of 8
Published Ahead of Print 25 May 2012.
10.1128/AEM.08008-11.
2012, 78(15):5368. DOI:
Appl. Environ. Microbiol.
Nicholas R. Ballor and Jared R. Leadbetter
Lignocellulose-Feeding Higher Termites
the Gut Microbial Communities of
Patterns of [FeFe] Hydrogenase Diversity in
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Patterns of [FeFe] Hydrogenase Diversity in the Gut Microbial
Communities of Lignocellulose-Feeding Higher Termites
Nicholas R. Ballor
and
Jared R. Leadbetter
Biochemistry and Molecular Biophysics, California Institute of Technology, Pasadena, California, USA
Hydrogen is the central free intermediate in the degradation of wood by termite gut microbes and can reach concentrations ex-
ceeding those measured for any other biological system. Degenerate primers targeting the largest family of [FeFe] hydrogenases
observed in a termite gut metagenome have been used to explore the evolution and representation of these enzymes in termites.
Sequences were cloned from the guts of the higher termites
Amitermes
sp. strain Cost010,
Amitermes
sp. strain JT2,
Gnathamit-
ermes
sp. strain JT5,
Microcerotermes
sp. strain Cost008,
Nasutitermes
sp. strain Cost003, and
Rhyncotermes
sp. strain Cost004.
Each gut sample harbored a more rich and evenly distributed population of hydrogenase sequences than observed previously in
the guts of lower termites and
Cryptocercus punctulatus.
This accentuates the physiological importance of hydrogen for higher
termite gut ecosystems and may reflect an increased metabolic burden, or metabolic opportunity, created by a lack of gut proto-
zoa. The sequences were phylogenetically distinct from previously sequenced [FeFe] hydrogenases. Phylogenetic and UniFrac
comparisons revealed congruence between host phylogeny and hydrogenase sequence library clustering patterns. This may re-
flect the combined influences of the stable intimate relationship of gut microbes with their host and environmental alterations in
the gut that have occurred over the course of termite evolution. These results accentuate the physiological importance of hydro-
gen to termite gut ecosystems.
H
ydrogen plays a pivotal role in the digestion of wood by ter-
mites (
7
,
11
,
39
). Concentrations in the guts of some species
exceed those measured for any other biological system (
18
,
41
,
43
). The turnover of the gas in the gut has been measured in some
species at daily fluxes as high as 33 m
3
/m
3
gut volume (
39
). The
environment is also spatially complex, comprising a matrix of
microenvironments characterized by different hydrogen concen-
trations (
14
,
18
,
26
,
39
).
Termites can be classified as belonging to one of two phyloge-
netic groups, higher termites and lower termites (
25
). Higher ter-
mites characteristically lack protozoa, which are abundant in the
guts of lower termites, in their guts and have more highly seg-
mented gut structures than do lower termites (
17
,
34
,
35
). Of the
over 2,600 known species of termites, over 70% are higher ter-
mites (
25
,
49
). They represent the largest and most diverse group
of termites (
24
,
49
). Yet, most of what we know about termite gut
microbes comes from work done with lower termites, and com-
paratively little work has been done with the communities of
higher termites (
8
10
,
12
). The primary reason for this is that it
was believed until recently that the gut microbes of higher termites
played only a minor role in wood digestion (
42
,
46
,
47
). This
changed with the recent publication of the gut metagenome of a
higher termite where it was found that the gut microbial commu-
nity harbors genes for reductive acetogenesis, polysaccharide deg-
radation, and an abundance of [FeFe] hydrogenases, all pointing
in the direction of a more active role in wood degradation (
47
).
This previously underacknowledged role for the gut microbes has
also found support in the findings of Tokuda and Watanabe (
46
).
