Department of Biology, University of Michigan
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Abstract |
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Introduction |
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Recent studies using morphological characters (e.g., Eernisse, Albert, and Anderson 1992
), molecular sequences (e.g., Ghiselin 1988
; Lake 1990
; Garcia-Machado et al. 1999
), and fossil evidence (i.e., the halkieriids; see Morris and Peel 1995
) have reinvigorated an alternative view, that mollusks and annelids are the most closely related pair. The primary character uniting this group (the Eutrochozoa; Ghiselin 1988
) is the shared presence of a trochophore larval form in some species of each of these phyla, whereas such larvae are unknown among arthropods. Deducing the pattern of evolution among these three protostome phyla hinges largely on the subjective interpretation of whether overt body segmentation or a trochophore larval form is the more reliable phylogenetic character (see Eernisse, Albert, and Anderson 1992
).
The phylum Annelida is traditionally divided into three classes: Oligochaeta (e.g., earthworms), Hirudinida (leeches), and Polychaeta (marine annelids). There is strong evidence for uniting the first two as the most closely related pair (the Clitellata; see Rouse and Fauchald 1995
and references therein). The third class, Polychaeta, is the most diverse and speciose, and some have concluded that polychaetes are a paraphyletic assemblage (e.g., McHugh 1997
; Kojima 1998
).
Another phylum, the Pogonophora ("beard worms"), are also vermiform animals with a trochophore larva. Pogonophorans live in thin-walled tubes anchored in the oceans sediment, often at great depths. Opinions about both the phylogenetic placement and the taxonomic level of pogonophorans have differed widely, but most now regard them as a protostome phylum related to the Annelida (see Rouse and Fauchald 1995
and references therein). However, some molecular sequence comparisons (McHugh 1997
) and more recent morphological comparisons (Rouse and Fauchald 1997
) have found support for their inclusion as a family within the Annelida. The work presented here supports this latter view, that the Pogonophora are not an independent phylum and should most properly revert to the name Siboglinidae Caullery, 1914, as a family within Annelida in accordance with the proposal by Rouse and Fauchald (1997)
.
Our proposal of the relationships among annelids, mollusks, arthropods, and pogonophorans is based on the comparisons of the sequences and gene arrangements for homologous segments constituting about 50% of the mitochondrial genomes of the polychaete Platynereis dumerii, the hirudinid Helobdella robusta, the pogonophoran Galathealinum brachiosum, the oligochaete Lumbricus terrestris (Boore and Brown 1995
), and homologous portions of several published mollusk, arthropod, and chordate species (for a list and summary descriptions of studied mitochondrial genomes, see Boore 1999
and links at http://biology.lsa.umich.edu/~jboore).
Metazoan mitochondrial genomes are usually unicircular DNA molecules of about 16 kb that encode the same set of 37 genes (for 2 rRNAs [rns, rnl], 22 tRNAs [trnX, with anticodon shown when more than one tRNA specifies the same amino acid], and 13 proteins [cox13, cob, atp6, atp8, nad16, nad4L]). (For historical reasons, these genes are sometimes named differently in animal mitochondrial genomes; see Boore 1999
for a table of synonymous gene names.) This set of 37 genes can potentially be rearranged in an enormous number of combinations, and the large number of different arrangements found among (and occasionally within) metazoan phyla suggest that this character is relatively unconstrained. Major rearrangements of genes, here defined as translocations and/or inversions of one or more multigene tracts, appear to be infrequent on a geological timescale, although minor rearrangements, such as exchanges of position or polarity between neighboring tRNA genes, are encountered with greater frequency. Therefore, with the possible exclusion of some minor rearrangements, identical, convergent rearrangements in independent lineages are highly unlikely, and arrangements promise to be a reliable character to use for determining very ancient relationships, such as those that exist between major taxonomic categories (e.g., phyla, classes) (see Boore and Brown 1998
). Indeed, comparisons of mitochondrial gene arrangements have proven especially informative in several recent phylogenetic studies (Smith et al. 1993
; Boore et al. 1995
; Boore, Lavrov, and Brown 1998
).
