* National Institute of Agrobiological Sciences, Tsukuba, Japan
Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Sezione di Patologia Generale e Parassitologia, Università di Milano, Italy
Department of Entomology, North Carolina State University
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Abstract |
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Key Words: symbiosis molecular clock cockroach fossil termite Blattabacterium
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Introduction |
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Cockroaches that have had their endosymbionts removed via antibiotic treatment grow poorly, die easily, and lay fewer and less viable eggs (Guthrie and Tindall 1968). The interactions between host and symbiont have not yet been fully established, but they appear to include the recycling of uric acid by the bacteria (Cochran 1985; Wren and Cochran 1987), presumably in return for accommodation and metabolic products provided by the host. This suggests that the relationship is one of mutualism (Douglas 1989). Such a relationship is expected to lead to cocladogenesis of host and symbiont, though horizontal transfer eventswhich can be mediated by parasitesmay lead to different phylogenetic topologies. Evidence has been found for phylogenetic congruence between Blattabacterium spp. and members of the genus Cryptocercus (Clark et al. 2001); however, a formal examination of whether cocladogenesis has occurred at deeper taxonomic levels of Dictyoptera has not yet been performed. Reasons for this have been (1) the absence of a suitable outgroup for rooting Blattabacterium spp. topologies; (2) the absence of a host phylogeny that included a variety of cockroaches, termites, and mantids; (3) the fact that Blattabacterium spp. from each of the five traditionally recognized lineages of cockroaches (McKittrick 1964) have not yet been examined.
The situation regarding suitable outgroups for Blattabacterium symbionts has improved with the discovery of CFB intracellular bacteria from ladybird beetles (Hurst et al. 1997). The 16S rDNA sequences of these endosymbionts share 92% identity with those from various Blattabacterium spp., compared with
80% in the previously known closest relatives (Bandi et al. 1995). With regard to dictyopteran phylogeny, two recent studies included members of each of the five cockroach families, as well as multiple termite and mantid representatives: that of Klass (1995), based on morphology, and that of Lo et al. (2000), based on molecular sequences. Both studies found evidence that mantids were the earliest branching lineage in the group, with termites sister to Cryptocercus spp. and nested within a paraphyletic cockroach grade. An alternative scenario is that cockroaches (including Cryptocercus) are monophyletic and a sister group to the mantids, with termites basal. This topology has found support from both morphological (Thorne and Carpenter 1992) and molecular (Kambhampati 1995) analyses. In addition, cockroach monophyly has been supported by a number of studies (Grandcolas 1996; Kambhampati 1996; Maekawa and Matsumoto 2000).
This recent accumulation of data provides an opportunity to examine whether the relationships among Blattabacterium spp. are congruent with those of their hosts. In this study, we performed the first outgroup-rooted, multifamily phylogeny of these bacteria and compared it to a phylogeny of host taxa based on a combined analysis of sequences from four genes. 16S rDNA sequences have been determined from Blattabacterium representatives of various cockroach families, including the Polyphagidaethe only cockroach family (sensu McKittrick 1964) that has not previously been examined.
Of particular interest with regard to dictyopteran phylogeny is the fossil record for these insects. Cockroach-like fossils are well known from late Carboniferous deposits (300 Myr old). The key characteristic setting these fossils apart from extant cockroaches is the presence of a long, external ovipositor. Cockroach fossils which lack an ovipositor and that fit into extant families appear in early Cretaceous deposits (
130 Myr old). It is during this period when mantid and termite fossils also appear for the first time. Some authors have asserted that modern cockroaches and those from late Carboniferous deposits are monophyletic, which would mean that mantids and termites also existed during the Carboniferous epoch (Balderson 1991; Kukalova-Peck 1991; Jarzembowski 1994). An alternative hypothesismore in line with the fossil recordis that the stem group of extant cockroaches, mantids, and termites was a roach-like insect with a reduced ovipositor (a characteristic of all dictyopterans) that radiated during the late Jurassic/early Cretaceous (Grimaldi 1997; Nalepa and Bandi 2000; Thorne, Grimaldi, and Krishna 2000). Divergence dates based on the rate of molecular evolution of endosymbiont genes (Moran et al. 1993) could provide evidence for or against the hypotheses mentioned above. We used data from cockroach and termite fossils to examine the rate of molecular evolution of the 16S rDNA gene in Blattabacterium spp., and we provide estimates of the splitting times between various lineages. Also, 16S rDNA rate evolution from endosymbionts of the CFB bacterial assemblage is of interest because it provides a comparison to previously determined fossil-based rate estimates, which, to our knowledge, have all been from the proteobacterial assemblage (Ochman, Elwyn, and Moran 1999).
