying multiple nests simultaneously (polydomy) and reuniting at a Emigration single location (monodomy), but little is known about how colonies achieve a consensus around these dynamics. To investigate the factors underpinning the splitting-reunification dynamics of ants, we manipulated the availability and quality of nest sites for the ant Temnothorax rugatulus and measured the likelihood and speed of reunification from contrasting starting conditions. We found that pursuing reunification was more likely for smaller colonies, that rates of initial splitting were lower when colonies could coordinate their activity from a central hub, and that diluting colonies among additional sites did not impair reaching consensus on a single nest. We further found mixed support for a specific threshold of social density that prevents reunification (i.e., prolonged polydomy) and no evidence that nest quality influences reunification behavior. Together our data reveal that consensus driven decisions can be influenced by both external and intrinsic group-level factors and are in no way simple stereotyped processes.

1. Introduction ants generally dwell within fragile pre-formed cavities (i.e., in rotting acorns, plant galls, sticks, etc.) that are prone to damage or total an- The capacity to jointly evaluate and unanimously choose between nihilation through environmental or animal disturbance. Over the multiple mutually exclusive options is common in social animals course of a year, more than half of all occupied nests can be destroyed (Conradt and Roper, 2005). In eusocial insects, the group’s selection of (Yamaguchi, 1992). Nest relocation is therefore frequent. a nest-site is a well-studied example of such consensus building Although the behavioral routines underpinning emigration help to (Visscher, 2007). Ants and bees, for example, can move their entire avoid colony fragmentation in the face of repeated nest destruction colonies to a new home when their current dwelling is damaged or (Pratt et al., 2002; Seeley, 2003; Cronin, 2012; Kolay and Annagiri, when a superior alternative becomes available (Möglich, 1978; 2017; Pratt, 2019), consensus is not guaranteed. Temnothorax will oc- Smallwood, 1982; Dornhaus et al., 2004; Seeley, 2010; Pratt, 2019). A casionally split their colonies between multiple discrete nest sites. The fundamental similarity in most of these insects’ selection processes is split colonies may remain socially connected with ants, brood, and re- the ability to integrate information from multiple individuals to reach a sources shared and exchanged between colony sites (Stuart, 1985; collective choice. In the ant genus Temnothorax, individuals dis- Herbers, 1986). Split colonies are, however, still capable of later re- criminate nest quality based on several features (e.g., light level in the unifying at a single site. In fact, predictably timed cycles of in- nest cavity) and begin to recruit nestmates to candidate homes with a tentionally splitting in the spring only to later reunify in autumn are a probability that depends on their assessment of the new location’s ap- characteristic feature of Temnothorax colonies (Headley, 1943; peal (Mallon et al., 2001; Robinson et al., 2011; Pratt, 2019). Once the Partridge et al., 1997; Foitzik and Heinze, 2001; Strätz and Heinze, number of ants committed to a site surpasses a quorum, workers will 2004; Kramer et al., 2014). initiate brood transport and carry the remaining population from the When a colony distributes itself across multiple nests, this is termed old nest to the new one (Pratt et al., 2002; Pratt, 2019). Temnothorax polydomy. Polydomy is thought to reduce predation risk (Van

⁎ Corresponding author. E-mail address: [email protected] (G.N. Doering). https://doi.org/10.1016/j.beproc.2019.103994 Received 20 September 2019; Received in revised form 24 October 2019; Accepted 28 October 2019 Available online 02 November 2019 0376-6357/ © 2019 Elsevier B.V. All rights reserved. G.N. Doering, et al. Behavioural Processes 170 (2020) 103994

Wilgenburg and Elgar, 2007) and to allow improved resource acquisi- establish nests inside rock crevices and in soil under stones, and nests tion (Debout et al., 2007; Robinson, 2014; Stroeymeyt et al., 2017b). usually range in size from 50 to 400 workers (Bengston, 2018). Like However, prolonged polydomy can have fitness costs too, such as a many other species in its genus (Forel, 1874; Wheeler, 1910; Creighton, colony being dispersed too thinly to effectively manage colony re- 1965), T. rugatulus is thought to forage on dead insect prey and hon- sources (Cook et al., 2013) or through queen-worker conflict over the eydew (Bengston and Dornhaus, 2013). production of males (Snyder and Herbers, 1991; Banschbach and All colonies used for this study had a single queen (monogynous), Herbers, 1996; Heinze et al., 1997). Furthermore, like emigration, re- but there was variation in the numbers of workers (min: 60, max: 425, unification itself can be dangerous as ants need to temporarily relin- mean: 148.8, standard deviation: 75.2) and brood (min: 28, max: 245, quish shelter while they consolidate nests (Rettenmeyer et al., 1978; mean: 116.6, standard deviation: 53.9). Each colony resided in a “poor” Franks and Sendova-Franks, 2000; Bouchet et al., 2013). Thus, there are style nest (see below) both before and between the experimental trials. likely trade-offs in a colony’s choice of whether to switch from Colonies were maintained in lidded plastic boxes (11 × 11 × 3 cm) and polydomy to monodomy or vice versa. Although the circumstances that were provided with an ad libitum supply of water and their food (pro- foster colony fission have received some attention (Alloway et al., 1982; tein: mealworms; sugar: maple syrup) was replenished weekly. Stuart, 1985; Franks et al. 1992; Lanan et al., 2011; Scharf et al., 2012; Cao, 2013; Stroeymeyt et al., 2017b), less is known about the conditions that facilitate reunification or the behaviors underlying the pro- 2.2. Nest designs cess (Herbers and Tucker, 1986; Kaur et al., 2012; Doering and Pratt, 2016, 2019; Sahu et al., 2019). All nests consisted of a balsa wood slat (2.4 mm thick) with a 38 mm In Temnothorax rugatulus, the choice of where to reunify is guided by diameter circular hole drilled through the center that acted as a cavity a preference for the highest quality site and, if all nests are identical in in which the ants could live. Each slat was sandwiched between two quality, for the nest inhabited by the queen (Doering and Pratt, 2016). glass microscope slides (50 × 75 mm) which created a roof and floor for Mechanistically, colony reunification is achieved through the actions of the nests. A 2 mm wide slit was cut into one side of each nest, providing a small team of hyperactive workers, or keystone individuals (Robson the ants with an entrance to the nest cavity. Since T. rugatulus colonies and Traniello, 1999; Modlmeier et al., 2014), whose behavior de- prefer darker nest cavities (Visscher, 2007), we set the attractiveness of termines a colony’s decision (Doering and Pratt, 2019). Yet, how ex- each experimental nest to one of three different brightness levels by ternal circumstances and group-level traits influence the