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BMC Evolutionary Biology
BioMed Central
Open Access
Research article
Patterns of interaction specificity of fungus-growing termites and
Termitomyces symbionts in South Africa
Duur K Aanen*1,2, Vera ID Ros1,2,6, Henrik H de Fine Licht2,
Jannette Mitchell3, Z Wilhelm de Beer4, Bernard Slippers4, Corinne RoulandLeFèvre5 and Jacobus J Boomsma2
Address: 1Laboratory of Genetics, Plant Sciences Group, Wageningen University and Research Center, Arboretumlaan 4, 6703 BD Wageningen,
The Netherlands, 2Department of Population Biology, Institute of Biology, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen,
Denmark, 3Agricultural Research Council-Plant Protection Research Institute, Rietondale Research Station, Private Bag X134, Queenswood,
Pretoria 0121, South Africa, 4Forestry and Agricultural Biotechnology Institute (FABI), Faculty of Agricultural and Biological Sciences, Department
of Microbiology and Plant Pathology, University of Pretoria, Pretoria, South Africa, 5UMR-IRD 137 Biosol Laboratory of Tropical Soils Ecology
(LEST) – Centre IRD d'Ile de France, 32 avenue Henri Varagnat 93 143 – Bondy Cedex, France and 6Evolutionary Biology, Institutefor Biodiversity
and Ecosystem Dynamics, University of Amsterdam, P.O. Box 94062, 1090 GB Amsterdam, The Netherlands
Email: Duur K Aanen* - [email protected]; Vera ID Ros - [email protected]; Henrik H de Fine Licht - [email protected];
Jannette Mitchell - [email protected]; Z Wilhelm de Beer - [email protected];
Bernard Slippers - [email protected]; Corinne Rouland-LeFèvre - [email protected];
Jacobus J Boomsma - [email protected]
* Corresponding author
Published: 13 July 2007
BMC Evolutionary Biology 2007, 7:115
doi:10.1186/1471-2148-7-115
Received: 30 March 2007
Accepted: 13 July 2007
This article is available from: http://www.biomedcentral.com/1471-2148/7/115
© 2007 Aanen et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: Termites of the subfamily Macrotermitinae live in a mutualistic symbiosis with basidiomycete fungi
of the genus Termitomyces. Here, we explored interaction specificity in fungus-growing termites using samples
from 101 colonies in South-Africa and Senegal, belonging to eight species divided over three genera. Knowledge
of interaction specificity is important to test the hypothesis that inhabitants (symbionts) are taxonomically less
diverse than 'exhabitants' (hosts) and to test the hypothesis that transmission mode is an important determinant
for interaction specificity.
Results: Analysis of Molecular Variance among symbiont ITS sequences across termite hosts at three hierarchical
levels showed that 47 % of the variation occurred between genera, 18 % between species, and the remaining 35
% between colonies within species. Different patterns of specificity were evident. High mutual specificity was
found for the single Macrotermes species studied, as M. natalensis was associated with a single unique fungal
haplotype. The three species of the genus Odontotermes showed low symbiont specificity: they were all associated
with a genetically diverse set of fungal symbionts, but their fungal symbionts showed some host specificity, as none
of the fungal haplotypes were shared between the studied Odontotermes species. Finally, bilaterally low specificity
was found for the four tentatively recognized species of the genus Microtermes, which shared and apparently freely
exchanged a common pool of divergent fungal symbionts.
Conclusion: Interaction specificity was high at the genus level and generally much lower at the species level. A
comparison of the observed diversity among fungal symbionts with the diversity among termite hosts, indicated
that the fungal symbiont does not follow the general pattern of an endosymbiont, as we found either similar
diversity at both sides or higher diversity in the symbiont. Our results further challenge the hypothesis that
transmission-mode is a general key-determinant of interaction specificity in fungus-growing termites.
Page 1 of 11
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Background
Mutualistic interactions between species are common and
have played a central role in the diversification of life [1].
Interactions range from temporal, facultative encounters
to obligate permanent symbioses. Mutualistic symbioses
also often represent major and ecologically highly successful transitions in evolution [2]. Co-evolution of mutualistic taxa involves reciprocal evolutionary change through
natural selection [3]. If co-evolutionary interactions persist through speciation events, whole clades of different
species can co-speciate [4]. However, co-cladogenesis is
only an extreme outcome of coevolution as specificity can
also exist at other levels than the species level. For example, when symbionts regularly switch between different
host species, they may still be specific to a particular host
genus.
