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facult y of s ci ence

uni vers i t y of copenhagen


centre f or s oci al evoluti on
department of bi ology
Fungal adaptations to mutualistic life with ants
This thesis dissertation has been submitted in accordance with the requirements
for the degree of PhD, at the PhD School of The Faculty of Science, University
of Copenhagen, Denmark to be defended publicly before a panel of examiners.
by
Pepijn W. Kooij
December 2013
Academic advisors
Prof. Jacobus J. Boomsma
Dr. Morten Schitt
Cover
Front: Atta sexdens workers carrying leaves down a tree. Picture: P. W. Kooij,
Location: Santa Cruz, Gamboa, Panama
Back: incipient colony of Atta colombica with queen on top. Picture: P. W. Kooij,
Location: Gamboa, Panama
This thesis is the result of a three-year PhD project carried out at the Cen-
tre for Social Evolution (CSE), Department of Biology, University of Copenhagen
in Denmark under the supervision of Professor Jacobus J. Boomsma and Dr. Morten
Schitt. During the project I spent fve weeks at the Laboratory of Genetics, Wagenin-
gen University and Research Center, The Netherlands, hosted by Dr. Duur K. Aanen. I
additionally carried out a total of seven weeks of feldwork at the Smithsonian Tropical
Research Institute in Panama. I was funded by the Danish National Research Founda-
tion and the Department of Biology.
The thesis is comprised of a general introduction on the current understand-
ing of life-time mutualistic interactions and how this can be applied to my model
system where the fungal symbiont adapted to a life-time commitment with ants. I also
present the objectives of the thesis research, and conclude with a summary of the main
results in a broader conceptual framework. This is followed by fve chapters of origi-
nal emperical work, one of which is in press in a peer-reviewed journal, one has been
submitted to a peer-reviewed journal and three that are in preparation for submission.
PREFACE
Pepijn W. Kooij
Copenhagen, December, 2013
Summaries.....................................................................................................................................................7
General introduction...................................................................................................................................13
Mutualism, commitment and co-adaptation.............................................................................................14
The fungus-growing ants as model system................................................................................................15
Open questions and objectives of this thesis.............................................................................................17
The infuence of domestication on fungal genetics.......................................................................................17
Incompatibility between fungal crops of different ant genera.......................................................................18
Decomposition enzymes in the fungus-growing ant symbiosis....................................................................19
Special Acromyrmex echinatior fungal symbiont enzymes...........................................................................20
Summary and future perspectives.............................................................................................................21
References....................................................................................................................................................27
Chapter 1:....................................................................................................................................................33
Advanced farming ants rear polyploidy crop fungi
Chapter 2:....................................................................................................................................................61
Somatic incompatibility of fungal crops in sympatric Atta and Acromyrmex leaf-cutting ants
Chapter 3:....................................................................................................................................................85
Differences in forage-acquisition and fungal enzyme activity contribute to niche segregation in Panamanian
leaf-cutting ants
Chapter 4:...................................................................................................................................................109
Leucoagaricus gongylophorus uses leaf-cutting ants to vector proteolytic enzymes towards new plant sub-
strate
Chapter 5:...................................................................................................................................................133
Cellulose degradation patterns in Acromyrmex echinatior colonies
Acknowledgements...................................................................................................................................153
CONTENTS
6
7
ENGLISH, DANSK, ESPAOL, NEDERLANDS
SUMMARIES
8
Fungus-growing ants (Attini) feed off a fungus they cultivate in a mutualistic symbiosis in under-
ground chambers by providing it substrate they collect outside the colony. The tribe of Attine ants ranges
from small colonies of the paleo- and basal Attine species with a few hundred workers that forage on crude
substrates such as insect frass and dry plant material, to large colonies of the leaf-cutting ants with several
thousands to several million workers that provide live plant material to their fungus gardens. Leaf-cutting
ants are the dominant herbivores of the Neo-tropics, and have a major contribution to cycling of nitrogen
and phosphorus in their direct environment and are, furthermore, considered pest species as they have a
large impact on human agriculture. These factors make leaf-cutting ants an ideal study subject to better
understand the mechanisms that make this mutualistic symbiosis so successful.
To understand the evolutionary development of domestication of the fungus over the phylogeny of
the Attine ants, I compared the average number of nuclei per cell for the fungal symbionts, for each of the
different groups of fungus-growing ants. I found that the fungal symbionts of the paleo- and basal Attine
ants, which have a relative low level of domestication, have two nuclei per cell, the standard for Basidiomy-
cete fungi, but that the average number increased to 7-17 nuclei per cell in the highly domesticated fungi of
the higher Attine ants and leaf-cutting ants. Furthermore, I was able to estimate that approximately half of
these nuclei were represented by different genomes, giving the fungus a ploidy level of 5n-6n.
In mutualistic symbioses it is important the partners stay true to each other. In fungus-growing
ants, new founding queens bring a piece of fungus to build up their new colony. However, in rare occa-
sions fungal symbionts might come into contact with symbionts from other colonies. I showed that in both
leaf-cutting ant genera incompatibility reactions between fungal strains can avoid intermixing of different
strains, and that these reactions strengthen when genetic distance is increased. This pattern, however, be-
comes distorted when fungal symbionts are contested across ant genera.
The most important mechanism in the succession of this mutualism of leaf-cutting ants is the con-
trolled degradation of plant material. I show that in the area of Gamboa, Panama, the two leaf-cutting ant
genera forage for rather different plant material, with Atta species specializing on tree-leaves and Acromyr-
mex focusing more on fower material and herbal plant material. This difference is refected in the overall
enzyme activity patterns in the fungus gardens, with Atta specializing more on specifc enzyme groups and
Acromyrmex having an overall high enzyme activity.
Finally, I show that the fungal symbiont of the leaf-cutting ant Acromyrmex echinatior produces
large amounts of biodegrading enzymes in special structures called gongylidia. The ants eat these structures,
but enzymes pass the ant gut without being digested, and are excreted by the ants in their fecal fuid which
they mix with freshly foraged plant material placed on the top of the fungus garden. The enzymes are still
active and have therefore an important role in the biodegradation of the plant material. With this I show that
the fungus evolved some incredible adaptations to a mutualistic life with the ants.
SUMMARY
9
Svampedyrkende myrer lever af en svamp de dyrker i en mutualistisk symbiose i underjordiske
kamre ved at forsyne den med substrat som de henter udefra. Underfamilien af svampedyrkende myrer (At-
tini) spnder over paleo- og basale arter med f hundrede arbejdere der fouragerer p trret plantemateriale
og insekt ekskrementer, til store kolonier af bladskrermyrer med fere tusinde til millioner af arbejdere
der indsamler friskt plantemateriale til deres svampehaver. Bladskrermyrer er den dominante herbivor i
neotroperne hvor de bidrager vsentligt til nitrogen og fosfor kredslbet i deres nrmilj og bliver yder-
mere betragtet som et skadedyr idet de har stor effekt p landbruget. Disse faktorer gr det ideelt at benytte
bladskrermyrer til bedre at forst mekanismerne der har gjort denne mutualistiske symbiose s succesfuld.
For at forst udviklingen af domesticeringen af svampen over hele den Attine fylogeni, sam-
menlignede jeg det gennemsnitlige antal kerner per celle i svampesymbionten hos forskellige grupper af
svampedyrkende myrer. Jeg fandt at svampesymbionten hos myrerne fra paleo- og basale arter, som har
relativ lavt niveau af domesticering, havde to kerner per celle som er standard for Basidimyceter, hvorimod
gennemsnittet steg til 7-17 kerner per celle hos de meget domesticerede svampesymbionter fra de hjere
udviklede svampedyrkende myrer og bladskrermyrerne. Ydermere kunne jeg estimere at ca. halvdelen af
disse kerner havde forskellige genomer hvilket gav svampen et ploidi niveau p 5-6n.
I en mutualistisk symbiose er det vigtigt at parterne er tro mod hinanden. Hos svampedyrkende
myrer medbringer de nye dronninger et stykke svamp til at grundlgge nye kolonier, men i sjldne tilflde
kan denne svampesymbiont komme i kontakt med symbionten fra en anden koloni. Jeg pviste at i begge
bladskrermyrerslgter srger inkompatibilitets reaktioner imellem varianter af svampe for, at symbionter
ikke bliver blandet og at dette styrkes nr den genetiske variation forges. Dette mnster bliver dog for-
vrnget nr svampesymbionter fra forskellige slgter af myrer testes imod hinanden.
Den vigtigste mekanisme i successionen af denne mutualisme hos bladskrermyrer er den kontrol-
lerede nedbrydning af plantemateriale. Jeg pviser at i omrdet omkring Gamboa, Panama, fouragerer de
to bladskrermyrerslgter p forskelligt plantemateriale, Atta er specialiseret p trblade og Acromyrmex
mere p blomster og urter. Denne forskel refekteres i enzymaktiviteten i svampehaverne, hvor Atta er spe-
cialiseret indenfor specifkke grupper af enzymer og Acromyrmex overordnet har en hj enzymaktivitet.
Endelig viser jeg, at svampesymbionten hos bladskrermyren Acromyrmex echinatior producer-
er store mngder bionedbrydende enzymer i specielle strukturer kaldet gongylidia. Myrerne spiser disse
strukturer, men enzymerne passerer ufordjet igennem tarmsystemet og ender i myrernes fkalievske
som de blander med friskt indsamlet plantemateriale og placerer verst i svampehaven. Enzymerne er sta-
dig aktive og spiller derfor en vigtig rolle i den biologiske nedbrydning af plante materialet. Dette viser at
svampen har udviklet nogle utrolige tilpasninger til en mutualistisk eksistens med myrerne.
RESUM
10