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Materials and Methods |
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Tree topologies were estimated by MP using heuristic searches in PAUP*, treating gaps as a fifth base. Branch lengths were fitted using ML criteria. Support for internal nodes was estimated by bootstrap analysis (1,000 replicates, with 5 random addition replicates per bootstrap replicate) and by Bayesian inference using the program MrBAYES 2.01 (Huelsenbeck and Ronquist 2001). For Bayesian analyses, parameters for the selected model of substitution were estimated from the data, and a total of 12,000 trees were obtained (,
). The first 6,000 of these were considered as the "burn in" and discarded, and a 50% majority-rule consensus tree of the remaining trees was produced. Clock-like evolution in the 16S rDNA of the taxa examined was tested for using the option in Tree-Puzzle 5.0.
To test for congruence, a phylogeny for the hosts was estimated based on four genes that have been sequenced for various taxa (nuclear 18S rDNA, mitochondrial 12S rDNA, 16S rDNA, and cytochrome oxidase II; see Supplementary Material). The rDNA sequences were aligned based on secondary structure considerations as outlined previously (Kambhampati 1996; Lo et al. 2000; Thompson et al. 2000). Regions that could not be aligned unambiguously were discarded from analyses. To check that the four genes were suitable to be combined with one another, 50% majority-rule consensus trees were obtained via MP bootstrap analysis for each individual data set. Upon comparison of these trees, no conflicting clades were found, and thus combined analyses were performed using the methods described above for endosymbiont analyses. Taxa included were those which had at least three of the four genes available in GenBank. For two genera (Blattella and Blaberus), genes from closely related species within the one genus were combined. Four mantid species, for which only two genes were available, were also included. Two Orthopteran taxa were used as outgroups. Data unavailable for genes of some taxa were coded as missing.
Three statistical tests of phylogenetic congruence between Blattabacterium spp. and their hosts were performed. The first tested whether there was a greater than random correspondence between reconstructed nodes for host and symbiont. This was performed in Component Lite (R. Page, University of Glasgow, UK) using the "compare tree with" function, with 1,000 randomized trees and the four available tree-comparison metrics. The second test examined the null hypothesis that the endosymbionts have undergone cocladogenesis with their hosts. The most-parsimonious tree and its length were first calculated for each of the host and endosymbiont data sets. The MP tree of one data set was then forced onto the other data set, and the tree-lengths were calculated. The two tree-lengths calculated for a single data set were then compared using the Templeton (1983) test. Significantly different scores can be interpreted as rejection of the null hypothesis. The third test was similar to the second test, except that ML criteria were used. ML trees were estimated using the successive approximation method (Swofford et al. 1996), and the different scores for the one data set were compared using the Shimodaira and Hasegawa (1999) test. This test is similar to that used by Peek et al. (1998) and Clark et al. (2000).
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Results and Discussion |
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Figure 1 shows the most-parsimonious tree recovered from 16S rDNA analysis. The 50% majority-rule consensus tree obtained from Bayesian analysis was identical to figure 1, with the exception that one node was not resolved. Consistent support was found for the monophyly of Blattabacterium spp. from each of the cockroach families Cryptocercidae, Blattidae, Polyphagidae, and Blaberidae (for Blattellidae, only one sequence was available). A clade containing endosymbionts from the termite M. darwiniensis and from Cryptocercus spp. was well supported in both bootstrap (90% of replicates) and Bayesian (92% posterior probability) analyses. Sequences from Blaberidae and Blattellidae were found to cluster together, in agreement with results from several studies of cockroach relationships (McKittrick 1964; Klass 1995; Grandcolas 1996; Kambhampati 1996; Maekawa and Matsumoto 2000). The exclusion of the few gap positions present in the alignment did not alter the topologies recovered from MP or Bayesian analysis, and similar support values were found (data not shown).
Examining Congruence Between Symbiont and Host Topologies
Figure 2 shows a comparison of the phylogeny of a subset of endosymbionts with that of their hosts (for which molecular data are available). From the four host genes, 2,991 characters were analyzed, of which 1,067 were variable and 674 were parsimony informative. Significant phylogenetic structure was found for each of the individual data sets (data not shown), as well as the combined data set (;
). For ML analyses, the model of substitution chosen by Modeltest was
. We note that PAUP* does not permit the application of different models for individual data sets within a combined data set. Attempts with a program that allows for this (PAML; Yang 1997) proved computationally intractable.
