Phylogenetic relationships among 18 species of orthopteroid insects (Blattaria: cockroaches, Isoptera: termites, Mantodea: mantids, Grylloblattodea: grylloblattids, Phasmatodea: stick-insects, Orthoptera-Caerifera: locusts, Orthoptera-Ensifera: crickets, and Dermaptera: earwigs), were estimated based on DNA sequencing of the mitochondrial cytochrome oxidase II gene. Our results drew attention to the need for caution in using third codon positions for tree construction, since it was likely that base pair substitutions of third codon positions in the COII gene were saturated among taxa used in the present study. We also detected that there were many phylogenetically informative sites in first codon positions. Phylogenetic trees using first and second codon positions based on both the neighbor-joining method and parsimony analysis indicated that the topology was nearly identical to each other. The phylogenetic relationships among these taxa differ from the current classification based on morphological characters. The inferred trees showed that grylloblattids were not a primitive group, but closely related to the Dictyoptera. Stick-insects were closely related to the Dictyoptera and grylloblattids, not to crickets. Locusts and crickets formed a monophyletic group. Earwigs were only distantly related to the Dictyoptera. Within the Dictyoptera, cockroaches and termites constituted a monophyletic group, with mantids as a sister group to that complex.
INTRODUCTION
Several phylogenetic hypotheses based on morphological characters have been proposed for orthopteroid insects (reviewed in Kristensen, 1995). Hennig (1969, 1981) suggested that the “lower Neoptera” might have two entities, Embioptera (web-spinners) and the “Orthopteromorpha”, which includes Grylloblattodea (grylloblattids), Dermaptera (earwigs), Mantodea (mantids), Blattodea (cockroaches), Isoptera (termites), Orthoptera-Ensifera (crickets), Orthoptera-Caelifera (locusts) and Phasmatodea (stick-insects). Furthermore, he suggested that the Orthopteromorpha might be represented as (Grylloblattodea + (Dermaptera + (Mantodea + (Blattodea + Isoptera))) + (Orthoptera-Ensifera + (Orthoptera-Caelifera + Phasmatodea))). The relationships among the monophyletic subgroups of the Orthopteromorpha, however, are still unresolved (Boudreaux, 1979; Hennig, 1969, 1981). Kukalová-Peck and Brauckmann (1992) and Kukalová-Peck and Peck (1993) has advocated the monophyly of a lineage comprising the Embioptera + (Isoptera + Orthoptera-Caelifera) + Phasmatodea, and that comprising the Zoraptera + Grylloblattodea + Dermaptera + (Blattodea + Mantodea). There were also suggestions that stick-insects were more closely related to the cockroach-like insects rather than to the Orthoptera (Ragge, 1955; Ross, 1955). Additionally, it is possible that crickets and locusts may not be monophyletic group (Hennig, 1969, 1981; Kristensen, 1995), and were each given ordinal status by Kevan (1986). The systematic position of grylloblattids is also controversial. In general, based on anatomical data, grylloblattids are considered to be a relic species and an intermediate form between cockroaches and the Orthoptera, and one of the primitive groups among orthopteroid insects (Walker, 1914, 1933, 1938; Crampton, 1915, 1935; Imms, 1945). Nagashima (1982) studied grylloblattid head anatomy in detail in comparison with other orthopteroid insects and indicated that grylloblattids were not a primitive group among these insects. The precise topology of the tree that includes cockroaches, termites and mantids also remains a topic of active discussion. Kristensen (1975) suggested the same relationships as Hennig (1969, 1981), but later Kristensen (1981) grouped all three taxa into the same order “Dictyoptera” and waited for further work to resolve the phylogeny. In this study, we use Dictyoptera as this meaning. Another phylogenetic hypothesis based on morphological characters is (termite + (cockroach + mantid)) proposed by Boudreaux (1979). Thorne and Carpenter (1992) recently did a parsimony analysis using 70 morphological and ecological characters, and found a single most parsimonious tree which supported the hypothesis of Boudreaux. Kukalová-Peck and Peck's (1993) analysis of wing structures also supported this relationship. Kambhampati (1995) constructed a phylogeny of cockroaches and related insects based on mitochondrial 16S and 12S rRNA genes, and found that termites were the sister group to a clade consisting of cockroaches and mantids. The primary goal of his study, however, was not to investigate the phylogeny of the Dictyoptera. Therefore, the relationships of these groups were only briefly discussed in his study.
In this study, we investigated the phylogenetic relationships among the Orthopteromorpha based on the complete nucleotide sequence of the mitochondrial cytochrome oxidase subunit II (COII). We chose this system for analysis because the lengths of COII genes of each group are nearly identical, thus facilitating the alignment and comparison of the sequences, and because a phylogenetic analysis of the Orthopteromorpha using modern methodology and based on sufficient numbers of characters is lacking.