Specificity in symbiotic interactions has two sides, the
host and the symbiont. From the symbiont perspective of
specificity, usually the term 'host specificity' is used,
which is defined on the basis of the range of hosts that a
symbiont can utilize as a partner. Analogously, from the
host perspective of specificity we introduce the term 'symbiont specificity' which is defined on the basis of the range
of symbionts that a host can utilize. In Figure 1, the different possibilities are summarized. As an umbrella term for
the different types of specificity we use the general term
'interaction specificity'.
Numerous factors have been hypothesized to influence
interaction specificity. For example, Law and Lewis [5]
noticed that the taxonomic diversity and the frequency of
sex are generally lower in the 'inhabitant' (the 'symbiont')
than in the 'exhabitant' (the 'host') of mutualistic interactions. Another important variable for patterns of specificity is the transmission mode of symbionts. Vertical
transmission greatly limits the frequency by which new
combinations of symbiotic partners arise and may therefore lead to a higher degree of coevolution than horizontal transmission, although many exceptions to this
prediction have been found [6].
The fungus-growing social insects are interesting and ecologically important examples of mutualistic symbiosis
with variable degrees of specificity. Growing fungi for
food has independently evolved in the old-world macrotermitine termites (c.f. [7-9]) and in the new-world attine
ants (c.f. [10,11]). Interaction specificity and co-evolution
have been studied intensively in the fungus-growing ants
[e.g. [10-12]], but much less is known about the fungusgrowing termites. However, the opportunities provided
by the fungus-growing termites and their Termitomyces
symbionts to obtain insight in the co-evolution of interaction specificity are substantial because the transmission
modes and reproductive systems of the termite symbionts
http://www.biomedcentral.com/1471-2148/7/115
vary much more than those of the fungus-growing ants
[9,13,14].
Natural history of fungus-growing termites
The fungus-growing termites have evolved into approximately 330 extant species, belonging to ca. 12 genera [15].
The termites maintain their fungal symbiont on special
structures in the nest, the fungus combs, which are housed
in specially constructed chambers, either inside a mound
or dispersed in the soil. Workers feeding on dry plant
material produce fecal pellets (primary feces) which are
added continuously to the top of the comb and fungal
mycelium rapidly develops in the newly added substrate.
After a few weeks, the fungus starts to produce vegetative
structures, nodules (which are modified unripe mushrooms) that are consumed by the termite workers. At a
later stage, the entire comb structure permeated with mycelium is consumed [8].
Direct and indirect evidence indicates that symbiont
transmission between colonies and across generations is
normally sexual and horizontal [[16-20], for a review, see
[21]]. This implies that new colonies will usually start
their existence without a fungus. The most likely route to
acquire the fungal symbiont is via sexual spores produced
by mushrooms from other nests. The single study performed so far on the genetic population structure of the
fungal symbiont of the species M. natalensis [18] has
shown a freely-recombining population structure of the
fungus, which is consistent with sexual horizontal transmission as the main transmission mode. At least two independent transitions to clonal, vertical and uniparental
transmission have occurred [9]: one involving a single
species, Macrotermes bellicosus (via the male sexuals; [17])
and one possibly involving the entire genus Microtermes
(via the female sexuals; based on the five studied species;
[17,22]). With vertical transmission, sexuals of one of the
two sexes ingest asexual spores before the nuptial flight
and use these as inoculum for the new fungus comb after
colony foundation.
The symbiosis between termites and Termitomyces fungi is
'symmetric' since both partners are obligatorily interdependent, and this dependence has a single evolutionary
origin with no known reversals to non-symbiotic states
[9,23,24]. Furthermore, specificity in interactions occurs
at higher taxonomic levels, i.e. (combinations of) genera
tend to rear different clades of Termitomyces [9]. This
higher-level specificity strongly suggests that coevolution
between termites and fungi, has occurred. Within genera,
however, generally no strong association between the evolutionary histories of the termites and their fungal symbionts has been found [9]. At the species level, opposite
specificity patterns have been observed. In some cases, the
sampled fungal symbionts of a single termite species did
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BMC Evolutionary Biology 2007, 7:115
Host side
http://www.biomedcentral.com/1471-2148/7/115
Symbiont side
a
High symbiont specificity, high host specificity
b
Low symbiont specificity, high host specificity
c
These conflicting patterns of interaction specificity indicate that further work is necessary before any general conclusions about interaction specificity at lower taxonomic
levels can be drawn. The present paper offers a detailed
comparative study of the interaction specificity of South
African fungus-growing termites. We investigated how
specific the interactions between termites and fungal symbionts in South Africa are at the genus, species and population level and how the different levels of specificity
might relate to inferred co-evolutionary dynamics and
known modes of transmission. To address these questions
we obtained sequence data from 101 colonies belonging
to eight species in three genera of South African fungusgrowing termites and estimated interaction specificity at
the genus, species and colony level.
