FEMS Microbiology Letters 171 (1999) 147^153
Phylogenetic relationships of symbiotic methanogens
in diverse termites
Moriya Ohkuma a; *, Satoko Noda
a
a;b
, Toshiaki Kudo
a
Microbiology Laboratory, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-0198, Japan
b
Bioengineering Laboratory, Department of Applied Chemistry, Toyo University, Kawagoe, Saitama 350, Japan
Received 25 September 1998; received in revised form 8 December 1998; accepted 11 December 1998
Abstract
Termites harbor symbiotic microorganisms in their gut which emit methane. The phylogeny of the termite methanogens was
inferred without cultivation based on nucleotide sequences of PCR-amplified 16S ribosomal RNA genes. Seven methanogen
sequences from four termite species were newly isolated, and together with those previously published, these sequences were
phylogenetically compared. The termite methanogen sequences were divided into three clusters. Two clusters of sequences,
derived from the gut DNA of so-called higher termites, were related to methanogens in the orders Methanosarcinales or
Methanomicrobiales. All of the sequences in the case of lower termites were closely related to the genus Methanobrevibacter.
However, most of the termite symbionts were found to be distinct from known methanogens. They are not dispersed among
diverse methanogen species, but rather formed unique lineages in the phylogenetic trees. z 1999 Federation of European
Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Symbiosis ; Methanogen; Termite ; Phylogeny ; 16S ribosomal RNA
1. Introduction
Termites harbor methanogenic archaea (methanogens) in their gut which emit methane and not many
terrestrial arthropods do so [1]. Since termites exist
in high biomass densities, particularly in tropical regions, they have been cited as a potentially signi¢cant source of atmospheric methane, although their
precise contribution to global methane emissions is
uncertain, with estimates ranging from less than 5%
* Corresponding author. Tel.: +81 (48) 462 1111, ext. 5724;
Fax: +81 (48) 462 4672;
E-mail: [email protected]
to more than 40% of the total annual global methane
production ([2], and references therein).
Although methanogens are important and often
cited components of the termite microbiota, there
have been only a few studies on these termite gut
symbionts. Partial rRNA sequences from uncultivated methanogens have been obtained by PCR ampli¢cation of DNA from the gut contents of xylophagous termites Reticulitermes speratus [3] and
Cryptotermes domesticus [4]. Methanogens in both
termites were found to be related to the genus Methanobrevibacter but were phylogenetically distinct
from other known members of this genus. Such a
strategy, which does not rely on the cultivation of
resident microorganisms, has already been applied to
0378-1097 / 99 / $19.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 5 9 3 - X
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show the microbial diversity in the gut of termites
[5^8]. Recently, three distinct types of methanogen
species were isolated from the hindgut of the xylophagous termite Reticulitermes £avipes and identi¢ed as
three new species of the genus Methanobrevibacter
[9,10]. These methanogens utilize primarily CO2
and H2 , which are produced in intermediate steps
of the metabolic pathway for lignocellulose degradation.
Termites are comprised of a complex assemblage
of species, roughly divided into so-called higher and
lower termites [11]. Especially higher termites show
considerable variation in their feeding behavior,
which is not limited to xylophagy. Some feed exclusively on soil, presumably deriving nutrition from the
humic compounds therein, and others cultivate and
consume cellulolytic fungi. Nevertheless, all known
termites have a dense and diverse hindgut microbial
community, which aids in digestion [12,13]. The only
termites whose symbiotic methanogens have been
phylogenetically identi¢ed are xylophagous (woodfeeding) lower termites. It is well known that soilfeeding and fungus-growing higher termites emit
more methane than wood-feeding termites [2]. Comparison of methanogen phylogenies in diverse termites of various feeding guilds is of great interest.
In this study, methanogens in three higher termite
species comprising three feeding guilds and in one
wood-feeding lower termite belonging to a di¡erent
termite family from the species whose symbiotic
methanogens have been characterized were phylogenetically identi¢ed based on the sequences of PCRampli¢ed 16S rRNA genes from DNA in the gut
microbial community of these termites. We also discuss the phylogenetic relationships between diverse
termites and the symbiotic methanogens reported
so far.
2. Materials and methods
2.1. Termites
The lower termite Hodotermopsis sjostedti (previously H. japonica), and three higher termites Odontotermes formosanus, Nasutitermes takasagoensis and
Pericapritermes nitobei were used in this study. H.
sjostedti was collected at Yakushima Island, Japan
in July 1997, and O. formosanus, N. takasagoensis
and P. nitobei were collected at Iriomote Island, Japan in April 1997. Their families, subfamilies and
feeding behaviors are shown in Table 1.