MATERIALS AND METHODS
Insects
The species investigated are listed in Table 1. The sequences of termite (Zootermopsis angusticollis), locust (Schistocerca gregaria), cricket (Acheta domesticus) and milkweed bug (Oncopeltus fasciatus) genes were obtained from GenBank Data Libraries (Accession nos. M83968, M83966, M83961, and M83959, respectively; Liu and Beckenbach, 1992). The phylogeny of insect orders based on morphology (Kristensen, 1991) and of the holometabolous insect orders inferred from 18S and 28S ribosomal DNA sequences and morphology (Fig. 9 and Fig. 10 in Whiting et al., 1997) showed that the Hemiptera was the sister group of the orthopteroid insects. Consequently, we used milkweed bugs as an outgroup.
Table 1
List of the species used in this study
DNA extraction
For termites, total genomic DNA was extracted from a total body with the hindgut removed; in all other insect leg tissue was used. Fresh or frozen materials, or samples preserved in acetone were used for DNA extraction. The procedure of the extraction was modified from Laird et al. (1991). Tissue was homogenized with a pair of dissecting scissors in a 1.5 ml microcentrifuge tube containing 500 μl of lysis buffer (100 mM Tris-HCl, 5 mM EDTA, 0.2% SDS, 200 mM NaCl) and 50 μg of Proteinase K (Wako Chemicals). The mixture was incubated overnight at 56°C. 10 μg of RNase A (Boeringer Mannheim) was added to the mixture, and incubated at 37°C for 30 min. Following a series of phenol-chloroform and chloroform extractions, DNA was precipitated with an equal volume of isopropanol. Pellets were rinsed with 70% ethanol, dried under vacuum and dissolved in 40 μl of TE buffer (10 mM Tris-HCl, 1 mM EDTA).
DNA amplification
The COII region of mitochondrial DNA was amplified using polymerase chain reaction (PCR; Saiki et al., 1985). Two pairs of primers were used for the amplification. The primer sequences are shown in Table 2. A-tLEU and B-tLYS, and C1-J-2773 followed Liu and Beckenbach (1992) and Miura et. al. (1998), respectively. Another internal primer (A-COII) was chosen in regions of sequence conservation between Drosophila yakuba and other insects and were based on partial sequences as they were obtained. The reaction was performed in an ASTEC PC-700 programmable temperature control system or GeneAmp 2400 thermal cycler (Perkin-Elmer) under the following conditions: 35 cycles of denaturing at 94°C for 1min, annealing at 50°C for 1min and extending at 70°C for 2 min (Liu and Beckenbach, 1992). The reaction mix was performed in a final volume of 40 μl of the following solution: 30 μl of distilled water, 4 μl of 10 × PCR buffer (Takara, TaKaRa Taq: containing 100 mM Tris-HCl (pH8.3), 500 mM KCl, 15 mM MgCl2, 0.01% (w/v) gelatin), 4 μl of dNTP mix (1 mM of each dNTP), 0.2 μl of each primer (100 pM), 0.7 U of Taq polymelase (Takara, TaKaRa Taq) and 2 μl of template DNA.
Table 2
Primer sequences used in this study
DNA purification and sequencing
PCR products were electrophoresed in a 1% agarose gel, and purified using Prep-A-Gene DNA Purification Kit (BIO RAD). The purified products were used as a template for the sequencing reaction. The sequencing reaction was performed using a Dye-Terminator Cycle Sequencing Kit (Perkin-Elmer) and GeneAmp 2400 thermal cycler. Electrophoresis and data collection were performed using an automatic DNA sequencer (Perkin-Elmer, model 373S) with 6% polyacrylamide gel (TOYOBO, Super Reading DNA Sequence Solution), following the recommended procedure. Both strands of the amplified PCR product were sequenced.
Phylogenetic analysis
Sequences were aligned using the Clustal W program package (Thompson et al., 1994), and confirmed with aligned sequences of 10 orders of insects (Liu and Beckenbach, 1992). The number of nucleotide substitutions were estimated according to Kimura's two-parameter methods (Kimura, 1980). Distance matrices were analyzed by the neighbor-joining method (Saitou and Nei, 1987) to construct phylogenetic trees using Clustal W. All alignment positions where there was a gap in any sequence were included in the distance analysis. A bootstrap analysis (Felsenstein, 1985) of 1000 replications was carried out. We also used PAUP 3. 1. 1. program package (Swofford, 1993) to carry out non-weighted parsimony analysis using the heuristic search option with tree bisection-reconnection. In the parsimony analyses, gaps were treated as a fifth base. The data set was also bootstrapped for 1000 replications with five random addition sequence replicates for each bootstrap replicate using PAUP. The numbers of base pair substitution in pairwise comparisons of each codon position were counted. We performed our analyses using (1) the complete data set, and restricted subsets of the data, namely (2) first codon positions, (3) second codon positions and (4) first and second codon positions.
