Insect Molecular Biology (1996)5(4), 229-238
Phylogenetic relationship among termite families
based on DNA sequence of mitochondrial 16s
ribosomal RNA gene
S. Kambhampatl,' K. M. Kjer' and 6. L. Thorne3
' Department of Entomology, Kansas State University,
Manhattan, Kansas:
Department of Zoology, Brigham Young University,
Provo, Utah, and
Department of Entomology, University of Maryland,
College Park, Maryland, USA
*
Abstract
Termites (Order isoptera: Class Insecta), are comprised of a complex assemblage of species, with
considerable variation in life history, morphology,
social behaviour, caste development and ecology. At
present, isoptera is divided into seven families, fourteen subfamliies, 270 genera and over 2000 species.
Phyiogenetic hypotheses currently available for
termlte families and genera are based on a limited
number of morphologicalcharacters and lack rigorous
cladistic analysis. In this paper we report on phylogenetic relationshipsamong ten termite genera of five
families based on a DNA sequence analysis of a
portion of the mitochondriai 16s rRNA gene. Parsimony and distance analysis of DNA sequences supported the existing hypothesis that Mastotermitidae is
the basal lineage among extant termites. Kalotermitidae was not found to be a sister taxon of Mastotermitidae as exlsting hypotheses suggest, but was most
closely related to Rhinotermitidae and Termitidae.
Representatives of Termopsidae were more basal
relative to those of Kaiotermitidae. The utility of 16s
rRNA nucieotide sequence analysis for inferring
phyiogenetic relationships among termlte families,
subfamilies and genera is discussed.
-
Keywords: termites, phyiogenetics, 16s rRNA,
mtDNA, isoptera.
Received 25 September 1995; accepted 11 March 1998. Correspondence:
Dr S. Kambhampati, Department of Entomology, Kansas State University,
Manhattan, KS 66506, USA.
01996 Blackwell Science Ltd
Introduction
Termites (Isoptera) are a diverse group of eusocial
insects. lsoptera is presently divided into seven
families, fourteen subfamilies, -270 genera and more
than 2000 fossil and extant species (Krishna &
Weesner, 1969, 1970; Pearce & Waite, 1994). There
has long been agreement among insect systematists
that cockroaches, mantids and termites are phylogenetically closely related (Boudreaux, 1979; Hennig,
1981; Kambhampati, 1995; Kristensen, 1995). However,
differing relationships have been proposed for the
three groups of insects (McKittrick, 1964; Boudreaux,
1979; Hennig, 1981; Kristensen, 1981) and the precise
topology of the tree that includes cockroaches, mantids
and termites remains a topic of active discussion
(Thorne & Carpenter, 1992; Kambhampati, 1995; Kristensen, 1995).
Several phylogenetic hypotheses have been proposed for relationships within Isoptera, all of which
are based on morphological characters and none
including cladistic analysis (reviewed in Krishna &
Weesner, 1969, 1970). Major proposals of termite
phylogeny at the family and the subfamily level
include those of Hare (1937), Snyder (1949), Grasse
(1949), Emerson (1952; 1955), Sands (1972), Emerson &
Krishna (1975), Ampion & Quennedey (1981), Prestwich (1983) and Noirot (1995). Relationships among
genera within certain families of lsoptera have also
been proposed (e.g. Krishna, 1961; Prestwich &
Collins, 1981; Miller, 1986; reviewed in Krishna, 1970).
The various hypotheses differ concerning the number
of families and subfamilies within lsoptera and the
evolutionary relationships among families, subfamilies and genera (Ahmad, 1950; Grasse 8, Noirot, 1959;
Roonwal & Sen-Sarma, 1960; Krishna, 1970).
