Novel Rearrangements of Arthropod Mitochondrial DNA Detected with Long-PCR: Applications to Arthropod Phylogeny and Evolution

R. L. Roehrdanz2, M. E. Degrugillier and W. C. Black, IV

*Biosciences Research Laboratory, Red River Valley Agricultural Research Center, Agricultural Research Service, United States Department of Agriculture, Fargo;
{dagger}Department of Microbiology, Colorado State University


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Rearrangements of mitochondrial DNA gene order have been suggested as a tool for defining the pattern of evolutionary divergence in arthropod taxa. We have employed a combination of highly conserved insect-based polymerase chain reaction (PCR) primers with long-PCR to survey 14 noninsect arthropods for mitochondrial gene rearrangements. The size of the amplified fragments was used to order the primer containing genes. Five chelicerates exhibit amplicons that are consistent with the presumptive ancestral arthropod mtDNA gene order. These five species comprise two soft ticks, two prostriate hard ticks, and an opilionid. Six other chelicerates, all metastriate hard ticks, have a different arrangement that was originally discovered by this procedure and has been previously detailed in a complete mtDNA sequence. Three new arthropod mtDNA gene arrangements are described here. They were discovered in a terrestrial crustacean (Isopoda) and two myriapods (Chilopoda, centipede; Diplopoda, millipede). These rearrangements include major realignments of some of the large coding regions and two possible new positions for the tRNAMet (M) gene in arthropods. The long-PCR approach affords an opportunity to quickly screen divergent taxa for major rearrangements. Taxa exhibiting rearrangements can be targeted for DNA sequencing of gene boundaries to establish the details of the mtDNA organization.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Major arthropod lines have long been recognized (insects, crustaceans, chelicerates, diplopods, chilopods, etc.), but the phylogenetic relationships among these groups remain controversial. Within the arthropods the historical view based on morphological characters has favored an arrangement that combined the myriapods and insects as the atelocerates. Crustaceans were considered the sister group to the atelocerates, and the chelicerates seemed furthest removed. The application of molecular techniques has challenged parts of the traditional arrangement. Nucleotide and amino acid sequences from mitochondrial genomes have been used extensively for closely related taxa, and the arrangement of the genes on the mtDNA has been informative at different phylogenetic levels. Complete mtDNA sequences or substantial portions of the gene organization are known for at least 10 insects, six of which are Diptera (Clary and Wolstenholme 1985Citation ; Beard, Hamm, and Collins 1993Citation ; Crozier and Crozier 1993Citation ; Mitchell, Cockburn, and Seawright 1993Citation ; Flook, Rowell, and Gellissen 1995Citation ; Lewis, Farr, and Kaguni 1995Citation ; Lessinger et al. 2000Citation ; Spanos et al. 2000Citation ; Nardi et al. 2001Citation ; Shao, Campbell, and Barker 2001Citation ), five crustaceans (Valverde et al. 1994Citation ; Van Raay and Crease 1994Citation ; Garcia-Machado et al. 1996Citation ; Crease 1999Citation ; Garcia-Machado et al. 1999Citation ; Hickerson and Cunningham 2000Citation ; Wilson et al. 2000Citation ), and five chelicerates (Staton, Daehler, and Brown 1997Citation ; Black and Roehrdanz 1998Citation ; Campbell and Barker 1999Citation ; Lavrov, Boore, and Brown 2000Citation ). Sequence data from key regions have been obtained from additional species, and the database continues to expand (reviewed by Boore 1999Citation ). Much of this evidence favors the insects and crustaceans as sister taxa. Questions have been raised, but not resolved, about the monophyly of both the myriapods and crustaceans.

