* Biology Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts; and Department of Biological Sciences, Auburn University, Auburn, Alabama
Correspondence: E-mail: ken{at}auburn.edu.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: phylogeny gene order mitochondria genome Annelida
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Despite the importance of Annelida (segmented worms) with over 12,000 described species and its dominance as the most abundant macrofaunal group in the deep sea (69% of the planet), only two complete annelid mitochondria have been sequenced (the nereid Platynereis dumerilii and the oligochaete Lumbricus terrestris). These genomes differ only slightly in gene order. In addition, partial genomes of the siboglinid Galathealinum brachiosum and the leech Helobdella robusta (Boore and Brown 2000) match the L. terrestris gene order exactly. (Note that Siboglinidae was previously referred to as Pogonophora and Vestimentifera [McHugh 1997; Rouse and Fauchald 1997; Halanych, Feldman, and Vrijenhoek 2001]). Some mtDNA genome data are available for allied Lophotrochozoan taxa; most relevant are mollusks (e.g., Hoffman, Boore, and Brown 1992; Boore and Brown 1994; Hatzoglou, Rodakis, and Lecanidou 1995; Terrett, Miles, and Thomas 1996; Wilding, Mill, and Grahame 1999; Kurabayashi and Ueshima 2000; Grande et al. 2002; Tomita et al. 2002; Serb and Lydeard 2003; Boore, Medina, and Rosenberg 2004; Dreyer and Steiner 2004; DeJong, Emery, and Adema 2004), brachiopods (Stechmann and Schlegel 1999; Noguchi et al. 2000; Helfenbein, Brown, and Boore 2001), phoronids (Helfenbein and Boore 2004) and sipunculans (Boore and Staton 2002). Of these taxa, the sipunculan Phascolopsis gouldii is the most similar to the known annelid arrangements with 16 of the 19 sipunculan genes examined in the same order as in L. terrestris (but in two separate blocks). For this reason, Boore and Staton (2002) hypothesized a close relationship between annelids and sipunculans. Mollusks are notable because their mitochondrial genomes appear to have experienced numerous large-scale rearrangements and some taxa have even lost the atp8 gene. Brachiopods also seem to have undergone numerous rearrangements. Of the three complete genomes currently available, Laqueus rubellus and Terebratalia transversa share 14 gene boundaries composed in nine blocks; L. rubellus and Terebratulina retusa share only eight gene boundaries in eight separate blocks (Helfenbein, Brown, and Boore 2001).
Recent views of annelid phylogeny have moved away from the traditional view of two main groups, Clitellata (Oligochaetes and Hirudineans) and Polychaeta. Although morphological cladistic analyses have supported this hypothesis (Rouse and Fauchald 1995), multiple sources of data clearly show that the Clitellata, Echiuridae, and Siboglinidae are within the polychaete radiation (reviewed in McHugh 2000, Halanych, Dahlgren, and McHugh 2002, and Halanych 2004). Such potential for morphological adaptation is not surprising, given the enormous amount of diversity in annelids' body plans, habitats, and life histories. A comprehensive molecular phylogeny of Annelida is wanting, and, currently, our best understanding of annelid evolutionary history comes from morphological cladistic analyses (Rouse and Fauchald 1997; Rouse and Pleijel 2001), which suggest annelids contain three major groups, Scolecida, Aciculata, and Canalipalpata. Unfortunately, the Clitellata are not considered in these treatments.