2.2. DNA extraction, PCR ampli¢cation, and cloning
Approximately 50^100 termites were collected
and, after their exterior surfaces were washed with
distilled water, their entire guts were removed with
forceps. The guts were homogenized and DNAs
from the mixed population in the whole gut were
extracted as described previously [3,5]. Genes encoding the 16S rRNA of the termite methanogens were
ampli¢ed from the extracted DNA by PCR using
ExTaq DNA polymerase (Takara) according to the
manufacturer's directions. The PCR primers used
were M23FB and 1392RH, which have been described previously [3]. These sequences were deduced
from the consensus regions of archaeal 16S rRNA.
The reaction conditions were for 35 cycles at 94³C
for 30 s, 48³C for 45 s, 72³C for 2 min. Because the
ampli¢cation of DNA from O. formosanus failed in
the ¢rst 35 cycles, we used the forward primer 350F
(5P-CTACGGGGCGCAGCAG-3P)
instead
of
M23FB in a second round of 30 PCR cycles under
the same conditions. PCR products corresponding to
the expected size of the archaeal rRNA gene were
size-fractionated on a low melting agarose gel and
puri¢ed by means of the Wizard PCR preps DNA
puri¢cation system (Promega). The puri¢ed PCR
products were ligated into a pGEM-T vector (Promega) according to the manufacturer's directions and
then introduced into Escherichia coli.
2.3. RFLP analysis, nucleotide sequencing and
phylogenetic analysis
The insertion of DNA fragments of appropriate
sizes was con¢rmed by PCR ampli¢cation using universal and reverse primers (Takara) which corresponded to both sides of the cloning site on the
vector. The ampli¢ed product was examined by
RFLP (restriction fragment length polymorphism)
analysis using the restriction enzymes, HapII, AluI,
HhaI and Sau3AI. The nucleotide sequence was determined by means of a Dye Terminator Cycle Sequencing Kit (Applied Biosystems) using an auto-
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149
Table 1
The 16S rRNA gene sequences of the methanogens from diverse termites
Termite (feeding guild or family)
Clone name
RFLPc group
Number of clones
A¤liationd
4
4
1
12
5
1
mic
sar
bac
3
20
sar
1
6
1
11
mic
mic
2
5
bac
Accession number
Higher termites (family Termitidae)a
Pericapritermes nitobei (soil-feeding)
MPn1
AB009825
MPn4
AB009826
MPn19
AB009827
Odontotermes formosanus (fungus-growing)
MOf1
AB009822
Nasutitermes takasagoensis (wood-feeding)
MNt1
AB009823
MNt2
AB009824
Lower termites (wood-feeding)b
Hodotermopsis sjostedti (family Termopsidae)
MHj4
AB009821
Cryptotermes domesticus (family Kalotermitidae)
Cd30e
AB008900
Reticulitermes speratus (family Rhinotermitidae)
M4f
D64027
Reticulitermes £avipes (family Rhinotermitidae)
M. curvatusg
U62533
M. cuticularisg
U41095
M. ¢liformish
U82322
bac
bac
bac
bac
bac
a
The higher termites are comprised of only one family, Termitidae.
All lower termites are wood-feeders.
c
The RFLP group indicates the number of groups showing di¡erent RFLP patterns which includes the groups having the same sequence but
di¡erent insertion directions relative to the vector sequence.
d
Abbreviations of the methanogen orders are: mic, Methanomicrobiales; sar, Methanosarcinales; bac, Methanobacteriales.
eÿh
Sequences were not sorted by RFLP and the pertinent references are: e [4]; f [3]; g [9]; h [10].
b
matic sequence analyzer (ABI model 377). Sequencing primers 519FB [5], 1100R (5P-CGGGTCTCGCTCGTTG-3P), 925R (5P-CAATTCCTTTAAGTTTC-3P), 925F complementary to 925R, 800R
(5P-CTACCCGGGTATCTAAT-3P), 700R (5P-ATTTCACTCCTACCCC-3P) and 325F (5P-CTACGGGGCGCAGCAG-3P), the sequence of which were
deduced from consensus regions of archaeal 16S
rRNA, were used. The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL
and GenBank nucleotide sequence databases under
the accession numbers shown in Table 1.
Sequence data were aligned using the CLUSTAL
W package [14] and checked manually. Nucleotide
positions of ambiguous alignments were omitted
from the subsequent phylogenetic analysis. The program fastDNAml [15] was used to create maximum
likelihood trees using empirical base frequency,
jumbled order for taxa addition, global branch swapping and bootstrapping options. The bootstrap [16]
with 100 replicates was used to estimate the robustness of branches in these analyses.
