Ant-plant sociometry in the Azteca-Cecropia mutualism

Abstract

A holistic understanding of superorganism biology requires study of colony sociometry, or the quantitative relationships among growth, nest architecture, morphology, and behavior. For ant colonies that obligately nest within plant hosts, their sociometry is likely intertwined with the plant, which has implications for the evolution, strength, and stability of the mutualism. In the Azteca-Cecropia mutualism, plants provide ants with food rewards and hollow stems for nesting in return for protection from herbivores. Several interesting questions arise when considering ant-plant sociometry: are colony growth and plant growth synchronized? How do colonies distribute themselves within the stem of their host plant? How do plant traits influence worker morphology? How is collective personality related to tree structure, nest organization, and worker morphology? To address these questions, we investigated patterns within and relationships among five major sociometric categories of colonies in the field – plant traits, colony size, nest organization, worker morphology, and collective personality. We found that colony sociometry was intimately intertwined with host plant traits. Colony and plant growth rates were synchronized, suggesting that positive feedback between plant and colony growth stabilizes the mutualism. The colony’s distribution inside the host tree tended to follow leaf growth, with most workers, brood, and the queen in the top half of the tree. Worker morphology correlated with plant size instead of colony size or age, which suggests that plant traits influence worker development. Colony personality was independent of colony distribution and tree structure but may correlate with worker size such that colonies with smaller, less variable workers had more aggressive personalities. This study provides insights into how ant-plant structural relationships may contribute to plant protection and the strength of mutualisms.

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Ant-plant sociometry in the Azteca-
Cecropia mutualism
Peter R. Marting
1,2, Nicole M. Kallman1, William T. Wcislo2 & Stephen C. Pratt
1
A holistic understanding of superorganism biology requires study of colony sociometry, or the
quantitative relationships among growth, nest architecture, morphology, and behavior. For ant
colonies that obligately nest within plant hosts, their sociometry is likely intertwined with the plant,
which has implications for the evolution, strength, and stability of the mutualism. In the Azteca-
Cecropia mutualism, plants provide ants with food rewards and hollow stems for nesting in return for
protection from herbivores. Several interesting questions arise when considering ant-plant sociometry:
are colony growth and plant growth synchronized? How do colonies distribute themselves within
the stem of their host plant? How do plant traits inuence worker morphology? How is collective
personality related to tree structure, nest organization, and worker morphology? To address these
questions, we investigated patterns within and relationships among ve major sociometric categories
of colonies in the eld – plant traits, colony size, nest organization, worker morphology, and collective
personality. We found that colony sociometry was intimately intertwined with host plant traits. Colony
and plant growth rates were synchronized, suggesting that positive feedback between plant and
colony growth stabilizes the mutualism. The colony’s distribution inside the host tree tended to follow
leaf growth, with most workers, brood, and the queen in the top half of the tree. Worker morphology
correlated with plant size instead of colony size or age, which suggests that plant traits inuence worker
development. Colony personality was independent of colony distribution and tree structure but may
correlate with worker size such that colonies with smaller, less variable workers had more aggressive
personalities. This study provides insights into how ant-plant structural relationships may contribute to
plant protection and the strength of mutualisms.
To understand how social insect colonies function as superorganisms, it is essential to quantify patterns of colony
growth, nest architecture, and morphology, a eld of study known as insect sociometry1. e relationships and
scaling between colony traits give insight about development, collective physiology, evolutionary constraints, and
plasticity. Such basic natural history is oen scarce or lacks depth because data can be hard to collect.
For ant colonies that obligately nest within plant hosts, aspects of their sociometry are likely intertwined
with their host plant, which may have interesting implications for the strength and stability of the mutualism.
We studied ant-plant sociometry in Azteca constructor colonies nesting in Cecropia trees in the lowland tropics
of central Panama (Fig.1). Cecropia trees provide hollow internodes for nesting and glycogen-rich food bodies
for the ants2,3, which in return protect the trees from herbivores and vines4,5 and provide nitrogen enrichment6–8.
is system provides a unique and interesting view of insect sociometry because the complex environmental
factors that typically shape sociometry – habitat structure, resource abundance, territory size, interactions with
intruders, microclimate – are simplied through the colony’s interaction with their host plant. e host plant is
their environment; a biotic environment possibly shaped by coevolution with the ants themselves (but see9). We
investigated patterns of and relationships among ve major categories of sociometry; tree size, colony size, nest
structure, ant morphology, and collective personality. In the following paragraphs, we outline driving questions
for each sociometric category through the lens of the mutualism.
