Molecular Ecology (2006) 15, 3131– 3138
doi: 10.1111/j.1365-294X.2006.03008.x
Presumptive horizontal symbiont transmission in
the fungus-growing termite Macrotermes natalensis
Blackwell Publishing Ltd
H . H . D E F I N E L I C H T , J . J . B O O M S M A and D . K . A A N E N *
Department of Population Biology, Institute of Biology, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen, Denmark,
*Laboratory of Genetics, Wageningen University and Research Center, the Netherlands
Abstract
All colonies of the fungus-growing termite Macrotermes natalensis studied so far are
associated with a single genetically variable lineage of Termitomyces symbionts. Such
limited genetic variation of symbionts and the absence of sexual fruiting bodies (mushrooms) on M. natalensis mounds would be compatible with clonal vertical transmission, as
is known to occur in Macrotermes bellicosus. We investigated this hypothesis by analysing
DNA sequence polymorphisms as codominant SNP markers of four single-copy gene
fragments of Termitomyces isolates from 31 colonies of M. natalensis. A signature of free
recombination was found, indicative of frequent sexual horizontal transmission. First, all
31 strains had unique multilocus genotypes. Second, SNP markers (n = 55) were largely in
Hardy–Weinberg equilibrium (90.9%) and almost all possible pairs of SNPs between
genetically unlinked loci were in linkage equilibrium (96.7%). Finally, extensive intragenic
α fragment. Substantial genetic variation
recombination was found, especially in the EF1α
and a freely recombining population structure can only be explained by frequent horizontal
and sexual transmission of Termitomyces. The apparent variation in symbiont transmission
mode among Macrotermes species implies that vertical symbiont transmission can evolve
rapidly. The unexpected finding of horizontal transmission makes the apparent absence
of Termitomyces mushrooms on M. natalensis mounds puzzling. To our knowledge,
this is the first detailed study of the genetic population structure of a single lineage of
Termitomyces.
Keywords: mutualism, population structure, recombination, symbiont transmission, symbiosis,
Termitomyces
Received 18 December 2005; revision received 12 April 2006; accepted 2 May 2006
Introduction
Fungus-growing termites (Macrotermitinae: Termitidae:
Isoptera) live in an obligate symbiosis with fungi of
the genus Termitomyces (Basidiomycota). The fungus is
cultured on special structures inside the termite nest
(fungus combs) and depends on the termites for substrate
provisioning and protection. The comb is constructed from
finely fragmented plant material, which is inoculated with
asexual spores of the fungal symbiont (Leuthold et al.
1989). After a few weeks, the inoculated fungus comb
produces nitrogen-rich basidiocarp primordia (nodules)
which contain conidia with asexual spores. The mycelium
Correspondence: Henrik H. De Fine Licht, Fax: 45 35321250,
E-mail: hhdefinelicht@bi.ku.dk.
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd
at the surface, the nodules with asexual spores, and (later)
the entire comb are eaten by the termites (Rouland-Lefevre
2000; Rouland-Lefevre & Bignell 2002; Hyodo et al. 2003).
The few available studies of intranest genetic diversity of
symbionts of the genus Termitomyces have shown that the
fungus is kept as a single-strain monoculture within a
single colony (Aanen et al. 2002; Katoh et al. 2002; Shinzato
et al. 2005). This may be due to vertical uniparental transmission, which implies that either the female or the male
reproductive brings fungal mycelium and asexual conidiospores from the natal colony when dispersing, and
that this inoculum is used to establish a new fungus garden
after pair formation. Such vertical transmission would
thus be associated with clonal reproduction of the
Termitomyces fungus (Korb & Aanen 2003). However, the
alternative, horizontal symbiont transmission, is much
3132 H . H . D E F I N E L I C H T , J . J . B O O M S M A and D . K . A A N E N
more widespread in the fungus-growing termites (Korb &
Aanen 2003), and is almost certainly the ancestral transmission mode (Aanen et al. 2002). Horizontal transmission
depends on fruiting bodies (mushrooms) growing out of
the exterior of the termite mound to release spores. Uniparental vertical transmission has only been observed in
species of the termite genus Microtermes (always via the
female in the five species studied) and (via the male) in the
species Macrotermes bellicosus (Johnson et al. 1981). However, other Macrotermes species [M. subhyalinus ( Johnson
et al. 1981) and M. michaelseni (Sieber 1983)] have been
shown to acquire their fungal symbiont horizontally and
the same is true for a few studied species of Ancistrotermes,
Pseudoacanthotermes and Odontotermes (Korb & Aanen 2003).