The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and GenBank nucleotide sequence databases with the following accession nos: AB005459, AB005461, AB005463, AB005470, AB005905, AB006435, AB006436, AB011233 and AB011234.
RESULTS
Nucleotide sequences
The COII genes ranged from 670 bp in stick-insects to 690 bp in milkweed bugs. The aligned sequences are shown in the Appendix. The sequences of Salganea taiwanensis and O. fasciatus have TAG and TAA at the 3′ end, potentially encoding the entire terminator. All of other sequences end in T. The transcripts of such mitochondrial genes that have incomplete stop codons contain a stop codon created by posttranscriptional polyadenylation (Wolstenholme, 1992).
Several internal insertion/deletion events could be seen. In the sequences of crickets, an insertion of three nucleotides appeared at positions 349–351, and a deletion of two codons occured at positions 394–399. In the stick-insect sequences, a deletion of two codons also occured at positions 373–378 (see Appendix). A total of 549 of the 693 nucleotide sites were variable, including 225 first, 101 second and 223 third codon positions. It was shown that the numbers of variable sites in the first codon position were as many as those in the third codon position. Average numbers of nucleotide substitution counted based on pairwise comparisons of each codon position are shown in Fig. 1. On the first, second and third codon positions, 5.6–36.4% (average 27.3%), 1.3–22.1% (average 15.6%) and 24.7–61.0% (average 44.8%) were variable, respectively. Average transition (Ti) and transversion (Tv) rates at each codon position are 12.8 and 15.0% (first position), 8.8 and 7.0% (second position) and 16.3 and 29.8% (third position).
Phylogenetic inference
Firstly, phylogenetic trees were inferred based on the neighbor-joining method and on parsimony analysis using complete nucleotide sequences, first codon positions and second codon positions (trees not shown). In the bootstrap parsimony trees, tree length, consistency index, rescaled consistency index and retention index are as follows; 2160 steps, 0.40, 0.14 and 0.34 (complete nucleotide), 584 steps, 0.46, 0.22 and 0.49 (first position), 302 steps, 0.52, 0.31 and 0.59 (second position). In all trees, monophyly of each order excluding cockroaches and termites was supported in 50% or more of the bootstrap replicates. Monophyly of cockroaches and termites was not supported in the parsimony trees using all data sets and first codon positions, and using second codon positions, respectively. Although some monophyletic groups (cockroach + termite in the trees using first codon position, and cockroach + termite + mantid + grylloblattid in the trees using second codon positions) were supported, phylogenetic relationships among the other orders were supported in less than 50% bootstrap replicates.
Secondly, bootstrap trees were inferred based on the first and second codon positions (Fig. 2). Fig. 2A is the neighbor-joining tree, while Fig. 2B is the parsimony tree. Monophyly of each order was supported in both trees. Monophyly of the cockroach + termite clade was also supported. Earwig was shown to be the basal taxon. Grylloblattids were shown to be more closely related to the Dictyoptera. Monophyly of the clade consisting of cockroaches, termites, mantids and grylloblattids, however, was supported in less than 50% bootstrap replicates in the neighbor-joining tree. Monophyly of the Dictyoptera was supported in Fig. 2A, but relationships among the cockroach-termite, mantids and grylloblattids clade were not resolved in Fig. 2B. Stick-insects were shown to be more closely related to the monophyletic group consisting of the Dictyoptera and grylloblattids in the neighbor-joining tree. The phylogenetic position of stick-insects, however, is not clear in the parsimony tree.
DISCUSSION
This study is the first comprehensive study focused on the phylogeny among orthopteroid insects based on the molecular chracters. Especially, molecular phylogenetic relationships between grylloblattids and other orthopteroid insects have not been studied until the present study. Although Liu and Beckenbach (1992) discussed about the evolution and characteristics of COII genes of 10 orders of insects, they used only 4 species belonging to 3 orders for orthopteroid insects.