What is the relationship between colony growth and plant growth? Comparing colony growth rate to that of the
host plant reveals potential strains in the mutualism. If plant growth outpaces colony growth, ants may not be able
to keep up with herbivory pressure, and plants suer from leaf damage and a reduction in tness10–12. One possi-
ble solution to this problem is the evolution of secondary polygyny through colony budding, where new queens
mate intracolonially and do not disperse, allowing the colony to live and grow faster13,14. However, A. constructor
display secondary monogyny15 where many queens establish the colony together but eventually ght to the death,
1School of Life Sciences, Arizona State University, Tempe, AZ, 85281, USA. 2Smithsonian Tropical Research Institute,
Balboa, Panama. Correspondence and requests for materials should be addressed to P.R.M. (email: pmarting@asu.edu)
Received: 8 August 2018
Accepted: 19 November 2018
Published: xx xx xxxx
OPEN
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so this solution is not likely in place. On the other hand, if colony growth outpaces plant growth, the benet from
ant protection diminishes as costs of housing and feeding them increase16,17. To avoid such imbalances, growth
rates may be equalized by positive feedback between colony and plant growth, reinforcing the mutualism18. We
can probe these interactions by comparing the scaling coecients over a large size range, allowing us to esti-
mate how the rate of worker production changes as A. constructor colonies grow, how the rate of leaf production
changes as Cecropia trees grow, and whether colonies produce new workers as fast as trees produce new leaves.
How do colonies structure and organize their nest inside the host plant? The physical nest architecture of
plant-ants is determined by hollow nesting spaces called domatia. Colonies make decisions about which doma-
tia to occupy, how to distribute themselves within the plant, and whether to add structural elements like carton
galleries. How a colony is distributed and organized may inuence the colonies’ ability to forage, tend brood,
respond to threats, and communicate eectively. Little is known about how plant-ant colonies distribute and
organize themselves within their host plant, and what forces inuence these patterns. e dissection of a large,
mature Cecropia tree revealed that the majority of the A. constructor colony is centralized in a large bulge in the
main trunk19, suggesting that the colony’s distribution may remain static as the tree grows. We investigated a
larger sample that includes smaller trees, and measured how colony components – specically workers, queen,
brood, commensal scale insects, refuse piles, carton, and entrances – are distributed in the tree, how these com-
ponents are spatially related to one another, and how their distribution changes with tree growth.
How do plants traits influence worker morphology? Worker size and polymorphism are often associated
with sociometric measures, such as colony size, age, and annual cycle20–23. Worker morphology within a col-
ony depends on intrinsic factors (genotype and development), external factors (environment and enemies) or a
combination of both (nutrition and social environment)24. In ant-plant mutualisms, worker morphology might
be related to mutualism dynamics or physical traits of the host plants themselves, especially since colony per-
formance feeds back into plant tness. In the Sonoran desert, ant species with larger body size are associated
with more myrmecophyte species25, suggesting that they can take advantage of a wider range of resources. A
comparison of two plant-ants found that the species with larger body size and greater variation in body size was
associated with the host plant species that has larger domatia and prostoma26, suggesting that worker morphology
may coevolve with plant traits. In addition to plant morphology, worker size may match the size of the dominant
herbivores threatening their host. Ant species that invest in smaller workers may be better at scrutinizing the
surface of their host plant and removing small sap sucking insects27, but worse at fending o larger insects and
vertebrates. In addition to plant dimensions, worker morphology may depend on food resources provided to the
colony via food bodies2 or nutritious pith called parenchyma28,29 – plants providing more nutrition may produce
larger workers. Morphometric analysis of the non-Cecropia-inhabiting congener Azteca trigona revealed that
Figure 1. A photographic overview of the Azteca-Cecropia mutualism. (A) A view from below the crown
of a juvenile Cecropia obtusifolia. (B) Azteca constructor havesting Müllerian food bodies from a trichilium.
(C) Azteca workers attacking an enchroching vine. (D) A cross-section of the central stem shows the queen,
workers, and brood residing in carton galleries inside the hollow internodes. All photos were taken by Peter
Marting.
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workers were dimorphic30, but worker size and allometry and their relation to plant traits have not been formally
described in A. constructor until the present study.
How is colony personality related to tree structure, nest organization, and worker morphology? While colony
personality is typically independent of colony size31–34, colony growth has been correlated with colony behavio-
ral traits35,36 or variation therein33,37. Sociometric traits beyond colony size and growth likely help shape colony
personality and are rarely examined (but see38). Colonies of A. constructor display collective personalities along a
docile-aggressive axis for a suite of behavioral traits34. e sources of behavioral variation are yet unclear, but are
likely to lie at the intersection of genotype and the environment39,40. Semi-permanent traits like nest architecture
likely eect colony behavior over long periods of time. e physical attributes of nest entrance chambers inu-
ence collective behavior by aecting worker encounter rates41 or ability to exit the nest in a state of alarm42. In the
context of an ant-plant mutualism, colony personality and plant traits may be related. Plants provide two major
resources for their ant colonies – nesting space and food bodies, both of which are correlated with plant height43.
Higher resource availability may increase energy reserves, fueling higher activity and aggression44. However, the
causality may ow in the opposite direction. In the Azteca-Cecropia mutualism, colonies with more aggressive
personalities live in trees with less leaf damage34, which may increase plant growth. Finally, colony aggression
and plant growth may inuence each other in a positive feedback loop, stabilizing the relationship. Colony per-
sonality may also interact with worker morphology. While body size and colony behavior were independent in
Temnothorax longispinosus ants33, larger workers of Cataglyphis niger ants were more aggressive toward conspe-
cics in staged encounters45. If this trend holds true in A. constructor, we might expect that at the colony level,
colonies with larger average workers are more aggressive.
