Molecular Biology (1996) 5(4), 229-238

Phylogenetic relationship among families based on DNA sequence of mitochondrial 16s ribosomal RNA gene

S. Kambhampatl,' K. M. Kjer' and 6. L. Thorne3 Introduction ' Department of Entomology, Kansas State University, (Isoptera) are a diverse group of eusocial Manhattan, Kansas: . lsoptera is presently divided into seven Department of Zoology, Brigham Young University, * families, fourteen subfamilies, -270 genera and more Provo, Utah, and Department of Entomology, University of Maryland, than 2000 fossil and extant species (Krishna & College Park, Maryland, USA Weesner, 1969, 1970; Pearce & Waite, 1994). There has long been agreement among insect systematists that , mantids and termites are phylo- Abstract genetically closely related (Boudreaux, 1979; Hennig, Termites (Order isoptera: Class Insecta), are com- 1981; Kambhampati, 1995; Kristensen, 1995). However, prised of a complex assemblage of species, with differing relationships have been proposed for the considerable variation in life history, morphology, three groups of insects (McKittrick, 1964; Boudreaux, social behaviour, caste development and ecology. At 1979; Hennig, 1981; Kristensen, 1981) and the precise present, isoptera is divided into seven families, four- topology of the tree that includes cockroaches, mantids teen subfamliies, - 270 genera and over 2000 species. and termites remains a topic of active discussion Phyiogenetic hypotheses currently available for (Thorne & Carpenter, 1992; Kambhampati, 1995; Kris- termlte families and genera are based on a limited tensen, 1995). number of morphologicalcharacters and lack rigorous Several phylogenetic hypotheses have been pro- cladistic analysis. In this paper we report on phylo- posed for relationships within Isoptera, all of which genetic relationships among ten termite genera of five are based on morphological characters and none families based on a DNA sequence analysis of a including cladistic analysis (reviewed in Krishna & portion of the mitochondriai 16s rRNA gene. Parsi- Weesner, 1969, 1970). Major proposals of termite mony and distance analysis of DNA sequences sup- phylogeny at the family and the subfamily level ported the existing hypothesis that Mastotermitidae is include those of Hare (1937), Snyder (1949), Grasse the basal lineage among extant termites. Kalotermiti- (1949), Emerson (1952; 1955), Sands (1972), Emerson & dae was not found to be a sister taxon of Mastotermi- Krishna (1975), Ampion & Quennedey (1981), Prest- tidae as exlsting hypotheses suggest, but was most wich (1983) and Noirot (1995). Relationships among closely related to and . genera within certain families of lsoptera have also Representatives of were more basal been proposed (e.g. Krishna, 1961; Prestwich & relative to those of Kaiotermitidae. The utility of 16s Collins, 1981; Miller, 1986; reviewed in Krishna, 1970). rRNA nucieotide sequence analysis for inferring The various hypotheses differ concerning the number phyiogenetic relationships among termlte families, of families and subfamilies within lsoptera and the subfamilies and genera is discussed. evolutionary relationships among families, subfami- lies and genera (Ahmad, 1950; Grasse 8, Noirot, 1959; Keywords: termites, phyiogenetics, 16s rRNA, Roonwal & Sen-Sarma, 1960; Krishna, 1970). mtDNA, isoptera. 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 and Termopsidae Received 25 September 1995; accepted 11 March 1998. Correspondence: Dr S. Kambhampati, Department of Entomology, Kansas State University, [formerly included in (Grasse, 1949)] Manhattan, KS 66506, USA. are relatively ancient families (Ahmad, 1950; Emerson,

0 1996 Blackwell Science Ltd 229 230 S. Kambhampati, K. M. Kjer and 6. L. Thorne

1952;Krishna, 1970;Watson & Sewell, 1985). According +P. corniceps I 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 1. snyderi I qualitative observations of a single morphological system, namely the mandibles of imagos, workers Mac. barneyi I 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 r 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 A. wroughtoni genetic basis, is lacking. We report here on a prelimin- IV 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 M. darwiniensis V ribosomal RNA subunit gene (16srRNA) as a means of exploring the utility of DNA sequence analysis for 26-9