There is general agreement among termite systematists that Mastotermitidae is the most ancient
lineage among extant termites (reviewed in Pearce &
Waite, 1994),and that Kalotermitidae and Termopsidae
[formerly included in Hodotermitidae (Grasse, 1949)]
are relatively ancient families (Ahmad, 1950; Emerson,
229
230
S. Kambhampati, K. M. Kjer and 6. L. Thorne
1952;Krishna, 1970;Watson & Sewell, 1985).According
to Krishna (1970),“The Kalotermitidae...have evolved
from Mastotermitidae” (p. 132) and the two families
share several morphological synapomorphies. Some
of the major phylogenetic hypotheses are based on
qualitative observations of a single morphological
system, namely the mandibles of imagos, workers
and/or soldiers (e.g. Ahmad, 1950;Hare, 1937;Krishna,
1961, 1970).These contributions represent the only
phylogenetic hypotheses available for termites today,
and existing phylogenetic hypotheses differ in their
proposed relationships among families. Specifically,a
rigorous cladistic analysis of evolutionary relationships among various termite families, based on phylogenetically-informative characters with a known
genetic basis, is lacking. We report here on a preliminary analysis of phylogenetic relationships among ten
genera of termites belonging to five families based on
DNA sequence of a portion of the mitochondrial large
ribosomal RNA subunit gene (16srRNA) as a means of
exploring the utility of DNA sequence analysis for
inferring phylogenetic relationships among termites.
Results
The length of the sequenced fragment of 16s rRNA
gene from the ten termite taxa ranged from 408 to 429
bp. The average (fSSE) base composition for the
termite taxa was: A: 24.9k0.6, C: 12.0f0.3, G:
21.1k0.4,T: 42.0f0.7. A bias toward adenine and
thymine (67% of total) is consistent with the base
composition of mtDNA sequences of other insects
(Simon ef a/., 1994).The overall transition and transversion rates were 8.8% and 14.0%, respectively.
Among transitions, 54% were C c-) T transitions and
the remainder A ++ G transitions. The relative proportions of the eight types of transversions were A c--) T:
46.4%, A ++C:5.0%, G c-) T: 45.0% and G C* C: 2.7%.
The alignment of the RNA sequence resulted in a
total of 450 characters, including gaps (Appendix). Of
the 450 characters, 192 (43%) were variable and 109
(24%) were parsimony-informative among the termites. Parsimony analysis identified a single tree of
376.82steps (decimal point due to down-weighted gap
characters; see Experimental procedures) with a
monophyletic grouping of the termites (Fig. 1). The
termites included in this study were grouped in accordance with the family level designations presently
recognized. Mastotermitidae, represented by the sole
extant species, Mastotermes darwiniensis,was found
to be the basal taxon among the termites. At the family
level, Kalotermitidae and (Rhinotermitidae + Termitidae) were most closely related to one another,
followed by Termopsidae and Mastotermitidae.
r
+P.
corniceps
I
1. snyderi
I
Mac. barneyi
I
A. wroughtoni
IV
M. darwiniensis
V
26-9
99-9
~
33-16
Flgura 1. Phylogenetic relationship among representativesof termite
families based on parsimony analysis of a portion of the mitochondrial16s
rRNA gene sequence and rooted by the outgroups B. vaga and M. religiose.
Tree length: 376.82 steps; consistency index: 0.7; retention index: 0.5.
Numbers above the branches are total number of supporting nucieotide
charactersand the number of supporting nucleotide characters without
homoplasy (excluding gap characters), respectively. Numbers below the
branches representbootstrap values in percent and decay index,
respectively.Decay indices Indicatethat a particular node was supported in
trees that were longer than the most parsimonious tree by the number of
steps Indicated (Donoghue eta/., 1992; Bremer, 1988,1994)and thus
represent progressively relaxed parsimony. The strength of an inferred
branch 1s directly proportional to the decay index. Family designations lor
termites are as follows: I, Kalotermitidae;11, Termitidae; ill, Rhinotermitidae;
IV, Termopsidae; V, Mastotermttidae. See text lor further details.
