Department of Biology, University of Michigan;
DOE Joint Genome Institute and Lawrence Berkeley National Laboratory, Walnut Creek, California
Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina
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Abstract |
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Introduction |
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Comparisons of mitochondrial gene arrangements have been very effective for reconstructing evolutionary relationships (Smith et al. 1993
; Boore et al. 1995
; Boore and Brown 1998, 2000
; Boore, Lavrov, and Brown 1998
; Dowton 1999
; Stechmann and Schlegel 1999
; Kurabayashi and Ueshima 2000
). Despite the occasional finding of mtDNAs with "scrambled" gene arrangements, the data set remains nearly free of homoplasy (Boore 1999, 2000
). Since the rate of rearrangement varies among lineages, these gene order characters may provide resolution at differing taxonomic levels. Within rapidly changing lineages, the signal may be best at lower levels, with anciently shared arrangements being eroded. Conversely, in more slowly evolving lineages the more ancient signal may be retained, but without rearrangements to resolve more recent branching. The merits of using gene rearrangements for recovering phylogenetic patterns lie not in their infrequency, as has been suggested (e.g., Saccone et al. 1999
; Le et al. 2000
), but in their complexity (i.e., there are a very large number of potential arrangements) and their near irreversibility (as judged by the infrequency of identified homoplasy). Thus, the ability to resolve any particular phylogenetic relationship may depend on the rate of gene rearrangement, but the confidence in a clade that is defined by a rearrangement, as analyzed by phylogenetic methods, does not.
In addition to providing gene order characters for inferring phylogeny, this data set also allows comparison of sequences as an independent estimator of evolutionary relationships. Furthermore, mitochondrial genome comparisons serve as a model system for genome evolution, where we can examine such factors as the following: What determines whether genes are all encoded by the same DNA strand versus being distributed between the two strands? How does mutation bias influence A+T-richness, dinucleotide frequency, codon usage, strand-skew between purines and pyrimidines, and amino acid composition of proteins? How do the structures of tRNAs evolve?
We describe here the partial mitochondrial genome of Phascolopsis gouldii, the first representative of the phylum Sipuncula to be so examined. The placement of this phylum within metazoan evolution has varied since its recognition as a taxon. Lamarck (1816)
confused sipunculans with sea cucumbers (Holothuroidea), and they were later considered to be a derived group of annelids (Delle Chiaje 1823
). In his reorganization of metazoans, de Quatrefages (1847)
created the Gephyrea, or "bridge group," which contained sipunculans, echiurans, sternapsid annelids, and priapulans. With their lack of segmentation and simple internal anatomies, de Quatrefages envisioned these groups as the transitional forms from "lower" to "higher" metazoan archetypes. Sipunculans were later elevated to the phylum level (Sedgwick 1898
) and associated with other spiralians either as a sister taxon to Annelida on the basis of biochemical properties (Florkin 1976
; Henry 1987
) or as sister to mollusks based on similarities in development (Scheltema 1993, 1996
).
The developmental lynchpin that supports a sipunculan/molluscan affiliation is a shared "molluscan cross" cleavage pattern in the blastomeres (Heath 1899
; Gerould 1907
; Baba 1951
; van Dongen and Geilenkirchen 1974
; Rice 1975, 1985
; Verdonk and van den Biggelar 1983
), which has been interpreted as a synapomorphy uniting these two groups (Scheltema 1993, 1996
). Annelids and echiurans possess a different arrangement of the blastomeres, termed an "annelid cross" (Gerould 1907
). As an extension of this proposed relationship, Scheltema (1996)
hypothesized further homology of sipunculan larval characteristics to those of larval and adult mollusks, including the ventral buccal organ of sipunculan larvae with the odontophore of mollusks, the ciliated lip below the mouth of sipunculan pelagosphera stage larvae with the foot of a molluscan pediveliger larva, and the lip glands of sipunculan larvae with the pedal glands of larval chitons and adult neomenioid aplacophorans.
We report the sequence of about half (7,470 nt) of the mtDNA of the first representative of the phylum Sipuncula, P. gouldii. We analyze genomic features in comparison with those of other animal mtDNAs and compare both gene arrangement and inferred amino acid sequences to resolve the phylogenetic placement of this phylum. These comparisons strongly support the closer relationship of Sipuncula with Annelida to the exclusion of Mollusca and other taxa.
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Materials and Methods |
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Initially, primers designed to match generally conserved regions of the mtDNA were used in the PCR to amplify four short (450710 nt) fragments from cox1 (primers LCO1490 and HCO2198; Folmer et al. 1994
), cox3 (primers COIIIF and COIIIB; Boore and Brown 2000
), cob (primers CytbF and CytbR; Boore and Brown 2000
), and rrnL (primers 16SARL and 16SBRH; Palumbi 1996
). DNA sequences obtained from these fragments were used to design oligonucleotides that were then employed in "long-PCR" (Barnes 1994
) to amplify the portions of the mtDNA spanning cox1cox3, cox3cob, and cobrrnL. Reaction conditions and results, fragment purification, DNA sequence determination and assembly, and gene identifications were as in Boore and Brown (2000)
. All sequence was determined for both strands using synthetic oligonucleotides to "primer-walk" through the long-PCR-amplified fragments. This 7,470-nt sequence was deposited in GenBank under accession number AF374337.