~ inferring phylogenetic relationships among termites. 99-9 33-1 6

Flgura 1. Phylogenetic relationship among representatives of termite Results 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. The length of the sequenced fragment of 16s rRNA Tree length: 376.82 steps; consistency index: 0.7; retention index: 0.5. gene from the ten termite taxa ranged from 408 to 429 Numbers above the branches are total number of supporting nucieotide characters and the number of supporting nucleotide characters without bp. The average (fSSE) base composition for the homoplasy (excluding gap characters), respectively. Numbers below the termite taxa was: A: 24.9k0.6, C: 12.0f0.3, G: branches represent bootstrap values in percent and decay index, 21.1k0.4,T: 42.0f0.7. A bias toward adenine and respectively.Decay indices Indicate that a particular node was supported in trees that were longer than the most parsimonious tree by the number of thymine (67% of total) is consistent with the base steps Indicated (Donoghue eta/.,1992; Bremer, 1988,1994)and thus composition of mtDNA sequences of other insects represent progressively relaxed parsimony. The strength of an inferred (Simon ef a/., 1994). The overall transition and trans- branch 1s directly proportional to the decay index. Family designations lor version rates were 8.8% and respectively. termites are as follows: I, Kalotermitidae; 11, Termitidae; ill, Rhinotermitidae; 14.0%, IV, Termopsidae; V, Mastotermttidae. See text lor further details. 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: acajutlae and barneyi

46.4%, A ++C: 5.0%, G c-) T: 45.0% and G C* C: 2.7%. were not included in a single clade. These two termites The alignment of the RNA sequence resulted in a presently are included in separate subfamilies within total of 450 characters, including gaps (Appendix). Of the family Termitidae. However, the node separating N. the 450 characters, 192 (43%) were variable and 109 acajuflae and M. barneyi was not supported in 50% or (24%) were parsimony-informative among the ter- more of the bootstrap replicates and collapsed in a mites. Parsimony analysis identified a single tree of consensus of four trees of length 377.82 steps, one step 376.82steps (decimal point due to down-weighted gap longer than the shortest tree. Most of the other relation- characters; see Experimental procedures) with a ships inferred in the parsimony tree were supported in monophyletic grouping of the termites (Fig. 1). The a majority of the bootstrap replications (Fig.1). termites included in this study were grouped in accor- Pairwise absolute nucleotide differences and dance with the family level designations presently Tajima-Nei distances are given in Table 1. The tree recognized. Mastotermitidae, represented by the sole based on the distance analysis was essentially iden- extant species, , was found tical in topology to the one based on parsimony to be the basal taxon among the termites. At the family analysis (Fig. 2). The only difference between the two level, Kalotermitidae and (Rhinotermitidae + Termiti- trees was that M. barneyiand N. acajutlae (Termitidae) dae) were most closely related to one another, formed a monophyletic clade. As in the parsimony tree, followed by Termopsidae and Mastotermitidae. 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 2 3 4 5 6 7 8 9 10 11 12

~ ~~ 1. f.corniceps 0.13 0.14 0.15 0.16 0.17 0.16 0.18 0.16 0.22 0.28 0.28 2. 1. snyderi 41 0.16 0.16 0.17 0.16 0.18 0.18 0.19 0.23 0.27 0.28 3. N. mona 47 53 0.19 0.20 0.19 0.20 0.21 0.19 0.23 0.30 0.29 4. M. barneyl 49 52 63 0.08 0.11 0.07 0.20 0.18 0.18 0.27 0.26 5. N. acajuflae 53 56 65 27 0.09 0.07 0.19 0.18 0.17 0.29 0.26 6. R. flavipes 53 52 63 36 32 0.07 0.21 0.20 0.19 0.27 0.29 7. C. formosanus 50 57 64 26 26 26 0.18 0.17 0.18 0.26 0.25 8. A. wroughtoni 51 57 66 64 60 66 58 0.11 0.18 0.23 0.25 9. Z.angusticullis 51 61 61 58 57 63 58 37 0.24 0.27 10. M. derwiniensis 69 71 71 59 55 60 47 57 57 0.21 0.26 11. M. religiosa 82 81 88 81 86 83 80 72 74 67 0.23 12. 8. V8Q8 83 83 86 79 78 86 76 77 81 79 73

