Department of Evolutionary Biology, University of Siena, Siena, Italy
Department of Environmental Science, Policy and Management, Division of Insect Biology, University of California at Berkeley
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Abstract |
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Introduction |
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The relatively small size and the ease with which this molecule can be handled make it a good model for studying various aspects of genomic structure and function, such as the evolution of the genetic code, the use of different synonymous codons, transcription and RNA maturation, and disequilibrium in base frequencies. Several genes encoded in the mitochondrion (particularly cox1, cox2, and rrnS) are widely used in molecular phylogenetics (Simon et al. 1994
; Caterino, Cho, and Sperling 2000
). More recently, there has been growing attention paid to the order with which the genes appear along the molecule (gene order) as a very informative genetic marker with which to resolve phylogenetic relationships between distantly related taxa (Boore, Lavrov, and Brown 1998
; Blanchette, Kunisawa, and Sankoff 1999
; Boore and Brown 2000
; Kurabayashi and Ueshima 2000
; Scouras and Smith 2001
).
So far, the mtDNA of eight hexapodan species has been sequenced completely. These species include the dipterans Cochliomiyia hominivorax (Lessinger et al. 2000)
, Drosophila yakuba (Clary and Wolstenholme 1985
), Drosophila melanogaster (Lewis, Farr, and Kaguni 1995
), Anopheles gambiae (Beard, Hamm, and Collins 1993
), Anopheles quadrimaculatus (Mitchell, Cockburn, and Seawright 1993
), and Ceratitis capitata (Spanos et al. 2000)
, the hymenopteran Apis mellifera (Crozier and Crozier 1993
), and the orthopteran Locusta migratoria (Flook, Rowell, and Gellissen 1995
). Despite the potential usefulness of mitochondrial gene order in inferring phylogenetic relationships among Arthropoda (Staton, Daehler, and Brown 1997
; Boore, Lavrov, and Brown 1998
), only seven nonhexapodan arthropods have been sequenced completely: Ixodes hexagonus and Rhipicephalus sanguineus (Black and Roehrdanz 1998
), Artemia franciscana (Valverde et al. 1994
), Daphnia pulex (Crease 1999
), Penaeus monodon (Wilson et al. 2000)
, Pagurus longicarpus (Hickerson and Cunningham 2000)
, and Limulus polyphemus (Lavrov, Boore, and Brown 2000)
. Unfortunately, also in the Hexapoda, the sampling is not uniform and is highly biased toward the more derived holometabolous orders (six sequences from the Diptera, one from the Hymenoptera), with only one sequence available from the Hemimetabola (L. migratoria). No information on mtDNA gene arrangement is available for basal wingless taxa, in spite of the recognized need for a more homogeneous sampling among hexapods in order to use mtDNA information to interpret their evolution (Flook, Rowell, and Gellissen 1995
).
Tetrodontophora bielanensis (Onychiuridae) is a representative of the apterygotan order Collembola. Members of this order, whose earliest fossil evidence dates back to the Devonian (Whalley and Jarzembowski 1981
), are believed to be among the first hexapods that appeared on the earth, and hence they represent a crucial taxon for the understanding of the evolution of the whole hexapodan assemblage. However, the phylogeny of basal orders constitutes one of the most controversial issues in hexapodan evolution (Kristensen 1997
; Bitsch and Bitsch 2000
; Carapelli et al. 2000
).
In this paper, we report the complete sequence of the mitochondrial genome of T. bielanensis and the consequent arrangement of its genes in the molecule.
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Materials and Methods |
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Protein-coding genes (PCGs) were located using the Profile alignment mode of Clustal X (Thompson et al. 1997
), aligning some representative arthropodan sequences plus Lumbricus terrestris for each gene first and then using this alignment as a "probe" on the sequence of T. bielanensis. This procedure was repeated for each PCG. The corresponding open reading frame (ORF) was identified, and the extremities of the sequence were checked by eye to locate the exact beginning and termination. Exact locations of tRNAs were found at the boundaries of the genes by searching for sequences capable of forming the cloverleaf structure typical of these molecules. Clustal X (multiple-alignment mode with Gonnet weight matrix series; gap separation distance = 8) was then used to align the amino acid sequences obtained by conceptual translation of the DNA sequences using the D. melanogaster (de Brujin 1983
) mitochondrial code, as implemented in GeneDoc (written by K. B. Nicholas and H. B. Nicholas). Phylogenetic analyses were performed on the complete amino acid sequences of all PCGs. The Lumbricus sequence was set as an outgroup in all analyses. Positions experiencing gaps were excluded. Maximum-parsimony (MP) and maximum-likelihood (ML) approaches were used for phylogenetic analyses. Parsimony-based analyses were performed assigning equal weights to all sites as implemented in PAUP* (Swofford 1998
; branch-and-bound search, simple addition sequence, TBR branch swapping); likelihood analyses were performed with the utility ProtML of the MOLPHY package, version 2.3b3 (Adachi and Hasegawa 1996
), on amino acid sequences using the mtREV24 model (Adachi and Hasegawa 1996
).