Phylogenetic Analysis of Sequences and Gene Arrangements
The invertebrate mitochondrial genetic code was used to infer the amino acid sequences of the six protein-encoding genes identified in the studied portion of P. gouldii mtDNA. Those of Cob, Cox1, Cox2, and Cox3 were aligned to the homologs of 16 other, phylogenetically diverse animals (listed, along with citations, in table 1
) using ClustalW as incorporated in MacVector 6.5 (Accelrys). The other two inferred protein sequences, Atp8 and Nad6, were judged to be too divergent to align with confidence and thus were not used in this phylogenetic analysis. The BLOSUM matrix was used to weight shared amino acids, with gap and extension penalties of 10 and 1, respectively. Amino acid alignments were then adjusted manually; these alignments can be viewed at EMBL accessions ALIGN_000119 and ALIGN_000121ALIGN_000123.
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Additional analyses specifically tested the effect of grouping P. gouldii with the two annelids versus with the mollusk/brachiopod group, first using a Kishino-Hasegawa test (Kishino and Hasegawa 1989
) as implemented in Tree-Puzzle (http://www.tree-puzzle.de/) and second using a nonparametric Wilcoxon signed-ranks test (Templeton 1983
) as implemented in PAUP*. For the partially determined mtDNA sequences of two other annelids, Galathealinum brachiosum and Helobdella robusta (Boore and Brown 2000
), only the partial sequences of the cob genes are known, along with the complete sequences of cox1, cox2, and cox3. These were included in a separate analysis to verify that the phylogenetic placement of P. gouldii was not affected.
We also compared the gene arrangement of this portion of P. gouldii mtDNA with those completely determined for 13 other animals (table 1
). These taxa were the same as those included in the sequence analysis except for three: Daphnia pulex and Ixodes hexagonus, were omitted since they had gene arrangements identical to those of Drosophila yakuba and Limulus polyphemus, respectively, and Locusta migratoria was omitted because it differed from the gene arrangement of D. yakuba by only a single tRNA position. A matrix was constructed that scored 36 characters, each as "upstream of X" or "downstream of X," where "X" refers to each of the 19 genes identified in the studied portion of P. gouldii mtDNA. Coded character states were the 5' or 3' end of the adjacent gene. This matrix of scored gene adjacencies was analyzed using parsimony criteria with the programs PAUP*, version 4.0b4a (Swofford 2000
), and MacClade (Maddison and Maddison 1992
).
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Results and Discussion |
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Base Composition and Codon Usage
Overall, the 7,470 nt determined for P. gouldii mtDNA are 63.1% A+T, similar to the percentage found for the whole mtDNA sequence of L. terrestris (61.6%; Boore and Brown 1995
). As is common among mtDNAs, CG is the least frequent dinucleotide, occurring at only 53% of expectation given the proportion of G and of C observed in this sequence.
Leucine is inferred to be the most frequent amino acid, present 243 times in these six inferred amino acid sequences, followed by four amino acids in nearly equal numbers, each about half as common as leucine: alanine (124), isoleucine (123), phenylalanine (135), and serine (125). Cysteine is the least frequently used amino acid, occurring only 14 times. These values are all similar to those for the amino acid usage of L. terrestris homologs (see table 2 ).
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Mitochondrial genes often terminate with abbreviated stop codons, where a single T or TA is presumably completed by polyadenylation to a TAA stop codon (Ojala, Montoya, and Attardi 1981
). This appears to be the case for three genes in P. gouldii mtDNA. The least certain of these is for atp8, since the next nucleotide (A), otherwise part of trnY, would complete the stop codon (fig. 2 ). If cob were to extend to the first complete stop codon, it would overlap trnP by 41 nt, and if nad6 were to do so, it would overlap cob by 20 nt. In each case, homologous genes of related animals are similar in predicted amino acid sequences to the abbreviated forms in P. gouldii and have no similarity to the overlapping extensions.
All genes are in a compact arrangement, with a total of only 13 noncoding nucleotides. These are distributed in six regions of 14 nt each. There is a CC between trnY and trnG, but otherwise, all are A or T. No genes are inferred to overlap except for the possibility of a single nucleotide shared between atp8 and trnY (see above).
Transfer RNAs
There are 11 sequences identified with the potential for folding into tRNA-like structures (fig. 3
). Each has an anticodon matching exactly one tRNA in L. terrestris mtDNA. Each has a seven-member amino-acyl acceptor stem (four with a single mismatch each) and a five-member anticodon stem (again, four with a single mismatch each). Two have 5 nt in the extra arm, and all others have 4 nt. All but four have A immediately preceding the anticodon arm. For all except tRNA(N) and tRNA(S2), there are three to five nucleotide pairs in both the DHU and the TC arms. All but four tRNAs have TA immediately preceding the DHU arm. The nucleotides preceding the anticodon are YT for all tRNAs, and the nucleotide following the anticodon is A for all but tRNA(P).