.091 Ne. mom .054 P. Codcap8 - .068

90 ~ I. rnydrri .018 .024 c. fOlrmo8aU8 68 €2. fladpe8

.012 N. acajutlae .039 .004 W.C. b-e* .057 .028 I A. wroughtoni .052 97 I 2. allglZ8tiCOlli8 .078 I bl. dahdeMi8 .122 8. vrga .112 m. rm1igio.a

Scale: each - is approximately equal to the distance of 0.002386 Fbure 2. 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 the mitochondrial 16s rRNA gene, is presented. Most consisting of Rhinotermitidae and Termitidae than it of the inferred relationships had strong quantitative was to Mastotermitidae. All inferred relationships, with support as indicated by bootstrap analysis and decay the exception of the P. corniceps and N. mona branch, indices. The relationships among taxa inferred from were supported in 50% or greater of the 1000 bootstrap the parsimony and the distance analyses were nearly replicates (Fig. 2). identical to one another, but only partially congruent with presently accepted phylogenetic relationships among termite families (e.g. Krishna, 1970). Whereas Dlscusslon the bootstrap support for some of the basal nodes was In this paper a phylogenetic analysis of relationships relatively weak (Figs 1 and 2), the fact that the relation- among ten genera of termites, belonging to five ships inferred from the parsimony and distance families, based on the DNA sequence of a portion of analyses were congruent and that the inferred