Nasutitermes acajutlae and Macrotermes barneyi
were not included in a single clade. These two termites
presently are included in separate subfamilies within
the family Termitidae. However,the node separating N.
acajuflae and M. barneyi was not supported in 50% or
more of the bootstrap replicates and collapsed in a
consensus of four trees of length 377.82steps, one step
longer than the shortest tree. Most of the other relationships inferred in the parsimony tree were supported in
a majority of the bootstrap replications (Fig.1).
Pairwise absolute nucleotide differences and
Tajima-Nei distances are given in Table 1. The tree
based on the distance analysis was essentially identical in topology to the one based on parsimony
analysis (Fig. 2).The only difference between the two
trees was that M. barneyiand N. acajutlae (Termitidae)
formed a monophyleticclade. As in the parsimony tree,
Termitidae was the sister group to Rhinotermitidae,
01996 Blackwell Science Ltd, Insect Molecular Biology 5: 229-238
Phylogenetic relationship among termite families
231
Table 1. Pairwise Tajima-Nei (above diagonal) and absolute nucleotide distances (below diagonal) among taxa used in this study.
1
3
2
5
4
6
7
8
9
10
11
~
1. f .corniceps
2. 1. snyderi
3. N. mona
4. M. barneyl
5. N. acajuflae
6. R. flavipes
7. C. formosanus
8. A. wroughtoni
9. Z.angusticullis
10. M. derwiniensis
11. M. religiosa
12. 8.V 8 Q 8
0.14
0.16
0.13
41
47
49
53
53
50
51
51
69
82
83
53
52
56
52
57
57
61
71
81
83
63
65
63
64
66
61
71
88
86
0.15
0.16
0.19
27
36
26
64
58
59
81
79
0.16
0.17
0.20
0.08
32
26
60
57
55
86
78
0.17
0.16
0.19
0.11
0.09
26
66
63
60
83
86
0.16
0.18
0.20
0.07
0.07
0.07
0.18
0.18
0.21
0.20
0.19
0.21
0.18
58
58
37
57
72
77
47
80
76
0.16
0.19
0.19
0.18
0.18
0.20
0.17
0.11
0.22
0.23
0.23
0.18
0.17
0.19
0.18
0.18
57
74
81
67
79
12
~~
0.28
0.27
0.30
0.27
0.29
0.27
0.26
0.23
0.24
0.21
73
.091
N e . mom
.054
90
P. Codcap8
.068
I. rnydrri
~
.024
.018
68
c.
fOlrmo8aU8
€2.
.012
.004
.028
97
N. acajutlae
W.C. b-e*
I
I
A. wroughtoni
.052
2. a l l g l Z 8 t i C O l l i 8
bl. d a h d e M i 8
.122
.112
Scale:
fladpe8
.057
.078
I
.039
0.28
0.28
0.29
0.26
0.26
0.29
0.25
0.25
0.27
0.26
0.23
8 . vrga
m.
rm1igio.a
each - i s approximately equal to the distance of 0.002386
Fbure2. Phylogenetic relationship among representatives of termite families based on neighbur-joininganalysis of a portion of the mitochondrial 16s rRNA gene
sequence and rooted by the outgroups 8. vagaand M. religiosa. Whole numbers (in italics) are bootstrap values in percent and fractions are branch lengths in
Tajima 8 Nei (1984) distance. See Fig. 1 for family designations of termites and Table 1 for pairwise Tajirna-Nei distances.
and Kalotermitidae was more closely related to a clade
consisting of Rhinotermitidae and Termitidae than it
was to Mastotermitidae.All inferred relationships, with
the exception of the P. corniceps and N. mona branch,
were supported in 50% or greater of the 1000 bootstrap
replicates (Fig. 2).