Fungal transmission modes have not been studied
directly for M. natalensis. However, it has recently been
shown that the Termitomyces symbiont associated with this
species grows as a heterokaryon in the fungus comb and
has a heterothallic mating system (De Fine Licht et al. 2005).
This implies that horizontal transmission of the fungus
necessarily has to be associated with sexual reproduction,
resulting in recombination and ample population-wide
genetic variation (Orr-Weaver & Szostak 1985; Stumpf
& McVean 2003; Halkett et al. 2005). Frequent recombination would mean that alleles at single loci are in Hardy–
Weinberg equilibrium (HWE) and that genetically unlinked
loci are in linkage equilibrium. In contrast, vertical transmission and clonal reproduction of the fungus would result
in deviations from HWE and in significant linkage disequilibrium (LD) across loci (Tibayrenc 1997; Vilgalys et al.
1997; Brown 1999; Maynard Smith 1999; Taylor et al. 1999;
Awadalla 2003). Basidiocarps (mushrooms) have not been
observed on Macrotermes natalensis mounds, although it
has been shown that functional basidiocarps, producing
sexual spores, can be formed on comb fragments in the
laboratory (De Fine Licht et al. 2005). Furthermore, a recent study of symbiont genetic variation showed that
M. natalensis cultures only a single lineage of Termitomyces
symbionts, in contrast to most other fungus-growing termite species (Aanen et al., in preparation). These observations
would be suggestive of vertical symbiont transmission, but the decisive tests of HWE and linkage equilibrium
at marker loci have not been carried out. Our present study
is the first to address these fundamental characteristics
of the genetic population structure in a single lineage of
Termitomyces. We show that the symbiont breeding system
is consistent with horizontal transmission. This unexpected result brings up the question why mushrooms
on M. natalensis mounds have so far not been observed.
Materials and methods
Sampling
Termite mounds of Macrotermes natalensis (n = 31) were
excavated in South Africa during January 2003 and
January–March 2004 (Fig. 1). Macrotermes natalensis has the
most southern range within the genus Macrotermes and is
the most widespread species of southern Africa (Ruelle
et al. 1975). The termites feed predominantly on dead plant
Fig. 1 Map of the seven sampling localities
of Macrotermes natalensis in South Africa.
Numbers refer to the number of sampled
colonies at each site. All samples were
collected in January–February 2004, except
for the three northernmost colonies and
seven of the colonies from Pretoria (point
13), which were collected in January 2003.
The dashed line represents the approximate
southwestern distribution limit of the
genus Macrotermes (Uys 2002).
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd
S Y M B I O N T T R A N S M I S S I O N I N M . N A T A L E N S I S 3133
material such as wood and leaf litter and are a serious pest
of wood in buildings (Uys 2002). The sampled mounds
were 0.3–2 m high and primarily present in grassland
habitats, but some were located at the edge of woodland
vegetation. The mounds were opened and fragments of
Termitomyces fungus comb collected and transported to the
laboratory in plastic bags. The fungus was isolated by
aseptically placing nodules on malt-yeast agar in Petri
dishes under a dissecting microscope (MYA; 20 g/L malt
extract, 2 g/L yeast extract and 15 g/L agar). When a
mycelium developed without contaminants, the fungus
was further subcultured on a new agar Petri dish. Liquid
cultures were prepared with nodules and mycelium from
these secondarily cultivated plates (20 g/L malt extract
and 2 g/L yeast extract). All isolates were incubated at
25 °C in complete darkness.
Sequence analysis
DNA was extracted from the 31 liquid cultures after
4 weeks of growth. Approximately 1.5 mL of fungal
mycelium was transferred to 2-mL Eppendorf tubes and
freeze-dried on a SpeedVac connected to a Lyovac GT 2
(Savant, Leybold-Heraus). We used a DNeasy Plant Mini
Kit (cat. no. 69104, QIAGEN GmbH) and measured the
DNA concentration of each sample with a NanoDrop ND1000 Spectrophotometer (NanoDrop Technologies).