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Materials and MethodsMolecular Analysis |
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Initially, three small fragments were amplified by PCR from each of Platynereis, Helobdella, and Galathealinum mtDNA using the following oligonucleotide pairs: (1) for a 710-nt fragment of cox1, LCO1490 (GGT CAA CAA ATC ATA AAG ATA TTG G) and HCO2198 (TAA ACT TCA GGG TGA CCA AAA AAT CA) (Folmer et al. 1994
); (2) for a 540-nt fragment of cox3, COIIIF (TGG TGG CGA GAT GTK KTN CGN GA) and COIIIR (ACW ACG TCK ACG AAG TGT CAR TAT CA); and (3) for a 450-nt fragment of cob, CytbF (GGW TAY GTW YTW CCW TGR GGW CAR AT) and CytbR (GCR TAW GCR AAW ARR AAR TAY CAY TCW GG) (see fig. 1
for primer placement). Reactions used Taq polymerase with supplied buffer (Fisher Scientific or Qiagen); Mg++ ion concentration and cycling conditions were optimized as necessary. Each reaction produced a single band when visualized under UV light following ethidium bromide staining on a 1% agarose gel. PCR reaction products were purified by three serial passages through an Ultrafree (30,000 NMWL) spin column (Millipore). This purified DNA (50300 ng) was used in a dye-terminator cycle sequencing reaction according to manufacturers (Perkin-Elmer) recommendations. Unincorporated nucleotides were removed by ethanol precipitation, and the purified product was analyzed on an ABI 377 automated DNA sequencer.
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The corresponding fragment was very difficult to amplify in Platynereis, with the reactions generally yielding multibanded products, perhaps because of the presence of signaling elements within the large noncoding region (see Shadel and Clayton 1997
). This region was amplified in shorter, overlapping fragments by using oligonucleotides designed to sequences conserved among the other annelid species (WormCOIF-3' [TAC TAC GTA GTA GCA CAC TTT CAC TA], WormCOIR-3' [TAR TCT GAG TAT CGT CGD GGT ATT CC], and WormCOIIR [GCT CCG CAA ATT TCT GAA CAT TGT CC]) or specific to sequence obtained (PlatCOIF [CCG AAA CCT AAA CAC TGC GTT CTT TGA TCC TGC], PlatCOIIF [GTG TAC TAG TAT CGG CTG CTG ACG], and PlatCOIIIR [GCA CTC TAA ATG GGT TGA TAG GGG TC]). Each of these amplifications that did not include the large noncoding region gave single-banded products in good quantity with minimum optimization efforts; however, the primer pair flanking this region (PlatCOIIF and PlatCOIIIR) continued to produce a multibanded product. One of these bands was of significantly greater quantity than the others and corresponded in size to the length of sequence obtained by using sequencing primers internal to the amplifying primers. This is evidence that this band corresponds to the actual mtDNA sequence, along with the presence within it of the expected genes. Although it is possible that the multiple bands were legitimate products representing multiple states of the mtDNA genome, the supernumerary bands were neither consistent in appearance among various attempts nor of regular size variation as expected of tandem repeat sequences that have been identified in the large noncoding regions of some mtDNAs.
All long-PCR reactions used rTth XL polymerase (Perkin-Elmer) with supplied buffer. Reactions were optimized for Mg++ concentration and cycling conditions as required. Each of these amplification products was purified and sequenced as above with additional oligonucleotides obtained as necessary (Gibco-BRL) for primer walking through each fragment. All nucleotides were determined on both strands except for a few short regions that were less than 200 nt in length, within 400 nt of the sequencing primer, and without any hint of ambiguity on the sequenced strand.
Sequence Analysis
Sequences were produced and assembled using the ABI suite of programs (e.g., Sequencing Analysis, Sequence Navigator, Autoassembler). Subsequent manipulations used MacVector 6.5 and GCG (Oxford Molecular Group).