Wood-feeding insects have shared a stable and intimate mutu-
alism with their respective gut microbial communities for at least
20 million years (
48
). It has been proposed that the gut microbes
of lower termites may “coevolve” with their respective hosts (
4
,
19
,
21
,
50
). Past analyses of
nifH
genes from the guts of higher and
lower termites and
Cryptocercus
have shown that genes from the
same genus or family of termite tend to be more similar to one
another than to those from more distantly related termites (
19
,
37
,
50
). Hongoh et al. found that the gut community composition is
consistent within a genus of termites (
21
). Analyses of spirochete
diversity in higher and lower termites have demonstrated a ten-
dency of spirochete 16S sequences from the same genus of termite
to cluster with one another, supporting the hypothesis that “spi-
rochaetes are specific symbionts that have coevolved with their
respective species of termites” (
4
,
36
). Moreover, the composition
of termite gut communities has been shown to vary substantially
with host feeding habits, which are closely linked with phylogeny
(
32
,
38
,
44
). In the case of wood roaches, a very close correspon-
dence of host and symbiont phylogenies has lent strong support
for the cospeciation of a
Cryptocercus
endosymbiont with its hosts
(
16
,
19
,
28
). Noda et al. have reported the cospeciation of intesti-
nal microorganisms with their termite hosts, thereby demonstrat-
ing the high stability of the association between a termite and its
gut microbiota (
33
).
Here we report a phylogenetic analysis of [FeFe] hydrogenase
genes cloned from the guts of higher termites. We have focused on
family 3 [FeFe] hydrogenases, first defined by Warnecke et al.,
because they comprise the most highly represented group of hy-
drogenases observed in a
Nasutitermes
hindgut metagenome se-
quence (
47
). They were also the only group of hydrogenases ob-
served in the
Nasutitermes
hindgut metagenome whose
in situ
translation was verified by mass spectroscopy (
47
). The objective
was to better understand the diversity, adaptation, and evolution
Received
29 December 2011
Accepted
17 May 2012
Published ahead of print
25 May 2012
Address correspondence to Jared R. Leadbetter, jleadbetter@caltech.edu.
Supplemental material for this article may be found at
http://aem.asm.org/
.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AEM.08008-11
5368
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of these genes in these hydrogen-metabolizing ecosystems. More-
over, the influence of host ecosystem variations on the hydroge-
nase sequence composition of their associated microbial commu-
nities was investigated through cross-comparisons with sequence
libraries reported previously for lower termite and wood roach
samples (
2
).
MATERIALS AND METHODS
Termites.
Nasutitermes
sp. strain Cost003 and
Rhyncotermtes
sp. strain
Cost004 were collected in the INBIO forest preserve in Guápiles, Costa
Rica. Cost003 was collected at a height of 1.2 m from a
Psidium guajaba
tree and was believed to be feeding on deadwood. Cost004 was collected
from a nest located under a bromeliad. Feeding trails leading from this
nest to a pile of decaying wood and plant material suggested litter feeding.
Microcerotermes
sp. strain Cost008 was collected from the base of a palm
tree about 100 m from the beach at Cahuita National Park in Costa Rica
and appeared to be feeding on the palm tree.
Amitermes
sp. strain Cost010
was collected from the roots of dead sugarcane plants at a plantation in
Costa Rica.
Amitermes
sp. strain JT2 and
Gnathamitermes
sp. strain JT5
were collected from subterranean nests at Joshua Tree National Park in
the United States (permit no. JOTR:2008-SCI-002). Termites were iden-
tified in a previous study (
38
) using insect mitochondrial cytochrome
oxidase subunit II (COXII) gene sequences and morphology.
DNA extraction and cloning.
For each termite sample, DNA was ex-
tracted from single whole dissected guts and quantitated as described
elsewhere (
2
,
31
). Degenerate primers reported in a previous study (
2
) for
the specific amplification of family 3 [FeFe] hydrogenases were used for
the cloning of gut sequences as described previously (
2
). The degenerate
primer sequences, which were ordered from IDT DNA, were WSI CCI
CAR CAR ATG ATG G and CCI CKR CAI GCC ATI ACY TC for the
forward and reverse primers, respectively, where “I” represents inosine.
Restriction fragment length polymorphism (RFLP) analysis and se-
quencing.
Clones were selected for sequencing, and sequences were ed-
ited and verified as encoding hydrogenase as described elsewhere (
2
).
Sequences that analyses aligned poorly with other cloned hydrogenase
sequences in our database were resequenced and analyzed manually for
frameshift mutations or internal stop codons. Frameshift mutations were
identified and manually corrected at the DNA level for three clones (see
the footnotes to Table S1 in the supplemental material).
