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Home , Blattella vaga, Blattellinae, Blattoidea, Cockroach, Cryptocercus, Cryptocercus punctulatus

Proc. Natl. Acad. Sci. USA Vol. 92, pp. 2017-2020, March 1995

A phylogeny of and related based on DNA sequence of mitochondrial ribosomal RNA genes (/mitochondrial DNA/molecular phylogenetics) SRINIvAs KAMBHAMPATI Department of Entomology, Kansas State University, Manhattan, KS 66506 Communicated by Charles D. Michener, University of Kansas, Lawrence, KS, December 12, 1994 (received for review June 2, 1994)

ABSTRACT Cockroaches are among the most ancient termites (11). It is the sole living representative of the family winged insects, the earliest dating back to about 400 and is found in northern (12). The million . Several conflicting phylogenies for close relationship between M. darwiniensis and is families, subfamilies, and genera have been proposed in the based on several apparent synapomorphies (13-16). past. In addition, the relationship of Cryptocercidae to other A phylogeny of mantids (one ), cockroaches (two cockroach families and the relationship between the cock- species), and termites (three species) based on previously roach, , and the , published morphological characters has recently been pro- darwiniensis, have generated debate. In this paper, a phylogeny posed (6). A conclusion of that study was that Cryptocercus for cockroaches, mantids, and termites based on DNA se- punctulatus is not closely related toM. darwiniensis but is a part quence of the mitochondrial ribosomal RNA genes is pre- of (= Blattaria), which is a sister group to Man- sented. The results indicated that cockroaches are a mono- toidea. A phylogenetic study (12) that included four termite phyletic group, whose sister group is Mantoidea. The inferred species and one each of cockroaches and mantids, and was relationship among cockroach families was in agreement with based on DNA sequence of a portion of the nuclear 18S rRNA the presently accepted phylogeny. However, there was only gene, indicated a sister group affinity of M. darwiniensis to partial congruence at the subfamil and the generic levels. The other termites. In contrast, a study (17) that included C. phylogeny inferred here does not support a close relationship punctulatus, M. darwiniensis, orientalis, and Reticulo- between C. punctulatus and M. darwiniensis. The apparent termes flavipes and was based on DNA sequence of the entire synapomorphies ofthese two species are likely a manifestation 18S rRNA gene suggested that C. punctulatus and M. darwini- of convergent evolution because there are similarities in ensis are closely related. This led the author to conclude that biology and habitat. "Mastotermitidae is considered to belong to Blattodea, instead of Isoptera" (ref. 17, p. 132). The conflicting conclusions of the Cockroaches (order: ; suborder: Blattaria) are among above studies (6, 12, 17) suggest a need to verify their findings the oldest winged insects known, dating back to the Carbonifer- by including a more diverse range of cockroach taxa and ous (1). About 4000 species of cockroaches have been described employing a DNA sequence from a different gene because of (2). A number of conflicting classifications exist for cockroaches, the issue of gene trees and species trees (18, 19). Thus, the the most widely accepted ofwhich is that of McKittrick (1), based primary objective of this study was to infer a phylogeny for on morphological characters. She considered the order Dic- cockroaches, mantids, and termites based on the DNA se- tyoptera to include cockroaches, mantids, and termites, each with quence of mitochondrial large (16S rRNA) and small ribo- its own suborder. She divided the suborder Blattaria into two somal (12S rRNA) subunit genes. The specific objectives were superfamilies, Blaberoidea and , and five families, to (i) compare the molecular phylogeny with that proposed by Polyphagidae, Blattellidae, and (all Blaberoidea), and McKittrick (1), (ii) infer the relationship between Cryptocer- and Cryptocercidae (both Blattoidea). Three other cidae and other cockroach families, and (iii) infer the rela- major cockroach classifications, based on morphological charac- tionship between C. punctulatus and M. darwiniensis.