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 T. Nobre, P. Eggleton and D. K. Aanen Madagascar by fungus-growing termites?Vertical transmission as the key to the colonization of  

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* Autho

Electron1098/rsp

doi:10.1098/rspb.2009.1373

Published online 14 October 2009

ReceivedAccepted

Vertical transmission as the keyto the colonization of Madagascar

by fungus-growing termites?T. Nobre1,*, P. Eggleton2 and D. K. Aanen1

1Laboratory of Genetics, Wageningen University and Research Center, Droevendaalsesteeg 1,

Radix West, Building 107, 6708 PB Wageningen, The Netherlands2Soil Biodiversity Group, Entomology Department, The Natural History Museum,

Cromwell Road, London SW7 5BD, UK

The mutualism between fungus-growing termites (Macrotermitinae) and their mutualistic fungi

(Termitomyces) began in Africa. The fungus-growing termites have secondarily colonized Madagascar

and only a subset of the genera found in Africa is found on this isolated island. Successful long-distance

colonization may have been severely constrained by the obligate interaction of the termites with fungal

symbionts and the need to acquire these symbionts secondarily from the environment for most species

(horizontal symbiont transmission). Consistent with this hypothesis, we show that all extant species of

fungus-growing termites of Madagascar are the result of a single colonization event of termites belonging

to one of the only two groups with vertical symbiont transmission, and we date this event at approximately

13 Mya (Middle/Upper Miocene). Vertical symbiont transmission may therefore have facilitated long-

distance dispersal since both partners disperse together. In contrast to their termite hosts, the fungal

symbionts have colonized Madagascar multiple times, suggesting that the presence of fungus-growing

termites may have facilitated secondary colonizations of the symbiont. Our findings indicate that the

absence of the right symbionts in a new environment can prevent long-distance dispersal of symbioses

relying on horizontal symbiont acquisition.

Keywords: symbiont transmission mode; long-distance dispersal; mutualism;

Microtermes; Termitomyces

1. INTRODUCTIONThe establishment of a mutualistic relationship between

two species allows both species to find a faster adaptive

solution to a problem than would be possible on their

own, owing to the synergistic combination of their

traits (Maynard Smith & Szathmary 1997). However,

obligate mutualistic interactions also constrain dispersal,

as that requires either that both partners disperse together

or that they are able to find each other after independent

dispersal. Here, we explore long-distance dispersal to

the island of Madagascar by a mutualistic symbiosis

between insects and fungi: macrotermitine termites and

Termitomyces fungi.

The mutualism between fungus-growing termites and

their fungal symbionts originated in Africa (Aanen &

Eggleton 2005) and is obligate for both partners (Wood &

Thomas 1989; Aanen et al. 2002; Froslev et al. 2003).

The termites (Isoptera, Termitidae, Macrotermitinae)

provide a constant, highly regulated, growth environment

for the fungi, and the fungi (genus Termitomyces) provide

the termites with a processed food source. Entering the

symbiosis has allowed the fungi to overcome unfavourable

ecological conditions (such us temperature and moisture

fluctuations) and the termites to efficiently exploit

complex and diverse plant substrates (such as

r for correspondence (tania.mesquitanobre@wur.nl).

ic supplementary material is available at http://dx.doi.org/10.b.2009.1373 or via http://rspb.royalsocietypublishing.org.

31 July 200918 September 2009 359

lignocellulose). The Termitomyces fungi are reared on a

special structure, the fungus comb, built from dry dead

plant material that has been chewed and quickly passed

through the termite gut. Within a nest, a single strain

monoculture of Termitomyces is present and the fungal

propagation is always asexual, via vegetative spores

(Leuthold et al. 1989; Aanen 2006; Aanen et al. in press).

In contrast to this asexual fungal propagation within a

colony, the between-nest fungal symbiont transmission

mode is horizontal for most species of fungus-growing

termites. This means that, to establish the fungus garden

of a new colony, foraging workers have to collect

Termitomyces sexual spores (actively or passively) from the

environment around the nest (Korb & Aanen 2003).

