Colony size and initial conditions combine to shape colony reunification dynamics

Journal Pre-proof COLONY SIZE AND INITIAL CONDITIONS COMBINE TO SHAPE COLONY REUNIFICATION DYNAMICS Grant Doering

PII:

S0376-6357(19)30363-8

DOI:

https://doi.org/10.1016/j.beproc.2019.103994

Reference:

BEPROC 103994

To appear in:

Behavioural Processes

Received Date:

20 September 2019

Revised Date:

24 October 2019

Accepted Date:

28 October 2019

Please cite this article as: Doering G, COLONY SIZE AND INITIAL CONDITIONS COMBINE TO SHAPE COLONY REUNIFICATION DYNAMICS, Behavioural Processes (2019), doi: https://doi.org/10.1016/j.beproc.2019.103994

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COLONY SIZE AND INITIAL CONDITIONS COMBINE TO SHAPE COLONY REUNIFICATION DYNAMICS

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GRANT DOERING* [email protected],

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MCMASTER UNIVERSITY, L8S 4K1, HAMILTON

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HIGHLIGHTS

Ant colonies often live in a single nest but can also split between several nests concurrently.

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We investigated what group traits and initial conditions promote or deter the reunification of multi-nest colonies. Larger colonies and a lack of a central information hub both diminished the likelihood of colony reunification.

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Colonies were no worse at reunifying when split between additional nests.

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ABSTRACT

Group cohesion and collective decision-making are important for many social animals, like social insects, whose societies depend on

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the coordinated action of individuals to complete collective tasks. A useful model for understanding collective, consensus-driven decision-making is the fluid nest selection dynamics of ant colonies. Certain ant species oscillate between occupying multiple nests

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simultaneously (polydomy) and reuniting at a 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

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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

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that consensus driven decisions can be influenced by both external and intrinsic group-level factors and are in no way simple

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stereotyped processes.

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INTRODUCTION

The capacity to jointly evaluate and unanimously choose between multiple mutually exclusive options is common in social animals

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(Conradt and Roper 2005). In eusocial insects, the group’s selection of a nest-site is a well-studied example of such consensus building (Visscher 2007). Ants and bees, for example, can move their entire colonies to a new home when their current dwelling is

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damaged or when a superior alternative becomes available (Möglich 1978; Smallwood 1982; Dornhaus et al. 2004; Seeley 2010; Pratt 2019). A fundamental similarity in most of these insects’ selection processes is the ability to integrate information from multiple individuals to reach a collective choice. In the ant genus Temnothorax, individuals discriminate nest quality based on several features (e.g., light level in the nest cavity) and begin to recruit nestmates to candidate homes with a probability that depends on their

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assessment of the new location’s appeal (Mallon et al. 2001; Robinson et al. 2011; Pratt 2019). Once the number of ants committed to a site surpasses a quorum, workers will initiate brood transport and carry the remaining population from the old nest to the new one (Pratt et al. 2002; Pratt 2019). Temnothorax ants generally dwell within fragile pre-formed cavities (i.e., in rotting acorns, plant galls,

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sticks, etc.) that are prone to damage or total annihilation through environmental or animal disturbance. Over the course of a year, more than half of all occupied nests can be destroyed (Yamaguchi 1992). Nest relocation is therefore frequent.

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Although the behavioral routines underpinning emigration help to avoid colony fragmentation in the face of repeated nest

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destruction (Pratt et al. 2002; Seeley 2003; Cronin 2012; Kolay and Annagiri 2017; Pratt 2019), consensus is not guaranteed. Temnothorax will occasionally split their colonies between multiple discrete nest sites. The split colonies may remain socially

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connected with ants, brood, and resources shared and exchanged between colony sites (Stuart 1985; Herbers 1986). Split colonies are,

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however, still capable of later reunifying at a single site. In fact, predictably timed cycles of intentionally splitting in the spring only to later reunify in autumn are a characteristic feature of Temnothorax colonies (Headley 1943; Partridge et al. 1997; Foitzik and Heinze

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2001; Strätz and Heinze 2004; Kramer et al. 2014).