Four gene fragments were amplified using standard
polymerase chain reactions (PCRs): the internal transcribed spacer (ITS) region between the 18 S and the 25 S
nuclear RNA genes, partial sequences of the two largest
nuclear RNA Polymerase II subunits, RPB1 and RPB2,
and partial sequences of the nuclear Elongation Factor 1
alpha (EF1α). The following primer combinations were
used: ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and
ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) (White et al.
1990), RPB1-AF (5′-GARTGYCCDGGDCAYTTYGG-3′)
and Frpb1-CR (5′-CCNGCDATNTCRTTRTCCATRTA-3′)
(Matheny et al. 2002), bRPB2 – 6F (5′-TGGGGYATGGTNTGYCCYGC-3′) and bRPB2-7.1R (5′-CCCATRGCYTGYTTMCCCATDGC-3′) (Matheny 2005), and EF595F
(5′-CGTGACTTCATCAAGAACATG-3′) and EF1160R (5′CCGATCTTGTAGACGTCCTG-3′) (Kauserud & Schumacher
2001). To improve amplification of isolates, which were
difficult to amplify with standard primers for EF1α, we
developed specific primers for this gene for the Termitomyces
associated with M. natalensis: EF634F-Mnat (5′-AGGCTGACTGCGCTATCCTTAT-3′) and EF1127R-Mnat (5′GGTTCGATGGCATCGATGGCAT-3′).
PCRs were run on a Hybaid PCR Express Thermal
Cycler or on a Hybaid Omn-E Thermal Cycler (Thermo
Molecular Biology). PCR was performed in 20-µL reactions containing 2 µL MgCl2, 2 µL PCR buffer (Applied
Biosystems), 2 µL of each primer, 8 µL GATC mix, 0.1 µL
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd
AmpliTaq Gold polymerase (Applied Biosystems) and
3.6 µL ddH2O. PCR programmes consisted of an initial
denaturing step of 10 min at 95 °C followed by 35 cycles
(30 s at 95°, 30 s at 57° and 30 s at 72°) finished by a final
elongation step of 5 min at 72 °C, except for the RPB1 primers.
Here, an initial step of 5 min at 95 °C was followed by
35 cycles (30 s at 95°, 30 s at 55°, 1° increase per 5 s until 72°
and then 30 s at 72°), finished by a final elongation step of
10 min at 72 °C. The PCR products were checked on a 2.0%
agarose gel with EtBr before purification with a QIAquick
PCR purification kit (cat. no. 28106, QIAGEN). Sequencing
was performed by MWG Biotech.
For all four gene fragments, PCR products were directly
sequenced. DNA sequences of different strains were
aligned and analysed with sequencher version 3.1.1 for
Macintosh (Gene Codes Corporation). Double peaks with
similar height were present in the sequence chromatogram
at multiple positions indicating intrastrain variation and
allowing us to obtain codominant unphased data for all
single nucleotide polymorphisms (SNPs) for ITS, RPB1
and RPB2. In addition, for the EF1α gene, separate DNA
sequences of the two alleles were determined. The purified
PCR products were cloned with a TOPO TA cloning kit
(Invitrogen A/S). Procedures were according to the manual, except that all reaction amounts were three times less
than stated. One cloned EF1α fragment from each strain
was purified using the QIAquick PCR purification kit (cat.
no. 28106, QIAGEN) and sequenced. By comparing the
cloned sequences with the direct sequences of the PCR
products, the two alleles of the EF1α locus of each isolate
were obtained. Sequences have been deposited in GenBank with accession nos DQ436938–DQ437074.
Population-genetic and phylogenetic analysis
SNPs were tested for deviations from HWE using the exact
probability test (Guo & Thompson 1992) implemented in
arlequin version 2.0 (Slatkin & Excoffier 1996), with 1000
steps in Markov chain and 1000 dememorization steps.
Observed and expected heterozygosity and F-statistics
(FIS) were estimated using popgene version 1.32 (Yeh &
Boyle 1997). The presence of LD between all pair-wise
comparisons of these SNPs in the four unlinked genes was
analysed with a likelihood-ratio test in arlequin version
2.0 with 20 000 permutation steps. The SNPs within the
EF1α gene were analysed for LD with an exact test based
on a Markov chain in arlequin version 2.0 with 1000 steps
in Markov chain and 1000 dememorization steps.