Results
High symbiont specificity, low host specificity
d
Low symbiont specificity, low host specificity
Figure 1
istic host-symbiont interaction
The theoretically possible patterns of specificity in a mutualThe theoretically possible patterns of specificity in a mutualistic host-symbiont interaction. a. Mutually high specificity.
One host lineage is exclusively associated with a single symbiont lineage. b. Low symbiont specificity, high host specificity.
A host lineage can be associated with three different symbiont lineages (low symbiont specificity), which each are specialized on that single host lineage (high host specificity). c.
High symbiont specificity, low host specificity. Three different
host lineages are each associated with the same symbiont lineage. d. Mutually low specificity. Three host lineages can all
be associated with three different symbiont lineages.
not form a monophyletic group (e.g. the fungal symbionts
of Macrotermes bellicosus and Odontotermes latericius) [9],
but the opposite pattern, where the termite hosts of a single symbiont did not form a monophyletic group, has also
been found. An extreme example of the latter was provided by the single fungal lineage shared between all sampled colonies in western Africa of the three divergent
genera Microtermes, Ancistrotermes and Synacanthotermes
[9]. Recently, it was found that Macrotermes natalensis has
a very specific association as it was exclusively associated
with a single fungal lineage [18], showing that strong specificity can also exist at the species level.
Sequence analyses
Complete ITS sequences were obtained for 101 strains
(Table 1; Table 1 in Additional file 1) and sequence length
ranged from 597 to 690 nucleotides. Of the 794 positions
in the final alignment, 300 were variable and 179 of these
were parsimony informative. Gap positions were coded as
missing data. In Figure 2a the unrooted neighbor-joining
phylogram is given, using uncorrected pairwise distances
as a distance measure. Using MAFFT [25] to align the
sequences instead of ClustalW resulted in an almost identical neighbor joining topology (not shown).
A rooted phylogeny was estimated in a second analysis,
using the more conserved nuclear 25S and the mitochondrial 12S on a single representative of each ITS haplotype,
while using the non-symbiotic fungus Tephrocybe rancida
as an outgroup [9,24,26]. In Figure 2b the majority rule
consensus tree of the trees sampled in the Bayesian analysis is given. Maximum parsimony and neighbour-joining
analyses gave similar results.
Within the sampled termites of the genus Microtermes,
four main COI lineages were found. We tentatively consider these to represent different species (designated as I,
II, III and IV).
Levels of interaction specificity
Analysis of Molecular Variance of the fungal sequence variation across various taxonomic levels showed that on
average 47 % of the symbiont sequence variation occurred
between termite genera, 18 % between termite species,
and 35 % between colonies within species (Table 2). High
bilateral specificity existed at the genus level, as no symbiont lineages were shared between the three genera Macrotermes, Microtermes and Odontotermes and the symbionts
of these genera formed three separate clusters in the phylogram of the aligned ITS sequences (indicated with different colours; Figure 2a). In the rooted analysis (Figure
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Table 1: The termite samples collected at the different locations and the associated genetic diversity of their Termitomyces symbionts
(See Figure 1 for further details)
Termite species
site
Macrotermes natalensis
ZA1
ZA3
ZA5
ZA6
ZA7
ZA8
ZA9
ZA3
ZA5
ZA3
ZA4
Se1
Se2
ZA5
ZA4
ZA2
ZA3
ZA5
ZA9
ZA10
ZA7
ZA4
ZA5
ZA7
ZA5
ZA3
Odontotermes badius
Odontotermes latericius
Odontotermes transvaalensis
Microtermes sp. I
Microtermes sp. II
Microtermes sp. III
Microtermes sp. IV
Microtermes?
number of samples
ITS haplotypes
1
14
3
1
9
3
1
5
1
14
1
8
6
1
4
1
14
1
1
1
2
1
1
4
2
1
1
1
1
1
1
1
1
4, 5, 6, 15, 16
4
10, 11, 12, 13, 18
10
2, 8, 9, 17, 19
2, 17
7
3, 14
24
20, 22, 23, 24, 25
20
25
24
21, 25
24
24
24, 25
20, 21
24
Genbank accession numbers for fungal ITS, nuclear 25S, mitochiondrial 12S and the COI sequences of Microtermes sp. I, II, III and IV are given in the
online supplementary table.