probability of shading the nest cavities with neutral density light filters. Nest type 1 reuni fication is unknown. was a “poor quality” nest with no light filter. Nest type 2 was a Here we evaluate how colony size, number of available nests, and “mediocre quality” nest. These nests were identical to the poor style initial splitting conditions combine to shape reunification in ants. nests, except a single light filter of 1-stop power (i.e., blocking 50% of Colony size is likely to be influential because differences in worker incoming light) covered each roof (Salvaggio, 2013). Nest type 3 was a populations can impact many dimensions of ant society (Michener, “good quality” nest and was also identical to the poor style nests except 1964; Karsai and Wenzel, 1998; Mailleux et al., 2003; Dornhaus et al., for three 3-stop filters (i.e., blocking 99.8% of incoming light) that 2012; Kramer et al., 2014), and some species strive to maintain a spe- covered each roof. Further details on these kinds of nests can be located cific density of workers within the nests (Franks et al., 1992). When a elsewhere (Sasaki et al., 2015). colony grows beyond this target, workers will attempt to expand the volume of their nest or relocate to a bigger one. If neither of these routes are possible, colonies can become polydomous (Cao, 2013). 2.3. Experimental design Likewise, larger colonies might have less reason to reunify if they have already split, and small colonies (or colonies split between many nests) Colonies were randomly assigned to one of three treatment groups, might have a stronger incentive to reunite. The route that colonies take which differed in the way that colonies were encouraged to become to being split may also impact reunification. When a Temnothorax polydomous. For the first treatment (Equal split), colonies were split colony emigrates to a new nest, its speed of relocation, accuracy, level equally between isolated nests that were later placed within a single of worker engagement, and resulting cohesiveness all depend on whe- arena together. In the second treatment (Forced emigration), colonies ther its original nest remains intact (Franks et al., 2003, 2013; Pratt and were induced to relocate to a new nest by damaging their original nest Sumpter, 2006). Similarly, rates of reunification could hinge upon how and presenting the ants with multiple identical options, encouraging damaged a colony’s home nest becomes before the colony first splits. splitting (Pratt and Sumpter, 2006; Franks et al., 2013). Colonies in the Finally, increasing the number of available or currently occupied nests third group (Scattered) had their entire population scattered in an area may make reunification more challenging. In theoretical models of containing potential new nests. opinion dynamics, more subgroups can slow convergence to a con- These three conditions were chosen because they differ in the sensus (Becchetti et al., 2015, 2016; Ghaffari and Parter, 2016). How- amount of damage inflicted on colonies’ nests before they split between ever, at least in typical nest relocation, Temnothorax can incorporate new nests. A colony from the equal split and scattered groups will be multiple options into its collective decision-making scheme without completely removed from its original nest, potentially signaling to the incurring any measurable loss of accuracy (Sasaki and Pratt, 2012). We ants that they are in greater danger or in a poor environment. This therefore aimed to explore how the circumstances of initial fractiona- contrasts with a colony in the forced emigration group, which will only tion, colony size, and the number of available nests interact to either experience the removal of its nest’s roof. When a nest falls apart in the promote or discourage the restoration of colony cohesion in T. ruga- wild, it is possible that portions of the workers and brood fall out in the tulus. process since T. rugatulus ants cling to the rocks concealing their nests (Möglich, 1978). Some nest relocations in the field therefore plausibly 2. Methods involve situations were ants must emigrate without all starting at the home nest. The dynamics of nest relocation in the scattered group may 2.1. Study colonies also differ from the forced emigration group. Ants in the scattered group will be robbed of any centralized location from where they can co- The 48 colonies of T. rugatulus Emery used in this study were col- ordinate their activity at the start of each trial, potentially leading to lected in February 2018 at Madera Peak [33.317 N, 110.876 W], part of more initial splitting than in the forced emigration group. This difference the Pinal Mountains of Arizona. T. rugatulus is widespread across wes- is not relevant for the equal split group because splitting is guaranteed tern North America and is abundant in many parts of its range. Colonies for that group

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2.3.1. Treatment I – equal split combinations. After introducing the new nests into an arena, all brood 12 colonies were randomly selected to be in the equal split treatment items and workers from a colony were lifted from their starting nest group. On each day of the experiment, each colony had its entire with a soft paintbrush and haphazardly dispersed around the arena. We worker and brood populations manually and evenly divided into one of then monitored thedecision-making of these colonies in a manner six possible conditions in a 2 (nest #) x 3 (nest quality) design: (1) two identical to the other treatment groups. poor nests, (2) two mediocre nests, (3) two good nests, (4) three poor nests, (5) three mediocre nests, or (6) three good nests. Each colony 2.4. Data collection & analysis experienced every condition exactly once, and the order in which colonies received each condition was randomized so that each colony in The number of worker ants inhabiting the nest in each photograph the group received the treatments in a unique order. Colonies were was determined by manually marking and counting all individuals in given two days of rest between each condition. The procedure for ImageJ (Schneider et al., 2012). Of the 2160 nest photographs, 61 were evenly splitting colonies has been described elsewhere (Doering and missing or unusable. If a trial contained any missing images from its Pratt, 2016, 2019), and our methods mirror those. Briefly, after re- sequence, then we excluded the trial from the analysis (27/288 trials). moving the colony’s glass roof, and destroying the nest, ants and brood For all three time periods during a 12-hr. run, we computed the were moved individually into isolated plastic arenas (24 cm diameter, level of cohesion exhibited by each colony by using an entropy-based 9 cm height) each of which contained a single new nest. After providing metric that was introduced by Franks et al. (2013) (Eq. 1): the ants an hour to move into each nest, all nests were transplanted H together into one new arena. When split in half, the two nests were C =−1 placed, respectively, in the left and right portions of the arena, facing Hmax (1) each other and lying 30 mm from the center. When a colony was split H represents the Shannon entropy index (Shannon, 1948; between three