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 com- prised 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, four- teen subfamliies, - 270 genera and over 2000 species. Phyiogenetic hypotheses currently available for termlte families and genera are based on a limited number of morphological characters and lack rigorous cladistic analysis. In this paper we report on phylo- genetic relationships among ten termite genera of five families based on a DNA sequence analysis of a portion of the mitochondriai 16s rRNA gene. Parsi- mony and distance analysis of DNA sequences sup- ported the existing hypothesis that Mastotermitidae is the basal lineage among extant termites. Kalotermiti- dae was not found to be a sister taxon of Mastotermi- tidae 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.

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 phylo- genetically 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; Kris- tensen, 1995).

Several phylogenetic hypotheses have been pro- posed 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), Prest- wich (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, subfami- lies and genera (Ahmad, 1950; Grasse 8, Noirot, 1959; Roonwal & Sen-Sarma, 1960; Krishna, 1970).

There is general agreement among termite sys- tematists 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,

0 1996 Blackwell Science Ltd 229

230 S. Kambhampati, K. M. Kjer and 6. L. Thorne

26-9 99-9

~

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 relation- ships among various termite families, based on phylo- genetically-informative characters with a known genetic basis, is lacking. We report here on a prelimin- ary 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 (16s rRNA) as a means of exploring the utility of DNA sequence analysis for inferring phylogenetic relationships among termites. 33-1 6

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 trans- version rates were 8.8% and 14.0%, respectively. Among transitions, 54% were C c-) T transitions and the remainder A ++ G transitions. The relative propor- tions 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 ter- mites. Parsimony analysis identified a single tree of 376.82 steps (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 accor- dance 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 + Termiti- dae) were most closely related to one another, followed by Termopsidae and Mastotermitidae.

r + P . corniceps I

1. snyderi I

I - Mac. barneyi

A. wroughtoni

M. darwiniensis

I V

V

Flgura 1. Phylogenetic relationship among representatives of termite families based on parsimony analysis of a portion of the mitochondrial 16s 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 characters and the number of supporting nucleotide characters without homoplasy (excluding gap characters), respectively. Numbers below the branches represent bootstrap values in percent and decay index, respectively. Decay indices Indicate that 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 377.82 steps, one step longer than the shortest tree. Most of the other relation- ships 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 iden- tical 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 monophyletic clade. As in the parsimony tree, Termitidae was the sister group to Rhinotermitidae,

0 1996 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. 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. V8Q8

1

41 47 49 53 53 50 51 51 69 82 83

2

0.13

53 52 56 52 57 57 61 71 81 83

3 4 5 6 7 8 9 10

0.14 0.15 0.16 0.17 0.16 0.18 0.16 0.22 0.16 0.16 0.17 0.16 0.18 0.18 0.19 0.23

0.19 0.20 0.19 0.20 0.21 0.19 0.23 63 0.08 0.11 0.07 0.20 0.18 0.18 65 27 0.09 0.07 0.19 0.18 0.17 63 36 32 0.07 0.21 0.20 0.19 64 26 26 26 0.18 0.17 0.18 66 64 60 66 58 0.11 0.18 61 58 57 63 58 37 71 59 55 60 47 57 57 88 81 86 83 80 72 74 67 86 79 78 86 76 77 81 79

11 12 ~ ~~

0.28 0.28 0.27 0.28 0.30 0.29 0.27 0.26 0.29 0.26 0.27 0.29 0.26 0.25 0.23 0.25 0.24 0.27 0.21 0.26

0.23 73

.012

.018

68

.028

.091 N e . mom

.054 P. Codcap8 - .068

.024 90 ~ I. rnydrri

c. f O l r m o 8 a U 8

€2. f ladpe8

N. aca jutlae .039

.004 W.C. b-e* .057

I A. wroughtoni

I .052 97 2. a l l g l Z 8 t i C O l l i 8

.078 I bl. d a h d e M i 8

.122

.112 8. vrga

m. r m 1 i g i o . a

Scale: each - i s approximately equal to the distance of 0.002386 Fbure 2. Phylogenetic relationship among representatives of termite families based on neighbur-joining analysis 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. Al l 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

0 1996 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 relation- ships 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, pro- vided 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 analy- sis, Termopsidae is more basal than Kalotermitidae. Kalotermitidae and a clade comprised of (Rhinotermi- tidae + 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 Serri- termitidae and Hodotermitidae, their phylogenetic relationship to other termite families cannot be dis- cussed at the present time.