Las hormigas atinas se alimentan de un hongo que cultivan en simbiosis mutualista en cmaras subterrneas al que
proveen con sustrato colectado fuera de la colonia. La tribu Attini presenta colonias que oscilan en tamao desde las
pequeas colonias de las especies basales, con unos pocos cientos de trabajadores que forrajean substratos crudos como
heces de insectos y material vegetal seco, hasta las grandes colonias de las hormigas cortadoras de hojas con varios
miles a millones de trabajadores que forrajean material vegetal vivo. Las hormigas cortadoras de hojas son los herbvo-
ros dominantes del Neotrpico, y contribuyen extensamente a los ciclos de nitrgeno y fsforo en su entorno directo.
Por otra parte, estas hormigas son consideradas una especie peste debido a su fuerte impacto en la agricultura humana.
Estos factores hacen de las hormigas cortadoras de hojas un grupo de estudio ideal para intentar comprender mejor los
mecanismos que hacen tan exitosa esta simbiosis mutualista.
Para entender la sucesin de la domesticacin a travs de la flogenia de las hormigas Attini, se compar el
nmero promedio de ncleos por clula para los simbiontes fngicos de cada uno de los diferentes grupos de hormigas
cortadoras de hojas. En este estudio encontramos que el hongo simbionte de las hormigas atinas basales, las cuales
presentan un nivel relativamente bajo de domesticacin, tienen dos ncleos por clula; lo estndar para hongos basidio-
micetos. Sin embargo, este nmero incrementa a un rango de 7 a 17 ncleos por clula en los hongos altamente domes-
ticados de los grupos ms recientes de hormigas atine y cortadoras de hojas. Asimismo se estim que aproximadamente
mitad de estos ncleos son representados por diferentes genomas, dndole al hongo un nivel de ploida de 5n-6n.
En simbiosis mutualistas, es importante que las partes sean feles entre s. En hormigas cultivadoras de hon-
gos, nuevas reinas fundadoras traen un pedazo de hongo para construir su nueva colonia. No obstante, en raras oca-
siones, los simbiontes fngicos puede entrar en contacto con simbiontes de tras colonias. Aqu demostramos que en
ambos gneros de hormigas cortadoras de hojas, reacciones de incompatibilidad entre cepas de hongos pueden prevenir
combinaciones de diferentes cepas, y que estas reacciones se fortalecen con el incremento de la distancia gentica. Sin
embargo, este patrn se distorsiona cuando los simbiontes fngicos se oponen a travs de los gneros de hormigas.
El mecanismo ms importante en la sucesin de este mutualismo de las hormigas cortadoras de hojas es la de-
gradacin controlada del material vegetal. Se encontr que en el rea de Gamboa, Panam, los dos gneros de hormigas
cortadoras de hojas forrajean material vegetal bastante diferente; las especies Atta se especializan en hojas de rboles
y las Acromyrmex en material de fores y hierbas. Esta diferencia se refeja en los patrones generales de la actividad
enzimtica en los cultivos de hongos, donde Atta se enfoca ms en grupos enzimticos especializados y Acromyrmex
posee una alta actividad enzimtica generalizada.
Finalmente, en este estudio demostramos que el simbionte fngico de las hormigas cortadoras de hojas Ac-
romyrmex echinatior produce grandes cantidades de enzimas biodegradadoras en estructuras especiales denominadas
gongilidios. Las hormigas comen estas estructuras y las enzimas pasan a travs del sistema digestivo de la hormiga,
aunque sin ser digeridas. Luego, estas son excretadas en el lquido fecal, el cual las hormigas mezclan con el material
vegetal fresco que ha sido recientemente colectado y ubicado en la parte superior del jardn de hongo. Las enzimas
permanecen activas y por lo tanto juegan un papel importante en la biodegradacin del material vegetal. Con esto dem-
uestro que el hongo evolucion adaptaciones extraordinarias para una vida mutualista con las hormigas.
RESUMEN
11
Mieren van de geslachtengroep Attini eten een schimmel die ze in ondergrondse kamers kweken in een mu-
tualisme. Deze schimmel kweken ze door groeisubstraat te geven dat ze buiten het nest verzamelen. De Attine-mieren
zijn divers, met aan de ene kant kleine kolonies van een paar honderd werkers - bij de basale soorten - die ruw materiaal
verzamelen zoals insecten-faeces en dood plantenmateriaal, en aan de andere kant grote kolonies - bij de bladsnijmieren
- met enkele duizenden en soms miljoenen werkers die levend plantenmateriaal verzamelen. Deze bladsnijmieren zijn
de belangrijkste herbivoren in de Neotropen en hebben een grote rol in het recyclen van stikstof en fosfor. Daarnaast
worden ze gezien als ongedierte vanwege hun impact op de landbouw. Dit alles tezamen maakt bladsnijmieren ideaal
model om te onderzoeken wat de mechanismen zijn die de mutualisme tussen mieren en schimmel zo succesvol maakt.
Om te begrijpen hoe de domesticatie van de schimmel evolueerde over de fylogenie van de Attine-mieren,
heb ik van de schimmel het gemiddelde aantal celkernen per cel vergeleken voor de verschillende groepen van deze
mieren. Uit de resultaten kan ik concluderen dat de minder gedomesticeerde schimmels, die gecultiveerd worden door
de basale groep van Attine-mieren, altijd twee celkernen per cel hebben, de standaard voor Basidiomyceten. Maar bij
de meer gedomesticeerde schimmels van de hogere Attine-mieren en bladsnijmieren is dit aantal toegenomen tot een
gemiddelde van 7-17 celkernen per cel. Daarnaast ben ik in staat geweest om aan te tonen dat ongeveer de helft van deze
celkernen een verschillende genoom hebben, wat betekent dat de schimmel een plodie-niveau heeft van 5n-6n.
Voor een mutualisme is het belangrijk dat beide partners trouw aan elkaar blijven. Bij de Attine-mieren neemt
de nieuwe koningin een stukje schimmel mee om de nieuwe kolonie te beginnen. Het kan echter gebeuren dat de
schimmel van de ene kolonie in contact komt met de schimmel van een andere. Ik toon hier aan dat bij de schimmels
van twee soorten, van twee verschillende genera, van bladsnijmieren incompatibiliteitsreacties kunnen voorkomen dat
verschillende lijnen van schimmels met elkaar mengen, en dat deze reacties toenemen naarmate de genetische afstand
tussen deze lijnen toeneemt. Dit patroon verdwijnt echter wanneer schimmellijnen van mieren van verschillende genera
met elkaar in contact komen.
De belangrijkste reden waarom bladsnijmieren zo succesvol zijn is het gecontroleerd afbreken van planten-
materiaal. Ik laat hier zien dat in de omgeving van Gamboa, Panama, de twee bladsnijmierengenera verschillend plant-
enmateriaal verzamelen. De Atta-soorten zijn gespecialiseerd in boombladeren en de Acromyrmex-soorten richten zich
op bloem- en kruidachtig plantenmateriaal. Dit verschil is ook te zien bij de enzymactiviteiten in de schimmeltuinen,
waarbij tuinen van Atta-soorten zich specialiseren op specifeke enzymen, maar de tuinen van Acromyrmex-soorten in
het algemeen een hoge enzymactiviteit hebben.
Tot slot laat ik zien dat de schimmel van bladsnijmierensoort Acromyrmex echinatior grote hoeveelheden
enzymen, gespecialiseerd in het afbreken van plantenmateriaal, produceert in speciale organen die gongylidia worden
genoemd. De mieren eten deze organen, maar de enzymen daarin worden niet verteerd door de mier en komen in de
faeces van de mier terecht. De mieren mengen hun faeces met vers plantenmateriaal en plaatsen dit bovenop de schim-
meltuin. De enzymen in de faeces zijn daar nog steeds actief en hebben daardoor een belangrijke rol in het afbreken
van het plantenmateriaal. Hiermee laat ik zien dat de schimmel belangrijke adaptaties aan een mutualistisch leven met
mieren heeft gevolueerd.
SAMENVATTING
12
13
GENERAL INTRODUCTION
14
Fungus-growing ants of the tribe Attini live in a tight relationship with crop fungi. This relation-
ship is so tight that the one partner cannot live without the other, making this symbiosis an obli-
gate mutualism (Weber 1966; Howe 1984). For more than a century, substantial knowledge has
accumulated about this intriguing symbiosis. However, most of the research has been focused on
the natural history of the ants and the system in general (Wheeler 1907; Weber 1966; Mueller et
al. 2005), or on the adaptations of the ants to maintain their crops. Some of these studies have in-
vestigated differences in worker polymorphism (e.g. Weber 1966; Mehdiabadi and Schultz 2010),
the domestication of actinobacteria at the origin of fungus farming 50 MYA to protect the fungus
against parasites (e.g. Currie et al. 1999; Currie et al. 2003; Poulsen and Currie 2009), the devel-
opment of antibiotic-producing metapleural glands (see e.g. Do Nascimento et al. 1996; Hughes
et al. 2008; Yek et al. 2012), followed by studies on biological control by the ants versus these
evolutionary derived chemical controls (see e.g. Fernandez-Marin et al. 2007; Fernandez-Marin
et al. 2009).
However, far less is known about the fungal adaptations to a mutualistic life with ants, and the
research to date has tended to focus on the role of the fungus from the ants perspective (e.g. Martin
1970; Martin 1974; Quinlan and Cherrett 1979; Bass and Cherrett 1995). In recent years, research-
ers have shown an increased interest in how the fungal symbiont adapted to make this mutualism
one of the dominant herbivores in the neo-tropics (e.g. Schitt et al. 2008; Schitt et al. 2010;
Semenova et al. 2011; De Fine Licht et al. 2013; Aylward et al. 2013). Therefore, in this thesis I
attempt to further explore fungal adaptations by investigating fungal morphology, genetics and
biochemistry. The frst section will give an overview of the research presented in this thesis within
a broad perspective of mutualistic life-style, commitment to mutualistic partners and the need
for co-adaptations to sustain and elaborate mutualisms that have become obligate. Next, I will
introduce my model system of fungus-growing ants and what is already known about the roles of
the fungal symbiont in the mutualism. Finally, Ill conclude with an outline of how I investigated
fungal adaptations to mutualistic life with ants.
Mutualism, commitment and co-adaptation
Despite their different fundamental organization, ant colonies and mycelia of fungi exhibit strik-
ing similarities in their social organization. (Rayner and Franks 2003)
In order for a stable mutualistic symbiosis to be maintained it is necessary to have a high degree
of partner commitment and functional complementarity (Janzen 1985; Keeler 1985). This can
15
mostly be observed in endosymbioses where the levels of interdependency are so high that the
symbiont becomes part of the cells and tissues of the host. However, also in ectosymbioses like
the fungus-growing ant mutualism, where the fungus lives outside the ants, these high levels of
partner commitment and functional complementarity can be seen, because the fungus acts as an
ectosymbiont for the individual ants, but as an endosymbiont for the colony (Poulsen and Booms-
ma 2005). This can lead to a reduced genetic diversity and sometimes even asexuality of the endo-
symbiont compared to its host (Law and Lewis 1983; Law 1985), something that was later shown
to be the result of co-speciation, which is present in tight mutualisms (Herre et al. 1999). Another
condition to maintain a stable mutualism, is that the host should only beneft relatives of the other
partner, which will then stabilize the mutualism, but can also lead to reduced population-wide
genetic variability in the partner that becomes domesticated (Frank 1994; Doebeli and Knowlton
1998; Sachs et al. 2004; Foster and Wenseleers 2006).
Hosts and symbionts are potentially in confict over the direction of symbiont transmission (hori-
zontal vs vertical), and the magnitude of symbiont dispersal (Frank 1996a; Frank 1996b; Douglas
1998; Douglas 2008). Hosts may therefore reduce the production of sexual symbiont investments,
because this may incur cost to the host and because the host risks being confronted with intro-
ductions of new symbiont strains that may reduce host ftness (Frank 1996a; Frank 1996b; Leigh
2010). Symbiont internal conficts (antagonism or incompatibility) might be benefcial for hosts
to maintain control of symbiont acquisition (Frank 1996a; Frank 1996b; Bot et al. 2001b). Due to
these factors the symbionts can get so integrated with their hosts that their genomes reduce in such
manner that a life without the host is merely impossible (McCutcheon and Moran 2012). At this
point the host-symbiont conficts seem to be resolved, so that high levels of cooperation and low
levels of confict will lead the symbiosis towards organismality (Queller and Strassmann 2009)
which in turn is maintained by life-time commitments of both partners (Boomsma 2013).
The fungus-growing ants as model system
Among the multitudinous activities of insects, none are more marvelous than the fungus-growing
and fungus-eating habits of Attiine ants. (Wheeler 1907)
The tribe of fungus-growing ants, the Attini, which contains ca. 14 genera and >230 species,
evolved approximately 50 MYA from a hunter-gatherer ancestor towards obligate farming of a
fungal crop (Schultz and Brady 2008; Mehdiabadi and Schultz 2010) which they maintain by
providing growth substrate and by removing of any contaminations (Mller 1893; Wheeler 1907;
16
Weber 1966; Weber 1972; Mueller et al. 2005). The ants cultivate their crop in monoculture and by
default transmit their crop vertically with incipient queens to new colonies (Mller 1893; Wheeler
1907; Weber 1955; Weber 1972; Mueller et al. 2005; Poulsen and Boomsma 2005; Mueller et
al. 2010). However, horizontal transmission seems to be common between shallow phylogenetic
branches (Mikheyev et al. 2006; Mikheyev et al. 2007; Poulsen et al. 2009), but more constrained
between older lineages (Mueller et al. 1998; Johnson and Vilgalys 1998; Vo et al. 2009)
The ants can be divided in different groups of farming, with the lower agriculture attines at the
base of the phylogenetic tree, mainly growing fungi from the Leucocoprinaea tribe, including the
specialized yeast agriculture clade, which evolved to grow the same fungal species in yeast form,
and the coral fungus agriculture in the genus Apterostigma, which cultivate a fungal crop from the
Pterula family (Mueller et al. 1998; Munkacsi et al. 2004; Schultz and Brady 2008; Mehdiabadi
and Schultz 2010). This group of lower attine ants provide their fungal crops with crude substrates
such as insect frass, nectar, seeds and dead and dry plant material (De Fine Licht and Boomsma
2010). The ants and fungi in these clades do not show signs of tight coevolution, with still free-liv-
ing fungal relatives in both the Leucocoprinaea clades (Chapela et al. 1994; Mueller et al. 1998;
Mueller 2002; Vo et al. 2009) and the Pterula clades (Chapela et al. 1994; Mueller 2002; Munkacsi
et al. 2004; Villesen et al. 2004; Dentinger et al. 2009), and signs of horizontal transfer between
ant species (Mueller 2002; Green et al. 2002; Mikheyev et al. 2010; Kellner et al. 2013). But when
focused on terminal clades of the ant phylogeny, such as the Cyphomyrmex wheeleri group, a strict
coevolution between the ants and fungi can be seen (Mehdiabadi et al. 2012).
Several changes in the symbiosis occurred with the introduction of the higher attine ants of the
genera Trachymyrmex and Sericomyrmex 20 MYA(Schultz and Brady 2008; Mehdiabadi and
Schultz 2010). The colonies have an increased size of approximately ten fold (Fernandez-Marin
et al. 2004; Schultz and Brady 2008; Baer et al. 2009; Fernandez-Marin et al. 2009; Mehdiabadi
and Schultz 2010), the growth substrate for these genera now includes fresh leaves, fowers and
fruits (De Fine Licht and Boomsma 2010) accompanied by differences in fungus garden enzyme
activities (De Fine Licht et al. 2010). Furthermore, lack of free living close relatives shows in-
creased level of domestication (Mikheyev et al. 2006; Mikheyev et al. 2010), and specialized hy-
phal structures, gongylidia, which act solely as food for the ants found their origin in these fungal
strains (Mller 1893; Weber 1955; Quinlan and Cherrett 1978; Quinlan and Cherrett 1979; Bass
and Cherrett 1995).
The fnal step was the evolution of the leaf-cutting ant genera Atta and Acromyrmex 10 MYA
17
(Schultz and Brady 2008; Mehdiabadi and Schultz 2010), which are considered major herbivores
in the neo-tropics and are major contributors of nitrogen and phosphorus recycling, but are also
often seen as pest species (Cherrett and Peregrine 1976; Fowler et al. 1989). The switch to primar-
ily live plant material with the addition of fowers and fruits (De Fine Licht and Boomsma 2010)
caused another shift in overall fungus garden enzyme activity towards plant cell wall degradation
(De Fine Licht et al. 2010) and allowed the colonies to grow to sizes of several thousands of work-
ers up to several millions (Fernandez-Marin et al. 2004; Schultz and Brady 2008; Baer et al. 2009;
Fernandez-Marin et al. 2009; Mehdiabadi and Schultz 2010). Along with this increase in colony
size, came an increase in worker polymorphism (Weber 1966; Mehdiabadi and Schultz 2010) and
queen mating frequency (Villesen et al. 2002; Baer et al. 2009; Mehdiabadi and Schultz 2010) to
allow the colonies to become this large. This together shows that the ecological footprint increased
with every single step in the evolution of the fungus growing ants, which was supported by the
arrival of the gongylidia 20 MYA and a recent horizontal sweep of the cultivars in the leaf-cutting
ants 2-4 MYA replacing all fungal symbionts in the ant clade with a new one (Mikheyev et al.
2010).
Open questions and objectives of this thesis
The infuence of domestication on fungal genetics
The study of the Attini and other fungus-growing insects has only just begun, and further ad-
vance in this fascinating subject will be more diffcult for the mycologist than for the entomolo-
gist. (Wheeler 1910)
Domestication of crop fungi can bring complications for the fungal symbiont such as degenera-
tion of genetic diversity due to asexuality caused by vertical transmission (Mller 1893; Wheeler
1907; Weber 1955; Weber 1972; Mueller et al. 2005; Poulsen and Boomsma 2005; Mueller et al.
2010), or mushroom castration by the ants (Fisher et al. 1994a; Fisher et al. 1994b; Mueller 2002),
personal observations M Schitt, HH De Fine Licht and PW Kooij). However, there are signs of
horizontal transfer in the fungal symbionts of the higher attine ants (Mikheyev et al. 2006; Mikhe-
yev et al. 2007; Poulsen et al. 2009), and recent studies showed signs of polyploidy in these fungi
(Scott et al. 2009; De Fine Licht et al. 2013), but no further research has been done specifcally
on the latter.
For comparison, in the human-domesticated Agaricus bisporus an increase in the number of hap-
loid nuclei (up to 26)(Saksena et al. 1976) was shown compared to its free-living sister species
18
Agaricus bitorquis (Raper 1976; Hintz et al. 1988). A similar increase in number of nuclei can be
seen in the termite-domesticated fungus Termitomyces with up to 12 nuclei per cell (De Fine Licht
et al. 2005). The fungus-growing ants have different levels of domestication of fungal crops, from
the paleo- and basal agriculture with free living relatives of the fungal symbionts (Mueller et al.
1998; Mueller 2002; Munkacsi et al. 2004; Schultz and Brady 2008), to more complex domesti-
cated agriculture by the higher attine ants and aggressive herbivore agriculture by the leaf-cutting
ants with no free living relatives of the symbionts (Mikheyev et al. 2006; Schultz and Brady 2008;
Mikheyev et al. 2010). Because of these different levels of domestication these ants are a perfect
subject to investigate the infuence of domestication on increasing number of nuclei in the fungal
cells and the level of ploidy of the crop fungi. Thus, the questions remain, how is the average
number of nuclei correlated with increased domestication? And, does the level of ploidy correlate
with increased domestication?
Incompatibility between fungal crops of different ant genera
A leaf-cutter ant colony is a mutualist to on large plant (its fungus), parasite to many others, and
commensal to yet others (Janzen 1985)
Each individual colony of leaf-cutting ants maintains a single strain of the fungal symbiont Leu-
coagaricus gongylophorus in monoculture (Acromyrmex, Poulsen and Boomsma 2005; Atta,
Mueller et al. 2010). The maintenance of the monoculture is done though behavioral adaptations
of the ants, e.g. weeding of foreign strains, (Bot et al. 2001b; Ivens et al. 2009) or by somatic in-
compatibility reactions by the fungal strain itself (Poulsen and Boomsma 2005). In Basidomycete
fungi, such as the fungal symbiont of the ants, such stepwise incompatibility reactions regulate the
allorecognition, by rejecting a foreign strain using programmed cell death and cell pigmentation at
the meeting point of the two different fungal strains (Rayner et al. 1984; May 1988; Rayner 1991;
Worrall 1997). The exact genetic mechanisms involved in these reactions are still unknown, but
multiple loci are involved, and the reactions are correlated with genetic distance (Worrall 1997).
The ants often kill alien strains that are genetically different from their native strain, but this antag-
onism disappears after force feeding the ants with a foreign strains (Bot et al. 2001b; Ivens et al.
2009). Furthermore, when the ants are almost entirely depleted from their native fungus, and pro-
vided with a new foreign fungal strain, the original native strain will grow back and take over the
whole colony again within two weeks (Seal et al. 2012). It has been suggested that this is caused
by imprinting of the ants by the native fungal strain (Seal et al. 2012), which could be
19
done by using chemical recognition profles (Richard et al. 2007). However, it was shown that the
fungal symbionts of Acromyrmex show somatic incompatibility on agar plates, which gradually
increases with genetic distance (Poulsen and Boomsma 2005). The same study showed that the
fecal fuid of the ants showed increasing levels of incompatibility on foreign fungi with increasing
genetic distance, but that these reactions disappeared when the ants were force fed with the foreign
fungus, suggesting the incompatibility reactions are of fungal origin. The somatic incompatibility
in this system could therefore be explained by the lifetime commitment of the symbiont to live
as a monoculture with the ants, and to avoid conficts within the symbionts (Boomsma 2013).
However, the questions, remaining are: Does the incompatibility in Atta fungal crops show sim-
ilar incompatibility mechanisms as Acromyrmex fungal crops? And, how are the incompatibility
mechanisms between fungal symbionts across the two ant genera?
Decomposition enzymes in the fungus-growing ant symbiosis
I believe that they are, in reality, mushroom growers and eaters. (Belt 1874)
The leaf-cutting ant mutualism is based in the degradation of fresh plant material, transported by
the ants to the colony, by the fungal symbiont, which in turn provides the ants with nutrients (We-
ber 1966; Bass and Cherrett 1995; De Fine Licht and Boomsma 2010). However, the degradation
of fresh plant material brings many complications due to the complex structure of the plant cell
walls, such as the plants defense mechanism in the shape of phenolic compounds (Coley et al.
1985; De Fine Licht et al. 2013), strong cellulose fbers that require a complex mix of decompo-
sition enzymes (Bguin 1990; Martin et al. 1991), and pectins and hemicelluloses that bind the
cellulose fbers together into a strong matrix (Selvendran 1984). Furthermore, a fungivore lifestyle
for the ants (Davidson 2004) as well as a herbivore lifestyle in general is likely to be nitrogen
limited (Mattson 1980; Behmer 2009) which might explain why the ants normally collect highly
nutritious plant material (Mundim et al. 2009) and the presence of nitrogen fxing bacteria in the
fungus gardens (Pinto-Toms et al. 2009).
However, the fungus-growing ants with their fungal symbiont seem to have found ways to over-
come these complications and become one of the dominating herbivore systems in the Neotropics.
This is refected in the patterns of forage material acquisition that are found throughout the phy-
logeny of the Attine tribe, with more crude materials at the basal lineages and mainly fresh plant
material in the higher attine clades (De Fine Licht and Boomsma 2010). This pattern corresponds
with the differences that can be found in enzyme activities of the fungus gardens in each of these
20
groups (De Fine Licht et al. 2010) as well as more specifc changes in protease activities for
the fungal symbionts (Semenova et al. 2011). Even though these patterns have been found, the
leaf-cutting ants acquire forage material from a wide range of plant species which correlate with
the availability during the different seasons (Wirth et al. 2003).
It was shown in colonies of Atta cephalotes that the fungus gardens are able to deal with this due to
a fast and high rate of plasticity in enzyme activity towards different substrates (Kooij et al. 2010).
But because these were laboratory colonies rather than colonies in the feld, the question remains,
how are acquired plant substrate and fungus garden enzyme activities correlated in feld colonies?
Furthermore, in the locality of Gamboa, Panama, six species of leaf-cutting ants co-occur, three
from each of the two genera. Within each type of habitat one species of each genus can be found:
Atta cephalotes and Acromyrmex volcanus as canopy foragers, Atta colombica and Acromyrmex
octospinosus as forest edge foragers, and Atta sexdens and Acromyrmex echinatior as open sunlit
area foragers. This raises the question; do the leaf-cutting ant genera Atta and Acromyrmex that
live in the same area forage for the same material? And, do the fungus gardens in both genera
express the same enzyme activity?
Special Acromyrmex echinatior fungal symbiont enzymes
As to leaf-cutting ants, I have always held the same view as which is proposed by Mr. Belt, viz.
that they feed upon the fungus growing on the leaves, they carry into their nests, though I had not
yet examined their stomachs. Now I fnd that the contents of the stomach are colourless, showing
under the microscope some minute globules, probably the spores of the fungus. I could fnd no
trace of vegetable tissue which might have been derived from the leaves they gather; and this, I
think, confrms Mr. Belts hypothesis. (Mller 1874)
Aside from the general enzyme patterns of the fungus gardens described above, it has been
shown that the fungal symbiont alone too has made incredible adaptations to live their mutu-
alistic life with the ants. It was shown that both xylanase and cellulase activities are particu-
larly high in the bottom layers of fungus gardens of Acromyrmex echinatior and Atta cepha-
lotes (Schitt et al. 2008; Moller et al. 2011; Aylward et al. 2013; Grell et al. 2013). To
show that these activities came from the fungal symbiont ant not from other organisms,
a xylanase gene was isolated from the fungus and shown to be functional when expressed in
yeast (Schitt et al. 2008). Furthermore, fungal genes for cellulases were expressed most-
ly in the in the bottom layer of the fungus garden (Aylward et al. 2013; Grell et al. 2013).
21
Recently, a total of 33 proteins were identifed and sequenced in the fecal fuid of the leaf-cutting
ant Acromyrmex echinatior (Schitt et al. 2010). The ants mix this fecal fuid as manure with fresh
leaf material is chewed to pulp and some tufts of mycelium taken from elsewhere in the fungus
garden and put this mixture on top of the fungus garden to ensure new fungal growth (Weber
1966). Of these 33 proteins, seven proved to be pectinases, important in the breakdown of the
plant cell wall, of which at least fve showed to be active in the fecal fuid of the ants. Further-
more, genes for six of these pectinases were upregulated in the gongylidia compared to normal
mycelium (Schitt et al. 2010). Later, similar patterns were shown for one laccase, important for
the breakdown of phenolic compounds, the chemical defense mechanism of plants, present in the
fecal fuid, which was both active in the fecal fuid and the gene for this enzyme was upregulated
in the gongylidia compared to normal mycelium (De Fine Licht et al. 2013).
Because herbivore systems are nitrogen limited, it is to be expected that proteases are present in
the fecal fuid as well, as part of the plant degrading enzyme package. Thus, the raised questions
are: Are fungal proteases also found in the fecal fuid? If so, are these proteases active in the fecal
fuid? And, are the genes encoding these enzymes upregulated in the gongylidia when compared
to normal mycelium? Also, because cellulose is the main component in plant material (Manzoni
et al. 2010; Bell et al. 2014), are cellulases present in the fecal fuid and what is the role of these
cellulases? And, what is the role of cellulases in the different layers of the fungus gardens?
Summary and future perspectives
The infuence of domestication on fungal genetics
Indeed, crop domestication in the context of coevolving and codomesticated microbial consortia
may explain the 50-million year old agricultural success of insect farmers. (Mueller et al. 2005)
I show that the level of domestication in the fungus-growing ants infuences the average number
of nuclei per fungal cell, with the fungal symbionts of the paleo- and basal agriculture always
having two nuclei, the standard for Basidiomycete fungi. However, when domestication intensi-
fes in the higher attine ants, the fungal symbionts have between 7-17 nuclei per cell on average.
Furthermore, using microsatellite markers, we were able to estimate that these higher attine fungal
symbionts have an increased level of ploidy of approximately 5n-6n, meaning that about half the
nuclei present in these strains have different genomes. This sudden increase coincides with the
arrival of gongylidia as well as the obligate commitment of being a domesticated crop without
free-living relatives (Mikheyev et al. 2006; Schultz and Brady 2008; Mikheyev et al. 2010).
22
The increase in number of nuclei and ploidy level seems to be a clever solution to resolve compli-
cations of asexuality due to loss of possible gene fow with free-living relatives and the increased
lack of viable fruiting bodies, which are, though rarely, generally only seen at colonies of the
paleo- and basal attine ants (Mueller et al. 1998; Pagnocca et al. 2001; Mueller 2002). However,
it would be interesting to obtain a better estimate of the ploidy level in the fungal symbiont, as
well as the nuclear ratio of the different nuclear types, i.e. different genomes, by sequencing the
nuclei individually. The challenge for this is to fnd the right techniques to make this possible.
One way could be to create protoplasts of the fungal strains, which is a technique that removes
the cell walls of the fungal cells to leave each nucleus separate in a suspension (Sonnenberg et al.
1988). This suspension can then be plated out on a growth medium and subsequently single pro-
toplast colonies can be isolated and sequenced. Another techniques is the newly developed laser
capture microdissection of single nuclei, which then can be isolated a sequenced using single cell
sequencing methods (Guo et al. 2012).
In the fungus-growing wasps Sirex, the fungus is asexually and vertically transmitted as in the
fungus-growing ants, but recent results have shown that the fungus maintained the possibility for
sexuality (Van der Nest et al. 2013). The diversity in recognition genes in the fungal symbiont of
the wasps is relatively high compared to other genes, which indicates that the fungal symbiont
has not lost its ability for sexual reproduction. However, the genetic variation in these genes is
signifcantly lower than similar genes in free-living sister species. It would be interesting to see
whether the same is true for the fungal symbionts of the ants, because even though it is extremely
rare fruiting bodies in colonies of the higher attines can be observed occasionally, though mainly
in lab colonies (Fisher et al. 1994a; Fisher et al. 1994b; Pagnocca et al. 2001; Mueller 2002), P.W.
Kooij, M. Schitt, H.H. de Fine Licht, personal observations).
Finally, polyploidy generally has an infuence in genetic aspects such as gene redundancy (Comai
2005), higher gene expression diversity (Groth and Christ 1992) and other stress related advantag-
es (Lidzbarsky et al. 2009). It would therefore obvious to investigate what the phenotypic conse-
quences are of the increase in ploidy level found in this system. One might expect to fnd increased
levels of expression as well as diversity of biodegrading enzymes when comparing transcriptomes
of the higher attine ant fungal symbionts with those of the paleo- and basal attine ants, due to a shift
in growth substrate from more crude substrates, e.g. insect frass and dry plant material, in the latter
group to more complex substrates, i.e. fresh plant material (De Fine Licht and Boomsma 2010).
23
Incompatibility between fungal crops of different ant genera
Nevertheless, on our functional grounds, the interests of ants and fungi in a colony seem best
viewed as organismally merged. (Queller and Strassmann 2009)
Following up on the incompatibility study on Acromyrmex fungal symbionts by Poulsen & Booms-
ma (2005), I present here that the Atta fungal symbionts show similar incompatibility reactions.
Even though the strength of the incompatibility reactions is increasing with increasing genetic
distance for Atta fungal symbionts, they tend to be lower in general than in Acromyrmex fungal
symbionts. Furthermore, this signal is distorted once the fungal symbionts are in confrontations
with strains across the ant genus. To achieve a better insight in the differences found between the
Atta symbionts and Acromyrmex symbionts, phylogenies based on microsatellite markers and on
AFLP were compared. This not only revealed that the symbionts, which are supposed to be the
same species (Mikheyev et al. 2010), separate in each of the ant genera, but also revealed that the
phylogeny for Acromyrmex symbionts is much more strict and less variable than that of the Atta
symbionts.
There must be interesting differences between Atta and Acromyrmex symbionts, which may be
due to conditional gene expression as these are essentially the same cultivars. This could be relat-
ed to the fact that Atta ant species have claustral colony founding, where the queen never leaves
the colony and closes of the entrance until the frst workers open it to go out foraging 80-100 days
later (Weber 1966), securing life time commitment. In contrast, the Acromyrmex queens go out
to forage and are therefore most likely to exchange cultivars in this founding phase of the col-
ony (Poulsen et al. 2009). Another possible explanation might lie in the possibility that the Atta
symbionts might use imprinting of the ants (Seal et al. 2012), e.g. with chemical cues (Richard
et al. 2007), rather than the incompatibility reactions shown in Acromyrmex symbionts (Bot et al.
2001b; Poulsen and Boomsma 2005). A fnal explanation might lie in the extra AFLP bands that
were found in the Atta symbionts compared to those of Acromyrmex, which indicate there might
be a presence of RNA/DNA from other organisms such as mycovirusses (Pearson et al. 2009),
bacteria (Suen et al. 2010) or prions (Wickner et al. 2007). However, no DNA was amplifed with
bacteria specifc 16S primers, which would exclude the possibility of any bacteria left to explain
the extra AFLP bands.
24
In order to get a better understanding of how this incompatibility system works it would be inter-
esting the measure the overall gene expressions in the interaction zones of both compatible as well
as incompatible reactions, by sequencing the full transcriptome in these interaction zones. This
has proven to be insightful in the fungus-growing wasp mutualism, and showed clear upregula-
tions of multiple genes involved in recognition, stress response as well as programmed cell death
(Van der Nest et al. 2011). Furthermore, similar analyses might give an insight in the difference in
incompatibility reactions between the Atta and Acromyrmex symbionts at the gene level.
Decomposition enzymes in the fungus-growing ant symbiosis
Virtually every facet of the ants behavior and life cycle has been shaped by their association
with the fungus they culture. (Martin 1970)
Foraging behavior in May 2011 by the six sympatric species of leaf-cutting ants in Gamboa, Pan-
ama, was different between the two genera, however, it was similar between the species within
these genera. The Atta species focus their foraging primarily on tree-leaves, whereas the Acromyr-
mex species focused more on collecting fower parts and herbal leaves. This separation in foraging
behavior might help explain why the six Panamanian leaf-cutting ant species can be found in strict
groups of two species per habitat in this locality (Atta cephalotes and Acromyrmex volcanus as
canopy foragers, Atta colombica and Acromyrmex octospinosus as forest edge foragers, and Atta
sexdens and Acromyrmex echinatior as open sunlit area foragers), because competition for forage
material is higher between species rather than between genera. The question that raises now is
whether similar separation between genera can be found in other smaller communities.
Furthermore, the fungus garden enzyme activities in the colonies of Atta and Acromyrmex cor-
related with the forage material collection by the ants, showing that even though these ant species
grow the same species of crop fungus, the expression of enzymes is being adjusted according to
the substrate provided. A more striking fnding is that the overall enzyme activity in Acromyrmex
fungus gardens is higher than that in Atta fungus gardens. Aside from that, Atta fungus gardens
seem to have a more specialized activity than Acromyrmex. These difference could be a possible
explanation why it seems that Acromyrmex colonies produce less waste material than Atta colo-
nies (Bot et al. 2001a; Hart and Ratnieks 2002), and it would therefore be interesting to investigate
whether the amount of generated waste material is the same when the genera are provided with
the same forage material.
25
Special Acromyrmex echinatior fungal symbiont enzymes
it is not their biochemical machinery which the ants are contributing to this mutualistic as-
sociation, but rather their capacity to serve as vehicles of transport. (Boyd and Martin 1975b)
In the frst proteomic analysis of the fecal fuid of the leaf-cutting ant Acromyrmex echinatior six
proteases and one cellulase were identifed and sequenced. The protease fall into three groups of
biodegrading enzymes, metalloendoproteases, serine proteases and aspartic proteases, of which
the frst two groups showed activity in the fecal fuid of the ants. Furthermore, the genes coding
for fve of these enzymes (peptidyl-Lys metallopeptidase I and II, subtilisin, grifolisin and sac-
charopepsin) showed to be upregulated in the gongylidia compared to normal mycelium. This
shows that the fungal symbiont made a special adaptation to use the ants as vehicles to transport
enzymes from the middle part of the fungus garden where there is enough mycelium to produce
the gongylidia as well as enzymes (De Fine Licht et al. 2013) to the top of the fungus garden to
degrade new substrate. The new techniques used confrm results from earlier studies, though with
much more detail (Boyd and Martin 1975a; Boyd and Martin 1975b). Furthermore, it shows that
the tightly regulated intake of proteins by insects in this nitrogen limited environment (Behmer
2009), has been taken over by the fungal symbiont, something that is refected in the fact that the
fungus growing ants are the only ants with extremely high protease activities in the rectum rather
than in their midgut (Martin 1970).
The single cellulase that was identifed and sequenced was identifed as an endocellulase, Lg-
Cel12, from the Glycoside Hydrolase family 12 (GH12), and has previously not been described
(Aylward et al. 2013; Grell et al. 2013). Endocellulases are the frst in a series of three cellulases
that complete the degradation of cellulose (Bguin 1990; Martin et al. 1991), the main component
in plant cell walls (Manzoni et al. 2010; Bell et al. 2014). Even though activity was present for all
types of cellulases in the fecal fuid of the ants, this particular enzyme showed to be upregulated in
the gongylidia compared to normal mycelium. And other than previously studied genes (Aylward
et al. 2013; Grell et al. 2013), this gene was mostly expressed in the middle layer of the fungus
garden rather than the bottom layer. This shows that the LgCel12 has an important function in the
fecal fuid of the ants, even though the highest cellulase activity can be found in the bottom layer
of the fungus garden and the debris material (Moller et al. 2011; Aylward et al. 2013; Grell et al.
2003; Chapter 5 of this thesis).
26
Together with previously studied pectinases (Schitt et al. 2010) and laccases (De Fine Licht et al.
2013), now proteases and a cellulase proved to have an important role in the initial stages of plant
cell wall degradation in this mutualistic symbiosis, which all together show interesting parallels to
the enzyme package used by phytopathogens to attack plants (St Leger et al. 1997). Furthermore,
because this system is most likely nitrogen limited due to the herbivore way of living (Mattson
1980; Behmer 2009), it is of interest that the combination of proteases and cellulases is in balance.
The proteases are necessary to utilize as much nitrogen as possible, but the cellulases also play an
important role in keeping the C:N ratio in balance. When this balance gets disrupted the effciency
of utilizing both carbon and nitrogen decreases (Manzoni et al. 2010; Hartman and Richardson
2013; Sinsabaugh et al. 2013), which in turn would have a detrimental effect on the development
of the colony. A reason to keep the cellulase activities particularly high in the bottom layer of the
fungus as suggested by Grell et al. (2013) is to maintain the water balance in this section, so to
keep the fungus healthy and prevent it from obtaining any diseases before it is discarded by the
ants.
These results however, only test the presence and activity of biodegrading enzymes in the leaf-cut-
ting ant Acromyrmex, but earlier studies show increased protease levels in the fecal fuid in ants
from the whole phylogeny of fungus-growing ants when compared to other ant species (Martin
1970; Martin and Martin 1971; Martin 1974). An obvious next step would thus be to investigate
how these fungal enzymes are regulated in the fecal fuid of the leaf-cutting ant genus Atta, which
have even larger colonies (Schultz and Brady 2008; Mehdiabadi and Schultz 2010), in the higher
attine genera Trachymyrmex and Sericomyrmex, which feed their fungal crop cruder substrates but
where gongylidia are present (De Fine Licht and Boomsma 2010), and in lower attines, which do
not have gongylidia, but where the ants too produce fecal fuid (Martin and Martin 1971).
27
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33
CHAPTER 1
ADVANCED FARMING ANTS REAR POLYPLOID CROP FUNGI
PEPIJN W KOOIJ, DUUR K AANEN, MORTEN SCHITT, JACOBUS J BOOMSMA
(MANUSCRIPT)
34
Advanced farming ants rear polyploid crop fungi
Pepijn W. Kooij
1
, Duur K. Aanen
2
, Morten Schitt
1
and Jacobus J. Boomsma
1
1
Centre for Social Evolution, Department of Biology, University of Copenhagen, Universi-
tetsparken 15, DK-2100 Copenhagen, Denmark
2
Laboratory of Genetics, Wageningen University, PO Box 309, 6700 AH, Wageningen, The Neth-
erlands
Phone: +45 35321239
Fax: +45 35321250
35
Abstract
Polyploidy is common in plants, presumably because advantages of increased functional heterozy-
gosity often surpass costs of destabilized mitosis or epigenetic instability, but rare in fungi, where
diploid cells retain the two parental haploid nuclei rather than merging them into a zygote. Such
dikaryotic mycelia are actively maintained in Basidiomycota where clamp connections ensure
honest propagation of nuclei during cell division, but the same mechanism may constrain the evo-
lution of multinucleate cells. Previous research has indicated that the domesticated basidiomycete
crop-fungus of leaf-cutting ants, which lacks clamp connections, is a functional polyploid, but
without pursuing this further. Here, we used microscopy to estimate the mean number of nuclei
per somatic cell for 42 fungal symbionts reared by 14 species (eight genera) of fungus-growing
ants in Panama, and mapped these numbers on a fungal symbiont phylogeny generated by ITS
and LSU sequencing. This showed that all higher attine ants that rear specialized fungi without
free-living close relatives had 7-17 nuclei per cell, whereas non-specialized fungal crops of the
basal paleo- and lower attines were dikaryons. Analysis with ten microsatellite markers revealed
that ca. 40% of the additional nuclei represented novel haplotypes, yielding an estimate of average
ploidy in higher attine crop fungi of 5-6. The fungal symbionts reared by Atta and Acromyrmex
leaf-cutting ants were distinct from the clade reared by other higher attine ants, but we found no
differences in nuclei number between these lineages. We hypothesize that functional polyploidy
in these asexual and vertically transmitted crop symbionts evolved because benefts of increased
heterozygosity outweighed costs that could be met by the farming ants. Polyploid crops may thus
represent a form of symbiont chimerism for the sake of enhanced genetic diversity, similar to mul-
tiple queen-mating that evolved in the leaf-cutting ants to generate higher heterozygosity among
colony workers. The transition to crop-polyploidy coincided with at least an order of magnitude
increase in colony size of farming ants.
Keywords
Attini, mutualism, co-evolution, multi-nucleate, Leucoagaricus, Basidiomycota, fungus-growing
ants
36
Introduction
Polyploidy, the possession of more than two sets of haploid chromosomes, is widespread in fow-
ering plants (Masterson 1994; Otto and Whitton 2000), with 15% of speciation events having
likely been facilitated by polyploidization, a fgure that is even twice as high (31%) in ferns (Wood
et al. 2009). Ploidy levels can be as high as 10x in Fragaria (Hummer et al. 2009) and 12x in
Rhodondendron (Jones et al. 2007), but have almost always remained even. Polyploidy may be
advantageous because it increases functional heterozygosity, which helps masking the phenotypic
effects of deleterious recessives, but this may come at the cost of more likely chromosome loss
during mitosis, spindle irregularities and other imbalances during meiosis, and more epigenetic
instability (Comai 2005). In contrast, polyploidy is rare in metazoans (Wertheim et al. 2013),
suggesting that the benefts of polyploidy outweigh the costs more consistently in plants, which
retained modular growth in spite of sharing zygote formation and embryonic development with
animals (De Mendoza et al. 2013).
Polyploidy is also rare in fungi, but this may be primarily related to fungal cells not being haploid
or diploid in the normal sense, but having one (monokaryon) or two (dikaryon) haploid nuclei per
cell, with just a few exceptions such as the unicellular Saccharomyces cerevisiae and the multi-
cellular Armillaria mellea (honey fungus) which occasionally have merged diploid nuclei (Rizzo
and May 1994; Carvalho et al. 1995). Monokaryon hyphae emerging from germinating spores
produced after meiosis are thus analogous to gametophytes in vascular plants, so the sexual pro-
cess remains uncompleted until two monokaryons merge to form a dikaryon. When such contact
is initiated and proves to be compatible, the nuclei of the parental monokaryons distribute them-
selves evenly throughout the hyphal system so all cells end up having both types of nuclei that
will henceforth be passed jointly when cells divide. This makes dikaryons functionally analogous
to diploid vascular plant sporophytes (Raper 1955; Ellingboe and Raper 1962).
As the original haploid nuclei of sexual spores do not become life-time committed in a zygote,
their association needs to be actively reinforced with every new cell division of a dikaryon to en-
sure that nuclei retain equal probabilities of taking part in sexual reproduction wherever mycelia
will initiate the formation of sexual organs. The Basidiomycota that occur mostly as dikaryons
have resolved this challenge by evolving clamp connections, specialized bridges between the new
cell, which has received a copy of one nucleus, and the old cell that is about to become separated
by a new cell wall (Buss 1987). This septum forms simultaneously in the clamp connection where
it separates the two copies of the second nucleus, so one of these is predictably returned to the old
cell (Moore et al. 2011). Once a basidiomycete dikaryon has formed, incompatibility mechanisms
37
will normally prevent that additional nuclei from another monokaryon or dikaryon can be accept-
ed unless they are genetically identical or very closely related (Adaskaveg and Gilbertson 1987;
Wilson 1991; Marais et al. 2000; Poulsen and Boomsma 2005).
Multinucleate hyphal cells have only rarely been observed in Basidiomycota. In some cases such
as Agaricus bisporus there may be up to 25 nuclei per cell, but these are all clonal copies of the
two original nuclei (Saksena et al. 1976) and thus an example of autopolyploidy. However, rare
examples of allopolyploidy are documented for Heterobasidion species that have three (James
et al. 2009) or four types of nuclei in a single mycelium (Johannesson and Stenlid 2004). These
studies used neutral microsatellites markers to estimate the number of haplotypes in excess of the
standard two and achieved high resolution, but there is only indirect evidence that the usual expla-
nation of increased heterozygosity might apply. The particularly demanding necrotrophic life his-
tory of Heterobasidion phytopathogens, relative to saprotrophic fungi, might offer unusual ftness
rewards to the higher gene expression diversity that allopolyploidy would likely allow (Groth and
Christ 1992), but stress-related advantages of this kind have only been documented for polyploid
yeasts (Lidzbarsky et al. 2009).
In a recent genome sequencing project of the basidiomycete fungal symbiont of the leaf-cutting
ant Acromyrmex echinatior, we found strong indications for functional polyploidy (De Fine Licht
et al. 2013). This was consistent with earlier fndings (Scott et al. 2009) and effectively precluded
that a draft genome could be assembled. This fungus, Leucoagaricus gongylophorous, is a special-
ized domesticated crop organism that the ants rear as their almost exclusive food source in special
underground gardens (Mueller et al. 2005). The evolutionary history of fungus farming goes back
ca. 50 million years when the ancestor of all extant attine ants gave up its hunter-gatherer life
style to start farming vertically transmitted leucocoprinaceous fungi for food (Schultz and Brady
2008). However, it took 30 million years until a single lineage of these crop fungi was irrevers-
ibly domesticated, and evolved specialized hyphal tips (gongylidia) to feed the ants (Quinlan and
Cherrett 1978; Quinlan and Cherrett 1979; Bass and Cherrett 1995) while losing genetic exchange
with free-living relatives (Mueller et al. 2005; Schultz and Brady 2008; De Fine Licht et al. 2010).
This gave rise to four genera of so called higher attine ants that all rear cultivars belonging to
this clade of gongylidia-bearing crop fungi, of which the most advanced Atta and Acromyrmex
leaf-cutting ants arose only 10 million years ago and rear a single, genetically polymorphic spe-
cies of crop fungus (Mikheyev et al. 2010).
Given the rarity of basidiomycete polyploidy, and the fact that the human domesticated mushroom
38
Agaricus bisporus has multinucleate cells while its free-living close relative Agaricus bitorquis
has normal dikaryons (Raper 1976; Hintz et al. 1988), we decided to test the hypothesis that do-
mestication might have induced functional polyploidy, to compensate for the absence or extreme
rarity of sexual reproduction (Pagnocca et al. 2001; Mueller 2002; Mikheyev et al. 2006) and pos-
sibly also to meet novel requirements of enzyme production (Schitt et al. 2010; De Fine Licht et
al. 2013). We therefore set out to estimate the average number of nuclei per cell across the fungal
symbionts of 14 species of attine ants from Panama and used internal transcribed spacer (ITS)
and large subunit rRNA (LSU) ribosomal RNA sequencing and microsatellite genotyping to map
nuclei-numbers on the symbiont tree and to estimate functional ploidy.
Materials and methods
Biological material
Fungal symbionts of attine ants were isolated on Potato Dextrose Agar (PDA) from 42 lab-col-
onies collected in Panama from 2004 to 2012. Samples included representatives of the different
stages of ant fungus farming (see Table S1, for details on species and laboratory nest numbers),
such that we had 25 samples of higher attine ants rearing domesticated gongylidia- bearing fun-
gi, and 17 samples of lower- and paleoattine ants that rear fungi without obvious adaptations to
being crops (Mueller et al. 1998; Schultz and Brady 2008). Plates were maintained at ca. 25C,
and DNA was obtained with 5% Chelex extractions when suffcient biomass had been produced.
Nuclei counting
Small pieces of mycelium from each colony were stained with DAPI on a microscope slide, after
which we counted the number of nuclei per cell in fve randomly chosen cells under a fuores-
cence microscope. For analysis the data were grouped in fve categories of fungus-farming that
are generally recognized as characterizing the diversifcation and progression of attine fungus
farming: coral fungus agriculture, yeast agriculture, lower agriculture, domesticated agriculture,
and leaf-cutting agriculture (Schultz and Brady 2008; Mehdiabadi and Schultz 2010). Differences
in number of nuclei per cell were subsequently analyzed with a General Linear Model in SAS with
colonies as random factor nested within species, species nested within genera, and genera nested
within the fve categories of fungus farming.
Internal transcribed spacer (ITS) and large subunit rRNA (LSU) sequencing
To make sure that the plated fungi were the same strains as the ones maintained by the fungus
growing ants and to verify whether the two known clades of lower agriculture crops (Clade 1
and Clade 2)(Mueller et al. 1998; Schultz and Brady 2008; Mehdiabadi and Schultz 2010) were
39
both represented, we amplifed and sequenced two conserved regions: Internal transcribed spacer
(ITS4: 5-TCC TCC GCT TAT TGA TAT GC-3; ITS5: 5-GGA AGT AAA AGT CGT AAC AAG
G-3) and the nuclear large subunit rRNA (LR0R: 5-ACC CGC TGA ACT TAA GC-3; LR5: 5-
TCC TGA GGG AAA CTT CG-3), using PCR with 8L dNTPs, 2L gold buffer, 2L 10mM
MgCl