The order of mitochondrial genes, especially the positions of tRNA genes, has been used to support a phylogenetic tree of the major arthropod groups. A unique location of tRNALeu(UUR)(L2) between the COI and COII genes is shared by the crustaceans and the insects and supports their sister taxa status. The ancestral position for tRNA(L2), between 16SrRNA and ND1, is found in chelicerates and myriapods and also some annelids, mollusks, and chordates (Boore et al. 1995Citation ; Boore, Lavrov, and Brown 1998Citation ; Boore 1999Citation ). If rearrangements in mtDNAs are relatively rare in evolutionary history, these events can be useful synapomorphies. Convergent or parallel translocations and reversions in nonrelated lineages would be expected to be uncommon. Because more taxa are examined, new rearrangements are being discovered that have the potential to add detail to our view of arthropod phylogeny. Major rearrangements of mitochondrial genes have been observed in a subgroup of chelicerates, the metastriate ticks (Black and Roehrdanz 1998Citation ; Campbell and Barker 1998, 1999Citation ), in a crustacean, the hermit crab Pagurus (Hickerson and Cunningham 2000Citation ), and in several hemipteroid insects, including the wallaby louse, thrips, and some psocopterans (Shao, Campbell, and Barker 2001; Shao et al. 2001Citation ). Lesser rearrangements, involving the reshuffling of tRNA genes, have been documented in honey bees (Crozier and Crozier 1993Citation ), other hymenopterans (Dowton 1999Citation ; Dowton and Austin 1999Citation ), mosquitoes (Beard, Hamm, and Collins 1993Citation ; Mitchell, Cockburn, and Seawright 1993Citation ), collembolans (Nardi et al. 2001Citation ), and an anostracan crustacean (Valverde et al. 1994Citation ).

We report the finding of three new mtDNA arrangements in the arthropods. The approach used long-polymerase chain reaction (PCR) and conserved insect-based primers in a manner described previously (Roehrdanz 1995Citation ; Roehrdanz and Degrugillier 1998Citation ). The rearrangements are detected as amplicons of altered size when compared with a standard insect mtDNA sequence. The validity of using this approach to detect major rearrangements is supported by sequence data that are consistent with the PCR results (Black and Roehrdanz 1998Citation ). This technique provides an avenue for sampling a large number of taxa to find those with novel gene orders that would be prime candidates for a more detailed examination.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Arthropods used in this study and abbreviations used in the text are listed in table 1 . The lowest taxa that we could identify with certainty are indicated. The oplionid, centipede, millipede, and isopod specimens were all collected in Fargo, ND, and voucher specimens exist for the latter two. The tick specimens originated in various locations. Collection details can be found in Norris, Klompen, and Black (1999)Citation .


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Table 1 Arthropod Taxa Used for mtDNA Comparisons

 
The high salt procedure of Cheung, Hubert, and Landry (1993)Citation was used to liberate total DNA from fresh or frozen centipede, millipede, isopod, and oplionid. Tick DNA was prepared by a CTAB protocol (Black, Klompen, and Keirans 1997Citation ) or by CsCl centrifugation. The latter procedure enriches for mtDNA and was used for four of the metastriate ticks. The amplicons of the same size were obtained from all the metastriate ticks, regardless of DNA preparation protocol. PCR amplification used the Gene-Amp XL PCR Kit (Perkin-Elmer, Roche Molecular Systems, Branchburg, NJ) which relies on rTth polymerase. The manufacturer's recommendations for the amounts of tricine buffer, enzyme, magnesium acetate, and nucleotides were followed. Primers were added to a final concentration of 0.5–1.0 µM. Reaction volumes were 100 µl. "Hot start" at 80°C was employed, and the reactions were overlaid with mineral oil. PCR reactions were carried out in a Perkin-Elmer Thermal Cycler (model 480), as described by Roehrdanz (1995)Citation and Roehrdanz and Degrugillier (1998)Citation . The two-temperature cycling parameters were: 93°C 1 min; 15 cycles of 93°C 1 min, 60°C 12 min; 25 cycles of 93°C 1 min, 60°C 12 min with 15 s auto extend; 72°C 7 min; 5°C hold. Successful amplification was determined by electrophoresing 5–10 µl of the PCR reaction on 1% agarose gels (1 x TBE) and visualizing with ethidium bromide staining. Commercial molecular size markers were included on the agarose gels.