01996 Blackwell Science Ltd, lnsect Molecular Blology5: 229-238 232 S. Kambhampati, K. M. Kjer and 6. L. Thorne relationships did not decay in trees that were several regions excluded from the analysis is a subjective steps longer than the most parsimonious tree, pro- decision, the use of secondary structure substantially vided confidence in the inferred relationships. The reduces the subjectivity involved in the alignment by most notable difference in family level relationships anchoring positions near the excluded regions. For between phylogenies inferred from molecular data and example, the largest of the excluded regions were two from morphological characters is as follows. Krishna highly A-U rich regions located within stem 75 (see (1970) proposed that Kalotermitidae evolved from Appendix), which is highly variable in a wide range of Mastotermitidae (implying a sister group relationship taxa (e.g. Hay et a/.. 1995; Kambhampati, 1995). The and/or a relatively close phylogenetic relationship excluded positions in stem 75 begin with nucleotides between the two families) and our data contradict this that can not be shown to be involved in hydrogen inference. Although both parsimony and distance bonding and are interrupted by a conserved section in analyses indicated that Mastotermitidae is the basal the middle of the unpaired loop. Parsimony analysis lineage among the five termite families included in the that included all characters including those that were study, neither analysis supported its sister group excluded because they were unalignable, resulted in relationship to Kalotermitidae. According to our analy- tree that was identical in topology to the one shown in sis, Termopsidae is more basal than Kalotermitidae. Fig. 1, but had a length of 545.82 steps. Additionally, Kalotermitidae and a clade comprised of (Rhinotermi- parsimony analysis based on the direct alignment of tidae + Termitidae) are sister groups. In this regard, DNA sequence using CLUSTALV (Higgins & Sharp, our results support the proposal by Noirot (1995, p. 1989) and alignment by the eye also resulted in a tree 223), who, based on observations of gut anatomy, that was identical in topology to the one in Fig. 1 (data stated “Kalotermitidae might be the sister group of not shown). Rhinotermitidae + + Termitidae.” lnsect mtDNA has a base composition that is Because we did not include representatives of Serri- strongly biased toward adenine and thymine (Simon termitidae and Hodotermitidae, their phylogenetic etal., 1994). The termites included in this study were no relationship to other termite families cannot be dis- exception with an average of 67% adenine and cussed at the present time. thymine among the ten taxa. However, the A + T bias Thorne & Carpenter (1992) proposed a phylogeny of within termite mtDNA is on the lower end for the 16s termites, cockroaches and mantids based on an analy- rRNA gene of insects studied to date (see Table 2 in sis of previously published morphological, develop- Simon etal., 1994). For example, the homologous 16s mental and anatomical characters, Of the three termite rRNA fragment from thirty-two species of cockroaches families included in the study (Kalotermitidae, Masto- was found to contain, on average, 72% adenine and termitidae and Termopsidae), Thorne 8, Carpenter thymine (Kambhampati, 1995). Similarly, in a study of (1992) found Mastotermitidae and Kalotermitidae to be leafhoppers of the family Cicadellidae, Fang et a/. sister families. Thorne & Carpenter (1992) concluded (1993) reported an average of 73% adenine and that Mastotermitidae may not be the most basal termite thymine content for a 16s rRNA fragment that was family and cautioned that a more comprehensive slightly larger than the one used in the present analy- analysis including all termite families is required sis. DNA sequence of the homologous fragment from before a conclusion concerning the position of Masto- the hymenopteran family Aphidiidae and the Homo- termitidae can be firmly established. The position of pteran family Lachnidae revealed an A+T content of Kalotermitidae as a relatively apical group in our -75% (S. Kambhampati, unpubl. data). The overall analysis and as a relatively basal group in the morpho- transition rate of 8% for the termites was similar to the logical analyses (Ahmad, 1950; Krishna, 1970; 7% reported for cockroaches (Kambhampati, 1995). Emerson & Krishna, 1975) suggests a need for further However, whereas the C u T transitions in cock- study of these relationships. roaches were almost twice as frequent as the A ++ G Several hydrogen-bonded stems described by transitions (66% and 34%, respectively), in termites Maidak eta/.(1994) could not be satisfactorily located the relative proportions of the four types of transitions in our rRNA data. Although there does appear to be were nearly equal. The observed transversion rate hydrogen bonding in stems 66 and 82, the guidelines among termites was lower (14%) than that reported presented by Kjer (1995) could not be unambiguously for cockroaches (21%; Kambhampati, 1995). In ter- assigned. Stem 73 was not located because the 3 half mites the relative proportion of the A H T transver- of this stem was not included in the amplified fragment. sions was considerably lower (46%) than that in Stem 84 was present in the insect 16s rRNA, whereas cockroaches (71YO) and that of the G +-) T transversions stem 86 could not be located. Stem 88 was not (46%) considerably higher than that in cockroaches supported by our data. Although the selection of (4%; Kambhampati, 1995). Our data suggest that sub-