To address these questions, we harvested trees containing colonies with known personality scores34 and meas-
ured the number of workers, queen, brood, scale insects, refuse piles, carton, and entrances in each internode to
determine how colonies were vertically distributed. We then measured the morphology of a subset of workers
from each colony. In addition, we measured key features of host tree morphology, including tree height, diameter,
number of internodes, number of leaves, and leaf area.
We rst use these data to describe the patterns of each separate sociometric category (plant size, colony size,
colony organization, ant morphology, and collective personality), then we explore the relationships among them,
focusing on the degree to which colony sociometry is intertwined with host plant biology.
Methods
Focal species and study site. Cecropia trees are diecious pioneer plants with a single central stem that
produces a new hollow, leaf-baring internode every 2–4 weeks46. e giant, radial leaves produce Müllerian food
bodies at specialized sites called trichilia at the petiole-stem juncture. Leaf lifespan is typically 3–6 months, but
food body production peaks a few weeks aer the leaf emerges43. Aer 3–5 years, branches grow out from the
central stem and bifurcate annually to produce a candelabra structure47–49. Workers chew entrances to individual
internodes and holes through the septa that separate internodes, creating a nearly complete, internal passageway
throughout the length of the tree19. Workers can further partition the available volume by constructing carton gal-
leries inside the internodes50, made from a combination of regurgitated plant materials including parenchyma, a
so, white tissue lining the inside of newly formed internodes28. In a related species, Azteca brevis, carton material
is structurally reinforced by a multi-species network of fungal hyphae51. Dark brown “refuse piles” can be found
throughout the internal structure, harboring nematodes52 and fungus29,53. Colonies display distinct behavioral
tendencies, or personalities, in that they dier repeatably in a suite of behavioral traits that are independent of
colony size and age34.
We located 14 A. constructor colonies along a 12 km stretch of Pipeline Road in and around the lowland trop-
ical rainforests of Soberania National Park, Colón, Panama between March and May 2013. At this site, there
are four common Cecropia species (C. peltata, C. obtusifolia, C. longipes, and C. insignis) and three common
Cecropia-inhabiting Azteca species (A. constructor, A. alfari, and A. isthmica). All pairings of ant and tree species
can be found, but C. peltata, C. longipes, and A. alfari tend to be found in large disturbed areas, while the others
tend to be found in forest gaps (PM, personal observation) – a trend that may be driven by humidity limitation.
For the purposes of this study, we focused on a single Azteca species (A. constructor) that occupied C. obtusifolia
(n = 10), C. peltata (n = 2), and C. insignis (n = 2).
Colony founding in Azteca involves secondary monogyny, meaning multiple queens cooperate in the incipient
stages, and eventually ght to the death until one queen remains15,29. To avoid these complex intracolony dynam-
ics, we selected trees old enough to have a single queen (above 2 m tall). Trees can reach over 20 m in height and
have many branching points, but we used shorter trees (below 8 m tall) with single stems for assay standardization
and ease of access. erefore, our sampling reects the sociometry of juvenile trees.
Tree size. We measured tree height, diameter, and number of leaves upon harvesting the colonies. To assess
total leaf area, all leaves were separated, photographed against a light background, and measured using ImageJ
soware. Cecropia internodes have a consistent growth-periodicity internode branching pattern that allows for
accurate estimation of plant age54: we counted the number of internodes between branching points of larger,
mature trees to estimate an average annual internode output for each Cecropia species (C. peltata: n = 11;
C. obtusifolia: n = 10; C. longipes: n = 10; and C. insignis: n = 4). We divided the number of internodes from our
focal plants by the annual output to estimate plant age. Azteca ants colonize Cecropia trees as saplings15, so while
plants are slightly older than colonies, their ages are likely tightly correlated. To estimate the total internal volume
of the plant, we measured the internal height and width to calculate the volume of a cylinder (V = πr2h) for each
internode and summed all cylinders per plant.
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Colony size, nest organization, and vertical distribution. Aer completing the behavioral trials
(described below), we harvested the host trees and extracted entire colonies in August of 2013. To subdue the
ants and minimize disturbance to their internal distribution, we used internal and external insecticides in quick
succession. e ants chew through most of the internode septa19, providing a path for the insecticide to trav-
erse the internal height of the tree. We drilled a hole into the base of each tree and inserted the nozzle of a
propane-powered insecticide fogger (active ingredient: resmethrin) and discharged the insecticide for several
minutes. e tree was then cut at the base, laid on a large plastic tarp, and sprayed with a liquid insecticide exter-
nally (active ingredients: pyrethrins, piperonyl butoxide, and permethrin). While some ants exited the domatia
during the harvest, this method provides the best estimate for relative abundance inside the stem. Stems were cut
in meter-long segments and split vertically to access the internal colony. For each internode, we quantied the
internal domatium dimensions, the number of workers, brood (larvae and pupae not distinguished), scale insects,
and refuse piles, and noted the presence of the queen, entrances to the exterior, carton material, and leaf-baring
petioles. Aer we quantied the internal distribution of the colony, we collected all workers from the stems,
leaves, tarps, and bags and immediately placed them in 95% ethanol. To survey colony size, workers were spread
out on grid paper, photographed, and counted using ImageJ soware.