* ters, have been published during the past four decades (3-5). In addition to the overall phylogenetic relationships among MATERIALS AND METHODS cockroaches, two other issues have generated debate. The first is the relationship of Cryptocercidae to other cockroach Insects. The species included in this study are as follows: families. Cryptocercidae consists of one (Cryptocercus) Blaberidae: Archimandrita tessellata, atropos, Blabe- and three species (6) and is generally considered a sister group rus craniifer, , , Byrsotria of Blattidae (1). However, it was recently proposed that fumigata, punctata, azteca, Cryptocercidae be merged with Polyphagidae (7). The second posticus, portentosa, Nauphoeta cinerea, Pan- issue concerns the relationship among cockroaches, mantids, chlora nivea, Phoetalia pallida, Phortioeca phoraspoides, Pyc- and termites. Three major schemes have been proposed: noscelus surinamensis, Rhyparobia maderae, Schultesia lampy- Blattaria and Mantoidea are sister groups and Isoptera is a ridiformis; Blattellidae: vaga, Nahublattella fraterna, sister group of the Blattaria-Mantoidea complex (8), cock- Nahublattella nahu, Nyctibora azteca, Nyctibora lutzi, Parco- roaches and termites belong to the order Blattodea and blatta pensylvanica, pallens; Blattidae: Blatta orien- mantids are a sister group to that order (9), and all three talis, Melanozosteria soror, americana, Periplaneta groups belong to Dictyoptera (1, 10). Of particular interest is australasiae, Periplaneta brunnea, Periplaneta fuliginosa, Shel- the presumed close phylogenetic relationship between Cryp- fordella lateralis; Cryptocercidae: C. punctulatus; : tocercus and the termite, . M. darwini- religiosa; : formosanus, ensis has been considered the most archaic living termite Reticulotermesflavipes; Mastotermitidae: M. darwiniensis. One species and the "missing link" between cockroaches and or more live specimens or DNA of the organism (M. darwini- ensis and M. religiosa) were obtained from colleagues. In most The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in *The sequences reported in this paper have been deposited in the accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession nos. U17761-U17832). 2017 Downloaded by guest on October 6, 2021 2018 Evolution: Kambhampati Proc. Natl Acad. Sci USA 92 (1995) cases, at least one individual was preserved as a voucher Table 1. Summary statistics for the DNA sequences of the specimen. mitochondrial rRNA genes DNA Extraction, PCR, and DNA Sequencing. DNA was Parameter 16S rRNA 12S rRNA extracted from a small portion of the fat body of frozen specimens and PCR was set up as described (20). The PCR Base composition (mean % ± SE) conditions were an initial denaturation step of 94°C for 3 min Adenine 39.1 ± 0.42 39.6 ± 0.39 followed by 35 cycles of 94°C for 30 sec, 50°C for 1 min, and Cytosine 17.9 ± 0.27 17.8 ± 0.21 72°C for 1.5 min. The amplification product was electropho- Guanine 10.6 ± 0.15 10.9 ± 0.13 resed on a 2% low-melting-point agarose gel and purified using Thymine 32.4 ± 0.50 31.7 ± 0.46 minicolumns (Wizard PCRpreps, Promega). DNA sequence Transition rate (%) was obtained directly from 3 ,u of the purified PCR product Overall 7.9 11.3 using the cycle sequencing method (fmol Sequencing System, C ++ T 65.5 66.2 Promega). The reaction mixtures were electrophoresed on 6% A * G 34.5 33.8 polyacrylamide denaturing gels. Both strands of the PCR Transversion rate (%) product were sequenced. Overall 22.0 24.1 Oligonucleotide Primers. The primers for the amplification A +-> C 23.4 22.3 of a 415-bp fragment of the 16S rRNA gene