2
, 0.1L BSA, 1L forward and reverse primer each, 0.2L gold Taq polymerase, 4.7L
ddH
2
O and 1L DNA. The PCR program had 10 min denaturing at 94C followed by 35 cycles
of 1 min denaturing at 94C, 30 sec annealing at 54C and 1 min extension at 72C, and fnally
a 10 min extension at 72C. All PCR products were sequenced at BGI Europe in Copenhagen
and sequences were deposited in GenBank (see Table S1 for accession numbers). In cases of bad
quality sequences due to multiple divergent copies with small length mutations, PCR products
were cloned in pCR4-TOPO before sequencing them again using the TOPO TA cloning method
(Invitrogen, Carlsbad, CA, USA). A phylogenetic tree similar to Mueller (1998) was constructed
using Maximum Likelihood calculations with 500 bootstrap replicates, using MEGA5.1 software
(Tamura et al. 2011).
Microsatellite screening
As we were fnding multinucleate cells throughout the higher attine ant symbionts, we decided
to screen these fungi for allelic variation at ten microsatellite loci: A128, A1030, A1132, A1151,
B12, B447, C101, C126, C647 and D115 taken from Scott (2009), using PCR with 5L VWR Red
Taq DNA polymerase Master Mix (VWR International, Haasrode, Belgium), 0.25L forward and
reverse primer each, 1.5L ddH
2
O and 1L DNA, and a program of 5 min denaturing at 95C, fol-
lowed by 14 cycles of 30 sec denaturing at 95C, 30 sec annealing at 68-58C with a touchdown
of -0.5C per cycle, and 30 sec extension at 72C followed by 20 cycles of 30 sec denaturing at
95C, 30 sec annealing at 58C and 30 sec extension at 72C, and fnally a 15 min extension at
72C. 1L of each PCR product was then mixed with 8.75L formaldehyde and 0.25L LIZ500
and analyzed on an ABI3130xl (Applied Biosystems, Nrum, Denmark) sequencer. Chromato-
grams were analyzed with Genemapper 4.0 (Applied Biosystems, Nrum, Denmark) to obtain
specifc allele scorings, after which a phylogenetic tree was constructed using pairwise F
st
values,
followed by Neighbor Joining analysis with 500 bootstrap replicates using Populations 1.2.32
software (Langella 2001).
Estimating ploidy
The microsatellite screenings often showed more than two allelic peaks, indicating that excess
nuclei were not all copies of dikaryon nuclei, but at least partly represented allopolyploidy. If we
would have had infnite variation at a single microsatellite marker, the number of alleles observed
40
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41
would have given us exact estimates of ploidy. However, the limited cumulative detection power
of our 10 moderately variable marker loci necessitated that we estimated the detection error of not
observing genetically different haplotypes (because alleles are equal by chance) before estimating
the average rate at which haplotype numbers increase with the number of nuclei per cell. To do
this, we frst plotted the number of different alleles observed against the mean number of nuclei
for each of the ten markers and calculated the slopes of regression lines forced through the origin.
As our dikaryon data for lower attine fungi showed that cells were sometimes counted after having
initiated division, we use that quantifed error rate of each nucleus being counted as 1.03 to cor-
rect scores along the x-axis (Fig. S2). Because the origin was the dikaryon expectation of two
nuclei giving an expected allele number score of 1+h, where h is the expected heterozygosity at
that particular locus, we subtracted these values from the respective x and y scores before running
the analyses. Samples with null alleles were excluded, since they provide incomplete information
about heterozygosity. As expected the positive slopes of these regression lines increased with lo-
cus-specifc heterozygosity for the eight most variable marker loci, so we next plotted these slopes
against the locus-specifc expected heterozygosity (based on observed allele frequencies). As the
slopes entered could only vary between zero and one, we ftted a logistic regression through these
data points, while weighting them according to their standard error, to obtain an overall estimation
of the number of haplotypes we should have detected if we would have had a marker locus with
infnite allelic variation.
Figure 1
Ancestry, diversifcation and mean number of nuclei per cell in garden symbionts of 14 Panamanian fungus-growing
ants. A) Phylogenetic tree of attine ant fungal symbionts based on ITS and LSU sequences and Maximum Likelihood
analysis with 500 bootstrap replicates and Agaricus bisporus (ITS: JX684008.1, LSU: AY635775.1) as outgroup (black
branch). Different levels of agriculture are presented in different colors: coral fungus agriculture gray, lower agriculture
yellow, yeast agriculture red, domesticated agriculture blue, and leaf-cutting agriculture green. Numbers at nodes are
percentages consensus support and colony numbers with species names are given next to branches. Because Apterostig-
ma dentigerum grows a completely different (secondarily acquired) coral fungus its tree was generated in a separate
analysis with the same human domesticated A. bisporus as outgroup. B) Histogram of the mean ( SE) number of nuclei
per cell of the fungal symbionts, showing a clear separations between the three basal levels in the phylogeny (2.40
0.29 nuclei per cell for coral fungus farming, 2.07 0.05 for lower attine fungus farming, and 2.00 0.00 for yeast
farming) and the two advanced levels of fungus farming that both rear clades of gongylidia-bearing fungi (10.80 0.43
for Trachymyrmex and Sericomyrmex symbionts and 12.46 0.41 for Atta and Acromyrmex symbionts). Representative
pictures of stained nuclei are given towards the right: C) Atta sexdens (14 nuclei per cell), D) Sericomyrmex amabilis
(8 nuclei per cell), E) Cyphomyrmex costatus (2 nuclei per cell), and F) Apterostigma dentigerum (2 nuclei per cell).
Arrows indicate the septae that separate the cells.
42
Results
The ITS and LSU sequences of the strains obtained from the different colonies were compared
to the NCBI GenBank database using blastn, which showed that all sequences had their highest
similarities with previously sequenced fungal symbionts from each of the different attine ant spe-
cies. Consistent with these earlier results, the phylogenetic tree showed distinct clades among the
fungal symbionts comparable to those published previously (Mueller et al. 1998; Munkacsi et al.
2004; Vo et al. 2009; Mikheyev et al. 2010), with all leaf-cutting agriculture strains clustering
together, all other gongylidia-bearing domesticated strains clustering together, and all lower- and
paleoattine ant strains appearing as basal branches when using the closely related human domes-
ticated Agaricus bisporus as outgroup (Figure 1A). A separate analysis with the same outgroup
provided a complementary tree for the three pterulaceous fungal symbionts of Apterostigma denti-
gerum. Although microsatellite markers are known to evolve much faster than ribosomal sequenc-
es, these markers also recovered the split between the gongylidia-bearing fungi reared by Atta
and Acromyrmex on one hand, and those reared by Trachymyrmex and Sericomyrmex on the other
(Figure S1). This indicates that there is no gene fow between these fungal clades at our study site
in Panama.
Practically no variation was found in the number of nuclei per cell across the lower agriculture
lineages. With only two exceptions we always observed a constant number of two, with only
two slightly higher counts (Cyphomyrmex longiscapus [Cylo005] and Apterostigma dentigerum
[Aden003]) that were likely due to hyphal tips being observed while cells were dividing so that
nuclei were already duplicated but a new septum had not yet been formed. Mapping the number
of nuclei per cell on the branches of the phylogenetic tree produced a clear pattern, with all low-
er- and paleo-attine strains, coral fungus strains and yeast strains having two nuclei per cell, and
all higher attine ant strains having high numbers (7-17) of nuclei per cell (Figure 1B). This sharp
transition thus coincided with the origin of the fungal gongylidia, the uniquely modifed hyphal
tips whose only known purpose is to be eaten by the ants, an irreversible adaptation to obligate
symbiotic life. Statistical analyses showed that the number of nuclei per cell differed between
higher (domesticated) fungus-farming on one hand and lower fungus-farming on the other (colors
in Figure 1A: F
4,27
= 74.46, p < 0.0001), but not between the ant genera within each of these farm-
ing types (F
4,27
= 1.64, p = 0.1941) or between species within ant genera (F
6,27
= 1.71, p = 0.1560)
(see also Figure S2).
Microsatellite allele numbers per locus varied between 2 and 10 (Table S2) and all loci produced
higher allele number scores when the number of nuclei per cell was higher, although the slopes for
43
the two least variable loci (B447 with 3 alleles and C126 with a total of 4 but maximally 2 alleles
per fungal clone) were very shallow and thus considered to be hardly informative (Figure S3). The
regression line predicting the rate at which haplotypes would increase with the number of nuclei
per cell for an ideal marker locus with an infnite allele number (h = 1) produced an estimate of
0.37 0.21 (95% CL 0.09 0.77)(Figure 2). As this regression slope estimated the rate of ploidy
increase beyond the dikaryon level of two, this indicates that ca. two out of every fve nuclei in
excess of the dikaryon starting point represented genetically distinct haplotypes. This implies that
the average ploidy of the higher attine fungal symbionts was estimated to be 6.2 (95% CL 3.0
10.8).
Discussion
Our results corroborate that the emergence of fungal gongylidia ca. 20 MYA is an even more
pronounced irreversible transition on the attine fungus farming symbiosis than previously appre-
ciated. It characterized the true domestication of fungal crops and produced four new genus-level
branches in the ant phylogenetic tree, of which one split into the two extant genera of leaf-cutting
ants 10 MYA (Schultz and Brady 2008; Mehdiabadi and Schultz 2010). Constant high levels
of cultivar auto- and allopolyploidy throughout the higher attine ants suggests that evolutionary
innovation in the crown group of the attine ants has been driven by signifcant improvements of
crop quality related to dosage advantages and chimeric genetic diversity. It seems reasonable to
hypothesize that autopolyploidy evolved frst as that remains compatible with occasional sex-
ual reproduction (Udall and Wendel 2006), but that later allopolyploidy effectively precluded
most if not all meiotic cell division. The emergence of gongylidia induced about an order of
magnitude increase in scale of operations, with colony sizes increasing to maximally ca. 3000 in

0.5 0.6 0.7 0.8 0.9 1.0


0
.
0
0
.
2
0
.
4
0
.
6
0
.
8
Heterozygosity
P
l
o
i
d
y

c
o
e
f
f
i
c
i
e
n
t
0
.
0
0
.
2
0
.
4
0
.
6
0
.
8
Figure 2
Regression coeffcients ( SE) for the locus-specif-
ic increase of alleles scored with increasing number
of nuclei plotted against the expected locus-specifc
heterozygosities. The ftted logistic regression line is
based on the eight loci with the highest allelic diver-
sity: p(x) = e
-11.077+10.553x
/ (1 + e
-11.077+10.553x
) (QAIC =
13.150). The two excluded loci, shown in light grey,
had standard errors that overlapped with the 95% c.l.
of the ftted regression line, corroborating that their
contribution just lacked information rather than devi-
ating from the overall trend in a signifcant manner.
44
Trachymyrmex and Sericomyrmex higher attine ants, quite possibly because the truly domesticat-
ed cultivars were better able to decompose freshly shed leaves and fowers with higher nutritious
value (De Fine Licht and Boomsma 2010). Leaf-cutting agriculture allowed a further increase in
scale of operations by 1-2 orders of magnitude (Schultz and Brady 2008; Baer et al. 2009; Meh-
diabadi and Schultz 2010), but how much of this was driven by evolutionary changes in the ants
or the acquisition of a novel clade of crop symbionts 2.3 MYA (Mikheyev et al. 2010) remains
unknown.
As far as mushrooms originating from attine fungus gardens have been found, they seem to be
most prevalent in the lower attine ants (Mueller 2002), which still have closely related free-living
sister lineages (Mueller et al. 1998; Pagnocca et al. 2001). This implies that the capacity to prog-
ress towards sexual reproduction, particularly in abandoned fungus gardens, is unlikely to have
been lost (Mueller 2002). It has been suggested that higher attine fungi have retained the capacity
for meiosis (Mikheyev et al. 2006), but observations of mushrooms are extremely rare, although
the initiation of primordia can occasionally be observed in lab colony where they generally appear
to fail (Fisher et al. 1994; Mueller 2002; P.W. Kooij, M. Schitt, H.H. de Fine Licht, personal ob-
servations). Whether consistent combinations of auto- and allopolyploidy would allow occasional
recombination without the production of sexual mushrooms remains an open question, but seems
a possibility as unrelated fungi are not always somatically incompatible when grown on the same
agar plate (Poulsen and Boomsma 2005; Lind et al. 2007). However, without regular recombina-
tion fungal clones may accumulate deleterious mutations, which will tend to affect phenotypic
performance in spite of polyploidy and will thus retain selection for occasional horizontal transfer
(Mikheyev et al. 2007; Poulsen et al. 2009) in a system where vertical transmission via virgin
queens is the norm (Weber 1972).
While the absence or rarity of recombination may be a long-term challenge for maintaining op-
timal phenotypic performance even in a well-protected crop symbiont, this problem is further
aggravated by monoculture farming, a mutualism-stabilizing principle that convergently evolved
in both the fungus-growing ants and termites via very different mechanisms (Poulsen and Booms-
ma 2005; Aanen et al. 2009). Long-lived colonies thus expose the same fungal phenotypes to
possible parasites, which would tend to increase disease pressure over time (Hamilton 1982).
Allopolyploidy may be a helpful mechanism to alleviate this type of vulnerability, as it will allow
the expression of many more resistance factors than a functionally diploid dikaryon could achieve.
Analogies of this chimerism principle can be found in the expression of many MHC alleles in
otherwise diploid vertebrate immune systems (Potts and Wakeland 1993) and in the evolution of
45
multiple queen-mating in hymenopteran eusocial insects, which allows the number of haplotypes
expressed at the colony level to increase from three (under haplodiploid monogamy) to 10 when
a queen mates with eight males. Recent studies have shown that this increase in colony-level het-
erozygosity indeed reduces vulnerability towards infections (Seeley and Tarpy 2007; Hughes et
al. 2010).
Apart from ameliorating the absence of recombination and disease pressure, allopolyploidy in
higher attine crop fungi may also have offered advantages that ultimately allowed the attine ants
to abandon the brown decomposition food chain (Shik and Kaspari 2010) to become functional
herbivores. This transition started with the Trachymyrmex and Sericomyrmex higher attines that
can use modest amounts of soft leaves and petals to manure their fungus gardens, and culminated
in the leaf-cutting ants that almost exclusively collect fresh plant material (Weber 1972; Hervey
et al. 1977; De Fine Licht and Boomsma 2010). This chain of events implied progressing from
litter substrate to live substrate actively defended by secondary plant compounds (De Fine Licht et
al. 2013), a challenge that may have been easier to meet with an allopolyploid chimeric spectrum
of fungal haplotypes that express larger arrays of complementary enzymes (Soltis et al. 1993),
increase biochemical fexibility in other ways (Roose and Gottlieb 1976), or merely allow better
growth (Otto and Whitton 2000). The latter may also be achieved with autopolyploidy, as gene
dosis advantages may generally enhance performance of multinucleate cells even though nuclei
are identical copies (Albertin and Marullo 2012). Fungal analogies of these plant examples could
possibly also facilitate the degradation of more complex or recalcitrant substrates, as shown in the
allopolyploid brewing yeast Saccharomyces pastorianus (Pope et al. 2007; Minato et al. 2009;
Libkind et al. 2011). It is interesting that the only known other examples of fungal allopolyploidy
are Heterobasidion species that attack live conifer trees (Hodges 1969; Johannesson and Stenlid
2004; James et al. 2009) and that the spectrum of leaf-cutting fungal decomposition enzymes
evolved to become similar to that of many plant pathogens after the attine ants became herbivores
(Schitt et al. 2010; De Fine Licht et al. 2013).
How the 7-17 alleles that we found are distributed across the multiple nuclei in the crop fungi of
higher attine ants remains to be resolved, but two hypotheses for cases like this have been proposed
(Scott et al. 2009): 1. The same primer pair amplifes the same set of multiple loci in each haploid
nucleus, which would be likely if L. gongylophorus underwent recent genome duplications, and
2. There is genetic variation among the haploid nuclei in multinucleate mycelia. The consistent
polyploidy across all lineages of higher attine cultivars suggests that recent duplications are un-
likely to explain more than a small fraction of the variation, but explicit studies would be needed
46
to verify the validity of this, or the alternative explanation. This will not be an easy task as similar
observations of multinucleate somatic cells in arbuscular mycorrhizal fungi have led to different
interpretations. Also here multiple alleles can be found in single mycelia, which has been claimed
to imply that a single mycelium could have genetically diverse nuclei (e.g. Kuhn et al. 2001; Hijri
and Sanders 2005). However, evidence is now accumulating that this type of sequence divergence
within mycelia may derive from differences within single nuclei rather than from differences be-
tween nuclei (Pawlowska and Taylor 2004; Lin et al. in press). It is interesting that the occurrence
of sex has also remained ambiguous in the arbuscular mycorrhizal fungi (Sanders 2011).
Concluding remarks
The adoption of fungus farming 50 MYA by the ancestor of all attine ants implied a radical aban-
donment of the normal hunter-gatherer life style of all ants, but our documentation of functional
allopolyploidy evolving in the ant crops 20 MYA strongly suggests that the domesticated, gon-
gylidia-bearing fungal symbionts drove later evolutionary developments towards large scale fun-
gus farming in complex societies of leaf-cutting ants (Bass and Cherrett 1995; Schultz and Brady
2008; Mehdiabadi and Schultz 2010). Our results do not allow us to differentiate between the
various selection forces (compensation for lack of recombination, reducing disease vulnerability,
performance advantages owing to multiple identical nuclei or disease resistance owing to genetic
chimerism) that could have maintained high degrees of crop allopolyploidy, but it is interesting
to note that the attine ants domesticated a lineage of fungi that lack clamp connections (Vellinga
et al. 2003), so the main constraint for evolving polyploidy was absent, and that this potential
was realized as soon as the gongylidia bearing obligate crop-lineage evolved. It is also intriguing
that essentially all human agricultural crops are polyploid or originated from polyploid ancestors
(Udall and Wendel 2006). Human cultural evolution of farming practices also started with largely
vertically transmitted crops and gradual improvement of their quality, to be replaced by global
horizontal transmission of industrially innovated cultivars only during the last half century.
Acknowledgements
We thank the Smithsonian Tropical Research Institute (STRI), Panama, for providing logistic help and facilities to work
in Gamboa, the Autoridad Nacional del Ambiente y el Mar (ANAM) for permission to sample ant colonies in Panama,
R.M.M Adams, A. Illum, J. Liberti, B. Baer, H.H. De Fine Licht, S.P.A. Den Boer and W. Hughes for help with col-
lecting the ant colonies or making some of their own collected colonies available, Gsta Nachmann for suggesting the
statistical analysis for ploidy estimation, and the Danish National Research Foundation for funding (DNRF57).
47
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i
Figure S2
The mean number of nuclei per cell was signifcantly different for the fve different types of fungus farming with the
gongylidia bearing symbionts of the higher attine ants (including leaf-cutting ants) having consistently higher numbers
of nuclei per cell than the symbionts reared by the lower- and paleo-attine ants (including the yeast growers)(F
4,27
=
74.46, p < 0.0001). No signifcant differences were found between the genera within these types of farming (F
4,27
= 1.64,
p = 0.1941) or between ant species within genera (F
6,27
= 1.71, p = 0.1560). Bars represent means SE and colors are
as in Figure 1.
53
Figure S3
Regression plots for the number of dif-
ferent alleles observed per symbiont
sample against the average number of
nuclei per cell for each of the ten micro-
satellite loci. Samples with null alleles
were excluded from the analysis, since
they provide incomplete information
about heterozygosity. Regressions were
forced through the origin (in this case
2,1+h, the expected number of nuclei
in a dikaryon and the expected number
of alleles to be observed for two nuclei,
which is equal to 1 plus the expected het-
erozygosity), by subtracting these values
from the respective x and y values before
entering them in the analysis. Regression
slopes were expected to vary between
zero (all additional nuclei are copies of
the two unrelated nuclei of the original
dikaryon) and one (the dotted line that
would be obtained if every additional
nucleus would represent an indepen-
dent unrelated haplotype and if a mark-
er locus would have an infnite number
of alleles so that all these nuclei would
also be scored as genetically different).
Text boxes give locus name (Scott et al.
2009), proportion of explained variation
(R
2
), F and p-values, and the slopes of
the regression lines.

Locus A128
R = 0.4964
F = 16.76
p = 0.0007575
Coef = 0.13373
2
1, 17

Locus A1030
R = 0.7363
F = 50.25
p = 1.312e-06
Coef = 0.23212
2
1, 18

Locus A1132
R = 0.6018
F = 27.21
p = 5.83e-05
Coef = 0.1273
2
1, 18

Locus A1151
R = 0.6078
F = 37.19
p = 2.678e-06
Coef = 0.29174
2
1, 24

Locus B12
R = 0.2323
F = 4.54
p = 0.05007
Coef = 0.05812
2
1, 15


Locus B447
R = 0.5229
F = 17.53
p = 0.0006969
Coef = 0.06881
2
1, 16


Locus C101
R = 0.5391
F = 28.07
p = 1.956e-05
Coef = 0.07107
2
1, 24


Locus C126
R = 0.0695
F = 1.793
p = 0.1931
Coef = -0.01431
2
1, 24

0
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5
Locus C647
R = 0.3775
F = 10.31
p = 0.005126
Coef = 0.11224
2
1, 17