@ 1996 Blackwell Science Ltd, lnsect Molecu/arBiologyS: 229-238 Phylogenetic relationship among termite families 233 stantive differences exist in the evolutionary dynamics Some of the sequences used in this study were published of the 16s rRNA gene in these two phylogenetically previously as indicated in Table 2. closely related insect groups. DNA extraction, polymerase chain reaction and DNA In summary, the phylogeny for ten termite genera of sequencing five families based on the DNA sequence of a portion of the mitochondria1 16s rRNA gene was only partially A small portion of the thoracic muscle tissue was dissected out from individual workers or soldiers of each species and congruent with presently accepted relationships. transferred to a sterile 1.5 ml centrifuge tube containing 50 pI Mastotermitidae was found to be the basal lineage of lysis buffer (10 mM Tris-HCI, pH 8.0, 1 mM EDTA, 1% Nonidet among extant termites as existing hypotheses suggest; P-40,lOO pglml Protelnase-K; Sigma Chemical Co.). The use of however, our results did not support a sister-group thoracic tissue minimized the risk of inadvertent amplification relationship between Mastotermitidae and Kalotermi- of DNA of symbiotic microbes in termite guts. The tissue was tidae. A more extensive sampling of genera, especially macerated with a sterile pipette tip and the tube was incubated those belonging to Kalotermitidae, Termopsidae and 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- Hodotermitidae, may be required to confirm our fuged for 10 s to pellet debris. The DNA was used either present findings. Some of the problems we have immediately in polymerase chain reaction (PCR) or stored at encountered in the use of the 16s rRNA gene fragment -20°C. for inferring relationships among termite families (e.g. PCR was set up in 50 pI volume as described by Kambham- ambiguities in alignment of some portions of the pati efal. (1992) and Kambhampati (1995). Briefly, the reaction sequenced fragment, relatively low bootstrap support mix was made up in 500 pI quantities, sufficient to carry out ten for some nodes and relatively small number of synapo- individual reactions, by pipetting 430 pI of sterile water into a 1.5 ml tube and adding 50pl of 10 x TaqDNA polymerase buffer morphic sites) may be avoided by using the DNA (Promega Corp.), 5 pl of dNTPs (Promega) to a final concentra- sequence of a gene that is more conserved than that tion of 200 nM, 10 pI of each primer (500 PM; see below) and 3 pI of the 16s rRNA gene. Nonetheless, our results demon- of Ta9 DNA polymerase (Promega). Aliquots of 50 pI were strate that DNA sequence of genes that are not likely to pipetted into sterile 0.5 ml centrifuge tubes. Template DNA (3-5 vary in function among the different castes is useful for pl; see above) was added. The reaction mix was layered with 2 inferring termite phylogeny. A robust and well-suppor- drops of mineral oil (Sigma) and the tubes were placed in a ted phylogeny is a prerequisite for a more thorough 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, understanding of the complex social organization, (3) 50°C for 1 min, (4) 72°C for 1.5 min. Steps 24were repeated developmental patterns and behaviours that underlie thirty-four more times for a total of thirty-five cycles. The termite evolution. 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 Experlrnental procedures ATC AAA AAC AT-3 (Simon et a/., 1994). The amplification product was electrophoresed on a 2% low melting point Insects agarose gel. The band corresponding to the amplification Ten termite genera representing five families, Mastotermiti- product was excised from the gel using a sterile razor blade, dae, Kalotermitidae, Termopsidae, Termitidae and Rhino- placed in a sterile 1.5 ml tube and incubated at 70°C for 5 min. termitidae, were included in this study (Table 2). All insects, The resulting solution was purified using minicolumns (Wizard except M. darwiniensis, were preserved in 80% ethanol; total PCRpreps, Promega) according to the manufacturer's instruc- genomic DNA of M. darwiniensis, was provided by L. Vawter. tions. 3 pI of this DNA was used in sequencing reactions. DNA sequence was obtained directly from double-stranded

Table 2. List of termite taxa used in this study. PCR products using the cycle sequencing method. The reac- tions were carried out according to the manufacturer's instruc- Species Family' Subfamily' tions (fmol Sequencing System, Promega). The forward and Mastotermes darwiniensis Mastoterrnitidae - the reverse primer employed for PCR amplification were end- Archotermopsis wroughtoni Terrnopsldae Terrnopainae labelled with y-[32P]ATP(6000 Cilmole; NEN-DuPont) and used angusticollis Terrnopsidae Termopsinae to sequence the PCR product. The reaction mixtures were Macrotermes barneyi Terrnitidae Macrotermltlnae electrophoresed on 6% polyacrylamide + urea denaturing Nasutitermes acajutlae Terrnitldae Nasutiterrnitlnae gels for 6 h, with two loading approximately 3 h apart. Both Rhinotermitidae Coptotermitinae Coptotermes formosanus strands of the PCR product were sequenced. Reticulitermes Navipes Rhinotermitidae Heteroterrnitinae lncistitermes snyderi Kalotermltidae - Neotermes mona Kalotermltidae - Sequence alignments and phylogenetic inference Procryptotermescorniceps Kaloterrnitidae - The sequences were read manually from the autoradiographs ' Family and subfamily designations follow those of Pearce 8 Waite into a computer and converted to RNA sequence. The align- (l?). ment of RNA sequences based on the secondary structure The 16s rRNA sequences of these termites were published by predictions wascarried out asdescribed by Kjer (1995). Brlefly, Kambhampati (1995). the secondary structural proposal for Drosophila rnelanoga-