Ant morphology. For each colony, we selected a subset of 100 workers from a large vial of ethanol containing
the entire colony. To reduce size bias selection as much as possible, we mixed the ethanol into a vortex with for-
ceps and selected workers haphazardly. For each ant, we separated head, mesosoma, gaster, and legs, and arranged
them on an index card using double-sided tape. With a camera mounted on a dissection scope, we photographed
each ant using SPOT imaging soware (www.spotimaging.com, Sterling Heights, MI). We calibrated the images
with a micrometer scale that was included in each photograph, and measured head width and mesosoma length
using ImageJ soware.
Behavioral traits. We related the sociometric measures described above to previous analyses demonstrating
collective personalities in these colonies. For detailed methods see34, but here we provide a brief description. To
characterize colony-level behavior, colonies were subjected to ve bioassays: patrolling behavior, vibrational dis-
turbance, response to intruder, response to leaf damage, and exploratory tendency. Colonies received each assay
at least two times to assess behavioral consistency (patrolling behavior assay was repeated four times per colony).
To standardize behavioral measurements across dierent tree sizes, we focused on the central stem at the lowest
leaf’s internode, which we estimated to be the location of median colony distribution based on four preliminary
tree dissections. For patrolling behavior, vibrational disturbance, and response to intruder, we scored activity by
counting the number of times we saw a worker completely traverse the lower septum line on the external surface
of the focal internode, regardless of direction or identity. For leaf damage assays, we focused on an entire leaf
instead of the stem and counted the number of workers on that leaf every minute. Trials were recorded with an
HD camcorder (Panasonic HC-X900M) between May and August of 2013.
Statistical analyses. Data were analyzed with linear correlation and regression, ANOVA, and paired t-tests.
We log-transformed colony and tree size variables to evaluate allometric scaling by testing if the observed scaling
coecient (log-log slope) diered from the scaling coecient predicted in the case of isometry with a Wald test.
We square-root-transformed total leaf area before evaluating scaling relationships so that the predicted scaling
coecient for isometry was 1 in all cases. us, observed scaling coecients that were indistinguishable from 1
indicate isometric relationships, below 1 indicate negatively allometry, and above 1 indicate positively allometry.
We used principal component analysis to simplify the characterization of each of the ve major categories
of sociometrical data (tree size, colony size, colony structure, worker morphology, and colony personality). We
performed separate unrotated PCA for each category, to reduce several dening traits to summary variables.
Only eigenvalues greater than the mean eigenvalue were used in subsequent analyses55. Summary variables were
then used to investigate relationships among the categories. All statistical analyses were performed in Stata 12.1.
Results
Plant size, colony size, and growth scaling. e Cecropia trees we sampled ranged from 2.42–7.95 m
tall with 55–144 hollow internodes that provided a total internal volume of 0.23–5.65 L with an estimated age
range of 1–4.5 years. Only the oldest tree bore inorescences during the study (C. insignis, 2 inorescences). Leaf
area and tree height scaled with marginally signicant negative allometry such that every 10-fold increase in
height produced a 7-fold increase in leaf area (regression, r2 = 0.79, scaling coecient (log-log slope) = 0.77, Wald
test for comparing the scaling coecient to 1, p = 0.073). Total leaf area was driven more by an increase in leaf size
rather than leaf number (Fig.2). Tree height and estimated age were not correlated (regression, n = 14, p = 0.47).
All colonies were identied as A. constructor, monogynous, and ranged in size from 1,880–13,534 workers,
with 73–93% of the workforce on the external surface of their tree at the time of harvesting. Alate production was
low, with only 2 of the larger colonies producing 1–22 males and no females. e number of brood and number of
workers scaled with negative allometry such that with every 10-fold increase in workers, there was only a 4-fold
increase in brood (regression, r2 = 0.22, scaling coecient = 0.41, Wald test p = 0.019).
The scaling of brood-to-workers and leaf area-to-tree height was not significantly different, i.e., the
log-log slope of number of brood vs. workers did not dier from the log-log slope of leaf area vs tree height
(t-Value = 1.466, p = 0.155, Fig.3A). e total number of workers scaled isometrically with tree height (regres-
sion, r2 = 0.36, scaling coecient = 1.18, Wald test p = 0.70, Fig.3B), meaning every 10-fold increase in tree
height produces a 10-fold increase in the number of workers in the colony. Furthermore, the number of external
workers increased isometrically with total leaf area (regression, r2 = 0.29, scaling coecient = 1.26, Wald test
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Figure 2. Leaf features contributing to total leaf area. (A) e relationship between number of leaves and total
leaf area. (B) e relationship between average leaf area per leaf and total leaf area. e dashed lines represent
linear regressions.