We report here the complete mitochondrial sequence of a bamboo worm Clymenella torquata (Maldanidae) and an estimated 80% of the genome of the deep-sea tubeworm Riftia pachyptila (Siboglinidae). Clymenella torquata and the other members of Maldanidae are called bamboo worms because the shape of their segments gives them a bamboo-like appearance. Clymenella torquata is common in sandy intertidal/subtidal estuaries of the Atlantic coast of the United States, where it builds tubes from the surrounding sand and ingests sediment and the associated interstitial organisms (Mangum 1964). Riftia pachyptila inhabits the hydrothermal vents of the East Pacific Rise and obtains energy from the chemosynthetic endosymbiotic bacteria in a specialized structure called the trophosome (Southward and Southward 1988). Although annelid phylogeny has not been well resolved, available molecular evidence (Halanych, unpublished data) places these two annelids in very distant parts of the annelid tree. By including these two taxa, we provide representatives for all major clades outlined by Rouse and Fauchald (1997). Our goals in presenting and analyzing these new genomes are (1) to further characterize the evolution of mitochondrial genome structure among annelids and (2) to explore the potential of mitochondrial genomes in resolving annelid phylogeny.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Clymenella torquata
All mtDNA amplifications of C. torquata employed 1 µl EXL Polymerase (Stratagene), as well as 5 µl EXL buffer, 25 pmol dNTPs, 200 ng each primer, 1 µl stabilizing solution, and approximately 10 ng genomic DNA per 50 µl reaction. The sections mLSUcox1 (using primers 16Sar-L/HCO2198), cox1cox3 (LCO1498/COIIIr), cox3cob (COIIIf/CytbR), and cobmLSU (CytbF/16Sbr-H) all generated single-banded products. The mLSU primers are from Palumbi (1996); cob and cox3 primers are from Boore and Brown (2000), and the COI primers are from Folmer et al. (1994). PCR protocols for these fragments are found in the Supplementary Material online. Products were verified on an agarose gel, purified using the QiaQuick kit (Qiagen), eluted in 40 µl water, and sheared separately in a HydroShear DNA shearer (GeneMachines) to generate random fragments of 1 to 2 kb in length. The sticky ends were polished with the Klenow fragment and were A-tailed using Taq polymerase, an excess of dATP, and incubation at 72°C for 10 min. DNA was then repurified with the QiaQuick kit, and cloned into pGEM-T Easy (Promega). Sequencing reactions were performed using Big Dye (versions 2 and 3) chemistry on an ABI 377 (Applied Biosystems). Fifteen mLSUcox1 clones (average coverage 5.3X), nine cox3cob clones (average 2.9X) and 7 cobmLSU clones (average 2X) were sequenced in both directions using T7 and SP6 and then assembled to generate contigs. Combined, the assemblies contained approximately 90% of the sequence of C. torquata's mt-genome. Three clones could not be entirely sequenced using plasmid primers. To complete sequencing on these clones, 19 walking-primers were designed (see Supplementary Material online). No clones were recovered containing the largest noncoding region (i.e., the control region or UNK) or the approximately 3 kb surrounding it (roughly including regions of the atp6 and nad4L genes and all of nad5, trnW, trnH, trnF, trnE, trnP, and trnT). This region was sequenced by amplification with flanking primers (Ctatp6f2 and Ctnad4r2) and direct sequencing using the walking primers.
Riftia pachyptila
mtDNA amplification for R. pachyptila was adapted from the procedure of Boore and Brown (2000). Standard primers were used to amplify short sections of cox1 (LCO1490 and HCO2198 [Folmer et al. 1994]) and cob (CytbF and CytbR [Boore and Brown 2000]) with Taq polymerase (Promega) in standard 25 µl PCRs. Products were purified using the QiaQuick Gel Extraction Kit (Qiagen) and sequenced on an ABI 377 automated sequencer. These sequences were used to design Riftia-specific primers for long PCR. In cox1, the primers Rp1536 and Rp2161 were designed, and, in cob, the primer CytBRp was designed. Information for all primers can be found in the Supplementary Material online.
These primers were then used to amplify long segments of the mt-genome in conjunction with the primers mentioned above: 16Sar-L and Rp1536 amplified the region spanning mLSUcox1, Rp2161 and COIIIr amplified cox1cox3, and COIIIf and CytBRp amplified cox3cob. These long PCR reactions consisted of 5 µl 10X rTth buffer, approximately 10 ng template DNA, 25 pmol dNTPs, 30 pmol of each primer, 0.4 µl (1 U) rTth polymerase, and 1 µl of Vent polymerase diluted 1:100 (0.02 U) per 50 µl. Both polymerases are from Applied Biosystems. PCR products were verified and, when necessary, size selected using 1% agarose gels. Single-banded products were purified and single A-overhangs added as above. A-tailed fragments were cloned into the pGEM-T Easy vector (Promega). Initial clone sequencing used the plasmid primers T7 and SP6; complete bidirectional sequencing was accomplished by primer-walking, resulting in an average sequencing coverage of 7.8X.
Amplification of the cobmLSU region in Riftia, which presumably contains UNK, was difficult. Part of this remaining region was sequenced by designing degenerate primers to nad4 sequences obtained from the complete genomes of Lumbricus terrestris, Platynereis dumerilii, and Katharina tunicata. These primers (nad4f, TGR GGN TAT CAR CCN GAR CG and nad4r, GCY TCN ACR TGN GCY TTN GG) amplified a short region of nad4 and allowed the design of primers specific to R. pachyptila (Rpnad4bf and Rpnad4br). Using EXL polymerase (Stratagene), the primer combination Rpnad4bf/16Sbr-H (Palumbi 1996) amplified the region spanning nad4mLSU, but the region between cob and nad4, which again was presumed to contain UNK, was still difficult to amplify and could not be cloned successfully after amplification. Three clones containing spliced PCR amplicons for this fragment (see Results) were partially sequenced and provided the remainder of cob as well as complete trnW and atp6 genes. For simplicity, the R. pachyptila fragment will, henceforth, be referred to as the R. pachyptila genome.