nests, the nests were arranged in an approximately Spellerberg and Fedor, 2003): equilateral triangle. Because queens bias decision-making during re- i=1 unification (Doering and Pratt, 2016), queens were captured during Hpp=− log ∑i=1 ii2 (2) splitting and kept apart from their colonies by placing them in their original nest-boxes. Queens were returned to their source colonies at This index quantifies the extent to which a group is aggregated at a the end of each trial. Although excluding queens from the reunification single site when multiple options are available. In Eq. (2), n signifies the process was necessary for the current study, it was not ideal as this number of nests available to a colony, and pi is the percentage of in- fi removes a possibly important part of how colonies make such decisions. dividuals from a colony present at nest i. Hmax conveys a con guration However, Temnothorax colonies may occupy three or more nests si- where a colony’s workers are evenly divided between every nest option. multaneously in the field (Alloway et al., 1982), and colonies can The values of C will range from 0 to 1 regardless of how many options persist after their queen has perished because workers can begin laying are available. When C = 1, all ants in a colony have decided on a single male-destined eggs in her absence (Heinze et al., 1997) or eventually nest, and when C = 0, a colony’s population is equally present in each merge with an unrelated colony (Foitzik and Heinze, 1998). Analyzing available nest. Any scouting of foraging ants that were patrolling their reunifications between queenless nests therefore retains some ecolo- arena when nest photographs were taken were excluded from the co- gical relevance (Doering and Pratt, 2019). hesion calculations. Following the approach used in previous re- Experimental arenas were housed within cabinets (Height: 57 cm, unification studies (Doering and Pratt, 2016, 2019), we also classified Width: 108 cm, Depth: 47 cm) whose interiors were homogeneously colonies as being “unified” and monodomous at the end of each trial if a illuminated with identical arrays of LEDs. After colonies had been split, minimum proportion of a colony’s workers resided in just a single nest. all nests were photographed approximately 1 h, 6 h, and 12 h after their For this study, we set 90% as the threshold for being “unified”. This initial forced division. After the final photographs were taken, the most corresponds to a minimum cohesion score of C = 0.531 in colonies populous nest in each arena was placed in its colony’s nest-box, and the given two nests and C = 0.641 in colonies given three nests. less populous nest(s) were dismantled and any ants inhabiting the cavity were placed back in their nest-box. Arenas and glass slides were 2.5. Statistical analysis reused between trials and were cleaned with ethanol to remove any residual chemical markings (Sasaki and Pratt, 2011; Franks et al., We used linear mixed-effects models (LMM) to evaluate the effects 2013). Only 12 colonies of this treatment group were established owing of nest quality, polydomy treatment, and colony size on cohesion, with to the time and space consuming nature of this treatment group. colony ID always set as a random factor acting on the intercept. We used likelihood ratio tests to assess the effect of each variable by se- 2.3.2. Treatment II – forced emigration quentially comparing models without a particular term to a full model 18 colonies were randomly selected to be in the forced emigration with each term present. We compared the cohesion values of different treatment group. These colonies experienced the same 2 × 3 design polydomy treatments to each other using simultaneous general linear above, except that colonies were presented with the nests in the circular hypothesis tests (GLHT). In our analyses, colony size was scaled so that arenas and had their starting nests destroyed by the removal of its roof, it was mean centered and had unit-variance in order to improve the fit stimulating an emigration to one or more of the target options. Starting of the models and the interpretability of regression coefficients (Table nests were positioned approximately 30 mm from the arena centers, S1). Through an inspection of the plots of model residuals and fitted creating either a triangle pattern (when given two nests) or a diamond values, we verified that the assumption of the independence of errors pattern (when given three nests). Because colonies in the equal split was approximately satisfied. However, the residuals from most of our group had 1 h to acclimate, colonies in the forced emigration group were LMM models were not normally distributed. We proceeded to use these analogously left in empty arenas for one hour to acclimate before being models despite this since the relaxation of this assumption is not con- induced to emigrate. Colonies were similarly photographed 1 h, 6 h, sidered highly problematic if one is seeking an assessment of general and 12 h after being stimulated to emigrate. trends and not precise quantitative predictions of individual points (Gelman and Hill, 2006), and our models’ results were consistent with 2.3.3. Treatment III – scattered the trends in our figures. The remaining 18 colonies were assigned to the scattered treatment A consequence of the cohesion metric used in this study is that the group. At the start of each trial, colonies were left to acclimate in empty distributions of cohesion will tend towards being bimodal in the 2-nest arenas for one hour before receiving one of the 2 × 3 nest selection condition but will be more evenly distributed for colonies that were

3 G.N. Doering, et al. Behavioural Processes 170 (2020) 103994 given 3-nests as there are more possible sites to occupy. The actual treatments after 1 h (Fig. 1;2–nest group: L ratio = 26.757, values of the cohesion metric will also be automatically higher for co- p < 0.0001; 3-nest group: L ratio = 38.810, p < 0.0001). These dif- lonies in the 3-nest condition than for colonies in the 2-nest condition ferences (at the 1 -h time point) were due to higher levels of cohesion in even when referring to the same population configuration. For example, the forced emigration group compared to the scattered (2-nest group a colony that is initially split into three nests and ends its trial evenly General linear hypothesis test [GLHT]: β = -0.2457, z = -5.029, split in two nests will have a final cohesion score of C = 0.369, but the p < 0.0001; 3-nest group GLHT: β = -0.1437, z = -4.663, same state will have a score of C = 0 for colonies in the 2-nest treat- p < 0.0001) and equal split (2-nest group GLHT: β = -0.2888, z = ment. Due to these inherent differences, we analyzed the cohesion re- -5.130, p < 0.0001; 3-nest group GLHT: β = -0.2673, z = -7.675, sults from the 2-nest group separately from the 3-nest group. However, p < 0.001) groups. At the 1 -h time point, colonies that received three in order to compare the behavior of colonies given two nests to those nests in the forced emigration group had a peak in their cohesion dis- given three nests, we assessed the number of “unified” colonies over tribution located roughly at C = 0.369 (Fig. 1), which corresponds to a time