When a colony distributes itself across multiple nests, this is termed polydomy. Polydomy is thought to reduce predation risk (Van Wilgenburg and Elgar 2007) and to allow improved resource acquisition (Debout et al. 2007; Robinson 2014; Stroeymeyt et al. 2017b). However, prolonged polydomy can have fitness costs too, such as a colony being dispersed too thinly to effectively manage

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colony resources (Cook et al. 2013) or through queen-worker conflict over the production of males (Snyder and Herbers 1991; Banschbach and Herbers 1996; Heinze et al. 1997). Furthermore, like emigration, reunification itself can be dangerous as ants need to temporarily relinquish shelter while they consolidate nests (Rettenmeyer et al. 1978; Franks and Sendova-Franks 2000; Bouchet et al. 2013). Thus, there are likely trade-offs in a colony’s choice of whether to switch from polydomy to monodomy or vice versa.

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Although the circumstances that foster colony fission have received some attention (Alloway et al. 1982; 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

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reunification or the behaviors underlying the process (Herbers and Tucker 1986; Kaur et al. 2012; Doering and Pratt 2016, 2019; Sahu

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et al. 2019).

In Temnothorax rugatulus, the choice of where to reunify is guided by a preference for the highest quality site and, if all nests

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are identical in quality, for the nest inhabited by the queen (Doering and Pratt 2016). Mechanistically, colony reunification is achieved

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through the actions of a small team of hyperactive workers, or keystone individuals (Robson and Traniello 1999; Modlmeier et al. 2014), whose behavior determines a colony’s decision (Doering and Pratt 2019). Yet, how external circumstances and group-level

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traits influence the probability of reunification is unknown. Here we evaluate how colony size, number of available nests, and initial splitting conditions combine to shape reunification in ants. Colony size is likely to be influential because differences in worker populations can impact many dimensions of ant society (Michener 1964; Karsai and Wenzel 1998; Mailleux et al. 2003; Dornhaus et al. 2012; Kramer et al. 2014), and some species strive to

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maintain a specific density of workers within the nests (Franks et al. 1992). When a 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). Likewise, larger colonies might have less reason to reunify if they have already split, and small colonies (or colonies split between many nests) might have a stronger incentive to reunite. The route that colonies take to being split may also

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impact reunification. When a Temnothorax colony emigrates to a new nest, its speed of relocation, accuracy, level of worker engagement, and resulting cohesiveness all depend on whether its original nest remains intact (Franks et al. 2003, 2013; Pratt and

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Sumpter 2006). Similarly, rates of reunification could hinge upon how damaged a colony’s home nest becomes before the colony first

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splits. Finally, increasing the number of available or currently occupied nests may make reunification more challenging. In theoretical models of opinion dynamics, more subgroups can slow convergence to a consensus (Becchetti et al. 2015, 2016; Ghaffari and Parter

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2016). However, at least in typical nest relocation, Temnothorax can incorporate multiple options into its collective decision-making scheme without incurring any measurable loss of accuracy (Sasaki and Pratt 2012). We therefore aimed to explore how the

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circumstances of initial fractionation, colony size, and the number of available nests interact to either promote or discourage the

METHODS

Study colonies

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restoration of colony cohesion in T. rugatulus.

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The 48 colonies of T. rugatulus Emery used in this study were collected in February 2018 at Madera Peak [33.317N, 110.876W], part of the Pinal Mountains of Arizona. T. rugatulus is widespread across western North America and is abundant in many parts of its range. Colonies establish nests inside rock crevices and in soil under stones, and nests usually range in size from 50-400 workers

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(Bengston 2018). Like many other species in its genus (Forel 1874; Wheeler 1910; Creighton 1965), T. rugatulus is thought to forage on dead insect prey and honeydew (Bengston and Dornhaus 2013).

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All colonies used for this study had a single queen (monogynous), but there was variation in the numbers of workers (min: 60, max: 425, mean: 148.8, standard deviation: 75.2) and brood (min: 28, max: 245, mean: 116.6, standard deviation: 53.9). Each colony

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3 cm) and were provided with an ad libitum supply of water and their food (protein: mealworms; sugar: maple syrup)

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boxes (11

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resided in a “poor” style nest (see below) both before and between the experimental trials. Colonies were maintained in lidded plastic

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was replenished weekly.