Figure 3. (A) A comparison of the scaling coecients ± condence intervals for the relationship between
brood-to-workers and leaf area-to-tree height. (B) e relationship between total number of workers and tree
height. e dashed line represents an allometric regression (log-log relationship). “Slope” indicates the observed
scaling coecient and “Slope-p” indicates the p-value resulting from a Wald test comparing the predicted and
observed scaling coecients. e slope of this line (the scaling coecient) was not signicantly dierent from
1, indicating an isometric relationship. (C) e relationship between the number of workers on the external
surface of the plant and total leaf area. e dashed line represents an allometric regression (log-log relationship).
e scaling coecient was not signicantly dierent from the 1, indicating an isometric relationship.
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p = 0.66, Fig.3C) meaning the overall density of ants remains constant across the range we sampled. e total
number of workers was not correlated with estimated tree age (correlation, n = 14, p = 0.918).
Nest organization and vertical distribution. We detailed nest structure and vertical distribution for
an exemplar colony in Fig.4. Colonies occupied 27–62% of the available internodes. While worker distribution
was oen patchy, nearly all the upper stem was inhabited. To compare vertical distribution patterns across dier-
ent tree and colony sizes, we rendered the proportion of each nest component by tree height decile, i.e., in 10%
increments starting at the top of the tree (Fig.5). Internal tree volume was not evenly distributed vertically, but
steadily increased with decile height because newer, herbaceous internodes are larger and more spacious. e
internal dimensions of the internodes do not change, but woody growth slowly increases the external diameter
so older, lower internodes have a much smaller internal space with the same external diameter. Nearly all leaves
were in the top half of the tree, with leaf proportion steadily increasing with decile height therein. e proportion
of workers, brood, scale insects, and refuse piles peaked around the second and third height decile. Carton was
more evenly distributed, tapering o in the lowest deciles, while the proportion of entrances steadily increased
with decile height. e vertical distribution of workers diered by the Cecropia species they inhabit (ANOVA
for proportional height of median workers, p < 0.05, Fig.6), with C. peltata supporting a low, broad distribution,
C. insignis supporting a high, narrow distribution, and C. obtusifolia ranging between the other two.
Nest component heights were correlated with tree and worker heights (Fig.7A). e relative median height
(percent of tree height) of these components is independent of tree height, i.e., the various tree components are
at the same proportional location in the tree, regardless of the tree’s absolute height (Fig.7B). Median worker
Figure 4. e distribution of colony nest components within an exemplar Cecropia tree. Each bar in the central
column represents an internode from the central stem, and the dimensions are scaled to the height and width
of the internal volume of each internode (width is doubled relative to height to show the components more
clearly). e width of the bars to the le represent the number of brood and the bars to the right represent the
number of workers. e shading of each internode indicates the hemipteran density. e shaded area near
the top of the tree represents internodes that bore leaves. e location of the queen is indicated by the golden
diamond, and entrances are indicated by black circles.
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distribution height was below median leaf height, but above brood, carton, and refuse median height. ere was
no dierence between median worker height and scale insect height or queen height (paired t-test, Fig.8).
While less than half of the total internodes contain carton (32 ± 4% mean ± s.e.), more than half of the total
workers (66 ± 5%) and brood (82 ± 4%) reside in internodes with carton.
To determine the relationship among several nest components in individual ant-occupied internodes, we
entered nest variables (the presence of entrances, queens, and carton, and the number of brood, scale insects, and
refuse piles) for each internode of 14 trees (n = 1194 internodes total) into a principal component analysis. e
rst two principal components had eigenvalues greater than the mean and together explain 55% of the variation.
us, each internode varied along two axes: a “resource management” score (PC1, entrance-hemipteran-refuse
axis) and a “nursery” score (PC2, brood-queen-carton axis) (Table1). Most internodes scored low on both, sev-
eral scored high on one but not the other, and very few scored high on both.
We analyzed how the royal chamber (the internode containing the queen) diered from other internodes
by comparing the condence intervals for the presence of each nest component (entrances, carton, brood, scale
insects, and refuse piles) for all ant-occupied internodes to their presence in the royal chambers. Compared to an
average ant-occupied internode, the royal chamber was more likely to contain carton and brood, and less likely to
contain refuse piles. ere was no signicant dierence for scale insect or entrances.
Ant morphology. Workers varied in size with head widths ranging from 0.57–1.29 mm and were positively
allometric (workers from all colonies pooled together, log head width-log mesosoma length slope = 1.13, Fig.9).
To analyze variation among colonies in worker size, for each colony we calculated the mean worker head width,
Figure 5. e mean proportion of each nest component as a function of tree height decile. Error bars indicate
95% condence intervals. e box plot represents the decile where the queen was located.
Figure 6. e distribution of workers within Cecropia trees. e proportion of internal workers are rendered by
tree height decile for each tree. Colonies are arranged by Cecropia species, then by the proportional height of the
median worker distribution. e proportional height of the median worker distribution diered signicantly
among Cecropia species (ANOVA, F = 7.17, p = 0.01).
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the maximum head width, the size rangefactor (max/min head width), and degree of allometry (log head-log
mesosoma scaling coecient). We report further PCA analysis on these measurements in the next section.