across these two groups. Because being “unified” at just one nest colony being evenly split between two out of the three nests. Likewise, reflects the same condition for all colonies, this measure avoids the colonies in the forced emigration group that received two nests have a issues of comparing cohesion scores for colonies initially split between secondary peak at C = 1, which is when all workers occupy a single different numbers of nests. Differences between the 2-and 3-nest groups nest. This implies that many colonies in the forced emigration group in the number of “unified “colonies were analyzed independently for often avoided fully breaking up to begin with. each polydomy treatment using generalized linear models of a binomial Cohesion increased over time for all three splitting treatments; the distribution family (Table S1). distribution of colony cohesion scores shifted towards C = 1 after each Since some colonies avoided splitting at the beginning of their trials time step (Fig. 1). At the 6 -h time point, colonies that were given two in the forced emigration group, we also analyzed the effects of nest nests show statistically significant differences in cohesion across the quality, quantity, polydomy treatment, and colony size using a reduced three polydomy treatments (L ratio = 25.574, p < 0.0001), with co- data set that excluded colonies that lacked low cohesion scores lonies in the forced emigration group exhibiting higher cohesion than (C < 0.1) at the 1 -h time point. Using LMM models, we also evaluated both the equal split (GLHT: β = -0.3116, z = -4.588, p < 0.0001) and whether colonies had any biases towards occupying particular sites scattered (GLHT: β = -0.3137, z = -5.341, p < 0.0001) groups. After based on the nests’ spatial position in the arenas (See Table S1). All 12 h the differences between polydomy treatments remained significant statistical calculations were performed in R version 3.4 (https://www.r- for colonies given two nests (L ratio = 12.889, p = 0.002). Colonies project.org). LMM models were fit using the package nlme (Pinheiro that experienced three nests showed less prominent differences be- et al., 2019), and the GLMM models used to compare the number of tween the splitting treatments than colonies that were given two nests. “unified” colonies in the 2 and 3 nest treatments were fit using the In 3-nest colonies, cohesion did significantly differ between splitting package lme4 (Bates et al., 2015). General linear hypothesis tests were groups at 6 -hs (L ratio = 7.507, p = 0.023) but did not meaningfully conducted using the package multcomp (Hothorn et al., 2008). differ at 12 -hs (L ratio = 3.19 p = 0.203). The cohesion distributions of the equal split and scattered treatments qualitatively match each other very closely (Fig. 1). When only colonies that exhibited low levels of 3. Results cohesion at the beginning of their trials are included (i.e., had a cohesion score of C < 0.1 at the 1 -h time point), the significant differ- ff 3.1. E ect of polydomy treatment ences in cohesion between polydomy treatments at 12 -hs disappears for colonies in the 2-nest group (Figure S1; L ratio = 5.755, p = 0.056) Regardless of whether colonies were given two or three nests, there and remains nonsignificant in the 3-nest group (Figure S1; L were significant differences in cohesion across the three polydomy

Fig. 1. Histograms of colony cohesion from all 261 trials. Each row of the ﬁgure corresponds to one of the three polydomy treatments, and each column corresponds to one of the three time periods at which cohesion was measured. Blue bars depict the cohesion of colonies who received two nests, and orange bars depict the cohesion of colonies that received three nests. The cohesion distributions of all polydomy treatments move towards C = 1 over 12 h, but this process is faster for colonies in the forced emigration treatment group.

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Fig. 2. The relationship between colony size and cohesion after 12 h for the three polydomy treatments. Larger colonies show lower cohesion after 12 h than smaller colonies when split between either two (a) or three nests (b). ratio = 2.095, p = 0.351). not make colonies worse at reaching consensus on a single nest. These results extend the idea that consensus-decision making in reunifying ’ 3.2. Effects of colony size and nest quality & quantity ants is a dynamic process that shifts based on a colony s circumstances (Doering and Pratt, 2016, 2019). We found no significant association between nest quality and Our observation that colonies who experienced forced emigration average cohesion at 12 h for either the 2-nest group (L ratio = 0.382, also exhibited higher rates of cohesion is intuitive; colonies are likely ff p = 0.826) or the 3-nest group (L ratio = 0.390, p = 0.823), and no more e ective at staying integrated when scouts can return to the old significant association between nest quality and average cohesion at the nest to recruit their sisters. Temnothorax colonies are known to tune beginning (i.e., degree of initial splitting) of the forced emigration trials their decision strategies based on prior experience and current condi- fl (2-nest: L ratio = 0.767, p = 0.681; 3-nest: L ratio = 1.819, p = 0.403). tions to respond exibly to their environment (Pratt and Sumpter, Quality also did not predict differences in cohesion at either the 1 -h (2- 2006; Healey and Pratt, 2008; Doran et al., 2015). For example, co- nest condition: L ratio = 1.745, p = 0.418; 3-nest condition: L lonies will move more rapidly into a new nest depending on how da- ratio = 4.792, p = 0.091) or 6 -h (2-nest condition: L ratio = 0.651, maged their current home is. This led to us to hypothesize that, if the fi p = 0.722; 3-nest condition: L ratio = 0.016, p = 0.992) time points. process of reuni cation carries risk, then colonies in the scattered and fi Overall, we found no evidence that colonies are biased towards re- equal split groups may be reluctant to launch into a reuni cation if they unifying at either the left or right nest when split between two nests (L sense that they are in a harsh or dangerous environment. However, the fi ff ratio = 1.206, p = 0.272), nor did we find any biases when colonies lack of a signi cant di erence between splitting treatments after the were split between three nests (L ratio = 0.489, p = 0.783). exclusion of colonies that did not initially fully split suggests that the Colony size strongly impacted cohesion. Larger colonies showed higher levels of cohesion at 12 -hs in the forced emigration group is ’ “ ” lower levels of cohesion after 12 h in both the 2-nest (Fig. 2a, L primarily caused by this group s head start in cohesion. Any initial ratio = 17.217, p < 0.0001) and 3-nest conditions (Fig. 2b, L population asymmetries between nests may serve as a cue to help co- ratio = 31.015, p < 0.0001). The number of colonies that were unified lonies decide where to unify, shortening the time to reach consensus. at a single nest after 12 h was not affected by the number of nests Indeed, cohesion increased over time for all three groups. If left long available for colonies in the forced emigration (Fig. 3; β = 0.1376, enough, the variation