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Testing for Clock-Like Behavior in Blattabacterium spp. 16S rDNA, and Estimating Its Rate of Evolution
The evidence for cocladogenesis and the presence of fossil data for termites and cockroaches prompted us to investigate the rate of evolution in the 16S rDNAs of Blattabacterium spp. If the sequences evolve in a roughly constant manner, the rate determined for a subset of sequences using fossil data can be used to date the divergences of other taxa. Clock-like evolution in the sequences used to generate figure 1 was tested for using the likelihood-ratiobased test available in Tree-Puzzle 5.0. The null hypothesis that the sequences have evolved in a clock-like manner was rejected at the 5% level (data not shown). However, it was apparent from the branch lengths in figure 1 that such rejection may have been caused by a minority of lineages that have undergone changes in rate. Indeed, when the lineages leading to Blaberidae/Blattellidae and C. relictus were removed from the analysis (5 of 17 taxa), the null hypothesis of clock-like evolution could not be rejected. The log likelihood score of the tree where branch lengths were free to vary was -3906.11, whereas that constraining them to be clocklike was -3914.22. These values were not significantly different at the 5% level (). We note that when taxa other than Blaberidae/Blattellidae and C. relictus were removed singly or in combination, the null hypothesis of clock-like evolution was consistently rejected. Thus we conclude that these two lineages are responsible for all significant among-lineage rate heterogeneity.
Because the Mastotermes/Cryptocercus spp. clade is supported by morphological (Klass 1995) and molecular data from the hosts (fig. 2; Lo et al. 2000), as well as endosymbiont molecular data (fig. 1), it is a good candidate for calibration of the 16S rDNA clock. Though no Cryptocercus fossils are known, the earliest known fossils for termites are from the early Cretaceous (Thorne, Grimaldi, and Krishna 2000). We can therefore assume that the latest possible time of splitting between the lineages leading to Cryptocercus spp. and M. darwiniensis was 130 MYA. The average ML distance between the 16S rDNAs from M. darwiniensis and Cryptocercus species (excluding C. relictus) is
(
;
). Thus, with the assumption of a molecular clock, 0.0225 substitutions per site have occurred in each of these lineages since their last common ancestor. Based on a minimum divergence date of 130 myr, we can estimate that the maximum rate of evolution is 0.0087 substitutions per site per 50 myr. This rate is within the range of 0.0076 to 0.0232 substitutions per site per 50 myr previously reported for the 16S rDNA sequences of aphid symbionts (Moran et al. 1993), despite being from a phylogenetically diverse bacterial group. Using the rate calculated in our study, the latest time of divergence between the termite/Cryptocercus lineage and Blattidae (for which endosymbiont 16S rDNA ML distances average
[
]) is 144 MYA. The appearance of Blattidae fossils in the early Cretaceous is in agreement with this value (Labandeira 1994). Similar results are found when examining distances between various taxa and Polyphagidae members, for which early Cretaceous fossils are also known (Labandeira 1994).
The branch lengths in figure 1 suggest that 16S rDNAs from the lineage comprising Blattellidae/Blaberidae have evolved in a slower manner compared with those from the other lineages. Because of these different rates, it is difficult to estimate the period when the last common ancestor to all families existed. Based on the short branch separating Blaberidae/Blattellidae from the other taxa (fig. 1), it could be speculated that this stem group was present at a time similar (but slightly earlier) to the value of 140145 MYA calculated for the split between termites/Cryptocercus, Blattidae, and Polyphagidae based on ML distances. Other genes that have evolved in a clock-like manner for all taxa will need to be examined to test this idea.
The above evolutionary rate calculation and its corroboration from other fossil taxa relies on the assumption that the fossil record adequately reflects the first appearance of the lineages examined. In support of this assumption is the combination of three observations: (1) The oldest termite, Polyphagidae, Blattidae, and Blattellidae fossils are all from the early Cretaceous. (2) Roach-like fossils are found from the Carboniferous up to the late Jurassic, and contain ovipositors that gradually decrease in length over time (Thorne, Grimaldi, and Krishna 2000). If ovipositorless roaches resembling modern taxa also existed during these periods, it is difficult to explain why their fossils are not found as well. (3) The branch lengths that separate the main modern lineages are short in both the endosymbiont and host phylogenetic trees (indicative of a relatively rapid radiation from a common ancestor).
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Conclusions |
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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Diethard Tautz, Associate Editor
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