We analyzed phylogenetic relationships using the following data sets; all nucleotide sites, first codon positions, second codon positions (trees not shown), and first and second codon positions (Fig. 2). Not surprisingly, there were many variable sites in the third codon positions (96.5%). By contrast, there were more variable sites in the first codon positions (97.4%). These high values in the first codon positions are noteworthy, because percentages of variable sites in the first and third codon positions of COII genes among 10 orders of insects are 70.0 and 91.3%, respectively (Liu and Beckenbach, 1992). The average numbers of nucleotide substitutions of the first codon positions were, however, shown to be much less than those of the third codon positions (Fig. 1). When the first codon positions were used for the phylogenetic analysis, monophyly of all orders (excluding cockroaches in the parsimony tree) was supported. Phylogenetic trees using only third codon positions did not support the monophyly of these groups (trees not shown). Furthermore, the ratio of Ti to Tv at the third codon positions (0.55) were shown to be much smaller than those of the first (0.85) and second codon positions (1.23). The ratio of Ti to Tv can decrease as a result of multiple substitutions at a sites within a gene (Simon et al., 1994). It is clear that nucleotide substitutions of the third codon positions of COII gene are saturated in pairwise comparisons between orders. Edwards et al. (1991) and Irwin et al. (1991) found that the inclusions of third position changes can obscure relationships in deep phylogenies. Moreover, we thought that there were many informative sites for phylogenetic analyses in the first codon positions. When only second codon positions were used for analyses, there were only 101 variable characters. Consequently, we used the first + second codon positions to construct phylogenetic trees.
Our phylogenetic trees using the first and second codon positions indicate that stick-insects are not closely related to locusts or crickets. More likely, they are more closely related to the Dictyoptera and grylloblattids. There was a relatively high genetic difference between locusts and crickets, but it was shown that they were a monophyletic group. Earwigs were the most phylogenetically distant from the Dictyoptera, suggesting that they are basal among the Orthopteromorpha used in the present study. The above-mentioned relationships are different from relationships presently accepted based on morphological characters (Hennig, 1969, 1981; Boudreaux, 1979; Kristensen, 1981, 1995; Kukalová-Peck and Brauckmann, 1992; Kukalová-Peck and Peck, 1993). Ross (1955), however, suggested that stick-insects were more closely related to cockroaches and mantids, based on egg morphology. A monophyly of locusts and crickets also has been suggested (Boudreaux, 1979; Kukalová-Peck and Brauckmann, 1992; Kukalová-Peck and Peck, 1993). However, earwigs were always thought to be closely related to the Dictyoptera until the present study (Hennig, 1969, 1981; Boudreaux, 1979; Kukalová-Peck and Brauckmann, 1992; Kukalová-Peck and Peck, 1993; Kristensen, 1995).
Grylloblattids were shown not to be a basal taxon among orders used in this study, but to be more closely related to the Dictyoptera. Nagashima (1982) pointed out that the extrinsic muscles of the antenna of grylloblattids were like those of mantids and web-spinners, and that those of the mandible resembled those of cockroaches, termites, mantids and stick-insects. He also showed that the muscles of the maxilla resembled those of earwigs. However, Ando and Nagashima (1982) pointed out that the pattern of embryonic development in Galloisiana nipponensis differed from that of earwigs. These morphological data were in agreement with the results of our present study. Unfortunately, we could not use web-spinners in this study, so phylogenetic relationships among grylloblattids, Dictyoptera and web-spinners still remains ambiguous.
In the trees using the first and second codon positions, cockroaches were shown to be more closely related to termites than mantids, although bootstrap confidence is not high (64% in the neighbor-joining tree, 57% in the parsimony tree). There has not hitherto been molecular data which supports this relationship. Cryptocercus spp. and Mastotermes darwiniensis, which are generally recognized to be most primitive cockroaches and termites, as well as mantid species belonging to other families than the Mantidae need to be included in further molecular studies of the Dictyoptera to resolve relationships within this group.
Although COII gene is a relatively conserved gene in the mitochondrial DNA (Clary and Wolstenholme, 1985; Liu and Bechenbach, 1992), this gene is probably not the best gene for revealing robust phylogenetic relathionships among orthopteroid insects. However, COII gene is clearly useful for the phylogeny to some extent, and this study is the first step to analyze molecular phylogenetic relationships among orthopteroid insects. It is clear that the phylogenetic analyses of numerous taxa, including web-spinners and based on not only molecular data of other genes but also morphological data are needed to determine precise relationships among these groups.
Acknowledgments
We thank Dr. M. Maryati for her support in the field sampling, and Dr. T. Nagashima for providing Galloisiana nipponensis. Dr. R. Ueshima taught us the sequencing technique. We are also grateful to Drs. C. A. Nalepa, C. Bandi, M. Terayama and Mr. N. Lo, who gave us valuable comments on the manuscript. Mr. T. Miura helped and supported us at our field sampling and sequencing. This study was supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists for K. M., and in part by a Grant-in-Aid from the International Scientific Research Program (No. 08041136) and a Scientific Research Grant (no. 07454211, 10440231) from the Ministry of Education, Science, Sports and Culture of Japan.
REFERENCES
Appendices
APPENDIX
Aligned sequence of the mitochondrial cytochrome oxidase II gene of 18 species of orthopteroid insects and milkweed-bugs used as an outgroup. Dots indicate identity to the S. taiwanensis sequences. Gaps are indicated by dashes. See Table 1 for complete names of taxa.