Phylogenetic analysis.
Phylogenetic analyses were completed as de-
scribed elsewhere (
2
). Cloned sequences and their operational taxonomic
units (OTUs) used in these analyses are listed in Table S1 in the supple-
mental material. Trees were constructed using 173 unambiguously
aligned amino acid positions with distance matrix (Fitch), maximum par-
simony (Phylip PROTPARS), and maximum likelihood (PhylipProML)
treeing methods. Phylograms presented in this paper were drawn using
Phylip drawgram (
20
). The following sequences, all derived from gut sym-
bionts of termites, comprised the outgroup used to construct
Fig. 1
and
2
(
3
,
22
):
Pseudotrichonympha grassii
(AB331668), uncultured parabasilid
(AB331670),
Holomastigotoides mirabile
(AB331669),
Pseudotrichonym-
pha grassii
(AB331667),
Treponema primitia
ZAS-1 (HndA1, HQ020732),
Treponema primitia
ZAS-2 (HndA2, HQ020741),
T. primitia
ZAS-2
(HndA3, HQ020740), and
T. primitia
ZAS-1 (HydA1, HQ020748). The
following family 3 [FeFe] hydrogenase sequences reported elsewhere (
3
)
were also used to construct
Fig. 1
and
2
:
Treponema primitia
strain ZAS-2
(HndA1, HQ020737) and
Treponema azotonutricium
strain ZAS-9
(HndA, HQ020755).
Diversity and sequence richness calculations.
Chao1 sequence rich-
ness and Shannon diversity indices for each clone set were calculated using
EstimateS version 8.0.0 for Macintosh computers, written and made freely
available by Robert K. Colwell (
http://viceroy.eeb.uconn.edu/EstimateS
).
The Shannon evenness index (
30
) was calculated from the Shannon di-
versity index. OTUs and their respective sequence abundances were used
as inputs to the program.
Community comparisons.
UniFrac (
29
) was used for quantitative
comparisons of the higher termite [FeFe] hydrogenase sequence libraries
with each other or with those prepared from lower termites and
Crypto-
cercus punctulatus
reported elsewhere (
2
). Maximum likelihood trees were
constructed according to the methods described above and subsequently
used as the input for UniFrac. One hundred seventy-three unambiguously
aligned amino acids were used in treeing calculations. Each sequence li-
brary was designated a unique environment. The number of cloned se-
quences represented by each OTU was input to UniFrac to be used for
calculating abundance weights. The environments were compared using
the UniFrac jackknife and principal component analyses (PCAs). Nor-
malized abundance weights were used in all calculations. The jackknife
calculation was completed with 1,000 samplings and using 75% of the
OTUs contained in the smallest environment sample as the minimum
number of sequences to keep.
Nucleotide sequence accession numbers.
Sequences have been de-
posited in the GenBank, DDBT, and EMBL databases under accession
numbers
HQ020957
to
-1201
.
RESULTS
Sequences cloned.
Hydrogenase sequences representing as
many as 44 sequence OTUs were cloned from each of the higher
termites (
Table 1
). Table S1 in the supplemental material lists
all cloned sequences, and their corresponding OTUs, analyzed
in this study. The collector’s curves for each sequence library
are provided as Fig. S1 in the supplemental material.
Microcer-
otermes
was the only sample having 75% of all cloned sequences
distributed among fewer than 7 OTUs. The Shannon diversity
index and the Chao1 species richness index for each sequence
library are listed in
Table 1
.
Phylogenetic analysis.
In phylogenetic analyses comparing
the cloned sequences to publically available [FeFe] hydrogenase
sequences, all sequences in our database, with one exception (see
footnote to Table S1 in the supplemental material), clustered
within a single large clade to the exclusion of all nontermite bac-
terial sequences (data not shown). Comprising the sequences fall-
ing within the clade were family 3 [FeFe] hydrogenase sequences
from a
Nasutitermes
gut metagenome (
47
) and from the genome
sequences of two treponemes isolated from
Zootermopsis angusti-
colis
,
T. primitia
ZAS-2, and
T. azotonutricium
ZAS-9 (
3
). A max-
imum likelihood tree for all of the cloned [FeFe] hydrogenase
sequences is provided as
Fig. 1
.