3. Results
3.1. Analysis of 16S rRNA gene clones
Sequences of methanogen 16S rRNA genes were
ampli¢ed by PCR from the mixed-population DNAs
obtained from the gut of the termites, and clonally
isolated. First, we sorted the sequences by RFLP
analysis of the cloned fragments. Then, representative clones from among those showing the same
RFLP patterns were partially sequenced (more
than 400 bp). The sequences showing only a few
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Fig. 1. Phylogenetic relationships of symbiotic methanogens inhabiting the gut of termites among members of the orders Methanosarcinales and Methanomicrobiales. The tree was inferred by the maximum likelihood method on the basis of 912 unambiguously aligned nucleotide positions which corresponded to the Halobacterium halobium sequence 337^1339. The sequence of H. halobium was used as an
outgroup. The scale bar represents 0.05 substitutions per nucleotide position. Numbers at nodes indicate bootstrap values for each node
out of 100 bootstrap resamplings (values below 50 are not shown). The database accession numbers of the reference sequences are in parentheses. The a¤liations with orders are shown in the right of the tree.
base di¡erences (fewer than 1%) were grouped together. Such small sequence di¡erences may be due
to ampli¢cation errors or intraspeci¢c sequence variability. One clone representative of each group was
further sequenced over the region corresponding to
nucleotide positions 337^1339 of Halobacterium halobium. The sequences were compared with those of
known methanogens and were assigned to existing
families of methanogens based on sequence similarity. The termite methanogens were largely divided
into three clusters. These results are summarized in
Table 1.
Formation of chimeras is a problem in ampli¢cations of mixed-population DNAs by PCR [17]. Evaluation by the CHECK_CHIMERA program of the
Ribosomal Database Project [18] indicated that the
sequences reported in this study showed no obvious
evidence of chimeric artifacts. The absence of chimeric artifacts was also con¢rmed by inspection of
the predicted secondary structures of the sequences,
which contained compensatory base changes in the
stem regions that maintained known 16S rRNA secondary structures.
3.2. Phylogeny of the methanogen sequences
related to the orders Methanosarcinales
and Methanomicrobiales
Most of the archaeal 16S rRNA gene sequences
found in the gut DNA of higher termites were divided into two clusters and related to methanogens of
the orders Methanosarcinales or Methanomicrobiales. Fig. 1 shows the phylogenetic relationships
among representative members of these orders. The
sequences MPn4 and MOf1 showed 98% similarity
and clustered together. These two sequences were
grouped with the sequences of most members of
the order Methanosarcinales (except for the members
of the genus Methanosaeta) and a bootstrap value of
100% supported their monophyly. However, they
showed less than 90% similarity compared with the
sequences of known methanogens and formed a nov-
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151
Fig. 2. Phylogenetic relationships of symbiotic methanogens inhabiting the gut of termites among members of the order Methanobacteriales. The tree was inferred by the maximum likelihood method, using the sequence of Methanococcus jannaschii as an outgroup, based
on a total of 734 unambiguously aligned nucleotide positions corresponding to the H. halobium sequence 472^1339. The sequences from
the methanogens from termites are indicated by bold letters. M. curvatus, M. ¢liformis and M. cuticularis have been isolated from Reticulitermes £avipes [9,10]. The scale bar represents 0.05 substitutions per nucleotide position. Bootstrap values above 50 from 100 resamplings
are shown for each node. The database accession numbers and the ID names in Ribosomal RNA Database Project [18] of the reference
sequences are in parentheses.
el lineage within this order, indicating the presence of
a novel, as yet unknown methanogen genus in termites.
The three sequences, MPn1, MNt1, and MNt2,
showed more than 98% similarity and clustered together. In the phylogenetic tree (Fig. 1), these three
sequences formed a monophyletic lineage with the
sequence of Methanospirillum hungatei and a bootstrap value of 98% supported their monophyly.
However, these three sequences showed only 91^
92% sequence similarity when compared with the
sequence of M. hungatei and showed less than 89%
similarity compared with those of other methanogens.
3.3. Phylogeny of the methanogen sequences related
to the order Methanobacteriales
The archaeal 16S rRNA gene sequences found in
the gut mixed-population DNA of three lower termites (MHj4, RsM4 and Cd30) and from the soilfeeding P. nitobei (MPn19) were from methanogens
associated with the family Methanobacteriaceae
within the order Methanobacteriales. Fig. 2 shows
the phylogenetic relationships among the representatives of this order. These sequences showed more
than 94% similarity to the sequences of the four species belonging to the genus Methanobrevibacter, M.
arboriphilicus, M. cuticularis, M. ¢liformis and M.
curvatus, indicating that the symbiotic methanogens
in these termites were closely related to the genus
Methanobrevibacter. The latter three have been isolated from the gut of the termite R. £avipes [9,10].
The MPn19 sequence showed 98.1% similarity with
the sequence of M. arboriphilicus and 97.6% with
that of M. cuticularis. The three sequences, MHj4,
RsM4 and Cd30, sharing more than 95% sequence
similarity, formed a novel lineage within this genus
and a bootstrap value of 94% supported this monophyly, suggesting the presence of an as yet uncultivated new species in the gut of lower termites.