The following mitochondrial primers were used—MET (TM-N-193) 5'-TGGGGTATGAACCCAGTAGCTT-3'; C1 (C1-J-2195) 5'-TTGATTTTTTGGTCATCCAGAAGT-3'; C2 (C2-N-3662) 5'-CCACAAATTTCTGAACATTGACC-3'; C2R (C2-J-3684) 5'-GGTCAATGTTCAGAAATTTGTGG-3'; N4-87 (N4-N-8487) 5'-TCAGCTAATATAGCAGCTCC-3'; N4 (N4-J-8944) 5'-GGAGCTTCAACATGAGCTTT-3'; CB2H (CB-N-10920) 5'-TCCTCAAAATGATATTTGTCCTCA-3'; N1 (N1-N-12595) 5'-GTAGCATTTTTAACTTTATTAGAACG-3'; 16S (LR-N-12868) 5'-TTACATGATCTGAGTTCAAACC-3'; 16SR (LR-J-12883) 5'-CTCCGGTTTGAACTCAGATC-3'; 16S2 (LR-N-12945) 5'-GCGACCTCGATGTTGGATTAA-3'; 16SA (LR-J-13417) 5'-ATGTTTTTGATAAACAGGCG-3'; 12SF (SR-J-14241) 5'-CGGGCGATGTGTACATAATT-3'; 12S (SR-N-14588) 5'-AAACTAGGATTAGATACCCTATTAT-3'; 12SR (SR-J-14612) 5'-AGGGTATCTAATCCTAGTTT-3'; ISO (TI-N-24) 5'-ATTTACCCTATCAAGGTAA-3'; ISOR (TI-J-42) 5'-TTACCTTGATAGGGTAAAT-3'; N1B (N1-J-12248) 5'-AAGCTAATCTAACTTCATAAG-3'; TL2 (TL2-N-3014) 5'-TCCAATGCACTAATCTGCCATATTA-3'.

The primer positions are defined by their 3' end in the Drosophila yakuba (Dya) mtDNA sequence (Clary and Wolstenholme 1985Citation ). All the primers, except C2R which is a direct complement of C2, are described and referenced in the Appendix of Simon et al. (1994)Citation . Primers were synthesized by NBI (Plymouth, Minn.). Amplification products are described with the abbreviations of their flanking primers.

The mtDNA of Dya serves as a reference because many of the primers were derived from its sequence. Its arrangement of the large coding regions is the same as the arrangement of the presumed ancestral arthropod (also found in Limulus, Ixodes, Artemia, and Penaeus). Mitochondrial genomes with the same arrangement of major coding regions would be expected to return amplicons of approximately the same size as Dya. Because the primer combinations often span several genes, major rearrangements of the mtDNA relative to the standard insect gene order would show up as amplified products, with significant size differences relative to Dya. In addition to the size variation of the amplified products, two other outcomes of the amplification can be informative. Rearrangements that reorient primers so that they point in the same direction would fail to amplify a product. Conversely, primers that are repositioned so that they are now aligned in opposite directions can allow the amplification of a new product. The former can only be used after the fact to support the case for a rearrangement, but the latter can be diagnostic for a certain rearrangement.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
PCR fragments representative of the complete mitochondrial genome were obtained from the centipede, opilionid, and several ticks. Less than 100% of the mitochondrial genome was amplified from several other noninsect arthropods (table 2 ). Approximately 3/4 of the mt genome was amplified from the millipede. About 1/2 of the mtDNA from the isopod has been amplified, although no single PCR product is larger than about 5 kb.


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Table 2 PCR Fragments Obtained from Arthropod mtDNA with Pairs of Conserved Insect Primers

 
In table 2 the first two columns indicate the primer pair combinations and the distance between the primers in the Dya complete mtDNA sequence. Succeeding columns show the amplicon size for the various arthropods. Although the long-PCR regimen was used for all amplifications, not all of the resulting amplicons are long by common definition of the term. Figure 1 contains the maps of the mtDNAs that were based on the sizes of the amplified fragments in table 2 . The comparative maps of the major mitochondrial coding regions are aligned in figure 2 . The mtDNA maps are shown with the COI gene at the left end. This arrangement corresponds to the one used by Boore (1999)Citation and facilitates direct comparisons of arrangements here with those from other arthropods. Only the genes that contained successful primer pairs for each taxon are shown in figures 1 and 2 . At the top of figure 2 is the ancestral arrangement of the large coding regions found in all insects examined so far as well as some chelicerates and crustaceans (see review by Boore 1999Citation ). The complete array of large coding regions is also shown for the prostriate and metastriate ticks because the full mtDNA sequence is known for one species in each group. The tRNA genes are excluded, except where positions are directly determined in this work, tRNAMet(M), tRNALeu(UUR)(L2), and tRNAIle(I). The locations of those three tRNAs is also shown where a complete sequence is known.