Dlscusslon
In this paper a phylogenetic analysis of relationships
among ten genera of termites, belonging to five
families, based on the DNA sequence of a portion of
01996 Blackwell Science Ltd, lnsect Molecular Blology5: 229-238
the mitochondrial 16s rRNA gene, is presented. Most
of the inferred relationships had strong quantitative
support as indicated by bootstrap analysis and decay
indices. The relationships among taxa inferred from
the parsimony and the distance analyses were nearly
identical to one another, but only partially congruent
with presently accepted phylogenetic relationships
among termite families (e.g. Krishna, 1970). Whereas
the bootstrap support for some of the basal nodes was
relatively weak (Figs 1 and 2), the fact that the relationships inferred from the parsimony and distance
analyses were congruent and that the inferred
232
S. Kambhampati, K. M. Kjer and 6. L. Thorne
relationships did not decay in trees that were several
steps longer than the most parsimonious tree, provided confidence in the inferred relationships. The
most notable difference in family level relationships
between phylogenies inferred from molecular data and
from morphological characters is as follows. Krishna
(1970) proposed that Kalotermitidae evolved from
Mastotermitidae (implying a sister group relationship
and/or a relatively close phylogenetic relationship
between the two families) and our data contradict this
inference. Although both parsimony and distance
analyses indicated that Mastotermitidae is the basal
lineage among the five termite families included in the
study, neither analysis supported its sister group
relationship to Kalotermitidae. According to our analysis, Termopsidae is more basal than Kalotermitidae.
Kalotermitidae and a clade comprised of (Rhinotermitidae
Termitidae) are sister groups. In this regard,
our results support the proposal by Noirot (1995, p.
223), who, based on observations of gut anatomy,
stated “Kalotermitidae might be the sister group of
Rhinotermitidae + Serritermitidae + Termitidae.”
Because we did not include representatives of Serritermitidae and Hodotermitidae, their phylogenetic
relationship to other termite families cannot be discussed at the present time.
Thorne & Carpenter (1992) proposed a phylogeny of
termites, cockroaches and mantids based on an analysis of previously published morphological, developmental and anatomical characters, Of the three termite
families included in the study (Kalotermitidae, Mastotermitidae and Termopsidae), Thorne 8, Carpenter
(1992)found Mastotermitidae and Kalotermitidae to be
sister families. Thorne & Carpenter (1992) concluded
that Mastotermitidae may not be the most basal termite
family and cautioned that a more comprehensive
analysis including all termite families is required
before a conclusion concerning the position of Mastotermitidae can be firmly established. The position of
Kalotermitidae as a relatively apical group in our
analysis and as a relatively basal group in the morphological analyses (Ahmad, 1950; Krishna, 1970;
Emerson & Krishna, 1975) suggests a need for further
study of these relationships.
Several hydrogen-bonded stems described by
Maidak eta/.(1994) could not be satisfactorily located
in our rRNA data. Although there does appear to be
hydrogen bonding in stems 66 and 82, the guidelines
presented by Kjer (1995) could not be unambiguously
assigned. Stem 73 was not located because the 3 half
of this stem was not included in the amplified fragment.
Stem 84 was present in the insect 16s rRNA, whereas
stem 86 could not be located. Stem 88 was not
supported by our data. Although the selection of
+
regions excluded from the analysis is a subjective
decision, the use of secondary structure substantially
reduces the subjectivity involved in the alignment by
anchoring positions near the excluded regions. For
example, the largest of the excluded regions were two
highly A-U rich regions located within stem 75 (see
Appendix), which is highly variable in a wide range of
taxa (e.g. Hay et a/.. 1995; Kambhampati, 1995). The
excluded positions in stem 75 begin with nucleotides
that can not be shown to be involved in hydrogen
bonding and are interrupted by a conserved section in
the middle of the unpaired loop. Parsimony analysis
that included all characters including those that were
excluded because they were unalignable, resulted in
tree that was identical in topology to the one shown in
Fig. 1, but had a length of 545.82 steps. Additionally,
parsimony analysis based on the direct alignment of
DNA sequence using CLUSTALV (Higgins & Sharp,
1989) and alignment by the eye also resulted in a tree
that was identical in topology to the one in Fig. 1 (data
not shown).
lnsect mtDNA has a base composition that is
strongly biased toward adenine and thymine (Simon
etal., 1994).The termites included in this study were no
exception with an average of 67% adenine and
thymine among the ten taxa. However, the A + T bias
within termite mtDNA is on the lower end for the 16s
rRNA gene of insects studied to date (see Table 2 in
Simon etal., 1994). For example, the homologous 16s
rRNA fragment from thirty-two species of cockroaches
was found to contain, on average, 72% adenine and
thymine (Kambhampati, 1995). Similarly, in a study of
leafhoppers of the family Cicadellidae, Fang et a/.