Upon inspection of phylogenetic groupings, the hydrogenase
sequences appeared to cluster in a manner congruent with the
phylogeny of their hosts. Moreover, hydrogenase sequences from
a given termite sample tended to cluster with one another.
Sequence library cross-comparisons.
A maximum likelihood
tree comparing all of the family 3 hydrogenases cloned from the
higher termite samples to those cloned previously from
C. punctu-
latus
and lower termite gut samples (
2
) is provided as
Fig. 2
.An
apparent congruence between the phylogenetic clustering of the
cloned hydrogenases and that of their respective hosts was ob-
served in the UniFrac jackknife clustering of the samples (
Fig. 3A
).
In this analysis, the clustering of the hydrogenase sequences was
congruent with the phylogeny of their respective hosts reported by
Legendre et al. and Inward et al. (
23
,
24
,
27
). Specifically, three
unique coclustering groups could be distinguished corresponding
to the three unique phylogenetic groupings sampled in this study,
including
Cryptocercus
, lower termites, and higher termites. For
instance, lower termites and
C. punctulatus
library sequences
grouped with one another within clusters that appeared in all
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three treeing methods. Nodes appearing in all three treeing meth-
ods tended to have bootstrap values above 50% in parsimony
analyses, whereas clusters appearing in only two methods or one
method tended to have less bootstrap support. In only 3 instances
was there a clustering appearing in all treeing methods that
formed between higher termite library sequences and sequences
originating from
C. punctulatus
, and this was never the case for
any of the lower termite sequences. Moreover, the
C. punctulatus
and lower termite library sequences each had a tendency to fall
within clusters appearing in multiple treeing methods that contain
only
C. punctulatus
or lower termite sequences, respectively. This
clustering was further supported by the UniFrac PCA of the se-
quences (
Fig. 3B
). There was a distinguishable separation between
sequences from each of the three groups representing higher termites,
lower termites, and
C. punctulatus
. Principal component 1, which
accounted for the separation of higher termites from lower termites
and
C. punctulatus
, explained 34.87% of the variation.
A UniFrac principal component analysis of the [FeFe] hydro-
genase sequences cloned from higher termites is provided as
Fig.
3C
. Sequences from
Amitermes
sp. Cost010,
Amitermes
sp. JT2,
and
Gnathamitermes
sp. JT5 clustered together. These samples
could be distinguished from the others according to principal
component 1, which explained 30.68% of the variation.
DISCUSSION
High [FeFe] hydrogenase sequence diversity in higher termites.
The abundance of [FeFe] hydrogenases cloned from the guts of
higher termites, representing as many as 44 OTUs in the case of
Rhyncotermes
sp. Cost004, emphasizes the physiological impor-
tance of these enzymes to these complex ecosystems. Moreover,
these cloned sequences were found to belong to the largest family
of [FeFe] hydrogenase sequences observed in a higher termite gut
metagenome. There is good reason to believe that this is only a
sampling of a much larger diversity, because only one of a total of
9 families reported in the
Nasutitermes
gut metagenome sequence
was targeted in this analysis. The grouping of the sequences with
one another to the exclusion of all other non-termite-associated
bacterial [FeFe] hydrogenase sequences in our database may im-
ply unique evolutionary responses to the termite gut ecosystem.
Similar community-wide evolutionary adaptations of [FeFe] hy-
FIG 1
Phylogram for family 3 [FeFe] hydrogenases cloned from the guts of higher termites. The tree was calculated using a maximum likelihood (Phylip ProML)
method with 173 unambiguously aligned amino acid positions. Open circles designate groupings also appearing in either parsimony (Phylip PROTPARS, 1,000
bootstraps) or distance matrix (Fitch) trees. Closed circles designate groupings appearing in trees constructed by all three methods. Each leaf represents an OTU.
The termite host corresponding to each OTU is indicated by a shape or lack thereof:
Amitermes
sp. Cost010, triangle;
Amitermes
sp. JT2, solid black star;
Gnathamitermes
sp. JT5, polygon with white center;
Microcerotermes
sp. Cost008, solid black polygon;
Nasutitermes
sp. Cost003, no shape;
Rhyncotermes
sp.