Nest designs

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All nests consisted of a balsa wood slat (2.4 mm thick) with a 38 mm diameter circular hole drilled through the center that acted as a cavity in which the ants could live. Each slat was sandwiched between two glass microscope slides (50

75 mm) which created a roof

and floor for the nests. A 2 mm wide slit was cut into one side of each nest, providing the ants with an entrance to the nest cavity. Since T. rugatulus colonies prefer darker nest cavities (Visscher 2007), we set the attractiveness of each experimental nest to one of

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three different brightness levels by shading the nest cavities with neutral density light filters. Nest type 1 was a “poor quality” nest with no light filter. Nest type 2 was a “mediocre quality” nest. These nests were identical to the poor style nests, except a single light filter of 1-stop power (i.e., blocking 50% of incoming light) covered each roof (Salvaggio 2013). Nest type 3 was a “good quality”

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nest and was also identical to the poor style nests except for three 3-stop filters (i.e., blocking 99.8% of incoming light) that covered

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each roof. Further details on these kinds of nests can be located elsewhere (Sasaki et al. 2015).

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Experimental design

Colonies were randomly assigned to one of three treatment groups, which differed in the way that colonies were encouraged to

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become polydomous. For the first treatment (Equal split), colonies were split equally between isolated nests that were later placed within a single arena together. In the second treatment (Forced emigration), colonies were induced to relocate to a new nest by

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damaging their original nest and presenting the ants with multiple identical options, encouraging splitting (Pratt and Sumpter 2006;

nests.

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Franks et al. 2013). Colonies in the third group (Scattered) had their entire population scattered in an area containing potential new

These three conditions were chosen because they differ in the amount of damage inflicted on colonies’ nests before they split between new nests. A colony from the equal split and scattered groups will be completely removed from its original nest, potentially

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signaling to the ants that they are in greater danger or in a poor environment. This contrasts with a colony in the forced emigration group, which will only experience the removal of its nest’s roof. When a nest falls apart in the wild, it is possible that portions of the workers and brood fall out in the process since T. rugatulus ants cling to the rocks concealing their nests (Möglich 1978). Some nest relocations in the field therefore plausibly involve situations were ants must emigrate without all starting at the home nest. The dynamics of nest relocation in the scattered group may also differ from the forced emigration group. Ants in the scattered group will Doering et al. 8

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be robbed of any centralized location from where they can coordinate their activity at the start of each trial, potentially leading to more initial splitting than in the forced emigration group. This difference is not relevant for the equal split group because splitting is

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guaranteed for that group

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Treatment I – Equal split: 12 colonies were randomly selected to be in the equal split treatment group. On each day of the experiment,

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each colony had its entire worker and brood populations manually and evenly divided into one of six possible conditions in a 2 (nest #) x 3 (nest quality) design: (1) two poor nests, (2) two mediocre nests, (3) two good nests, (4) three poor nests, (5) three mediocre

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nests, or (6) three good nests. Each colony experienced every condition exactly once, and the order in which colonies received each condition was randomized so that each colony in the group received the treatments in a unique order. Colonies were given two days of

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rest between each condition. The procedure for evenly splitting colonies has been described elsewhere (Doering and Pratt 2016, 2019), and our methods mirror those. Briefly, after removing the colony’s glass roof, and destroying the nest, ants and brood were moved individually into isolated plastic arenas (24 cm diameter, 9 cm height) each of which contained a single new nest. After providing the ants an hour to move into each nest, all nests were transplanted together into one new arena. When split in half, the two nests were

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placed, respectively, in the left and right portions of the arena, facing each other and lying 30 mm from the center. When a colony was split between three nests, the nests were arranged in an approximately equilateral triangle. Because queens bias decision-making during reunification (Doering and Pratt 2016), queens were captured during splitting and kept apart from their colonies by placing them in their original nest-boxes. Queens were returned to their source colonies at the end of each trial. Although excluding queens Doering et al. 9

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from the reunification process was necessary for the current study, it was not ideal as this removes a possibly important part of how colonies make such decisions. However, Temnothorax colonies may occupy three or more nests simultaneously in the field (Alloway

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et al. 1982), and colonies can persist after their queen has perished because workers can begin laying male-destined eggs in her

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absence (Heinze et al. 1997) or eventually merge with an unrelated colony (Foitzik and Heinze 1998). Analyzing reunifications between queenless nests therefore retains some ecological relevance (Doering and Pratt 2019).