2b) the symbionts of the genera Macrotermes and Microtermes formed two monophyletic groups, while the symbionts of Odontotermes consisted of two distinct non-sister
clades.
Specificity was generally low at the level of termite species,
with the exception of Macrotermes natalensis where all 31
sampled mounds had the same unique ITS lineage of Termitomyces [18]. However, since Macrotermes natalensis was
the only species sampled in this genus, we could not
determine whether the ITS lineage that we found is specific for this particular Macrotermes species. The three species in the genus Odontotermes were all associated with a
highly diverse set of fungal symbionts, which did not
form a monophyletic group for any of these species (Figure 2b). However, AMOVA showed that significant differences occurred between the species (Table 2). Pairwise
comparisons revealed that these differences were mainly
due to the symbionts of O. latericius being different from
the symbionts of the two other Odontotermes species, O.
badius and O. transvaalensis (the symbionts of O. badius
and O. transvaalensis were not significantly different: P =
0.19). Interestingly, although the three Odontotermes species were all associated with a wide range of fungal symbionts, none of the ITS haplotypes were shared between
species. To test the significance of this result, we also made
pairwise comparisons between the Odontotermes species,
using only the frequency of the different haplotypes
within species, and not the actual sequence information
[27,28]. This revealed that the differentiation of fungal
symbionts between species was indeed significant for all
three pair-wise comparisons. However, this result may be
due to our sampling effort: a high overall diversity of
Odontotermes-associated Termitomyces in combination
with spatial autocorrelation may explain this result. In
line with this, only two haplotypes (4 and 10) were found
at more than one locality.
A different pattern was found for the variation in Termitomyces symbionts between species of the genus Microtermes,
as most of the six fungal haplotypes were shared between
species (Figure 2a) and there were no significant differences between species (p = 0.08; Table 2).
Discussion
Grassé [29] proposed that every genus of fungus-growing
termites was associated with a single species of Termitomyces. However, it was recently shown [9,24] that this
hypothesis is invalid and that different morphospecies of
Termitomyces can be associated with the same genus of ter-
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Ma. natalensis
1(32)
Od. latericius *
2(4)
3(3) Od. transvaalensis
0.01 substitutions/site
Mi.III (3)25(3)
?
(1)
Mi.III (3)
Od. badius
4(2)
Mi.I
5 Od. badius
(1)
24 (9)
II
6 Od. badius
Mi.I
7 Od. transvaalensis
Mi.I 23(2)
92
99
100
Mi.I 22
8 Od. latericius*
95
98
87
62
21
Mi.IV (1)(1)
9(4) Od. latericius *
89
Mi.II
91
10 Od. latericius
82
100
100
11(6)
Od. latericius
100
100
15
Od.
Od. badius badius
Mi.IV(1)
16
20
(3)
Mi.I
13(5)
12
Od. latericius Od. latericius
14 Od. transvaalensis
Ma. natalensis (1)
100
Mi. I, III (23, 24)
Mi. I (22)
100
56
69
Mi. II, IV (21)
97
Mi. I, IV (20)
Mi. I, II, III (25)
99
Od. transvaalensis (14)
95
19(2)
77
Od. latericius *
100
Od. badius (15)
95
Od. latericius (12, 13)
93
18
Od. latericius
17(4)
Od. badius (16)
Od. latericius (8*, 9*, 10, 11)
Od. badius, transvaalensis (5,6,7)
77
Od. latericius (18)
100
65
Od. latericius (17*)
62
Od. latericius *
Od. transvaalensis (3)
Od. latericius (19*)
100
Od. latericius (2*)
Od. badius (4)
T. rancida
Figure 2
gus growing neighbor-joining phylogram species in three genera)
a. Unrooted termites (belonging to eight of the 25 ITS haplotypes found among the Termitomyces symbionts of 101 nests of funa. Unrooted neighbor-joining phylogram of the 25 ITS haplotypes found among the Termitomyces symbionts of 101 nests of fungus growing termites (belonging to eight species in three genera). The three genera are indicated with different colours, and
the species within the genera in different intensities of these colours. The Senegalese samples of O. latericius are indicated with
an asterisk. Within each circle the haplotype number is indicated and, in brackets, the number of that haplotype found (if more
than one). The area of circles is proportional to the observed frequency of particular haplotypes. Red: genus Macrotermes;
green: genus Odontotermes; blue: genus Microtermes. Within the genus Microtermes four tentative species were distinguished
(labelled I-IV). For one Microtermes termite sample no sequence was obtained, which is indicated with a '?'. The numbers on the
branches are the percentage bootstrap values (> 50) based on 1000 bootstrap replicates. b. Phylogenetic relationships
between the Termitomyces symbionts of the fungus growing termites included in this study. The cladogram is the majority rule
consensus tree of trees sampled in a Bayesian analysis of combined partial nuclear 25S sequences and mitochondrial 12S
sequences. The free-living fungus Tephrocybe rancida was used as an outgroup, based on previous analyses (Hoffstetter et al.