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Results and Discussion |
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Thirteen PCGs (cox1cox3, nad1nad6, 4L, cob, atp6, and atp8) and the two genes coding for the large (rrnL) and small (rrnS) ribosomal RNA subunits were unambiguously identified. A long (955) noncoding region is present between trnQ and trnI, which is presumed to be homologous to the A+T-rich region of other hexapods by positional homology and shared features. A closer analysis of this region evidenced the presence of tandemly repeated sequences, the features of which will be described below. The positions and orientations of PCGs, ribosomal RNAs, tRNAs, and the A+T-rich region differ from those of Drosophila and Daphnia, which have been proposed to be the ancestral arrangement for the crustacean-hexapod clade (Crease 1999
), for the translocation of the trnQ and the trnS(ucn). Genes and tRNAs are closely assembled one after the other, leaving a total of only 105 nt (excluding the A+T-rich region) in unassigned intergenic spacers, ranging in size from 1 nt (between atp6 and atp8 and between nad6 and cob) to 33 nt (between trnS(ucn) and trnM). This value is similar to that of Locusta (100 bp) and well in the range of other insects, where Apis has the largest (620 bp) and the two Anopheles species have the smallest (4347 bp) values. The extremities of some genes overlap by a few nucleotides. Six overlaps, ranging in size from 1 nt (between trnK and trnD and between trnE and trnF) to 5 nt (between trnY and cox1), were inferred, giving a total of 22 overlapping nucleotides. A detailed diagram of the genome organization of Tetrodontophora is shown in figure 2 .
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Complete termination codons TAG and TAA were found in two and six PCGs, respectively. The remaining five genes are supposed to end with a single T, which may be completed into a proper TAA stop codon by RNA polyadenylation (Ojala, Montoya, and Attardi 1981
): four of them are exactly adjacent to tRNAs, and one (nad4L) is exactly adjacent to the beginning of another gene (nad4). Thus, it is highly probable that during mRNA processing the U is exposed at the end of an mRNA molecule and polyadenylated, forming the complete UAA termination signal.
Transfer RNAs
As described above, transfer RNAs were located by searching intergenic spacers for sequences capable of forming the typical cloverleaf structure. The whole set of 22 tRNAs typical of metazoan mitochondrial genomes was found, and secondary structures were drawn for each one (fig. 3
). Anticodons were identified in the anticodon arms and were identical to those in Drosophila. The acceptor stem is always composed of seven paired nucleotides plus the discriminator nucleotide at its 3' end. In 10 tRNAs, this nucleotide was found in regions of sequence overlap, and in 5 of these cases, adjacent sequences overlapped for only this nucleotide. This suggests that, at least in some tRNAs, this discriminator nucleotide may be added after the splicing of the primary transcript (Yokobori and Pääbo 1997
) in such a way that no alternative splicing would be needed in order to obtain the complete tRNA. The DHU arm is composed by a stem of 35 bp enclosing an unpaired loop of 38 nt. The anticodon arm consistently showed a 5-bp stem and seven unpaired nucleotides comprising the anticodon. The T
C stem was 35 bp long, and its apical loop was 28 nt. Between anticodon and T
C arms, an unpaired region of 25 nt was present. Mismatches were found in many tRNAs. Even considering AC and GU as possible pairings and setting the trnC aside, 12 mismatches were found in the acceptor stem, 1 in the DHU arm, 9 in the anticodon stem, and none in the T
C stem.
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There is growing evidence that RNA editing could also be involved in the maturation of tRNAs, as demonstrated in land snails by Yokobori and Pääbo (1995)
and suggested also for opistobranchs (Kurabayashi and Ueshima 2000)
. However, the application of the mechanism outlined by Yokobori and Pääbo (1995)
to our sequences could correct only a few of the mismatches, namely those having U's or C's in the 5' end of the acceptor arm and no A's in the 3' end. This could be the case for trnC and trnR, and, for one base only, trnM and trnS(agy). In general, T. bielanensis tRNAs appear to be subject to structural constraints to a lesser extent on both sequence and size.