between groups could vanish as more colonies in z = 0.326, p = 0.745) or equal split group (Fig. 3; β = -0.1737, z = the equal split and scattered groups reunify. Worker stress, disorienta- -0.307, p = 0.759), but the scattered treatment saw a significantly tion, confusion, or an evaluation of risk therefore seem less likely as ff higher number of their colonies unify at one nest if three sites were explanations for the cohesive di erences between splitting methods. provided (Fig. 3; β = -1.0064, z = -2.354, p = 0.019). When only Colonies in the scattered group likely have greatly reduced quorum considering colonies that showed low cohesion (C < 0.1), these non- thresholds, and this increases the chances of splitting (Pratt and significant differences remain true for the forced emigration (Figure S2; Sumpter, 2006). Scouts from a scattered colony might immediately fi β = 0.2877, z = 0.351, p = 0.726) and equal split groups (Figure S2; β launch into recovering stray workers and brood once they nd any nest, = -0.1094 z = -0.193, p = 0.847), but the effect of nest number be- which could account for the higher rate of initial splitting in this group comes non-significant in the scattered group (Figure S2; β = -0.6585, z compared to forced emigration colonies. Prior work has shown that co- = -1.238, p = 0.216). lonies have even lower levels of fragmentation when they emigrate from a fully intact nest (Franks et al., 2013). The reason that forced emigration colonies from the 3-nest group did not exhibit higher levels 4. Discussion of cohesion compared to other polydomy treatments at 12 h is not completely obvious. One possible explanation is that colonies with ac- Our study explored several factors that could alter the tendency of cess to two nests should naturally possess a greater imbalance towards ant colonies to reunify after a disturbance to their nest. Colonies that just one site compared to when more nests are available because any split during forced emigration were faster to reunify, but, contrary to asymmetry in nest populations for a two-nest scenario must favor ex- results from prior work (Doering and Pratt 2019), we found no evidence actly one nest. If more nests are involved, multiple nests could have ff fi that nest quality a ected colony cohesion. We did, however, nd a higher populations than competing sites. A greater imbalance towards a strong negative association between group size and cohesion for all single nest could help beak the symmetry between sites, leading to a treatments. We also found that splitting between additional nests did

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Fig. 3. Unification rates of 2-nest vs. 3-nest colonies for the three polydomy treatments. Each bar represents the proportion of colonies in either the 2-nest or 3- nest conditions that were “unified” with at least 90% of workers in a single nest. Colonies in the forced emigration group can avoid splitting at the start of their trials, and colonies in the forced emigration and equal split groups are no worse at reunifying when presented with three options. Scattered colonies have a higher reunification rate when given three nests. faster reunification. However, this is difficult to reconcile with our reunification versus prolonged polydomy. Future comparative studies finding that colonies in the equal split group reach consensus on a single that address polydomy across species would be particularly valuable, by nest equally well regardless of the number of starting subpopulations. providing insights into the species traits and ecological conditions that We anticipated that colonies would either have a more difficult time cause the evolution of contrasting nesting strategies and consensus agreeing when there were more competing factions or that imbalances building in the fission versus fusion of nests (Pratt, 2005; Stroeymeyt towards a single nest would induce faster reunifications for the 2-nest et al., 2017a). The data herein convey that the processes underlying condition, yet this was not what we observed. The protocol that co- multi-nest population dynamics are unlikely to be simple, and hint that lonies used to reunify when faced with three nests could resemble the the selection pressures driving these behaviors may vary considerably one used in two-nest divisions (Doering and Pratt, 2019). Depending on among even closely related species. how efficient this method is, splitting between three nests instead of two may only have a marginal impact on reunification speed. Splitting Declarations of Competing Interest colonies among an even larger quantity of nests, along with data on how colonies can break symmetry and reunify when there are more None. options, would help address these questions. Our results are consistent with earlier reports of elevated rates of Acknowledgements polydomy in larger colonies (Stuart, 1985; Cao, 2013; Stroeymeyt et al., 2017b), although the presence or absence of a key “threshold density” This work was financially supported by the National Institutes of that enables polydomy remains ambiguous. Social density in occupied Health (GM115509 to JNP). We thank Stephen C. Pratt, David N. nests is argued to be important for spontaneous polydomy (Stuart, Fisher, and David J. D. Earn for helpful comments and discussion. We 1985; Cao, 2013). Likewise, we found there to be a strong negative also thank Michelle Gertsvolf for support with managing the experi- association between colony size and final cohesion when colonies were ments. split between nests. We failed to observe a specific social density that fosters stable polydomy vs. reunification in our data, yet, consistent Appendix A. Supplementary data with the size density threshold hypothesis, we observed that in the two- nest trials every colony with more than 200 workers (with one excep- Supplementary data associated with this article can be found, in the tion) remained divided after 12 h. This does provide some circum- online version, at https://doi.org/10.1016/j.beproc.2019.103994. stantial evidence for a key social density that triggers stable polydomy in this system — though the combined evidence is clearly mixed for References now. This study had too few large (> 200 workers) colonies to resolve this question. Alloway, T.M., Buschinger, A., Talbot, M., et al., 1982. Polygyny and polydomy in three The collective decision-making process of reunification is not a north american species of the ant genus Leptothorax mayr (Hymenoptera: formicidae). Psyche J. Entomol. 89, 249–274. https://doi.org/10.1155/1982/64124. stereotyped routine that unfolds deterministically for all colonies. Banschbach, V.S., Herbers, J.M., 1996. Complex colony structure in social insects: II. Instead, our data reveal that a combination of both intrinsic factors reproduction, queen-worker conflict, and levels of selection. Evolution 50, 298–307. (e.g., colony size) and extrinsic factors (e.g., circumstances preceding https://doi.org/10.1111/j.1558-5646.1996.tb04493.x. fi Bates, D., Mächler, M., Bolker, B., Walker, S., 2015. Fitting linear mixed-effects models the uni cation process) mingle to determine the speed and likelihood of using lme4. J. Stat. Softw. 67, 1–48. https://doi.org/10.18637/jss.v067.i01.