Genomic Assembly
Assembled sequences were checked by Blast (Altschul et al. 1990) searches against GenBank. Those sequences that returned strong Blast hits to mitochondrial protein-coding genes were translated into amino acids using the Drosophila mitochondrial code and aligned in ClustalX (Thompson et al. 1997) with other available lophotrochozoan genome sequences (table 1) obtained from GenBank to ensure correct identification. The full genomes were assembled by resolving ambiguous sequence reads in AutoAssembler (Applied Biosystems), checking against the amino acid alignments, and concatenating the individual alignments to make the complete genome alignment in MacClade version 4.03 (Maddison and Maddison 2000).
Candidate tRNA genes were found using the tRNAScan-SE Web server (http://www.genetics.wustl.edu/eddy/tRNAscan-SE); this identified all but four tRNAs in C. torquata and one in R. pachyptila. Stretches of mtDNA that did not code for protein genes and were in a similar position to tRNAs in previously published annelid genomes were scanned by eye for potential tRNA secondary structure and the presence of the anticipated anticodon sequence. The tRNA structures reported here are proposed based on the tRNAScan-SE foldings, keeping in mind the general forms suggested by Dirheimer et al. (1995). rRNA genes were identified by sequence homology with Blast entries, and 5' and 3' ends were assumed to be directly adjacent to upstream and downstream genes. The boundaries of the C. torquata UNK were similarly inferred from the ends of the upstream and downstream tRNAs.
Phylogenetic Analysis
Table 1 lists the taxa and their GenBank accession numbers used for phylogenetic inference. Outgroups were chosen based on knowledge of Lophotrochozoan evolutionary history (Halanych 2004). Because we hoped to develop a better understanding of the utility of mtDNA in constructing annelid phylogeny, we chose to subsample available lophotrochozoan mtDNA genomes for use as outgroups. For mollusks, we chose the polyplacophoran Katharina tunicata for its basal position, the two pulmonate gastropods Albinaria caerulea and Cepaea nemoralis because they were more easily aligned than other gastropods, and the cephalopod Loligo bleekeri to achieve a broader representation of mollusks. Several other molluscan genomes contained large insertions and deletions in several genes relative to annelids, greatly complicating attempts at adjusted. All three available brachiopods (Terebratalia transversa, Terebratulina retusa, and Laqueus rubellus) were included in the analyses. To create the final adjusted DNA from protein-coding genes was aligned in MacClade 4.03 using ClustalX alignments of the corresponding amino acids; rRNA genes were aligned manually using secondary structure as a guide, employing phylogenetic conservation diagrams obtained from the RNA database at the University of Texas's Institute for Cellular and Molecular Biology (http://www.rna.icmb.utexas.edu/topmenu.html). tRNAs, UNK, and noncoding DNA were not included in the alignments because of high variability (see below). This produced a single multipartitioned alignment in MacClade 4.03, which is available at TreeBase (http://www.treebase.org) and in the Supplementary Material online.
Two sequence-based data sets and one gene-order data set were created. One sequence-based data set contained nucleotide sequences from protein-coding and rRNA genes, and the second contained only inferred amino acid sequences. Regions that could not be unambiguously aligned and all third codon positions were removed. The amino acids of three protein-coding genes (atp6, atp8, and nad6) exhibited high variation, which made alignment difficult, and, thus, were excluded from both data sets.
All non-annelid taxa herein were treated as outgroups; however, brachiopods are drawn basally for illustrative purposes. Although mollusks, annelids, brachiopods, and sipunculans are closely related, the relationships between them are not well resolved (Halanych 2004). PAUP* version 4.0b10 (Swofford 2002) was used for parsimony and maximum-likelihood (ML) analyses. For both data sets, gaps were treated as missing data. For the DNA data set, ML models and their parameters were determined with hierarchical likelihood ratio tests (hLRTs) using the program MODELTEST version 3.5 (Posada and Crandall 1998). Heuristic searches in PAUP* under both parsimony and ML employed random sequence addition (parsimony:100 replicates; likelihood:10 replicates) to obtain starting trees, and Tree Bisection Reconnection (TBR) swapping. Bootstrapping with character resampling was performed with 1,000 replicates for parsimony and 500 replicates for ML. Decay indices (also called Bremer support [Bremer 1994]) were also calculated for the parsimony trees using constraints in PAUP*.