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Experimental arenas were housed within cabinets (Height: 57 cm, Width: 108 cm, Depth: 47 cm) whose interiors were

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homogeneously illuminated with identical arrays of LEDs. After colonies had been split, all nests were photographed approximately 1 hour, 6 hours, and 12 hours after their initial forced division. After the final photographs were taken, the most populous nest in each arena was placed in its colony’s nest-box, and the less populous nest(s) were dismantled and any ants inhabiting the cavity were

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placed back in their nest-box. Arenas and glass slides were reused between trials and were cleaned with ethanol to remove any residual chemical markings (Sasaki and Pratt 2011; Franks et al. 2013). Only 12 colonies of this treatment group were established

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owing to the time and space consuming nature of this treatment group.

Treatment II – Forced emigration: 18 colonies were randomly selected to be in the forced emigration treatment group. These colonies experienced the same 2 x 3 design above, except that colonies were presented with the nests in the circular arenas and had their starting nests destroyed by the removal of its roof, stimulating an emigration to one or more of the target options. Starting nests were positioned approximately 30 mm from the arena centers, creating either a triangle pattern (when given two nests) or a diamond pattern Doering et al. 10

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(when given three nests). Because colonies in the equal split group had 1 hour to acclimate, colonies in the forced emigration group were analogously left in empty arenas for one hour to acclimate before being induced to emigrate. Colonies were similarly

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photographed 1 hour, 6 hours, and 12 hours after being stimulated to emigrate.

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Treatment III – Scattered: The remaining 18 colonies were assigned to the scattered treatment group. At the start of each trial,

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colonies were left to acclimate in empty arenas for one hour before receiving one of the 2 x 3 nest selection combinations. After introducing the new nests into an arena, all brood items and workers from a colony were lifted from their starting nest with a soft

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identical to the other treatment groups.

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paintbrush and haphazardly dispersed around the arena. We then monitored the decision-making of these colonies in a manner

Data collection & Analysis

The number of worker ants inhabiting the nest in each photograph was determined by manually marking and counting all individuals in ImageJ (Schneider et al. 2012). Of the 2160 nest photographs, 61 were missing or unusable. If a trial contained any missing images

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from its sequence, then we excluded the trial from the analysis (27/288 trials). For all three time periods during a 12-hr. run, we computed the level of cohesion exhibited by each colony by using an

entropy-based metric that was introduced by Franks et al. (2013) (eq. 1):

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𝐶

𝐶=1−𝐶

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(1)

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H represents the Shannon entropy index (Shannon 1948; Spellerberg and Fedor 2003):

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𝐶 = − ∑𝐶 𝐶=1 𝐶𝐶 log2 𝐶𝐶

(2)

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This index quantifies the extent to which a group is aggregated at a single site when multiple options are available. In eq. 2, n signifies the number of nests available to a colony, and 𝑝𝑖 is the percentage of individuals from a colony present at nest 𝑖. 𝐻max conveys a

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configuration where a colony’s workers are evenly divided between every nest option. The values of C will range from 0 to 1

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regardless of how many options are available. When C = 1, all ants in a colony have decided on a single nest, and when C = 0, a colony’s population is equally present in each available nest. Any scouting of foraging ants that were patrolling their arena when nest

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photographs were taken were excluded from the cohesion calculations. Following the approach used in previous reunification studies (Doering and Pratt 2016, 2019), we also classified colonies as being “unified” and monodomous at the end of each trial if a minimum proportion of a colony’s workers resided in just a single nest. For this study, we set 90% as the threshold for being “unified”. This

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corresponds to a minimum cohesion score of C = 0.531 in colonies given two nests and C = 0.641 in colonies given three nests.