01996 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 References 60s taurus structure from the Ribosomal Database Project (Maidak et a/., 1994). By using the general features of these Ahmad, M. (1950) The phylogeny of termites based on imago-worker models, potentially base-paired regions were identified for mandibles. BullAmer Mus Nat Hist95 37-86. each taxon as a preliminary alignment. Modifications were Ampion, M. and Quennedey, A. (1981) The abdominal epidermal made to the preliminary alignment by aligning structural glands of termites and their phylogenetic significance. Biosyste- features. Within stems, each paired nucleotide was considered maticsofSocial/nsects(Howse. P.E. and Clement, J.L., eds), pp. an anchor point and gaps were inserted in order to maintain 249-261. Academic Press, London these proposed homologous portions. The aligned RNA Boudreaux, H.B. (1979) Phylogeny with Special Refer- sequences for all taxa included in this study are shown in the ence to Insects. Wiley and Sons, New York. Appendix. The data were analysed using parsimony analysis Bremer, K. (1988) The limits of amino acid sequence data in (PAUP 3.1.1; Swofford, 1993) with the multiple equally parsimo- angiosperm phylogenetic reconstruction. Evolution42: 795-803. nious exhaustive search option, tree bisectiowreconnection Bremer, K. (1994) Branch support and tree stability. Cladistics 10: and 100 random addition sequences. Gaps were treated as 295-304. single binary characters and gaps of variable length (G2 and Donoghue, M.J., Olmstead, R.G., Smith, J.F. and Palmer, J.D. (1992) G4 in the Appendix) were down-weighted so that each indel Phyiogenetic relationships of Dipsacales based on rbcl was equivalent to a single character. Four regions of the sequences. Ann Missouri Bot Gard79: 333-345. sequence data (total of fifty-seven characters) were excluded Emerson, A.E. (1952) The biogeography of termites. BullAmer Mus from the analysis because they could not be aligned unam- NatHistW 217-225. biguously. These regions are indicated in the Appendix. The Emerson, A.E. (1955) Geographical origins and dispersions of data set was bootstrapped for 1000 replications (fifty random termite genera. Fieldiana Z00137: 465-521. addition sequences per replicate) using PAUP. The aligned Emerson, A.E. and Krishna, K. (1975) The termite family Serritermi- sequence was also analysed by the neighbour-joining method tidae (Isoptera). Amer Mus Noviates2570: 1-31. (Saitou 8 Nei, 1987) based on the Tajima 8 Nei (1984) distance Fang, Q.,Black, W.C., IV, Blocker, H.D. and Whitcomb, R.F. (1993) A using MEGA 1.01 (Kumar eta/., 1993). The same fifty-seven phylogeny of New World Deltocephalwlike leafhopper genera characters that were excluded from the parsimony analysis based on mitochondrial 16s ribosomal DNA sequences. Mol were also excluded from the distance analysis. However, the fhylo Evol2: 119-1 31. characters used in the distance analysis were unweighted and Grasse, P.