Relationships among sociometric categories. Each of the ve major categories of sociometrical data –
tree size, colony size, nest structure, antmorphology, and colony personality – are complex with several variables,
so we sought to simplify each category by an unrotated PCA. We then used the simplied descriptions to explore
relationships among categories. For every PCA, the rst principal component (PC1) had the only eigenvalue
greater than the mean and explained a substantial majority of the variation. Furthermore, the nature of the load-
ings on PC1 were easily interpreted and given intuitive summary descriptors we outline below.
Figure 7. e relationship among the median height of tree and colony components. (A) e absolute height of
each component. (B) e proportional height of each component relative to absolute tree height.
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Tree size. Height, total internal volume, total leaf area, and stem diameter all loaded strongly positive and PC1
explained 90% (Table2). We named PC1 “tree size” because higher values indicate taller trees with greater diam-
eter, internal volume, and leaf area.
Colony size. Total workers, brood, hemipterans, and refuse piles all loaded strongly positive and PC1 explained
71% (Table2). We named PC1 “colony size” because higher values indicate colonies with more workers, brood,
hemipterans, and refuse piles.
Colony distribution breadth. Queen, median worker, and median brood height loaded strongly positive, while
the percent of total internodes with worker and brood present loaded strongly negative and PC1 explained 74%
(Table2). We named PC1 “colony distribution breadth” because higher values indicate that the colony nest com-
ponents have narrower distribution and are located higher in the tree.
Worker size. Allometry slope, size range factor, max head width, and average head width all loaded strongly
positive and PC1 explained 80% (Table2). We named PC1 “worker size” because colonies with higher values have
larger workers, greater size disparities, and steeper allometries.
Colony personality. e results for colony behavior were published in34, but we include them here for congru-
ency (Table2). Vibrational disturbance, leaf damage, intruder, and patrolling all loaded strongly positive and PC1
Figure 8. e distance in meters between median nest component heights and median worker height. e
median height of workers is the height up the stem at which half of the internal ants reside above and half reside
below. e same height was calculated for each nest component, e.g., the point at which half of the entrances
are above and half are below. e distance in meters between the median worker height and each of the nest
components were calculated for each tree and are represented here as box plots. Positive values indicate the
median nest component was higher in the stem than the median worker height, while negative values indicate it
was lower. An asterisk indicates a signicant dierence from the median worker height.
PC1 “Resource
management score” PC2 “Nursery
score”
Eigenvalue 1.72 1.57
Variance Explained 28.7% 26.2%
Loading Scores
Number of brood — 0.65
Number of refuse piles 0.61 —
Number of hemipterans 0.57 —
Entrance present 0.50 —
Carton present — 0.46
Queen present — 0.57
Table 1. A summary of the principal component analysis for the nest components in each internode (n = 613).
Dashes indicate loading scores below 0.2.
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explained 48%. We named PC1 “colony personality” and colonies with higher values were more active, aggressive,
and responsive.
e colony scores for PC1 of each sociometrical category are summarized in Fig.10. We tested for correlations
among all sociometrical categories using these PC1 scores (Table2). Signicant and trending correlations are
shown in Fig.11; larger trees supported larger colonies (p = 0.02, Fig.11A), larger colonies promoted broader nest
distributions (p = 0.008, Fig.11B), larger trees supported larger, more allometric worker morphologies (p = 0.02,
Fig.11C), and colonies with larger, more allometric worker morphologies tended to be less aggressive (p = 0.06,
Fig.11D). ere was no correlation between ant morphology and colony size (p = 0.47).
Discussion
Our results support the notion that the growth, nest organization, andmorphology of Azteca constructor colo-
nies are intertwined with their Cecropia host plants. Costs to the host plant can accrue if tree growth outpaces
colony growth10–12 or vice versa16,17, but our results show that, over the size range that we sampled, colony and
plant growth rates are similar. Furthermore, colony size increased isometrically with tree height, but not with
tree age. Older trees were not necessarily taller, which likely reects that some plants are growing in unfavorable
conditions, e.g., poor soil nutrients43 or low light56, which in turn likely aects colony growth. is provides
further evidence that there is positive feedback between colony and plant growth rates that stabilizes the mutu-
alism. Additionally, the number of workers on the external surfaces, i.e., the stem, leaves, and petioles, increased
isometrically with total host plant leaf area, suggesting that ant density remains consistent as the tree grows. Leaf
damage did not increase with tree size, as it does with Cordia plants10, but rather decreased with colony-level
aggression34, suggesting that colony behavior is more important for preventing herbivory than colony size. For
colonies to eectively reduce herbivory, they must successfully search leaves, communicate threats, and recruit
workers appropriately. e optimal strategy for collective search and deployment may depend on threat level57,
colony size58, or territory size and shape59,60. Given that individual leaf size increases with tree height (Fig.2B),
the most eective patrolling strategy may shi as the colony and the plant grow. Further research is merited to
test whether colonies employ dierent collective search strategies as their host plant surfaces increase. Some
plant-ants have evolved secondary polygyny as a possible solution to diminishing growth rates relative to their
host plant13,14. However, the more synchronized growth rates in the Azteca-Cecropia system may negate the ben-
ets of secondary polygyny, leading to the evolution of secondary monogyny instead15.