For the most variable locus, EF1α, for which we had
obtained the two allele sequences for each strain, intragenic recombination was checked with a parsimony analysis of the sequences using paup* 4.0b10 (Swofford 2001).
In the absence of intragenic recombination and recurrent
mutation, the ancestry of the alleles can be represented as
3134 H . H . D E F I N E L I C H T , J . J . B O O M S M A and D . K . A A N E N
Table 1 The characteristics of the four gene sequences obtained
for each isolate: the number of isolates for which it was possible to
amplify the four sequences is given with the base-pair length of
each fragment, the number of single nucleotide polymorphisms
(SNPs), and the percentage of these that did not depart
significantly from Hardy–Weinberg proportions (exact probability
test implemented in arlequin version 2.1)
Gene
Sequenced
isolates
Length SNPs
Hardy–Weinberg
equilibrium
ITS
EF1α (in phase)
RPB1
RPB2
29
31
21
31
17 (100%)
16 (89%)
9 (75%)
8 (100%)
586 bp
444 bp
716 bp
552 bp
17
18
12
8
Table 2 Observed and expected heterozygosity and Wright’s
fixation index (FIS) of single nucleotide polymorphisms (SNPs)
from the four gene sequences of Termitomyces sp. associated with
Macrotermes natalensis
Locus ITS (n = 29) EF1α (n = 31) RPB1 (n = 21) RPB2 (n = 31)
HO
HE
FIS
0.087
0.083
−0.047
0.208
0.241
0.073
0.202
0.271
0.180
0.129
0.126
−0.030
a tree with a length that is equal to the number of SNPs and
with a consistency index of 1 (e.g. Burt et al. 1996). In contrast, if intragenic recombination is frequent, the tree will
be poorly resolved and rife with homoplasy (see, e.g. Burt
et al. 1996). In paup*, a heuristic search was performed [settings: 10 random addition sequences; mulpars on; treebisection–reconnection (TBR) branch swapping]. To test
whether recombination is completely free, the length of the
shortest trees was compared to the length of 1000 randomized data sets [using the permutation tail probability
(PTP) test in paup*].
Results
SNPs in the four sequenced genes concerned either single
base-pair substitutions (76.4%) or indels (23.6%) in introns
of the gene fragments. Adjacent double and triple SNPs
made up 9.1% of the total variation (Table 1). At all SNP
sites only two of the possible five character states (the four
nucleotides plus gap) were observed, except one with
three states. This indicates that mutations at those sites are
rare so that any changes in combinations of character states
are likely to be due to recombination rather than to
recurrent mutations. All SNPs of ITS, RPB1, RPB2 and
EF1α were treated as single loci and checked for deviation
from Hardy–Weinberg proportions with an exact
probability test (Guo & Thompson 1992). The genotypic
distribution of the large majority (90.9%) of these SNPs was
Table 3 Percentages of Termitomyces single nucleotide polymorphisms (SNPs) that were in linkage equilibrium in pairwise
comparisons within and across loci. The presence of LD was analysed with a likelihood-ratio test with 20 000 permutation steps,
except for comparisons within the EF1α gene where an exact
test based on 1000 steps in Markov chain and 1000 dememorization steps was used
% SNPs
ITS
EF1α
RPB1
RPB2
ITS
EF1α
RPB1
RPB2
89.7
94.2
100
96.4
72.5
96.5
96.9
87.3
99.0
92.9
not significantly different from HW proportions (Table 1).
Observed and expected heterozygosity and Fis values
are shown in Table 2. A small heterozygote deficit was
observed for ITS and RPB2, whereas a small heterozygote
excess was observed for EF1α and RPB1 (Table 2). Almost
all possible pairs of SNPs between the four genetically
unlinked loci were in linkage equilibrium (96.7%; Table 3).
Also most of the pairs of SNPs within the gene regions
(89.7% for ITS, 87.3% for RPB1, 92.9% for RPB2 and, 72.5%
for EF1α) were in linkage equilibrium (Table 3), showing
that frequent intragene recombination has occurred. The
distribution of SNPs in ITS, RPB1, RPB2 and EF1α is
presented in Table S1 (Supplementary material).