2002; Aanen et al., 2002). Abbreviations used: Ma.: Macrotermes; Mi.: Microtermes; Od.: Odontotermes.
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Table 2: Results of AMOVA of Termitomyces symbionts at three hierarchical levels.
Source of variation
d.f.
Sum of squares
Variance component
% variation
Fixation indices
P value
Between genera
Among species within genera
Within species
Total
Within Odontotermes: ΦST = 0.25 (p < 0.001)
Comparison
latericius & transvaalensis
latericius & badius
transvaalensis & badius
Within Microtermes: ΦST = 0.13 (p = 0.08)
Comparison
Sp. I & sp. II
Sp. I & sp. III
Sp. I & sp. IV
Sp. II & sp. III
Sp. II & sp. IV
Sp. III & sp. IV
Sp. I & sp. III
Sp. I & sp. IV
Sp. II & sp. III
Sp. II & sp. IV
Sp. III & sp. IV
2
5
77
84
2064.85
439.53
1677.90
4182.27
28.72
11.18
21.79
61.68
47
18
35
100
ΦCT = 0.47
ΦSC = 0.34
ΦST = 0.65
0.0029
0.0020
< 0.0001
Pairwise ΦST
0.34
0.23
0.08
Pairwise ΦST
0.21
0.01
0.27
0.10
-0.36
0.37
0.01
0.27
0.10
-0.36
0.37
mites and sometimes with single species. Here, we detail
this further by showing that many species of fungus-growing termites are associated with highly divergent strains of
Termitomyces that probably represent different morphospecies. The symbiont diversity that we have found in
Odontotermes and Microtermes species is the highest
reported for any fungus-growing termite so far. Compared
to the results reported here, Katoh et al. [30] found only
minor intraspecific symbiont diversity (two ITS haplotypes) in SE Asian Odontotermes formosanus. This is consistent with Africa being the core area where the association
between termites and fungi arose and radiated, whereas
SE Asia represents the edge of the present distribution of
this symbiosis [31].
Interpreting differences in interaction specificity
Genus-level symbiont specificity was high, as symbiont
lineages formed completely separate clusters without any
overlap. This is consistent with the patterns of specificity
above the species level that were documented previously
[9]. At the species level, however, we found differences in
interaction specificity between the three genera: specificity
was high for both sides in Macrotermes natalensis (situation a in figure 1) and much lower in the other two genera. Although AMOVA showed that significant differences
occurred between species within the genus Odontotermes,
pairwise comparisons revealed that this difference was
due to the symbionts of O. latericius being different from
the symbionts of the two other Odontotermes species, O.
badius and O. transvaalensis, which themselves were not
significantly different. Nevertheless, none of the ITS hap-
P value
p < 0.001
p = 0.002
p = 0.19; n.s.
0.37
P value
p = 0.11; n.s.
p = 0.58; n.s.
p = 0.13; n.s.
p = 0.36; n.s.
p = 0.99; n.s.
P = 0.04
p = 0.58; n.s.
p = 0.13; n.s.
p = 0.36; n.s.
p = 0.99; n.s.
P = 0.04
lotypes were shared between these three Odontotermes species, and the overall differentiation between species was
significant. One possible interpretation of this result is
that symbionts are exchanged between Odontotermes species, because each species would otherwise be associated
with a monophyletic group of symbionts, but that this
exchange is rare enough to allow the evolution of some
differentiation between species. However, an alternative
explanation is that this result is a sampling artifact: a high
overall diversity of Odontotermes-associated Termitomyces
in combination with spatial autocorrelation may explain
this result. In line with this interpretation, only two haplotypes (4 and 10) were found at more than one locality.