Statistical Analysis

We used linear mixed-effects models (LMM) to evaluate the effects of nest quality, polydomy treatment, and colony size on cohesion, with colony ID always set as a random factor acting on the intercept. We used likelihood ratio tests to assess the effect of each

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variable by sequentially comparing models without a particular term to a full model with each term present. We compared the cohesion values of different polydomy treatments to each other using simultaneous general linear hypothesis tests (GLHT). In our

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analyses, colony size was scaled so that it was mean centered and had unit-variance in order to improve the fit of the models and the

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interpretability of regression coefficients (Table S1). Through an inspection of the plots of model residuals and fitted values, we verified that the assumption of the independence of errors was approximately satisfied. However, the residuals from most of our LMM

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models were not normally distributed. We proceeded to use these models despite this since the relaxation of this assumption is not considered highly problematic if one is seeking an assessment of general trends and not precise quantitative predictions of individual

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points (Gelman and Hill 2006), and our models’ results were consistent with the trends in our figures. A consequence of the cohesion metric used in this study is that the distributions of cohesion will tend towards being bimodal in

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the 2-nest condition but will be more evenly distributed for colonies that were given 3-nests as there are more possible sites to occupy. The actual values of the cohesion metric will also be automatically higher for colonies in the 3-nest condition than for colonies in the 2-nest condition even when referring to the same population configuration. For example, a colony that is initially split into three nests and ends its trial evenly split in two nests will have a final cohesion score of C = 0.369, but the same state will have a score of C = 0

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for colonies in the 2-nest treatment. Due to these inherent differences, we analyzed the cohesion results from the 2-nest group separately from the 3-nest group. However, in order to compare the behavior of colonies given two nests to those given three nests, we assessed the number of “unified” colonies over time across these two groups. Because being “unified” at just one nest reflects the same condition for all colonies, this measure avoids the issues of comparing cohesion scores for colonies initially split between Doering et al. 13

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different numbers of nests. Differences between the 2-and 3-nest groups in the number of “unified “colonies were analyzed independently for each polydomy treatment using generalized linear models of a binomial distribution family (Table S1).

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Since some colonies avoided splitting at the beginning of their trials in the forced emigration group, we also analyzed the

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effects of nest quality, quantity, polydomy treatment, and colony size using a reduced data set that excluded colonies that lacked low cohesion scores (C < 0.1) at the 1-hour time point. Using LMM models, we also evaluated whether colonies had any biases towards

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occupying particular sites based on the nests’ spatial position in the arenas (See Table S1). All statistical calculations were performed in R version 3.4 (https://www.r-project.org). LMM models were fit using the package nlme (Pinheiro et al. 2019), and the GLMM

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models used to compare the number of “unified” colonies in the 2 and 3 nest treatments were fit using the package lme4 (Bates et al.

RESULTS

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2015). General linear hypothesis tests were conducted using the package multcomp (Hothorn et al. 2008).

Effect of polydomy treatment

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Regardless of whether colonies were given two or three nests, there were significant differences in cohesion across the three polydomy treatments after 1 hour (Figure 1; 2–nest group: L ratio = 26.757, p < 0.0001; 3-nest group: L ratio = 38.810, p < 0.0001). These differences (at the 1-hour time point) were due to higher levels of cohesion in the forced emigration group compared to the scattered (2-nest group General linear hypothesis test [GLHT]: β = -0.2457, z = -5.029, p < 0.0001; 3-nest group GLHT: β = -0.1437, z = Doering et al. 14

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4.663, p < 0.0001) and equal split (2-nest group GLHT: β = -0.2888, z = -5.130, p < 0.0001; 3-nest group GLHT: β = -0.2673, z = 7.675, p < 0.001) groups. At the 1-hour time point, colonies that received three nests in the forced emigration group had a peak in their

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cohesion distribution located roughly at C = 0.369 (Figure 1), which corresponds to a colony being evenly split between two out of the

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three nests. Likewise, colonies in the forced emigration group that received two nests have a secondary peak at C = 1, which is when all workers occupy a single nest. This implies that many colonies in the forced emigration group often avoided fully breaking up to

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begin with.

Cohesion increased over time for all three splitting treatments; the distribution of colony cohesion scores shifted towards C = 1

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after each time step (Figure 1). At the 6-hour time point, colonies that were given two nests show statistically significant differences in cohesion across the three polydomy treatments (L ratio = 25.574, p < 0.0001), with colonies in the forced emigration group exhibiting

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higher cohesion than both the equal split (GLHT: β = -0.3116, z = -4.588, p < 0.0001) and scattered (GLHT: β = -0.3137, z = -5.341, p < 0.0001) groups. After 12 hours the differences between polydomy treatments remained significant for colonies given two nests (L ratio = 12.889, p = 0.002). Colonies that experienced three nests showed less prominent differences between the splitting treatments than colonies that were given two nests. In 3-nest colonies, cohesion did significantly differ between splitting groups at 6-hours (L

