DOI: 10.5061/dryad.bvq83bk6x
title: Nest choice in arboreal ants is an emergent consequence of network creation under spatial constraints

description: Biological transportation networks must balance competing functional
 priorities. The self-organizing mechanisms used to generate such networks
 have inspired scalable algorithms to construct and maintain low-cost and
 efficient human-designed transport networks. The pheromone-based trail
 networks of ants have been especially valuable in this regard. Here, we
 use turtle ants as our focal system: In contrast to the ant species
 usually used as models for self-organized networks, these ants live in a
 spatially constrained arboreal environment where both nesting options and
 connecting pathways are limited. Thus, they must solve a distinct set of
 challenges which resemble those faced by human transport engineers
 constrained by existing infrastructure. Here, we ask how a turtle ant
 colony’s choice of which nests to include in a network may be influenced
 by their potential to create connections to other nests. In laboratory
 experiments with Cephalotes varians and Cephalotes texanus, we show that
 nest choice is influenced by spatial constraints, but in unexpected ways.
 Under one spatial configuration, colonies preferentially occupied more
 connected nest sites; however, under another spatial configuration, this
 preference disappeared. Comparing the results of these experiments to an
 agent-based model, we demonstrate that this apparently idiosyncratic
 relationship between nest connectivity and nest choice can emerge without
 nest preferences via a combination of self-reinforcing random movement
 along constrained pathways and density-dependent aggregation at nests.
 While this mechanism does not consistently lead to the de-novo
 construction of low-cost, efficient transport networks, it may be an
 effective way to expand a network, when coupled with processes of pruning
 and restructuring.
description: <strong>Study species</strong><br>Queenright
 colonies of <em>Cephalotes varians </em>were collected from
 state-managed Hammock forest in Key Largo, Florida, and queenright
 colonies of <em>Cephalotes texanus</em> were collected from
 live oaks on private property near the city of Gonzalez, Texas. Colony
 collections followed the procedures of Powell (2009). In brief extensive
 baiting was used to locate all nests in a focal tree, and a lack of
 aggression among ants from different nests was used to confirm membership
 of nests to the same polydomous colony. The first set of experiments,
 performed in June to July 2017, used three colonies of <em>C.
 varians</em> (V1, V2, and V3) and three colonies of <em>C.
 texanus </em>(T1, T2, and T3). The second set of experiments,
 performed in October to November 2017, used four colonies of <em>C.
 varians</em> (V4, V5, V6, and V7). The colony sizes (comprising
 workers, soldiers, and queens) at the start of each experiment for V1 to
 V7 and T1 to T3 were 190, 100, 140, 184, 64, 137, 56, 43, 55, and 82,
 respectively.  <strong>Experimental
 setup</strong><br>To examine how spatial constraints affect
 turtle ant nest choice, we constructed arenas that had two sections with
 different structural features and levels of constraint (Fig 1). One
 section had cavities that were fully connected to one another (F), while
 the other section had cavities that were linearly connected in sequence
 (L), with greater distances between the cavities. The F section had
 cavities that were closer to each other with more possible pathways
 between them, so it was less constrained than the L section. The arenas
 were made with boxes (dimensions: 11 x 11 x 3.75 cm high) in a grid
 arrangement as shown in Figure 1. Boxes were connected with arched paper
 bridges which went from the floor of one box to the floor of another, over
 their adjacent walls. Fluon was applied to the sides of each box to
 prevent ants from escaping. The artificial nest cavities all had the same
 entrance size and volume and were made as outlined in Powell and Dornhaus
 (2013, <em>Animal Behaviour</em>). In the first set of
 experiments (June to July 2017), there were three nest cavities in each
 section for a total of six cavities while in the second set of experiments
 (October to November 2017), there were four nest cavities in each section
 for a total of eight cavities. Additional cavities were added in the
 second set of experiments to allow us to assess potential nest preferences
 in larger colonies that may be finding and occupying all of the cavities.
 We refer to the two sets of experiments as the limited-cavity and
 additional-cavity experiments, respectively. 
 <strong>Nest Choice Experiments</strong><br>At
 the beginning of each experiment, we placed original cavities containing a
 single turtle ant colony in the box labeled O, equidistant from the
 closest cavities in the F and L sections (Figure 1). We then opened the
 original cavities to force the ants to move and occupy new cavities in the
 arena. Colony movement was filmed for the first 12 hours and cavity
 occupation was checked 4 times a day at 8:00, 12:00, 16:00, and 20:00.
 Cavity occupation was measured by lifting the outer cover of the cavities
 and counting the number of workers inside with minimal disturbance. Both
 workers and soldiers moved between cavities, but workers were counted for
 simplicity and because they were much more numerous. The presence of brood
 was also noted. Turtle ants rapidly allocate workers and brood to new
 cavities and differentiate between different cavity properties, so both
 the number of workers occupying a cavity and the presence of brood can be
 used as indications of their preference for the cavity. The limited-cavity
 experiments lasted 5 days and showed that colonies occupied new cavities
 within the first 12 hours, with the proportions of ants in each cavity
 remaining stable after the second day.  Thus, the additional-cavity
 experiments lasted only 3 days.
 <strong>Statistical analyses</strong><br>For
 all analyses, we considered the observation in the afternoon of the third
 day as being representative of the colony decision. Although later
 observations were in some cases available, standardizing on a single
 observation time makes all observations more comparable, and initial
 observations suggested that nest choice typically did not change after
 three days. For both models, cavity connectivity
 (whether a cavity was in section F or L) was included as a fixed effect
 since we were interested in the effects of structural features on nest
 choice. For the limited-cavity experiments, we also included species
 (<em>C. varians</em> or <em>C. texanus</em>) as a
 fixed effect, to account for consistent differences in colony size between
 the two species used in those experiments. For the model of worker number
 in the limited-cavity experiments, we also included the interaction
 between species and nest type, to determine whether the two species
 differed in the effect of structural features on nest choice. Within all
 models, the colony was included as a random effect to account for
 consistent differences between colonies, except in the case of the brood
 model for the additional-cavity experiments, where including it caused a
 singular model fit. For the Poisson models of the number of worker ants
 per cavity, an observation identifier (a unique identifier assigned each
 time occupation was counted for each cavity) was used as an additional
 observation-level random effect to account for overdispersion caused by
 the possibility that ants aggregate non-independently in cavities. We
 report the fixed effects along with their 95% confidence intervals,
 z statistics and <em>p</em>-values, estimated with the Wald
 method. We also used randomization tests to examine
 whether the observed effects of structural features on cavity occupation
 could have been due to chance. First, we chose just to consider the
 observation in the afternoon of the third day as being representative of
 the colony decision. The number of adult ants (workers, soldiers, and
 queens) was averaged across all cavities in each section (F and L) for
 each colony, and the test statistic was calculated as the difference
 between the two sections, averaged across all colonies in each experiment.
 In each randomization, the section label for each cavity (F or L) was
 randomly shuffled within each colony, and the test statistic was
 recalculated. For each experiment, 5000 randomizations were performed to
 calculate the proportion of randomizations with test statistics more
 extreme than the observed test statistic.  All
 statistical analyses were performed with R version 4.0.3; generalized
 linear mixed models were constructed using package lme4.
description: There are two different sections of each arena, with the nest
 type corresponding to the section it is in. Note that the nest types here
 labeled as "R" and "D" are referred to as
 "F" (fully connected section) and "L" (linearly
 connected section), respectively, in the accompanying paper.