Low specificity at both sides was found for the studied
species of the genus Microtermes, which essentially shared
a common pool of genetically diverse symbionts (situation d in figure 1). A completely different pattern has previously been found in species of the genera Microtermes,
Ancistrotermes and Synacanthotermes collected in western
Africa (Senegal and Cameroon), which were all associated
with a single fungal lineage with little sequence divergence (consistent with situation c in figure 1; [9]. The reason for this difference in Microtermes symbiont diversity
between the two African regions is presently unclear, but
the observed patterns do suggests that specificity and coevolutionary dynamics may differ both geographically
and between closely related termite species, as envisaged
in geographic mosaic models for co-evolution [1].
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Are the observed patterns consistent with the Law and
Lewis hypothesis?
Law and Lewis [5] argued that the exhabitant (host) in a
mutualistic symbiosis is generally more diverse and has a
higher frequency of sex than the inhabitant(s) (the symbiont(s)). They hypothesized that different host species may
converge, evolutionarily, on a single mutualist symbiont,
whereas they will disperse in phenotypic space to escape
the attacks of a parasite, thus encouraging speciation in
the latter [2]. The question is whether this inhabitantexhabitant distinction is equally valid for the symbiosis
between Macrotermitidae and Termitomyces, which is an
ectosymbiosis relative to the individual termites and an
endosymbiosis (sensu Law and Lewis) only for the entire
colony of termites. The patterns that we found are not
consistent with the Law and Lewis predictions for most of
the species of fungus-growing termites that we studied,
because they tend to be associated with a genetically
highly diverse set of symbionts. Thus, although the fungus-growing termites create a benign environment for the
fungus inside their nest, the fungus garden may not be as
well protected from external parasitic microorganisms as
a real endosymbiont would be, so that selection for symbiont genetic diversity at the population level is maintained in most groups of fungus-growing termites. This is
also consistent with the high frequency of sex in most of
the Termitomyces symbionts [18,21]. Interestingly, a similar pattern of high genetic diversity at the population level
has been found among the symbionts of fungus-growing
ants [12,32,33], but here the frequency of sex among symbionts is apparently much lower [34].
Is interaction specificity linked to transmission mode?
Symbiont transmission modes have been studied for a
rather limited number of macrotermitine species. For the
species included in this study, we have direct evidence that
Odontotermes badius and Macrotermes natalensis rely on
horizontal symbiont acquisition [18,19]; De Fine Licht,
Korb and Aanen, pers. obs.). For the other two included
species of Odontotermes we have indirect evidence,
because: 1. fruiting occurs frequently for both species; 2.
all other species of the genus that have so far been tested
have horizontal symbiont acquisition [17,20]. Also our
inferences for Microtermes have to be based on indirect evidence: fruiting bodies have never been observed for any of
the species included in the present study and five other
species of this genus are known to rely on vertical, uniparental symbiont transmission via the female sex [17,22].
Unexpectedly, the presumably vertically transmitted fungi
of the three Microtermes species were shared between species. This indicates that horizontal transmission must
occur frequently enough to prevent any host specificity at
the species level. In an earlier study [9] it was found that
the fungal symbionts of several west African Microtermes
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species were identical to the symbionts of the divergent
genera Ancistrotermes and Synacanthotermes, showing that
horizontal transmission also occurred between these genera in this other region. Obviously, the transmission
modes for these species need to be addressed directly in
future studies.
How does interaction specificity arise?
For the termite species with horizontal symbiont acquisition, it is still an open question how combinations
between termites and their fungal symbionts arise and
how specificity arises. One way in which interaction specificity could arise would be geographical isolation, but
we did not find strong geographical differentiation at the
scale of our study. For some species of fungus-growing termites it has been found that fungal fruiting is synchronized with the time that the first workers of founding
colonies of the typical host leave the nest to start foraging
[17]. Since dispersal on the wing varies between species,
but is highly synchronized within species, such temporal
segregation would be a possible way for interaction specificity to arise. Temporal segregation of this kind could
potentially explain that all three Odontotermes species in
our study were associated with a different range of fungal
symbionts, so that none of the ITS haplotypes were shared
between the species. A related question of crucial importance is how long basidiospores of Termitomyces remain
viable and whether a functional Termitomyces spore bank
exists. We hypothesize that active partner selection may
also play a role in the establishment of interaction specificity, as neither temporal nor geographical isolation seem
sufficient explanations to arrive at the levels of specificity
observed. Similarly, for species with predominantly vertical transmission, the question is how symbiont exchange
occurs. Detailed experimental studies will be needed to
investigate whether and how selection takes place during
the establishment and later maintenance of fungal symbionts in termite colonies.