(1993) reported an average of 73% adenine and
thymine content for a 16s rRNA fragment that was
slightly larger than the one used in the present analysis. DNA sequence of the homologous fragment from
the hymenopteran family Aphidiidae and the Homopteran family Lachnidae revealed an A+T content of
-75% (S. Kambhampati, unpubl. data). The overall
transition rate of 8% for the termites was similar to the
7% reported for cockroaches (Kambhampati, 1995).
However, whereas the C u T transitions in cockroaches were almost twice as frequent as the A ++ G
transitions (66% and 34%, respectively), in termites
the relative proportions of the four types of transitions
were nearly equal. The observed transversion rate
among termites was lower (14%) than that reported
for cockroaches (21%; Kambhampati, 1995). In termites the relative proportion of the A H T transversions was considerably lower (46%) than that in
cockroaches (71YO)
and that of the G +-) T transversions
(46%) considerably higher than that in cockroaches
(4%; Kambhampati, 1995). Our data suggest that sub@ 1996 Blackwell Science Ltd, lnsect Molecu/arBiologyS:229-238
Phylogenetic relationship among termite families
stantive differences exist in the evolutionary dynamics
of the 16s rRNA gene in these two phylogenetically
closely related insect groups.
In summary, the phylogeny for ten termite genera of
five families based on the DNA sequence of a portion of
the mitochondria1 16s rRNA gene was only partially
congruent with presently accepted relationships.
Mastotermitidae was found to be the basal lineage
among extant termites as existing hypotheses suggest;
however, our results did not support a sister-group
relationship between Mastotermitidae and Kalotermitidae. A more extensive sampling of genera, especially
those belonging to Kalotermitidae, Termopsidae and
Hodotermitidae, may be required to confirm our
present findings. Some of the problems we have
encountered in the use of the 16s rRNA gene fragment
for inferring relationships among termite families (e.g.
ambiguities in alignment of some portions of the
sequenced fragment, relatively low bootstrap support
for some nodes and relatively small number of synapomorphic sites) may be avoided by using the DNA
sequence of a gene that is more conserved than that
of the 16s rRNA gene. Nonetheless, our results demonstrate that DNA sequence of genes that are not likely to
vary in function among the different castes is useful for
inferring termite phylogeny. A robust and well-supported phylogeny is a prerequisite for a more thorough
understanding of the complex social organization,
developmental patterns and behaviours that underlie
termite evolution.
Experlrnental procedures
Insects
Ten termite genera representing five families, Mastotermitidae, Kalotermitidae, Termopsidae, Termitidae and Rhinotermitidae, were included in this study (Table 2). All insects,
except M. darwiniensis, were preserved in 80% ethanol; total
genomic DNA of M. darwiniensis, was provided by L. Vawter.
Table 2. List of termite taxa used in this study.
Species
Family'
Mastotermes darwiniensis
Archotermopsis wroughtoni
Zootermopsisangusticollis
Macrotermes barneyi
Nasutitermes acajutlae
Coptotermes formosanus
Reticulitermes Navipes
lncistitermes snyderi
Neotermes mona
Procryptotermescorniceps
Mastoterrnitidae
Terrnopsldae
Terrnopsidae
Terrnitidae
Terrnitldae
Rhinotermitidae
Rhinotermitidae
Kalotermltidae
Kalotermltidae
Kaloterrnitidae
Subfamily'
Terrnopainae
Termopsinae
Macrotermltlnae
Nasutiterrnitlnae
Coptotermitinae
Heteroterrnitinae
-
-
'
Family and subfamily designations follow those of Pearce 8 Waite
(l?).