Thorne & Carpenter (1992) proposed a phylogeny of termites, cockroaches and mantids based on an analy- sis of previously published morphological, develop- mental and anatomical characters, Of the three termite families included in the study (Kalotermitidae, Masto- termitidae 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 Masto- termitidae can be firmly established. The position of Kalotermitidae as a relatively apical group in our analysis and as a relatively basal group in the morpho- logical 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 analy- sis. DNA sequence of the homologous fragment from the hymenopteran family Aphidiidae and the Homo- pteran 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 cock- roaches 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 ter- mites the relative proportion of the A H T transver- sions was considerably lower (46%) than that in cockroaches (71 YO) 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/ar BiologyS: 229-238

Phylogenetic relationship among termite families 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 centri- fuged 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 Kambham- pati 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 concentra- tion 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 0.5 ml 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 instruc- tions. 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 reac- tions were carried out according to the manufacturer's instruc- tions (fmol Sequencing System, Promega). The forward and the reverse primer employed for PCR amplification were end- labelled 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.

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 Kalotermi- tidae. 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 synapo- morphic 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 demon- strate 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-suppor- ted 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, Mastotermiti- dae, Kalotermitidae, Termopsidae, Termitidae and Rhino- termitidae, were included in this study (Table 2). Al l 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' Subfamily'

Mastotermes darwiniensis Archotermopsis wroughtoni Zootermopsis angusticollis Macrotermes barneyi Nasutitermes acajutlae Coptotermes formosanus Reticulitermes Navipes lncistitermes snyderi Neotermes mona Procryptotermes corniceps

Mastoterrnitidae - Terrnopsldae Terrnopainae Terrnopsidae Termopsinae Terrnitidae Macrotermltlnae Terrnitldae Nasutiterrnitlnae Rhinotermitidae Coptotermitinae Rhinotermitidae Heteroterrnitinae Kalotermltidae - Kalotermltidae - Kaloterrnitidae -

' Family and subfamily designations follow those of Pearce 8 Waite

The 16s rRNA sequences of these termites were published by (l?).

Kambhampati (1995).

Sequence alignments and phylogenetic inference The sequences were read manually from the autoradiographs into a computer and converted to RNA sequence. The align- ment of RNA sequences based on the secondary structure predictions wascarried out asdescribed by Kjer (1995). Brlefly, the secondary structural proposal for Drosophila rnelanoga-

0 1996 Blackwell Science Ltd, Insect Molecular Biology 5: 229-238

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 parsimo- nious 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 unam- biguously. 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 eta / . , 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 homo- logous mitochondria1 16s rRNA gene fragment of the cock- roach, 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. darwinien- sisDNA 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 assis- tance. This is journal article no. 96-73J of the Kansas Agricultural Experiment Station.

References Ahmad, M. (1950) The phylogeny of termites based on imago-worker

mandibles. BullAmer Mus Nat Hist95 37-86. Ampion, M. and Quennedey, A. (1981) The abdominal epidermal

glands of termites and their phylogenetic significance. Biosyste- maticsofSocial/nsects(Howse. P.E. and Clement, J.L., eds), pp. 249-261. Academic Press, London

Boudreaux, H.B. (1979) Arthropod Phylogeny with Special Refer- ence to Insects. Wiley and Sons, New York.

Bremer, K. (1988) The limits of amino acid sequence data in angiosperm phylogenetic reconstruction. Evolution 42: 795-803.

Bremer, K. (1994) Branch support and tree stability. Cladistics 10: 295-304.

Donoghue, M.J., Olmstead, R.G., Smith, J.F. and Palmer, J.D. (1992) Phyiogenetic relationships of Dipsacales based on rbcl sequences. Ann Missouri Bot Gard79: 333-345.

Emerson, A.E. (1952) The biogeography of termites. Bull Amer Mus NatHistW 217-225.

Emerson, A.E. (1955) Geographical origins and dispersions of termite genera. Fieldiana Z00137: 465-521.

Emerson, A.E. and Krishna, K. (1975) The termite family Serritermi- tidae (Isoptera). Amer Mus Noviates2570: 1-31.

Fang, Q., Black, W.C., IV, Blocker, H.D. and Whitcomb, R.F. (1993) A phylogeny of New World Deltocephalwlike leafhopper genera based on mitochondrial 16s ribosomal DNA sequences. Mol fhylo Evol2: 1 19-1 31.