-P. (1949) Ordre des lsopteres ou termites. Traite de gaps were treated as fifth base. A bootstrap analysis of 1000 Zoologie. (Grasse, P.-P., ed.), Voi. 9, pp. 40&544. replications was carried out on the tree inferred from the Grasse, P.-P. and Noirot, Ch. (1959) L’evolutionde la symbiose chez neighbour-joining method. The DNA sequence of the homo- les Isopteres. Experientia 15:365472. logous mitochondria1 16s rRNA gene fragment of the cock- Gutell, R.R., Schnare, M.N. and Gray, M.W. (1990) A compilation of roach, Blaffella vaga (Blattellidae) and the mantid, Mantis large subunit ribosomal RNA sequences presented in a second- religiosa (Kambhampati, 1995), was used as outgroups. 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. Sequence availability Hay, J.M., Ruvinski, I., Hedges, S.B. and Maxson, L.R. (1995) Phylogenetic relationshipsof amphibian families inferred from The sequences reported in this study can be obtained from S.K. DNA sequences of mitochondrial 12s and 16s ribosomal RNA U50772-U50778. or from GenBank under accession numbers genes. MolBiolfvoll2: 92E937. The GenBank accession numbers for C. formosanus, M. Hennig, W. (1981) lnsectfhylogeny.Wiiey and Sons, New York. darwlniensisand R. flavipes 16s rRNA sequences are U17778, Higgins, D.M. and Sharp, P.M. (1989) Fast and sensitive multiple U17790 and U17824, respectively (Kambhampati, 1995). The sequence alignment on microcomputer. CABIOSI: 151-153. accession numbers for the and the mantid 16s rRNA Kambhampati, S. (1995) A phylogeny of cockroaches and related sequences were given by Kambhampati (1995). 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. Acknowledgements Kambhampati, S., Black, W.C., IV, and Rai, K.S. (1992) Random amplified polymorphic DNA of mosquito species and popula- We thank M. S. Akthar for supplying A. wroughtoni, J. tions: techniques, statistical analysis and applications. J Med Reeve for Z. angusticollis, L. Vawter for M. darwinien- Entomol29:939-945. sisDNA and M. W. J. Crosland for M. barneyi. Financial Kjer, K.M. (1995) Use of rRNA secondary structure in phylogenetic support for this study was provided by a seed grant studies to identify homologous positions: an example of align- from the Department of Entomology, Kansas State ment and data presentation from frogs. Mol fhylogen Evol4: University, to S.K. K.M.K. was supported during this 314-320. Kjer, K.M., Baldridge, G.D. and Fallon, A.M. (1994) Mosquito large J. study by NSF grant DEB 91-19091 to W. Sites, Jr. We subunit ribosomal RNA: simultaneous alignment of primary and thank A. L. Nus and L. J. Krchma for technical assis- secondary structure. Biochim Biophys Acta 1217:147-155. tance. This is journal article no. 96-73J of the Kansas Krishna, K. (1961) A generic revision and phylogenetic study of the Agricultural Experiment Station. 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