Phylogenetic analysis for EF1α
For the EF1α fragment, sequences of both alleles for each
strain were determined so that this fragment could be
studied in more detail. Forty-two different haplotypes
were found among the 62 sequenced alleles and all 31
isolates had distinct genotypes (see also Table S1). To check
for intragenic recombination within the EF1α fragment, we
performed a parsimony analysis of the 42 different EF1α
alleles. Parsimony analysis of the 18 SNPs resulted in
> 30 000 trees (length 48: consistency index 0.31: Fig. 2a), the
strict consensus tree of which is completely unresolved
(Fig. 2b). This shows that extensive intragenic recombination
has occurred in the EF1α gene. As expected, recombination
is not completely free as the length of the shortest trees is
still shorter than the length of trees fitted to randomized
data (Fig. 3).
The frequency of sexual reproduction
Our analyses show that Termitomyces sp. associated with
Macrotermes natalensis has a recombining population structure. However, a low frequency of sex in a population can
be sufficient to create a recombining population structure
in a sample of that population (Bengtsson 2003; Stumpf
© 2006 The Authors
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S Y M B I O N T T R A N S M I S S I O N I N M . N A T A L E N S I S 3135
Fig. 2 Phylogenetic analysis of the 42 different EF1α alleles. (a) One of the > 30 000 most parsimonious trees of length 48 (c.i. 0.31). (b) The
completely unresolved strict consensus tree of the > 30 000 most parsimonious trees, which indicates frequent intragenic recombination.
Fig. 3 Frequency distribution of tree lengths
for randomized data of the 42 different
EF1α alleles. The observed length (48) falls
well below the distribution of lengths of
the randomized data, indicating that recombination is not completely free.
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd
3136 H . H . D E F I N E L I C H T , J . J . B O O M S M A and D . K . A A N E N
Fig. 4 The expected number of clones (genotypes) in a population
with a low frequency of sex based on a sample of 31. The expected
number of clones was estimated from the absolute number of
sexual events, using the formula y = [(2σN/2σN) + (2σN/2σN
+ 1) + ··· + (2σN/2σN + r − 1)], where y is the expected number of
clones in a sample of size r, N is the population size and σ is the rate
of sexuality (see text for details). A few sexual events rapidly
increase the expected number of clones, even in a population with
a low frequency of sex.
& McVean 2003; Halkett et al. 2005). With a low frequency
of sex, the expected number of different genotypes in
a sample can be calculated as a function of the absolute
number of sexual events per generation (Ewens 1979;
as described in Bengtsson 2003). In Fig. 4, we plotted the
expected number of different genotypes in a sample of
31 as a function of the absolute number of sexual events per
generation (i.e. the product of the fraction of sexually
reproducing individuals and the total population size). As
all 31 samples represent different genotypes, this figure
indicates that the minimum number of sexually reproducing individuals per generation is > 100.
Discussion
The samples analysed in this study indicate that the
Termitomyces strains associated with Macrotermes natalensis
have a sexual population structure with frequent recombination, which is consistent with horizontal transmission (see also Korb & Aanen 2003; De Fine Licht et al.
2005). All genes used as markers in this study are present
in the genome as single-copy genes, except for the ITS,
which is part of the ribosomal RNA repeat unit, and occurs
in multiple copies per genome. Theoretically therefore
the intrastrain variation in the ITS sequences that we found
could also represent variation between copies within
the repeat (Selosse et al. 2002; Okabe & Matsumoto 2003).
However, previous studies of basidiomycete fungi have
shown that multiple ITS types only occur in heterokaryons and never in homokaryons and that the different
ITS variants from a heterokaryon always segregate in the
homokaryotic offspring (Aanen et al. 2001; Kauserud &
Schumacher 2003). This has also been shown for
Termitomyces (De Fine Licht et al. 2005). This implies that
the ITS gene in heterothallic basidiomycetes effectively
behaves as a single locus with Mendelian segregation. The
distinct genotypes of all the isolates clearly suggest that
Termitomyces recombines and creates novel genotypes each
generation. Because nearly all SNPs were in HWE and LD
between loci was rare, the accumulation of mutations is
unlikely as an alternative explanation for the observed
population structure.