Conclusion
Interaction specificity between fungus-growing termites
and Termitomyces fungi is highly variable and ranges from
mutually high specificity to mutually low specificity (Figure 1). This implies that the hypothesis that inhabitant
mutualistic symbionts should be less diverse than the
exhabitant hosts is not supported by the data, as we found
either similar diversity at both sides or higher diversity in
the symbiont. Our results further challenge the hypothesis
that transmission-mode is a general key-determinant of
interaction specificity in fungus-growing termites.
Methods
Samples and identification of termites
Eighty six nests of fungus-growing termites, belonging to
three genera and eight species were sampled in South
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Africa in January 2003 and January 2004 (Figure 3; Table
1; Table 1 in Additional File 1). Several worker and soldier
termites and some comb material were preserved in 96%
alcohol for each nest, and stored at 4°C for future reference or DNA extraction. The fungus of most colonies was
isolated in pure culture (Table 1 in Additional File 1) from
nodules on malt yeast extract agar (per liter: 20 g malt
extract, 2 g yeast extract and 15 g agar). For one species,
Odontotermes latericius, 15 additional samples were
obtained from four sites in Senegal in December 2000.
Here, only termites were sampled by collecting > 10 workers from foraging trails and storing them in 96% alcohol.
Fungal sequences were obtained from the guts of these termites using Termitomyces specific primers (see below). The
distance between foraging trails was at least 10 meters so
that the chance of sampling the same nest twice was low.
Nonetheless, in cases where multiple identical sequences
were found within a site, only one of these was included
in the analysis.
Most termite samples of the genera Macrotermes and
Odontotermes could be identified at the species level on the
basis of morphological characters. However, the taxonomy of the genus Microtermes is complex and awaits further study [35] so that the Microtermes samples could not
be identified. In order to classify the Microtermes samples
in "species groups", we sequenced a ca. 1000 bp region of
the mitochondrial cytochrome oxydase I (COI) gene,
using only the forward primer as a sequencing primer
(resulting in a readable sequence of at least 500 but usually more than 800 nucleotides). Sequences that differed
less than 4% were considered to belong to the same tentative species. PCR amplifications were done using previously
published
primers
(forward:
BL1834,
5'TCAACAAATCATAAAGATATTGG3'
or
TL1862,
5'TACTTCGTATTCGGAGCTTGA3' and reverse: a nondegenerate
version
of
TH2877,
5'TAGGTGTCGTGTAATACAATGTC3'; [9,31]. Sequences
of representatives of the recognised tentative species have
been submitted to Genbank (accession numbers in Table
1 in Additional File 1).
Fungal sequences and phylogenetic analyses
The analysis of the Termitomyces diversity in our samples
consisted of two steps. First, a hierarchical analysis of
interaction specificity was done using the highly variable
ITS sequences. Second, the phylogenetic position of the
different ITS haplotypes was determined by using more
conserved DNA regions and including an outgroup. The
high variability of the ITS region made alignment ambiguous and less suitable for estimating higher-order relationships. We therefore refrained from using the ITS
sequences to make phylogenetic inferences and used two
more conserved regions of nuclear 25S rDNA and mitochondrial 12S RNA instead (details given below).
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For all 101 samples, the nuclear ribosomal region including ITS1, 5.8S, and ITS2 was amplified and sequenced
using the primers ITS1 and ITS4 [36]). ITS sequences for
M. natalensis were determined in a previous study [18]
and included in the present analysis. In Senegal we had
only sampled termites, so that we obtained total fungal
DNA from termite abdomens (including gut contents; see
[9]), and amplified ITS sequences by using a Termitomyces
specific primer (ITS1FT: GTTTTCAACCACCTGTGCAC,
based on available sequences in GenBank), which was
used in combination with the universal primer ITS4 [36]).
Sequencing was done using the forward and backward
primer, and in some cases the internal primers ITS2 and
ITS3 [36]). For some strains, two ITS copies were present
in the PCR products, differing by a small length mutation,
giving chromatograms with double peaks for part of the
sequence. However, using both forward and reverse primers and sometimes internal primers, the two copies could
be recovered. Of these, we selected a single sequence for
the analysis, so that our data are effectively haplotypic.
Selection of the other copy did not affect the results (data
not shown). Sequences were aligned using ClustalW [37];
settings for both pairwise alignment and multiple alignment: gap insertion penalty 10, gap extension penalty 0.1.
An unrooted tree was obtained by cluster analysis of the
ITS sequences using the neighbor-joining algorithm with
uncorrected distances [38]). Bootstrap support for individual branches was assessed using 1000 replicates. An
alternative alignment was performed using the fast Fourier transform method implemented in MAFFT version
5.64 [25]). The most accurate option (L-INS-i) was chosen
(iterative refinement method incorporating local pairwise
alignments; gap opening penalty: 1.5 and gap extension
penalty 0.14).