were forward, A <-> T 70.8 69.6 5'-TTA CGC TGT TAT CCC TTA-3' (positions 13,000- G C 1.7 3.1 13,017 of Drosophila yakuba), and reverse 5'-CGC CTG TTT G T 4.1 5.0 ATC AAA AAC AT-3' (13,396-13,415 of D. yakuba). The Characters primers for amplification of a 431-bp fragment of the 12S Total (including gaps) 455 468 rRNA gene were forward, 5'-TAC TAT GTT ACG ACT Variable 276 287 TAT-3' (14,182-14,199 of D. yakuba), and reverse, 5'-AAA Invariable 179 181 CTA GGA TTA GAT ACC C-3' (14,594-14,612 of D. Parsimony informative 237 242 yakuba). The primers were derived from previously published The statistics represent the means for 36 taxa, excluding L. migra- mitochondrial sequences (21-24). Both sets of primers toria. result in the amplification of a homologous fragment from a wide range of insects. Internal primers (16S rRNA: 5'-TCT tified among cockroach taxa corresponding to the four families ATA GGG TCT TCT CGT C-3' and its reverse complement; in this study. Taxa within Blaberidae were subdivided into two 12S rRNA: 5'-TGC ACC TTG ACC TGA A-3' and its reverse subclades. One consisted of genera in Blaberinae, Oxyhaloi- complement) were used to obtain the sequence on the ends of nae, Panchlorinae, and Diplopterinae and the second con- the fragments. sisted of genera in Zetoborinae, Epilamprinae, and Pycnos- Sequence Alignments and Phylogenetic Inference. The se- celinae. Within Blattellidae, Bla. vaga and Par. pensylvanica quences were read manually from autoradiographs into a were shown to be sister taxa and joined to Sy. pallens, followed computer. They were aligned using CLUSTAL V (25) and then by the joining of Nyctibora spp. and Nahublattella spp. to the optimally aligned manually. The alignment parameters were above three genera. Within Blattidae, the four species of k-tuple score = 1, gap penalty = 3, and window size = 5 Periplaneta were found to be paraphyletic. The Periplaneta- (pairwise alignments); fixed and floating gap penalties = 10 was first joined to B. orientalis followed by (multiple alignments). Phylogenetic analysis was carried out in Me. soror. PAUP 3.1.1 (26) using the multiple equally parsimonious heu- At the family level, Blattellidae and Blaberidae were shown ristic search option with tree bisection-reconnection. The data to be more closely related to each other than either was to set was too large to be used with the exhaustive or the branch Blattidae. The sole representative of Cryptocercidae, C. and bound algorithms. Gaps were treated as a fifth base. The punctulatus, formed a separate clade that was joined to the sequences of the two genes were analyzed as a single data set above three families. This was followed by the joining of M (27) without character weighting. The data set was boot- religiosa to the cockroach clade. Among the three species of strapped for 1000 replications using PAUP. The DNA sequence termites included in this study, C. formosanus and R. flavipes of the 16S rRNA gene ofLocusta migratoria (24) was included were shown to be sister taxa. Next, M. darwiniensis was joined as the outgroup. The DNA sequence of the small ribosomal to the above two taxa. Most of the relationships shown in Fig. subunit gene of L. migratoria was not available; thus the 12S 1 were supported in 70-100% of the replications in a tree rRNA data for this were designated as missing. derived from bootstrap analysis (Fig. 2).