0 5 10 15 0 5 10 15
Locus D115
R = 0.4448
F = 19.23
p = 0.0001985
Coef = 0.15535
2
1, 24
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54
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61
CHAPTER 2
SOMATIC INCOMPATIBILITY OF FUNGAL CROPS IN SYMPATRIC ATTA AND
ACROMYRMEX LEAF-CUTTING ANTS
PEPIJN W KOOIJ, MICHAEL POULSEN, MORTEN SCHITT, JACOBUS J BOOMSMA
(MANUSCRIPT)
62
Somatic incompatibility of fungal crops in sympatric Atta and Acromyrmex leaf-cutting ants
Pepijn W. Kooij, Michael Poulsen, Morten Schitt, and Jacobus J. Boomsma
Centre for Social Evolution, Department of Biology, University of Copenhagen, Universi-
tetsparken 15, 2100 Copenhagen, Denmark
Phone: +45 35321239
Fax: +45 35321250
63
Abstract
Obligate mutualistic symbioses rely on mechanisms that secure host-symbiont commitments to
maximize host benefts and prevent symbiont cheating. Evolutionary theory predicts that incom-
patibility mechanisms between clonal symbiont lineages may enforce such commitments, but the
most advanced endosymbioses have exclusive uniparental and vertical transmission of symbionts
so that resident symbiont lineages are never challenged. Although attine ants also transmit their
fungus-garden symbionts vertically via colony founding queens, horizontal transmission is possi-
ble. Resident garden symbionts may therefore face competition by alternative symbionts, which
does not serve their ftness interests and is also unlikely to offer short-term benefts to the host
colony. Previous studies showed that somatic incompatibility mechanisms correlate with neutral
marker genetic distance between fungal symbionts in Panamanian Acromyrmex leaf-cutting ants,
but the extent to which this result applies more generally has remained unclear. We hypothesized
that somatic incompatibility mechanisms are only likely to evolve between fungal genotypes that
compete for the same niche, i.e. for being the clonal symbiont of one or a few ant species that
share the same clade of fungal symbionts. We test this by comparing the expression of somatic in-
compatibility among plated fungi reared by sympatric Acromyrmex and Atta colonies in Panama.
We show that genetic distances for neutral microsatellite and AFLP markers accurately predict
somatic incompatibility for Acromyrmex symbionts, but that these correlations are weaker (mi-
crosatellites) or absent (AFLP) in sympatric Atta colonies. Neither set of genetic markers explains
any incompatibility between combinations of symbionts from Atta and Acromyrmex on the same
plates. Further analysis showed that the symbiont clades maintained by Atta and Acromyrmex
are likely to represent separate gene pools, so that neutral markers are unlikely to be correlated
with incompatibility loci that have experienced different selection regimes. We discuss possible
reasons for somatic incompatibility among Atta symbionts being only modest, based on the like-
lihood of resident symbionts being challenged by competing symbionts during colony founding
and when colonies are mature.
Keywords
Leucoagaricus gongylophorus, commitment, mutualism, fungus-growing ants
64
Introduction
Endosymbionts are normally asexual and transmitted by uniparental vertical inheritance (Sachs
et al. 2011). Multicellular organisms thus have a single mitochondrial genotype and those that
have photosynthesis rely on a single clone of plastids. The evolution of such obligate symbiotic
mutualisms has strong elements of partner commitment driven by kin selection, because exclusive
association of hosts with a single symbiont genotype ensures that any services to growth and sur-
vival of the host will beneft clone mates of symbionts that are vertically transmitted when hosts
reproduce (Frank 1994; Doebeli and Knowlton 1998; Sachs et al. 2004; Foster and Wenseleers
2006). The same logic implies that hosts and symbionts are potentially in confict over the mode
of symbiont transmission (Frank 1996; Douglas 2008), as symbionts would always beneft from
additional horizontal transmission. However, hosts might suffer ftness losses from this form of
commitment disloyalty and therefore suppress symbiont investments in sexual reproduction even
if that incurs only a marginal cost (Frank 1996; Leigh 2010). When, in spite of such host efforts,
symbiont lineages manage to co-infect hosts and compete for host commitment, hosts will be un-
der selection to monitor this confict and eliminate it when symbiont mixing implies a net loss of
cumulative symbiont service to the host (Frank 1996).
The classical mitochondrial and plastid endosymbioses are so completely integrated with the
host that their reduced genomes preclude any form of non-symbiotic life, and the same is true
for many obligate and facultative endosymbionts with less reduced genomes (McCutcheon and
Moran 2012). Many of these interactions likely represent adaptive endpoints of host-symbiont
coevolution, where host-symbiont conficts were resolved in favor of the hosts (McCutcheon and
Moran 2012; Wernegreen 2012) or symbiont (Werren et al. 2008), but their advanced stage of
symbiosis normally precludes direct tests of evolutionary confict theory over symbiont mixing
because symbionts cannot be reared in vitro. However, the fungus-growing ants offer a feasible
model system to do such tests, because they have multiple obligate mutualisms including fungus
gardens (Schultz and Brady 2008; Mikheyev et al. 2010) and cuticular Actinobacteria (Cafaro et
al. 2011; Andersen et al. 2013) that are ectosymbionts for individual ants, but endosymbionts for
the ant colonies. This condition implies that partners can be separated and reared in vitro without
each others interference for suffcient periods of time to quantify antagonism between symbiont
clones, monitor host reactions to alternative symbionts, and relate observed differences to the
genetic characteristics of the interactants (Bot et al. 2001; Armitage et al. 2011; Seal et al. 2012).
As far as populations of fungus-growing ants have been studied, attine ant colonies have nev-
er been found to rear a multi-clone fungus garden (Acromyrmex, Poulsen and Boomsma 2005;
65
Apterostigma, Dentinger et al. 2009; Atta, Mueller et al. 2010; Cyphomyrmex, Green et al. 2002,
Mehdiabadi et al. 2012). In all of these studies, there was substantial genetic variation among
fungus-garden clones across sympatric colonies, consistent with the normal patterns of variation
of mitochondrial and plastid organelles across individual animals and plants (e.g., Embley and
Martin 2006). However, in contrast to these cellular endosymbionts, there may be considerable
horizontal transfer of symbionts between attine colonies that have similar ecologies, with species
belonging to the same genus often (but not always) sharing clades of symbionts (Green et al. 2002;
Poulsen and Boomsma 2005; De Fine Licht and Boomsma 2011; Mehdiabadi et al. 2012), while
sympatric attine ant genera normally rear distinct fungal symbiont clades (Mueller and Gerardo
2002; Vo et al. 2009; Dentinger et al. 2009; Mehdiabadi et al. 2012; Kooij et al. Chapter 1, in
prep).
Horizontal swaps of fungus-garden symbionts between colonies of the same or closely related at-
tine ant species reduce the effciency of co-evolutionary adaptation at the lowest taxonomic level,
but may allow ant lineages to replace any asexual crop symbiont that is compromised by genetic
load or other forms of maladaptation to prevailing ecological conditions. However, as long as fun-
gus-garden clones are thriving, they will also be under selection to actively defend their ant-care
monopoly (Bot et al. 2001). Such defenses are expected to evolve when the threat to be replaced
is real, i.e. any hostility of this kind should target non-self symbiont genotypes belonging to the
clade of symbionts that can in fact partake in a viable symbiosis with a focal attine ant species.
Sympatry and co-exploitation of the same clade of fungus-garden symbionts characterize the Ac-
romyrmex echinatior and Acromyrmex octospinosus populations in Gamboa, Panama (Bot et al.
2001; Richard et al. 2007a; Poulsen et al. 2009), where resident fungus gardens have been shown
to maintain their clonal integrity by a combination of behavioral adaptations in the ants to remove
and kill alternative fungus clones (Bot et al. 2001; Ivens et al. 2009) and the expression of somatic
incompatibility reactions between clones from different Acromyrmex colonies reared together on
the same agar plates (Poulsen and Boomsma 2005). These expressions of incompatibility correlat-
ed with AFLP genetic distances between pairs of fungal symbionts, a pattern that also applied to
the fecal fuid of Acromyrmex large workers fed with fungus from their own and other colonies
(Poulsen and Boomsma 2005). However, founding queens of these Acromyrmex species readily
accepted fungal clones from other colonies, suggesting that horizontal transfers may be successful
at this stage even though new combinations may imply reduced symbiont performance (Poulsen et
al. 2009). This study also suggested that incompatibility mechanisms might be different for sym-
patric Atta leaf-cutting ants, which tend to rear different fungal symbionts (Mikheyev et al. 2006;
66
Mikheyev et al. 2007), and whose queens never forage during colony founding and therefore have
negligible likelihood of encountering alternative symbionts at that stage.
In the present study, we focus explicitly on comparing sympatric mature colonies of Atta and
Acromyrmex leaf-cutting ants to address the following questions: 1. Do plated fungal symbionts
of Panamanian A. echinatior and Atta colombica express similar somatic incompatibility reac-
tions when they are confronted with symbionts from other colonies? 2. To what extent does the
intensity of these reactions differ within and between the two genera? 3. To what extent is the
intensity of these reactions correlated with genetic distance between the fungal symbionts? 4.
Are sympatric colonies of Acromyrmex and Atta rearing the same or overlapping set(s) of fungal
symbionts or are they associated with segregated lineages of Leucoagaricus gongylophorus? The
same set would be expected if horizontal symbiont transmission between genera is more frequent
than natural divergence of lineages via mutation and genetic drift. Segregated lineages would be
expected when horizontal transmission between genera is rare or absent because co-adaptations in
L. gongylophorus strains reared by Acromyrmex and Atta would preclude that horizontal symbiont
swaps between genera are viable. At the same time, we also compared two different sets of genetic
markers (AFLP and microsatellites) and different time spans between plate inoculation and scor-
ing of incompatibilities to evaluate the robustness of our conclusions.
Natural history summary of attine ants and their fungi
Attine ants evolved ca. 50 MYA from a hunter-gatherer ancestor to become obligate farmers of
fungal crops, with strict domestication of the cultivar fungus evolving ca. 20 MYA and aggressive
herbivory in leaf-cutting ants ca. 10 MYA ago (Mueller et al. 1998; Schultz and Brady 2008; Me-
hdiabadi and Schultz 2010). Similar to all other fungus-growing ants, leaf-cutting ants rely on the
successful maintenance of their fungus garden crop as main food source (Weber 1966; Quinlan
and Cherrett 1978; Quinlan and Cherrett 1979). The large workers of Panamanian Acromyrmex
species are known to actively remove fungal symbiont fragments from other congeneric colonies
when they are genetically different from their resident garden fungus (Bot et al. 2001; Ivens et al.
2009), but whether foragers or nurses of Atta do the same is unknown.
The fungal symbiont reared by Atta and Acromyrmex leaf-cutting ants is considered to be a single
species (Leucoagaricus gongylophorus; Agaricales, Basidiomycota), specifcally associated with
these two higher attine genera that appear to have obtained this symbiont via a continent-wide hor-
izontal selective sweep ca. 2-3 MYA (Mikheyev et al. 2010). As all other fungal symbionts of the
higher attine ants, L. gongylophorus is vertically and asexually transmitted by newly inseminated
67
colony-founding queens (Mller 1893; Wheeler 1907; Weber 1955; Weber 1972). Horizontal ge-
netic exchange has been documented (Mikheyev et al. 2006; Mikheyev et al. 2007) but has only
been studied systematically for Panamanian Acromyrmex (Poulsen et al. 2009) and not for Atta
(see Green et al. 2002 and Mehdiabadi et al. 2012 for comparable studies in the lower attine genus
Cyphomyrmex).
Somatic (in)compatibilities between plated fungi of Panamanian Acromyrmex are expressed in a
gradual manner that correlates with AFLP genetic distances between pairs of clones (Poulsen and
Boomsma 2005). In basidiomycete fungi, such somatic incompatibilities regulate allorecognition
so that strains are increasingly likely to be incompatible when they are more genetically different
(May 1988; Worrall 1997). These reactions tend to be stepwise (Rayner et al. 1984; Rayner 1991;
Worrall 1997) and usually involve dark pigmentation in the interaction zone (Rayner et al. 1984),
changes in septal maintenance, or blockage of septa precluding the movement of cytoplasma, and
can lead to programmed cell death (Rayner 1991). The underlying genetic mechanisms remain
largely unknown, but multiple loci appear to be involved (Worrall 1997) and their expression may
be linked to sexual incompatibility genes (Van der Nest et al. 2009; Van der Nest et al. 2011). Sex
may be present but very rare in the fungal symbionts of the higher attine ants (Fisher et al. 1994a;
Fisher et al. 1994b; Pagnocca et al. 2001; Mueller 2002; Mikheyev et al. 2006) and recent work
indicates that these symbionts are invariably functionally polyploid (Kooij et al. Chapter 1, in
prep), which likely explains why somatic incompatibility patterns between fungal symbionts of
Panamanian Acromyrmex colonies appear to be gradual (Poulsen and Boomsma 2005).
Materials and methods
Biological material
Fungal cultivars were isolated from nine A. echinatior colonies (Ae150A, Ae160, Ae168, Ae263,
Ae266, Ae322, Ae356, Ae394, Ae488) and nine A. colombica colonies (Ac-2006-27, Ac-2009-
42, Ac-2009-46, Ac-2011-2, Ac-2011-3, Ac-2012-1, Ac-2012-2, Ac-2012-8, Ac-2012-31) living
sympatically in Gamboa, Panama, and grown on 39 g/L Potato Dextrose Agar (Sigma-Aldrich)
with the addition of 5 g/L yeast extract, 15 mg/L Tetracycline and 12 mg/L Streptomycin. DNA of
each fungal strain was extracted using the Qiagen DNeasy Plant Tissue extraction kit and stored
at -20C until further analysis. The Gamboa sampling site was the same as where most previous
colonies of the Copenhagen fungus-growing ant research program have been collected, including
the Acromyrmex colonies studied by Bot et al. (2001), Poulsen & Boomsma (2005), Mikheyev et
al. (2007), Richard et al. (2007a), Ivens et al. (2009) and Poulsen et al. (2009).
68
Genetic analyses
To calculate the genetic distance between each of the fungal strains we used two different methods:
Amplifed Fragment Length Polymorphism (AFLP) and ten microsatellite markers (A128, A1030,
A1132, A1151, B12, B447, C101, C126, C647 and D115 developed by Scott et al. (2009)). AFLP
was performed as described by Vos et al. (1995) with two selective primer combinations (Eco-
ACC + Mse-CAT and Eco-ACC + Mse-CAC). Microsatellite markers were analyzed using PCR
with 5 L VWR Red Taq DNA polymerase Master Mix (VWR International, Haasrode, Belgium),
0.25 L forward and reverse primer each, 1.5 L ddH2O and 1 L DNA, and a program of 5 min
denaturing at 95C, followed by 14 cycles of 30 sec denaturing at 95C, 30 sec annealing at 68-
58C with a touchdown of -0.5C per cycle, and 30 sec extension at 72C followed by 20 cycles
of 30 sec denaturing at 95C, 30 sec annealing at 58C and 30 sec extension at 72C, and fnally
a 15 min extension at 72C.
Both AFLP and microsatellite amplifcation products were analyzed on an ABI 3130xl (Applied
Biosystems) sequencer. Specifc allele scorings were obtained by analyzing chromatograms in
Genemapper 4.0 (Applied Biosystems). The program Populations 1.2.32 (Langella 2001) was
used to calculate F
st
values for the microsatellite data and Neis standard genetic distance (D
s
) for
the AFLP data, followed by Neighbor Joining phylogenetic analyses with 500 bootstrap replicates
for each of the two types of markers.
Somatic incompatibility
To test whether plated fungi showed (in)compatibility, cultures of all 18 fungi were paired in all
possible (171) combinations with four replicate pairings for each combination. For each pair,
small tufts of mycelium (ca. 2 mm
3
) were placed at a distance of 1.5 cm from each other on a 5
cm Petri dish with 39 g/L Potato Dextrose Agar (Sigma-Aldrich) with the addition of 5 g/L yeast
extract and 35 g/L Agar. The growth medium was selected in a pilot study testing somatic incom-
patibility reactions for control (self) encounters on this and three alternative media, which showed
that the used PDYA medium most consistently avoided any unexpected discolorations in controls
(Figure S1).
Incompatibility reactions were assessed after six, eight and ten weeks and scored using the
semi-quantitative scale described by Poulsen and Boomsma (2005): 0 = demarcation zone absent,
1 = demarcation zone weak but present, 2 = demarcation zone broad and distinct, and 3 = strong
demarcation zone with consistent brown or black coloration of mycelium. These four scores are
consistent with the variation in somatic (in)compatibility reactions that are typically found in
69
free-living basidiomycetes, although usually in a more stepwise manner as explained above (Wor-
rall 1997). All scorings were done blindly by randomly assigning numbers to each plate, after
which two of the authors did the initial assessment and the third author blindly checked combina-
tions for which the frst two authors did not agree on the score. Degrees of (in)compatibility were
subsequently compared with genetic distances between pairs of fungal clones with R (R Core
Team 2013) using Mantel and Partial Mantel Tests for Dissimilarity Matrices (mantel) with
99999 permutations in the Community Ecology Package: Ordination, Diversity and Dissimilar-
ities vegan (Oksanen et al. 2013). Figures were created using Plot With Repeated Symbols by
Size (sizeplot) in the Plotrix package (Lemon 2006).
Results
For Acromyrmex symbionts, consistent scorings of somatic incompatibility were obtained eight
weeks after agar plates were inoculated, as coloration contrasts had not fully developed after six
weeks, so Mantel correlation coeffcients between incompatibility and genetic distance after six
weeks remained low (Figure S2). Beyond eight weeks, Mantel correlation coeffcients continued
to improve, but contaminations and medium desiccation problems affected scoring accuracy 10
weeks after inoculation (Figure S2) so that we lost almost 5% of the replicates. As eight weeks
was approximately the same as the two-months period between inoculation and scoring in Poulsen
& Boomsma (2005), we decided to present our main results for the eight weeks observation pe-
riod, to remain as comparable as possible with that previous study on somatic incompatibilities
between A. echinatior and A. octospinosus fungal symbionts from the same site. However, for
Atta symbionts we only obtained a comparable result when using microsatellite markers, as AFLP
markers produced Mantel correlation coeffcients close to zero for all observation periods (Figure
S2).
Using the eight weeks data, somatic incompatibilities increased with increasing AFLP genetic
distances between fungi (Mantel r = 0.463, p = 0.003, Figure 1B) in Acromyrmex symbionts, but
not in Atta symbionts (Mantel r = -0.164, p = 0.792, Figure 1D). When using genetic distances
based on microsatellite markers, both Acromyrmex (Mantel r = 0.469, p = 0.003, Figure 1A) and
Atta (Mantel r = 0.312, p = 0.032, Figure 1C) symbionts had incompatibilities that increased with
genetic distance, but less of the incompatibility variance was explained in Atta than in Acromyr-
mex. The A. echinatior results were consistent with the results obtained by Poulsen & Boomsma
(2005), but the variance explained by the Mantel coeffcient was larger in that study (r = 0.855; p
< 0.0001). However, when we used our ten-weeks scorings for Acromyrmex symbiont pairings we
obtained a correlation closer to the one obtained after two months by Poulsen & Boomsma (2005)
70
(Microsatellites: Mantel r = 0.596, p < 0.001; AFLP: Mantel r = 0.654, p < 0.001).
Overall, the Atta fungi had smaller genetic distances when calculated from the microsatellite
marker data (0.09 0.01 SE), but larger genetic distances when using AFLP markers (0.26 0.03
SE) compared to Acromyrmex (0.13 0.01 SE and 0.17 0.02 SE, respectively), which is refect-
ed in the average number of AFLP bands observed (Atta: 37.3 1.8 SE; Acromyrmex 30.9 1.5
SE; t
15.49
= -2.724, p = 0.015). This implied that AFLP and microsatellite genetic distances (F
st
)
Microsatellite markers AFLP
A
c
r
o
m
y
r
m
e
x
A
t
t
a

0
.
0
1
.
0
2
.
0
3
.
0

0.0 0.1 0.2 0.3


0
.
0
1
.
0
2
.
0
3
.
0

0.0 0.2 0.4 0.6


A B
C D
Genetic distance
S
o
m
a
t
i
c

i
n
c
o
m
p
a
t
i
b
i
l
i
t
y
Figure 1.
Mutual somatic incompatibility reactions for Acromyrmex and Atta-associated fungal symbionts plotted against genetic
distances calculated from genetic variation at ten microsatellite markers and AFLPs, with the size of each circle rep-
resenting the number of times a particular combination was found. Correlations were signifcant for Acromyrmex with
both microsatellites (Mantel r = 0.469, p = 0.003) and AFLP markers (Mantel r = 0.463, p = 0.003), and for Atta with
microsatellites (Mantel r = 0.312, p = 0.032) but not AFLP markers (Mantel r = -0.164, p = 0.792). All Mantel tests were
performed with 99999 permutations.
71
were only comparable for Acromyrmex symbionts (r = 0.932; Figure S3), whereas correlations
decreased in comparisons with Atta symbionts and became very low in comparisons involving
only colonies of A. colombica (Figure S3). Independent of the type of genetic marker used, mean
genetic distances were higher in comparisons across the ant genera (Microsatellites: 0.20 0.01
SE; AFLP: 0.36 0.02 SE) than in comparisons within genera (means SEs given above; mi-
crosatellite markers: F
2,168
= 45.127, p < 0.0001; AFLP: F
2,168
= 22, p < 0.0001). Also the average
incompatibility scores were different for comparisons within ant species and comparisons across
ant species (genera) (
2
= 12.236; Df = 2; p < 0.01). This difference was mostly due to less strong-
ly expressed somatic incompatibilities between Atta symbionts, as the stronger mean reactions
among Acromyrmex symbionts alone and between Acromyrmex and Atta symbionts were not sig-
nifcantly different from each other (W = 1635, p = 0.338). After pooling data across this entire
range of genetic distances, the increase in somatic incompatibility with genetic distance was no
longer signifcant (Microsatellites: Mantel r = 0.153, p = 0.213, Figure 2A) or absent (AFLP:
Mantel r = -0.045, p = 0.595, Figure 2B).
Microsatellite markers AFLP
A
c
r
o
m
y
r
m
e
x

v
s

A
t
t
a

0.0 0.2 0.4 0.6

0.0 0.1 0.2 0.3


0
.
0
1
.
0
2
.
0
3
.
0
A B
Genetic distance
S
o
m
a
t
i
c

i
n
c
o
m
p
a
t
i
b
i
l
i
t
y
Figure 2.
Somatic incompatibilities in confrontations between fungal symbionts across the Acromyrmex and Atta ant-genus-level
barrier plotted against genetic distances calculated using microsatellite and AFLP markers, respectively. Each circle
represents the presence of a combination of incompatibility score and genetic distance with the size of the circles rep-
resenting the number of times a particular combination was found. Correlations were not signifcant for microsatellites
(Mantel r = 0.1518, p = 0.2064) or AFLP (Mantel r = -0.1034, p = 0.7063). All mantel tests were performed with 99999
permutations.
72
Specifc evaluation of the microsatellite genetic differences between the fungal symbionts of the
sympatric Atta and Acromyrmex colonies showed that they were completely separated (Figure
3A), consistent with earlier fndings of Mikheyev et al. (2007) for the same sampling site. Rooting
the symbiont tree with a sympatric fungal symbiont of Trachymyrmex zeteki confrmed that the
symbionts belonged to separate clades although bootstrap values were low (Figure 3B). Mirror im-
aging of trees obtained by microsatellite and AFLP markers showed almost complete congruence
for the Acromyrmex fungi, but more noisy correspondence for the Atta fungal symbionts (Figure
S4), confrming that these markers are less reliable when used to characterize Atta symbionts.
Discussion
The results of our study show that sympatric Panamanian colonies of A. echinatior and A. colom-
bica rear genetically different lineages of the leaf-cutting ant garden symbiont L. gongylopho-
rus and that microsatellite markers appear to predict genetic (in)compatibility better than AFLP
markers. However, even when using microsatellite markers, the correlation between somatic in-
compatibility and neutral marker genetic distance in Atta is noisier than in Acromyrmex. These
0.1
Ae394
Ae168
Ae263
Ae356
64
63
90
Ae322
Ae150A
Ae266
53 Ae160
Ae488
69
24
61
Ac-2006-27
Ac-2011-2
Ac-2012-8
Ac-2009-46
Ac-2012-1
45
43
23
Ac-2011-3
Ac-2009-42
Ac-2012-31
Ac-2012-2
57
88
16
40
Acromyrmex-
associated fungi
Atta-associated
fungi
Tzet028
Ae322
Ae150A
Ae266
54
Ae160
Ae488
71
26
49
Ae394
Ae168
Ae263
Ae356
63
61
84
Ac-2012-8
Ac-2009-46
Ac-2012-1
53
34
Ac-2006-27
Ac-2011-2
Ac-2011-3
Ac-2009-42
Ac-2012-31
Ac-2012-2
47
85
52
29
21
31
26
B A
Figure 3.
(A) Circular phylogenetic tree based on Fst values estimated from the ten microsatellite loci showing a clear separation
between fungal strains associated with Acromyrmex and Atta. (B) Rooted tree of the same Acromyrmex and Atta fungal
symbionts with the symbiont of a sympatric colony of Trachymyrmex zeteki as outgroup. Both trees were calculated
with Neighbor Joining and the numbers at nodes are percentage consensus support for 500 bootstrap replicates.
73
incompatibility reactions correlate only with genetic distances among fungal strains that have a
realistic probability of being horizontally transferred, and not between more distant clades that
are apparently unsuitable as symbionts for the sister genus of leaf-cutting ants. We discuss these
fndings in more detail below.
Somatic incompatibility only between fungal symbionts that can be exchanged?
The results of our study confrm the earlier fndings by Poulsen and Boomsma (2005) showing
that somatic (in)compatibility of Panamanian Acromyrmex symbionts is predictable from pairwise
genetic distances for AFLP markers (Figure 1A, B) and show that the same result can be obtained
with more specifc microsatellite markers. They also indicate that incompatibility reactions be-
tween separate clades of fungal symbionts, maintained by different genera of leaf-cutting ants,
can no longer be predicted by neutral genetic markers. This may be due to incompatibility being
ultimately caused by allelic variation at unknown loci (Worrall 1997) that only correlate with
neutral markers when there is recent common ancestry. This is only likely for local lineages of
fungal symbionts that are exploited by a single or a few populations of attine ants that share the
same pool of symbionts because each ant colony can in principle establish a viable symbiosis with
each fungal genotype.
It is only in this situation that we expect somatic incompatibility to be actively maintained, be-
cause resident fungus-garden symbionts are selected to defend their monopoly and farming ants
loose ftness when maintaining multiple lineages of the same symbiont that are equally viable but
compete rather than serve their hosts (Frank 1996; Bot et al. 2001). When symbionts belong to
different clades that no longer mix or exchange genes, each symbiont lineage is only a viable sym-
biont for one lineage of ant farmers or the other, but not for both. Our fnding that A. colombica
and A. echinatior maintain 100% separated clades of fungal symbionts (Figure 3) suggests that
L. gongylophorus has in fact been split into an Atta and Acromyrmex clade after its monophyletic
origin 2-3 MYA (Mikheyev et al. 2010), and that the two species of leaf-cutting ants that we in-
vestigated rear representatives of these symbiont lineages that are adapted to being, respectively,
an Atta and Acromyrmex symbiont.
These fndings are consistent with the results of a recent study that experimentally swapped fun-
gus-garden symbionts between sympatric Trachymyrmex septentrionalis and Atta texana from
Texas, USA (Seal et al. 2012). Although these ants are at the northern edge of the attine ant
distribution (Mueller et al. 2011a), and likely to have lower genetic variation among their symbi-
onts, they share even less common ancestry than the Panamanian fungal symbionts of our present
74
study (Mueller et al. 2011b). The swapped fungal symbionts thus could only serve as viable mu-
tualists for either Trachymyrmex or Atta, but not both, which was confrmed in the experiments
showing that: 1. Alternative fungus gardens were not always rejected by the ants, but they were
never adopted as a viable alternative symbiont, because the ants were able to grow their original
symbiont back from minuscule remnants that the authors had been unable to remove. 2. None of
the T. septentrionalis colonies ever produced virgin queens when they maintained an A. texana
fungal symbiont, consistent with the new combination being non-viable for transmitting ant of
fungal genes to future generations. A follow up study transplanting A. texana fungus to colonies
of both T. septentrionalis and T. turrifex confrmed that these Trachymyrmex cannot enter into
viable symbiosis with L. gongylophorus symbionts and that, even though some virgin queens
were produced on swapped gardens, they had poor fat reserves making it unlikely that they could
successfully found colonies (Seal and Mueller 2013).
To make further progress, it would be highly desirable to identify the genes that are directly re-
sponsible for somatic incompatibility, as this would allow direct studies on the signatures of selec-
tion and specialization across the clades of higher attine ant symbionts. In another, non-eusocial
model system of fungus-growing insects, the Sirex woodwasp, a range of genes are involved in
somatic incompatibility reactions, including fusion and recognition genes, and genes that mediate
cellular damage, stress response, and programmed cell death (Van der Nest et al. 2011). Whether
these genes have homologs in attine ant fungal symbionts remains to be seen, as the Sirex symbi-
ont Amylostereum areolatum belongs to a distantly related clade of basidiomycete fungi (Binder
and Hibbett 2002), and their respective domestication histories may have implied that recognition
systems were lost and gained over evolutionary time.
Why do Atta fungal symbionts express weaker incompatibility reactions?
Incompatibility reactions among Atta symbionts were signifcantly weaker and less predictable
from neutral marker F
st
values than similar reactions between Acromyrmex-associated fungi. One
possible explanation for this difference could be that there is a fundamental difference in colony
founding in the sense that Acromyrmex queens forage during colony founding similar to all more
basal attine ants, whereas Atta queens have secondarily evolved claustral colony founding. This
implies that newly-mated Atta queens close off their nest cavity to raise the frst worker cohort
purely on their body reserves, so that new colonies will only be opened by these workers 80 to
100 days after they were founded (Weber 1966; Fernandez-Marin and Wcislo 2005). As a con-
sequence, Acromyrmex queens may not only forage for leaf fragments to manure their incipient
fungus garden, but also for gardens of other incipient colonies whose queens are out foraging
75
(Poulsen et al. 2009), an option that is unavailable for founding Atta queens. Although swapping
of incipient fungus gardens with a fungus garden fragment from a mature Acromyrmex colony
seems relatively unconstrained during early colony founding, it is likely that stronger mutual com-
mitment between a founding queen and her resident fungus garden builds up in a matter of weeks,
including relatively strong incompatibility reactions in case a queen or one of her frst workers
bring in an unrelated fungus garden fragment from a neighboring nest (Poulsen et al. 2009). This
can never happen in the 80-100 days during which Atta colonies remain closed, removing selec-
tion for expressing incompatibility mechanisms during colony founding.
Why neutral markers should be less reliable predictors of somatic incompatibility in Atta colonies
after workers start foraging remains unclear. Imprinting of workers on the odor of a resident fun-
gus garden is a possibility (Seal et al. 2012), but it seems unclear why such mechanisms should
differ between Atta and Acromyrmex symbionts and why that should reduce selection for more
direct defenses by fungal symbionts against being replaced. Fungus gardens of Panamanian Acro-
myrmex colonies differ in chemical profles (Richard et al. 2007a; Richard et al. 2007b), but these
differences do not correlate with genetic distances and comparable data for sympatric Atta colo-
nies are lacking. The study by Richard et al. (2007a) also showed that feeding Acromyrmex work-
ers with the fungus of another colony increased their chance of being accepted by members of
that other colony. A compelling hypothesis may also be that mature Atta colonies in Panama have
hundreds of fungus gardens, whereas sympatric mature Acromyrmex colonies have one or a few
at best. This may make a difference in the likelihood of a resident fungus garden symbiont being
replaced by an accidentally imported small fragment of fungus garden from a neighboring colony,
so that less accurate recognition systems suffce in mature colonies of Atta but not in Acromyrmex.
Finally, there could also be technical explanations for the incompatibility differences between the
fungal symbionts of Atta and Acromyrmex. First, specifc growth conditions may be needed to
switch on incompatibility genes and the agar medium that we used may have approached these
conditions for Acromyrmex better than for Atta symbionts. Second, we found similar variation for
the Panama-symbiont-specifc microsatellite markers (Scott et al. 2009) and the general AFLP
markers for Acromyrmex symbionts, but enhanced variation in AFLP peaks for Atta symbionts,
relative to Acromyrmex symbionts (Figure S3). This suggests that DNA from other organisms may
have been amplifed with the AFLPs and that such other organisms were only present in Atta sym-
biont cultures. In principle, this could be viral (Pearson et al. 2009), bacterial (Suen et al. 2010) or
prion (Wickner et al. 2007) DNA. However, universal bacterial 16S primers did not amplify the
DNA samples (P.W. Kooij, unpublished data), making bacterial contaminations highly unlikely.
76
Acknowledgements
We thank the Smithsonian Tropical Research Institute (STRI), Panama, for providing logistic help and facilities to work
in Gamboa, the Autoridad Nacional del Ambiente y el Mar (ANAM) for permission to sample ant colonies in Panama,
a STENO grant from The Danish Council for Independent Research | Natural Sciences to MP, and the Danish National
Research Foundation (DNRF57) and the ERC (323085) for funding.
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80
PDA PDA+ PDYA PDYA+
S
o
m
a
t
i
c

i
n
c
o
m
p
a
t
i
b
i
l
i
t
y

s
c
o
r
e
0.0
0.5
1.0
1.5
2.0
2.5
3.0
A B C A B C A B C A B C
Figure S1.
Average somatic incompatibilities on different media for the two intraspecifc controls, Atta vs Atta (A) and
Acromyrmex vs Acromyrmex (C), and the cross-confrontations between Atta and Acromyrmex symbionts
(B). All media were Potato Dextrose Agar (PDA) with either 5 g/L Yeast Extract (PDYA) or 35 g/L Agar
(PDA+) or both (PDYA+). The average incompatibilities of the intraspecifc controls were signifcantly
lower than the scores obtained in the interspecifc test combinations Ac-Ae (F
2, 21
= 5.88, p < 0.01). The
PDYA medium was selected for the remaining experiments because it had consistent zero scores for the in-
traspecifc controls, even though there were no signifcant overall differences between the four media (F
3, 21
=
1.49, p = 0.25). Also the medium x type-of-confrontation interaction term was not signifcant (F
6, 21
= 1.19, p
= 0.35). Although we selected the PDYA for our further experiments, the results that we obtained should be
reproducible in other media, with the possible exception of PDA.
81
6 8 10 6 8 10

0
.
2
0
.
0
0
.
2
0
.
4
0
.
6
0
.
8
Number of weeks between inoculation and scoring
M
a
n
t
e
l

r

c
o
e
f
f
i
c
i
e
n
t
AFLP
Acromyrmex
Atta

0
.
2
0
.
0
0
.
2
0
.
4
0
.
6
0
.
8
Microsatellite markers
all
cross
all
cross
Acromyrmex
Atta
Figure S2.
Mantel correlation coeffcients (r) for the level of incompatibility against genetic distance for each of the three time
points (six, eight and 10 weeks) at which incompatibilities were scored. Correlations increased over time for the mi-
crosatellite markers, indicating stronger relationships between genetic distance and level of incompatibility, but this
was only true for Acromyrmex when AFLP markers were used, and cross-confrontations never produced signifcant
correlations. We decided to base our main analyses on the week 8 data, because in week 10 a total of 32 plates (4.7%)
had to be discarded due to infections (6) or because they had dried out (26), so that fungal interactions could no longer
be assessed.
82
Figure S3.
Comparisons of genetic distances calculated with AFLP and microsatellite markers. Pearsons correlation coeffcients
were 0.580 for all comparisons (A), 0.932 for comparisons between Acromyrmex symbionts (B), 0.400 for comparisons
between Atta symbionts (C), and 0.418 for the cross-comparisons between Acromyrmex and Atta symbionts (D).
Microsatellite markers
A
F
L
P
B A
C D