Cost004, star with white center. Hydrogenase sequences taken from
T. primitia
ZAS-2 and
T. primitia
ZAS-9 are labeled as ZAS-2 (HndA1) and ZAS-9 (HndA),
respectively.
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drogenase sequences from unique ecosystems, as evidenced by
sequence similarity and uniqueness, have been reported by Ballor
and Leadbetter and by Boyd et al. (
2
,
5
,
6
).
The hydrogenases cloned from the higher termites tended to
have a more even distribution and broader sequence diversity
than sequences cloned from
C. punctulatus
or lower termites by
Ballor and Leadbetter (
2
). For example, the higher termites ana-
lyzed in this study had an average of 29 OTUs per library and an
average Shannon mean of 2.87 compared to the same parameters
measured for the
C. punctulatus
and lower termite libraries re-
ported previously as together having an average of 18 OTUs per
library and an average Shannon mean of 2. A
t
test indicated that
the Chao1 and Shannon diversity indices and OTU counts for the
higher termite libraries were all greater than those for the lower
termite and
C. punctulatus
libraries with a confidence greater than
95%. The Shannon evenness index too is found by a
t
test to be
significantly higher at a confidence of over 95% in the higher ter-
mite libraries. This means that in the guts of higher termites not
only there is a greater diversity of hydrogenase sequences than in
lower termites or
C. punctulatus
but there is also a more evenly
distributed representation of each individual OTU.
The
Microcerotermes
gut hydrogenase sequence library had the
lowest diversity among the higher termite samples analyzed, and
the
Rhyncotermes
sequence library had the highest. Both of these
observations are in agreement with a study of formyltetrahydro-
TABLE 1
Analysis of diversity of [Fe] hydrogenase sequences cloned in
this study
Host species
No. of
RFLPs
a
No. of
OTUs
b
Chao1 index
c
Shannon index
Mean
95% CI
Lower
bound
Upper
bound Mean
d
Evenness
e
Amitermes
sp.
Cost010
60
31
45
35.02 79.77 2.97
0.865
Amitermes
sp.
JT2
33
22
32.67 24.18 74.18 2.65
0.857
Gnathamitermes
sp. JT5
44
30
f
40.29 32.75 68.49 3.05
0.906
Microcerotermes
sp. Cost008
36
21
29.1 22.84 56.57 2.33
0.765
Nasutitermes
sp.
Cost003
38
25
43
29.54 96.38 2.69
0.836
Rhyncotermes
sp. Cost004
54
44
68.05 52.73 110.24 3.53
0.933
a
Number of unique RFLP patterns observed. Ninety-six clones were selected from each
clone library in each RFLP analysis.
b
The number of OTUs was calculated using the furthest-neighbor method and a 97%
amino acid sequence similarity cutoff.
c
Chao1 species richness index calculated using the classic method in EstimateS. OTUs
representing family 3 [FeFe] hydrogenases with their respective abundances were used
as program inputs. 95% CI, 95% confidence interval.
d
Shannon diversity index calculated using EstimateS. OTUs representing family 3
[FeFe] hydrogenases with their respective abundances were used as program inputs.
e
The Shannon evenness index has been calculated by dividing the Shannon mean by
the natural log of the number of family 3 OTUs.
f
One of these OTUs represented family 7 [FeFe] hydrogenase sequences (see Table S1 in the
supplemental material) and was not used in the calculation of the diversity indices.
FIG2
Phylogram comparing family 3 [FeFe] hydrogenases cloned from higher termites to sequences cloned previously from
C. punctulatus
and lower termites. The
Fig.
1
caption describes the open and closed black circles and tree construction methods. Each leaf represents an OTU. Leaves and branches representing OTUs cloned from
lower termites have a star at their end, those from
C. punctulatus
have a polygon at their end, and those from higher termites have nothing at their end. Hydrogenase
sequences taken from
T. primitia
ZAS-2 and
T. primitia
ZAS-9 are labeled as ZAS-2 (HndA1) and ZAS-9 (HndA), respectively, with a star following the names.
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folate synthetase gene diversity reported by Ottesen and Leadbet-
ter on the very same two gut samples analyzed in this study (
38
).
