Nature © Macmillan Publishers Ltd 1998 8 Washington, DC, 1996). 8. Panasik, N., Jr, Eberhardt, E. S., Edison, A. S., Powell, D. R. & Raines, R. T. Int. J. Pept. Protein Res. 44, 262–269 (1994). 9. Eberhardt, E. S., Panasik, N., Jr & Raines, R. T. J. Am. Chem. Soc. 118, 12261–12266 (1996). 10. Semisotnov, G. V. et al. Biopolymers 31, 119–128 (1991). 11. Shah, N. K., Ramshaw, J. A. M., Kirkpatrick, A., Shah, C. & Brodsky, B. Biochemistry 35, 10262–10268 (1996). 12. Werkmeister, J. A. & Ramshaw, J. A. M. (eds) Collagen Biomaterials (Elsevier Science, Barking, Essex, 1992). inferred from the terminal 39 nucleotides of the L(UUR) gene. These nucleotides are in frame with the actual start site of the ND1 gene and match 5/13 of the corresponding residues of the ND1 gene in Drosophila. The most parsimonious explanation for the gene arrangement data (Fig. 1) is that a single translocation of the L(UUR) gene occurred in a common lineage that led, after it split from the other lineages shown, to crustaceans and insects. This signature of common evolutionary history persists in the mtDNAs of these groups today. If myriapods and insects were sister groups, either this tRNA translocation would need to have occurred twice identically in the lineages leading to insects and crustaceans, or it would need to have reverted to its prim- itive state in the myriapods. Each of these explanations would require an identical complex process. Furthermore, this gene has translocated to a position remote from the original one. So the process is not a simple exchange of positions between neighbouring genes, nor does it involve genes adjacent to a large non-coding region, either of which might increase the frequency of gene rearrangement. Another argument against the change being convergent is the infrequency of rearrangements among arthropod mito- chondrial DNAs. Complete arrangements of all 37 mitochondrial genes have been deter- mined for six arthropod genera: one che- licerate (Limulus 2 ); one crustacean (Artemia 3 ); and four insect (Drosophila 4 , Locusta 5 , Anopheles 6 , Apis 7 ). The Drosophila arrangement differs from that of Limulus only in the location of L(UUR), from that of Artemia only in the location of the tRNA gene block I–Q, and from those of Locusta, Anopheles, and Apis by one, two and eight tRNA translocations, respectively. There is no evidence of ‘hot spots’ for tRNA gene translocations in these genera, although the sample size is small. Rearrangements of the 37 genes typical of metazoan mtDNA appear to be unique, rare events, unlikely to be duplicated by convergence, stable once they have occurred, and easily recognized because of the homology of mitochondrial genes across the Metazoa 1 . Our phylogenetic interpretation requires no convergence in any taxon for which data are available (more than 200 taxa representing 8 phyla). We believe that this synapomorphy scientific correspondence NATURE | VOL 392 | 16 APRIL 1998 667 The evolutionary relationships among the four major lineages of arthropods remain controversial, despite extensive study. We report here a derived gene rearrangement common to insects and crustaceans but absent in the other arthropod groups. This finding strongly supports an insect– crustacean evolutionary lineage that is sepa- rate from those leading to myriapods and chelicerates. The four major arthropod groups are Chelicerata (such as scorpions and horse- shoe crabs), Crustacea (such as crabs and brine shrimp), Myriapoda (such as cen- tipedes and millipedes), and Insecta (such as flies and beetles). Much of arthropod evolution remains contentious but, until recently, there has been general agreement that myriapods are the closest relatives of insects, forming a group known as the Ate- locerata. However, several recent morphological and molecular comparisons suggest that crustaceans, rather than myriapods, are the sister group to insects. If this is the case, some characteristics shared by insects and myriapods (such as a tracheal system for respiration, Malpighian tubules for excre- tion, and unbranched legs) then become examples of convergent evolution, perhaps as adaptations to life on land. Similar fea- tures are found among terrestrial chelicer- ates, where their occurrence is already viewed as convergent. In a previous study 1 , we reported that insects and crustaceans share a derived loca- tion for the gene encoding mitochondrial leucine transfer RNA, designated L(UUR), as compared with its primitive location in a chelicerate, an onychophoran, and several non-arthropod metazoans. However, in that study we were unable to associate a myria- pod (Thyropygus) with either group. Now, further mitochondrial DNA (mtDNA) sequence for Thyropygus and for three other myriapod species allows us to make that association. These four myriapods share the gene arrangement LrRNA- L(CUN)–L(UUR)–ND1, which is almost certainly primitive 1 . Our earlier misinterpre- tation of the Thyropygus sequence was due to a similarity of the amino-acid sequence Gene translocation links insects and crustaceans 4 Crustaceans (4 orders) 134 Insects (10 orders) 2 Chelicerates (2 orders) 4 Myriapods (2 orders) Onychophoran Tardigrade Pogonophoran, 3 annelids (S–A rather than A–S in Platynereis) Echiuran Gastropod, polyplacophoran molluscs Arthropoda COI L(UUR) COII LrRNA L(CUN) L(UUR) L(UUR) L(UUR) L(CUN) L(CUN) L(CUN) L(CUN) L(UUR) L(UUR) L(UUR) L(UUR) L(CUN) L(CUN) NDI NDI NDI LrRNA LrRNA LrRNA LrRNA LrRNA NDI NDI NDI NDI I A S A S Figure 1 Relative location of the L(UUR) gene for 153 taxa. The primitive location is identified in many non- arthropods and is retained in the mitochondrial genomes of the chelicerates and myriapods. An insect–crustacean clade is identified by the shared translocation of L(UUR) to the position between COI and COII . For 49 of these insect taxa, only the gene arrangement L(UUR)–COII has actually been determined 8 . For the chelicerates, myriapods, onychophoran, tardigrade, and echiuran, COI–COII are directly adjacent without any intervening tRNA genes; other taxa have an unrelated tRNA here. The three crustaceans and ten insects share the gene arrangement LrRNA–L(CUN)–ND1. For all arthropods for which the relative locations of these two gene blocks have been determined 2–7 , they are separated by more than 2.5 kilobases and are encoded on opposite DNA strands. Data are from published sources for three crustaceans (Homarus 1 , Daphnia 1 , Artemia 3 ), the insects 8 (additional citations available from JLB), a chelicerate (Limulus 2 ), onychophoran (Euperipatoides 1 ), annelid (Lumbricus 9 ), gastropod (Plicopurpura 1 ) and polyplacophoran (Katharina 10 ). Sequences determined here are for a remipede crustacean (Speleonectes), chelicerate (Pandinus), four myriapods (Thyropygus, Lithobius, Spirostrephon, Narceus), tardigrade (Thulinia), pogonophoran (Galathe- olinum), two annelids (Helobdella, Platynereis) and echiuran (Urechis). Sequences were determined from DNA fragments amplified using the polymerase chain reaction with primers made to conserved gene regions. Mitochondrial DNA typically contains 37 genes, only a subset of which is shown here (gene abbre- viations are as published 9 ).