Krishna, K. (1970) , phylogeny and distribution of drial gene sequences and a compilation of conserved polymer- termites. Biology of Termites, (Krishna, K. and Weesner, F.M., ase chain reaction primers. Ann EntomolSocAmer87: 651-701. eds), Vol. 2, pp, 127-152. Academic Press, London. Snyder, T.E. (1949) Catalog of the termites of the world. Smithsonian Krishna, K. and Weesner, F.M. (eds) (1969) Biology of Termites, MiscCollll2: 1490. Vol. 1. Academic Press, London. Swofford, D.L. (1993) PAUP: Phylogenetic Analysis Using Parsi- Krishna, K. and Weesner, F.M. (eds) (1970) Biology of Termites, mony, Vcr.3.1.1. Package distributed by Smithsonian Institution, Vol. 2. Academic Press, London. Washington, D.C. Kristensen, N.P. (1981) Phylogeny of insect orders. Annu Rev Tajima, F. and Nei, M. (1984) Estimation of evolutionary distance Entomol26: 135-157. between nucleotide sequences. Mol BiolEvoll: 269-285. Kristensen, N.P. (1995) Forty years of phylogenetic systematics. Thorne, B.L. and Carpenter, J.M. (1992) Phylogeny of the ZoolBetrN.F. 36: 83-124. . Syst Entomol17: 2%268. Kumar, S., Tamura, K. and Nei, M. (1993) MEGA: Molecular Evolu- Watson, J.A.L. and Sewell, J.J. (1985) Caste development in Masto- tionary Genetics Analysis, Ver. 1.01. Pennsylvania State Uni- and Kalotermes: which is primitive? Caste Differentia- versity. tion in Social lnsects (Watson, J.A.L., Okot-Kotber, B.M. and Maidak, B.L., Larsen, N.. McCaughey, M.J., Overbeek, R., Olsen, Noirot, Ch., eds), pp. 27-40. Pergamon Press, New York. G.J., Fogel, K., Blandy, J. and Woese, C.R. (1994)The ribosomal database project. Nucl Acids Res22: 348M487. Appendix McKittrick, F.M. (1964) Evolutionary studieson cockroaches. Cornell UnivAgr Expt Stn Mem389: 1-197. Aligned sequence of a portion of the mitochondria1 large Miller, L.R. (1986) The phylogeny of Nasutitermitnae. Sociobiology subunit (16s rRNA) sequence from termites, cockroach and 11: 203-214. mantid in a format that includes secondary structure. The Noirot, C. (1995) The gut of termites (Isoptera): comparative sequence for the P. corniceps RNA is given at the top, In anatomy, systematics and phylogeny. 1. Lower termites. Ann sequences of other taxa, nucleotides identical to those of P. Soc EntomolFr 31: 197-226. cornicepare Indicated by dots. Gaps are indicated by dashes. Pearce, M.J. and Waite, B.S. (1994) A list of termite genera with Square brackets represent regions involved in base pairing comments on taxonomic changes and regional distribution. where the regions are separated by other hydrogen-bonded Sociobiology 23: 247-263. stems in the molecule. Unprimed numbered half-stems pair Prestwich, G.D. (1983) Chemical systematics of termite exocrine with their prlmed downstream complementary counterparts. secretions. Annu Rev Ecol Systl4: 287-311. Stem sequences narrowly separated from their complemen- Prestwich, G.D. and Collins, M.S. (1981) Chemotaxonomy of Sub- tary sequences are Indicated by parentheses. Stems are litermes and Nasutitermes termite soldier defense secretions: numbered above the sequence as in Larson eta/. (1992) and evidence against the hypothesis of diphyletic evolution of the Kjer et a/. (1994). Nucleotides that are paired in a stem are . Biochem Syst €cola: 83-88. underlined whereas bulges and single-stranded loops are not. Roonwal, M.L. and Sen-Sarma, P.K. (1960) Contributions to the No inferences can be made about base pairing where comple- systematics of oriental termites. Entomological Monograph mentary sequence is unavailable (e.g. block 3, stem 61’). In No.1, Indian Council for Agricultural Research. such cases the nucleotides are not underlined even though Saitou, N. and Nei, M. (1987) The neighbor joining method: a new they are probably involved in base pairing. In regions where method for reconstructing phylogenetic trees. Mol Biol Evol4: stems could not be unambiguously assigned, potential base 406425. pairs were underlined but no parentheses were Inserted (e.9. Sands, W.A. (1972) The soldierless termites of Africa (Isoptera: stem 66). Characters excluded from the phylogenetic analyses Termitidae). BullBritMus NatlHistSuppl18: 1-244. because they could not be aligned unambiguously are indi- Simon, C., Frati, F., Beckenbach, A., Crespi, B., Liu, H. and Flook, P. cated. See Table 2for complete names and family designations (1994) Evolution, weighting and phylogenetic utility of mitochon- of taxa.

8 1996 Blackwell Science Ltd, Insect Molecular BiologyS: 22S238 236 S. Kambhampati, K. M. Kjer and B. L. Thorne

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