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ratio = 7.507, p = 0.023) but did not meaningfully differ at 12-hours (L ratio = 3.19 p = 0.203). The cohesion distributions of the equal split and scattered treatments qualitatively match each other very closely (Figure 1). When only colonies that exhibited low levels of cohesion at the beginning of their trials are included (i.e., had a cohesion score of C < 0.1 at the 1-hour time point), the significant

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differences in cohesion between polydomy treatments at 12-hours disappears for colonies in the 2-nest group (Figure S1; L ratio =

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5.755, p = 0.056) and remains nonsignificant in the 3-nest group (Figure S1; L ratio = 2.095, p = 0.351).

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Effects of colony size and nest quality & quantity

We found no significant association between nest quality and average cohesion at 12 hours for either the 2-nest group (L ratio = 0.382,

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p = 0.826) or the 3-nest group (L ratio = 0.390, p = 0.823), and no significant association between nest quality and average cohesion at the beginning (i.e., degree of initial splitting) of the forced emigration trials (2-nest: L ratio = 0.767, p = 0.681; 3-nest: L ratio = 1.819,

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p = 0.403). Quality also did not predict differences in cohesion at either the 1-hour (2-nest condition: L ratio = 1.745, p = 0.418; 3-nest condition: L ratio = 4.792, p = 0.091) or 6-hour (2-nest condition: L ratio = 0.651, p = 0.722; 3-nest condition: L ratio = 0.016, p =

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0.992) time points. Overall, we found no evidence that colonies are biased towards reunifying at either the left or right nest when split between two nests (L ratio = 1.206, p = 0.272), nor did we find any biases when colonies were split between three nests (L ratio = 0.489, p = 0.783).

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Colony size strongly impacted cohesion. Larger colonies showed lower levels of cohesion after 12 hours in both the 2-nest (Figure 2a, L ratio = 17.217, p < 0.0001) and 3-nest conditions (Figure 2b, L ratio = 31.015, p < 0.0001). The number of colonies that were unified at a single nest after 12 hours was not affected by the number of nests available for colonies in the forced emigration (Figure 3; β = 0.1376, z = 0.326, p = 0.745) or equal split group (Figure 3; β = -0.1737, z = -0.307, p = 0.759), but the scattered treatment saw a significantly higher number of their colonies unify at one nest if three sites were provided (Figure 3; β = -1.0064, z = Doering et al. 16

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2.354, p = 0.019). When only considering colonies that showed low cohesion (C < 0.1), these non-significant differences remain true for the forced emigration (Figure S2; β = 0.2877, z = 0.351, p = 0.726) and equal split groups (Figure S2; β = -0.1094 z = -0.193, p =

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0.847), but the effect of nest number becomes non-significant in the scattered group (Figure S2; β = -0.6585, z = -1.238, p = 0.216).

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DISCUSSION

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Our study explored several factors that could alter the tendency of ant colonies to reunify after a disturbance to their nest. Colonies that split during forced emigration were faster to reunify, but, contrary to results from prior work (Doering and Pratt 2019), we found no evidence that nest quality affected colony cohesion. We did, however, find a strong negative association between group size and

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cohesion for all treatments. We also found that splitting between additional nests did not make colonies worse at reaching consensus on a single nest. These results extend the idea that consensus-decision making in reunifying ants is a dynamic process that shifts based on a colony’s circumstances (Doering and Pratt 2016, 2019).

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Our observation that colonies who experienced forced emigration also exhibited higher rates of cohesion is intuitive; colonies

are likely more effective at staying integrated when scouts can return to the old nest to recruit their sisters. Temnothorax colonies are known to tune their decision strategies based on prior experience and current conditions to respond flexibly to their environment (Pratt and Sumpter 2006; Healey and Pratt 2008; Doran et al. 2015). For example, colonies will move more rapidly into a new nest

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depending on how damaged their current home is. This led to us to hypothesize that, if the process of reunification carries risk, then colonies in the scattered and equal split groups may be reluctant to launch into a reunification if they sense that they are in a harsh or

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dangerous environment. However, the lack of a significant difference between splitting treatments after the exclusion of colonies that did not initially fully split suggests that the higher levels of cohesion at 12-hours in the forced emigration group is primarily caused by

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this group’s “head start” in cohesion. Any initial population asymmetries between nests may serve as a cue to help colonies decide

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where to unify, shortening the time to reach consensus. Indeed, cohesion increased over time for all three groups. If left long enough, the variation between groups could vanish as more colonies in the equal split and scattered groups reunify. Worker stress,

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disorientation, confusion, or an evaluation of risk therefore seem less likely as explanations for the cohesive differences between splitting methods.