Ribosomal RNAs
As in all other mitochondrial genomes sequenced so far, two genes for ribosomal RNAs were present in T. bielanensis, one for the small and one for the large ribosomal subunit. These were located between trnQ and trnV and between trnV and trnL(cun), respectively (fig. 1
). Their exact boundaries could not be identified by unambiguously analyzing the sequence alone, due to the high sequence variability at the extremities of rRNAs, but their lengths were confirmed by the reconstruction of their secondary structures. A detailed analysis of the structure and variability of rRNAs in T. bielanensis and other hexapods will be given elsewhere (unpublished data).
A+T-Rich Region: Structure and Heteroplasmy
The A+T-rich region in T. bielanensis mtDNA, comprising the region between trnI and trnQ, was 955 nt long. This length was well in the range of other arthropods, which show a remarkable variability, from 263 nt for Rhipicephalus to 4,601 nt for D. melanogaster (Zhang and Hewitt 1997
). The structure of this sequence, outlined in figure 4a,
was remarkable in that a close array of repeats could be found at its 5' end. At the beginning of the region, 1 nt apart from the beginning of trnQ, three tandem repeats extended for 550 nt, accounting for more than a half of the whole A+T region. The first two repeats were identical in sequence, 193 nt long, and were called R-repeats. The third repeat was slightly different in sequence, 164 nt long, and was called a D-repeat. The D-repeat was nearly identical to the R-repeats in 87 nt at the beginning and 46 nt at the end, while the middle part was completely different. An additional sequence resembling the repeats, called a pseudorepeat, could be found directly after the end of the D-repeat but was only 38 nt long and will not be considered further. We searched the entire A+T-rich region for sequences capable of forming stable secondary structures using the program RNAdraw (Matzura and Wennborg 1996
). A close array of secondary structures was predicted by the algorithm, but due to the uncertainty of extending to DNA results that are based on RNA energetic values, only four structures were identified as possible candidates. These structures are presented in figure 5
: the first one (fig. 5a
) can be found in each repeat (both R and D), while the second (fig. 5b
) encompasses the junction between any repeats. These structures are thus repeated three and two times, respectively, in the shorter haplotype and more times as the number of repeats increases (see below). Two additional inferred secondary structures are present in single copy: one at the junction between the D-repeat and the nonrepeated sequence (fig. 5c
), and one in the unrepeated sequence (fig. 5d
). Neither of these, nor any of the other possible stem-forming sequences, is flanked by the conserved sequences identified in many hexapods by Zhang, Szymura, and Hewitt (1995)
; thus, it is impossible to assess the homology between any of the structures of T. bielanensis and those identified by these authors.
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Gene Order and its Evolutionary Significance
The order and orientation of genes and tRNAs along the mitochondrial genome of Tetrodontophora (fig. 2
) differs from that of Drosophila in the translocation of two tRNAs. trnQ, which is located in Drosophila between trnI and trnM, is found in Tetrodontophora between rrnS and the A+T-rich region. trnS(ucn) (between ND1 and cob in Drosophila) is translocated between trnM and trnI in Tetrodontophora. In both cases, the orientation does not change.
Both translocations are unprecedented among arthropods and thus are to be considered autapomorphic features of some restricted taxa comprising Tetrodontophora. The remaining genes have the same order as in Drosophila, which has been proposed to carry the ancestral hexapodan arrangement (Crease 1999
). Preliminary unpublished data in another collembolan species from the same suborder (Arthropleona), but a different family, show that trnS(ucn) is located, as in Drosophila, between ND1 and cob. Therefore, the autapomorphic feature represented by the translocation of this tRNA could be limited to a single family.
All gene translocations found so far in insect mitochondrial genomes are useless for reconstructing phylogenetic relationships among Hexapoda, either because they are autapomorphic features of restricted groups (Crozier and Crozier 1993
; Mitchell, Cockburn, and Seawright 1993
) or because they are clearly examples of homoplasy (Flook, Rowell, and Gellissen 1995
). However, sampling is still very scarce and biased toward a few holometabolous orders, and additional taxa might provide further insights into the mechanisms of evolution of mitochondrial gene order. Nevertheless, the finding of different gene arrangements restricted to groups of lower taxonomic levels, such as within the order Diptera (Clary and Wolstenholme 1985
; Beard, Hamm, and Collins 1993
), the Collembolan suborder Arthropleona, the acarine family Ixodidae (Black and Roehrdanz 1998
; Campbell and Barker 1999
), and the crustacean order Decapoda (Hickerson and Cunningham 2000)
, suggests that gene order data may be more informative at lower taxonomic ranks than previously hypothesized (Boore, Lavrov, and Brown 1998
; Boore and Brown 2000
).
