Proc. Natl. Acad. Sci. USA Vol. 92, pp. 2017-2020, March 1995 Evolution A phylogeny of cockroaches and related insects based on DNA sequence of mitochondrial ribosomal RNA genes (termites/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 fossils dating back to about 400 Mastotermitidae and is found in northern Australia (12). The million years. Several conflicting phylogenies for cockroach close relationship between M. darwiniensis and Cryptocercus 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 species), cockroaches (two cockroach families and the relationship between the cock- species), and termites (three species) based on previously roach, Cryptocercus punctulatus, and the termite, Mastotermes 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 Blattodea (= 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, Blatta 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: Dictyoptera; 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 Blattoidea, and five families, to (i) compare the molecular phylogeny with that proposed by Polyphagidae, Blattellidae, and Blaberidae (all Blaberoidea), and McKittrick (1), (ii) infer the relationship between Cryptocer- Blattidae 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 genus (Cryptocercus) Blaberidae: Archimandrita tessellata, Blaberus atropos, Blabe- and three species (6) and is generally considered a sister group rus craniifer, Blaberus discoidalis, Blaberus giganteus, Byrsotria of Blattidae (1). However, it was recently proposed that fumigata, Diploptera punctata, Epilampra azteca, Eublaberus Cryptocercidae be merged with Polyphagidae (7). The second posticus, Gromphadorhina 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: Blattella 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, Symploce pallens; Blattidae: Blatta orien- mantids are a sister group to that order (9), and all three talis, Melanozosteria soror, Periplaneta 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; Mantidae: tocercus and the termite, Mastotermes darwiniensis. M. darwini- Mantis religiosa; Rhinotermitidae: Coptotermes 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- insect 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 Shelfordella clade was first joined to B.