These spores (basidiospores) are produced from sexual

fruiting bodies (basidiocarps or mushrooms) that

occasionally arise from nodules and are wind-dispersed.

This mode of symbiont transmission is likely to rep-

resent the ancestral state (Aanen et al. 2002). Only two

exceptions are known where the termites show vertical

transmission of their symbiont (i.e. where Termitomyces

vegetative spores are transported in the gut of alates

from a parental colony and used to inoculate the fungus

comb of the newly founded colonies). This vertical trans-

mission mode is uniparental in the fungus-growing

termites: (i) via the female alate in Microtermes species

and (ii) via the male alate in the species Macrotermes

bellicosus (Grasse & Noirot 1955; Johnson 1981; Johnson

et al. 1981; Aanen et al. 2002).

This journal is q 2009 The Royal Society

360 T. Nobre et al. Origin of Malagasy termite fungiculture

on May 3, 2010rspb.royalsocietypublishing.orgDownloaded from

We hypothesize that horizontal transmission presents

a severe constraint on long-distance colonization as it

implies that the right fungal symbiont must be available

in the new environment and that it is acquired by the

termite colonizer upon arrival. Long-distance dispersal

is a stochastic process and the likelihood that the

disperser arrives, survives and reproduces to establish

a new population is even smaller if the two symbiotic

partners start the journey independently. Dispersing as

a unit (with vertical symbiont transmission) is thus

expected to enhance the chance of successful

colonization.

A further challenge for successful colonization by a

sexually reproducing species is the need of mate-finding

after dispersal. Termites provide a clear contrast to

other social insects in this respect. For example, in the

fungus-growing ants, mate-finding after dispersal is not

a critical constraint as the female disperses and founds a

nest on her own, after having been inseminated. The

case of the fungus-growing ant population of Guadeloupe

is a good example, as it seems to have been founded by a

single introduction of a lone multiply mated female, or

perhaps a group of sisters (Mikheyev 2008). However,

in the case of fungus-growing termites, finding a suitable

mate in the new environment poses an additional severe

constraint, as colonies are founded by a pair of

reproductives.

Several successful out-of-Africa migrations by fungus-

growing termites have occurred. Only 3 of the 11

genera of fungus-growing termites were previously

reported in Madagascar: Odontotermes, Ancistrotermes

and Microtermes (Eggleton & Davies 2003). Madagascar

broke off from Gondwana around 165 Ma, reached its

current position with respect to Africa at least 118 Ma

and the most widely accepted date for the breaking off

from India is 80 Ma (Yoder & Nowak 2006). While the

proximity to Africa has allowed multiple colonizations,

the distance separating these two landmasses, and the

prevalent ocean currents that minimize transport on float-

ing trees and other flood debris, has meant that

colonizations have been rare, allowing Madagascar to

develop a highly distinctive flora and fauna. The extant

flora and fauna of Madagascar is a mixture of groups

resulting from ancient vicariant events and groups orig-

inating from transoceanic dispersal (Monaghan et al.

2005; Kodandaramaiah & Wahlberg 2007; Orsini et al.

2007). For fungus-growing termites, previous work

suggests that their presence on Madagascar is not due

to vicariance but to secondary transoceanic dispersal

(Aanen & Eggleton 2005). This is also consistent with the

estimates obtained by Brandl et al. (2007), who dated the

origin of fungus-growing termites to around 62 Ma (30–

108 Myr as credibility interval). Given this age estimate,

all extant genera have evolved after the separation of

Madagascar from Africa and probably even from India.

Here we investigate (i) the number and timing of

colonization events of Madagascar by fungus-growing

termites and their fungal symbionts, and (ii) the degree

of co-speciation between termite and fungus colonizers.

The presence of fungus-growing termites on this isolated

island provides us with a unique opportunity to study

long-distance dispersal by organisms living in obligate

symbiosis and showing variation in symbiont transmission

mode.