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Fig. 1.—PCR amplicons and sizes used to determine the gene order and spacing. Major coding regions containing successful primers are shown in boxes. tRNAs with successful primers are shown above the boxes. The successful primer pairs and the sizes of the amplicons are from table 2

 


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Fig. 2.—Map of mitochondrial gene arrangements in various arthropods. All of the major coding regions are shown as boxes for the basic arthropod map at the top and for the prostriate and metastriate ticks (see Boore 1999Citation for references to complete sequences of chelicerate, crustacean, and insect mtDNAs). For the remainder of the species, major coding regions in boxes indicate the positions of those genes that contained primers that were successful for PCR amplification (see fig. 1 ). Underlined genes are in the opposite orientation relative to the ancestral arthropod. "Cont" is the control region or the A+T-rich region. "M," "L2", and "I" are the identified positions of the tRNAs Met, Leu(UUR), and Ile, respectively. In one insect, Apis (not shown), I remains between Cont and ND2; however, the relative positions of M and I are reversed. The position of Cytb in the isopod is tentative. The normally circular mtDNAs are linearized and aligned with the COI-COII gene pair at the left end. The major coding regions are shown as the same size for ease of alignment even though there may be some slight species differences in size. Similarly, not all the mtDNAs are of the same total length

 
The position of seven genes has been determined for the arachnid opilionid, and all of them are consistent with the basal arthropod organization (figs. 1E and 2 ). Similarly, the two soft tick and two prostriate hard tick species' gene positions obtained from the PCR match that of the basal arthropod (figs. 1F and 2 ), although fewer gene positions were actually fixed. No difference among these four species of ticks was observed (table 2 ).

The other arachnid group, comprising the metastriate hard ticks, is significantly different (figs. 1C and 2 ). The 12S-C2R, C2-N4, and MET-N4 amplicons are substantially smaller in each of the three genera of metastriate ticks than they are in Dya. In addition, the MET-12SR segment is much larger in the three tick genera. The inferred arrangement was confirmed in the previously published complete sequence of one of these six species, Rsa (Black and Roehrdanz 1998Citation ).

In the centipede, ND1 has moved to the opposite side of ND4, leaving Cytb adjacent to 16SrRNA (figs. 1A and 2 ). There is not enough room between ND4 and Cytb for ND6, so it must also have moved, although the data do not assign it a position. The tRNA(M) has been inserted between ND1 and ND4. None of the four genes (ND1, tRNA(M), ND4, Cytb) was inverted in the process. The space between COII and ND1 can accommodate the Atp8/6-COIII-ND3-ND5 gene block, and there is adequate space to the right of 16SrRNA for the 12SrRNA-control region-ND2 block, so there is no requirement for additional rearrangements; however, they cannot be ruled out.

Fewer amplified products were obtained from the millipede, but the results suggest another reshuffling of the arthropod mtDNA gene organization (figs. 1B and 2 ). The 16S-ND4 distance is similar to the basal arthropod, whereas the small MET-16SA segment is consistent with tRNA(M) being relocated to a new position between 12SrRNA and 16SrRNA. Cytb is now to the left of ND4 on the map. The ISO-C2 and ISOR-12SF amplicons indicate that tRNA(I) has a reverse orientation relative to most arthropods. The size of ISO-C2 suggests that ND2 is not located between tRNA(I) and COI.

The amplicons spanning the recovered portion of the isopod mtDNA total only about 8.6 kb and do not contain any large stretches devoid of defined genes (figs. 1D and 2 ). ND1 and possibly Cytb move close to COII. The 12SrRNA and 16SrRNA genes are inverted relative to ND1. As in the millipede, tRNA(M) is inserted between 12SrRNA and 16SrRNA. ND4 has moved closer to 16SrRNA and retains its orientation relative to that gene, which means it is inverted relative to the COI-ND1 block. No amplification products were recovered that span the long region from ND4 to COI.