Amino acid sequences were inferred for all protein-encoding genes determined for Galathealinum, Helobdella, and Platynereis, along with the homologous genes of L. terrestris (Annelida: Oligochaeta) (Boore and Brown 1995
), Katharina tunicata (Mollusca: Polyplacophora) (Boore and Brown 1994a, 1994b
), Artemia fransiscana (Arthropoda: Crustacea) (Valverde et al. 1994
), Drosophila yakuba (Arthropoda: Hexapoda) (Clary and Wolstenholme 1985
), Cyprinus carpio (Chordata) (Chang, Huang, and Lo 1994
), and Squalus acanthias (Chordata) (Rasmussen and Arnason 1999
) using the genetic code for Drosophila or vertebrate mtDNA, as appropriate. All proteins were inferred to initiate with formyl-methionine regardless of the DNA sequence of the designated start codon (Smith and Marcker 1968
). Each protein-encoding gene and ribosomal RNA gene was easily identified by comparison with homologs in Lumbricus mtDNA. Transfer RNA genes were identified generically by their potential secondary structures and specifically by anticodon sequence.
Phylogenetic Analysis of Inferred Protein Sequences
Sequences were aligned using CLUSTAL W, as implemented in MacVector 6.5 (Oxford Molecular Group); the BLOSSUM matrix was used to weight shared amino acids, with gap and extension penalties of 5 and 1. Nad6 could not be aligned with confidence and so was not used in the analyses. Gap placement is sometimes ambiguous near the ends of each gene alignment due to occasional variation in the lengths of some genes, poor conservation of the sequences in these regions, or uncertainty in the actual initiation and/or termination codons. To deal with this, some positions were eliminated from phylogenetic analysis according to the following criterion: In any case where gaps were introduced in the alignment of the gene ends, positions were eliminated progressively until the first occurrence of a residue conserved in at least eight of the nine taxa. This resulted in elimination of up to 3 positions at the carboxyl end of Atp8, the first 14 and the last 612 of Cox1, the last 38 of Cox2, the first 47 of Cox3, the first 712 of Cob, the first 1419 and the last 1625 of Nad1, the first 1023 and the last 3855 of Nad2, and the first 2124 and the last 25 of Nad3. This left 1,948 aligned positions, which constituted the "whole" data set. Because lesser, but still significant, ambiguities remained in the alignments of Nad2 and Nad3, those genes were omitted from some analyses. We refer to the 1,579 aligned positions from this more conserved set of genes as the "limited" data set.
PAUP* 4.0 (Swofford 1998
) was used for phylogenetic analyses. For maximum parsimony, all characters were unordered and of equal weight, and all searches employed the exhaustive search algorithm. The accelerated transformation option (ACCTRANS) was used to determine branch lengths. In separate analyses of both the whole and the limited data sets, gaps were considered "missing data" or "21st amino acids," and a subset of taxa that omitted the chordates and arthropods was analyzed. Unrooted trees were also produced by the neighbor-joining method (Saitou and Nei 1987
). Confidence estimates included consistency, retention, and rescaled consistency indices and bootstrap analysis with 1,000 replicates of a heuristic search with random order of taxon entry. Trees were rooted by designating as outgroup taxa the vertebrates C. carpio (Chang, Huang, and Lo 1994
) and S. acanthias (Rasmussen and Arnason 1999
), or, for the limited taxon analysis, the mollusk K. tunicata (Boore and Brown 1994a
).
The sequences of tRNA genes were analyzed as a separate data set. Each of the sequences for the 12 tRNAs determined for all of Katharina, Platynereis, Galathealinum, Helobdella, and Lumbricus was aligned by eye, using potential secondary structure as a guide. The resulting 810 aligned nucleotide positions were analyzed by parsimony and neighbor-joining, as above. Maximum-likelihood analysis used quartet puzzling with empirically derived nucleotide frequencies, a 2:1 assumed ratio of the rate of transitions to transversions, and the HKY85 model (Hasegawa, Kishino, and Yano 1985
). Gaps were separately treated as "missing data" or "fifth nucleotides." The mollusk Katharina was used as the outgroup to root these trees.
Phylogenetic Analysis of Gene Arrangements
The mitochondrial gene arrangements of Platynereis, Lumbricus, and Katharina were also compared with those previously inferred to be primitive for Arthropoda (Staton, Daehler, and Brown 1997
; Boore, Lavrov, and Brown 1998
) and Chordata (Boore 1999
; Boore, Daehler, and Brown 1999
) (see below). We assumed that Galathealinum, Helobdella, and Lumbricus share all of the same synapomorphies, since their gene arrangements are identical for the regions determined.
The gene arrangements were analyzed using the minimum-breakpoint method (Sankoff and Blanchette 1998
; Blanchette, Kunisawa, and Sankoff 1999
). Briefly, this method compares each pair of arrangements and determines the number of breakpoints required to change one arrangement into the other. To simplify calculations, an early analysis had then applied distance methods to a matrix of these differences (Sankoff et al. 1992
), but this approach was unsatisfactory due to problems in handling unequal rates of rearrangement and due to information lost by not identifying the specific genes involved in translocations. An improved method is now available which bases phylogeny reconstructions on parsimony and specifies genes involved in translocations (Sankoff and Blanchette 1998
; Blanchette, Kunisawa, and Sankoff 1999
). We employed the latter method.
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ResultsGene Content and Organization |
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Initiation/Termination Codons
Alternatives to ATG start codons are very common among metazoan mtDNAs, so it is unusual to find an ATG codon at the beginning of all but one of the protein-encoding genes among these species (appendices 13). The cox3 gene of Helobdella is the only exception and is most probably initiated by TTG. Although the ATA preceding it could be the start codon, this would cause a 3-nt overlap with the preceding trnG.
Many of the protein gene sequences in this study appear to end with a single T that is directly adjacent to the downstream gene. It is common for termination codons to be truncated (to T- or TA-) in metazoan mtDNAs; such codons are converted to complete (UAA) stop codons by polyadenylation after transcript processing (Ojala, Montoya, and Attardi 1981
). Several of the genes we sequenced have complete, in-frame stop codons that would require short overlaps with the downstream genes, sometimes of only 1 or 2 nt. We speculate that these are normally unused; that normal cleavage of the polycistronic transcript yields incomplete stop codons, subsequently completed by polyadenylation; and that the encoded TAA codons function (if at all) only as "backups" to prevent translational readthrough if the transcripts are not properly cleaved.
Transfer RNAs
There are 12 tRNA genes in the sequenced portions of Galathealinum and Helobdella mtDNAs, each identical in relative position and polarity to its homolog in Lumbricus. The same set of 12 tRNA genes is also found in the sequenced portion of Platynereis mtDNA, along with two additional tRNA genes, trnC and trnM. figure 1
shows the relative location of each of these genes, and figure 2
shows their potential secondary structures and compares them with their homologs in Lumbricus.
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There are 10 cases in which the sequences of adjacent tRNA genes overlap (marked with boldface nucleotides in fig. 2
and shown in the appendices). No other gene overlaps occur, assuming that all stop codons have been correctly inferred (see below). For the structures in figure 2
to form, complete individual tRNA gene transcripts are required. Given the overlaps, however, these are not possible unless (1) transcription of each originates from a different promoter; (2) polycistronic transcript processing alternates, yielding sometimes one or the other complete tRNA; or (3) transcript editing restores the nucleotides that are lost in processing. For 6 of these 10 overlapping pairs, the overlap involves only the discriminator nucleotide. It is possible that this nucleotide is not encoded by these genes but is added posttranscriptionally either by polyadenylation (demonstrated for some mt tRNAs; Yokobori and Pääbo 1997
) or by a mechanism similar to the one that adds CCA to the 3' ends of tRNAs. There are four cases of 2-nt overlap, involving the adjacent gene pairs trnA-trnS2(tga) and trnY-trnG in both Lumbricus and Helobdella; since these two are the most closely related pair of taxa (see below), it is possible that they share some mechanism for resolving these larger overlaps of these identical gene pairs.
As is found in many other mitochondrial systems, the DHU arms of the serine tRNAs cannot be folded into standard stem-loop structures (fig. 2
). In some cases, alternative folding yields a structure with a DHU stem having only 1 nt between the acceptor and DHU stems and 2 nt between the DHU and anticodon stems. (Similar alternative folding is possible for the serine tRNAs of Katharina; see Boore and Brown 1994a.
)
Nucleotide and Amino Acid Composition
These mtDNA sequences like those of most metazoans, are AT-rich, ranging from 61% to 76% (table 1
). Platynereis and Lumbricus have similar biases in codon usage (table 2
), and their values for these are not greatly different from those found in the mollusk Katharina (Boore and Brown 1994a
). Codon usage in Helobdella is somewhat different, generally reflecting its greater A+T richness, and is most extreme for the codons ACG and CGG, neither of which is used within the portion of the mtDNA sequenced.
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Although selection for A+T richness could account, in part, for the infrequency of G and C at third codon positions, it cannot account specifically for the bias against G relative to C. It is possible that an anti-G bias results from selection for translational efficiency or, alternatively, that it results from a bias in mutational tendency.
The higher A+T bias in Galathealinum mtDNA may account for the differences in the amino acid compositions of some of its proteins from those of the other species (table 3 ). Alanine, valine, and threonine, each encoded by a G- or C-containing codon, are underrepresented in Galathealinum, and isoleucine and phenylalanine, each encoded by AT-rich codons, are overrepresented. Interestingly, the ratios of nonpolar to polar amino acids are very similar among these four mtDNAs, indicating the importance of physicochemical characteristics on substitution patterns. Also reflecting Galathealinums higher A+T bias is its greater usage of TTR than CTN to encode leucine (TTR:CTN ratio = 1.67); CTN is more commonly used in the other three species (TTR:CTN ratios of 0.75, 0.49, and 0.79). Finally, further emphasizing Galathealinums anti-G bias, all but three of the 206 TTR codons are TTA.
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Unassigned Nucleotides
Metazoan mitochondrial genomes typically contain at least one relatively large region devoid of structural genes. In vertebrates, this region contains elements that control replication and transcription (see Shadel and Clayton 1997
), and analogous regions may function similarly in other taxa. No such region is found in the sequenced portions of Galathealinum or Helobdella mtDNAs, but there is a 1,091-nt noncoding region in Platynereis mtDNA. This region is somewhat higher in A+T (72%) than an analogous 384-nt region in Lumbricus (64%) and is located between trnG and trnY in Platynereis and between trnR and trnH in Lumbricus. The primitive position for this region in Annelida is uncertain, but the presence in Platynereis of apparently translocated tRNA genes (see below) flanking it, along with the frequent movement of such regions in conjunction with other translocations (Boore 1999
), suggests that its position in Platynereis is derived.
Proportional to their individual frequencies, all four homodinucleotides and all four homotrinucleotides, except for CCC in Lumbricus, are overrepresented for the noncoding regions of both Platynereis and Lumbricus. The dinucleotide CG, normally one of the least common in metazoan mtDNAs (Cardon et al. 1994
), is also present at higher frequencies than expected in both Platynereis and Lumbricus noncoding regions. The dinucleotide TA occurs 151 times in the Platynereis and 34 times in the Lumbricus noncoding region. This is not greatly different from expectation, given the high A+T composition, but is noteworthy because many of these dinucleotides are found in runs of alternating TA pairs. In the noncoding regions of Platynereis and Lumbricus mtDNAs, respectively, 61 and 14 of the TA pairs occur adjacent to at least one other TA pair, with the longest runs being 14 and 6 consecutive TA pairs for each of the two mtDNAs. This has also been observed in other mtDNA noncoding regions (e.g., Katharina mtDNA contains a run of 36 TA pairs; Boore and Brown 1994a
). Finally, no blocks of significant sequence similarity were identified between the noncoding regions of Lumbricus and Platynereis, although short T, A, and G homopolymer runs occur in the noncoding regions in both species.
There are totals of only six noncoding nucleotides in the sequenced portion of Galathealinum mtDNA and only two for Helobdella. In Platynereis mtDNA, in addition to the large noncoding region described above, there are numerous intergenic nucleotides: 5 (CACAT) between trnL1(tag) and trnS2(tga), 1 (T) between trnC and cox1, 61 between cox2 and trnG, 60 between trnY and atp8, 3 (AAT) between trnM and trnD, 32 between trnD and cox3, and 9 (GGATATCCT) between trnQ and nad6 (appendix 1). All except the 9 nt separating trnQ and nad6 are adjacent to tRNAs whose positions appear to be derived. The presence of a block of intergenic nucleotides is a condition often associated with a recent gene translocation (Boore 1999
). The tRNA(Q) of Platynereis is quite dissimilar to those of the other animals and is shorter; it is tempting to speculate that these nine adjacent noncoding nucleotides are the vestige of a recent shift in the sequences coding for this tRNA. None of these noncoding sequences of Platynereis mtDNA are similar to any portion of Lumbricus mtDNA.
Phylogenetic Reconstruction
The whole data set of 1,948 amino acid positions for nine taxa was analyzed by parsimony, using PAUP* 4.0 (Swofford 1998
). The limited data set, which omits the two most variable inferred proteins (Nad2 and Nad3), was subjected to the same analysis. Gaps introduced to maximize alignment similarity were alternatively scored as additional characters or as missing data. Trees were rooted using the two vertebrate species as an outgroup. All four of these analyses (using whole or limited data sets with two modes of gap scoring) yielded the same most-parsimonious tree, with the next-shortest tree being at least three steps longer (fig. 3
). An alternative method, neighbor-joining analysis (Saitou and Nei 1987
), yielded trees with the same topology.
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As an independent source of phylogenetic information, mitochondrial gene arrangements were also compared. The mitochondrial gene arrangements analyzed include those of the mollusk K. tunicata (Boore and Brown 1994a
), the oligochaete L. terrestris (Boore and Brown 1995
), the polychaete P. dumerii (with missing genes in positions to match their arrangement in Lumbricus), and those inferred to be primitive for Arthropoda and Chordata (see fig. 1B
).
In order to infer the primitive arrangement for Arthropoda, we consider the two most distantly related arthropod groups so far studied, Cheliceriformes, represented by the horseshoe crab Limulus polyphemus (Staton, Daehler, and Brown 1997
) and Insecta, represented by Drosophila (Clary and Wolstenholme 1985
). The mitochondrial gene arrangements of these two animals differ by only a single tRNA gene position, that of trnL2(taa). It is parsimonious to infer that their common ancestor had one or the other of these two arrangements, which can easily be determined by considering those of less related taxa. The position of trnL2(taa) in Limulus mtDNA (i.e., rnl-trnL1-trnL2-nad1) is shared by many outgroup taxa (Boore et al. 1995
; Boore, Lavrov, and Brown 1998
), including the mollusk Katharina, as shown in figure 1B,
and so it is the gene arrangement found in Limulus that must be primitive for Arthropoda or, more exactly, for whatever arthropods diverged after the split of Cheliceriformes and Insecta.
The same type of logic allows inference of the primitive arrangement for Chordata. The early-branching cephalochordate Branchiostoma (Spruyt et al. 1998
; Boore, Daehler, and Brown 1999
) has a gene arrangement differing from that of a fish (Chang, Huang, and Lo 1994
) (which is, itself, identical to the arrangements found in dozens of other diverse vertebrates; see Boore 1999
) by only four tRNA positions. For one of these four differences, the Branchiostoma arrangement (trnN-trnW-trnA-trnC-trnY) is also found in the even more distantly related hemichordate Balanoglossus (Castresana et al. 1998b
), so this must be the primitive arrangement for the common ancestor of the Chordata. For the other three cases (positions of trnF, trnM, and trnG), similar or identical positions are shared between the mtDNAs of the fish and outgroups such as arthropods and/or echinoderms, so it is most parsimoniously inferred that the fish arrangement is primitive, with separate, later translocations in the lineage leading to Branchiostoma (see further discussion in Boore, Daehler, and Brown 1999
).
We applied the minimum-breakpoint method for reconstructing patterns of gene rearrangement (Sankoff and Blanchette 1998
; Blanchette, Kunisawa, and Sankoff 1999
). Given a tree topology with gene arrangements associated with each branch, this method reconstructs ancestral gene arrangements for each node in such a way that the total number of breakpoints between neighboring nodes is minimized. A particular ancestral reconstruction at a node may not be unique and can range from being identical to one descendent arrangement (with the branch to the other descendent having all differences) to the opposite condition. To overcome this, the search is run iteratively with random trials of ancestral reconstructions, each searching for the minimum number of breakpoints. All possible unrooted trees are scored this way for the total number of breakpoints; the shortest corresponds to a parsimony analysis of gene arrangements and forms a phylogenetic hypothesis.