0
.
0
0
.
2
0
.
4
0
.
6
0
.
8

0.0 0.1 0.2 0.3 0.4


0
.
0
0
.
2
0
.
4
0
.
6
0
.
8

0.0 0.1 0.2 0.3 0.4


All Acromyrmex
Atta Acromyrmex vs Atta
83
Figure S4.
Comparison of unrooted phylogenetic trees based on F
st
values estimated from the ten microsatellite loci (left; the same
data as in Figure 3) and Neis standard genetic distances (D
s
) calculated from AFLP markers (right), indicating that
phylogenies are almost identical for Acromyrmex-associated fungal strains, but less consistent between the two markers
for Atta-associated strains.
45
43
23
64
63
90
53
Ac-2012-2
Ac-2012-31
Ac-2009-42
Ac-2011-3
Ac-2011-2
Ac-2012-8
Ac-2009-46
Ac-2012-1
Ac-2006-27
Ae394
Ae168
Ae263
Ae356
Ae322
Ae150A
Ae266
Ae160
Ae488
69
24
61
60
40
16
88
57
65
Ac-2012-31
Ac-2009-42
Ac-2012-2
25
23
60
44
19
15
46
19
74
Ac-2009-46
Ac-2012-1
Ac-2006-27
Ac-2012-8
Ac-2011-2
Ac-2011-3
Ae168
Ae263
Ae356
Ae322
Ae150A
Ae266
Ae160
Ae394
43
24
67
24
3
3
6
Microsatellite markers AFLP
Ae488
84
85
CHAPTER 3
DIFFERENCES IN FORAGE-ACQUISITION AND FUNGAL ENZYME ACTIVITY
CONTRIBUTE TO NICHE SEGREGATION IN PANAMANIAN LEAF-CUTTING ANTS
PEPIJN W. KOOIJ, JOANITO LIBERTI, KONSTANTINOS GIAMPOUDAKIS, MORTEN SCHITT,
JACOBUS J. BOOMSMA
(SUBMITTED)
86
Differences in forage-acquisition and fungal enzyme activity contribute to niche segregation
in Panamanian leaf-cutting ants
Pepijn W. Kooij*, Joanito Liberti, Konstantinos Giampoudakis, Morten Schitt, Jacobus J.
Boomsma
Centre for Social Evolution, Department of Biology, University of Copenhagen, Universi-
tetsparken 15, 2100 Copenhagen, Denmark
Phone: +45 35321239
Fax: +45 35321250
*Corresponding author: pkooij@bio.ku.dk
87
Abstract
The genera Atta and Acromyrmex are often grouped as leaf-cutting ants for pest management
assessments and ecological surveys, although their mature colony sizes and foraging niches may
differ substantially. Few studies have addressed such interspecifc differences at the same site,
which prompted us to conduct a comparative study across six sympatric leaf-cutting ant species in
Central Panama. We show that foraging rates during the transition between dry and wet season dif-
fer about 60 fold between genera, but are relatively constant across species within genera. These
differences appear to match overall differences in colony size, especially when Atta workers that
return to their nests without leaves are assumed to carry liquid food. We confrm that Panamanian
Atta specialize primarily on tree-leaves whereas Acromyrmex focus on collecting fowers and
herbal leaves and that species within genera are similar in these overall foraging strategies. Spe-
cies within genera tended to be spaced out over the three habitat categories that we distinguished
in Central Panama (forest, forest edge, open grassland), but each of these habitats normally had
only a single predominant Atta and Acromyrmex species. We measured activities of twelve fungus
garden decomposition enzymes, belonging to the amylases, cellulases, hemicellulases, pectinases
and proteinases, and show that average enzyme activity per unit of fungal mass in Atta gardens is
lower than in Acromyrmex gardens. Expression profles of fungal enzymes in Atta also appeared
to be more specialized than in Acromyrmex, possibly refecting variation in forage material. Our
results suggest that species- and genus-level identities of leaf-cutting ants and habitat-specifc
foraging profles may give predictable differences in the expression of fungal genes coding for
decomposition enzymes.
Keywords
AZCL-assay, behavior, mutualism, plant degradation, Acromyrmex, Atta, Leucoagaricus gongy-
lophorus
88
Introduction
Maximizing the acquisition of high quality food under varying ecological conditions is expected
to be under continuous natural selection. This notion has inspired many studies addressing opti-
mal foraging strategies [1] and the extent to which related species realize niche segregation [2]
and character displacement [3] to avoid interspecifc competition. These processes often lead to
(sub)habitat segregation [4-6] or food specialization, but few comparative studies have focused
on generalist insect herbivores as it often remains unclear whether specialization within gener-
alist strategies does in fact occur and what the decisive axes are along which niches and habitats
may segregate [7, 8]. This question is particularly relevant for social insects, as they are central
place foragers and often have a large impact on their surrounding communities. For wood eating
termites that live in their food, pest management agencies will automatically accumulate compara-
tive data on habitat and niche segregation among species and genera [9, 10], but such comparative
studies have remained rare in the leaf-cutting ants.
Atta [FABRICIUS, 1804] and Acromyrmex [MAYR, 1865] leaf-cutting ants originated between 8
and 12 million years ago as the most specialized crown-group of the fungus growing ants (Attini
[EMERY, 1913]) [11]. Their extant distribution ranges from warm-temperate South America up
to the southern regions of the United States [12-14]. Throughout this range these ants are import-
ant (often dominant) herbivores and signifcant accelerators of nitrogen and phosphorus cycling
[15]. They decompose the harvested live plant material through the mutualistic services provided
by their fungus-garden symbiont Leucoagaricus gongylophorus [SINGER, 1986], which feeds
the ants in exchange for the plant substrate provided [16]. Weber [14] estimated that ca. two kg
of fresh plant material is needed to build one fungus garden in an Atta cephalotes [LINNAEUS,
1758] colony and that almost 6000 kg of fresh vegetation had been processed by the collective
fungus gardens of a 6.5 year old colony of Atta sexdens [LINNAEUS, 1758].
Many species of leaf-cutting ants are considered pests in agricultural and urban areas [17]. For
economic damage assessments, the genera Atta and Acromyrmex are often considered indiscrim-
inately, in spite of large differences in colony size [14, 18], degree of worker polymorphism [18-
20], fungus garden enzyme activity [21], and foraging behavior [14, 19, 22, 23]. For example,
Cherrett [24] showed that forage material of an Atta cephalotes colony in Guyana consisted mostly
of leaves with fowers as a distinct minority class, similar to a later studied colony of Atta colombi-
ca [GURIN-MNEVILLE, 1844] in Panama [25], whereas forage of Costa Rican Acromyrmex
octospinosus [REICH, 1793], Acromyrmex coronatus [FABRICIUS, 1804] and Acromyrmex vol-
canus [WHEELER, 1937] is known to consist of leaves, fowers and some fruit fragments [26].
89
However, to our knowledge no studies have been done to quantify differences of this kind simulta-
neously at the same site for an entire local guild of leaf-cutting ants. This implies that habitat-spec-
ifcity, foraging effciency, and leaf-processing in fungus gardens have not been compared with
formal statistical analyses.
So far, 40 species of leaf-cutting ants have been described [11, 18], and they have all been hy-
pothesized to rear the same polymorphic species of L. gongylophorus as fungal symbiont [27],
despite the enormous distribution range mentioned above [12, 28] and the highly variable habitats
and forage availabilities [18, 22, 24, 28-32]. A recent study [33] has indicated that the extant L.
gongylophorus species is only 2-3 million years old, inferring that it must have swept through
all leaf-cutting ant species while replacing the original fungus garden symbiont(s) that they had
retained after coming into existence 8-12 million years ago. Other recent studies have shown that
the L. gongylophorus fungus garden symbiont is highly plastic in its enzymatic responses to the
various leaf-substrates that the ants deposit on their fungus gardens [21, 34], suggesting that for-
age type may systematically affect the expression of decomposition enzymes.
The objective of our study was to design a sampling scheme that allows the key characteristics
of forage acquisition and processing to be compared across an entire guild of leaf-cutting ants.
To achieve that goal, we quantifed the diversity of forage material and the absolute and relative
foraging rates for six sympatric leaf-cutting ants in the month of May, around the start of the
rainy season, in Gamboa, Panama: Atta cephalotes, Atta sexdens, Atta colombica, Acromyrmex
echinatior [Schultz, Bekkevold & Boomsma, 1998], Acromyrmex octospinosus, and Acromyrmex
volcanus. Based on feld surveys at this mosaic landscape of secondary growth forest and subur-
ban areas for a period of two decades the following generalizations of habitat differentiation [35]
appear to apply in Gamboa: Atta cephalotes and Acromyrmex volcanus are forest canopy foragers,
whereas Acromyrmex octospinosus forages on the forest-foor. Atta colombica occurs both in the
forest (usually at lower elevations) and in moist open grassland habitats, while Atta sexdens and
Acromyrmex echinatior prefer open and sunlit nesting habitats for foraging. The latter two species
extend their distributions towards the Pacifc coast where annual rainfall is less than in Gamboa
and natural habitat resembles savannas rather than a mosaic of forest patches [12, 28], matching
their preference for open habitat in Gamboa. We supplemented our comparative data on foraging
rates and substrate diversity with feld measurements on the activity of extracellular enzymes in
the fungus gardens maintained by the six leaf-cutting ant species to assess whether foraging pref-
erences might be related to specifc garden processing activities.
90
Material and methods
Ant foraging behavior
In May 2011 we located 9 11 foraging trails each for fve of the six ant species (Table 1), always
< 30m from the nest for Atta and < 5 m from the nest for Acromyrmex. The sixth species, Acro-
myrmex volcanus, was so rare that only one trail was found. We observed Acromyrmex trails for
15 to 30 min and Atta trails for 2 min (or 4 times 0.5 min when trails were very busy) to obtain
comparable data when counting ants that passed an imaginary line perpendicular to the trail. We
replicated observations by sampling either trails of different colonies or multiple trails of the
same colony going in different directions so they could be considered as independent samples of
foraging habitat (Table S1). Diversity of forage material was classifed in six categories: (parts
of) fowers, (pieces of) fruit, herbaceous leaves, tree-leaves, other material (always rare), and ants
carrying nothing on their way back to the colony. When in doubt, we verifed the origin of forage
particles by back-tracking the trail to the source. Observations were repeated across parts of the
day (morning 9 AM-12 PM, afternoon 12 PM-5 PM, evening in the dark 10 PM 11 PM) and
compared statistically to see whether this made any difference.
To assess rates of foraging, we expressed our records in numbers of workers on trails per hour,
counting both workers carrying material back to the nest and those without. Data were log-trans-
formed to approximately equalize variances and analyzed with R [36], using Linear Mixed-Ef-
fects Models (lme) [37] and generating p-values with General Linear Hypotheses (glht) in
the package multcomp [38]. Proportional distributions of forage types (tree-leaves, herbaceous
leaves, fowers, fruit, other) were analyzed with the same tests, with separate trails being consid-
ered as a random factor in both analyses. To test for heterogeneity across trails within species we
created a sub dataset consisting of four Atta trails from two colonies (one Atta colombica and one
Atta cephalotes) and compared the foraging category scores between trails and species (with trails
nested within species, and 0.5 min replicate observations for each trail). This was only possible
for these Atta species as we did not have replicate samples within single trails for Acromyrmex
(Table S1).
Results for the proportional distribution of forage types were visualized using heatmap.2 in the
R package gplots [39]. Dendrograms were calculated with the pvclust package [40] using
1000000 bootstrap iterations. Final clustering plots were based on the overall similarities in mean
proportions (p) between species and supplemented by estimates of the inverse Simpson Diversity
index (D = 1/[p
i
2
]) to allow an explicit analysis of the degree of evenness (high D) between the
different forage or expressed enzyme categories across species and genera of leaf-cutting ants.
91
The denominator of the index decreases when more categories (p) enter the equation, but when the
number of categories is constant (as in our analyses) more even distributions will give lower sums
in the denominator and thus higher values of D. For the purpose of our study, D therefore func-
tions as an index of generalist foraging or equal enzyme expression, so that high values indicate
that all categories are important and low values indicate specialization either on a subset of forage
categories or on a subset of expressed enzymes that were most active.
AZCL enzyme activity assays
For each of the six ant species the garden enzyme profles were analyzed for fve different colo-
nies, with the exception of Acromyrmex volcanus for which only one colony was available, but
where we could add data for another colony obtained in the previous year by H. H. De Fine Licht
(pers. com.). For each colony, fungus gardens were dug up, and about equal size fragments (ca 80
mg) from top, middle and bottom layers of fungus gardens were collected and immediately ho-
mogenized together to obtain representative average enzyme activity measures per colony. These
measurements were subsequently performed using previously published methods, which are easi-
ly applicable in the feld and give repeatable results [21, 34, 41]. In short, fungus garden material
(240 mg) was crushed with a pestle in a 1.5 ml eppendorf-tube containing 1000 l 0.05 M TRIS-
HCl buffer (pH 7.0), vortexed immediately and then centrifuged for 15 minutes (15000g) after
which the supernatant was removed and applied immediately to each of 12 different assay-plates
containing 0.1g/L of the Azurine-Crosslinked (AZCL) substrates: amylose, arabinoxylan, barley
-glucan, casein, collagen, debranched arabinan, galactan, galactomannan, HE-cellulose, rham-
nogalacturonan, xylan and xyloglucan that were chosen because they yielded positive enzyme
activities in an earlier study [21].
The assay-plates of 6 cm diameter were prepared separately for each substrate using an agarose
medium (1% agarose, 23 mM phosphoric acid, 23 mM acetic acid, 23 mM boric acid), and pH
adjusted according to the manufacturers description (Megazyme, Bray, Ireland). After the medi-
um had solidifed, round wells (area of ca. 0.1 cm) were made in each plate with a cut-off pipette
tip and 12 l of the supernatant was applied to each well in triplicate. After 22 hours of incuba-
tion at 25C the plates were photographed and the area of the blue halo surrounding each well (a
quantitative measure for the absolute amount of enzyme activity [21, 34]) was measured using the
software program ImageJ ver. 1.43u for Macintosh. Enzyme activity measurements were grouped
into categories based on which plant cell wall component is the main target of the enzymes ([42]
and Megazyme, Bray, Ireland): amylases (measured with amylose), cellulases (measured with
barley -glucan and HE-cellulose), hemicellulases (measured with arabinoxylan, galactomannan,
92
xylan and xyloglucan), pectinases (measured with debranched arabinan, galactan and rhamnoga-
lacturonan), and proteases (measured with casein and collagen). We present these data grouped
for fve categories of enzymes, implying that each of these categories had data from 2-3 enzymes,
except for amylases that were represented by only a single enzyme amylose.
The enzyme activity scores were analyzed using a General Linear Model in SAS, with colony
nested within species and species nested within genus. Colony was then treated as having
a random enzyme class activity, composed of specifc enzyme activities nested within enzyme
activity class. This procedure implied that, we had to omit amylase, because we only had a single
substrate (amylose = starch) for testing activity and because starch is not a primary challenge in
the degradation of plant material [34]. We also left out rare Acromyrmex volcanus and thus report
original mean values for enzyme activity in Figure 2 for all species and enzyme classes, whereas
statistics given in the text refer to the reduced data set of four enzyme classes and the gardens of
fve ant species. In our fnal analyses (Figure 3) we combined the enzyme and foraging datasets
and visualized patterns of association with the plot.PCA function after Principal Component Anal-
ysis (PCA) using the FactoMineR package [43].
Results
The three Atta species had an average foraging rate of 5014 555 SE ants/h, with 3374 476
SE ants (67%) returning to the nest carrying forage material and 1640 203 returning unloaded,
whereas the three Acromyrmex species had an average foraging rate of 80 14 SE ants/h (t
52.444
=
-19.347, p < 0.0001) and no workers returning without forage (Table 1). Separate analyses, using
the probability of a foraging trail having loaded ants, showed that the genus-level differences in
loaded and unloaded returning foragers per hour were highly signifcant (
2
1
= 7.567, p < 0.01),
but that the differences in loaded returning workers between species within genera were not sig-
nifcant (
2
4
= 1.257, p = 0.87).
Across genera, Atta foragers harvested signifcantly more tree-leaves than Acromyrmex workers (z
= 6.420, p < 0.0001), while Acromyrmex foragers collected signifcantly more (pieces of) fowers
than Atta workers (z = -4.894, p < 0.0001). However, there were also differences in the most abun-
dant forage category within genera. All Acromyrmex species preferred some combination of fow-
ers and herbaceous leaves, but Acromyrmex volcanus was more fower-biased and Acromyrmex
echinatior more herbaceous-leaves-biased (Figure 1A). Similarly, while Atta cephalotes primarily
harvested tree-leaves (sexdens vs cephalotes, z = -4.987, p < 0.001; cephalotes vs colombica, z =
6.782, p < 0.0001), Atta colombica brought in more herbaceous leaves (cephalotes vs colombica,
93
z = -7.109, p < 0.0001), and Atta sexdens had approximately equal shares of all forage categories
(Figure 1A), which confrmed earlier fndings by De Vasconcelos [44].
We validated the statistical independence of our trail samples, using a subset of two Atta colonies
(one Atta colombica and one Atta cephalotes), for which we had four replicated samples of the
same trails (0.5 min each) and two separate trails per colony (Table S1). This recovered our ear-
lier result that the two Atta species have different fractions of forage categories (F
4,60
= 89.48, p <
0.0001), but also showed that different trails of the same colony yielded similar results in spite of
covering non-overlapping fractions of the colonys foraging habitat (F
8,60
= 0.75, p = 0.65). Fur-
ther ANOVA showed that frequencies of forage types between the different times of the day were
signifcantly different for Atta species (F
8,218
= 4.451, p = 0.0001), and a post-hoc test indicated
this was due to a higher share of herbaceous leaves in the afternoon compared to the evening (z
= -3.929, p = 0.009). Acromyrmex species did not show any activity in the dark (evening) and
frequencies of forage types between morning and afternoon observations were not different (F
4,76
= 1.457, p = 0.224).
Ranking the six species according to the diversity of forage material (Figure 1B) gave no signif-
cant difference between species within genera in evenness of forage category use (F
5,45
= 1.776, p
= 0.137), but the pooled Atta species had a lower evenness in forage category use (D = 1.420.08
SE) than the pooled Acromyrmex species (D = 1.86 0.13 SE; F
1,49
= 5.435, p = 0.024). After
excluding Acromyrmex volcanus where sample size was very small and two forage categories
completely missing, the evenness trends in Figure 1B corresponded fairly well with the relative
proportional forage acquisition data in Figure 1A with, from left to right, a clearly increasing
Genus Species Trails Observation Total ants Loaded ants per hour Unloaded ants per hour
Acromyrmex
echinatior 10 (10) 285 min 265
80 14
58 10
0 0
0 0
octospinosus 10 (10) 285 min 458 102 26 0 0
volcanus 1 (1) 30 min 43 86 NA 0 NA
Atta
cephalotes 11 (6) 26 min 1519
3374
476
2740 456
1640 203
2408 373
colombica 10 (7) 24.5 min 1724 5383 1003 1115 188
sexdens 9 (7) 22 min 801 1173 447 1012 301
Table 1. Differences in foraging rate between loaded and unloaded foragers.
Differences in foraging rate between loaded and unloaded foragers of Atta and Acromyrmex species in Gamboa, Pan-
ama, with summary statistics on the number of trails observed per species (number of colonies in brackets), the total
number of minutes of observation per species, the total number of ants counted while returning to their nests, and the
foraging rates for loaded and unloaded returning workers: means ( SE) per genus and per species (see Table S1 for
details).
94
trend in tree-leaf use, a decreasing trend in the use of fowers, and hump-shaped trends in the use
of herbaceous leaves and fruit. These inferences were supported by moderately high overall Ap-
proximately Unbiased (AU) p-values and Bootstrap Probability (BP) values (Figure 1). As night
foraging tended to decrease the acquisition of herbaceous leaves by Atta species, we probably
underestimated the difference in dependence on tree leaves between Acromyrmex and Atta by
obtaining most of our Atta observations at daytime.
In absolute quantities, fungus gardens of Acromyrmex showed a higher overall enzyme activity
than gardens of Atta (F
1,20
= 8.54, p < 0.01)(Figure 2A), but species within genera did not show
signifcant additional differences (F
3,20
= 1.32, p = 0.29). To further investigate the differences in
0 0.3 0.9 0.6
Value
0.512
0.491
0.104
0.140
0.062
0.031
0.352
0.105
0.115
0.083
0.027
0.008
0.036
0.016
0.149
0.093
0.188
0.121
0.054
0.032
0.002
0.001
octo volc echi sex cep col
0.349
0.007
0.007
0.000
0.000
0.252
0.099
0.323
0.116
0.790
0.054
tree
other
0.000
0.298
0.081
0.417
0.100
0.565
0.098
0.251
0.093
0.000
0.000
herbaceous
0.000
0.142
0.082
0.115
0.073
0.121
0.057
0.089
0.058
0.153
0.051
fruit
flowers
A
Forest
Forest edge
Open area
B
A
c
r
o
m
y
r
m
e
x

v
o
l
c
a
n
u
s
A
t
t
a

c
e
p
h
a
l
o
t
e
s
A
t
t
a

c
o
l
o
m
b
i
c
a
A
t
t
a

s
e
x
d
e
n
s
A
c
r
o
m
y
r
m
e
x

e
c
h
i
n
a
t
i
o
r
A
c
r
o
m
y
r
m
e
x

o
c
t
o
s
p
i
n
o
s
u
s
1.37 0.14 1.77 0.19 1.63 0.22 1.88 0.18 2.48 1.36 0.08 D =
D = 1.86 0.13 D = 1.42 0.08
67 71
1
99 83
2
98 71
3
89 75
4
au bp
edge #
100
92
au
21
30
bp
2
3
edge #
100
76
83
41
1
4
Figure 1. Differences in forage diversity.
Differences in forage diversity between leaf-cutting ant species (nested within genera), using solid lines for Atta and
dotted lines for Acromyrmex, and with typical foraging habitat indicated with dark green (forest), yellow (forest edge),
and orange (open sunlit areas): (A) Heatmap showing differences between species and genera in the use of forage cat-
egories, with numbers representing the mean proportions SE of the forage types. Darker colors indicate higher mean
acquisition proportions, with the top-dendrogram illustrating similarities between species/genera across means of the
fve forage categories (vertical axis). Ant species names are given as abbreviations (volc, octo, echi, col, sex, cep).
(B) Dendrogram based on the Inverse Simpson Diversity Index of the fve forage categories, indicating the degree of
evenness across foraging categories (numbers below the branches are mean D-values SE per species and means per
genus), showing that Acromyrmex has a broader (more even) spectrum (D = 1.86 0.08 SE) of forage material than
Atta (D = 1.42 0.13 SE; F
1,49
= 5.435, p < 0.05). Numbers above the branch nodes represent Approximately Unbi-
ased p-values (AU, red) and Bootstrap Probability values (BP, green).
95
enzyme activity between the two genera, we decomposed the signifcant interaction term of ant
genus and AZCL category (F
10,780
= 4.95, p < 0.0001). This revealed signifcant differences be-
tween Acromyrmex and Atta for all substrates except rhamnogalacturonan. The spectrum of rel-
ative enzyme activities, as expressed by the inverse Simpson indices (Figure 2B), showed that
Acromyrmex species tend to have more evenly distributed enzyme activities (D = 4.55 0.05 SE)
than Atta species who tend to specialize more on the expression of specifc classes of enzymes
(D = 4.18 0.07 SE; F
1,52
= 15.006, p < 0.0001). No signifcant differences were observed for the
evenness of the enzyme activity spectra for species within genera (Atta: F
2,12
= 1.618, p = 0.239;
Acromyrmex: F
2,9
= 0.111, p = 0.896).
B A
D = 4.55 0.05 D = 4.18 0.07
A
t
t
a

c
o
l
o
m
b
i
c
a
A
t
t
a

s
e
x
d
e
n
s
A
c
r
o
m
y
r
m
e
x

e
c
h
i
n
a
t
i
o
r
A
c
r
o
m
y
r
m
e
x

v
o
l
c
a
n
u
s
A
t
t
a

c
e
p
h
a
l
o
t
e
s
D = 4.15 0.10 3.98 0.14 4.54 0.09 4.64 0.25 4.42 0.07 4.54 0.05
A
c
r
o
m
y
r
m
e
x

o
c
t
o
s
p
i
n
o
s
u
s
proteinases
pectinases
hemicellulases
cellulases
amylases
1.716
0.031
1.372
0.163
1.322
0.140
1.670
0.134
1.261
0.159
1.938
0.113
0.980
0.108
1.652
0.086
1.205
0.087
1.426
0.103
1.581
0.109
1.038
0.094
1.074
0.097
0.942
0.073
1.014
0.074
1.823
0.109
0.915
0.135
1.081
0.071
0.736
0.076
0.968
0.097
1.382
0.081
0.549
0.116
0.826
0.054
0.611
0.064
0.650
0.097
1.454
0.150
0.551
0.075
0.992
0.047
0.741
0.065
0.907
0.086
octo volc echi sex cep col
0.5 1 1.5 2
Value
100
100
66
66
au
44
44
59
59
bp
1
2
3
4
edge #
99 99
28
66
au
51 22
21
41
bp
1 2
3
4
edge #
Figure 2. Differences in fungus garden enzyme activity.
Differences in fungus garden enzyme activity between species grouped as in Figure 1 with solid lines for Atta and dot-
ted lines for Acromyrmex, and with dark green, yellow and orange indicating the same habitat categories: (A) Heatmap
showing differences between species and genera in fungus garden activity of enzyme classes, expressed as the mean
area in cm
2
SE of colored halos on AZCL plates across all assays for enzymes belonging to the amylases (1), cellulases
(2), hemicellulases (4), pectinases (3) and proteinases (2). Darker colors in the heatmap indicate higher means, and the
top-dendrogram illustrates similarities between species across all means for the fve groups of enzymes, estimated by
pvclust with 1000000 bootstraps. (B) Dendrogram based on the inverse Simpson Diversity Index of proportional
enzyme activity showing that Acromyrmex fungus gardens have more even secretions across enzyme categories (D =
4.55 0.05 SE) than Atta (D = 4.180.07 SE, F
1,52
= 15.006, p < 0.0001). Numbers above the branch nodes represent
Approximately Unbiased p-values (AU, red) and Bootstrap Probability values (BP, green).
96
Comparative analyses (PCA), with either fungus garden enzyme expression as a predictor variable
and forage diversity as a response variable (Figure 3A and B) or vice versa (Figure 3C and D),
confrmed a separation between the genera Atta and Acromyrmex (Figure 3B and D). Taking the
fungus garden enzyme activities as predictor variables produced a frst axis explaining 69.04% of
the variation and a second axis explaining 12.02% of the variation. The frst axis corresponded to
overall enzyme activity and illustrates that general fungus garden enzyme activity is lower towards
the left (predominantly Atta) and higher towards the right (predominantly Acromyrmex) (Figure
3A and B), confrming the results given in Figure 2. The vertical axis refects higher amounts of
pectinases (positive scores) versus higher amounts of proteases (negative scores). However, this
does not correspond in any obvious way with genus-level differences, but may correlate with
colony-level differences in the proportions of fowers and fruit in the forage (Figure 3A and B).
A similar pattern was obtained when the predictor and response variables were reversed, (Figure
3C and D). The frst axis (explaining 33.11% of the variation) illustrates a preference for tree-
leaves (mostly Atta) towards the left and a preference for herbaceous leaves (mostly Acromyrmex)
towards the right (Figure 3C and D). The second axis (explaining 25.54% of the variation) indi-
cates higher intake of fruit (negative scores) in weak association with cellulases, and of fowers
(positive scores) mostly in association with pectinases. Here the leaf-cutting genera are also sepa-
rated to some extent (centroid squares) with Acromyrmex having some preference for fowers and
Atta for fruit, confrming the results depicted in Figures 1 and 2.
The PCA comparisons also revealed correlations between forage preference and fungus garden
enzyme expression, as both PCA analyses showed a negative correlation between extent of acqui-
sition of tree-leaves and overall intensity of enzyme activity. Kendalls rank test for correlation
showed that this negative trend was signifcant for most enzymes: amylases (z = -3.185, p < 0.01);
pectinases (z = -2.608, p < 0.01); proteases (z = -3.465, p < 0.001), but not cellulases (z = -1.429,
p = 0.153) and hemicellulases (z = -1.953, p = 0.051). The same analyses also found a negative
correlation between fruit foraging and expression of amylases (z = -2.311, p < 0.05) and positive
correlations between foraging on herbaceous leaves and expression of amylases (z = 3.643, p <
0.001), pectinases (z = 2.130, p < 0.05) and proteases (z = 3.404, p < 0.001) and between fower
foraging and expression of the same enzymes: amylases (z = 2.712, p < 0.01), pectinases (z =
3.066, p < 0.01) and proteases (z = 3.769, p < 0.001). Foraging on other materials was only (pos-
itively) correlated with the expression of pectinases (z = 2.366, p < 0.05).
97
A B
C D
-1.0 -0.5 0.0 0.5 1.0
-
1
.
0
-
0
.
5
0
.
0
0
.
5
1
.
0
Variables factor map
Dim 1 (69.04%)
D
i
m

2

(
1
2
.
0
2
%
)
Amylases
Cellulases
Hemicellulases
Pectinases
Proteinases
Flowers
Fruit
Herbacous
Other
Tree
-2 0 2 4
-
4
-
2
0
2
4
Individuals factor map
Dim 1 (69.04%)
D
i
m

2

(
1
2
.
0
2
%
)
Acromyrmex
Atta
Acromyrmex
Atta
-1.0 -0.5 0.0 0.5 1.0
-
1
.
0
-
0
.
5
0
.
0
0
.
5
1
.
0
Variables factor map
Dim 1 (33.11%)
D
i
m

2

(
2
5
.
5
4
%
)
Flowers
Fruit
Herbacous
Other
Tree
Amylases
Cellulases
Hemicellulases
Pectinases
Proteinases
-4 -2 0 2
-
3
-
2
-
1
0
1
2
3
Individuals factor map
Dim 1 (33.11%)
D
i
m