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
A common arrangement for the major mitochondrial coding blocks is a characteristic of a diverse assemblage of arthropods, including two chelicerates, Limulus and Ixodes, the crustaceans, Artemia, Daphnia, and Penaeus, and all insects that have been studied (see Boore 1999Citation ). Within this basal arthropod pattern the smaller tRNA genes have undergone a number of repositionings. The crustacean Penaeus and the insect Drosophila differ from the presumed ancestral arrangement of Limulus and Ixodes by a single translocation of tRNA(L2), and a number of other tRNA rearrangements are known within insects and crustaceans (Beard, Hamm, and Collins 1993Citation ; Crozier and Crozier 1993Citation ; Mitchell, Cockburn, and Seawright 1993Citation ; Valverde et al. 1994Citation ; Staton, Daehler, and Brown 1997Citation ; Dowton 1999Citation ; Dowton and Austin 1999Citation ; Wilson et al. 2000Citation ; Nardi et al. 2001Citation ). Larger-scale rearrangements of gene order have been delineated in a group of chelicerates (Black and Roehrdanz 1998Citation ; Campbell and Barker 1998, 1999Citation ), a crustacean (Hickerson and Cunningham 2000Citation ), and some hemipteroid insects (Shao, Campbell, and Barker 2001; Shao et al. 2001Citation ). Our results demonstrate lability in the gene organization of myriapod, chelicerate, and crustacean mtDNAs.

The primers were chosen because they had been successfully used with insects. However, even in insects it was impossible to predict whether a particular primer pair would be successful from one insect species to the next (Roehrdanz and Degrugillier 1998Citation ). This situation is exacerbated when insect primers are applied to a broad range of arthropods.

The long-PCR method described here was used to obtain the material for sequencing tick mtDNA. The PCR results, from the beginning, indicated that ND4 and the 12S-16SrRNA genes had exchanged places relative to the rest of the mtDNA in the metastriate ticks (figs. 1C and 2 ). This did not match the known patterns of mtDNA organization in arthropods. The complete sequence of the metastriate tick, Rsa, mtDNA confirmed our interpretation based on long-PCR data and added some additional details, e.g., tRNA shuffles and duplicated control region (Black and Roehrdanz 1998Citation ).

The centipede included a translocation of tRNA(M) and translocations that result in new neighbors for at least four other genes, ND4, ND6, Cytb, and ND1. Although the position of ND6 was not fixed, there does not seem to be enough room for it to be retained between ND4 and Cytb. At minimum, it seems two more translocation events would be required to arrive at the final arrangement. As an example, translocation of ND1 to a position immediately to the left of ND4 would give the order ND1-ND4-ND6-Cytb-16SrRNA. A translocation of ND6 to another site would leave the ND1-ND4-Cytb-16SrRNA that was observed. Other scenarios that either move different genes or move them in a different sequence but arrive at the same final arrangement of genes also involve at least two additional translocations.

The millipede has two identified translocations of tRNAs. As in the centipede, tRNA(M) has moved. Here, it has inserted between the 12S- and 16SrRNAs. It retains the basal arthropod orientation with respect to the rRNA genes, which means that it would be transcribed from the opposite DNA strand as the 12S- and 16SrRNA genes. tRNA(I) has moved closer to COI and has switched strands in the process. A very similar position for tRNA(I) has been reported for the crustacean, Artemia franciscana, where both tRNA(I) and tRNA(Q) have moved to the opposite side of ND2, with tRNA(I) being inverted in the process (Valverde et al. 1994Citation ). The position of tRNA(I) relative to ND2 was not determined here. Also, in the millipede, Cytb has been relocated to the other side of ND4. Because the distance between ND4 and 16SrRNA is relatively unchanged, there must have been a corresponding movement of a similar sized block of DNA in the opposite direction. The double switch means that at least two translocations are required, combined with the two tRNA translocations; a minimum of four translocations must have occurred to arrive at the current gene arrangement.

The isopod rearrangement is both complicated and the least well defined. The small TL2-C1 amplicon indicates that this isopod has tRNA(L2) between COI and COII that has been found in nearly all insects and crustaceans. The isopod has tRNA(M) inserted between 12SrRNA and 16SrRNA, the same position observed for the millipede. An approximation of the isopod order for this half of the mt genome can be obtained with a translocation and two inversions, in addition to the tRNA(M) translocation. Some information from the other half of the mtDNA is needed for a better understanding of how this arrangement might have originated.