The order of genes in the mitochondria was used as a third data set for phylogenetic analysis. Although breakpoint analysis (Blanchette, Kunisawa, and Sankoff 1999) has proved useful in many cases, we prefer a newer parsimony framework (described in Boore and Staton 2002), which does not condense the data into pairwise distance measures and allows partial genomes to be included. Briefly, 74 multistate characters were created ("upstream of gene X" and "downstream of gene X" for each of the 37 genes), and character states were coded as "beginning of gene Y" and "end of gene Y," for a total of 74 states (although, obviously, a gene cannot appear upstream or downstream of itself). The matrix was then analyzed in PAUP* under parsimony as previously outlined. Because the gene orders of four taxa (P. gouldii, G. brachiosum, H. robusta, R. pachyptila) are incompletely known, missing and ambiguous characters (52) were removed before searching for trees, leaving 22 characters. The brachiopods were again placed as the basal-most outgroup. For comparative purposes, breakpoint and inversion distances were calculated using GRAPPA version 1.6 (Bader, Moret, and Yan 2002).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
The genome of C. torquata contains the standard 37 genes found in mtDNAs: 13 protein-coding genes, two genes for rRNAs, and 22 genes for tRNAs (Boore 1999). The R. pachyptila fragment contains nine complete protein-coding genes (atp8, cox1, cox2, cox3, cob, nad1, nad2, nad3, and nad6) and portions of two others (atp6 and nad4), as well as both rRNA genes (mLSU and mSSU) and 16 tRNA genes (trnA, trnC, trnD, trnG, trnI, trnK, trnL1, trnL2, trnM, trnN, trnQ, trnS1, trnS2, trnV, trnW, and trnY); the remaining genes (nad4L, nad5, and trnE, trnF, trnH, trnP, trnR, and trnT) and the UNK are presumably in the unsequenced portion. As seen in all other annelids to date, all genes in both genomes are encoded on a single strand.
Start and stop codon usage also shows patterns of bias. Start codons in protein-coding genes are highly biased towards ATG over ATA; ATG is observed in 12 of 13 coding genes in C. torquata (nad4 uses ATA) and all 10 R. pachyptila coding genes for which the 5' end is known. In addition, overlap typically exists between the presumptive stop codon (TAA or TAG) and the 5' end of the next gene. In other words, some stop codon bases appear to be part of the transcript of the downstream gene (illustrated in Supplementary Material online). For the purposes of annotation, the stop codon in all such cases is assumed to be incomplete (see Ojala, Montoya, and Attardi 1981) and the shared bases assigned to the downstream gene.
There is considerable codon usage bias in both genomes as well, with some codons within a group being used more than an order of magnitude more frequently than others (table 4). In codons that exhibit fourfold degeneracy, triplets ending in G tend to be the least used, as expected from overall nucleotide frequencies. However, codons ending in A tend to be the most common within a codon group, despite the slightly higher prevalence of T's in nucleotide frequency. In twofold codon groups, the use of XXG tends to be considerably less than XXA, and use of XXC is somewhat less than XXT. CCG (Pro) and CGG (Arg) were never observed in R. pachyptila.
|
|
|
|
The gene-order analysis produced 15 equally parsimonious trees of 112 steps. The strict consensus of these trees (see Supplementary Material online) contained far less resolution than the trees derived from nucleotide or amino acid sequences, with only three supported nodes. Consistent with other analyses, the two gastropods clustered together with 100% bootstrap support. Ninety-one percent support was also recovered for the node containing all annelids and P. gouldii. A grouping of this clade as sister to L. rubellus had weak support (53%). To determine whether this lack of resolution was caused by the parsimony method of analyzing gene order or was intrinsic to the data, GRAPPA version 1.6 breakpoint and inversion distances were also calculated. However, in these trees, Brachiopoda and Mollusca interdigitated to a large degree (data not shown). Neither algorithm can handle partial genomes; thus, P. gouldii, G. brachiosum, H, robusta, and R. pachyptila had to be excluded from these analyses, further reducing the phylogenetic inferences that could be made. It, thus, appears that all of these gene order algorithms are sensitive to the disparate rates of change present in our data set.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Phylogenetic Relationships
The AA parsimony and DNA likelihood phylogenetic analyses are consistent with previous findings that place Clitellata (McHugh 1997; Rota, Martin and Erséus 2001; Bleidorn, Vogt and Bartolomaeus 2003) and siboglinids (McHugh 1997; Rouse and Fauchald 1997; Kojima 1998; Halanych, Lutz, and Vrijenhoek 1998; Halanych, Lutz, and Vrijenhoek 2001) as derived "polychaetes". Thus, the last common ancestor of "Polychaeta" and Annelida are one and the same. However, bootstrap values (67% likelihood, <50% for AA parsimony) for this result were weak and Shimodaira-Hasegawa tests (Shimodaira and Hasegawa 1999) fell short of significant values (in both cases, P = 0.14, 1,000 replicates with RELL option). An alternative topology in the nucleotide parsimony analysis was not well supported. Clearly, considerably more taxa need to be sampled to understand the robustness of these results and placement of these groups within annelids. The groupings R. pachyptila + G. brachiosum and H. robusta + L. terrestris were highly supported in all sequence analyses, in agreement with morphological expectations. An additional result consistently recovered by sequenced-based analyses was placement of the sipunculan as sister to or inside Annelida (Shimodaira-Hasegawa test, P = 0.003). Boore and Staton (2002) first reported this result using many of the same mtDNA sequences used herein. Thus, although gene order may be uninformative in this case, there is high support from both DNA and amino acid sequences for an Annelida/Sipuncula clade to the exclusion of mollusks and brachiopods. Interestingly, nuclear large ribosomal subunit data also weakly support sipunculans as the sister clade to annelids (Passamaneck and Halanych, unpublished data). The likelihood tree provides the first suggestion that Sipuncula is within Annelida, but this finding requires additional verification.