Earlier studies of termite symbiont transmission modes
have used direct experimental tests with incipient colonies
and/or dissection of alates (Lüscher 1951; Sands 1960;
Johnson 1981; Johnson et al. 1981; Sieber 1983; Leuthold
et al. 1989). Compared to these studies, our genetic evidence for horizontal transmission remains indirect. In
particular, the frequency by which horizontal transmission
occurs in the field is unknown and this is important
because even a low frequency of recombination can result
in an apparently fully recombining population structure in
a random sample from the population (Bengtsson 2003;
Stumpf & McVean 2003; Halkett et al. 2005). As all 31 samples represented different genotypes, the recombination
frequency analysis indicated that the minimum number of
sexually reproducing individuals per generation was > 100
(Fig. 4). The alternative clonal dispersal mode of Termitomyces would rely on vertical dispersal via the winged termites,
which is known to be much less effective than windborne spore dispersal (Nutting 1969; Korb & Linsenmair
2001). Therefore, clonally related fungal genotypes
would be expected to be clustered in space. Several of
the collected samples were from neighbouring termite
mounds (from four sites we sampled: 13, 9, 3 and 3
mounds, respectively, see also Fig. 1). We did not find any
clonally related (i.e. identical) genotypes within a single
locality, which corroborates our conclusion that transmission is horizontal and sexual. Furthermore, a low
frequency of sex would also be at odds with the extensive
intragenic recombination that we found in this study.
Two interesting new questions arise from our results.
First, it now seems beyond reasonable doubt that symbiont
transmission within the genus Macrotermes is variable.
This suggests that transmission modes can evolve rapidly
quickly, which implies that generalizations for entire genera may not hold even when based on several case studies.
For example, the five species of Microtermes that have now
been shown to rely on vertical symbiont transmission via
the female sex (Johnson 1981; Johnson et al. 1981) do not
preclude that there may be Microtermes species that have
either retained or have reverted to horizontal transmission.
Second, although our Fig. 4 shows that sex and recombination do not need to occur every generation, it remains
puzzling that field observations have so far failed to find
symbiont mushrooms on M. natalensis mounds. One
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S Y M B I O N T T R A N S M I S S I O N I N M . N A T A L E N S I S 3137
explanation could be that fruiting only happens in some
years and that basidiospores are relatively long lived
after dispersal. However, it is also possible that M. natalensis
workers actively suppress fruiting of their symbiont, which
is a waste of resources for the termites (Aanen, 2006).
Reproductive conflicts of this kind have been extensively
treated elsewhere (Korb & Aanen, 2003; Aanen, 2006;
Aanen & Boomsma, 2006). Worker suppression of fruiting
bodies can persist as an evolutionary stable strategy if
M. natalensis in reality ‘parasitizes’ on other sympatric
Macrotermes species where the symbiont is known to
produce fruiting bodies (Aanen & Boomsma, 2006), such as
M. subhyalinus (Johnson et al. 1981) and M. michaelseni
(B. Slippers & W. de Beer, personal observation). This
hypothesis remains to be tested by comparing Termitomyces
genotypes of all three species. Because the fungal transmission mode is variable across species, the genus Macrotermes
may therefore be the most suitable genus to look for the
expression of these conflicts, and M. natalensis would be a
promising species to start.
Acknowledgements
We thank Sylvia Mathiasen, Pia Friis and Tina Brand for assistance
in the laboratory, Jannette Mitchell and Wilhelm de Beer for help
during fieldwork and Fons Debets for comments on an earlier
version of this manuscript.
Supplementary material
The supplementary material is available from http://
www.blackwellpublishing.com/products/journals/suppmat/
MEC/MEC3008/MEC3008sm.htm
Table S1 Distribution of single nucleotide polymorphisms
(SNPs) of the internal transcribed nuclear rDNA spacer sequence
(ITS), and partial EF1á, RPB1 and RPB2 sequences of Termitomyces
sp. associated with Macrotermes natalensis. Variable positions in
the sequences are indicated (vertical numbers). In addition to
direct sequencing of the PCR product, EF1á was also sequenced
after cloning to obtain the two alleles when polymorphism
occurred (see text for details)
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This work is part of H.H. de Fine Licht’s M.Sc. research on the
transmission and specificity of fungal symbionts in termites. D.K.
Aanen is interested in the evolution of conflict and cooperation in
basidiomycete fungi and has focused on the mutualistic symbiosis
between fungus-growing termites and Termitomyces in recent
years. J.J. Boomsma works on a wide array of evolutionary questions, mainly in social insect biology.
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd