* Department of Biology, University of Michigan, Ann Arbor
DOE Joint Genome Institute and Lawrence Berkeley National Laboratory, Walnut Creek, California
Correspondence: E-mail: jlboore{at}lbl.gov.
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Abstract |
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Key Words: scaphopod bivalve mollusk mitochondria evolution genome
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Introduction |
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For some phyla of animals, mitochondrial gene arrangements seem seldom to have changed. For example, with few notable exceptions, those vertebrates studied have identical gene arrangements, as do most studied arthropods. (In the latter case, exceptions include the highly rearranged mtDNAs found for wallaby louse [Shao, Campbell, and Barker 2001], hermit crab [Hickerson and Cunningham 2000], and metastriate ticks [Black and Roehrdanz 1998; Campbell and Barker 1998].) Mollusks differ, with many gene rearrangements noted for the molluscan taxa listed above. High levels of rearrangement have also been noted for nematodes and brachiopods (see Boore 1999, 2002). Gene rearrangements have been shown to be very powerful characters for reconstructing evolutionary relationships (see Boore and Brown 1998), and the rapidity of rearrangement within a lineage determines the level at which rearrangements are likely to be phylogenetically informative.
The phylogenetic relationships among the different extant molluscan classes are not well established, and anatomical studies have proposed multiple alternatives to this issue. A common proposal is a gradist scenario where chitons (Polyplacophora) and solenogasters and caudofoveates (Aplacophora) are the basal lineages to a grade of valve-bearing taxa (Gastropoda, Bivalvia, Cephalopoda, Monoplacophora, and Scaphopoda), collectively known as the Conchifera (Salvini-Plawen 1985; Salvini-Plawen and Steiner 1996; Haszprunar 2000) (fig. 1A). Some propose that chitons and aplacophorans form a monophyletic clade rather than a grade (Scheltema 1993, 1996), and some view Conchifera as split into a cephalopod/gastropod clade and a scaphopod/bivalve clade with monoplacophorans as the basal conchiferan lineage (Runnegar and Pojeta 1974) (each as in fig. 1B). Recent evaluations of morphological and paleontological data (Waller 1998) as well as 18S rRNA sequences (Steiner and Dreyer 2002) alternatively conclude that Scaphopoda is the sister group to Cephalopoda (fig. 1C). The many ribosomal RNA sequences have so far only poorly resolved molluscan phylogeny, rendering some taxa paraphyletic (e.g., bivalves; Steiner and Müller 1996), making it difficult to assess whether the proposed anatomical interpretations are identifying true synapomorphies (Steiner and Müller 1996; Steiner and Hammer 2000). Consequently, we are in need of additional characters that can help address phylogenetic relationships among major molluscan lineages. As the mitochondrial genome database continues to grow, we will be able to incorporate both gene order and sequence data into this analysis. Here we present the first complete mitochondrial genome sequence for a member of the Scaphopoda (Graptacme eborea), a previously unsampled class, and the completed sequence of the F-type mitochondrial genome of the bivalve Mytilus edulis.
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Materials and Methods |
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A set of two oligonucleotide primers was designed facing "out" from this fragment, matching to positions that are separated by only 27 nts of the G. eborea cox1 sequence. These were used to amplify 14,465 nt, nearly the entire mtDNA, in a single reaction. This PCR used rTth-XL polymerase (Perkin-Elmer) with 1.3 mM MgOAc, and was otherwise performed according to supplier's instructions. Reaction volume was 100 µl and conditions were 94°C for 45 sec, followed by 37 cycles of: (94°C, 10 sec; 55°C, 20 sec; 65°C, 12 min, with an additional 15 sec per cycle after the 16th), then an incubation at 72°C for 12 min. An aliquot yielded a single band on a 1% agarose gel stained with ethidium bromide.
Approximately 2 µg of this product was digested separately with the restriction enzymes MboI and TaqI, each of which recognizes 4-nt sites. Several fragments were selected from each digest and gel purified as above, then they were ligated into the compatible BamHI and ClaI sites, respectively, of pBluescript plasmid (Stratagene), followed by DNA preparation and sequence determination as above. Additional oligonucleotide primers were designed for determining the sequence "out" from each of these cloned fragments. The 14,465 nt PCR product was passed three times through an Ultrafree Spin Column (30,000 NMWL; Millipore) to eliminate amplification primers and PCR reagents and then used directly as a template for sequencing reactions as above. Using a combination of oligonucleotides matching the ends of the amplified fragments with those matching internal sequences obtained from the cloned MboI and TaqI fragments greatly reduced the time required to "primer walk" through this fragment. All sequence was determined in both directions.
Completing the mtDNA Sequence of Mytilus edulis
Most (13.9 kb) of the F-type mtDNA sequence of M. edulis has been previously reported (Hoffmann, Boore, and Brown 1992). Although this was sufficient to determine the gene content and arrangement, it omitted the sequences of the central portions of many genes. To complete this, we designed oligonucleotide primers for PCR that match the ends of the previously reported sequences and used these to amplify the undetermined portions using DNA preparations of the appropriate M. edulis clones (Hoffmann, Boore, and Brown 1992) as templates. Each PCR reaction yielded a single band on a 1% agarose gel when visualized by ethidium bromide staining and UV irradiation. DNA was purified and the DNA sequence was determined as for G. eborea, using the amplifying or internal primers as necessary. All sequence was determined in both directions and was assembled with that previously reported into a complete mtDNA sequence.
Gene Annotation and Gene Order Comparison
Protein-encoding genes of each mtDNA were identified by sequence similarity of open reading frames to mitochondrial gene sequences of Katharina tunicata (Boore and Brown 1994a). Ribosomal RNA genes were identified by sequence similarity and potential secondary structures. As a class, tRNA genes were identified by their potential to form tRNA-like secondary structures; specific identifications were made according to anticodon sequence.
A search for shared gene arrangements was conducted against all mitochondrial sequence data available in GenBank that included sequence from three or more genes (3,376 entries). This search employed a PERL script that decomposed the query genome into all binary gene arrangements, searched for shared gene orders, and then reassembled any overlapping pairs for each comparison.
Phylogenetic Analysis of Protein Data
We included 27 taxa in the phylogenetic analysis (table 1), 15 of which are mollusks or other lophotrochozoans and 12 of which are metazoan outgroups (five non-lophotrochozoan protostomes, six deuterostomes, and one cnidarian). (One taxon, Inversidens japanensis, is represented by two sets of sequences, one F-type and one M-type.) We performed multiple sequence alignments for each protein using the pileup program in the GCG package. Each alignment was then refined by eye and subsequently combined into a concatenated data set. Because atp8 is missing in several of the taxa, it was excluded from all analyses. Regions of ambiguous alignment were also excluded; table 2 shows the regions corresponding to each gene in the concatenated alignment, the total number of positions per protein, and the number of amino acid sites included in the final analysis. Maximum parsimony (MP) reconstructions were conducted with PAUP*4.0b (Swofford 2001), with branch support estimated from 1,000 bootstrap pseudoreplicates. Quartet-puzzling (QP) was performed with Tree-Puzzle (Strimmer and Haeseler 1997) using both the mtREV24 and Blosum62 models with 100,000 quartet-puzzling steps, with a gamma correction and eight rate categories, and estimating amino acid frequencies from the data set. Bayesian reconstructions (MB) used MrBayes 3.0 (Huelsenbeck and Ronquist 2001). Exploratory Markov Chain Monte Carlo runs were performed starting with different amino acid substitution priors (i.e., mixed models, Poisson). Subsequently, we ran the heated MCMC chain for 1,000,000 generations, which was sampled every 100 updates using the models with higher posterior probabilities (mtREV and Blossum) from the mixed model prior. We discarded 1,000 cycles as burn-in before estimating joint posterior probabilities. We also analyzed each gene individually using MP and QP as above, and MB with mixed and mtRev amino acid models. The Nexus-formatted file of the alignment is available as online Supplementary Material.
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Results and Discussion |
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There have been extensive, unique rearrangements involving nearly every gene (fig. 5) of both of these mtDNAs. G. eborea and M. edulis mtDNAs have only a few gene boundaries in common with any other animal studied to date. An examination of all 3,376 entries in GenBank of sequences having three or more mitochondrial genes reveals that G. eborea shares the arrangement rrnL, trnM, rrnS with the Yesso scallop, Mizuhopecten yessoensis (GenBank accession AB052599), and nad1, P, nad6 with the squid, Loligo bleekeri. The first is an interesting potential synapomorphy that would exclude, among the sampled mollusks, only the polyplacophoran Katharina tunicata (Boore and Brown 1994a) and the cephalopod Loligo bleekeri (Sasuga et al. 1999); others have these genes in autapomorphic arrangements. It is interesting that the sampled gastropods have the arrangement trnM, rrnS, although rrnL is elsewhere. The inferred basal group, Polyplacophora, is represented by K. tunicata, which has an arrangement similar to the second case, nad1, -P, nad6; the other studied mollusks have further rearrangements of these genes. The same analysis of M. edulis mtDNA reveals that it shares the arrangement trnL1, trnL2, nad1 with the hemichordate Balanoglossus carnosus (Castresana et al. 1998) and with K. tunicata. Other than these arrangements, neither mtDNA shares more than a single gene boundary with any other animal.
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Mutational Bias Is Reflected in Codon Usage Patterns and Protein Amino Acid Composition
G. eborea mtDNA is 14,492 bp in length and is 74.1% A+T, very high even for a mitochondrial genome. Strand skew measures (Perna and Kocher 1995) for the distribution of GC pairs [(GC)/(G+C)] and TA pairs [(TA)/(T+A)] between the two strands are nearly zero (0.02 and +0.002, respectively).
M. edulis mtDNA is 16,740 bp in length and is 61.8% A+T. GC skew is +0.246 and TA skew is +0.110, indicating that the strand containing the genes is quite rich in G and T relative to the other strand. This bias is very evident in comparisons of synonymous codon usage pattern between the two genomes (table 4); for every case where an amino acid can be specified by any NNR codon, M. edulis has a much greater proportion of NNG:NNA relative to G. eborea.
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The amino acid leucine can be specified by six different codons (TTR and CTN) and the proteins of these two mtDNAs have a very similar number of leucines. As reflects the mutation bias, M. edulis has a much greater proportion of leucines specified by TTG and CTG codons, mainly at the expense of TTA codons. The amino acid serine can be specified by eight different codons (TCN and AGN); the proteins of the two mtDNAs also contain similar numbers of this amino acid and, again, the distribution reflects the mutation bias of M. edulis toward higher G and T. All AGN codons are used much more frequently in M. edulis, especially AGG; TCG usage is also elevated, all at the expense of TCT, TCC, and TCA codons.
This bias is also reflected in patterns of amino acid substitutions between these two mtDNAs. G. eborea and M. edulis mitochondrial proteins contain nearly identical numbers of nonpolar (A, V, L, I, P, M, F, W) (2,082 and 2,020, respectively) and polar (G, S, T, C, Y, N, Q) (1,170 and 1,219, respectively) amino acids. For nonpolar amino acids, M. edulis proteins use many more alanines (GCN) and valines (GTN) at the expense of isoleucine (ATY), methionine (ATR), and phenylalanine (TTY). For polar amino acids, M. edulis proteins contain more glycine (GGN) at the expense of asparagine (AAY). Presumably, the bias toward G and T in the gene-containing strand of M. edulis has resulted in amino acid replacements within the tolerance of physio-chemical similarity.
Unassigned DNA
G. eborea mtDNA is very uncommon for lacking any large noncoding regions, as are usually inferred to contain the origin(s) of replication and transcription control signals. The largest noncoding region is only 24 nt between trnK and trnF. Next in size are the 19 nt gap between cox3 and trnG and the 18 nt between trnG and trnQ. Noncoding DNA of M. edulis mtDNA has been analyzed and described earlier (Hoffmann, Boore, and Brown 1992). There is no obvious conservation of either nucleotide identities or potential secondary structures between the mollusks' noncoding regions. Whatever regulatory elements may be present are apparently short, dispersed, and/or rapidly changing.
Phylogenetic Analysis
Figure 6A presents a 70% majority rule consensus tree of MP bootstrap analysis for the taxa outlined in table 1, which has a topology congruent with those from quartet puzzling (QP) and some of the Bayesian analysis. These analyses support the monophyly of the lophophorates, annelids, and brachiopods. Relationships among the major molluscan lineages, however, remain unresolved, as are those among mollusks, brachiopods, and annelids, despite using this relatively large data set of 2,420 confidently aligned amino acid positions. In contrast, relationships among the major groups of deuterostomes and of arthropods are well resolved and conforming to expectation from other analyses, bolstering the view that the relationships among the lophotrochozoan groups are especially difficult to resolve.
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However, this data set of concatenated protein sequences does give consistent results for support of many metazoan clades regardless of the type of analysis performed (table 5). In contrast, analyses using each individual gene recovered only the deuterostome and protostome nodes with high levels of confidence (>70%) in six cases and not all methods were consistent. The arthropod clade was recovered by only three individual genes and by only one or two methods in the best of cases. The Lophotrochozoa clade was recovered only by Nad2 and only in the case of using the mixed model prior analysis.
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Acknowledgements |
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Footnotes |
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Richard Thomas, Associate Editor
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