Phylogenetic Analysis
Phylogenetic analyses were performed on the concatenated amino acid sequences of all PCGs of T. bielanensis in conjunction with those from 15 other arthropodan sequences whose complete mtDNA sequences were available in GenBank. These sequences were as follows: D. yakuba (X03240; Clary and Wolstenholme 1985
), D. melanogaster (U37541; Lewis, Farr, and Kaguni 1995
), A. gambiae (L20934; Beard, Hamm, and Collins 1993
), A. quadrimaculatus (L04272; Mitchell, Cockburn, and Seawright 1993
), C. capitata (AJ242872; Spanos et al. 2000)
, C. hominivorax (AF260826; Lessinger et al. 2000)
, A. mellifera (L06178; Crozier and Crozier 1993
), L. migratoria (X80245; Flook, Rowell, and Gellissen 1995
), P. monodon (AF217843; Wilson et al. 2000)
, A. franciscana (X69067; Valverde et al. 1994
), D. pulex (AF117817; Crease 1999
), P. longicarpus (AF150756; Hickerson and Cunningham 2000)
, L. polyphemus (AF216203; Lavrov, Boore, and Brown 2000)
, and I. hexagonus and R. sanguineus (AF081828 and AF081829; Black and Roehrdanz 1998
). The sequence of the anellid L. terrestris (U24570; Boore and Brown 1995
) was used as an outgroup in all phylogenetic analyses. Preliminary parsimony and likelihood analyses showed that Apis always clustered with the two ticks. This abnormal result was correlated with the extreme nucleotide composition bias of the Apis sequence, which was thus removed from the subsequent analyses (as in Wilson et al. 2000)
.
The removal of Apis left a 16-taxon data set (3,329 characters after removal of gap-experiencing sites, of which 3,414 were variable and 1,919 were parsimony-informative), which was analyzed using MP and ML. The topologies of the ML and MP trees (shown in fig. 6 ) were very similar.
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A second important conclusion is that crustaceans appear paraphyletic in our reconstruction. The two classes of Crustacea represented in this analysis, Malacostraca and Branchiopoda, do not form a monophyletic group, with the former being more closely related to the winged insects than to the Branchiopoda. Not surprisingly, this result confirms that obtained with a similar data set by Wilson et al. (2000)
, and the inclusion of P. longicarpus reinforces their conclusion. The paraphyly of the Crustacea has been suggested by other molecular and morphological studies (see Wilson et al. [2000]
for a thorough discussion), and our results support the view that insects may have originated from a group of crustaceans.
The remaining relationships resulting from our analysis are fairly congruent with the classical interpretation of arthropodan phylogeny, including the monophyly of the winged insects and that of the two ticks. Within the winged insects, the basal position of L. migratoria is confirmed, while the dipteran sequences are clustered according to the Brachycera/Nematocera split. However, our MP and ML analyses are in conflict with the neighbor-joining analysis of Lessinger et al. (2000)
regarding the relative position of C. hominivorax and C. capitata with respect to Drosophila. The tree generated by our analysis, in fact, places C. hominivorax as the sister taxon of Drosophila, therefore rejecting the Acalyptrate taxon (Drosophila + Ceratitis).
Low or no support was found for the deeper nodes, including the monophyly of the chelicerates. Two nodes, Artemia salina + D. pulex and the node uniting all crustacean and hexapodan sequences, were well supported in the likelihood analysis but poorly supported (65% and 54%, respectively) in the parsimony analysis, suggesting that at least some of the relationships are sensitive to the methodological approach of phylogenetic reconstruction.
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Acknowledgements |
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Footnotes |
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1 Abbreviations: ML, maximum likelihood; MP, maximum parsimony; PCG, protein-coding gene.
2 Keywords: complete mitochondrial genome
Tetrodontophora bielanensis,
Collembola
heteroplasmy
tRNA translocations
arthropod phylogeny
3 Address for correspondence and reprints: Francesco Nardi, Department of Evolutionary Biology, University of Siena, via P.A. Mattioli 4, 53100 Siena, Italy. E-mail: nardi{at}unisi.it
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