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Colonies in the scattered group likely have greatly reduced quorum thresholds, and this increases the chances of splitting (Pratt and Sumpter 2006). Scouts from a scattered colony might immediately launch into recovering stray workers and brood once they find any nest, which could account for the higher rate of initial splitting in this group compared to forced emigration colonies.

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Prior work has shown that colonies 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 of cohesion compared to other polydomy treatments at 12 hours is not completely obvious. One possible explanation is that colonies with access to two nests should naturally possess a greater imbalance towards just one site compared to when more nests are available because any asymmetry in nest populations for a two-nest scenario must favor exactly one nest. If more nests are involved, multiple nests could have higher Doering et al. 18

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populations than competing sites. A greater imbalance towards a single nest could help beak the symmetry between sites, leading to a faster reunification. However, this is difficult to reconcile with our finding that colonies in the equal split group reach consensus on a

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single nest equally well regardless of the number of starting subpopulations.

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We anticipated that colonies would either have a more difficult time agreeing when there were more competing factions or that imbalances towards a single nest would induce faster reunifications for the 2-nest condition, yet this was not what we observed. The

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protocol that colonies used to reunify when faced with three nests could resemble the one used in two-nest divisions (Doering and

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Pratt 2019). Depending on how efficient this method is, splitting between three nests instead of two may only have a marginal impact on reunification speed. Splitting colonies among an even larger quantity of nests, along with data on how colonies can break symmetry

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and reunify when there are more options, would help address these questions. Our results are consistent with earlier reports of elevated rates of polydomy in larger colonies (Stuart 1985; Cao 2013; Stroeymeyt et al. 2017b), although the presence or absence of a key “threshold density” that enables polydomy remains ambiguous. Social density in occupied nests is argued to be important for spontaneous polydomy (Stuart 1985; Cao 2013). Likewise, we found

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there to be a strong negative association between colony size and final cohesion when colonies were split between nests. We failed to observe a specific social density that fosters stable polydomy vs. reunification in our data, yet, consistent with the size density threshold hypothesis, we observed that in the two-nest trials every colony with more than 200 workers (with one exception) remained divided after 12 h. This does provide some circumstantial evidence for a key social density that triggers stable polydomy in this

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system — though the combined evidence is clearly mixed for now. This study had too few large (>200 workers) colonies to resolve

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this question.

The collective decision-making process of reunification is not a stereotyped routine that unfolds deterministically for all

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colonies. Instead, our data reveal that a combination of both intrinsic factors (e.g., colony size) and extrinsic factors (e.g., circumstances preceding the unification process) mingle to determine the speed and likelihood of reunification versus prolonged

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polydomy. Future comparative studies that address polydomy across species would be particularly valuable, by providing insights into

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the species traits and ecological conditions that cause the evolution of contrasting nesting strategies and consensus building in the fission versus fusion of nests (Pratt 2005; Stroeymeyt et al. 2017a). The data herein convey that the processes underlying multi-nest population dynamics are unlikely to be simple, and hint that the selection pressures driving these behaviors may vary considerably

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among even closely related species.

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Figure legends Fig. 1. Histograms of colony cohesion from all 261 trials. Each row of the figure corresponds to one of the three polydomy

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treatments, and each column corresponds to one of the three time periods at which cohesion was measured. Blue bars depict the

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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 hours, but this process is faster for colonies in the forced

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emigration treatment group.

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Fig. 2. The relationship between colony size and cohesion after 12 hours for the three polydomy treatments. Larger colonies

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show lower cohesion after 12 hours than smaller colonies when split between either two (a) or three nests (b).

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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

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forced emigration group can avoid splitting at the start of their trials, and colonies in the forced emigration and equal split groups are

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no worse at reunifying when presented with three options. Scattered colonies have a higher reunification rate when given three nests.

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