The results here indicate two new positions for tRNA(M). In most arthropods tRNA(M) is one of a trio of tRNAs found between the control region and ND2 with the arrangement, control-tRNA(I)-tRNA(Q)-tRNA(M)-ND2. Rare exceptions have occurred by the addition or subtraction of tRNAs other than tRNA(M), by the repositioning of tRNA(I)-tRNA(M)-ND2 between ND3 and ATP6 in the hermit crab, and the complete reassortment of these genes in the wallaby louse. In the centipede we have found tRNA(M) situated between ND1 and ND4, a position not yet found in any other arthropod. We also found tRNA(M) between the 12S and 16SrRNA genes in two diverse groups, the diplopod millipede and the crustacean isopod (fig. 3 ). This second arrangement raises the possibility of convergent evolution, that the same translocation occurred more than once in arthropod history. The orientation of tRNA(M) relative to 12S and 16SrRNA is the same for both events. However, the PCR results do not prove that the two translocations are identical. All other arthropods, except the wallaby louse, have the order 16SrRNA-tRNA(V)-12SrRNA. The tRNA(M) could have inserted on either side of tRNA(V), or tRNA(V) could have moved elsewhere when tRNA(M) arrived, making the two translocations nonidentical. Duplication of part or all of tRNA(M) or the reassignment of tRNAs also should be considered. Precedent for duplication exists with identification of two slightly different copies of tRNA(M) in the mollusc, Mytilus (Hoffmann, Boore, and Brown 1992Citation ). In a PCR assay, only the 20+ bases of the primer binding site need to be present to favor amplification of shorter products.



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Fig. 3.—Arthropod phylogenetic tree showing the positions of mtDNA rearrangements. Taxa with representatives in this work are underlined. Other taxa are from the literature (Boore 1999Citation ). The PCR data indicate that multiple events are required to account for the new mitochondrial arrangements. Not all genes are identified; therefore, the number of events detected is a minimum. General summaries of the events are shown in the boxes leading to the respective taxa. Rearrangements are identified in the boxes as a specific gene order, or the position change of a specific gene, or an estimated minimum number of events necessary to achieve the new gene order. Trn, translocation; Dp, duplication. Arrow and "?" indicate events that may have occurred before or after the divergence of two centipede families. Lithobius has the ancestral ND1-L2-L1-16S gene block, but little else is known about its mt genome organization. The myriapods are shown as a monophyletic group. In order to simplify the tree, the insects are shown as a single terminal branch, although their level of rearrangement varies from none to nearly total reorganization. Underlined Trn 16S-M-12S indicates a possible homoplasy involving the translocation of M in a diplopod and an isopod. Because most of the unusual rearrangements have been identified in a very few widely diverse species, the details of internal branching patterns are not addressed, and branch lengths have no significance

 
Boore, Lavrov, and Brown (1998)Citation have shown that two millipedes, Spirostrephon and Narceus, and the centipede, Lithobius, all carry the primitive arthropod gene sequence ND1-tRNA(L2)-tRNA(L1)-16SrRNA. In the Scutigera centipede used here, ND1 has moved to a new position. Scutigera and Lithobius belong to separate orders (Scutigeromorpha and Lithobiomorpha). Ribosomal DNA sequence data suggest that these two orders are on different main lines of chilopod phylogeny (Giribet et al. 1999Citation ). The ND1 aspect of the Scutigera rearrangement must have occurred after these two clades had diverged (fig. 3 ). This does not preclude other elements of the complex rearrangement from being more ancient. Our millipede results and the Spirostrephon and Narceus data are not directly comparable because different genes were located. The observation of novel rearrangements in both the chilopod and diplopod lineages (fig. 3 ) raises the possibility that the myriapods are not monophyletic.

The isopods are a diverse group that have marine, freshwater, and terrestrial forms. The example used here is one of the terrestrial isopods (Oniscidea). RFLP results from another terrestrial isopod species have indicated that the mt gene array exists in two physical forms. One form is linear, and the other is a circular dimer composed of two monomers linked in opposite directions (i.e., head-to-head and tail-to-tail). Both forms apparently coexist in cells (Raimond et al. 1999Citation ). These conformations could hinder PCR amplification across a long stretch of the isopod DNA and offer one explanation for our failure to amplify a large block of the isopod mtDNA. Additional isopods need to be examined to determine if our novel gene arrangement of isopod mtDNA is restricted to the oniscideans or is representative of a broader array of isopods or malacostracans.