The
Rhyncotermes
termites were gathered from a colony that ap-
peared to be feeding on a compost pile containing a mixture of
woody and leaf detritus, which may have the consequence of a
broader diversity of bacteria within its gut environment. The re-
maining termites were gathered from subterranean nests and may
have had a less varied diet.
If we can assume that functional variation is directly correlated
with genetic variation, this observation of differences in evenness
and diversity may imply that the absence of protozoa in higher
termite guts has introduced important selective forces resulting in
a broadening of bacterial hydrogenase functionality and, thereby,
sequence diversity. This diversity may also stem from the more
complex anatomy and segmentation of the higher termite gut
than of lower termites or wood roaches, providing more ecologi-
cal niches and, thereby, a broader diversity of hydrogenase se-
quences associated with microbes that have adapted to myriad
microenvironments. Functional variation is known to be linked to
increased ecosystem function (
45
). In their study of hydrogenase
sequence diversity and phylogeny, Boyd et al. propose that an
increase in phylogenetic diversity that they observed in slightly
acidic geothermal springs may result in a more resilient commu-
nity able to “better respond to change in both physical and chem-
ical conditions in these environments due to seasonal hydrological
and chemical changes” (
5
).
Congruence of [FeFe] hydrogenase and host phylogeny.
[FeFe] hydrogenases cloned from closely related termites had a
tendency to cluster with one another in phylogenetic analyses (
Fig.
1
). For example, sequences from both
Amitermes
gut samples
tended to group together despite their being collected from loca-
tions separated by a great distance—California and Costa Rica.
FIG 3
UniFrac analyses of family 3 [FeFe] hydrogenase sequences. (A) Jackknife clustering analysis. The maximum-likelihood tree shown in
Fig. 2
and the OTUs
with their respective abundance weights as listed in Table S1 in the supplemental material to this work and in Table S1 in the work of Ballor and Leadbetter (
2
)
were used as inputs to UniFrac. The analysis was completed using normalized abundance weights and 1,000 samplings and keeping a number of sequences equal
to 75% of the number of OTUs represented by the smallest sample analyzed. Each insect sample was designated a unique environment. The gray box highlights
all higher termite environments. The numbers designate the percentage of samplings supporting a particular cluster. (B) UniFrac principal component analysis.
The program inputs were as described for panel A. Principal components were calculated using normalized abundance weights. Each termite or
C. punctulatus
sample was designated a unique environment. Higher termite environments, lower termite environments, and
C. punctulatus
environments are each individually
circled. P1, principal component 1; P2, principal component 2; Ca,
C. punctulatus
adult; Cn,
C. punctulatus
nymph; GA, a cluster of samples comprising
Amitermes
sp. Cost010,
Amitermes
sp. Cost003, and
Gnathamitermes
sp. JT5; I,
Incisitermes minor
isolate collection Pas1; M,
Microcerotermes
sp. Cost008; N,
Nasutitermes
sp. Cost003; R,
Reticulitermes hesperus
collection ChiA2; Rh,
Rhyncotermes
sp. Cost004; Z,
Zootermopsis nevadensis
collection ChiA1. (C) UniFrac
principal component analysis. The maximum-likelihood tree shown in
Fig. 1
and the OTUs with their respective abundance weights given in Table S1 in the
supplemental material were used as inputs to UniFrac. Principal components were calculated using normalized abundance weights. Each termite sample was
designated a unique environment. The circled environment corresponds to the most closely related higher termites analyzed in this study. P1, principal
component 1; P2, principal component 2; Ac,
Amitermes
sp. Cost010; Aj,
Amitermes
sp. JT2; G,
Gnathamitermes
sp. JT5; M,
Microcerotermes
sp. Cost008; N,
Nasutitermes
sp. Cost003; Rh,
Rhyncotermes
sp. Cost004.
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Sequence OTUs from a particular termite tended to group with
one another rather than with sequences from other termites. In a
phylogenetic analysis of the COII sequences used for molecular
characterization of the termite samples,
Gnathamitermes
sp. JT5
and
Amitermes
sp. JT2 were found to be the most closely related of
any of the higher termites used in this study (
38
). Correspond-
ingly, there was a tendency for sequences from
Gnathamitermes
sp. JT5 to group with those from the
Amitermes
sp. samples. As
one would expect, sequences taken from the genomes
T. primitia
ZAS-2 and
T. azotonutricium
ZAS-9, each isolated from the gut of
a lower termite, did not group strongly with any of the sequences
cloned from the higher termites (
Fig. 1
).