RESULTS DISCUSSION DNA Sequences of rRNA Genes. The sequences for taxa in A phylogeny of cockroaches inferred from DNA sequence of this study can be obtained directly from GenBank or from the the mitochondrial rRNA genes indicated that cockroaches are author. The average size of the sequenced portion of the 16S monophyletic. This is-in contrast to the suggestion (17) that the rRNA gene was 415.3 ± 0.75 bp (mean ± SE) and that of the group is perhaps paraphyletic. The relationship among families 12S rRNA gene was 431.4 ± 0.72 bp. Summary statistics for the inferred here was in agreement with the presently accepted two genes are presented in Table 1. phylogeny (1). However, there were significant differences at Phylogenetic Inference. The alignment of the sequences the subfamily and generic levels between McKittrick's (1) and resulted in a total of 923 characters, including gaps. Unam- the molecular phylogenies. Of particular note were taxa in biguous alignment was possible for most regions; six regions Blaberidae, within which relationships among genera did not totaling 140 characters were relatively more difficult to align. always reflect the subfamily designations of McKittrick. For When the data were analyzed without these regions, the example, D. punctata and Pho. pallida were placed in Diplop- topology of the tree identified by PAUP was nearly identical to terinae by McKittrick (1) and in Diplopterinae and Epilam- that of the tree generated with the full data set. Therefore, all prinae, respectively, by Princis (4). In this study, Pho. pallida further analyses were carried out with the full data set. was shown to be more closely related to Sc. lampyridiformis of PAUP identified a single most parsimonious tree of 3750 Zetoborinae than to D. punctata. steps (Fig. 1). The taxa in the various orders, suborders, and Two subfamilies of Blattellidae, and Nyctibori- families formed distinct . Four major clades were iden- nae, were represented in this study. Blattella, , and Downloaded by guest on October 6, 2021 Evolution: Kambhampati Proc. Natt Acad Sci USA 92 (1995) 2019

3 I Phor. phoraspoides 1 Phor. phoraspoides 8 Pho. pallida 2 Pho. pallida 2 Sc. lampyridiformis Sc. lampyridiformis 1 Py. surinamensis 31 Py. surinamensis 3 azteca 2 Ep. azteca 90Ep. A. tessellata 44 A. tessellata 4 BI. discoidalis 31 0 BI. discoidalls 4 BI. atropos 47 1 Bl. atropos 4 BI. craniifer 66 46 37 1 Bl. cranilfer 4 BI. giganteus 6162 BI. giganteus 4 By. fumigata By. fumigata 4 Eu. posticus 3434_ 86886 ~~~Eu. postlcus 4 Pa. nivea 41 108 D. punctata 5 Nau. cinerea 68 Nau. cinerea 6 R. maderae 51 42 32 69R. maderae 6 G. portentosa G. portentosa 6 D. punctata 82 Pn. nivea 7 Bla. vaga 85 Par. pensylvanica 32 BIa. vaga 8 36 61 Sy. pall ens Par. pensylvanica 8 Ny. lutzi 48 Sy. pallens 8 Ny. azteca 71 Ny. lutzi 9 Na. naha 58 41 Ny. azteca 9 Na. fraterna 118 10- - Na. naha 8 B. orientalis Na. fraterna 8 Sh. lateralis 68 B. orientalis 10 Pe. brunnea 46 X304 Sh. lateralis 10 46 Pe. americana 61 21 62 Pe. brunnea 10 Pe. ful i gi nosa 2 4 Pe. americana 10 II Pe. australasiae 22 Pe. fuliginosa 10 Me. soror 52 Pe.fuliginoPe.australasiae 1 0 C. punctulatus 72 Me. soror 11 M. religiosa 1 1 5 C. punctulatus 1 2 V R. flavipes _ 132 M. religiosa 13 V C. formosanus 73 85 R. 14 M. darwiniensis flavipes L. 55 C. formosanus 15 VI migratoria M. darwiniensis 16 106 1 74 FIG. 2. Bootstrap parsimony tree (1000 replications) based on L. migratoria OG DNA sequence of mitochondrial 16S rRNA and 12S rRNA genes of cockroaches, termites, and mantid, rooted by the outgroup, L. migra- FIG. 1. Single most parsimonious tree for DNA sequence of toria. Tree length, 3912 steps; consistency index, 0.34; retention index, mitochondrial 16S rRNA and 12S rRNA genes of cockroaches, 0.51. Numbers above the branches indicate percent times the branch termites, and mantid, rooted by the outgroup, L. migratoria. Tree was recovered. length, 3750 steps; consistency index, 0.35; retention index, 0.54. Numbers above the branches are branch lengths. Numbers accompa- nying scientific names are family or subfamily designations: 1, Zeto- made (7). Unfortunately, I could not include Polyphagidae to borinae; 2, Epilamprinae; 3, Pycnoscelinae; 4, Blaberinae; 5, Diplop- test its relatedness to Cryptocercidae. Further work is neces- terinae; 6, Oxyhaloinae; 7, Panchlorinae; 8, Blattellinae; 9, Nyctibori- sary to infer the relationship between Polyphagidae and nae; 10, Blattinae; 11, Polyzosteriinae; 12, Cryptocercinae; 