6 G.N. Doering, et al. Behavioural Processes 170 (2020) 103994

Becchetti, L., Clementi, A., Natale, E., et al., 2015. Plurality consensus in the gossip BF00299946. model. Proceedings of the Twenty-sixth Annual ACM-SIAM Symposium on Discrete Herbers, J.M., Tucker, C.W., 1986. Population fluidity in Leptothorax longispinosus Algorithms. Society for Industrial and Applied Mathematics, Philadelphia, PA, USA, (Hymenoptera: formicidae). Psyche J. Entomol. 93, 217–229. https://doi.org/10. pp. 371–390. 1155/1986/39768. Becchetti, L., Clementi, A., Natale, E., et al., 2016. Stabilizing consensus with many Hothorn, T., Bretz, F., Westfall, P., 2008. Simultaneous inference in general parametric opinions. Proceedings of the Twenty-seventh Annual ACM-SIAM Symposium on models. Biom. J. 50, 346–363. https://doi.org/10.1002/bimj.200810425. Discrete Algorithms. Society for Industrial and Applied Mathematics, Philadelphia, Karsai, I., Wenzel, J.W., 1998. Productivity, individual-level and colony-level flexibility, PA, USA, pp. 620–635. and organization of work as consequences of colony size. Proc. Natl. Acad. Sci. U. S. Bengston, S.E., 2018. Life-history and behavioral trait covariation across 3 years in A. 95, 8665–8669. https://doi.org/10.1073/pnas.95.15.8665. Temnothorax ants. Behav. Ecol. 29, 1494–1501. https://doi.org/10.1093/beheco/ Kaur, R., Anoop, K., Sumana, A., 2012. Leaders follow leaders to reunite the colony: ary101. relocation dynamics of an Indian queenless ant in its natural habitat. Anim. Behav. Bengston, S.E., Dornhaus, A., 2013. Colony size does not predict foraging distance in the 83, 1345–1353. https://doi.org/10.1016/j.anbehav.2012.02.022. ant Temnothorax rugatulus: a puzzle for standard scaling models. Insectes Soc. 60, Kolay, S., Annagiri, S., 2017. Deterioration in nest quality triggers relocation without 93–96. https://doi.org/10.1007/s00040-012-0272-4. affecting its dynamics in an ant. Insectes Soc. 64, 321–327. https://doi.org/10.1007/ Bouchet, D.C., Peeters, C., Fisher, B.L., Molet, M., 2013. Both female castes contribute to s00040-017-0546-y. colony emigration in the polygynous ant Mystrium oberthueri. Ecol. Entomol. 38, Kramer, B.H., Scharf, I., Foitzik, S., 2014. The role of per-capita productivity in the 408–417. https://doi.org/10.1111/een.12033. evolution of small colony sizes in ants. Behav. Ecol. Sociobiol. 68, 41–53. https://doi. Cao, T.T., 2013. High social density increases foraging and scouting rates and induces org/10.1007/s00265-013-1620-8. polydomy in Temnothorax ants. Behav. Ecol. Sociobiol. 67, 1799–1807. https://doi. Lanan, M.C., Dornhaus, A., Bronstein, J.L., 2011. The function of polydomy: the ant org/10.1007/s00265-013-1587-5. Crematogaster torosa preferentially forms new nests near food sources and fortifies Conradt, L., Roper, T.J., 2005. Consensus decision making in animals. Trends Ecol. Evol. outstations. Behav. Ecol. Sociobiol. 65, 959–968. 20, 449–456. https://doi.org/10.1016/j.tree.2005.05.008. Mailleux, A.-C., Deneubourg, J.-L., Detrain, C., 2003. How does colony growth influence Cook, Z., Franks, D.W., Robinson, E.J.H., 2013. Exploration versus exploitation in poly- communication in ants? Insectes Soc. 50, 24–31. https://doi.org/10.1007/ domous ant colonies. J. Theor. Biol. 323, 49–56. https://doi.org/10.1016/j.jtbi.2013. s000400300004. 01.022. Mallon, E.B., Pratt, S.C., Franks, N.R., 2001. Individual and collective decision-making Creighton, W.S., 1965. The habits and distribution of Macromischa Subditiva Wheeler during nest site selection by the ant Leptothorax albipennis. Behav. Ecol. Sociobiol. 50, (Hymenoptera: Formicidae). Psyche J. Entomol. Accessed 26 Aug 2019. https:// 352–359. www.hindawi.com/journals/psyche/1965/064181/abs/. Michener, C.D., 1964. Reproductive efficiency in relation to colony size in hymenopterous Cronin, A.L., 2012. Consensus decision making in the ant Myrmecina nipponica: house- societies. Insectes Soc. 11, 317–341. https://doi.org/10.1007/BF02227433. hunters combine pheromone trails with quorum responses. Anim. Behav. 84, Modlmeier, A.P., Keiser, C.N., Watters, J.V., et al., 2014. The keystone individual concept: 1243–1251. https://doi.org/10.1016/j.anbehav.2012.08.036. an ecological and evolutionary overview. Anim. Behav. 