To estimate a rooted phylogeny, we sequenced two more
conserved DNA regions for each ITS haplotype:
• Ca. 530 nucleotides of the nuclear 25S rDNA gene using
the primers 25S4R (acaagtgctgagttcctcag; which is specific
for Termitomyces; [9]) and ITS4R (GCATATCAATAAGCGGAGGA: the reverse complement of the universal primer
ITS4; [36]). A region of maximally 11 nucleotides was
excluded from the analysis, because it could not be unambiguously aligned.
• Ca. 320 nucleotides of the 12S mitochondrial RNA gene,
using the Termitomyces specific primer pair ssufw (specific
for Termitomyces, TCGCGTTAGCATCGTTACTAGT; [9])
and ssurev475 (specific for some Lyophylleae including
Termitomyces, GCCAGAGACGCGAACGTTAGTCG; [9]). A
region of maximally 28 nucleotides was excluded from
the analysis, because it could not be unambiguously
aligned.
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Figure 3
Sampling localities in Senegal and the Republic of South Africa
Sampling localities in Senegal and the Republic of South Africa. Abbreviations used: Se1 = Thiès, Senegal; Se2 = Kolda, Senegal;
Se3 = Tambacounda, Senegal; ZA1 = Kalahari (S27.10 E24.11); ZA2 = Pretoria surroundings (S25.42 E27.47); ZA3 = Pretoria
PPRI Rietondale (S25.43 E28.14); ZA4 = SABS Farm, Radium, South Africa (S25.00 E28.21); ZA5 = Pietersburg (S29.27 E23.54);
ZA6 = Sabie, South Africa (S25.33 E30.58); ZA7 = Vygeboomdam, South Africa (S25.53 E30.37); ZA8 = Amsterdam surroundings, South Africa (S26.50 E30.30); ZA9 = Kwazulu Natal, South Africa (S28.57 E29.48); ZA10 = Pietermaritzburg, South Africa
(S29.35 E30.20).
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The two sequences were manually aligned and analyzed
using Bayesian methods. First, the two fungal sequences
were tested for combinability using the Partition Homogeneity Test [39] implemented in PAUP*4.0b10 [40],
which showed that there was no significant incongruence
between the two data sets (1000 artificial data sets, p =
0.14). Bayesian analyses (MrBayes 3.0, [41]) were therefore performed on the combined data set. The two partitions were defined and for each partition a separate model
was used (MrModeltest; [42]): SYM + Γ [43]) (lset nst = 6
rates = gamma) for the nuclear 25S and the F81 model +
Γ [44]) (lset nst = 1 rates = gamma) for the mitochondrial
12S. Additional maximum parsimony and neighbor-joining analyses (using PAUP*4.0b10 [40]) were performed
to check for consistency with the Bayesian results.
Sequences have been submitted to Genbank (accession
numbers in Table 1 Additional File 1).
Statistical analysis of host specificity using AMOVA
We used Analysis of Molecular Variance (AMOVA) in
Arlequin vers.3.1 [45]) to partition sequence variation
among isolates at three hierarchical levels: between host
genera, between host species within genera, and between
colonies within species. We excluded the 15 allopatric
samples from Senegal in the AMOVA analysis, because of
the way these were sampled (only one of multiple identical samples was included, so that sampling could be
biased). As a measure of genetic distance between ITS haplotypes we used uncorrected pairwise distances. Significance was assessed through 100,000 random permutation
replicates. To study the exact source of between host-species symbiont variation, we also performed separate
AMOVAs within the two genera with more than a single
species, Odontotermes and Microtermes. Furthermore, we
made pairwise comparisons between species, using only
the frequency of the different fungal haplotypes of termite
species, and not sequence information [27,28]). The
rationale behind this was to statistically test the significance of the finding that none of the species in the genus
Odontotermes shared fungal haplotypes, despite having
closely related haplotypes. Since AMOVA takes the genetic
distance between the haplotypes into account, it will
underestimate differentiation between species in this specific case.
Authors' contributions
DKA and VIDR carried out the DNA work. DKA and JJB
conceived the study. DKA designed and coordinated the
study, and analyzed and interpreted the data and drafted
the manuscript. DKA, VIDR, HHdFL, CRL, JM, BS and
ZWdB contributed with the sampling work and revised
the manuscript. JJB revised the manuscript. All authors