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Phylogenetic Reconstruction
Analyses based on maximum likelihood (ML), maximum parsimony (MP), and neighbor-joining of pairwise distances (NJ) of inferred amino acids were in almost complete agreement (fig. 4
). In all cases, P. gouldii was sister to the two annelids, and the sipunculan, annelids, mollusks, and brachiopods were placed within a well-supported Eutrochozoa (Ghiselin 1988
). Furthermore, there was strong support for the monophyly of several traditionally recognized groups (at least as composed by this small sampling), such as Insecta, Chelicerata, Arthropoda, and Echinodermata. Of the 1,396 aligned inferred amino acids, 673 were parsimony-informative. A heuristic search with 1,000 random stepwise additions of taxa yielded a single tree (consistency index = 0.618, retention index = 0.366, and rescaled consistency index = 0.226), 4,552 steps in length, identical to that in figure 4
except for the resolution of two trichotomies.
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The tree shown in figure 4
was specifically compared with the alternative which repositions P. gouldii to be more closely related to mollusks than to annelids. Using ML, a Kishino-Hasegawa test (Kishino and Hasegawa 1989
) demonstrated that the grouping of P. gouldii with the two annelids was significantly better (at the 5% significance level) than the grouping of P. gouldii with the brachiopod/mollusk clade. Using MP, nonparametric tests (Templeton 1983
) of the single tree with P. gouldii as sister to annelids (4,552 steps) and the single most-parsimonious tree with P. gouldii sister to mollusks (4,582 steps) demonstrated that the (P. gouldii, P. dumerilii, L. terrestris) tree was significantly shorter (P < 0.0001). In case of bias from the inclusion of the ambiguously placed T. retusa among the mollusks, a second test omitting T. retusa was performed. The resultant sipunculan/annelid tree in this analysis was also significantly shorter (P < 0.0001) than (P. gouldii, L. bleekeri, K. tunicata).
Two other annelids, Galathealinum brachiosum and Helobdella robusta, were included in a separate analysis, since each lacks a complete sequence for cob (Boore and Brown 2000
). Their inclusion had no effect on the phylogenetic placement of P. gouldii, and these two taxa joined P. dumerilii and L. terrestris in a topology consistent with the results of the earlier study (not shown).
Gene arrangements were also compared for phylogenetic reconstruction by scoring gene adjacencies as characters for cladistic analysis. The strict consensus of 30 equally parsimonious trees is shown in figure 5 . The unusual placement of Alligator mississippiensis as basal to the other included deuterostomes is not well supported, since it is based on the sharing of a single gene boundary (trnI, trnM) between Branchiostoma floridae and Balanoglossus carnosus in contrast to an alternative arrangement (trnI, -trnQ, trnM) in A. mississippiensis and two outgroup taxa (D. yakuba and L. polyphemus). This shared gene boundary has evidently been created by two independent translocations of trnQ from its primitive position (between trnI and trnM), leading in one case to the arrangement nad1, -trnQ, trnI, trnM, nad2 in B. carnosus and in the other to nad1, trnI, trnM, -trnQ, nad2 in B. floridae.
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Our findings are in general agreement with several published studies (Winnepenninckx, Backeljau, and De Wachter 1995
[parsimony-based analysis]; Giribet et al. 2000
[18S-only tree]; Regier and Shultz 1998) in that sipunculans are closely associated with annelids to the exclusion of mollusks. Other studies, or portions thereof, give results to the contrary. Comparisons of 18S rDNA sequences have placed Sipuncula as basal and sister to an assemblage of worms and mollusks (Field et al. 1988
; Winnepenninckx, Backeljau, and De Wachter 1995
[neighbor-joining analysis]; Giribet et al. 2000
[when combined with morphological characters]) or as sister to only one of two ectoprocts analyzed (Mackey et al. 1996
). The conflicts among these studies indicate that comparisons of short DNA sequences lack the necessary resolving power at this level of relationship. Furthermore, some of the associations between sipunculans and annelids found in these past studies were probably discounted based on the problematica associated with these data (Maley and Marshall 1998
).
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Conclusions |
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Likewise, many characteristics that have been hypothesized to link sipunculans with mollusks, e.g., developmental pattern, lack of segmentation, etc., must be reevaluated. The presence of a "molluscan cross" may be ancestral in all eutrochozoans and subsequently lost in the annelids, or possibly is a convergent pattern that has independent origins in the two phyla. Recent reports of segmental development in chitons (Jacobs et al. 2000
) suggest that segmentation in some form may be present in all spiralians, so its loss in other mollusks and worm groups may not be surprising. Certainly, the detailed examination of hypothesized synapomorphic larval and adult morphology in the sipunculans and molluscan groups (Scheltema 1993, 1996
) will need careful reconsideration to exclude convergence or oversight of annelid larval and adult homologs.
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Acknowledgements |
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Footnotes |
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Keywords: Phascolopsis
mitochondria
evolution
phylogeny
genome
Annelida
Address for correspondence and reprints: Jeffrey L. Boore, DOE Joint Genome Institute and Lawrence Berkeley National Laboratory, 2800 Mitchell Drive, Walnut Creek, California 94598. jlboore{at}lbl.gov
.
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