Grasse, P.-P. (1949) Ordre des lsopteres ou termites. Traite de Zoologie. (Grasse, P.-P., ed.), Voi. 9, pp. 40&544.

Grasse, P.-P. and Noirot, Ch. (1959) L’evolution de la symbiose chez les Isopteres. Experientia 15: 365472.

Gutell, R.R., Schnare, M.N. and Gray, M.W. (1990) A compilation of large subunit ribosomal RNA sequences presented in a second- ary structure format. Nucl Acids Resl8: 2319-2330.

Hare, L. (1937) Termite phylogeny as evidenced by soldier mandible development. Ann fnt Soc Amer37: 459-486.

Hay, J.M., Ruvinski, I., Hedges, S.B. and Maxson, L.R. (1995) Phylogenetic relationships of amphibian families inferred from DNA sequences of mitochondrial 12s and 16s ribosomal RNA genes. MolBiolfvoll2: 92E937.

Hennig, W. (1981) lnsectfhylogeny. Wiiey and Sons, New York. Higgins, D.M. and Sharp, P.M. (1989) Fast and sensitive multiple

sequence alignment on microcomputer. CABIOSI: 151-153. Kambhampati, S. (1995) A phylogeny of cockroaches and related

Insects based on DNA sequence of mitochondrial ribosomal RNA genes. frocNatl Acad Sci USA 92: 2017-2020.

Kambhampati, S. and Smith, P.T. (1995) PCR primers for ampiifica- tion of four insect mitochondrial gene fragments. lnsect MolBiol 4: 233-236.

Kambhampati, S., Black, W.C., IV, and Rai, K.S. (1992) Random amplified polymorphic DNA of mosquito species and popula- tions: techniques, statistical analysis and applications. J Med Entomol29: 939-945.

Kjer, K.M. (1995) Use of rRNA secondary structure in phylogenetic studies to identify homologous positions: an example of align- ment and data presentation from frogs. Mol fhylogen Evol4: 314-320.

Kjer, K.M., Baldridge, G.D. and Fallon, A.M. (1994) Mosquito large subunit ribosomal RNA: simultaneous alignment of primary and secondary structure. Biochim Biophys Acta 1217: 147-155.

Krishna, K. (1961) A generic revision and phylogenetic study of the family Kalotermitidae. Bull Amer Mus Nat Hist122: 303-408.

0 1096 Biackwell Science Ltd, lnsect Molecular BiologyS: 229-238

Phylogenetic relationship among termite families 235

drial gene sequences and a compilation of conserved polymer- ase chain reaction primers. Ann EntomolSocAmer87: 651-701.

Snyder, T.E. (1949) Catalog of the termites of the world. Smithsonian MiscCollll2: 1490.

Swofford, D.L. (1993) PAUP: Phylogenetic Analysis Using Parsi- mony, Vcr.3.1.1. Package distributed by Smithsonian Institution, Washington, D.C.

Tajima, F. and Nei, M. (1984) Estimation of evolutionary distance between nucleotide sequences. Mol BiolEvoll: 269-285.

Thorne, B.L. and Carpenter, J.M. (1992) Phylogeny of the Dictyoptera. Syst Entomol17: 2%268.

Watson, J.A.L. and Sewell, J.J. (1985) Caste development in Masto- termes and Kalotermes: which is primitive? Caste Differentia- tion in Social lnsects (Watson, J.A.L., Okot-Kotber, B.M. and Noirot, Ch., eds), pp. 27-40. Pergamon Press, New York.

Krishna, K. (1970) Taxonomy, phylogeny and distribution of termites. Biology of Termites, (Krishna, K. and Weesner, F.M., eds), Vol. 2, pp, 127-152. Academic Press, London.

Krishna, K. and Weesner, F.M. (eds) (1969) Biology of Termites, Vol. 1. Academic Press, London.

Krishna, K. and Weesner, F.M. (eds) (1970) Biology of Termites, Vol. 2. Academic Press, London.

Kristensen, N.P. (1981) Phylogeny of insect orders. Annu Rev Entomol26: 135-157.

Kristensen, N.P. (1995) Forty years of phylogenetic systematics. ZoolBetrN.F. 36: 83-124.

Kumar, S., Tamura, K. and Nei, M. (1993) MEGA: Molecular Evolu- tionary Genetics Analysis, Ver. 1.01. Pennsylvania State Uni- versity.