13, Cryptocercidae. Mantidae; 14, ; 15, Coptotermitinae; 16, Mastoter- The results of this study suggested that Mantoidea is a sister mitidae. The roman numerals indicate ordinal, subordinal, or family group of Blattaria and that termites are a sister group of the designations: I, Blaberidae; II, Blattellidae; III, Blattidae; IV, Cryp- cockroach-mantid complex. Although it seems appropriate tocercidae; V, Mantoidea; VI, Isoptera; OG, outgroup. that cockroaches and mantids be retained in Dictyoptera with each group being assigned a subordinal status (8), future Symploce, all Blattellinae, were inferred to be closely related to studies that include a broader range of mantids and termites one another as previously suggested (1). However, because of are needed to confirm the relationships inferred here. the placement of Nahublattella relative to the above genera, The results of my analysis indicated that M. darwiniensis and Blattellinae as presently recognized is paraphyletic. Nyctibori- C. punctulatus are not closely related. The sister group affinity nae has been inferred to be a sister group of Blattellinae (1), of M. darwiniensis was clearly with other termites and that of a relationship confirmed by this analysis. C. punctulatus was with other cockroaches. At present, two Two subfamilies of Blattidae were included in this study: opposing views exist concerning the phylogenetic relationship Blattinae and Polyzosteriinae. As expected, all genera in of these two taxa. One states that they are no more closely Blattinae were sister taxa to one another and Polyzosteriinae related to each other than cockroaches in general are related was shown to be the sister group of Blattinae. Within Blattinae, to termites and any apparent synapomorphies of Cryptocercus Sh. lateralis was found to be a sister taxon of Pe. brunnea and and Mastotermes together are the result of convergent evolu- thus the genus Periplaneta is paraphyletic according to my tion (6, 9). The alternate view (1, 17) holds that the synapo- analysis. No consensus on the generic status of Sh. lateralis is morphies of the two taxa are a result of common ancestry. apparent. Historically, this species has been successively placed Specifically, the conclusions of morphological analysis (6) and in the genera Periplaneta, Blatta, and Shelfordella. Walker (28) earlier analyses of DNA sequences (17) are at odds concerning originally described this species as Periplaneta lateralis. The the relationship of C. punctulatus with M. darwiniensis. The relationship inferred here suggests that this species should present study, also based on DNA sequence analysis, but of perhaps be placed in the genus Periplaneta as originally different genes and with an extensive sampling of cockroach proposed rather than Blatta (4, 29) or Shelfordella as presently taxa, clearly supported the conclusions of the morphological recognized. analysis (6). In both trees (Figs. 1 and 2), M. darwiniensis was The results of my analysis indicated that Cryptocercidae is a sister taxon of the other termites. In the parsimony tree (Fig. most closely related to Blattidae. As mentioned, however, a 1) the affinity of C. punctulatus was clearly with other cock- proposal to merge Cryptocercidae and Polyphagidae has been roaches. Downloaded by guest on October 6, 2021 2020 Evolution: Kambhampati Proc. Natl. Acad Sci. USA 92 (1995) A significant synapomorphy on which a close phylogenetic ensis belongs to Blattaria instead of Isoptera (17) can be relationship of Cryptocercus with Mastotermes has been pro- ignored for the present. Finally, my analysis indicated that posed is the cellulolytic gut fauna that is present in both taxa cockroaches are monophyletic and are a sister group to but not in other cockroaches (1, 13-16). Previous studies (1, mantids. The issue of whether Isoptera is a sister group of the 30-33) and the present study indicate that C. punctulatus Blattaria-Mantoidea complex needs to be