2

(
2
5
.
5
4
%
)
Acromyrmex
Atta
Acromyrmex
Atta
E
n
z
y
m
e

a
c
t
i
v
i
t
y
F
o
r
a
g
e
Figure 3. Correlations between forage diversity and enzyme activity.
Principal Component Analyses (PCA) using either the fve enzyme groups of Figure 2 (A and B) or the fve forage
material categories of Figure 1 (C and D) as predictor variables (black arrows). Red arrows represent response vectors
for forage material (A) or enzymes (C). The B and D panels complement the respective A and C panels by plotting
PCAs scores across the fungus garden measurements (B, 45 for Atta and 32 for Acromyrmex) the sampled ant trails
(D, 30 for Atta and 21 for Acromyrmex; Table 1), largely separating the ant genera along the x-axes, confrming that
Atta primarily focuses on tree-leaf material (compare panels C and D) and Acromyrmex on herbaceous leaves, fowers
and (less pronounced) fruit. Comparison of the A and B panels illustrates that enzyme activity was generally higher in
Acromyrmex (towards the right).
98
Discussion
Genus-level niche segregation between Atta and Acromyrmex
Although it is widely appreciated that Atta and Acromyrmex differ by more than two orders of
magnitude in their scale of operations (see e.g. [14, 18, 26, 45]), systematic comparative studies
similar to the present analyses have to our knowledge not been done. Although our snapshot re-
sults for the month of May cannot be generalized, we believe to have achieved our objective of
demonstrating that larger scale studies like this can be done in principle. Our analyses illustrate
that the statistical tools to analyze such data are available and can easily be expanded for use in
more encompassing feld surveys, as extra seasons and/or sampling sites can be added as new
factors within which genera, species, and colonies can be nested. In the sections below, we offer
tentative interpretations and compare them with available literature.
Our results confrm that the two genera of leaf-cutting ants operate at different scales, show that
their foraging niches are different and that the enzymatic processing activities of fungus gardens
appear to refect these differences. The differences in foraging preferences quantifed our intuitive
expectations based on two decades of feldwork in Gamboa, but the enzymatic activity differences
were more substantial than we expected, because the two genera rear fungus-garden symbionts
that belong to a single species L. gongylophorus [33]. This suggests that studies of phenotypic
plasticity in enzyme gene expression will be worthwhile to enhance our understanding of the ver-
satility of the leaf-cutting ant symbiosis. We will return to this in more detail below.
The fnding that average genus-specifc foraging rates show a 42 fold difference in loaded-worker
return rates and a 63 fold difference in total worker return rates per hour, seems to match the ca.
two order of magnitude difference in colony size between Atta and Acromyrmex. The fact that the
differences do not quite reach 100 fold [14, 18, 26, 46] may be due to our primary focus on the
largest Acromyrmex colonies (smaller colonies have too little foraging activity making the type of
sampling that we did less feasible), whereas our selection of Atta colonies mostly contained me-
dium size colonies. It is also conceivable that the Atta workers that returned to their nests without
carrying plant material may have had their crops flled with plant sap as suggested by Littledyke
& Cherrett [47], Quinlan & Cherret [48] and Hlldobler & Wilson [19], but the present setup did
not allow any measurements on this. This suggests that considering only loaded workers may
underestimate foraging effort, and that larger scale comparative studies should include sampling
of liquid food in the crops of returning foragers. In spite of these limitations, we will also return
to tentative inferences on species- and genus-level niche segregation that our snapshot data for
Gamboa may allow.
99
Garden enzyme activity and forage material is there a connection?
It has long been known that the fungus is a major producer of decomposition enzymes for the
plant material that leaf-cutting ant foragers provide, and recent work has shown that these decom-
position services are supplemented by several other microorganisms that live in attine gardens
[49, 50]. Other recent studies have emphasized that the expression of enzymes can be remarkably
plastic and substrate dependent [21, 34]. This is consistent with earlier notions that there are active
feedback loops between forager supply and symbiont demand, such that foragers may discard
some forage material under specifc conditions where its excess processing would not be optimal
[51].
Our present results suggest that Atta and Acromyrmex represent ecologically distinct ant genera,
both with regard to forage acquisition/diversity and garden enzyme activity/diversity. The latter
confrms recent results showing differences in proteomes between Acromyrmex echinatior and
Atta cephalotes fungus gardens [52]. It is remarkable that our results indicate that Atta gardens
generally produce lower amounts of enzymes, even though these ants forage mostly on tree-leaves
(Figure 3), which one would expect to be more demanding to decompose. It also appears that Atta
gardens tend to overproduce two classes of enzymes, cellulases and pectinases, in addition to
amylases (Figure 2), whereas Acromyrmex tended gardens produce higher amounts of all enzyme
categories. This suggests that Atta gardens may somehow extract necessary nutrients more eff-
ciently, but further work will be needed to understand the details of these processes. An additional
factor to consider in this context is that Atta colonies produce conspicuous waste heaps or under-
ground compost chambers, whereas this is rare for Acromyrmex (J.J. Boomsma & P.W. Kooij pers.
obs.; CSE logbooks). This is consistent with Panamanian Atta discarding a much larger fraction
of not fully degraded older fungus garden biomass than Acromyrmex [53, 54], perhaps because
average enzyme activity per unit of fungus garden mass is lower and fresh tree leaves are more
abundantly available than fower parts.
An earlier study [21] has hypothesized that Atta species focus on the rapid degradation of starch
and proteins, but discard fungus garden material before most of the cellulose and hemicellulose is
degraded. This is consistent with other recent studies showing that high amounts of cellulose and
hemicellulose are still present in the bottom layer of the fungus gardens [50, 55, 56] and that only
cellulases from L. gongylophorus remain highly active in this bottom layer [52, 57]. The larger
scale and more wasteful substrate processing practiced by Atta may thus leave more substantial
niches for additional bacterial and/or yeast [49] decomposition, similar to the domestication of
specialized gut bacteria in large ungulates [58] that rely on residues of leaf-material that were
100
hard to digest even for ruminants. Focused, comparative transcriptomics to investigate conditional
gene expression in fungus gardens of the two leaf-cutting ant genera could shed further light on
possible differences of this kind and metagenome sequencing could identify the microbial com-
munities involved, similar an earlier yeast study on the fungus gardens of Acromyrmex and Atta
[49].
Niche partitioning in Panamanian Atta and Acromyrmex
The data provided in our study are a snapshot of year-round foraging, which is known to vary
across the seasons [25]. This implies that we cannot be sure that sampling in other seasons or at
other sites would have yielded similar results. However, we also note that the fve forage catego-
ries that we distinguished are very general and likely to be available throughout the year and that
medium-size colonies of Atta and large colonies of Acromyrmex are unlikely to move over sub-
stantial distances (but see [59]), so their central place for foraging would tend to cover the same
(sub) habitat over time. In spite of these caveats, our study shows that genus- and species-level
differences across leaf-cutting ants can be quantifed with the statistical tools we developed during
this study.
The results of our study suggest that direct competition for forage material between the two genera
of leaf-cutting ants is likely to remain limited, because Atta and Acromyrmex species target rather
different types of forage, in spite of some overlap consistent with earlier reports that mostly re-
port allopatrically collected data[18, 22, 24, 28-30, 32]. The correlations between garden enzyme
activity and genus-level difference in forage use that we uncovered for the Gamboa community
of leaf-cutting ants, may be reinforced or supplemented differences in salivary gland secretions
between the two ant genera [60], a variable we were unable to measure. However, comparisons
at species level suggested that both Atta and Acromyrmex species tend to have optimal habitats
that are largely mutually exclusive, with Acromyrmex volcanus and Atta cephalotes foraging in
the canopy, Acromyrmex octospinosus and (somewhat less specifcally) Atta colombica foraging
on the forest foor, and Acromyrmex echinatior and Atta sexdens foraging in the open landscape.
Although it is possible that these differences are less pronounced in other seasons or sites, these
results seem consistent with ecological theory predicting that interspecifc competition is more
pronounced when species are more similar, so that habitat partitioning may evolve [4-6].
The only case in which habitat segregation was somewhat less pronounced was between Atta
sexdens and Atta colombica, which often overlap in park-like and man-made habitats. Although
there is a clear gradient across the isthmus of Panama, Atta sexdens is the dominant Atta species
101
along the Pacifc coast and becomes less abundant towards Gamboa in central Panama, whereas
the pattern is opposite for Atta colombica [28]. It is interesting that these are the only two spe-
cies for which we once observed neighboring trails and active avoidance behavior, i.e. trails of a
colony stopping ca. one meter from the trail of another colony (P.W. Kooij, pers. obs.), behavior
expected for all Atta spp. For the two common Panamanian Acromyrmex species, of which our
research group has dug up ca. 500 colonies over the last two decades, habitat segregation (forest
for A. octospinosus and open grassland areas for A. echinatior) is so pronounced that they will
rarely encounter each other, similar to what is seen in Costa Rica [61]. In this way mature colonies
of these species are unlikely to compete for the same type of plant forage. As far as we are aware
distributions of incipient (founding) colonies are similar in Gamboa, but this is harder to quantify.
Acknowledgments
We thank the Smithsonian Tropical Research Institute (STRI), Panama, for providing logistic help and facilities to work
in Gamboa, the Autoridad Nacional del Ambiente y el Mar (ANAM) for permission to sample ant colonies in Panama,
Anders Illum, Mattias Lange Nielsen, Smi Schr and Nicholas Westwood for help with collecting the foraging data,
Henrik De Fine Licht for supplying data on enzyme activity in an Acromyrmex volcanus colony and for helping to
interpret the results, Gsta Nachmann for suggesting the statistical analysis of the enzyme experiment, and the Danish
National Research Foundation for funding (DNRF57). The collection of the observational data reported in this paper
was facilitated by the Gamboa Graduate Course in Tropical Behavioral Ecology and Evolution, hosted by the Copenha-
gen Centre for Social Evolution and the Smithsonian Tropical Research Institute.
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105
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Table S1.
Sample sizes for each colony.
106
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109
CHAPTER 4
LEUCOAGARICUS GONGYLOPHORUS USES LEAF-CUTTING ANTS TO VECTOR
PROTEOLYTIC ENZYMES TOWARDS NEW PLANT SUBSTRATE
PEPIJN W KOOIJ, ADELINA ROGOWSKA-WRZESINSKA, DANIEL HOFFMANN, PETER ROEPSTORFF,
JACOBUS J BOOMSMA, MORTEN SCHITT
(IN PRESS AT THE ISME JOURNAL)
110
Leucoagaricus gongylophorus uses leaf-cutting ants to vector proteolytic enzymes towards
new plant substrate
Running title: Protease activity in ant fungus gardens
Pepijn W Kooij*
a
, Adelina Rogowska-Wrzesinska
b
, Daniel Hoffmann
a
, Peter Roepstorff
b
, Jacobus
J Boomsma
a
, Morten Schitt*
a
a
Centre for Social Evolution, Department of Biology, University of Copenhagen, Universi-
tetsparken 15, DK-2100 Copenhagen, Denmark
b
Protein Research Group, Department of Biochemistry and Molecular Biology, University of
Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark.
*Corresponding authors: pkooij@bio.ku.dk and mschiott@bio.ku.dk at the above address
Accepted for publication at The ISME Journal, December 2013
111
Abstract
The mutualism between leaf-cutting ants and their fungal symbionts revolves around processing
and inoculation of fresh leaf pulp in underground fungus gardens, mediated by ant fecal fuid
deposited on the newly added plant substrate. As herbivorous feeding often implies that growth
is nitrogen-limited, we cloned and sequenced six fungal proteases found in the fecal fuid of the
leaf-cutting ant Acromyrmex echinatior and identifed them as two metalloendoproteases, two ser-
ine proteases and two aspartic proteases. The metalloendoproteases and serine proteases showed
signifcant activity in fecal fuid at pH values of 5-7, but the aspartic proteases were inactive
across a pH range of 3-10. Protease activity disappeared when the ants were kept on a sugar
water diet without fungus. Relative to normal mycelium, both metalloendoproteases, both serine
proteases and one aspartic protease were upregulated in the gongylidia, specialized hyphal tips
whose only known function is to provide food to the ants. These combined results indicate that the
enzymes are derived from the ingested fungal tissues. We infer that the fve proteases are likely
to accelerate protein extraction from plant cells in the leaf pulp that the ants add to the fungus
garden, but regulatory functions such as activation of proenzymes are also possible, particularly
for the aspartic proteases that were present but without showing activity. The proteases had high
sequence similarities to proteolytic enzymes of phytopathogenic fungi, consistent with previous
indications of convergent evolution of decomposition enzymes in attine ant fungal symbionts and
phytopathogenic fungi.
Keywords:
Acromyrmex echinatior/nutrition/mutualism/phytopathogens/proteases
112
Introduction
Obligate symbioses are characterized by high degrees of partner commitment and functional
complementarity (Janzen 1985). Such interactions normally show only minor remnants of the
potential reproductive conficts that needed to be overcome when these partnerships evolved,
allowing them to ultimately become single adaptive units with organismal properties (Queller &
Strassmann 2009; Boomsma 2013). Such high levels of interdependency are most characteristic
for endosymbioses where the symbiont lives in cells or tissues of the host, but may also occur in
ectosymbioses like the eusocial fungus-farming mutualisms of ants and termites where the fungus
gardens are ectosymbionts for the individual ants but endosymbionts for the colony (Poulsen &
Boomsma 2005; Aanen et al. 2009). We are intuitively inclined to consider the insect colony as
the active farming host and the fungus gardens as passive crops, but the contention that fungal
clones have contracted ant families to propagate them across generations appears to be equally
valid, especially in the attine ants where the fungal symbiont is vertically transmitted (Poulsen &
Boomsma 2005).
In terms of biomass conversion effciency, the functional complementarity between attine ants
and their fungus garden symbionts achieved spectacular progression, moving from initial stages
of ant-driven litter-based decomposition farming that evolved 50 Million Years Ago (MYA) to
advanced farming of irreversibly domesticated crop fungi 20 MYA (Mueller et al. 1998; Schultz
& Brady 2008; Mehdiabadi & Schultz 2010), culminating in aggressive herbivorous leaf-cutting
farming 10 MYA (De Fine Licht et al. 2010; Schitt et al. 2010). Particularly the latter transition
allowed the ants to evolve much larger and complex societies (Mueller et al. 1998; Villesen et al.
2002; Schultz & Brady 2008; Fernandez-Marin et al. 2009; Mehdiabadi & Schultz 2010) after
they had managed to overcome a series of challenges that are associated with a herbivorous life-
style (Schitt et al. 2008; 2010; De Fine Licht et al. 2010; 2013). Although depending on live veg-
etation rather than dead leaf litter undoubtedly implied better access to the proteins that normally
limit insect growth, an almost exclusive fungal diet is generally poorer in protein than the carnivo-
rous diets of many hunter-gatherer species in the Myrmicinae subfamily of ants (Davidson 2004).
Hosts and symbionts can thus be assumed to have been under consistent selection to maximize
protein yield as this will determine colony growth rate and the production of winged virgin queens
that transmit both their own genes and clonal copies of the fungal symbiont to future generations.
Specialized foraging on live plant material as growth substrate for the fungal symbiont Leucoag-
aricus gongylophorus implies that leaf-cutting ants are major herbivores in the Neo(sub)tropics
with substantial roles in recycling nitrogen and phosphorus (Fowler et al. 1989). A recent study
113
has confrmed that the leaf-cutting fungus-farming mutualism is likely to be nitrogen-limited
as Atta fungus gardens harbour substantial levels of nitrogen fxing bacteria (Pinto-Toms et al.
2009). Studies on the other genus of leaf-cutting ants, Acromyrmex, have shown that the fungal
symbiont uses the ants to vector pectinases and a laccase from the most productive middle layers
of fungus gardens to the top where they are most useful for the symbiosis but where fungal bio-
mass to produce these enzymes is in short supply (Schitt et al. 2010; De Fine Licht et al. 2013).
The fungal gongylidia, special hyphal tips produced only to feed the ants and their brood (Bass &
Cherrett 1995), are instrumental in this process as they upregulate the expression of genes produc-
ing these enzymes (Schitt et al. 2010; De Fine Licht et al. 2013). These studies indicate that the
fungal symbiont uses the ants for vector enzymes for the neutralization of phenolic compounds
(laccase) and for loosening up the cell wall matrix (pectinases) to new garden sections. However,
whether these initial decomposition steps are followed by a similar boost in protease activity to
decompose intracellular proteins has remained unknown.
The ants process freshly cut leaf fragments into a mass of leaf pulp while mixing it with droplets
of fecal fuid and subsequently deposit the new substrate on fungal ridges in the top of the garden
where they inoculate it with small tufts of mycelium from the older part of the garden (Weber
1966). In the 1970s, leaf-cutting ant fecal fuid was shown to contain active proteases (Martin
1970; Martin & Martin 1970a; Martin & Martin 1970b; Martin & Martin 1971; Martin 1974) with
similar chemical properties as enzymes originating from the fungal symbiont (Boyd & Martin
1975a; 1975b), and recent work has shown that fungus garden endo-protease activity in evolu-
tionarily derived leaf-cutting ants is much higher than in sister ant lineages that do not use fresh
leaves to make their gardens grow (De Fine Licht et al. 2010), activity that could subsequently be
assigned to metalloproteases and serine proteases (Semenova et al. 2011).
Recently, we obtained the proteome of Acromyrmex echinatior fecal fuid and were able to iden-
tify 33 proteins. Among these were seven pectinases (Schitt et al. 2010), one laccase (De Fine
Licht et al. 2013), and seven proteases. Only a single of these proteases is known to be produced
by the ants, whereas the other six are contributed by the fungal symbiont. We hypothesized that a
number of these proteases might serve to either process the intracellular proteins of freshly opened
plant cells or to assist in the breakdown of secondary plant defences (Christeller et al. 1992; Mer-
cado-Flores et al. 2003; Ievleva et al. 2006). The latter is a distinct possibility as plants are known
to use protease inhibitors to counteract the harmful effect of proteases produced by phytopathogic
fungi and to generally decrease the digestibility of their tissues to discourage herbivores (Habib &
Fazili 2007). The objectives of the present study were to: 1. measure the specifc protease activity
114
in fecal fuid of gardening A. echinatior workers, 2. confrm that they are derived from the fungal
symbiont and have different expression levels and pH optima, 3. assess the extent to which the
expression of genes coding for these enzymes was enhanced in the fungal gongylidia, as would
be expected when vectoring these enzymes to the top of fungus gardens is a specifc adaptation of
the symbiosis, and 4. discuss the implications of our results for understanding the co-adaptations
between partners that has allowed this symbiosis to evolve its substantial ecological footprint in
the Neo(sub)tropics.
Materials and methods
Biological material
We used seven colonies of A. echinatior (Ae263, Ae280, Ae322, Ae332, Ae335, Ae349 and
Ae370), collected in and around Gamboa, Panama, between 2004 and 2007 and kept in rearing
facilities in Copenhagen under controlled conditions of ca. 25C and ca. 70% humidity, where
they were fed twice a week with fresh bramble leaves, apple pieces and dry rice. Fecal fuid was
obtained by gently squeezing the abdomen of large workers with a forceps on a microscope slide.
Each fecal droplet was then mixed with 0.5 l demineralised water, collected with a micropipette,
and stored in an Eppendorf tube on ice. Sixty droplets from fve colonies each (Ae263, Ae280,
Ae322, Ae332 and Ae349) were collected this way, pooled per colony and diluted with deminer-
alised water to a fnal volume of 250 l.
For the gene expression measurements, gongylidia clusters (staphylae) and normal mycelium were
collected separately under a stereomicroscope at 40x magnifcation from each of fve colonies
(Ae263, Ae280, Ae322, Ae335 and Ae370) in 2 mL Eppendorf tubes foating in liquid nitrogen.
After collecting app. 100 L for each type of tissue, samples were stored at -80C for subsequent
RNA extraction.
Protein identifcation and gene cloning
SDS-PAGE and mass spectrometry were performed as described previously (Schitt et al. 2010;
De Fine Licht et al. 2013). RNA extraction from the fungal symbiont, followed by cDNA con-
struction, was done as in Schitt et al. (2010). Aspartic protease 2 (AspII), Metallopeptidase 1
(MetI), Serine protease 1 (SerI) and Serine protease 2 (SerII) were initially identifed by PCR
amplifcation from cDNA using degenerate primers constructed from the mass spectrometry data
using the PCR scheme: one cycle of 95C for 5 min, 35 cycles of 94C for 20 sec, 50C for 30
sec and 72C for 2 min, and ending with one cycle of 72C for 7 min. Aspartic protease 1 (AspI)
and Metallopeptidase 2 (MetII) were identifed at a later stage by a Blast search of the ca. 100x
115
coverage A. echinatior genome (Nygaard et al. 2011) and a low coverage genome sequence of
the A. echinatior fungal symbiont (De Fine Licht et al. 2013) using the mass spectrometry data as
queries. Sequencing of full-length gene transcripts was done using a RACE (Rapid Amplifcation
of cDNA Ends) strategy. 3 and 5-RACE libraries were made from ca. 1 g of purifed RNA
with the SMART RACE cDNA kit (CLONTECH, Mountain View, California, USA), and gene
sequences were PCR amplifed from these libraries using specifc primers designed from the PCR
amplifed gene fragments (AspII, MetI, SerI and SerII) or BLAST search identifed sequence reads
(AspI and MetII) along with the primers enclosed in the SMART RACE cDNA kit. In some cases
the 5RACE had to be done in several steps to get a full transcript sequence. The following PCR
scheme was used in the RACE experiments: one cycle of 95C for 5 min, 10 cycles of 94C for
20 sec, 72C for 30 sec (with a decrease in temperature of 0.5C in every cycle) and 72C for 2
min, followed by 35 cycles of 94C for 20 sec, 67C for 30 sec and 72C for 2 min, and ending
with one cycle of 72C for 7 min. All PCR products were cloned in pCR4-TOPO using the TOPO
TA cloning method (Invitrogen, Carlsbad, California, USA) and sequenced at Eurofns MWG Op-
eron (Ebersberg, Germany). Gene sequences are deposited in GenBank with accession numbers
KF571927-KF571932. Primer sequences for cloning of the genes are provided in Table S1.
Enzyme assays
Protease activity was measured for four different protease inhibitors and two controls (without
inhibitors, one of which incubated and the other not incubated) at eight different pH levels. The
inhibitors used in the experiment were 5mM 1,10-phenanthroline to inhibit metalloendoproteases,
2M pepstatin to inhibit aspartic proteases, 10mM phenylmethylsulfonyl fuoride to inhibit serine
proteases and 10M trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane to inhibit cysteine
proteases. In the assays, 4 l of the diluted fecal fuid was mixed with 1 l of inhibitor or distilled
demineralised water and incubated on ice for one hour. Next, 35 l of 0.1M Britton Robinson
buffer (Britton & Robinson 1931) with 2% azoalbumin was added and the mix was incubated at
30C for one hour. For the blank control (not incubated) the azoalbumin mix was added at the
last moment without incubation to prevent any protease activity to occur. To stop the activity of
the proteases, 120 l 10% trichloroacetic acid was added, after which the mix was vortexed and
incubated for 15 minutes before centrifugation for 5 minutes at 8000g. The supernatant (86 l)
was added to 100 l freshly prepared 1M NaOH on a 96 well cell culture plate.
Absorbance was measured at 440 nm on a Versamax ELISA microplate reader (Molecular Devic-
es, Sunnyvale, USA) and scored after subtracting the corresponding blank-control measurements
to correct for background absorbance. To fnd the enzyme activity in each of the four protease
116
classes, measurements obtained with specifc inhibitors for each protease class were subtracted
from the total protease activity in identical assays without addition of inhibitors. Protease activity
was defned as the amount of enzyme required to cause a unit increase at 440 nm across a 1 cm
path length (Sarath et al. 2001). The number of units of protease activity data were plotted and
analyzed with R (R Core Team 2013), using Linear Mixed-Effects Models (lme) (Pinheiro et
al. 2013), with Colony as random factor, and generating p-values with General Linear Hypoth-
eses (glht) in the Simultaneous Inference in General Parametric Models package multcomp
(Hothorn et al. 2008).
For measuring protease activity in control ants, 100-200 workers of the colonies Ae322, Ae332
and Ae349 were transferred to subcolonies with no access to fungus garden material, but with
an ad libitum diet of 10% sucrose and bramble leaves. After ca. two weeks protease activity was
measured in pooled samples of fecal droplets from two ants for each colony at pH 6 in three rep-
licates per colony.
Quantitive real-time PCR
Primers for the six different genes (Table S2) were designed by matching the obtained cDNA
sequences to a database of a partially sequenced genome of the A. echinatior fungal symbiont
(De Fine Licht et al. 2013) using BLASTn to identify intron and exon sequences. Primers were
designed to span an intron to avoid amplifcation of genomic DNA. The qPCRs were run on an
Mx3000P QPCR System (Agilent, Santa Clara, CA, USA) in 20 L reactions (0.5 L cDNA, 10
L 2x SYBR Premix Ex Taq [TaKaRa Bio Inc., Otsu, Japan] and 0.4 L of each primer [10M])
with the following PCR conditions: one cycle of 95C for 2 min, followed by 40 cycles of 95C
for 30 sec, 55C (AspI, AspII, MetII, SerI, SerII, and EF1-) or 57C (MetI, GAPDH, Ubc) for 30
sec, and 72C for 30 sec, and ending with a melting curve cycle of 95C for 30 sec, 55C or 57C
for 30 sec and 95C for 30 sec.
For each of the ten samples (one gongylidia and one mycelium sample from fve colonies), three
technical qPCR replicates were done for nine genes: three housekeeping genes (GAPDH, Ubc
and EF1-) and six target genes (MetI, MetII, AspI, AspII, SerI and SerII). All Ct values from the
RT-qPCR analysis were analyzed in R with packages ReadqPCR (Perkins & Kohl 2011) and
NormqPCR (Kohl & Perkins 2011) using averages across the three technical replicates. Stabili-
ty of housekeeping genes was assessed across the entire data set, which showed that GAPDH and
Ubc were more stable than EF1-, so the latter gene was discarded in the further analyses. GAP-
DH and Ubc were then used to calculate the normalized expression (2
Ct
) of the target genes in
117
Table 1
Peptide sequences of proteases in fecal fuid of A. echinatior and corresponding molecular weights (Da) of full-length
proteins. The letter J symbolises the amino acids leucin and isoleucin, which have the same molecular mass and are
therefore indiscernible by mass spectrometry. The single discrepancy between mass spectrometry data and sequence
data (letters D and H in bold) may have been caused by deamination of asparagine into aspartic acid.
Protein Mass spectrometry data Sequence data MW (Da)
Aspartic protease 1 DENDGGEATFGGJNPSSYR
NAYWEV
YFTVYDJGR
DENDGGEATFGGINPSSYR
NAYWEV
YFTVYDLGR
44788
Aspartic protease 2 FTGAJNFTPR
SSGFEGTDGJJGJGPVDJTR
GTJSPAVNSJVPTVTDNJFSSGR
TSTSPSSJFWGJNQSVR
VJDNNTGJJR
FTGALNFTPR
SSGFEGTDGILGIGPVDLTR
GTLSPAVNSLVPTVTDNLFSSGR
TSTSPSSLFWGLNQSVR
VLDNNTGLLR
43158
Metallopeptidase 1 TYASNAATYJSSHSSSSTR
YTTWFGTYTSAR
TYASNAATYLSSHSSSSTR
YTTWFGTYTSAR
37207
Metallopeptidase 2 TYASNAJJYJR TYASNALIYLR 37511
Serine protease 1 AWAGJHFAVAAGNDNR
GSDGSGSTSDVJAGVQWAAR
AADAGLHFAVAAGNDNR
GSDGSGSTSDVIAGVQWAAR
50114
Serine protease 2 ADJQTFFR
AGYJDEFANR
AJYNTVNYVPSQTSR
DGJGVAGYJDEFANR
GVAGYJDEFANR
GSVGGTSASSPTVAGVFAJJNDFR
FRPDAAGSSFTTVR
YFSTPSYQSAAVSR
ADLQTFFR
AGYLDEFANR
ALYNTVNYVPSQTSR
NGLGVAGYLDEFANR
GVAGYLDEFANR
GSVGGTSASSPTVAGVFALLNDFR
FRPDAAGSSFTTVR
YFSTPSYQSAAVSR
63388
Carboxypeptidase A
(EGI65848)
QNNPGVFFESGJHAR QNNPGVFFESGIHAR 46943
118
gongylidia and mycelium. Values of 2
-Ct
(Livak & Schmittgen 2001) for each gene subsequently
produced estimates of fold changes in relative gene expression between gongylidia and mycelium.
This sequence of procedures allowed us to identify genes with signifcantly different expression
levels and to obtain estimates of their normalized and relative expression.
Results
The mass spectrometry data of fecal fuid proteins (Table 1) described previously (Schitt et al.
2010) allowed us to clone and sequence six protease genes encoded by the fungal symbiont ge-
nome, and to identify an ant encoded protease (EGI65848) belonging to a family of M14 carboxy-
peptidases, which is signifcantly expanded in the genome of A. echinatior (Nygaard et al. 2011).
Comparison with the MEROPS database release 9.8 (http://merops.sanger.ac.uk/ (Rawlings et al.
2012)) (Table S3) showed that all six proteases (AspI, AspII, MetI, MetII, SerI and SerII) were
similar to peptidases known from other fungi; AspI with saccharopepsin (A01.018) from Laccar-
ia bicolor (78.57%, E-value = 1.10e-142); AspII with polyporopepsin (A01.019) from L. bicolor
(71.60%, E-value = 1.80e-125); MetI with peptidyl-Lys metallopeptidase (M35.004) from Pleuro-
tus ostreatus (56.55%, E-value = 1.50e-53); MetII with peptidyl-Lys metallopeptidase (M35.004)
from P. ostreatus (69.23%, E-value = 2.60e-42); SerI with an unassigned peptidase from the S8A
subfamily from L. bicolor (61.28%, E-value = 6.90e-111); SerII with grifolisin (S53.010) from L.
bicolor (66.67%, E-value = 4.40e-136).
To test the overall protease activity in the ant fecal fuid and to determine whether the fungus
garden is responsible for this activity, protease activity was measured in fecal droplets of worker
ants taken directly from their colonies and from purged ants that had been kept on sugar water
and bramble leaves for ca. two weeks with no access to fungal garden material. This showed that
protease activity is present in fecal fuid when the ants have their normal diet, but disappears al-
most completely when the ants are deprived of fungus garden material (t
17
= 14.8972, p < 0.0001),
indicating that the protease activity in the ant fecal fuid is caused by ingested proteases from the
fungal symbiont and not by proteases produced by the ants themselves (Figure 1).
Next, we tested the activity of the four main types of proteases using different inhibitors spe-
cifcally targeted at each category of proteases: metalloendoproteases, serine proteases, aspartic
proteases and cysteine proteases. Only the metalloendoproteases and serine proteases had sig-
nifcant activities, with respective peaks at pH 6 (z = -10.824, p < 0.0001) and pH 7 (z = 4.654,
p < 0.0001) and serine protease activity also being signifcantly enhanced at pH 6 (z = 2.004, p
< 0.05), consistent with fecal fuid also having pH 6 (Figure 2). In contrast to serine proteases,
119
metalloendoproteases were active at a very broad range of pH values and retained activities up to
pH 10. We found no activity for aspartic proteases (present in fecal fuid), and cysteine proteases
(absent in fecal fuid).
Relative changes in gene expression in gongylidia versus mycelium (fold-changes: 2
-Ct
) showed
that the expression of fve out of the six fungal protease genes was upregulated in the gongylidia
compared to the mycelium (Figure 3a): peptidyl-Lys metallopeptidase I and II (MetI and MetII) (z
= -3.979, p = 0.00004; z = -20.010, p = 0.00001), saccharopepsin (AspI) (z = -3.946, p = 0.00004),
subtilisin (SerI) (z = -8.927, p = 0.00001) and grifolisin (SerII) (z = -9.041, p = 0.00001), whereas
the expression of polyporopepsin (AspII) remained unchanged (z = 2.580, p = 0.99492). This is
consistent with all but one of these fungal proteases having their main function after being ingest-
ed by the ants, i.e. after they pass the ant guts unharmed to be deposited with the fecal fuid.
Plotting relative gene expression (fold change in gongylidia vs. mycelium) as a function of nor-
malized gene expression in the gongylidia (i.e. expression relative to housekeeping genes) (Figure
3b) showed proportionality for four of the proteases (saccharopepsin, subtilisin, grifolisin and
polyporopepsin). However, the two metalloendoproteases deviated from the 1:1 proportionality
represented by the diagonal in Figure 3b. One of them (MetII) combined a fairly low normalized
expression (Table S4) with a 17x upregulation in gongylidia, and the other (MetI) combined a high
normalized expression with a relatively low 5x fold upregulation in gongylidia. This implies that
Figure 1
Enzyme activity in milliunits of proteases
(mU) in fecal fuid of workers of the leaf-cut-
ting ant A. echinatior kept on natural fun-
gus-garden-diet and on a diet of only sugar
water and possibly plant sap as control (t
17
=
14.8972, p < 0.0001). Whiskers are SEs of the
mean.
120
the expression of MetII is much more gongylidia-specifc than the expression of MetI, which is
apparently also needed in high quantities in the undifferentiated mycelium. It also appeared that
the single non-differentially expressed protease (AspII) had a low normalized expression level
(Figure 3b), indicating that it may not play a very important role under normal conditions, but
might have an unknown adaptive function under specifc feld conditions that did not apply to our
lab colonies.
Discussion
In the present study we used mass spectrometry to identify seven proteases in the fecal fuid of
A. echinatior leaf-cutting ants. For all but one of these we could exclude that they are produced
by the ants, because we have a high quality reference genome for the ants (Nygaard et al. 2011),
to which only one protease matched, and a poorly assembled genome for the fungal symbiont
(De Fine Licht et al. 2013) to which the remaining six sequences matched. Our protease assays
showed both metalloendoprotease activity and serine protease activity in the ant fecal fuid, but
when we deprived the ants of their fungal symbiont by giving them a diet of sugar water and
bramble leaves, essentially all protease activity disappeared. We further showed that fve out of the
six protease genes were overexpressed in the fungal gongylidia that the ants eat. In combination,
the results strongly suggest that these proteases mostly belong to a distinct but restricted group
of proteins that have been selected to be ingested by the ants for the sole purpose of being passed
on to the fecal fuid where they provide functional mutualistic benefts for the farming symbiosis.
Figure 2
Activity of metalloendoproteases, serine proteases, aspartic proteases, and cysteine proteases in milliunits (mU). Both
metalloendoproteases (z = 10.824, p < 0.0001) and serine proteases (z = 2.004, p < 0.05) showed signifcantly enhanced
activity at pH 6 relative to the zero base line. Serine proteases had their peak activity at pH 7 (z = 4.654, p < 0.0001)
and activities of metalloendoproteases at pH levels >7 remained equally signifcant as those at pH 6. Whiskers are SEs
of the mean.
121
The most parsimonious hypothesis to explain this remarkable phenomenon is that the stationary
fungus uses the ants to vector crucial enzymes produced in the central parts of the garden, where
the ratio between fungal biomass and remaining substrate allows high gongylidia production, to
the upper parts of the garden where an abundance of new substrate is available but as yet without
much hyphal growth. These fndings and interpretations are consistent with earlier results ob-
tained for seven pectinases (Schitt et al. 2010) and a laccase enzyme (De Fine Licht et al. 2013).
It remains possible that the ingested fungal enzymes also interact with unknown other compounds
in the ant gut, produced either by the ants or their microbiome, but such additional complexities
appear unnecessary to explain our current or previous results obtained for other enzymes in the
gongylidia and fecal fuid (Rnhede et al. 2004; Schitt et al. 2010; De Fine Licht et al. 2013).
Figure 3
Average relative gene expression (fold change) across gongylidia collected from fungus gardens of fve colonies of A. ech-
inatior: dark grey = metalloendoproteases, grey = aspartic proteases, light grey = serine proteases. A) Relative expression