In contrast to the sequence-based data sets, the gene order analysis offers little resolution. This result is to be expected with the limited observed variation in annelid gene order. Nonetheless, annelids and the sipunculan cluster together because of identical arrangement of the 11 genes between cox1 and cob (inclusive) and the sequence mSSUtrnVmLSU. The latter sequence appears to be somewhat conserved across lophotrochozoan clades (it is found in 10 of the 23 lophotrochozoan taxa for which data are currently available in GenBank) and potentially in other protostomes as well. Further, the subsequence trnVmLSU is found in 16 of the 23 lophotrochozoan genomes and some other protostomes. In any case, based on the available data, gene order appears to be of limited utility for relationships within the annelids because of its highly conserved nature. All rearrangements seen so far are minor and found in single taxa only, although with greater taxonomic coverage, potential synapomorphic gene orders may emerge. Apparently, both within annelids and between annelids and other lophotrochozoans, there is no consistent mechanism controlling the rate or types of gene order modifications. In contrast to the lack of phylogenetic signal in gene order among annelids, the resolution offered by sequence-based analyses holds promise.
Mitochondrial Genome Organization and Structure
The two genomes presented here also exhibit the pattern of posttranscriptional modification and splicing described by Ojala, Montoya, and Attardi et al. (1981), in which many stop codons are incomplete in the transcript and are filled in by posttranscriptional editing machinery. This type of splicing is presumed to occur in several genes in both the C. torquata and the R. pachyptila genomes. In the majority of these cases, the overlap in question contains an in-frame stop codon (TAA or TAG), but it is not presumed to be functional. Moreover, in several cases, there is no in-frame stop codon at or near the end of the protein-coding gene, making posttranslational addition of a stop codon the only plausible mechanism (see figure 2 in Supplementary Material online). One example is the nad1/trnI junction in C. torquata, where nad1 presumably ends with T__, and trnI begins with GA, such that assigning more of the codon (TG_ or TGA) to nad1 still does not produce a stop codon. Additionally, in C. torquata, the last six bases of nad4 (GGCCCT) appear to be used as the first six of trnC; a seven-base overlap could give nad4 an incomplete TA_ stop codon, but the next base is a T, and, therefore, it is not possible to generate a full stop codon from the primary sequence.
The AT-bias seen in both genomes seems to be contributing to a strong codon bias in protein-coding genes. Although the R. pachyptila genome is incomplete, the absence of two GC-rich codons (CCG, encoding proline, and CGG, encoding arginine) may be linked to the low percentage of G and C. However, even given these low frequencies in the protein-coding genes as a whole, the probability of never observing CCG (Pro) in 160 proline codons, given an average G content of 12%, is (0.12)0(10.12)160 = 1.31 x 109, and the probability of never seeing CGG (Arg) in 53 arginine codons is (0.12)0(10.12)53 = .0011 (both assuming independence of codons). Thus, the amount of AT-bias alone does not adequately explain the lack of these two codons and suggests that some other mechanism(s) is responsible for the observed codon bias. Cardon et al. (1994) discuss the paucity of CG dinucleotides in metazoan mitochondrial genomes, regardless of their position in codons (i.e., positions (I, II), (II, III) and (III, I)) and overall low usage of arginine (CGN) in mitochondrial proteomes. Indeed, arginine is the least frequent of all amino acids possessing fourfold degenerate codons in both C. torquata and R. pachyptila and is even less frequent than some twofold degenerate amino acids. Based on the symmetrized odds-ratios (NN, where NN is the dinucleotide in question) of Cardon et al. (1994), R. pachyptila does show CG suppression (
CG = 0.5299; 0.78
NN
1.23 is considered the normal range). Suppression of CG dinucleotides in vertebrate nuclear genomes has been linked to mutation to TG by methylation of the C followed by deaminization to T. This cannot underlie CG suppression in mtDNAs, because mitochondria lack the methylation pathway and because mtDNAs do not usually contain an excess of TG dinucleotides (R. pachyptila
TG = 0.83). Although no simple explanation has been found, the authors suggest that CG suppression is correlated with small genome size and "streamlined" mtDNA organization.