This analysis yields a single shortest tree requiring a total of 76 breakpoints (fig. 5 ); the next-shortest tree requires a total of 80. The tree produced was rooted by designating Chordata as the outgroup.
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Discussion |
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All genes are identically arranged in the studied portions of the mtDNAs of the oligochaete Lumbricus, the hirudinid Helobdella, and the pogonophoran Galathealinum. Several tRNA genes differ in their locations in the mtDNA of the polychaete Platynereis. It is unclear which of these two gene arrangements is primitive for the Annelida. For three of the differently located tRNA genes, trnG, trnY, and trnD, there is a noncoding region flanking their positions in Platynereis mtDNA, as is often found for recently translocated genes (Boore 1999
). One portion of the gene arrangement for the clitellates and pogonophoran (trnL1-trnA-trnS2-trnL2) is also found in the echiuran Urechis caupo (Boore, Lavrov, and Brown 1998
). Some view echiurans as an outgroup to the Annelida (Brusca and Brusca 1990
); if this is correct, then the parsimonious reconstruction would be that this is the primitive annelid condition, with the change being derived for Platynereis. However, others view echiurans as the sister taxon to clitellates to the exclusion of both pogonophorans and the polychaete family Nereidae (McHugh 1997
), so this arrangement could, alternatively, be derived from the primitive one found in Platynereis (trnL1-trnS2-trnA-trnL2) for a pogonophoran-echiuran-clitellate clade. The positions of trnC and trnM are the same in the Katharina and the Lumbricus mtDNAs (trnC-trnM-rns), so this can be inferred as the primitive annelid condition, with translocations of these two genes in the lineage leading to Platynereis.
Comparisons of mitochondrial genomes using both gene arrangements and inferred amino acid sequences provide strong support for an Annelida-Mollusca clade that excludes Arthropoda, as has been found in other studies (e.g., Ghiselin 1988
; Lake 1990
; Eernisse, Albert, and Anderson 1992
; Morris and Peel 1995
; Garcia-Machado et al. 1999
). This revised view of the relationship of these phyla (i.e., accepting the Eutrochozoa rather than the Articulata as the correct superphylum group) compels a reinterpretation of the patterns of morphological evolution. For example, the parsimonious interpretation is that body segmentation is primitive for all three taxa. Traditionally, mollusks have been viewed as unsegmented, but some have serially arranged structures, notably Polyplacophora, Monoplacophora, and Nautiloidea (Vagvolgyi 1967
; Lemeche 1959; Wingstrand 1985
). These have often been interpreted as not being "true" segmentation, due partly to the lack of metamerism in groups thought to be primitive mollusks, the solenogasters and caudofoveates, and in groups thought to be closely related to mollusks, such as sipunculids and echiurans. It may also be that the long-standing description of mollusks arising from a nonsegmented ancestor, either the hypothetical ancestral mollusk (see Ghiselin 1988
for discussion) or a flatworm (see Haszprunar 1996
), has stifled the more straightforward interpretation of molluscan iterative structures as being segmental.
Finally, these data indicate that Pogonophora is more closely related to the Clitellata than is Platynereis (order Phyllodocida, family Nereidae). This is consistent with the results of comparing partial EF1- sequences (McHugh 1997
). Thus, the Pogonophora should no longer be considered an independent phylum, but rather a group within Annelida, and should revert to the name Siboglinidae Caullery, 1914 in accordance with the suggestion of Rouse and Fauchald (1997)
. The current status of the Polychaeta is uncertain, with recent studies pointing out the possibility that they are a paraphyletic group (McHugh 1997
, Kojima 1998
). With the inclusion of only a single representative, this study cannot address this specifically. Whether or not other polychaete groups would cluster with Platynereis awaits further study.
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Acknowledgements |
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Footnotes |
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1 Keywords: Galathealinum,
Helobdella,
Platynereis,
Lumbricus,
mitochondria,
evolution,
genome.
2 Address for correspondence and reprints: Jeffrey L. Boore, Department of Biology, University of Michigan, 830 North University Avenue, Ann Arbor, Michigan 48109-1048. E-mail: jboore{at}umich.edu
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