89, 53–62. https://doi.org/ Debout, G., Schatz, B., Elias, M., Mckey, D., 2007. Polydomy in ants: what we know, what 10.1016/j.anbehav.2013.12.020. we think we know, and what remains to be done. Biol. J. Linn. Soc. 90, 319–348. Möglich, M., 1978. Social-organization of nest emigration in Leptothorax (hym form). https://doi.org/10.1111/j.1095-8312.2007.00728.x. Insectes Soc. 25, 205–225. https://doi.org/10.1007/BF02224742. Doering, G.N., Pratt, S.C., 2016. Queen location and nest site preference influence colony Partridge, L.W., Partridge, K.A., Franks, N.R., 1997. Field survey of a monogynous lep- reunification by the ant Temnothorax rugatulus. Insectes Soc. 63, 585–591. https:// tothoracine ant (Hymenoptera, Formicidae): evidence of seasonal polydomy? Insectes doi.org/10.1007/s00040-016-0503-1. Soc. 44, 75–83. https://doi.org/10.1007/s000400050031. Doering, G.N., Pratt, S.C., 2019. Symmetry breaking and pivotal individuals during the Pinheiro, J., Douglas, B., Saikat, D., et al., 2019. nlme: Linear and Nonlinear Mixed Effects reunification of ant colonies. J. Exp. Biol. 222https://doi.org/10.1242/jeb.194019. Models. jeb194019. Pratt, S.C., 2005. Behavioral mechanisms of collective nest-site choice by the ant Doran, C., Newham, Z.F., Phillips, B.B., Franks, N.R., 2015. Commitment time depends on Temnothorax curvispinosus. Insectes Soc. 52, 383–392. https://doi.org/10.1007/ both current and target nest value in Temnothorax albipennis ant colonies. Behav. s00040-005-0823-z. Ecol. Sociobiol. 69, 1183–1190. https://doi.org/10.1007/s00265-015-1932-y. Pratt, S.C., 2019. Nest site choice in social insects. In: Choe, J.C. (Ed.), Encyclopedia of Dornhaus, A., Franks, N.R., Hawkins, R.M., Shere, H.N.S., 2004. Ants move to improve: Animal Behavior, Second Edition). Academic Press, Oxford, pp. 766–774. colonies of Leptothorax albipennis emigrate whenever they find a superior nest site. Pratt, S.C., Mallon, E.B., Sumpter, D.J.T., Franks, N.R., 2002. Quorum sensing, recruit- Anim. Behav. 67, 959–963. https://doi.org/10.1016/j.anbehav.2003.09.004. ment, and collective decision-making during colony emigration by the ant Dornhaus, A., Powell, S., Bengston, S., 2012. Group size and its effects on collective or- Leptothorax albipennis. Behav. Ecol. Sociobiol. 52, 117–127. ganization. Annu. Rev. Entomol. 57, 123–141. https://doi.org/10.1146/annurev- Pratt, S.C., Sumpter, D.J.T., 2006. A tunable algorithm for collective decision-making. ento-120710-100604. Proc. Natl. Acad. Sci. U. S. A. 103, 15906–15910. https://doi.org/10.1073/pnas. Foitzik, S., Heinze, J., 2001. Microgeographic genetic structure and intraspecific para- 0604801103. sitism in the ant Leptothorax nylanderi. Ecol. Entomol. 26, 449–456. https://doi.org/ Rettenmeyer, C., Topoff, H., Mirenda, J., 1978. Queen retinues of army ants(hyme- 10.1046/j.1365-2311.2001.00354.x. noptera-Formicidae-Ecitoninae). Ann. Entomol. Soc. Am. 71, 519–528. Foitzik, S., Heinze, J., 1998. Nest site limitation and colony takeover in the ant Robinson, E.J., 2014. Polydomy: the organisation and adaptive function of complex nest Leptothorax nylanderi. Behav. Ecol. 9, 367–375. https://doi.org/10.1093/beheco/9.4. systems in ants. Curr. Opin. Insect Sci. 5, 37–43. https://doi.org/10.1016/j.cois. 367. 2014.09.002. Forel, A., 1874. Les fourmis de la Suisse: systématique, notices anatomiques et physio- Robinson, E.J.H., Franks, N.R., Ellis, S., et al., 2011. A simple threshold rule is sufficient logiques, architecture, distribution géographique, nouvelles expériences et observa- to explain sophisticated collective decision-making. PLoS One 6, e19981. https://doi. tions de moeurs. Société Helvétique des Sciences Naturelles. org/10.1371/journal.pone.0019981. Franks, N.R., Dornhaus, A., Fitzsimmons, J.P., Stevens, M., 2003. Speed versus accuracy Robson, S.K., Traniello, J.F.A., 1999. Key individuals and the organisation of labor in in collective decision making. Proc. R. Soc. B-Biol. Sci. 270, 2457–2463. https://doi. ants. In: Detrain, C., Deneubourg, J.L., Pasteels, J.M. (Eds.), Information Processing in org/10.1098/rspb.2003.2527. Social Insects. Birkhäuser Basel, Basel, pp. 239–259. Franks, N.R., Richardson, T.O., Stroeymeyt, N., et al., 2013. Speed–cohesion trade-offsin Sahu, P.K., Kolay, S., Annagiri, S., 2019. To reunite or not: a study of artificially frag- collective decision making in ants and the concept of precision in animal behaviour. mented Diacamma indicum ant colonies. Behav. Process. 158, 4–10. https://doi.org/ Anim. Behav. 85, 1233–1244. https://doi.org/10.1016/j.anbehav.2013.03.010. 10.1016/j.beproc.2018.10.017. Franks, N.R., Sendova-Franks, A.B., 2000. Queen transport during ant colony emigration: Salvaggio, N.L., 2013. Basic Photographic Materials and Processes. Taylor & Francis. a group-level adaptive behavior. Behav. Ecol. 11, 315–318. https://doi.org/10.1093/ Sasaki, T., Colling, B., Sonnenschein, A., et al., 2015. Flexibility of collective decision beheco/11.3.315. making during house hunting in Temnothorax ants. Behav. Ecol. Sociobiol. 