R. pachyptila is a large tubeworm found at Eastern Pacific hydrothermal vent fields. Early genetic analyses on this species led to speculation that hydrothermal vent animals would harbor a high GC nucleotide composition because the extra hydrogen bond, when compared with AT base pairing, would confer additional stability in the potentially high-temperature and reducing environment (Dixon, Simpson-White, and Dixon 1992). Although high GC content has been documented in thermophilic microbes (Woese et al. 1991), R. pachyptila's low GC content (a pervasive feature of metazoan mtDNAs in general) argues against such temperature-driven evolution in R. pachyptila. Possibly, the higher GC content in R. pachyptila postulated by Dixon, Simpson-White, and Dixon (1992) is restricted to the nuclear genome; however, it is unclear why mitochondrial and nuclear genomes would respond in different ways to the same environmental pressure if this were true.
Genomic Amplification and Sequencing
Our difficulties in amplifying and cloning the UNK region of C. torquata and R. pachyptila likely stem from regulatory aspects of this region of the molecule. In R. pachyptila, our long PCR reactions for the region cobnad4 repeatedly generated three to five bands, even though the reactions employed two approximately 30mer species-specific primers. Attempts to clone the band of the expected size resulted in very low transformation efficiencies. Of three clones sequenced, each contained an apparent splice in a similar, but not exact, position just downstream of atp6, indicating host removal of the genes between atp6 and nad4 (presumably containing trnW, UNK, trnH, nad5, trnF, trnE, trnP, and trnT). Sequencing of the 3' end of these clones provided the complete gene sequences for trnW and atp6, but the splice prevented accurate determination of what lay farther downstream. A similar region was apparently unclonable in the sheared fragments of C. torquata's mt-genome and had to be obtained by direct sequencing. Boore and Brown (2000) had similar problems when obtaining the similar region in Platynereis dumerilii and suggested that the presence of signaling elements in UNK disrupted PCR. Our observations suggest the UNK region is identifiable as a foreign origin of replication and is spliced out by at least some E. coli cell types (in this case, DH5 and JM109, both of which are recA) in addition to possibly interfering with PCR. Alternative strategies may need to be developed to completely sequence large numbers of complete mitochondrial genomes to avoid the need to direct-sequence and primer-walk the region containing UNK.
![]() |
Conclusions |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403410.[CrossRef][ISI][Medline]
Bader, D. A., B. M. E. Moret, and M. Yan 2001. A linear-time algorithm for computing inversion distance between signed permutations with an experimental study. J. Comput. Biol. 8:483491.[CrossRef][ISI][Medline]
Blanchette, M., T. Kunisawa, and D. Sankoff. 1999. Gene order breakpoint evidence and animal mitochondrial phylogeny. J. Mol. Evol. 49:193203.[ISI][Medline]
Bleidorn, C., L. Vogt, and T. Bartolomaeus. 2003. A contribution to sedentary polychaete phylogeny using 18S rRNA sequence data. J. Zool. Syst. Evol. Res. 41:186195.[ISI]
Boore, J. L. 1999. Animal mitochondrial genomes. Nucleic Acid Res. 27:17671780.
Boore, J. L., and W. M. Brown. 1994. Complete DNA sequence of the mitochondrial genome of the black chiton, Katharina tunicata. Genetics 138:423443.
. 1998. Big trees from little genomes: mitochondrial gene order as a phylogenetic tool. Curr. Opin. Genet. Dev. 8:668674.[CrossRef][ISI][Medline]
. 2000. Mitochondrial genomes of Galathealinum, Helobdella, and Platynereis: sequence and gene rearrangement comparisons indicate the Pogonophora is not a phylum and Annelida and Arthropoda are not sister taxa. Mol. Biol. Evol. 17:87106.
Boore, J. L., and J. L. Staton. 2002. The mitochondrial genome of the Sipunculid Phascolopsis gouldii supports its association with Annelida rather than Mollusca. Mol. Biol. Evol. 19:127137.
