* Department of Microbiology and Parasitology, and Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia
Institute of Conservation Biology, Department of Biology, Wollongong University, Wollongong, Australia
Correspondence: E-mail: r.shao{at}mailbox.uq.edu.au.
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Abstract |
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Key Words: hemipteroid Psocoptera mitochondrial clock molecular evolution rate heterogeneity
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Introduction |
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The hemipteroid assemblage has four orders of insects: Hemiptera (bugs, cicadas, whiteflies, aphids, etc.), Phthiraptera (lice), Psocoptera (psocids, book lice, and bark lice), and Thysanoptera (thrips). The rate of mt gene rearrangement varies substantially in the hemipteroid assemblage. Compared with the inferred ancestral arrangement of mt genes of insects (Shao, Campbell, and Barker 2001), no rearrangements have been found in the Hemip-tera (Shao et al. 2001). In contrast, 24 genes have rearranged in the plague thrips, Thrips imaginis (Thysanoptera [Shao and Barker 2003]), and 31 genes have rearranged in the wallaby louse, Heterodoxus macropus (Phthiraptera [Shao, Campbell, and Barker 2001]). The nucleotide sequence of a 1.8-kb fragment of the mt genome of a lepidopsocid (Psocoptera) indicated a level of rearrangement in this species intermediate to that of the Hemiptera and the plague thrips (Shao et al. 2001). In the present study, we sequenced the entire mt genome of this lepidopsocid and found that eight genes have rearranged in this genome.
Other lineages of insects also vary in the rate of mt gene rearrangement. The three species of Coleoptera sequenced entirely have the ancestral gene arrangement of insects (see table 1). Five of the seven species of Diptera sequenced entirely also have the ancestral arrangement, whereas two species of Diptera, Anopheles quadrimaculatus and A. gambiae, have two tRNA genes rearranged. Four species of Lepidoptera and the locust, Locusta migratoria (Orthoptera), have a single tRNA gene rearranged, and the honeybee, Apis mellifera (Hymenoptera), has eight tRNA genes rearranged. Thus, the Insecta has lineages with low rates, intermediate rates, and high rates of mt gene rearrangement.
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Materials and Methods |
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There are six pairwise comparisons among the four hemipteroid insects and 45 pairwise comparisons among the 20 insects (table 2)species of insects that have identical gene arrangements and are in the same taxonomic order were treated as a single lineage in the relative-rate tests. These species are (1) the five dipterans that have the ancestral gene arrangement of insects, (2) the two dipterans (Anopheles spp.) that have two genes rearranged, (3) the four lepidopterans that have one gene rearranged, and (4) the three coleopterans that have the ancestral gene arrangement of insects. We compared Ks (number of synonymous substitutions per synonymous site), Ka (number of nonsynonymous substitutions per nonsynonymous site), and B4 (number of synomymous transversions per fourfold degenerate site).
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Tests that assess the relative rate of gene rearrangement between two taxa have not been developed. So we devised such a test, using the relative-rate test of nucleotide substitution as a framework (Li and Bousquet 1992). We assessed the relative rate of gene rearrangement between pairs of taxa by reference to a third taxon (an outgroup). We calculated the BP between the outgroup and each of the ingroup taxa. The relative rate of gene rearrangement (RRTGR) was then calculated as the difference between these two BP values, or
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Test for Significance of the Correlation Between Rates of Gene Rearrangement and Nucleotide Substitution
We tested the correlation between the rate of gene rearrangement and the rate of nucleotide substitution for statistical significance using parametric (Pearson) and nonparametric (Spearman Rank) tests. We only used the pairs of species that were independent from one another on the phylogenetic tree (table 3 and fig. 2); this is because the inclusion of nonindependent pairs of species may overestimate the degree of correlation by using the same data more than once (Felsenstein 1985). Among the 20 insects whose mt genomes have been sequenced entirely, there were nine pairs of insects that were suitable for this test. We sampled each branch in the phylogeny of these insects only once and purposefully sampled all branches along which rearrangements have occurred (fig. 2, dashed branches). Tribolium castaneum (Coleoptera) was used as an outgroup in four of the nine comparisons because it has the ancestral mt gene arrangement of insects and has a lower rate of nucleotide substitution compared with the other two species of Coleoptera sequenced entirely (data not shown). These properties made it an ideal outgroup since relative rate tests are most accurate when the mean distance of each taxon to the outgroup is smallest (Robinson et al. 1998).
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Results and Discussion |
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Relative Rates of Nucleotide Substitution and Gene Rearrangement
RRTree tests indicated that Ks (number of synonymous substitutions per synonymous site) was saturated in 53 of the 60 pairwise comparisons in tables 2 and 3. In the other seven comparisons in which Ks was not saturated, there was no significant variation in Ks between the two taxa compared. Saturation plots of corrected and uncorrected pairwise distances of first, second, and third codon positions also indicated extreme saturation at the third codon position, where corrected distances were frequently greater than 1.0 (data not shown). There was no significant variation in B4 (number of synonymous transversions per fourfold degenerate site) in any of the 60 pairwise comparisons. However, there was significant variation in Ka (number of nonsynonymous substitutions per nonsynonymous site) in 49 of the 60 comparisons. Together, these observations indicate that neither Ks nor B4 is a good proxy or measure for nucleotide substitution rate when examining highly divergent organisms (e.g., in our case, species across nine taxonomic orders of insects). Therefore, only the rates of non-synonymous substitutions were shown in tables 2 and 3.