resolved by includ- belongs to a primitive phyletic line from which the other ing other closely related taxa such as Zoraptera and represen- cockroaches have descended. This suggests two alternate tatives of diverse termite families. scenarios for the presence of cellulolytic symbionts in Crypto- in I thank the following: R. D. Bowling, J. Y. Bradfield, R. DeSalle, S. but not other cockroaches. First, M. darwiniensis and Gatti, D. E. Mullins, C. A. Nalepa, R. S. Patterson, L. M. Roth, S. Cryptocercus share gut fauna due to common ancestry. In this Starkey, B. Stay, and L. Vawter for insects/DNA; D. W. Alsop, W. J. case, it must be assumed that all other cockroaches (and Bell, W. C. Black IV, R. DeSalle, T. L. Hopkins, and L. M. Roth for mantids) that descended from a Cryptocercus-like ancestor advice; W. J. Bell, C. D. Michener, and two anonymous reviewers for have secondarily lost gut fauna (6). Alternatively, Cryptocercus comments on the manuscript; and A. L. Nus for technical assistance. acquired the symbionts through some mechanism (see below) This research was supported by a seed grant from the Department of after other cockroaches arose. In this scenario, a secondary Entomology, Kansas State University. This is journal article no. loss need not be invoked for the non-cryptocercid cockroaches. 94-540-J of the Kansas Agricultural Experiment Station. If secondary loss is not invoked, the shared gut fauna of 1. McKittrick, F. A. (1964) Mem-N. Y Agric. Exp. Stn. (Ithaca) 389, Cryptocercus and Mastotermes implies convergent evolution (9) 1-191. and perhaps interspecific transfaunation (34). It is not known 2. Bell, W. J. (1990) in Cockroaches as Models for Neurobiology, eds. which group first acquired the cellulolytic gut fauna. However, Huber, I., Masler, E. P. & Rao, B. R. (CRC, Boca Raton, FL), Vol. a scenario in which transfaunation could occur through inter- 1, pp. 7-12. 3. Rehn, J. W. H. (1951) Am. Entomol. Soc. Mem. 14, 1-134. specific predation has been proposed (34). Since Cryptocercus 4. Princis, K. (1960) Eos (Madrid) 36, 429-449. and Mastotermes were sympatric in the Americas before the 5. Huber, I. (1974) Univ. Kans. Sci. Bull. 50, 233-332. latter became extinct there (9, 12), the possibility exists that the 6. Thorne, B. L. & Carpenter, J. M. (1992) Syst. Entomol. 17, 253-268. gut fauna was obtained through interspecific predation. 7. Grandcolas, P. (1992) Syst. Entomol. 19, 145-158. Thorne (34) concluded that "Cryptocercus shares no unam- 8. Boudreaux, H. B. (1979) Phylogeny with SpecialReference to biguous morphological, behavioral or symbiotic synapomor- Insects (Wiley, New York). 9. Hennig, W. (1981) Insect Phylogeny (Wiley, New York). phies with the Isoptera that are not shared with other cock- 10. Kristensen, N. P. (1981) Annu. Rev. Entomol. 26, 135-157. roaches as well. Cryptocercidae are clearly primitive roaches, 11. Hennig, W. (1966) Phylogenetic Systematics (Univ. of Illinois Press, but it is more likely that the family is a sister group to other Urbana, IL). cockroach taxa rather than a sister group to modern termites." 12. DeSalle, R., Gatesy, J., Wheeler, W. & Grimaldi, D. (1992) Science Cogent arguments against the scenario in which transfaunation 257, 1933-1935. 13. Crampton, G. C. (1923) Brooklyn Entomol. Soc. Bull. 18, 85-93. could occur have been presented (35). Thus, the question of 14. Tilliyard, R. J. (1936) Nature (London) 137, 655. how Cryptocercus and Mastotermes came to share gut symbi- 15. Hill, G. F. (1925) R. Soc. Victoria Proc. 37, 119-124. onts is yet to be resolved. Further studies, directly on the DNA 16. Cleveland, L. R., Hall, S. R., Sanders, E. P. & Collier, J. (1934) of the gut fauna, may help resolve the issue of whether the gut Brooklyn Entomol. Soc. Bull. 13, 49-68. symbionts of Cryptocercus and Mastotermes are more closely 17. Vawter, L. (1991) Ph.D. Thesis (Univ. of Michigan, Ann Arbor). related to each other than a random pair of symbionts from 18. Nei, M. (1987) Molecular Evolutionary Genetics (Columbia Univ. Press, New York). xylophagous insects. With PCR, it is feasible to study symbiont- 19. Li, W.