69, Franks, N.R., Wilby, A., Silverman, B.W., Tofts, C., 1992. Self-organizing nest construc- 707–714. https://doi.org/10.1007/s00265-015-1882-4. tion in ants: sophisticated building by blind bulldozing. Anim. Behav. 44, 357–375. Sasaki, T., Pratt, S.C., 2012. Groups have a larger cognitive capacity than individuals. https://doi.org/10.1016/0003-3472(92)90041-7. Curr. Biol. 22, R827–R829. https://doi.org/10.1016/j.cub.2012.07.058. Gelman, A., Hill, J., 2006. Data Analysis Using Regression and Multilevel/Hierarchical Sasaki, T., Pratt, S.C., 2011. Emergence of group rationality from irrational individuals. Models. Cambridge University Press. Behav. Ecol. 22, 276–281. https://doi.org/10.1093/beheco/arq198. Ghaffari, M., Parter, M., 2016. A polylogarithmic gossip algorithm for plurality consensus. Scharf, I., Modlmeier, A.P., Fries, S., et al., 2012. Characterizing the collective personality Proceedings of the 2016 ACM Symposium on Principles of Distributed Computing. of ant societies: aggressive colonies do not abandon their home. PLoS One 7, e33314. ACM, New York, NY, USA, pp. 117–126. https://doi.org/10.1371/journal.pone.0033314. Headley, A.E., 1943. Population studies of two species of ants, Leptothorax longispinosus Schneider, C.A., Rasband, W.S., Eliceiri, K.W., 2012. NIH Image to ImageJ: 25 years of Roger and Leptothorax curvispinosus mayr. Ann. Entomol. Soc. Am. 36, 743–753. image analysis. Nat. Methods 9, 671–675. https://doi.org/10.1038/nmeth.2089. https://doi.org/10.1093/aesa/36.4.743. Seeley, T.D., 2010. Honeybee Democracy. Princeton University Press. Healey, C.I.M., Pratt, S.C., 2008. The effect of prior experience on nest site evaluation by Seeley, T.D., 2003. Consensus building during nest-site selection in honey bee swarms: the ant Temnothorax curvispinosus. Anim. Behav. 76, 893–899. https://doi.org/10. the expiration of dissent. Behav. Ecol. Sociobiol. 53, 417–424. 1016/j.anbehav.2008.02.016. Shannon, C.E., 1948. A mathematical theory of communication. Bell Syst. Technol. J. 27, Heinze, J., Puchinger, W., Hölldobler, B., 1997. Worker reproduction and social hier- 379–423. https://doi.org/10.1002/j.1538-7305.1948.tb01338.x. archies in Leptothorax ants. Anim. Behav. 54, 849–864. https://doi.org/10.1006/ Smallwood, J., 1982. Nest relocations in ants. Insectes Soc. 29, 138–147. https://doi.org/ anbe.1996.0511. 10.1007/BF02228747. Herbers, J.M., 1986. Nest site limitation and facultative polygyny in the ant Leptothorax Snyder, L.E., Herbers, J.M., 1991. Polydomy and sexual allocation ratios in the ant longispinosus. Behav. Ecol. Sociobiol. 19, 115–122. https://doi.org/10.1007/ Myrmica punctiventris. Behav. Ecol. Sociobiol. 28, 409–415.

7 G.N. Doering, et al. Behavioural Processes 170 (2020) 103994

Spellerberg, I.F., Fedor, P.J., 2003. A tribute to Claude Shannon (1916–2001) and a plea curvispinosus Mayr (Hymenoptera; Formicidae). Psyche J. Entomol. 92, 71–81. for more rigorous use of species richness, species diversity and the ‘Shannon–Wiener’ https://doi.org/10.1155/1985/29215. Index. Glob. Ecol. Biogeogr. 12, 177–179. https://doi.org/10.1046/j.1466-822X. Van Wilgenburg, E., Elgar, M.A., 2007. Colony characteristics inﬂuence the risk of nest 2003.00015.x. predation of a polydomous ant by a monotreme. Biol. J. Linn. Soc. 92, 1–8. https:// Strätz, M., Heinze, J., 2004. Colony structure and sex allocation ratios in the ant doi.org/10.1111/j.1095-8312.2007.00868.x. Temnothorax crassispinus. Insectes Soc. 51, 372–377. https://doi.org/10.1007/ Visscher, P.K., 2007. Group decision making in nest-site selection among social insects. s00040-004-0755-z. Annu. Rev. Entomol. 52, 255–275. https://doi.org/10.1146/annurev.ento.51. Stroeymeyt, N., Giurfa, M., Franks, N.R., 2017a. Information certainty determines social 110104.151025. and private information use in ants. Sci. Rep. 7. https://doi.org/10.1038/srep43607. Wheeler, W.M., 1910. Ants; Their Structure, Development and Behavior. Columbia uni- Stroeymeyt, N., Joye, P., Keller, L., 2017b. Polydomy enhances foraging performance in versity press, New York. ant colonies. Proc. R. Soc. B 284, 20170269. https://doi.org/10.1098/rspb.2017. Yamaguchi, T., 1992. Interspeciﬁc interference for nest sites between Leptothorax con- 0269. gruus and Monomorium intrudens. Insectes Soc. 39, 117–127. https://doi.org/10. Stuart, R.J., 1985. Spontaneous polydomy in laboratory colonies of the ant Leptothorax 1007/BF01249288.