fold-changes (mean SE) in gongylidia compared to mycelium, with the dashed horizontal line representing no change
in expression. Target genes were signifcantly upregulated in the gongylidia (P = 0.00004, 0.00001, 0.00004, 0.99492,
0.00001 and 0.00001 for MetI, MetII, AspI, AspII, SerI and SerII, respectively, marked with asterisk (*)), except for poly-
poropepsin (AspII). B) Relative expression fold-change plotted against gene expression normalized for the housekeeping
genes GAPDH and Ubc (means SE). The three upregulated serine (SerI and SerII) and aspartic (AspI) proteases had
fold-changes proportional to normalized gene expression (being very close to the dashed line diagonal), but peptidyl-Lys
metallopeptidase I (MetI) combined a high normalized gene expression with a relatively low gene fold-change, and pep-
tidyl-Lys metallopeptidase II (MetII) combined a low normalized gene expression with a relatively high gene fold-change.
122
It remains to be seen whether the proteases that we identifed may also have functions that are
restricted to the gongylidia. If that would be the case, the non-adaptive null hypothesis would be
that fnding them in the fecal fuid may merely imply that digestion was incomplete, but this seems
unlikely given that SDS-PAGE banding patterns of fecal fuid were always consistent, showing
a very specifc subset of proteins that escape digestion (Schitt et al. 2010). Together with our
previous work (Schitt et al. 2010; De Fine Licht et al. 2013) we have now shown that 12 of the
14 identifed fungal pectinase, laccase and protease enzymes in the A. echinatior fecal fuid have
upregulated gene expression in the gongylidia, which we believe underlines the key signifcance
of these special hyphal tips as fungal organs mediating a series of symbiotic adaptations.
As it appears, the main function of the fecal fuid proteases is enhanced digestion of the most valu-
able nitrogen-containing molecules of the fresh plant material harvested by the leaf-cutting ants.
Peptidyl-Lys metallopeptidases (MetI and MetII) and serine carboxyl-peptidases (SerII) have been
suggested to be involved in the extraction of nutrients from extracellular proteins (Heck 2012;
Oda 2012). As both MetI, MetII and SerII were active in the fecal fuid (Figure 2) and upregulated
in the gongylidia (Figure 3), it seems reasonable to hypothesize that these enzymes increase nitro-
gen extraction from the chewed-up leaf pulp after other enzymes such as the pectinases (Schitt et
al. 2010) have partially broken down the cell walls. The other active serine protease, subtilisin, is
structurally similar to several unassigned fungal proteases in the subfamily S8A (Table S3). Most
of the matches in protein databases for this enzyme were proteases from general pathogenic or
phytopathogenic fungi. This fnding is intriguing, because most of the subtilisins in subfamily S8A
are part of a large gene-family expansion in Angiosperms with functions roles in seed or fruit de-
velopment or manipulating cell walls in response to changes in biotic and abiotic factors (Schaller
2012; Schaller et al. 2012).
While subtilisins likely have defensive functions in plants, other studies have shown that subtil-
isins have important aggressive functions in saprotrophs, phytopathogens and other infectious
pathogens (Sreedhar et al. 1999; Dunaevskii et al. 2006; Ievleva et al. 2006; Bryant et al. 2009;
Li et al. 2010). One explanation for this could be that using proteins very similar to those present
in the hosts may allow pathogens to avoid detection by immune defences, a phenomenon referred
to as molecular mimicry (Elde & Malik 2009; Armijos Jaramillo et al. 2013). Although the fungal
symbionts of leaf-cutting ants hardly face any active immune defense in the severed plant material
that the ants bring in, they will be challenged by substantial amounts of secondary (defensive)
compounds, similar to any functional insect herbivores whose digestive systems need to cope
with plant defences that decrease digestibility (Chen 2008). Gongylidial subtilisin might therefore
123
have some role in the penetration and digestion of newly harvested plant material, consistent with
serine protease activity being also high in the gardens of other higher attine ants. Panamanian rep-
resentatives of the genera Trachymyrmex and Sericomyrmex that readily use soft fresh leaves as
substrate in the laboratory, tend to have very high levels of serine protease activity in their fungus
gardens, whereas these levels tend to be low in attine ant species that do not or only reluctantly
use fresh leaves as fungal substrate (Semenova et al. 2011, P. Kooij and M. Schitt, unpublished
observations).
We found two aspartic proteases in the fecal fuid, but none of them showed activity (Figure 2),
even though one of them, saccharopepsin, was upregulated in the gongylidia when compared to
mycelium (Figure 3). This suggests that this protein has a function, either for the gongylidia or
after ingestion by the ants. Saccharopepsin is known to be important in the activation of yeast
vacuolar hydrolases (Parr et al. 2007). This could explain the abundance of this enzyme in the
gongylidia as they always have a large vacuole, but it does not explain why this protease is passed
on to the fecal fuid. As non-upregulated polyporopepsin is also found in fecal fuid, it might be
that these proteases cleave very specifc target sequences so they would not show activity in our
general activity assays. Possible target sequences could be other fungal enzymes that are produced
as inactive precursors (zymogens or proenzymes), which are subsequently activated when regu-
latory proteases cleave off part of the precursor amino acid chain. Such an activation mechanism
would be particularly appropriate for abundant plant degrading enzymes that are also harmful to
the fungus garden but not to the ants. These enzymes could then be produced in large quantities in
the gongylidia, but only be activated in the ant gut to be mixed with the plant substrate via fecal
fuid before the fungal hyphae are inoculated.
The possibility of leaf-cutting ant fungi having independently evolved adaptations reminiscent
of phytopathogens is intriguing, so it is of interest to briefy evaluate recent studies addressing
evolutionary arms-races between phytopathogens and their host plants (Misas-Villamil & van
der Hoorn 2008). Also here, metalloproteases appear to have a role in the breakdown of cell wall
stabilizing proteins (Rauscher et al. 1995) and in targeting specifc plant resistance proteins (Xia
2004). Among serine proteases, trypsins appear to be most prevalent in phytopathogens, whereas
subtilisins are more important in saprotrophs (Dunaevskii et al. 2006; 2008). However, when they
occur, subtilisins may have an important role in the breakdown of cell wall structural proteins
(Murphy & Walton 1996), as in infections of Kentucky bluegrass, Poa pratensis (Sreedhar et al.
1999) and in Colletotricum fungi that apparently acquired a subtilisin gene by horizontal transfer
from their host plant, encoding a protease which is active during infection (Armijos Jaramillo et
al. 2013).
124
The possible similarities in attine fungal proteases with those of phytopathogens (St Leger et
al. 1997) has interesting parallels to our earlier studies on pectinases (Schitt et al. 2010), and a
laccase (De Fine Licht et al. 2013) in the fecal fuid of A. echinatior ants. Also here, indications
of convergent evolution were found, although the evidence remained largely indirect as in the
present evaluation of our proteases results. Although many of the molecular mechanisms remain
unknown (but see De Fine Licht et al. 2013), it seems beyond doubt that selection pressure on
herbivorous leaf-cutting ant fungi must have been largely in the same direction as in unrelated
lineages of necrotrophic fungi. The relatively simple fecal fuid proteome (a total of ca. 33 iden-
tifed proteins so far in A. echinatior) appears to be a valuable and relatively transparent window
through which the molecular adaptations that have shaped fungus-farming in the monophyletic
clade of the attine ants can be unravelled.
Acknowledgements
The authors thank the Smithsonian Tropical Research Institute (STRI), Panama, for providing logistic support and facil-
ities to work in Gamboa, the Autoridad Nacional del Ambiente y el Mar (ANAM) for permission to sample ant colonies
in Panama in to export them to Denmark, and the Danish National Research Foundation for funding (DNRF57).
Abbreviations
EF1 - Elongation factor 1-
GAPDH - Glyceraldehyde 3-phosphate dehydrogenase
SDS-PAGE - Sodium dodecyl sulphate polyacrylamide gel electrophoresis
Ubc Ubiquitin
Confict of interest
The authors declare that they have no competing interests related to this manuscript
Supplementary Information accompanies the paper on The ISME Journal website (http://www.natur.com/ismej)
125
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Table S1. List of primers used for gene cloning.
Gene
Primer
name
Primer function Primer sequences
AspI
Asp-F1 3 RACE primer 5-CGTTCCCTTCGTTGTTGGCCTCC-3
Asp-R1 5 RACE primer 5- GCAAGCTTGTGAAGTCTGAGGGTGTG-3
AspII
Pro-F4 Forward degenerate primer 5-ACNGGXAGNTCXAAYACNTGG-3
Pro-R2 Reverse degenerate primer 5-TTRTCNGTIACNGTIGGDAT-3
Pro-F5 3 RACE primer 5-CCGTCGATCTCACGCGAGGAACGCTG-3
Pro-R5 5 RACE primer 5-GCGACGCTGTTGCCGGTCTGCTGAC-3
MetI
Sp21-F2
Forward 3RACE degenerate
primer*
5-GGTGGTACYTAYGTNCAYGA-3
Met-R1 5 RACE primer 5-AGCGAGTGCCTTGGCGCCAGATTG-3
Met-R2 5 RACE primer 5-CGTAGTCTTGTGTTCCGCCATTTCTGAGG-3
Met-R3 5 RACE primer 5-AGGGCTTGAGAGGCTCGATTGAGTAAG-3
Met-R4 5 RACE primer 5-CCGGAGTTGGTGAAGTTGTACATTGTTGAC-3
Met-R5 5 RACE primer 5-CAAGAGCAACAGCACATCGC-3
MetII
MetII-full-F3 Forward primer 5-AATGTCAATTCTATCGAATTCAAG-3
MetII-R1 Reverse qPCR primer 5-CGTGGACGATTGTTCCACCTC-3
MetII-F1 Forward qPCR primer 5- GGCTATTACTGCAGCGAAGACC -3
MetII-full-R Reverse primer 5-CAATTCAAGAATTATATGGCAGG-3
SerI
Sub-F1 Forward degenerate primer 5-GTTGARTTYGARGGXMGNGC-3
Sub-R1 Reverse degenerate primer 5-TTRTCRTTACCXGCNGCNAC-3
Sub1-F1 3 RACE primer 5-GATCGCCGACAACAAGTATGAAGATGGTG-3
Sub1-R1 5 RACE primer 5-GCACAGTGAGTACCATGACCAACACC-3
SerII
Sp24-F3 Forward degenerate primer 5-GCTGACCTYCARACNTTYTT-3
Sp16-R2 Reverse degenerate primer 5-GAGAAACCACCNCCNGARAA-3
Ser-F1 3 RACE primers 5-TAGGCGGTGGCGACTGCCGTACTAATGAC-3
Ser-R2 5 RACE primer 5-GAAACTAGAACCTGCAGCATCGGGGCGG-3
Ser-R3 5 RACE primer 5-CGGAATCTGGGTCAAAAGGAGGGGAACCG-3
Ser-R4 5 RACE primer 5-GTCCATACCGTGCATGACCAGGATCGC-3
* This primer was used together with the UPM primers from the SMART RACE cDNA kit
Note: X symbolizes inosines
129
Table S2
The Real Time qPCR primers used in this study.
Enzyme Primer 5- sequence -3
Annealing
temp C
Size
Saccharopepsin
(AspI)
Pep-F2 CCG CCA TTG ACA CTG GTA CC 53.8
289bp
Pep-R2 GGC GGA GGA AAA CAT CAC CAA 54.4
Polyporopepsin
(AspII)
Pol-F2 CTC GTT GAC ACC GGG AGT TC 55.9
243bp
Pol-R3 GAC GGG GCC GAT GCC TAA 54.9
Peptidyl-Lys
metallopeptidase
(MetI)
Met-F2 CGG GAC CGA CTC TAA GGG 54.9
169bp
Met-R6 TTC ATT GTG AAT ACA TCA TAG ACT TC 51.7
Peptidyl-Lys
metallopeptidase
(MetII)
MetII-F1 GGC TAT TAC TGC AGC GAA GAC C 56.7
307bp
MetII-R1 CGT GGA CGA TTG TTC CAC CTC 56.3
Subtilisin (SerI)
Sub-F2 AGA AAT CTG CTC CAT GGG GC 53.8
300bp
Sub-R1 CTA CCA TCA GAA CCA AGC ACC 54.4
Grifolisin (SerII)
Ser-F3P GCT ACC TTC AAT GGT CTA TAC AAT AAA AC 56.0
310bp
Ser-R4P TAC CCA GGC CAG TGA CAG GAT 55.4
Elongation factor
1-a (EF1-a)
EF1a-F1 TTG GAG GAA TCT CCC AAC ATG 57.9
273bp
EF1a-R1 AAC GGA CTT GAC TTC AGT AGT C 58.4
Glyceraldehyde
3-phosphate
dehydrogenase
(GAPDH)
GAPDH-F4 TCA ACG GCA AGC TCA CTG GT 53.8
253bp
GAPDH-R4 ACA AAA TTC CCG TTC AAA GGA ATC 52.3
Ubiquitin (Ubc)
Ubc-F4 AAC GAT AAT GGG ACC CGG TG 53.8
239bp
Ubc-R4 GAT TGG GAT CAG TCA GCA TTG 52.4
130
Table S3
Comparison of the sequenced proteases with the MEROPS database showing similarities with known fungal
peptidases. For each protease the top ten blast results are shown.
Protease MEROPS
code
MEROPS
classifcation
Protease name Species % identity E-value
AspI MER122335 A01.018 saccharopepsin Laccaria bicolor 78.57% 1.10e
-142
MER309581 A01.018 saccharopepsin Serpula lacrymans 79.56% 4.40e
-141
MER107385 A01.018 saccharopepsin Coprinus cinereus 78.02% 1.70e
-139
MER309629 A01.018 saccharopepsin Schizophyllum
commune
77.36% 4.10e
-138
MER382986 A01.018 saccharopepsin Stereum hirsutum 76.88% 5.20e
-138
MER382978 A01.018 saccharopepsin Coniophora puteana 76.42% 8.40e
-138
MER382983 A01.018 saccharopepsin Punctularia
strigosozonata
77.67% 1.80e
-137
MER382989 A01.018 saccharopepsin Trametes versicolor 77.12% 180e
-135
MER382982 A01.018 saccharopepsin Dichomitus squalens 76.42% 4.80e
-135
MER382984 A01.018 saccharopepsin Fomitiporia
mediterranea
74.53% 1.30e
-132
AspII MER118464 A01.019 polyporopepsin Laccaria bicolor 71.60% 1.80e
-125
MER383691 A01.019 polyporopepsin Pholiota nameko 74.53% 2.70e
-122
MER383440 A01.019 polyporopepsin Trametes versicolor 71.60% 2.80e
-120
MER383572 A01.019 polyporopepsin Dichomitus squalens 71.74% 3.20e
-190
MER000940 A01.019 polyporopepsin Polyporus tulipiferea 69.04% 9.70e
-118
MER383537 A01.019 polyporopepsin Stereum hirsutum 70.15% 2.00e
-117
MER383829 A01.019 polyporopepsin Punctularia
strigosozonata
70.37% 4.20e
-117
MER062431 A01.019 polyporopepsin Phanerochaete
chrysosporium
67.70% 4.50e
-113
MER172923 A01.019 polyporopepsin Laccaria bicolor 65.43% 5.30e
-110
MER383761 A01.019 polyporopepsin Stereum hirsutum 64.71% 1.40e
-109
MetI MER003546 M35.004 Peptidyl-Lys
metallopeptidase
Pleurotus ostreatus 56.55% 1.50e
-53
MER005595 M35.004 Peptidyl-Lys
metallopeptidase
Armillaria mellea 57.49% 1.20e
-51
MER003545 M35.004 Peptidyl-Lys
metallopeptidase
Grifola frondosa 51.50% 7.90e
-50
MER187451 M35.003 EcpA peptidase Corallococcus
coralloides
50.00% 4.30e
-42
MER019534 M35.003 EcpA peptidase Xanthomonas
axonopodis
48.82% 3.60e
-38
MER255520 M35.003 EcpA peptidase Collimonas
fungivorans
45.51% 5.80e
-38
MER089593 M35.003 EcpA peptidase Shewanella loihica 47.56% 2.50e
-37
MER071568 M35.003 EcpA peptidase Shewanella
denitrifcans
48.45% 4.10e
-37
MER075809 M35.003 EcpA peptidase Shewanella
amazonensis
48.50% 5.20e
-37
MER062468 M35.003 EcpA peptidase Xanthomonas
campestris
47.62% 2.90e
-36
131
MetII MER003546 M35.004 Peptidyl-Lys
metallopeptidase
Pleurotus ostreatus 69.23% 2.60e
-42
MER003546 M35.004 Peptidyl-Lys
metallopeptidase
Pleurotus ostreatus 54.90% 2.60e
-42
MER003546 M35.004 Peptidyl-Lys
metallopeptidase
Pleurotus ostreatus 47.06% 2.60e
-42
MER003546 M35.004 Peptidyl-Lys
metallopeptidase
Pleurotus ostreatus 71.43% 2.60e
-42
MER003546 M35.004 Peptidyl-Lys
metallopeptidase
Pleurotus ostreatus 35.48% 1.60e
-21
MER005595 M35.004 Peptidyl-Lys
metallopeptidase
Armillaria mellea 69.23% 6.80e
-33
MER005595 M35.004 Peptidyl-Lys
metallopeptidase
Armillaria mellea 53.19% 6.80e
-33
MER005595 M35.004 Peptidyl-Lys
metallopeptidase
Armillaria mellea 48.48% 6.80e
-33
MER005595 M35.004 Peptidyl-Lys
metallopeptidase
Armillaria mellea 54.17% 4.60e
-24
MER255520 M35.003 EcpA peptidase Collimonas
fungivorans
59.18% 2.60e
-32
SerI MER169844 S08.UPA Subfamily S8A
unassigned
peptidases
Laccaria bicolor 61.28% 6.90e
-111
MER034994 S08.UPA Subfamily S8A
unassigned
peptidases
Coprinus cinereus 60.17% 3.10e
-108
MER032520 S08.UPA Subfamily S8A
unassigned
peptidases
Aspergillus oryzae 52.47% 3.30e
-100
MER032520 S08.UPA Subfamily S8A
unassigned
peptidases
Aspergillus oryzae 36.11% 3.30e
-100
MER236481 S08.UPA Subfamily S8A
unassigned
peptidases
Cryptococcus gattii 56.90% 5.80e
-98
MER087598 S08.UPA Subfamily S8A
unassigned
peptidases
Neosartorya fscheri 61.65% 9.00e
-94
MER087598 S08.UPA Subfamily S8A
unassigned
peptidases
Neosartorya fscheri 36.11% 9.00e
-94
MER003475 S08.UPA Subfamily S8A
unassigned
peptidases
Aspergillus fumigatus 61.65% 1.10e
-93
MER003475 S08.UPA Subfamily S8A
unassigned
peptidases
Aspergillus fumigatus 36.11% 1.10e
-93
MER006088 S08.UPA Subfamily S8A
unassigned
peptidases
Penicillium
chrysogenum
60.43% 4.90e
-93
SerII MER137045 S53.010 grifolisin Laccaria bicolor 66.67% 4.40e
-136
MER137535 S53.010 grifolisin Laccaria bicolor 60.05% 1.90e
-119
MER389943 S53.010 grifolisin Stereum hirsutum 56.77% 6.00e
-93
MER125099 S53.010 grifolisin Coprinus cinereus 46.44% 1.20e
-89
MER178229 S53.010 grifolisin Sclerotinia
sclerotiorum
44.78% 1.30e
-85
MER178229 S53.010 grifolisin Sclerotinia
sclerotiorum
40.80% 1.30e
-85
MER405414 S53.UPW Family S53
unassigned
peptidases
Agaricus bisporus 66.24% 1.50e
-82
MER078639 S53.010 grifolisin Grifola frondosa 45.70% 1.90e
-80
MER032166 S53.010 grifolisin Emericella nidulans 44.53% 1.10e
-79
MER030252 S53.010 grifolisin Aspergillus oryzea 43.91% 1.50e
-78
132
Table S4
Results of statistical analyses in R on normalized (relative to the housekeeping genes GAPDH and Ubc) and relative
gene expression measurements (gongylidia relative to mycelium)
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133
CHAPTER 5
CELLULOSE DEGRADATION PATTERNS IN ACROMYRMEX ECHINATIOR COLONIES
PEPIJN W KOOIJ, JEROEN W M PULLENS, JACOBUS J BOOMSMA , MORTEN SCHITT
(MANUSCRIPT)
134
Cellulose degradation patterns in Acromyrmex echinatior colonies
Pepijn W. Kooij
1
, Jeroen W.M. Pullens
1,2
, Jacobus J. Boomsma
1
, Morten Schitt
1
1
Centre for Social Evolution, Department of Biology, Copenhagen University, Universitetsparken
15, DK-2100 Copenhagen, Denmark
2
Laboratory of Genetics, Wageningen University and Research Centre, P.O. Box 309, 6700 AH
Wageningen, The Netherlands
135
Abstract
Cellulose is the dominant component in plant cell walls, but only few herbivorous animals can
digest this compound without help of microbial symbionts. Leaf-cutting ants are special in feeding
on a domesticated fungus that is provisioned with freshly harvested live plant material that the ants
chew up and mix with fecal fuid. In recent studies we have shown that fungal-derived fecal fuid
pectinases, laccases and proteases are particularly produced in the gongylidia, infated hyphal tips
that are the main food source for the ants. Here we complement these results by investigating the
presence, activity and gene regulation of a putative fungal cellulase enzyme (LgCel12) in the ant
fecal fuid. We show that this cellulase is only active in the fecal fuid when the ants have access to
their normal fungal diet, but remains less active than the fecal fuid pectinases and proteases. The
Lgcel12 gene is ca. 3-fold upregulated in the gongylidia that the ants ingest compared to normal
mycelium, consistent with overall gene expression being highest in the middle layer of fungus
gardens where most gongylidia are produced. However, overall cellulase activity was higher in
the older, bottom layers of gardens and in discarded debris material than in the upper and middle
layers of gardens. These results indicate that LgCel12 endocellulase plays a role in the initial
breakdown of plant substrate but without being a major participant in the decomposition process,
as overall cellulolytic activity is most prevalent in the bottom layer of the gardens, consisting of
material that is about to be discarded by the ants. We offer a stoichiometric interpretation of these
results emphasizing that the symbiosis between leaf-cutting ants and their garden symbiont is
almost certainly protein limited, so that the acquisition of nitrogen and phosphorus is prioritized
by the joint efforts of the mutualistic partners and excess carbon-based resources discarded in
considerable quantities.
Keywords:
Leaf-cutting ants, Leucoagaricus gongylophorus, gene expression, stoichiometry
136
Introduction
Cellulose is the dominant constituent of plant cell walls and is considered the most abundant or-
ganic compound on earth, but proteins with the capacity to degrade cellulose are encoded in the
genomes of very few animals (Watanabe and Tokuda 2001). Instead, most animals that specialize
on cellulose-rich food live in symbiosis with cellulose degrading microorganisms allowing them
to tap into this enormous nutritional resource. Symbioses of this kind are particularly prevalent
among the insects where feeding on dead wood and other plant material has evolved multiple
times independently (Breznak 1982; Douglas 2013). These gut symbiont microorganisms are usu-
ally bacteria, but protists fulfl similar roles in the lower wood-dwelling termites (Ohkuma 2003;
Noda et al. 2007). However, in some other cases, these services are provided by fungi, with gut
yeasts in several families of wood-ingesting beetles (Zhang et al. 2003; Grnwald et al. 2010),
ambrosia fungi in wood-boring Xyleborine beetles (Biedermann et al. 2009), and Termitomyces
fungi in fungus-growing termites (Aanen et al. 2002).
Also the lower attine fungus-growing ants use dead plant material to manure their fungus-gardens,
which likely implies that these fungi must have considerable cellulose degrading capacities (De
Fine Licht and Boomsma 2010). However, in contrast to any other insect-fungus symbiosis, the
attine ants evolved an evolutionarily derived lineage of active herbivores, which gained access to
vast amounts of fresh leaf substrate with an abundance of intracellular sugars and proteins that
would typically no longer be available in dead plant cells. Although the fungi of these leaf-cutting
ants have retained the capacity to degrade cellulose (De Fine Licht et al. 2010; Moller et al. 2011;
Aylward et al. 2013; Grell et al. 2013), the relative importance of cellulolytic enzymes for the
herbivorous leaf-cutting ant symbiosis has remained controversial (Abril and Bucher 2002; Erthal
et al. 2009; Nagamoto et al. 2011).
The two genera of leaf-cutting ants, Atta and Acromyrmex, live in obligate mutualistic symbiosis
with a single, genetically variable fungal species: Leucoagaricus gongylophorus (Weber 1966;
Mueller et al. 2005; Kooij et al., Chapter 1). These ants are dominant herbivores in the New World
tropics (Della Lucia et al. 2014; Kooij et al., Chapter 3, submitted) and signifcant accelerators of
nitrogen and phosphorus cycling (Fowler et al. 1989). Similar to their lower attine ant relatives
rearing saprotrophic fungus gardens, the leaf-cutting ants cultivate their symbiotic fungus in spe-
cial underground chambers, but their fungal crop belongs to a unique domesticated lineage that
has evolved special symbiotic organs, staphylae, that grow bundles of infated hyphal tips (gon-
gylidia) that provide essentially all nutrition for the ants and their brood (Quinlan and Cherrett
1978; Quinlan and Cherrett 1979; Bass and Cherrett 1995). After the larger foraging worker ants
137
have cut the leaf fragments and transported them back to the nest, smaller workers chew these
pieces into a pulp, reducing the crystallinity of the plant cellulose (Martin et al. 1991; Nagamoto
et al. 2011), and place this material on top of the fungus garden before manuring it with fecal fuid
and inoculating it with fresh tufts of mycelium.
The fecal fuid is used for the rapid maceration of cut leaves (Martin et al. 1975), and contains a
wide range of fungal plant degrading enzymes such as pectinases (Schitt et al. 2010), laccases
(De Fine Licht et al. 2013) and proteases (Kooij et al. in press). These enzymes have retained
activity in the fecal fuid after gut passage, and the genes encoding them were almost always
upregulated in the gongylidia when compared to the normal mycelium. However, gongylidia up-
regulation and fecal fuid expression for cellulases has not been investigated, as cellulases were
not particulatly common among the ca. 33 proteins found in the Acromyrmex echinatior fecal fuid
(Schitt et al. 2010). Only a single protein (LgCel12) belonging to the glycoside hydrolase family
12 (GH12) was identifed in the fecal fuid of Panamanian Acromyrmex echinatior (Schitt et al.
2010), but remained unconfrmed in a recent study on lignocellolytic enzymes in Leucoagaricus
gongylophorus from other sympatric colonies of the same ant species (Aylward et al. 2013).
Substrates targeted by the members of the GH12 enzyme family are cellulose, xyloglucan and
-1,3-1,4-glucan. As already indicated above, the importance of cellulose degradation for the
ant-fungus mutualism has been much debated (e.g. Martin and Weber 1969; Abril and Bucher
2002; Richard et al. 2005; Erthal et al. 2009; Moller et al. 2011; Nagamoto et al. 2011; Aylward
et al. 2013; Grell et al. 2013), which makes fnding a putative non-digested fungal cellulase in the
ant fecal fuid (but missed in the proteome of the fungus garden; Aylward et al. 2013) of interest,
as it may suggest that some steps in cellulose degradation in the top of fungus gardens (where
fecal fuid is deposited) may be important for the symbiosis even in low quantities. The present
study therefore set out to: 1. Measure cellulase activity in both fecal fuid and fungus gardens of
Acromyrmex echinatior, 2. Confrm that most of the cellulase activity in the fecal fuid is fungal
derived, 3. Measure gene expression levels of Lgcel12 in the different layers of the fungus garden
and in the fungal gongylidia relative to normal mycelium, and 4. Discuss the implications of our
fndings to the ongoing discussion on cellulose degradation by leaf-cutting ant fungus gardens.
Material and methods
Biological material
A total seven ant colonies of Acromyrmex echinatior (Ae226, Ae263, Ae266, Ae280, Ae322, Ae335
and Ae370), collected between 2004 and 2007 in Gamboa, Panama, were used for this study.
138
All colonies were kept under controlled conditions of ca. 25C and a relative humidity of ca. 70%,
and were fed twice a week with fresh bramble leaves (Rubus sp.), parboiled rice and pieces of ap-
ple. For enzyme activity measurements ant fecal fuid and fungus garden material were collected
from fve of these ant colonies (Ae226, Ae263, Ae266, Ae280 and Ae322).
For the collection of fecal fuid, the gaster of a large worker was gently pressed to a glass mi-
croscope slide using a forceps (Rodrigues et al. 2008). Each fecal droplet was mixed with 0.5l
demineralized ddH
2
O (the fecal fuid enzyme extract) and collected with a micropipette in an
Eppendorf tube kept on ice until further processing. Fecal fuid was collected from two different
groups of large workers in each colony, one having access to the fungus garden and thus likely
having ingested fungal gongylidia, and the other being deprived of their fungus garden and pro-
vided only with 10% sucrose water for 20 days. We collected 120 mg fungus garden material
from each of the three visible layers of fungus garden (Kooij et al. 2011), the fresh and dark top
layer, the gongylidia-rich middle layer (De Fine Licht et al. 2013) and the older bottom layer. We
supplemented this material with samples from the debris material, i.e. old fungus garden material
discarded by the ants, similar to a previous study (Kooij et al. 2011) and added 500 l of ddH
2
O
to each sample and crushed material with a pestle before vortexing and centrifuging for 15 min
(15000 g). The fungus garden supernatant and the fecal fuid extract were then kept on ice until
further processing the same day.
For gene expression measurements in gongylidia and mycelium, we collected from fve colonies
(Ae263, Ae280, Ae322, Ae335 and Ae370) ca. 100 l of gongylidia clusters (staphylae) and my-
celium in separate 2 ml Eppendorf tubes foating in liquid nitrogen using a stereomicroscope at
40x magnifcation. For gene expression measurements in the different fungus garden layers and
the debris pile, these samples were supplemented by 120 mg fungal material from colonies Ae226,
Ae263, Ae266, Ae280 and Ae322, after carefully removing any visible ants or ant parts, larvae and
eggs. All these gene expression samples were stored at -80C for RNA extraction at a later point.
Cellulase activity
Fecal fuid and fungus garden cellulase activities were measured for -glucosidase with a
pNPG-assay and for endo-1,4--glucanase with both a CMC/DNS-assay (Zhang et al. 2009) and
the AZO-CM-Cellulose-assay (Megazyme, Wicklow, Ireland). Exoglucanase assays require pu-
rifed enzymes to avoid interference with other cellulases and could therefore not be performed.
-Glucosidase activity was determined by incubating 3 l enzyme extract from fecal fuid or fun-
gus garden in 42 l mM 4-nitrophenyl--D-glucopyranoside (Sigma N7006) and 0.1 M sodium
139
acetate buffer, pH 4.8, for 30 minutes at 30C. We added 60 l 0.4 M glycine buffer, pH 10.8
and read absorbances at 430nm using a Versamax ELISA microplate reader (Molecular Devices,
Sunnyvale, USA). Absorbance measurements were converted to enzyme units using a standard
curve made with 4-nitrophenol (Sigma 241326), defning one unit as the amount of enzyme re-
quired to release 1 g nitrophenol per minute per fecal droplet or mg fungus garden.
Using the CMC/DNS-assay, endo-1,4--glucanase activity was determined by incubating 2.5 l
enzyme extract in 2 % (w/v) Carboxymethylcellulose (Sigma 21902) and 50 mM citrate buffer,
pH 4.8 for 30 minutes at 30C. A quantity of 15 l DNS solution (0.4 M NaOH, 0.04M 3,5-dini-
trosalicylic acid, 1 M potassium sodium tartrate) was added, and the mixture was heated at 99.9C
for 5 minutes in a PCR machine. Samples were put on ice to terminate the reaction and 100 l
distilled water was added, after which the absorbance at 540 nm was read in a Versamax ELISA
microplate reader (Molecular Devices, Sunnyvale, California, USA). The absorbance measure-
ments were converted to enzyme units using a standard curve made with glucose (Sigma G8270).
One unit was defned as the amount of enzyme required to release 1 g of glucose per minute per
fecal droplet or mg fungus garden.
Endo-1,4--glucanase activity was also determined using the AZO-CM-Cellulose-assay (Mega-
zyme, Wicklow, Ireland) using 50 l of enzyme extract added to 50 l pre-equilibrated substrate
solution, pH 5. The solution was vortexed and incubated for 30 minutes at 30C and the reaction
terminated by adding 250 l of precipitant solution. Samples were then vortexed and centrifuged
for 10 minutes at 1000 g, after which the supernatant was measured for absorbance at 590 nm in
the Versamax ELISA micro plate reader and converted to enzyme units using the standard curve
included in the protocol, once more defning a unit as the amount of enzyme required to release
1 g of glucose per minute per fecal droplet or mg fungus garden. All data for cellulase activity
were analysed with R (R Core Team 2013) using a Wilcoxon test for the fecal fuid data and a Kru-
skal-Wallis test for the garden-layer data followed by the multiple comparison test kruskalmc
from the package pgirmess (Giraudoux 2013).
AZCL enzyme activity assays
Fecal fuid enzyme profles were performed similar to previously published methods (Rnhede et
al. 2004; De Fine Licht et al. 2010). In short, 50 fecal droplets per colony were diluted 20x with
ddH2O and 1 ml of this suspension was applied immediately to 16 different assay-plates contain-
ing 0.1 g/l of the Azurine-Crosslinked (AZCL) substrates: amylose, arabinoxylan, barley -glu-
can, casein, chitosan, collagen, curdlan, debranched arabinan, dextran, galactan, galactomannan,
140
HE-cellulose, pachyman, pullulan, xylan and xyloglucan.
The assay-plates of 6 cm diameter were prepared separately for each substrate using an agarose
medium (1 % agarose, 23 mM phosphoric acid, 23 mM acetic acid, 23 mM boric acid) and were
pH adjusted according to the manufacturers description (Megazyme, Bray, Ireland). After the
medium had solidifed, round wells of ca. 0.1 cm were made in each plate with a cut-off pipette
tip and 10 l of the fecal fuid mixture was applied to each well in triplicate. After 22 hours of
incubation at 25C the plates were photographed and the area of the blue halo surrounding each
well (a quantitative measure for the absolute amount of enzyme activity) was measured using
the software program ImageJ ver. 1.43u for Macintosh (De Fine Licht et al. 2010; Kooij et al.
2011). Enzyme activity measurements were grouped into categories based on the main plant cell
wall target of each enzyme (Kacurkov et al. 2000 and Megazyme, Bray, Ireland): amylases
(measured with amylose), cellulases (measured with barley -glucan and HE-cellulose), hemi-
cellulases (measured with arabinoxylan, curdlan, galactomannan, pachyman, xylan and xyloglu-
can), pectinases (measured with debranched arabinan and galactan), proteases (measured with
casein and collagen), and a rest group (including the substrates chitosan, dextran and pullulan, to
measure chitosanases, dextranases and pullulanases respectively). The average halo values were
calculated for each triplicate of identical plates after which the data were log transformed and an-
alyzed with R, using Linear Mixed-Effects Models (lme) in the package nlme (Pinheiro et al.
2013) and posthoc analysis with General Linear Hypotheses (glht) in the package multcomp
(Hothorn et al. 2008). We present these data grouped for the six enzyme categories with colony
as random effect.
Protein identifcation and gene cloning
SDS-PAGE and mass spectrometry was performed as described previously (Schitt et al. 2010;
De Fine Licht et al. 2013; Kooij et al. in press). A mix of ffty fecal droplets was subjected to SDS-
PAGE, after which individual protein bands were manually excised from the gel. Proteins were
extracted from the gel plugs and digested with trypsin. De novo sequencing of the resulting pep-
tide fragments was performed on the basis of b and y fragment ions present in MS/MS spectra of
derivatized and underivatized samples. Amino acid sequences were obtained by manual analysis
of the spectra using the software program AminoCalc (Protana A/S, Odense, Denmark) as support.
The obtained amino acid sequences were used as queries in a Blast search of the assembled Ac-
romyrmex echinatior genome (Nygaard et al. 2011) and a low coverage genome sequence of the
Acromyrmex echinatior fungal symbiont (De Fine Licht et al. 2013).
141
RNA extraction and cDNA production was performed as described in Schitt et al. (2010). A full-
length gene transcript sequence of Lgcel12 was obtained using a RACE (Rapid Amplifcation of
cDNA Ends) strategy. 3 and 5-RACE libraries were made from ca. 1 g of extracted RNA with
the SMART RACE cDNA kit (CLONTECH, Mountain View, California, USA), and 3end and
5end gene sequences were PCR amplifed from these libraries using the gene specifc primers
SYT-F1 (CAG TCG ACA CTT CCC AGC ACT GTG) or SYT-R1 (GGT ATA CTG ACC AGC
AGT GAC GGT G) designed from the BLAST-search-identifed sequence reads along with the
UPM primer enclosed in the SMART RACE cDNA kit. The following PCR scheme was used in
the RACE experiments: one cycle of 95C for 5 min, 10 cycles of 95C for 20 sec, 72C for 30 sec
(with a decrease in temperature of 0.5C in every cycle) and 72C for 3 min, followed by 35 cycles
of 94C for 20 sec, 67C for 30 sec and 72C for 3 min, and ending with one cycle of 72C for 7
min. PCR products were cloned in pCR4-TOPO using the TOPO-TA cloning procedure (Invitro-
gen, Carlsbad, California, USA) and sequenced at Eurofns MWG Operon (Ebersberg, Germany).
The full length gene sequence will be deposited in GenBank after publication.
Quantitative real-time PCR
Primers for the Lgcel12 gene (Table S1) were designed by matching the obtained cDNA sequenc-
es to a partially sequenced genome of the Acromyrmex echinatior symbiont (De Fine Licht et al.
2013) using BLASTn. Matching sequencing reads were assembled and aligned to the cDNA se-
quence in question to identify intron and exon sequences. Primers were then designed to span an
intron to avoid amplifcation of genomic DNA. The qPCR was run on an Mx3000P QPCR System
(Agilent, Santa Clara, CA, USA) in a 20 l qPCR reaction (0.5 l cDNA, 10 l 2x SYBR Premix
Ex Taq [TaKaRa Bio Inc., Otsu, Japan] and 0.4 l of each primer [10M]) with the following PCR
conditions: one cycle of 95C denaturing for 2 min, followed by 40 cycles of 95C denaturing for
30 sec, 57C annealing for 30 sec, and 72C extension for 30 sec and ending with a melting curve
cycle of 95C for 30 sec, 57C for 30 sec and 95C for 30 sec.
We performed qPCR for three genes in total: the target gene Lgcel12 and two housekeeping genes
ubiquitin (Ubc, GenBank HQ174771) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH,
GenBank HQ174770) with three technical replicates for each sample. All Ct values from the
RT-qPCR analyses were analysed using R with packages ReadqPCR (Perkins and Kohl 2011)
and NormqPCR (Kohl and Perkins 2011). Average expression was calculated from the three
technical replicates of each sample for two datasets, one with Ct values for the different fungus
garden layers including the debris pile and the other with Ct values for the fungus garden myce-
lium and gongylidia.
142
Based on calculations for housekeeping gene stability with selectHKs from the NormqPCR
package, only GAPDH was used as reference housekeeping gene to determine the normalized
expression values for Lgcel12 in the fungus garden layer samples, which produced 2
Ct
values
(Schmittgen and Zakrajsek 2000) for each layer. These results were further analysed with R using
ANOVA to generate individual p-values with General Linear Hypotheses (glht) in the Simulta-
neous Inference in General Parametric Models package multcomp (Hothorn et al. 2008). For the
gene expression comparison between gongylidia and mycelium, both housekeeping genes (Ubc
and GAPDH) were used to calculate the relative expression levels of the Lgcel12 gene, resulting
in 2
-Ct
values (Livak and Schmittgen 2001).
Results
Both the Megazyme-assay (Figure 1A) and the CMC-assay (Figure 1B) for endo-1,4--glucanase
showed a substantial reduction in activity when ants had been feeding on sucrose water for 20
days (W = 23, p = 0.0159; W = 22, p = 0.0278), but low activity remained and was signifcantly
higher than zero for the CMC-assay (V = 15, p = 0.0313) but not for the Megazyme-assay (V = 10,
p = 0.0502). Also -Glucosidase activity in the fecal fuid (Figure 1C) was signifcantly different
between the two diets (W = 25, p = 0.0060), but with activity in the purged ants not being signif-
cantly different from zero (V = 6, p = 0.0907).
The cellulase activities were different among the layers of the fungus gardens with a trend of
increased activity from top to bottom/debris material (Figure 2). The Megazyme-assay for en-
do-1,4--glucanase (Figure 2A) showed signifcantly lower activity in the top and middle layer
compared to the bottom layer and debris material (overall:
2
3
= 85.979, p < 0.0001, see Figure 2
for post-hoc details). The CMC-assay for endo-1,4--glucanase (Figure 2B) showed similar re-
sults but only the activity in debris material was signifcantly higher than the middle layer (overall:

