Fungal adaptations to mutualistic life with ants

155 Pages·2014·2.13 MB·English

faculty of science
university of copenhagen
centre for social evolution
department of biology
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 Schiøtt
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
P
reface
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
Schiøtt. During the project I spent five 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 fieldwork 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 five 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.
Pepijn W. Kooij
Copenhagen, December, 2013

c
ontents
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 influence 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
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s
ummaries
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s
ummary
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 flower material and herbal plant material. This difference is reflected in the overall
enzyme activity patterns in the fungus gardens, with Atta specializing more on specific 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 fluid 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.
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r
esumé
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) spænder over paleo- og basale arter med få hundrede arbejdere der fouragerer på tørret plantemateriale
og insekt ekskrementer, til store kolonier af bladskærermyrer med flere tusinde til millioner af arbejdere
der indsamler friskt plantemateriale til deres svampehaver. Bladskærermyrer er den dominante herbivor i
neotroperne hvor de bidrager væsentligt til nitrogen og fosfor kredsløbet i deres nærmiljø og bliver yder-
mere betragtet som et skadedyr idet de har stor effekt på landbruget. Disse faktorer gør det ideelt at benytte
bladskærermyrer til bedre at forstå mekanismerne der har gjort denne mutualistiske symbiose så succesfuld.
For at forstå 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 højere
udviklede svampedyrkende myrer og bladskærermyrerne. Ydermere kunne jeg estimere at ca. halvdelen af
disse kerner havde forskellige genomer hvilket gav svampen et ploidi niveau på 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 grundlægge nye kolonier, men i sjældne tilfælde
kan denne svampesymbiont komme i kontakt med symbionten fra en anden koloni. Jeg påviste at i begge
bladskærermyrerslægter sørger inkompatibilitets reaktioner imellem varianter af svampe for, at symbionter
ikke bliver blandet og at dette styrkes når den genetiske variation forøges. Dette mønster bliver dog for-
vrænget når svampesymbionter fra forskellige slægter af myrer testes imod hinanden.
Den vigtigste mekanisme i successionen af denne mutualisme hos bladskærermyrer er den kontrol-
lerede nedbrydning af plantemateriale. Jeg påviser at i området omkring Gamboa, Panama, fouragerer de
to bladskærermyrerslægter på forskelligt plantemateriale, Atta er specialiseret på træblade og Acromyrmex
mere på blomster og urter. Denne forskel reflekteres i enzymaktiviteten i svampehaverne, hvor Atta er spe-
cialiseret indenfor specifikke grupper af enzymer og Acromyrmex overordnet har en høj enzymaktivitet.
Endelig viser jeg, at svampesymbionten hos bladskærermyren Acromyrmex echinatior producer-
er store mængder bionedbrydende enzymer i specielle strukturer kaldet gongylidia. Myrerne spiser disse
strukturer, men enzymerne passerer ufordøjet igennem tarmsystemet og ender i myrernes fækalievæske
som de blander med friskt indsamlet plantemateriale og placerer øverst 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.
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r
esumen

Las hormigas atinas se alimentan de un hongo que cultivan en simbiosis mutualista en cámaras subterráneas al que
proveen con sustrato colectado fuera de la colonia. La tribu Attini presenta colonias que oscilan en tamaño desde las
pequeñas 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 herbívo-
ros dominantes del Neotrópico, y contribuyen extensamente a los ciclos de nitrógeno y fósforo 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 sucesión de la domesticación a través de la filogenia de las hormigas Attini, se comparó el
número promedio de núcleos por célula para los simbiontes fúngicos 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 domesticación, tienen dos núcleos por célula; lo estándar para hongos basidio-
micetos. Sin embargo, este número incrementa a un rango de 7 a 17 núcleos por célula en los hongos altamente domes-
ticados de los grupos más recientes de hormigas atine y cortadoras de hojas. Asimismo se estimó que aproximadamente
mitad de estos núcleos son representados por diferentes genomas, dándole al hongo un nivel de ploidía de 5n-6n.
En simbiosis mutualistas, es importante que las partes sean fieles entre sí. 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 fúngicos puede entrar en contacto con simbiontes de tras colonias. Aquí demostramos que en
ambos géneros 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 genética. Sin
embargo, este patrón se distorsiona cuando los simbiontes fúngicos se oponen a través de los géneros de hormigas.
El mecanismo más importante en la sucesión de este mutualismo de las hormigas cortadoras de hojas es la de-
gradación controlada del material vegetal. Se encontró que en el área de Gamboa, Panamá, los dos géneros de hormigas
cortadoras de hojas forrajean material vegetal bastante diferente; las especies Atta se especializan en hojas de árboles
y las Acromyrmex en material de flores y hierbas. Esta diferencia se refleja en los patrones generales de la actividad
enzimática en los cultivos de hongos, donde Atta se enfoca más en grupos enzimáticos especializados y Acromyrmex
posee una alta actividad enzimática generalizada.
Finalmente, en este estudio demostramos que el simbionte fúngico 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 través del sistema digestivo de la hormiga,
aunque sin ser digeridas. Luego, estas son excretadas en el líquido fecal, el cual las hormigas mezclan con el material
vegetal fresco que ha sido recientemente colectado y ubicado en la parte superior del jardín de hongo. Las enzimas
permanecen activas y por lo tanto juegan un papel importante en la biodegradación del material vegetal. Con esto dem-
uestro que el hongo evolucionó adaptaciones extraordinarias para una vida mutualista con las hormigas.
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