Maidak, B.L., Larsen, N.. McCaughey, M.J., Overbeek, R., Olsen, G.J., Fogel, K., Blandy, J. and Woese, C.R. (1994)The ribosomal database project. Nucl Acids Res22: 348M487.

McKittrick, F.M. (1964) Evolutionary studieson cockroaches. Cornell UnivAgr Expt Stn Mem389: 1-197.

Miller, L.R. (1986) The phylogeny of Nasutitermitnae. Sociobiology 11: 203-214.

Noirot, C. (1995) The gut of termites (Isoptera): comparative anatomy, systematics and phylogeny. 1. Lower termites. Ann Soc Entomol Fr 31: 197-226.

Pearce, M.J. and Waite, B.S. (1994) A list of termite genera with comments on taxonomic changes and regional distribution. Sociobiology 23: 247-263.

Prestwich, G.D. (1983) Chemical systematics of termite exocrine secretions. Annu Rev Ecol Systl4: 287-311.

Prestwich, G.D. and Collins, M.S. (1981) Chemotaxonomy of Sub- litermes and Nasutitermes termite soldier defense secretions: evidence against the hypothesis of diphyletic evolution of the Nasutitermitinae. Biochem Syst €cola: 83-88.

Roonwal, M.L. and Sen-Sarma, P.K. (1960) Contributions to the systematics of oriental termites. Entomological Monograph No.1, Indian Council for Agricultural Research.

Saitou, N. and Nei, M. (1987) The neighbor joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol4: 406425.

Sands, W.A. (1972) The soldierless termites of Africa (Isoptera: Termitidae). BullBritMus NatlHistSuppl18: 1-244.

Simon, C., Frati, F., Beckenbach, A., Crespi, B., Liu, H. and Flook, P. (1994) Evolution, weighting and phylogenetic utility of mitochon-

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 complemen- tary 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 comple- mentary 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 indi- cated. See Table 2for complete names and family designations of taxa.

8 1996 Blackwell Science Ltd, Insect Molecular BiologyS: 22S238

236 S. Kambhampati, K. M. Kjer and B. L. Thorne

. . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -- ill : ? g

* r l : p t; 4 u)

L

d

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . !i 3 . . . . * . . . : 9 ? 5 - . . . . . . . I- I u)

c! . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . $ : : : : : 4 5 5 5 $ : i . . . . . . . . . . . . . . . . . . . . ..o . . . . . . . . . . . . . . . . . .

Y V Y Y Y Y Y Y Y Y V Y . . . . . . . . . . . . . . . . . . . . . .o . . . . . . . . . . " . . . . . . . . . . *r l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ' 0 . . . . . . . . . . . r ( . . . . . . . . . . . d . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Y Y U Y Y Y Y Y . . . . . . . .

. . . . . . . . . : :

g:::::: : . : : :

. . nnnnnn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

nn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . *I *I . . . . . . . . .

Y Y Y

. . . . . . . . . . . . . . : : : : : ? *

. . . . 4 : . * o . .ol ' 4 < . . . . . .

Y Y

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Y Y I Y Y Y Y

. . . . . . . . . . . . . . . . . . . . . . . . . . . .

0 1996 Blackwell Science Ltd, lnsect Moleculer Biology 5: 229-238

3 L N

N z 0

W

238 S. Kambhampati, K. M. Kjer and B. L. Thorne

t -B W

a YI Y

hl W I I A

IdJS $j : $ . . . 4 * * . . . . . .

4 4 4 0 In d . . . . . . . . . . . . . . . . . . . . . . .

E l : : : : : : : : : : :

t . . . . . . . . . : : 7

. . . . . . . . . . . Y Y V U Y Y Y Y Y Y V Y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . O . . . . . . . . . . .dl . . . . . . . . . d . . . . . . . . . . . . . . . . . . . . . . i .

n .I

I . . . . . . . . 8 . . . . . . . . E : : : : . . . . . . .o . . . . . . . , : ? : Z . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . j . . . . . . . . . . . . . . . . . . . . . . I : : :443 ' r ; :3

. . . . I I . . . . . . ggi . . . . . . . . . . . 3 [ [ + + + + + G r ; . . . . I I <

. . . . . . . . b 1

0 8 : : .- . . . . . . . . . . e m * m :I :I < . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I!- 4

Y Y Y Y Y Y Y Y Y Y

4 3 3

0 1996 Blackwell Science Lid, Insect Molecular Biology5: 229-238