Proc. R. Soc. B (2010)

2. MATERIAL AND METHODS(a) Taxa sampling and data collection

Fungus-growing termites were sampled in South Africa and

Madagascar, at a range of locations (see table S1, electronic sup-

plementary material) between 2000 and 2006. Most of the

Malagasy samples consisted of foragers collected from dead

wood while feeding in closed-canopy forest habitats, while

most of the South African and some of the Malagasy samples

were retrieved directly from the nest. Upon collection, they

were preserved in 100 per cent alcohol.

Termite workers were allowed to dry on filter paper

prior to DNA extraction. DNA was extracted separately

from individual heads and abdomens using the Chelex

method. The DNA extracted from the head capsules was

used for the termites’ analyses and, as no fungus combs were

sampled, Termitomyces DNA was obtained from the termites’

abdomen. All extraction products were stored at 2208C.

(b) Termite sequences and phylogenetic analyses

The mitochondrial gene cytochrome oxidase subunit 1 (COI)

and part of the nuclear ribosomal internal transcribe spacer

(ITS2) region (Jenkins et al. 2001; Aanen et al. 2002) were

amplified using a standard PCR reaction (for details, see elec-

tronic supplementary material). All PCR products were then

purified using the GenElute PCR clean-up kit (Sigma) and

directly sequenced with the amplification primers. Sequencing

was performed by MWG Biotech Germany.

The alignment of COI was straightforward, as no insertions/

deletions had to be inferred. For the ITS2 region an iterative

refinement method, which accounts for larger gaps in the

sequences (E-INS-i) as implemented in the program MAFFT

(Katoh & Toh 2008) was used to align the sequences. The

optimal model of evolution was selected using MRMODELTEST

v. 2.2 (Posada & Crandall 1998). Bayesian inference was

conducted using MRBAYES v. 3.0 (Huelsenbeck & Ronquist

2001; Ronquist & Huelsenbeck 2003). The default settings of

MRBAYES were used and with Markov chain Monte Carlo

(MCMC) runs being repeated twice as a safeguard against

spurious results. The first 1000 trees were discarded as

burn-in, and the remaining trees were used to calculate a

majority rule consensus tree. Stationarity was confirmed by

analysis of the log likelihoods and the consistency between runs.

We used a relaxed molecular clock to estimate the age of

the Malagasy fungus-growing termite node(s) by using the

penalized likelihood function implemented on the program

R8S v. 1.7 (Sanderson 2002). The analysis was based on

the topology of the majority rule consensus COI tree of the

trees sampled in the Bayesian analysis, with non-fungus-

growing termite taxa being pruned, prior to age calculations.

The Odontotermes node was constrained to a minimum age of

7 Ma according to the dating of the fossilized fungus comb

reported in Duringer et al. (2007). A second constrained

node was used simultaneously, with a minimum age of

3.4 Ma, and corresponding to the age for the most recent

common ancestor (MRCA) of Macrotermes jeanneli according

to Darlington (2005). The truncated Newton algorithm was

employed and a coarse scale cross-validation (with par-

ameters n1 ¼ 0, n2 ¼ 0.3 and k ¼ 12) was undertaken to

select the optimal smoothing parameter. The mean node

age and its s.d. were calculated using 50 non-parametric

bootstrap replicates of the dataset generated by PHYLIP

v. 3.68 (Felsenstein 1989), followed by re-estimation of

branch lengths and divergence times for each replicate

(Sanderson 2002).

Origin of Malagasy termite fungiculture T. Nobre et al. 361

on May 3, 2010rspb.royalsocietypublishing.orgDownloaded from

(c) Termitomyces sequences and

phylogenetic analyses

Part of the nuclear ribosomal region—including the first

internal transcribed spacer (ITS1), the 5.8S RNA gene and

the second internal transcribed spacer (ITS2)—and the

nuclear 25S RNA gene were amplified in a standard PCR

reaction, using the ITS1FT and 25S4R primers described

in Aanen et al. (2007; for details, see electronic supplemen-

tary material). Samples were selected for analysis on the

basis of the host COI-based phylogeny (i.e. a minimum of

two samples per clade were chosen for Termitomyces analysis).

All desired PCR products were then purified using the Gen-

Elute PCR clean-up kit (Sigma). Purified PCR products

were directly sequenced with both amplification primers.

For some Termitomyces strains, two copies were present in

the PCR products, differing by a small length mutation,

which has been found previously (De Fine Licht et al.

2005). Sequencing was performed by MWG Biotech

Germany, using both forward and reverse primers, and,

whenever needed, the intermediate primers ITS4 (White

et al. 1990) and/or its reverse complement.

Sequences were aligned in MAFFT (Katoh et al. 2005)

under the option L-INS-I (iterative refinement method

incorporating local pairwise alignments; gap opening penalty:

1.5 and gap extension penalty 0.14; 1PAM/k ¼ 2 scoring

matrix for nucleotide sequences). Added to the dataset were

sequences of Termitomyces symbionts of several Macrotermiti-

nae (table S2, electronic supplementary material) from Aanen

et al. (2002), whenever both ITS and 25S sequences were

available from the same specimen or the specimen was still

available for further analysis. A representative of each

Termitomyces haplotype found in Aanen et al. (2007) was

also included. The MCMC ran according to the models

inferred with MRMODELTEST and using the default settings of

MRBAYES as described for the termite analysis.

3. RESULTS AND DISCUSSION(a) The colonization of Madagascar by

fungus-growing termites and their fungi

The fungus-growing termites of Madagascar are mono-

phyletic on our tree (99% posterior probability), nested

within the genus Microtermes (rooted majority rule consen-

sus Bayesian tree; figure 1a). This analysis was based on the

aligned dataset, for 108 fungus-growing termites taxa, of

929 bp of the COI region. The monophyly of the Malagasy

Microtermes based on the mitochondrial COI gene, which is

inherited via the maternal lineage only, is corroborated by

the biparentally inherited ITS gene tree (figure 1b; total

of 298 positions sequenced). Consistent with the different

inheritance patterns of COI and ITS2, the tree topologies

based on these genes are not totally congruent (e.g. the

non-sister clades I and V in the COII tree form a monophy-

letic group with no substructure in the ITS2 tree), although

the intergeneric relationships are kept.

This result shows that the fungus-growing termites of

Madagascar all originate from a single colonization

event, estimated to have occurred at 13.18 Ma (boot-

strapped mean: 11.04+7.77 Ma). Given Madagascar’s

geographical history, with the last conceivable connection

to other landmasses being dated from 80 Ma (Yoder &

Nowak 2006), the current presence of Malagasy fungus-

growing termites must be the result of long-distance

dispersal and not of vicariance. The colonization of

Proc. R. Soc. B (2010)

Madagascar occurred in the Upper/Middle Miocene, a

time in which overseas dispersal would have been necess-

ary. Our finding that all extant species of fungus-growing

termites on Madagascar belong to a single clade within

the genus Microtermes contradicts the previous report

based on morphology that Odontotermes and Ancistro-

termes species are also present on the island (Eggleton &

Davies 2003). Whether the fungus-growing termites

from Madagascar originate from a single female, a

group of sisters or from multiple co-founding reproduc-

tives from a single species can not be inferred from the

present data.

In contrast to their termite hosts, the Termitomyces

symbionts of Madagascar are not monophyletic based

on our reconstructed phylogeny (figure 2). Moreover,

the degree of co-speciation between the two partners is

very low. These findings confirm earlier results showing

that specificity exists mainly at the generic level but not

within genera (Aanen et al. 2002, 2007). Termitomyces

symbionts associated with Malagasy Microtermes belong

to three different clades and have in general identical or

very similar haplotypes to known continental African

symbionts (figure 2): (I) a large well-supported cluster

containing the majority of the Madagascar haplotypes

belonged to a clade together with three South African

samples; (II) the only exclusively Madagascar clade is

closely related to South African haplotypes; and, finally,

(III) a single Madagascar haplotype is very similar to

South African representatives. Coalescence theory predicts

that ancestral haplotypes will be the most frequent

sequences sampled in a population-level study (Crandall &

Templeton 1993; Posada & Crandall 2001). If predictions

from coalescence theory are to be applied to this particular

case, then the first cluster (I) may represent the ancestral

Termitomyces. However, this clade has a low diversity,

which argues against this interpretation.

The generally low level of differentiation observed

between Termitomyces from Madagascar and from conti-

nental Africa is striking. Similar results (i.e. nearly

identical sequences of mixed Malagasy and continental

African origin) were also observed on a molecular phylo-

genetic study of fungi of the genus Lactarius in

Madagascar (Buyck et al. 2007). The very low endemism

observed for both fungi suggests regular exchange

between Madagascar and continental Africa preventing

strong genetic differentiation. We can infer a minimum

of three transoceanic migrations of the fungal symbionts

of Microtermes onto Madagascar to explain the recon-

structed Termitomyces phylogeny. The occurrence of

several dispersion events of Termitomyces into Madagascar

is not surprising, given what is known, mainly from

studies on plant pathogens, about long-distance dispersal

of fungi (Nagarajan & Singh 1990; Brown & Hovmoller

2002; Viljanen-Rollinson et al. 2007). Mainly the wind,

but also water, animals or plants can act as passive

spore dispersers.

(b) The significance of vertical transmission

for long-distance colonization

In all studied Microtermes species, the female reproductive

ingests conidia (asexual spores) from the comb before

leaving the nest and transports the conidia in her digestive

tube (Johnson 1981). This means that a colony can be

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362 T. Nobre et al. Origin of Malagasy termite fungiculture

Proc. R. Soc. B (2010)

on May 3, 2010rspb.royalsocietypublishing.orgDownloaded from

746188 Microtermes sp.

673064 Microtermes sp.

T55c Microtermes sp.

dka93 Odontotermes latericiusZA22 Odontotermes latericius

dka13 Odontotermes latericiusdka20 Odontotermes latericius

ZA51 Odontotermes transvaalensisZA19 Odontotermes badius

ZA158 Odontotermes badiusZA17 Odontotermes transvaalensisZA157 Odontotermes badius

ZA159 Odontotermes badiusZA7 Odontotermes badiusdka367567 Protermes minutusZA55 Odontotermes transvaalensis

dka28 Odontotermes latericiusZA23 Odontotermes latericius

ZA20 Odontotermes latericiusdka15 Odontotermes latericiusZA50 Odontotermes latericiusZA21 Odontotermes latericius

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673420 Microtermes sp.

673435 Microtermes sp.673449 Microtermes sp.746186 Microtermes sp.

746299 Microtermes sp.673060 Microtermes sp.673054 Microtermes sp.673068 Microtermes sp.677145 Microtermes sp.

673434 Microtermes sp.746163 Microtermes sp.673071 Microtermes sp.677124 Microtermes sp.673098 Microtermes sp.T42k Microtermes sp.ZA746340 Microtermes sp.ZA5 hap20 Microtermes sp.

100

ZA138 hap21 Microtermes sp.

746346 Allodontermes sp.

69

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99

dka38 Microtermes sp.dka103 Microtermes sp.

dka119 Synacanthotermes heterodonZA126 hap22 Microtermes sp.

dka46 Ancistrotermes cavithoraxdka69 Ancistrotermes crucifer

T40b Microtermes sp.

ZA34 hap23 Microtermes sp.ZA25 hap24 Microtermes sp.

100

9997100

I

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673452 Microtermes sp.

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ZA136 Macrotermes natalensisdka50 Macrotermes bellicosus

dka8 Macrotermes bellicosusdka14 Macrotermes bellicosusdka22 Macrotermes bellicosus

dka5 Macrotermes subhyalinusdka65 Macrotermes bellicosusdka19 Macrotermes bellicosusdka52 Macrotermes bellicosus

100

100

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100

8199

I

II

III

Figure 2. Phylogenetic relationship of the Termitomyces sym-bionts of Microtermes included in this study and from Aanenet al. (2002, 2007; for details, see table S3, electronic sup-plementary material). The phylogeny was rooted using the

free-living fungus Lyophyllum semitale (GenBank no.AF357049 and AF042581) as outgroup. The numbersabove the branches refer to the Bayesian posterior probabilityof the nodes (more than 50%) and were derived from 19 500Markov chain Monte Carlo-sampled trees. Scale bar, 0.5.

Origin of Malagasy termite fungiculture T. Nobre et al. 363

on May 3, 2010rspb.royalsocietypublishing.orgDownloaded from

established even if no fruiting fungus is present near the

new colony. Presumably, vertical transmission therefore

reduces the risk of fungus comb failure compared with

most species of fungus-growing termites, which rely on

the presence of spores in the environment of the new

nest. Our results are compatible with the hypothesis

that vertical transmission provided an advantage for

long-distance colonization. However, as we have docu-

mented only one colonization event, we cannot say that

the observed colonization by Microtermes does any more

than provide a single example of support for the vertical

transmission hypothesis.

Nonetheless, we can more generally hypothesize that

Microtermes species with vertical transmission were readily

able to disperse within Madagascar, and found there free

niche space that has allowed them to diversify on the

Proc. R. Soc. B (2010)

island into Ancistrotermes-like and Odontotermes-like mor-

photypes. This would explain the claims that members

of these genera occur on Madagascar (Eggleton & Davies

2003). It is well accepted that, given a suitable unoccu-

pied environment, the success of any given natural

colonization event will depend largely on the availability

of ecological space (Gillespie & Roderick 2002). The

hypothesis that Microtermes has undergone an adaptive

radiation on Madagascar needs to be tested in future

studies using detailed morphological data.

Even though all of the five species of Microtermes

studied so far have vertical transmission, earlier studies

(Aanen et al. 2002, 2007) have indicated that occasional

events of horizontal transmission also occur in the

genus Microtermes. Such occasional horizontal trans-

mission must explain the present result that multiple

non-sister lineages of Termitomyces are associated with

the fungus-growing termites of Madagascar.

Long-distance dispersal is an important strategy and the

ability of fungal spores to use atmospheric pathways for

rapid spread into new areas is a key factor for fungal suc-

cess (Viljanen-Rollinson et al. 2007). However, a severe

constraint for Termitomyces to be established on Madagas-

car (and thus to colonize it) is the need of spores to

colonize fungus gardens: presumably aided by foraging ter-

mites, the spores need to be brought into the colony and

become established in the fungus comb. A recent study

shows that positive frequency-dependent selection leads

to single-strain monocultures of Termitomyces within

single nests and prevents subsequent colonization by

other strains (Aanen et al. in press). Presumably, the

establishment of a fungus comb by a new, horizontally

transmitted, symbiont strain is therefore most likely to suc-

ceed in the initial stages of the colony formation, when the

fungus still has a low biomass. Future experimental studies

should focus on the details of symbiont transmission and

the frequency of horizontal transmission in this group of

fungus-growing termites.

4. CONCLUSIONSWe provide strong evidence that all extant fungus-growing

termites on Madagascar result from a single overseas

colonization event. This colonizer belongs to one of the

only two clades within fungus-growing termites known

to have vertical symbiont transmission, suggesting that

transmission mode is a relevant pre-adaptation for long-

distance dispersal of obligate symbioses. Vertical trans-

mission couples the dispersal of the partners, thereby

maximizing the probability of associating with a suitable

partner species. In contrast to the single colonization

event by their termite hosts, the symbiotic fungi have

repeatedly colonized Madagascar secondarily, suggesting

that the initial termite colonizer has facilitated subsequent

colonizations of fungal symbionts. Our findings indicate

that the absence of the right symbionts in a new environ-

ment can be a limiting factor for the success of

long-distance dispersal of symbioses that rely on

horizontal symbiont acquisition.

This research was supported by a Marie Curie Intra-European Fellowship within the 7th European CommunityFramework Programme (T.N.; IEF Project No. 220077)and by the Netherlands Organization for ScientificResearch (D.K.A.; VIDI).

364 T. Nobre et al. Origin of Malagasy termite fungiculture

on May 3, 2010rspb.royalsocietypublishing.orgDownloaded from

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