Mitochondrial gene order may be more labile than previously believed which calls for caution about putting too much phylogenetic weight on any one arrangement. Figure 3 shows that the major rearrangements discovered to date are nearly all out at the tips of the tree branches. Some of this is an artifact of limited sampling. However, only the tRNA(L2) translocation is verifiably on an internal branch of the arthropod tree. If the tRNA(M) translocations of the diplopod and isopod prove to be identical, they would most likely represent convergent events in the tree. The new arrangements described here remain out at the ends of branches until proven otherwise. What is striking is that such divergent taxa as the prostriate tick, Ixodes, and the horseshoe crab, Limulus, are identical, whereas major rearrangements appear in the metastriate ticks. Similarly, the gene array for the malacostracan decapod shrimp, Penaeus, matches the base insect-crustacean pattern and is identical to an insect, Dya (Wilson et al. 2000Citation ), yet eight rearrangements are found in another decapod hermit crab, Pagurus (Hickerson and Cunningham 2000Citation ), seven completely different rearrangements have been found in a group of braconid wasps (Dowton 1999Citation ; Dowton and Austin 1999Citation ), and all the tRNA genes and nine protein coding genes have been reordered in the wallaby louse, Heterodoxus (Shao, Campbell, and Barker 2001; Shao et al. 2001Citation ). Heterodoxus also exhibits an autapomorphy as the first example of an insect-crustacean, where tRNA(L2) is no longer adjacent to either COI or COII. It appears as if rearrangements are much more common in recent evolutionary history than they were eons ago when the major arthropod lineages diverged. This could reflect recent increases in the rate of gene rearrangement events, such as translocations or partial duplications followed by partial deletions. Alternately, it could be testimony to the rapid divergence of basal arthropod lineages. Although the focus here is on the putative new mt gene arrays, it should not be forgotten that representatives from two previously unexamined chelicerate taxa, soft ticks and opiliones, yielded PCR products that are consistent with the ancestral arthropod order.

Long-PCR can be used to give a quick indication of gross mt gene rearrangements by surveying a large number of taxa. Detailed sequencing should then be performed on selected taxa to confirm the details of the inferred novel arrangements. Our sequencing of the tick mtDNAs is an example where this was done. In another instance we reported some PCR anomalies, including a larger than expected C1-C2 amplicon in Thrips sp. (Thysanoptera) that suggested a possible rearrangement in that insect (Roehrdanz and Degrugillier 1998Citation ). Shao et al. (2001)Citation sequenced this region from Thrips and found ND3 inserted between COI and COII, an arrangement consistent with our larger PCR product. These follow-ups are essential because there is always the possibility that a PCR reaction can amplify a nonspecific target or nuclear resident mt gene copies. Although the insect conserved primers used here are widely available and thus convenient to use, the efficiency of the PCR could probably be improved by designing new primers closer to the taxon of interest. Confirmed sequences could serve as the basis for new primers to detect the new array in related taxonomic groups without extensive sequencing. In addition to describing new gene arrangements, large amplicons have been used for nucleotide sequencing (Black and Roehrdanz 1998Citation ; Hickerson and Cunningham 2000Citation ) and are ideal for RFLP analysis at the lower taxonomic and population levels (Szalanski et al. 1999Citation ; Roehrdanz 2001Citation ).


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors recognize the assistance of G. Fauske (North Dakota State University Entomology Department) in identifying some of the specimens. We thank A. Cockburn and A. Szalanski for comments on earlier versions of the manuscript. We are especially grateful to J. Boore for his detailed critique that improved the presentation of the data and helped focus the scope of the manuscript. We are also grateful to technical assistance provided by C. Mueller and S. Degrugillier.


    Footnotes
 
Richard Thomas, Reviewing Editor

Keywords: mtDNA long-PCR gene order arthropod phylogeny Back

Address for correspondence and reprints: Richard L. Roehrdanz, Biosciences Research Laboratory, 1605 Albrecht Blvd., Fargo, North Dakota 58105. roehrdar{at}fargo.ars.usda.gov . Back


    References
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

    Beard C. B., D. M. Hamm, F. H. Collins, 1993 The mitochondrial genome of the mosquito Anopheles gambiae, DNA sequence, genome organization and comparisons with mitochondrial sequences of other insects Insect Mol. Biol 2:103-124[Medline]

    Black W. C. IV,, J. S. H. Klompen, J. E. Keirans, 1997 A phylogeny of hard and soft tick taxa based on mitochondrial 16S ribosomal DNA sequences Mol. Phylo. Evol 7:129-144[ISI][Medline]

    Black W. C. IV,, R. L. Roehrdanz, 1998 Mitochondrial gene order is not conserved in arthropods: prostriate and metastriate tick mitochondrial genomes Mol. Biol. Evol 15:1772-1785[Abstract/Free Full Text]

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Accepted for publication January 15, 2002.