This congruence was further supported by phylogenetic com-
parisons of the higher termite sequences to lower termite and
Cryptocercus
sequences cloned previously (
2
). The observed lack
of coclustering of the higher termite hydrogenase sequences with
those from
Cryptocercus
or lower termites and the lack of clear
segregation of the lower termite sequences from those of
C.
punctulatus
is in agreement with the close evolutionary related-
ness of these insects (
23
,
24
,
27
). A UniFrac principal component
analysis using the maximum likelihood tree shown in
Fig. 2
fur-
ther supported these observations (
Fig. 3B
). Also, the jackknife
clustering of the [FeFe] hydrogenase communities closely approx-
imated previously proposed termite phylogenies (
23
,
24
,
27
).
UniFrac principal component and jackknife clustering analy-
ses of a maximum likelihood tree of all higher termite sequences
(
Fig. 3C
) revealed a close clustering of the
Amitermes
sp. and
Gnathamitermes
sp. JT5 samples. As mentioned above, these were
the most closely phylogenetically related termites analyzed in this
study. Their hydrogenase sequence libraries grouped with one an-
other in over 99.9% of the samplings used to construct the jack-
knife-clustering tree shown in
Fig. 3
. This clustering was also ap-
parent when the first and second principal components,
collectively explaining 57.22% of variation, were plotted against
each other.
The observed congruence between [FeFe] hydrogenase phy-
logeny and that of the host may imply that hydrogenases, and by
extension their respective gut communities, have coevolved in an
intimate relationship with their host termites. This provides fur-
ther experimental support for previous proposals that termite or
Cryptocercus
gut microbes have coevolved with their host (
1
,
4
,
19
,
21
,
36
,
37
,
40
,
50
). Perhaps this observation may be explained as a
consequence of the influence of environmental changes in the gut,
such as the presence or lack of protozoa or various anatomical
alterations (
34
,
35
), that have developed over the course of termite
evolution. In particular, the gut compartments of higher termites
facilitate dramatic changes in chemical composition along the
length of the gut; for example, the P1 and P3 segments found in
almost all higher termites and no lower termites are highly alkaline
(pH

10) and hydrogen concentrations reach maxima in the ms
and P3 segments (
13
15
). Schmitt-Wagner et al. have demon-
strated that the composition of the microbial community varies
from compartment to compartment in the guts of higher termites
(
40
). Interestingly, Boyd et al. in their study of hydrogenase se-
quence distribution and diversity in geothermal springs found
that geographic distance was the best predictor of phylogenetic
relatedness of sequence communities, resulting most probably as
a consequence of dispersal limitation (
5
). Care must be taken
when making this comparison, however, because the cases re-
ported by Boyd et al. are instances of covicariance explained en-
tirely by geological constraints, whereas in the case of a termite gut
there is an interaction of two biological entities where one influ-
ences the evolution of the other, which is an instance of coevolu-
tion (
51
). In this case, changes in the host are intimately linked to
changes in the associated microbial community such that the evo-
lution of one shapes the evolution of the other, hence, “coevolu-
tion.” In light of the geographically constrained geothermal
springs studied by Boyd et al. (
5
) and keeping in mind the above
caution, the termite gut may be thought of as a “host-constrained”
environment. In summary, surveying the representation of family
3 [FeFe] hydrogenase genes has begun to shed further light onto
the evolutionary physiology of H
2
metabolism by the gut bacterial
communities of termites.
ACKNOWLEDGMENTS
This research was supported by grants from the NSF (MCB-0523267, to
J.R.L.) and the DOE (DE-FG02-07ER64484, to J.R.L.), as well as by an
NSF Graduate Student Research Fellowship (to N.R.B.).
Specimens from Joshua Tree National Park were collected under a
National Park Service research permit (JOTR-2008-SCI-0002).
We thank our laboratory colleagues and anonymous reviewers for
their insightful comments during the preparation and review of the man-
uscript.
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