This study combined complete 18S with partial 28S ribosomal RNA gene sequences ( approximately 2,000 nt in total) to investigate the relations of basal hexapods. Ten species of Protura, 12 of Diplura, and 10 of Collembola (representing all subgroups of these three clades) were sequenced, along with 5 true insects and 8 other arthropods, which served as out-groups. Trees were constructed with maximum parsimony, maximum likelihood, Bayesian analysis, and minimum-evolution analysis of LogDet-transformed distances. All methods yielded strong support for a clade of Protura plus Diplura, here named Nonoculata, and for monophyly of the Diplura. Parametric-bootstrapping analysis showed our data to be inconsistent with previous hypotheses (P < 0.01) that joined Protura with Collembola (Ellipura), that said Diplura are sister to true insects or are diphyletic, and that said Collembola are not hexapods. That is, our data are consistent with hexapod monophyly and Collembola grouped weakly with "Protura + Diplura" under most analytical conditions. As a caveat to the above conclusions, the sequences showed nonstationarity of nucleotide frequencies across taxa, so the CG-rich sequences of the diplurans and proturans may have grouped together artifactually; however, the fact that the LogDet method supported this group lessens this possibility. Within the basal hexapod groups, where nucleotide frequencies were stationary, traditional taxonomic subgroups generally were recovered: i.e., within Protura, the Eosentomata and Acerentomata (but Sinentomata was not monophyletic); within Collembola, the Arthropleona, Poduromorpha, and Entomobryomorpha (but Symphypleona was polyphyletic); and in Diplura, the most complete data set (> 2,100 nt) showed monophyly of Campodeoidea and of Japygoidea, and most methods united Projapygoidea with Japygoidea.

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BackgroundThe phylogenetic position of the Protura, traditionally considered the most basal hexapod group, is disputed because it has many unique morphological characters compared with other hexapods. Although mitochondrial genome information has been used extensively in phylogenetic studies, such information is not available for the Protura. This has impeded phylogenetic studies on this taxon, as well as the evolution of the arthropod mitochondrial genome.ResultsIn this study, the mitochondrial genome of Sinentomon erythranum was sequenced, as the first proturan species to be reported. The genome contains a number of special features that differ from those of other hexapods and arthropods. As a very small arthropod mitochondrial genome, its 14,491 nucleotides encode 37 typical mitochondrial genes. Compared with other metazoan mtDNA, it has the most biased nucleotide composition with T = 52.4%, an extreme and reversed AT-skew of -0.351 and a GC-skew of 0.350. Two tandemly repeated regions occur in the A+T-rich region, and both could form stable stem-loop structures. Eighteen of the 22 tRNAs are greatly reduced in size with truncated secondary structures. The gene order is novel among available arthropod mitochondrial genomes. Rearrangements have involved in not only small tRNA genes, but also PCGs (protein-coding genes) and ribosome RNA genes. A large block of genes has experienced inversion and another nearby block has been reshuffled, which can be explained by the tandem duplication and random loss model. The most remarkable finding is that trnL2(UUR) is not located between cox1 and cox2 as observed in most hexapod and crustacean groups, but is between rrnL and nad1 as in the ancestral arthropod ground pattern. The "cox1-cox2" pattern was further confirmed in three more representative proturan species. The phylogenetic analyses based on the amino acid sequences of 13 mitochondrial PCGs suggest S. erythranum failed to group with other hexapod groups.ConclusionsThe mitochondrial genome of S. erythranum shows many different features from other hexapod and arthropod mitochondrial genomes. It underwent highly divergent evolution. The "cox1-cox2" pattern probably represents the ancestral state for all proturan mitogenomes, and suggests a long evolutionary history for the Protura.