e spatial distribution of colonies within their hosts also follows tree structure. Vertical worker distribu-
tion tended to be most dense near the top of the tree, which reects the distribution of available nesting space
and food-body-bearing leaves. While we did not measure how ants were distributed among leaves themselves,
previous work indicates that most ants occur in the upper third of the of the leaves despite most of the leaf area
occurring in the middle third61. e overabundance of workers on younger, newer leaves reects the contribu-
tion the leaf will make to plant growth61, which is likely driven by the fact that newer leaves produce the most
food bodies43. e median leaf height was consistently above the median worker height in the internal stem, and
Figure 9. e relationship between head width and mesosoma length for workers from all colonies (n = 1,300).
e dashed line represents an allometric regression (log-log relationship). e scaling coecient was
signicantly higher than the predicted isometric slope of 1, indicating a positive allometric relationship. e
histogram shows the frequency of workers by mesosoma length and head width on their respective axes.
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median brood height was below the median worker height. is suggests that as new nesting space and leaves
grow from the apical meristem, workers follow, then brood. Even though less than half of internodes contained
carton galleries, we found that the majority of workers and most of the brood resided in internodes with carton,
suggesting they serve as brood storage. e shape of vertical Azteca worker distributions resembled the distri-
bution patterns of several ground-nesting ant species21,62, which may reect comparable resource proximity or
available nest volume.
Given the distribution shape and height of each nest component, we posit a generalized hypothesis about how
the colony distributes itself as the tree grows. As trees grow upward, adding new leaves and larger internodes,
workers quickly chew entrances and move into the new space, harvest the new food bodies, and bring the scale
insects to feed on the soer tissues. Carton is built more slowly and eventually brood is deposited there. Lower
internodes are eventually abandoned, leaving behind used carton and sealed entrances (workers must actively
maintain the entrance sites by chewing, or the tree will eventually seal them, PRM, pers. obs.). is hypothesis is
limited to the range of tree sizes included in this study. It appears that colony distribution patterns may shi dra-
matically as tree’s central stem bifurcates into several branching points. In the dissection of a larger tree in Costa
PC1 “Tree size” PC2 (not used)
Eigenvalue 3.67 0.17
Variance Explained 91.8% 4.3%
Loading Scores
Height 0.50 —
Diameter 0.50 0.74
Interna l volume 0.49 —
Total leaf a rea 0.49 −0.65
PC1 “Colony size” PC2 (not used)
Eigenvalue 2.90 0.53
Variance Explained 72.6% 13.3%
Loading Scores
Total worker s 0.51 −0.28
Total brood 0.44 0.89
Number of refuse piles 0.51 —
Number of hemipterans 0.52 −0.30
PC1 “Colony distribution breadth” PC2 (not used)
Eigenvalue 3.71 0.67
Variance Explained 74.2% 13.2%
Loading Scores
Percent of internodes with workers −0.40 0.70
Percent of internodes with brood −0.45 0.31
Median proportional height of workers 0.43 0.57
Median proportional height of workers 0.49 0.25
Proportional height of the queen 0.42 —
PC1 “Worker size” PC2 (not used)
Eigenvalue 3.21 0.45
Variance Explained 80.2% 11.3%
Loading Scores
Mean head width 0.48 0.34
Max head width 0.52 −0.45
Size range factor (max head/min head width) 0.52 −0.46
Head-mesosoma scaling coecient (log-log slope) 0.46 0.67
PC1 “Colony personality” PC2 (not used)
Eigenvalue 1.934 1.065
Variance Explained 48.3% 26.6%
Loading Scores
Patrolling 0.620 0.236
Vibrational disturbance 0.351 0.731
Intruder response 0.511 0.262
Leaf D amage Resp onse 0.482 −0.610
Table 2. A summary of the principal component analyses for the each sociometric categories – tree size,
colony size, neststructure, and worker size. Dashes indicate loading scores below 0.2. e PCA data for colony
personality is from34, but is included here for completeness. See Fig.10 for a visualization of how colonies are
distributed along each PC1.
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Rica, the A. constructor colony distribution appeared to be very centralized, with the queen and all brood residing
in a large, permanent, carton-lled bulge near the center of the tree19. Such a centralized conguration may be
advantageous for workers patrolling and foraging across several distributed meristems. Future sampling should
include a larger range of tree sizes and structures to capture the transition from a more vertically distributed to a
more centralized nest structure.