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To test whether the correlation between the rate of gene rearrangement and the rate of nucleotide substitution in these hemipteroid insects is a more general phenomenon, we tested the relative rates of mt gene rearrangement and mt nucleotide substitution among all 20 species of insects whose mt genomes have been sequenced entirely. The crustacean Daphnia pulex was the outgroup (table 2). The hierarchy of rate of nucleotide substitution of these 20 insects was Heterodoxus macropus Thrips imaginis > Apis mellifera > lepidopsocid > Triatoma dimidiata > Locusta migratoria
Lepidoptera
Coleoptera
Diptera (Anopheles spp. excluded) > Anopheles spp. ("
" indicates no significant difference). This hierarchy is similar but not identical to the hierarchy of the rate of mt gene rearrangement of these insects: Heterodoxus macropus (35 breakpoints) > Thrips imaginis (30 breakpoints) > Apis mellifera (16 breakpoints) > lepidopsocid (15 breakpoints) > Locusta migratoria (3 breakpoints) = Lepidoptera (3 breakpoints) > Anopheles spp. (1 breakpoint) > Triatoma dimidiata (0 breakpoints) = Coleoptera (0 breakpoints) = Diptera (0 breakpoints). The major difference between the two hierarchies is due to the positions of the Anopheles spp. and Triatoma dimidiata (underlined in the hierarchy of the rate of mt gene rearrangement above). In 28 of the 45 comparisons where the two lineages differed by more than five breakpoints, the more rearranged lineage always had a statistically significantly higher rate of nucleotide substitution than the less rearranged lineage. The average difference for these 28 comparisons was 0.1680. In the 17 other comparisons where the two lineages differed by five or less breakpoints, the more rearranged lineage did not always have a higher rate of nucleotide substitution than the less rearranged lineage. The average difference for these 17 comparisons was 0.0280. In some cases, the less rearranged lineage had a significantly higher rate of nucleotide substitution than the more rearranged lineage (the comparisons with negative values of BP(1,3) BP(2,3) in table 2).
Correlation Between Rate of Gene Rearrangement and Rate of Nucleotide Substitution
Table 2 shows that the lineages with high rates of gene rearrangement generally have high rates of nucleotide substitution. However, table 2 does not show if the correlation between the rate of gene rearrangement and the rate of nucleotide substitution is statistically significant or not. We tested the statistical significance of the correlation between the rate of gene rearrangement and the rate of nucleotide substitution by comparing these rates in nine phylogenetically independent pairs of species (table 3).The rate of gene rearrangement and the rate of nucleotide substitution were positively correlated and the degree of correlation was statistically significant: Pearson R2= 0.73, P = 0.01 and Spearman Rank Rs = 0.67, P < 0.05 (fig. 3).
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Little is known about the factors that affect the rate of gene rearrangement in mt genomes of animals. The rate of mt gene rearrangement was thought to be universally low in animals since the arrangement of mt genes is identical in most lineages of vertebrates (Boore 1999). However, a low rate of mt gene rearrangement seems to be peculiar to vertebrates. The rate of mt gene rearrangement varies substantially in the Insecta (see above) and in other lineages of arthropods; for example, the Crustacea (Tigriopus japonicus [Machida et al. 2002]), the ticks (Chelicerata [Black and Roehrdanz 1998; Campbell and Barker 1998, 1999]) and the millipedes (Myriapoda [Lavrov, Boore, and Brown 2002]). Indeed, most groups of invertebrates have lineages with many rearrangements and lineages with few rearrangements; for example, Mollusca, Nematoda, Platyhelminthes, Cnidaria, Echinodermata, and Brachiopoda (Boore 1999).
So, why is the rate of mt gene rearrangement high in some lineages of animals but low in others? We propose that the statistically significant correlation between the rate of mt nucleotide substitution and the rate of mt gene rearrangement in insects may point to an answer to this question. Our hypothesis is that a high rate of nucleotide substitution may lead to a high rate of mt gene rearrangement. There are three steps in our hypothesis. First, one or more of the four biochemical and life history traits listed above evolves in a lineage; that is, there may be a decrease in efficiency of DNA repair, increase in metabolic rate, decrease in generation time, and/or decrease in body size. Second, these changes cause the rate of nucleotide substitution to increase in mt genomes. Third, a high rate of nucleotide substitution leads to an increase in mutation at the sites of initiation and termination of the mt genome replication and causes errors during replication of mt genomes. These errors then cause gene rearrangements through duplication and deletion mechanism (Boore 2000; Lavrov, Boore, and Brown 2002) and lead to an increase in the rate of mt gene rearrangement. It is possible that other mechanisms, in addition to the duplication and deletion mechanism, may also cause a high rate of gene rearrangement from a high rate of nucleotide substitution. For example, a high rate of endogenous DNA damage, due to a high rate of nucleotide substitution, may result in a high rate of double-strand breaks that may cause illegitimate recombination (Boore 2000) or intramitochondrial recombination (Dowton and Campbell 2001). Alternatively, the organisms with high substitution rates may have relaxed selection; thus mtDNA rearrangement are tolerated more than in species with low substitution rates.
Of course, other as yet undiscovered factors may also affect the rate of gene rearrangement in the mt genomes of animals. For example, we speculate that life histories and ecologies that tend to cause genetic founder effects, such as the life history of lice, psocopterans, and thrips, may increase the chance that rearranged mt genomes survive and become fixed in populations. Further studies on hemipteroid insects on such factors should be instructive.
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Acknowledgements |
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Footnotes |
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