The 16s rRNA sequences of these termites were published by
Kambhampati (1995).
01996 Blackwell Science Ltd, Insect Molecular Biology 5: 229-238
233
Some of the sequences used in this study were published
previously as indicated in Table 2.
DNA extraction, polymerase chain reaction and DNA
sequencing
A small portion of the thoracic muscle tissue was dissected out
from individual workers or soldiers of each species and
transferred to a sterile 1.5 ml centrifuge tube containing 50 pI
of lysis buffer (10 mM Tris-HCI, pH 8.0, 1 mM EDTA, 1% Nonidet
P-40,lOO pglml Protelnase-K; Sigma Chemical Co.). The use of
thoracic tissue minimized the risk of inadvertent amplification
of DNA of symbiotic microbes in termite guts. The tissue was
macerated with a sterile pipette tip and the tube was incubated
at 37°C for 30 rnin followed by 95°C for 3 min. 50 pI of sterile
water was added to the homogenate and the tube was centrifuged for 10 s to pellet debris. The DNA was used either
immediately in polymerase chain reaction (PCR) or stored at
-20°C.
PCR was set up in 50 pI volume as described by Kambhampati efal. (1992) and Kambhampati (1995). Briefly, the reaction
mix was made up in 500 pI quantities, sufficient to carry out ten
individual reactions, by pipetting 430 pI of sterile water into a
1.5 ml tube and adding 50pl of 10 x TaqDNA polymerase buffer
(Promega Corp.), 5 pl of dNTPs (Promega) to a final concentration of 200 nM, 10 pI of each primer (500 PM; see below) and 3 pI
of Ta9 DNA polymerase (Promega). Aliquots of 50 pI were
pipetted into sterile 0.5ml centrifuge tubes. Template DNA (3-5
pl; see above) was added. The reaction mix was layered with 2
drops of mineral oil (Sigma) and the tubes were placed in a
thermal cycler (MJ Research, Inc.). The following steps were
used for DNA amplification: (1) 95°C for 3 min, (2) 94°C for 30 s,
(3) 50°C for 1 min, (4) 72°C for 1.5 min. Steps 2 4 were repeated
thirty-four more times for a total of thirty-five cycles. The
primers for the amplification of a 415 bp fragment of the 16s
rRNA gene were: forward: 5'-TTA CGC TGT TAT CCC TAA-3'
(Kambhampati & Smith, 1995) and reverse 5'-CGC CTG TIT
ATC AAA AAC AT-3 (Simon et a/., 1994). The amplification
product was electrophoresed on a 2% low melting point
agarose gel. The band corresponding to the amplification
product was excised from the gel using a sterile razor blade,
placed in a sterile 1.5 ml tube and incubated at 70°C for 5 min.
The resulting solution was purified using minicolumns (Wizard
PCRpreps, Promega) according to the manufacturer's instructions. 3 pI of this DNA was used in sequencing reactions.
DNA sequence was obtained directly from double-stranded
PCR products using the cycle sequencing method. The reactions were carried out according to the manufacturer's instructions (fmol Sequencing System, Promega). The forward and
the reverse primer employed for PCR amplification were endlabelled with y-[32P]ATP(6000 Cilmole; NEN-DuPont) and used
to sequence the PCR product. The reaction mixtures were
electrophoresed on 6 % polyacrylamide + urea denaturing
gels for 6 h, with two loading approximately 3 h apart. Both
strands of the PCR product were sequenced.
Sequence alignments and phylogenetic inference
The sequences were read manually from the autoradiographs
into a computer and converted to RNA sequence. The alignment of RNA sequences based on the secondary structure
predictions wascarried out asdescribed by Kjer (1995). Brlefly,
the secondary structural proposal for Drosophila rnelanoga-
234
S. Kambhampati, K. M. Kjerand B. L. Thorne
sterwas obtained from Gutell eta/. (1QQO)and the most recent
60s taurus structure from the Ribosomal Database Project
(Maidak et a/., 1994). By using the general features of these
models, potentially base-paired regions were identified for
each taxon as a preliminary alignment. Modifications were
made to the preliminary alignment by aligning structural
features. Within stems, each paired nucleotide was considered
an anchor point and gaps were inserted in order to maintain
these proposed homologous portions. The aligned RNA
sequences for all taxa included in this study are shown in the
Appendix. The data were analysed using parsimony analysis
(PAUP 3.1.1; Swofford, 1993) with the multiple equally parsimonious exhaustive search option, tree bisectiowreconnection
and 100 random addition sequences. Gaps were treated as
single binary characters and gaps of variable length (G2 and
G4 in the Appendix) were down-weighted so that each indel
was equivalent to a single character. Four regions of the
sequence data (total of fifty-seven characters) were excluded
from the analysis because they could not be aligned unambiguously. These regions are indicated in the Appendix. The
data set was bootstrapped for 1000 replications (fifty random
addition sequences per replicate) using PAUP. The aligned
sequence was also analysed by the neighbour-joining method
(Saitou 8 Nei, 1987) based on the Tajima 8 Nei (1984) distance
using MEGA 1.01 (Kumar e t a / . , 1993). The same fifty-seven
characters that were excluded from the parsimony analysis
were also excluded from the distance analysis. However, the
characters used in the distance analysis were unweighted and
gaps were treated as fifth base. A bootstrap analysis of 1000
replications was carried out on the tree inferred from the
neighbour-joining method. The DNA sequence of the homologous mitochondria1 16s rRNA gene fragment of the cockroach, Blaffella vaga (Blattellidae) and the mantid, Mantis
religiosa (Kambhampati, 1995), was used as outgroups.
Sequence availability
The sequences reported in this study can be obtained from S.K.
or from GenBank under accession numbers U50772-U50778.
The GenBank accession numbers for C. formosanus, M.
darwlniensisand R. flavipes 16s rRNA sequences are U17778,
U17790 and U17824, respectively (Kambhampati, 1995). The
accession numbers for the cockroach and the mantid 16s rRNA
sequences were given by Kambhampati (1995).
Acknowledgements
We thank M. S. Akthar for supplying A. wroughtoni, J.
Reeve for Z. angusticollis, L. Vawter for M. darwiniensisDNA and M. W. J. Crosland for M. barneyi. Financial
support for this study was provided by a seed grant
from the Department of Entomology, Kansas State
University, to S.K. K.M.K. was supported during this
study by NSF grant DEB 91-19091 to J. W. Sites, Jr. We
thank A. L. Nus and L. J. Krchma for technical assistance. This is journal article no. 96-73J of the Kansas
Agricultural Experiment Station.
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Appendix
Aligned sequence of a portion of the mitochondria1 large
subunit (16s rRNA) sequence from termites, cockroach and
mantid in a format that includes secondary structure. The
sequence for the P. corniceps RNA is given at the top, In
sequences of other taxa, nucleotides identical to those of P.
cornicepare Indicated by dots. Gaps are indicated by dashes.
Square brackets represent regions involved in base pairing
where the regions are separated by other hydrogen-bonded
stems in the molecule. Unprimed numbered half-stems pair
with their prlmed downstream complementary counterparts.
Stem sequences narrowly separated from their complementary sequences are Indicated by parentheses. Stems are
numbered above the sequence as in Larson eta/. (1992) and
Kjer et a/. (1994). Nucleotides that are paired in a stem are
underlined whereas bulges and single-stranded loops are not.
No inferences can be made about base pairing where complementary sequence is unavailable (e.g. block 3,stem 61’). In
such cases the nucleotides are not underlined even though
they are probably involved in base pairing. In regions where
stems could not be unambiguously assigned, potential base
pairs were underlined but no parentheses were Inserted (e.9.
stem 66). Characters excluded from the phylogenetic analyses
because they could not be aligned unambiguously are indicated. See Table 2for complete names and family designations
of taxa.
236
S. Kambhampati, K. M. Kjer and B. L. Thorne
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