Despite the generalized pattern, there was a large amount of variation in how colonies distributed themselves
within their trees. is variation is partially explained by larger colonies having broader distributions, but other
factors not measured here may inuence colony distribution. In Temnothorax ants, colonies consistently vary
in how they structure their nests across time and contexts63. Our data were snapshots of colony distribution – it
would be interesting to test whether patterns of colony distribution are consistent across time or persist across
host plant transplants.
e close association between tree growth and colony structure extends to worker size. In many ant species
where workers vary in size, worker morphology correlates with colony size and age, with larger colonies produc-
ing larger workers, greater size variation, and steeper allometries24. is trend reects the natural progression
of resource acquisition, colony nutrition, and colony growth. Intriguingly, here we show that worker size is not
correlated with colony size or age, but rather host tree size. Worker morphology may be controlled by intrinsic
factors like nutrition; larger trees may produce more food bodies, more nutrition is invested per larvae, resulting
in larger workers. It is also possible that the nutrient ratios of the food bodies shi with tree height, resulting in
larger workers. Worker size may also be responding to external factors like available space, load size, or entrance
size. Larger trees naturally provide more voluminous chambers, greater surface area, and larger territory to patrol,
which could be more eciently traversed by larger workers. Perhaps the size of individual food bodies increases
with tree size and are more eciently carried by larger workers. Finally, larger trees may have larger prostomas –
the dedicated dimpled sites where ants chew entrances into the internal internode space. Larger-headed workers
may ll larger entrance gaps more appropriately to prevent intruders from entering the tree as in turtle ants64.
Figure 10. Score distributions for the 5 major sociometric categories. Plots display how colonies vary along
the PC1 axes for tree size, colony size, colony distribution breadth, worker size, and colony personality. e
illustrations on either side are visual interpretations of what the extreme values represent for each PC1. For
colony personality, higher values indicate more active, aggressive colonies.
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Colony personality was independent of colony size, tree size, and vertical distribution. However, an interesting
pattern may emerge with ant morphology. Colonies with more aggressive personalities tended to have smaller, less
allometric worker morphologies, which contradicts our hypothesis. Although the trend was weak, it is potentially
interesting and worth more exploration. e trend may reect some resource investment tradeo between collec-
tive aggression and worker size – perhaps colonies can either have an aggressive demeanor or larger workers, but
not both. Alternatively, worker size may be connected to task demand. Our measures of aggression are based on
the number of ants responding to a given stimulus. If the colony has larger workers, perhaps fewer ants need to
respond because they are more ecient at dealing with threats. A third possibility is that colonies fed more food
bodies can produce larger workers than colonies not fed enough food bodies. Colonies not fed enough may try
to compensate for their nutrient deciency by increasing prey consumption65, thus resulting in a more aggressive
collective personality. More experiments are needed to tease apart the correlation between worker size, colony
personality, and tree size, as well as a proper foodweb analysis.
Food body production likely plays an important role in ant-plant sociometry, and therefore our view is limited
by the fact that we were unable to quantify food rewards in this study. Food body production not only depends
on ontogenetic factors we measured like plant height and leaf area12, but also environmental factors like soil
nutrients43,66 and light availability56. It would be interesting to test how these factors contribute not only to the
number and mass of food bodies, but how the nutrient content and size of individual food bodies might change
as the plant grows. Food body production likely inuences many aspects of ant sociometry, such as colony size67,
distribution on leaves61, worker size, and colony behavior. Our study provides a good foundation to further test
hypotheses about how food rewards t in.
Our study on ant-plant sociometry is a comprehensive investigation on growth scaling, colony organization
and vertical distribution, worker morphology, and collective personality in an ant-plant mutualism. We show
the synchronization of plant growth and colony growth in the Azteca-Cecropia mutualism, a novel nding that
supports the idea that such synchronization is a crucial enabler of the stability of a mutualism. Azteca sociometry
is intimately intertwined with host plant biology and is an important consideration for mutualism dynamics. Our
study may be valuable for the interpretation of other mutualisms between plants and stem-nesting ants, shedding
light on convergent evolution and the unique strategies of these fascinating symbioses.
Data Accessibility
e data associated with this manuscript have been deposited at Dryad Digital Repository (doi link will be provided).
Figure 11. Correlations among sociometric categories. Solid lines indicate a signicant correlation (p < 0.05)
between traits and the dashed line indicates a nearly signicant trend (p < 0.1). (A) e relationship between
colony size and tree size. (B) the relationship between colony distribution breadth and colony size. (C) e
relationship between worker size and tree size. (D) e relationship between colony personality and worker size.
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Acknowledgements
We thank Taylor Mazzacavallo for the wonderful assistance in the eld and helping count thousands of workers.
anks to all others who helped with data collection in the eld: Gabe Patterson, Andrew Quitmeyer, Danielle
Hoogendijk, Maggie Raboin, Susanne Wiesner, Ummat Somjee, Megan Oconnell, Kara Fikrig, Hannah Bregulla,
Jullia Legeli, Stephen Orr, Hana Duckworth, Ted Carstensen, Evan Walton. anks to Raineldo Urriola and
Adriana Bilgray for logistical support. anks to May Boggess and Irene van Woerden for statistical advice.
We thank Martin Heil and an anonymous reviewer for their crutial comments that improved our manuscript.
Funding for this research was provided by the Smithsonian Tropical Research Institute, Arizona State University,
and the National Science Foundation (CCF-1012029).
Author Contributions
P.M. conceived of the study, designed it, conducted the eld work and video scoring, carried out the statistical
analysis, and drafted the manuscript; N.K. conducted all morphological measurements of the ants; W.W.
participated in the design of the study, helped troubleshoot eldwork, and helped edit the manuscript; S.P.
participated in the design of the study, helped with statistical analysis, and helped dra the manuscript. All
authors gave nal approval for publication.
Additional Information
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