Boore, J. L., M. Medina, and L. A. Rosenberg. 2004. Complete sequences of the highly rearranged molluscan mitochondrial genomes of the scaphopod Graptacme eborea and the bivalve Mytilus edulis. Mol. Biol. Evol. 21:14921503.
Bremer, K. 1994. Branch support and tree stability. Cladistics 10:295304.[CrossRef][ISI]
Cardon, L. R., C. Burge, D. A. Clayton, and S. Karlin. 1994. Pervasive CpG suppression in animal mitochondrial genomes. Proc. Natl. Acad. Sci. USA 91:37993803.[Abstract]
DeJong, R. J., A. M. Emery, and C. M. Adema. 2004. The mitochondrial genome of Biomphalaria glabrata (Gastropoda, Basommatophora), intermediate host of Schistosoma mansoni. J. Parasitol. 90:991997.[Medline]
Dirheimer, G., G. Keith, P. Dumas, and E. Westhof. 1995. Primary, secondary, and tertiary structures of tRNAs. Pp. 93126 in D. Söll and U. RajBhandary, eds. tRNA: structure, biosynthesis, and function. ASM Press, Washington, DC.
Dixon, D. R., R. Simpson-White, and L. R. J. Dixon. 1992. Evidence for thermal stability of ribosomal DNA sequences in hydrothermal-vent organisms. J. Mar. Biol. Assoc. UK 72:519527.
Dreyer, H., and G. Steiner. 2004. The complete sequence and gene organization of the mitochondrial genome of the gadilid scaphopod Siphonondentalium lobatum (Mollusca). Mol. Phylogenet. Evol. 31:605617.[CrossRef][ISI][Medline]
Folmer, O., M. Black, W. Hoeh, R. Lutz, and R. Vrijenhoek. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotech. 3:294299.[Medline]
Grande, C., J. Templado, J. L. Cervera, and R. Zardoya. 2002. The complete mitochondrial genome of the nudibranch Roboastra europaea (Mollusca: Gastropoda) supports the monophyly of opisthobranchs. Mol. Biol. Evol. 19:16721685.
Halanych, K. M. 2004. The new view of animal phylogeny. Ann. Rev. Ecol. Evol. Syst. 35:229256.[CrossRef]
Halanych, K. M., T. G. Dahlgren, and D. McHugh. 2002. Unsegmented annelids? Possible origins of four lophotrochozoan worm taxa. Integrat. Compar. Biol. 42:678684.[ISI]
Halanych, K. M., R. A. Feldman, and R. C. Vrijenhoek. 2001. Molecular evidence that Sclerolinum brattstromi is closely related to vestimentiferans, not to frenulate pogonophorans (Siboglinidae, Annelida). Biol. Bull. 201:6575.
Halanych, K. M., R. A. Lutz, and R. C. Vrijenhoek. 1998. Evolutionary origins and age of vestimentiferan tube-worms. Cah. Biol. Mar. 39:355358.[ISI]
Hatzoglou, E., G. C. Rodakis, and R. Lecanidou. 1995. Complete sequence and gene organization of the mitochondrial genome of the land snail Albinaria caerulea. Genetics 140:13531366.
Helfenbein, K. G., and J. L. Boore. 2004. The mitochondrial genome of Phoronis architecta comparisons demonstrate that phoronids are lophotrochozoan protostomes. Mol. Biol. Evol. 21:153157.
Helfenbein, K. G., W. M. Brown, and J. L. Boore. 2001. The complete mitochondrial genome of the articulate brachiopod Terebratalia transversa. Mol. Biol. Evol. 18:17341744.
Hoffmann, R. J., J. L. Boore, and W. M. Brown. 1992. A novel mitochondrial genome organization for the blue mussel, Mytilus edulis. Genetics 131:397412.
Knoll, A., and S. B. Carroll. 1999. Early animal evolution: emerging views from comparative biology and geology. Science 284:21292137.
Kojima, S. 1998. Paraphyletic status of Polychaeta suggested by phylogenetic analysis based on the amino acid sequences of elongation factor-1-alpha. Mol. Phylogenet. Evol. 9:255261.[CrossRef][ISI][Medline]
Kurabayashi, A., and R. Ueshima. 2000. Complete sequence of the mitochondrial DNA of the primitive opisthobranch gastropod Pupa strigosa: systematic implication of the genome organization. Mol. Biol. Evol. 17:266277.
Lavrov, D. V., W. M. Brown, and J. L. Boore. 2004. Phylogenetic position of the Pentastomida and (pan)crustacean relationships. Proc. R. Soc. Lond. B Biol. Sci. 271:537544.[CrossRef][ISI][Medline]
Maddison, D. R., and W. P. Maddison. 2000. MacClade. Sinauer Associates, Sunderland, Mass.
Mangum, C. P. 1964. Studies on speciation in Maldanid polychaetes of the North American Atlantic Coast. II. Distribution and competitive interaction of five sympatric species. Limnol. Oceanogr. 9:1226.[ISI]
McHugh, D. 1997. Molecular evidence that echiurans and pogonophorans are derived annelids. Proc. Natl. Acad. Sci. USA 94:80068009.
. 2000. Molecular phylogeny of the Annelida. Can. J. Zool. 78:18731884.[CrossRef][ISI]
Noguchi, Y., K. Endo, F. Tajima, and R. Ueshima. 2000. The mitochondrial genome of the brachiopod Laqueus rubellus. Genetics 155:245259.
Ojala, D., J. Montoya, and G. Attardi. 1981. tRNA punctuation model of RNA processing in human mitochondria. Nature 290:470474.[ISI][Medline]
Palumbi, S. R. 1996. Nucleic acids II: The polymerase chain reaction. Pp. 205248 in D. M. Hillis, C. Mortiz, and B. K. Mable, eds. Molecular systematics. Sinauer Associates, Sunderland, Mass.
Perna, N. T., and T. D. Kocher. 1995. Patterns of nucleotide composition at fourfold degenerate sites of animal mitochondrial genomes. J Mol. Evol. 41:353358.[ISI][Medline]
Posada, D., and K. A. Crandall. 1998. Modeltest: testing the model of DNA substitution. Bioinformatics 14:817818.[Abstract]
Rota, E., P. Martin, and C. Erséus. 2001. Soil-dwelling polychaetes: enigmatic as ever? Some hints on their phylogenetic relationship as suggested by a maximum parsimony analysis of 18S rRNA gene sequences. Contrib. Zool. 70:127138.[ISI]
Rouse, G. W., and K. Fauchald. 1995. The articulation of annelids. Zool. Scripta 24:269301.[ISI]
. 1997. Cladistics and polychaetes. Zool. Scripta 26:139204.[ISI]
Rouse, G. W., and F. Pleijel. 2001. Polychaetes. Oxford University Press, New York.
Serb, J. M., and C. Lydeard. 2003. Complete mtDNA sequence of the North American freshwater mussel, Lampsilis ornata (Unionidae): an examination of the evolution and phylogenetic utility of mitochondrial genome organization in Bivalvia (Mollusca). Mol. Biol. Evol. 20:18541866.
Shimodaira, H., and M. Hasegawa. 1999. Multiple comparisons of Log-likelihoods with applications to phylogenetic inference. Mol. Biol. Evol. 16:11141116.
Southward, A. J., and E. C. Southward. 1988. Pogonophora: Tube-worms dependent on endosymbiotic bacteria. Anim. Plant Sci. 1:203207.
Stechmann, A., and M. Schlegel. 1999. Analysis of the complete mitochondrial DNA sequence of the brachiopod Terebratulina retusa places Brachiopoda within the protostomes. Proc. R. Soc. Lond. B Biol. Sci. 266:2043.[CrossRef][ISI][Medline]
Swofford, D. L. 2002. PAUP*: phylogenetic analysis using parsimony (*and other methods). Sinauer Associates, Sunderland, Mass.
Terrett, J. A., S. Miles, and R. H. Thomas. 1996. Complete DNA sequence of the mitochondrial genome of Cepaea nemoralis (Gastropoda: Pulmonata). J. Mol. Evol. 42:160168.[ISI][Medline]
Thompson, J., T. Gibson, F. Plewniak, F. Jeanmougin, and D. Higgins. 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acid Res. 25:48764882.
Tomita, K., Yokobori, S. I., Oshima, T., Ueda, T. and Watanabe, K. 2002. The cephalopod Loligo bleekeri mitochondrial genome: multiplied noncoding regions and transposition of tRNA genes. J Mol. Evol. 54:486500.[CrossRef][ISI][Medline]
Wilding, C. S., P. J. Mill, and J. Grahame. 1999. Partial sequence of the mitochondrial genome of Littorina saxatilis: relevance to gastropod phylogenetics. J. Mol. Evol. 48:03480359.
Woese, C. R., L. Achenbach, P. Rouviere, and L. Mandelco. 1991. Archaeal phylogeny: reexamination of the phylogenetic position of Archaeoglobus fulgidus in light of certain composition-induced artifacts. Syst. Appl. Microbiol. 14:364371.[ISI][Medline]