-H. & Graur, D. (1990) Fundamentals of Molecular Evolution specific genes as has been done for other insect symbionts (36, (Sinauer, Sunderland, MA). 37). 20. Kambhampati, S., Black, W. C., IV, & Rai, K. S. (1992) J. Med. As with other insect mtDNA studied to date (21-24), the Entomol. 29, 939-945. base composition of sequences in this study was strongly biased 21. Crozier, R. H. & Crozier, Y. C. (1993) Genetics 133, 97-117. toward adenine and which of the 22. Clary, D. 0. & Wolstenholme, D. R. (1985) J. Mol. Evol. 22,252-271. thymine, comprised 72% 23. Mitchell, S. E., Cockburn, A. F. & Seawright, J. A. (1993) Genome 36, total. The observed transition and transversion rates were 1058-1073. comparable to those reported for other insect groups and the 24. Uhlenbusch, I., McCracken, M. & Gellissen, G. (1987) Curr. Genet. rate of transversions was significantly greater than that of 11, 631-638. transitions. A greater transversion rate relative to transition 25. Higgins, D. M. & Sharp, P. M. (1989) Comput. Appl. Biol. Sci. 5, rate has also been observed in these mtDNA genes: 16S rRNA 151-153. II 26. Swofford, D. L. (1993) PAU.: Phylogenetic Analysis Using Parsimony of leafhoppers (38) and black flies (39), cytochrome oxidase (Smithsonian Inst., Washington, DC), Version 3.1.1. of 10 insect orders (40), and NADH 1 and 16S rRNA of 27. Miyamoto, M. M., Kraus, F. & Ryder, 0. A. (1990) Proc. Natl. Acad. Drosophila spp. (41). The bias toward transversions in insect Sci. USA 87, 6127-6131. mtDNA is in contrast to the mtDNA of primates in which 92% 28. Walker, F. (1868) British Museum Catalog (British Museum, London). of the substitutions were transitions (42). It has been suggested 29. Gurney, A. B. (1978) Plant Rep. 3, 295. that the bias may be due to deficient mtDNA repair mecha- 30. Roth, L. M. & Willis, E. R. (1958) Trans. Am. Entomol. Soc. 83, 221-238. nisms and a tautomeric base pairing chemistry (43). As with 31. Roth, L. M. (1967) Ann. Entomol. Soc. Am. 61, 83-111. other insects (references above), >70% of transversions in 32. Roth, L. M. (1982) Proc. Entomol. Soc. Wash. 84, 277-280. cockroaches, termites, and mantid were A *-+ T transversions. 33. Roth, L. M. (1989) Proc. Entomol. Soc. Wash. 91, 441-451. In summary, the significant findings of this study are as 34. Thorne, B. L. (1990) Proc. R. Soc. London B 241, 37-41. follows. The molecular phylogeny was congruent at the family 35. Nalepa, C. A. (1991) Proc. R. Soc. London B 246, 185-189. level with the morphological phylogeny proposed by McKit- 36. O'Neill, S. L., Giordano, R., Colbert, A. M. E., Karr, T. L. & Rob- ertson, H. M. (1992) Proc. Natl. Acad. Sci. USA 89, 2699-2705. trick (1). However, significant differences were observed at the 37. Kambhampati, S., Rai, K. S. & Burgun, S. J. (1993) Evolution 47, subfamily and generic levels. DNA sequences from more 673-677. species need to be obtained to further delineate the relation- 38. Fang, Q., Black, W. C., IV, Blocker, D. & Whitcomb, R. F. (1993) ships at the subfamily level. The results of this study suggested Mol. Phylogenet. Evol. 2, 119-131. that Cryptocercidae is most closely related to Blattidae. 39. Xiong, B. & Kocher, T. D. (1991) Genome 34, 306-311. 40. Liu, H. & Beckenbach, A. T. (1992) Mol. Phylogenet. Evol. 1, 41-52. Whether it should be placed in the Polyphagidae needs to be 41. DeSalle, R. (1992) Mol. Phylogenet. Evol. 1, 31-40. resolved using appropriate taxa. My analysis did not lend 42. Brown, W. M., Prager, E. M., Wang, A. & Wilson, A. C. (1982)J. Mol. support to the hypothesis that C. punctulatus and M. darwini- Evol. 18, 225-239. ensis are closely related. Thus, the suggestion that M. darwini- 43. Topal, M. D. & Fresco, J. R. (1976) Nature (London) 263, 285-289. Downloaded by guest on October 6, 2021