2
3
= 11.480, p = 0.0094, see Figure 2 for post-hoc details). -Glucosidase activity (Figure 2C)
showed larger differences comparable to those for endo-1,4--glucanase in the Megazyme essay,
with signifcantly higher activity in the bottom layer and debris material compared to the top layer
(overall:
2
3
= 15.789, p = 0.0013, see Figure 2 for post-hoc details).
To better understand the signifcance of the cellulase activities in the fecal fuid and be able to
compare activities to those found in earlier studies of fecal fuid pectinases (Schitt et al. 2010)
and proteases (Kooij et al. in press) we measured the overall activities of all enzymes important
for plant material degradation with AZCL enzyme assays. This showed signifcant differences
among the enzyme groups (Figure 3, F
5,70
= 68.108, p < 0.0001), with pectinases having the
143
highest activity, followed by amylases and proteases, and cellulases and hemicellulases having
lowest activities. Other enzymes targeting the substrates chitosan, dextran and pullulan showed
no activity at all.
Mass spectrometry analysis of fecal fuid from Acromyrmex echinatior (Schitt et al. 2010; De
Fine Licht et al. 2013; Kooij et al. in press) identifed a peptide sequence (SYTNLNLNANAQR)
that matched the Lgcel12 gene in the genome sequence of Leucoagaricus gongylophorus, which
harboured the amino acid sequence SYTNLNLNANLNK. The two sequences deviate in the last
three amino acids, but as both combinations gave the exact same peptide mass, this discrepancy
can be attributed to diffculties in interpreting the mass spectra. BLAST search allowed us to
Fungal diet Sucrose water
0
2
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4
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6
0
8
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Figure 1
Enzyme activities for endo-1,4--glucanase (from two different
assays) and -glucosidase in fecal fuid of ants provided with
either a normal fungus garden diet or with sucrose water for 20
days. Wilcoxon rank sum test showed a signifcant reduction in
enzyme activity for endo-1,4--glucanase (A: Megazyme assay,
W = 23, p = 0.016; B: CMC-assay, W = 22, p = 0.028) and
-glucosidase (C: W = 25, p = 0.006) when ants were deprived
of fungus garden material. Enzyme activity levels are given as
milliunits per fecal droplet.
144
assign the Lgcel12 gene to glycosyl hydrolase family 12. This protein was the only one in the
obtained fecal fuid proteome that could function as a cellulase, so we inferred that this enzyme
mediates the cellulose activity measured in the fecal droplets. Relative changes in Lgcel12 gene
expression in gongylidia versus normal mycelium (fold-changes: 2
-Ct
) showed that the gene was
signifcantly more expressed (fold change = 3.1, min max SE = 0.33 5.82) in the gongylidia
than in normal mycelium (Figure 4A, z = -6.358, p < 0.00001), suggesting that the ingestion of
this enzyme by the ants for transfer to the fecal fuid represents a mutual adaptation that somehow
enhances symbiotic effciency. In order to correlate the cellulase activity measurements in the
fungus garden with the expression of Lgcel12, we measured the normalized expression levels of
this gene (Figure 4B) in the same layers of fungus garden that were used for the activity measure-
ments (Figure 2). This showed signifcantly higher normalized gene expression in the middle layer
(F
3,16
= 3.699, p = 0.0339) compared to the other layers and the debris material, consistent with the
middle layer having the largest amount of gongylidia (De Fine Licht et al. 2013).
A
AB
B
B
0 100 200 300 400
Glucosidase activity U/g
C B
A
AB
AB
B
100 200 300 400 500 600 700
Endo-1,4--glucanase activity U/g
A
A
B
B
B
o
t
t
o
m
D
e
b
r
i
s
M
i
d
d
l
e
T
o
p
0 5 10 15 20
Endo-1,4--glucanase activity U/g
A
(Megazyme assay) (CMC-assay)
Figure 2
Enzyme activities for endo-1,4--glucanase (from two different assays) and -glucosidase in three layers of fungus gar-
den (Top, Middle and Bottom) and debris material. (A) Endo-1,4--glucanase activity (Megazyme assay) was signif-
cantly higher in the bottom layer of the fungus garden and in the debris material (overall: Kruskal-Wallis
2
3
= 85.976,
p < 0.0001). (B) Endo-1,4--glucanase activity (CMC-assay) was signifcantly higher in the debris material compared
to the middle layer (overall: Kruskal-Wallis
2
3
= 11.480, p < 0.01). (C) -Glucosidase activity was signifcantly higher
in the bottom layer and debris material compared to the top layer (overall: Kruskal-Wallis
2
3
= 15.789, p < 0.001). En-
zyme activity levels are given as units per gram fungus garden material. Different letters above bars refer to differences
in post-hoc tests at P < 0.05.
145
Discussion
Since Martin and Weber (1969) found similar cellulose degrading capabilities in the fungal sym-
biont and fecal fuid of Atta leaf-cutting ants (see also Martin et al. 1975), the joint cellulose
degradation capacity of the ant fungus-farming mutualism has remained controversial. Cellulose
degrading capabilities were later confrmed for the fungus (Bacci et al. 1995a) and for bacteria
in the fungus garden (Bacci et al. 1995b), but the level of cellulose degradation was found to be
relatively low (De Siqueira et al. 1998). The debate continued with different techniques giving
varying results, some showing that the fungus was incapable of utilizing cellulose as a substrate
(Abril and Bucher 2002; Abril and Bucher 2004; Bucher et al. 2004), but most studies indicating
mediocre (DEttorre et al. 2002; Silva et al. 2006; Erthal et al. 2009; Kooij et al. 2011; Moller et
al. 2011) or relatively high cellulose degradation capabilities (Rnhede et al. 2004; Richard et al.
2005; Schitt et al. 2008; De Fine Licht et al. 2010; Suen et al. 2010; Nagamoto et al. 2011; Ayl-
ward et al. 2013; Grell et al. 2013) without reaching consensus.
The degradation of plant cell wall cellulose is a tedious process, which involves at least three
types of cellulases in order to degrade this polysaccharide to single glucose molecules: 1. en-
do-cellulases, which open up the matrix of the cellulose fbres and cleave the fbres at random
places, 2. exo-cellulases, which cut off short cellulose fragments mainly from the ends of the
cellulose chain, and 3. -glucosidases that break down these smaller chains into single glucose
molecules (Bguin 1990; Martin et al. 1991). We show here that both endo-1,4--glucanase and
-glucosidase activity increase towards the bottom layer of the fungus garden and in the debris
0.0
0.5
1.0
1.5
2.0
2.5
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s
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s
C
Figure 3
Enzyme activity of fecal fuid for six different enzyme classes
expressed as the mean area (cm
2
SE) of coloured halos on
AZCL plates. Differences in activity between the enzyme class-
es were signifcant (F
5,70
= 68.108, p < 0.0001), with pectinas-
es being the most active, followed by amylases and proteases
and cellulases and hemicellulases being least active (different
letters indicate differences in post-hoc tests at P < 0.05). The
substrates chitosan, dextran and pullulan were grouped together
in the class other, as no activity was observed for any of these.
146
material (Figure 2), which is consistent with earlier fndings (Suen et al. 2010; Moller et al. 2011;
Aylward et al. 2013; Grell et al. 2013). Although explicit data on exocellulase activity are lacking,
this suggest that the fungus garden has the ability to convert cellulose into glucose molecules that
can be taken up as nutrients by fungal cells, but that this activity is mostly expressed towards the
end of the substrate degradation process when other more easily accessible nutrients have already
been decomposed. Although many studies have pointed out that high cellulolytic activity in the
bottom of fungus gardens may also be due to bacteria (Bacci et al. 1995b; Suen et al. 2010; Ayl-
ward et al. 2012) or yeasts (Carreiro et al. 1997; Middelhoven et al. 2003; Mendes et al. 2012), the
present study and recent results by Aylward et al. (2013) and Grell et al. (2013) are consistent in
suggesting that a signifcant fraction of cellulose decomposition in the bottom of gardens is indeed
performed by the fungal symbiont. However, this appears to be less likely for the debris material
discarded by the ants, as most of the fungal tissue should have died when the ants stop maintaining
it, so only yeasts and bacteria are left.
0
.
1
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.
2
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Top Middle Bottom Debris
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(
2
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C
t
)
A
B B
B
A B
Figure 4
Gene expression in gongylidia relative to mycelium and normalized gene expression after scaling
for the expression of housekeeping genes in fungus garden layers and debris piles. (A) The Lgcel12
gene is upregulated in gongylidia compared to mycelium with an average 3.1x fold change (0.330 -
5.819 min - max SE, z = -6.358, p < 0.00001). (B) Normalized gene expression of the Lgcel12 gene
was signifcantly higher in the middle layer of the fungus garden compared to any other part of the
fungus garden (F
3,16
= 3.699, p = 0.034). Different letters above bars refer to differences in post-hoc
tests at P < 0.05.
147
In addition to confrming cellulolytic activity towards the end of the decomposition process, our
present study shows that early stages of cellulose decomposition are an explicit target in the top
of fungus gardens. A single putative endo-cellulase enzyme (LgCel12) of fungal origin apparently
escapes degradation in the ant gut to end up in the fecal fuid where it remains active (Figure 1 and
3). Although our earlier mass spectrometry results (Schitt et al. 2010) did not indicate the pres-
ence of any -glucosidases we nevertheless found these enzymes to be active in the fecal fuid and
to originate from the fungal symbiont as well (Figure 1). The expression of the Lgcel12 gene was
also upregulated in the gongylidia relative to the normal mycelium, indicating that this enzyme
has been selected to be ingested by the ants to function either in the ant gut or in the fecal fuid,
as has also been shown for many other fecal fuid enzymes (Schitt et al. 2010; De Fine Licht et
al. 2013; Kooij et al. in press). Overall fungal expression of the Lgcel12 gene was highest in the
middle section of the gardens (Figure 4), consistent with gardens producing most of the gongylid-
ia when fungal biomass is abundant and substrate not yet depleted (De Fine Licht et al. 2013).
Endo-cellulase enzyme (LgCel12) activity in the top of gardens would likely serve the purpose of
loosening up the cell walls in the leaf pulp macerated by the ants to allow hyphal access to the cell
interior with proteins and starch (Schitt et al. 2010; Moller et al. 2011; Grell et al. 2013; Kooij
et al. in press). However, we also obtained evidence for -glucosidase activity in the fecal fuid,
which would produce free glucose from oligosaccharides that could be absorbed by the myceli-
um. Recently, more advanced mass spectrometry (Schitt et al., unpublished) has now also shown
exocellulases in the fecal fuid of A. echinatior, indicating that all three steps of cellulose degra-
dation take place in the top of fungus gardens. However, compared to the activity of other fungal
enzymes such as proteinases, pectinases and amylase, fecal fuid cellulolytic activity appears to
remain relatively low in all three steps (endocellulases, exocellulases, -glucosidases)(Figure 3).
Overall, our results suggest a scenario in which the fungal symbiont is capable to degrade cellu-
lose but only at a relatively slow rate and not primarily for maximizing glucose production for
the symbiosis. As the bottom of fungus gardens hardly produces gongylidia (De Fine Licht et al.
2013), any glucose produced here remains unavailable to the ants and is thus unlikely to beneft
the symbiosis. As suggested by Grell et al. (2013), the main function of glucose production at
this stage may be to prevent structural fungus-garden collapse and pathogen invasion until the
ants discard this otherwise depleted material. At the top of gardens modest endocellulase activity
may be required to supplement xylanase breakdown (Schitt et al. 2008) to open up plant cells.
Proportional production of exocellulases and -glucosidases at that stage would then merely make
sure that complex sugars can beneft fungal growth rather than imposing risks of bacterial growth
148
that may damage symbiotic effciency. This hypothetical framework would remain consistent with
fungus-garden activity of leaf-cutting ants being ultimately focused on maximizing protein acqui-
sition per unit of foraging and processing effort by the ants in spite of this leaving behind consid-
erable amount of non-degraded cellulose material that is discarded by the ants from the bottom
layers of fungus gardens (Schitt et al. 2008; De Fine Licht et al. 2010; Moller et al. 2011; Grell
et al. 2013; Kooij et al. in press).
This interpretation follows the general idea that decomposition of organic matter takes place in a
stoichiometric manner, which keeps ratios of liberated carbon, nitrogen and phosphorus relatively
constant and releases any surplus (non-limiting) nutrients into the external environment (Man-
zoni et al. 2010; Hartman and Richardson 2013; Sinsabaugh et al. 2013). It is well documented
that plant diets offer an excess of carbon relative to nitrogen and phosphorus (Manzoni et al.
2010; Bell et al. 2014), so that decomposers (in this case the symbiotic collaboration between
leaf-cutting ants and fungal symbiont) should be under selection to invest primarily in enzymes
for acquiring the most limiting nutrients (nitrogen and possibly phosphorus), and to adjust carbon
decomposition/liberation to the level needed for achieving stoichiometric equilibrium. Indications
of the leaf-cutting ant symbiosis to be nitrogen-limited have emanated from the demonstration of
nitrogen-fxing bacteria in the fungus gardens of Atta leaf-cutting ants (Pinto-Toms et al. 2009)
and other nitrogen conserving bacteria in the guts of both Atta and Acromyrmex leaf-cutting ants
(Sapountzis et al. in review). Depending on the effciency of these additional nitrogen preserving
bacterial symbionts, it may thus turn out that the availability of phosphorus in the plant substrate
is ultimately the most limiting factor for the leaf-cutting ant symbiosis, but much further work will
be needed to substantiate these hypotheses.
Acknowledgements
We thank the Smithsonian Tropical Research Institute (STRI), Panama, for providing logistic help and facilities to work
in Gamboa, and the Autoridad Nacional del Ambiente y el Mar (ANAM) for permission to sample ant colonies in Pan-
ama, the Danish National Research Foundation (DNRF57) and the ERC (323085) for funding.
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ACKNOWLEDMENTS
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First of all I want thank the appr. 7000 (six-legged) girls whos butt I was allowed to pinch, with-
out them this thesis would not exist.
Special thanks go out for my two supervisors, Koos Boomsma and Morten Schitt, for all their
support during the last three and a half years. You both have been great teachers and showed me
how to see results in a bigger picture. Our discussions on various subjects have inspired me to
think beyond the results as they were and refect them to the greater aspects of the science of mu-
tualisms.
Duur Aanen, without whom I would not have found the Centre of Social Evolution. My Masters
project with you on fungus-growing termites led me to a great interest in fungus-growing insect
systems and mutualisms in general. Also thank you for our work together on entangling the won-
ders of the multiple nuclei in the ant fungi.
Michael Poulsen, for your expert help on incompatibility in ant-fungi, the long hours of squeezing
ants, staring at fungi, and analyzing data. And off course thank you for the interesting discussions
we had about fungus-growing ants, fungus-growing termites, and other topics, scientifc or not.
My other co-authors, Adelina Rogowska-Wrzesinska, Peter Roepstorff and Daniel Hoffmann, for
your help and expertise on various aspects of the proteases produced by the fungus and transport
to the right place by the ants.
The Smithsonian Tropical Research Institute, Panama, for allowing me to do feld work at the
Gamboa feld station and the scientifc support there.
The Department of Biology, University of Copenhagen, for giving me this opportunity to perform
and defend my PhD.
The Danish National Research Foundation for providing part of the money for this PhD.
I want to thank Henrik, for his great supervision during my internship at the Centre, which insti-
gated me to apply for a PhD position here. Also thanks for the various discussions we had on the
multitude of topics of fungus-growing ants.
My offce mate, Luigi. I had a great time sharing my offce with you these three and a half years,
155
from interesting discussions on politics, our both research topics or when to put up our Christmas
decorations.
Bernhardt and Luigi, for the long evenings of shouting at each other while playing FIFA, and still
fnd time together to discuss science.
Panos and Saria, for the after work beers at the end of these three years as a nice break in between
the writing, and reminding me there are other topics in science then the ones described in this
thesis.
And of course, Sylvia and Louise, the best lab-techs in the world! You both deserve a special place
here. You help has been fantastic! Especially to Sylvia, for listening to my whining about dirty
labs over and over and for the delicious banana-chocolate cakes.
My parents, Jos and Jos, for making it possible for me to study and make it possible for me to
stand where I stand today. And my parents as well as my brothers and sisters, Taco, Eelke, Sanne
and Jojanneke (and Jalke and Avine) for their support during these years.
My new family in Colombia, Edgar, Gladys, Paula and Juan, for welcoming me in their midst and
their support.
My friends who I left in the Netherlands or who moved away themselves, Remco&Dorine,
Roos&Freek, Silvan&Zuuz, Marijke, Jan-Willem, Ezra, Fam&Chris, Liset and others I might
have forgot. As well as all the new friends I found, Kostis&Laetitia, Claudia&Francesco, Danka,
Lilia&Martin, Rasmus, Marlene, Jesper, Caya, Vale, Lina&Lina, Alex, Vicky, and everyone else.
And everyone else at the Centre for Social Evolution, and the Section for Ecology and Evolution
in Copenhagen, Denmark, and the Laboratory of Genetics in Wageningen, The Netherlands.
Poes, welcoming me every morning with a happy meow and for keeping my lap warm while
writing.
And last but not least, Andrea, mi amor. Without this PhD we would probably never have met, and
without you this PhD would have been the most boring ever. You bring the light in my life, and I
will always be grateful for all your support and love during this years! I love you.