4. Discussions
Methanogens inhabiting diverse termite species
were categorized into three groups. The presence of
methanogens belonging to the orders Methanosarci-
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nales and Methanomicrobiales in termites is newly
described in this study, although the presence of
members of the order Methanobacteriales has been
reported before [3,4,9,10]. Most of the 16S rRNA
gene sequences of the termite symbiotic methanogens
were distinct from those of other known methanogens and these methanogens formed unique lineages
in the phylogenetic trees, suggesting the presence of
new, as yet uncultivated methanogens in the gut of
the termites. The termite gut may be a rich reservoir
of novel methanogen diversity. Furthermore, the termite methanogens were found to form discrete clusters in the phylogenetic trees, and were not dispersed
among diverse methanogen species. These results
suggest that only certain speci¢c types of methanogens are able to inhabit the gut of termites.
Three methanogen species, M. curvatus, M. cuticularis and M. ¢liformis have been isolated from the
hindgut of the termite R. £avipes [9,10]. These
methanogens utilize H2 plus CO2 but use other substrates poorly, yielding CH4 as the sole product. H2
and CO2 are liberated from intermediate steps of
lignocellulose decomposition in the termite gut. Considering that all known members of Methanobrevibacter species show the property of a narrow range
of utilizable substrates (CO2 reduction using H2 or
formate) [19], the termite symbiotic methanogen related to this genus may utilize them. The genus
Methanospirillum, to which the sequences MPn1,
MNt1 and MNt2 are most closely related, also
grow by CO2 reduction using H2 or formate [19].
However, the members of the order Methanosarcinales, to which the sequences MPn4 and MOf1 are
closely related, grow mainly on acetate and methyl
compounds such as methanol and metylamines [19].
Acetate is a major compound produced by microbial
fermentation in the termite hindgut [12,13] and it is
probable that methanol derives from methoxyl
groups of pectins. In animal rumen, hydrogenotrophic methanogens are responsible for the major part
of rumen methanogenesis, although Methanosarcina
can be present in low numbers [20]. The real substrates utilizable by the termite symbiotic methanogens cannot be clari¢ed until they have been cultivated and characterized.
More than 98% sequence similarity was evident
between MPn4 and MOf1, and among MPn1,
MNt1 and MNt2, indicating that closely related
methanogens were shared between P. nitobei and
O. formosanus, and between P. nitobei and N. takasagoensis, respectively. These sequences are thought
to be derived either from closely similar organisms or
from populations of the same species. This may be
explained by recent transfer of the methanogens
from one termite to another. Since these three higher
termites were collected from the same small island
(Iriomote island, located in the southernmost part
of Japan), such a regional restriction must be noted.
A regional diversity of methanogen composition in
R. £avipes has already been reported [9,10]. Analysis
of more termites inhabiting various geographic locations is necessary. On the other hand, the sequences
from termite methanogens related to the genus
Methanobrevibacter shared rather low sequence similarity (95^98%). This suggests that related but distinct species inhabit the gut of termites.
It has been reported that the soil-feeding and fungus-growing termites emit more methane than woodfeeders [2]. All lower termites feed on wood and
methanogens in the lower termites were related to
the genus Methanobrevibacter. A Methanobrevibacter-like sequence was not isolated from the
wood-feeding higher termite, N. takasagoensis. In
contrast to the lower termites, the methanogens in
higher termites were a rather diverse collection of
species. Especially the soil-feeder P. nitobei harbored
methanogens belonging to three di¡erent families. As
mentioned above, phylogenetically related methanogen species were shared between di¡erent feeding
guilds in higher termites. Thus, at present, it is di¤cult to say that termites harbor speci¢c types of
methanogen species depending on their feeding behavior.
In order to study the role and contribution of each
methanogen species identi¢ed here, measurement of
the rates of methane emission and quantitative analysis of their populations are necessary. The sequences reported here are bene¢cial to design species-speci¢c probes and/or primers for the termite symbiotic
methanogens. Such an oligonucleotide-probing experiment has appeared, in which the rRNAs of members of the family Methanobacteriaceae comprised
the most abundant of the archaeal rRNAs in the
guts of almost all termite species, regardless of their
diet [21]. The sequence-speci¢c probes are also useful
to detect in situ localization of the symbionts, since
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many types of symbiosis have been found in the gut
of termites: free-living, endosymbiosis in intestinal
anaerobic protists, and colonization of the surface
of the gut epithelium of the host [1,9,10,22,23].
[10]
[11]
Acknowledgments
We thank Y. Murayama for assistance and I. Yasuda for advice on the collection of termites. This
work was partially supported by grants for the Biodesign Research Program and the Genome Research
Program from RIKEN. One of us (S.N.) was supported by a grant for the Junior Research Associate
Program from RIKEN.
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