* Centre d'Océanologie de Marseille, Marseille, France; Laboratoire de Biologie Animale (Plancton), Université Aix-Marseille I, Marseille, France; and
Institut de Biologie du Développement de Marseille, Laboratoire de Génétique et Physiologie du Développement, Campus de Luminy, Marseille, France
Correspondence: E-mail: papillon{at}com.univ-mrs.fr.
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Abstract |
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Key Words: Chaetognatha mitochondria gene loss evolution phylogeny Spadella cephaloptera
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Introduction |
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However, the affinities with the deuterostomes have not been supported by most recent authors. Indeed, in the following years, Casanova (1986) has revived the idea of a relationship with mollusks, after the discovery of a hermaphrodite gland in deep benthoplanktonic species belonging to the Archeterokrohnia genus. Finally, Nielsen (2001) has regarded the phylum as a sister group of the gnathostomulids and the rotifers on the basis of the chitinous spines surrounding the mouth (hooks) and the innervations of the muscles from a vestibular ganglion that resemble the ganglion in rotifers and gnathostomulids.
More recently, the metazoan phylogeny surveys using molecular data, sometimes combined with morphological characters, suggested various affinities, mostly among protostomes: within lophotrochozoans with intermediate filament sequences (Erber et al. 1998) or with ribosomal RNA 18S sequences (18S) within ecdysozoans (Halanych 1996; Littlewood et al. 1998; Zrzàvy et al. 1998), basal ecdysozoans (Peterson and Eernisse 2001), between deuterostomes and protostomes (Giribet et al. 2000), or as an early offshoot of the bilaterian lineage (Telford and Holland 1993; Wadah and Satoh 1994). However, the sequences used in these works are extremely divergent, and most of these authors admitted that the phylogenetic position of Chaetognatha remains dubious because of long-branch attraction artifacts. Landmark studies of 18S in nematodes (Aguinaldo et al. 1997) and acoels (Ruiz-Trillo et al. 1999) clearly excluded Chaetognatha sequences from the analyses, and this long-branch attraction problem is well documented and reviewed in the study by Mallatt and Winchell (2002). Finally, the Hox gene survey we performed emphasized a basal position among Bilateria (Papillon et al. 2003), whereas other nuclear markers we isolated (myosin, elongation factor, and slow-evolving 18S from several genera [unpublished data]) did not allow us to propose an unambiguous position.
The mitochondrial DNA (mtDNA) genome is now widely used to infer deep phylogenetic relationships (Boore 1999). It is, then, of particular interest in deciphering the Chaetognatha phylogenetic position, and it gives us the opportunity to add phylogenetically useful genes to a growing metazoan data base. Metazoan mtDNA genome is usually considered a small extrachromosomal circular molecule ranging in size from about 13 to 18 kb and almost always containing the same 37 genes: two genes coding for rRNAs, 22 coding for tRNAs, and 13 coding for proteins. This gene content has been shown to vary in several metazoans (Boore 1999), such as nematodes (lack of atp8), mollusks (lack of atp8 and presence of one extra tRNA), or cnidarians (lost of nearly all tRNA genes and gain of genes not found in other mtDNAs). Phylogenetic analyses of the primary sequences of mitochondrial genes (Boore and Staton 2002; Stechmann and Schlegel 1999) support the new animal phylogeny based on 18S (Aguinaldo et al. 1997) and Hox genes (de Rosa et al. 1999). Moreover, mitochondrial gene arrangement is a powerful phylogenetic tool for several reasons. First, the gene content is almost invariant in the metazoans and, thus, provides a unique and universal pool of information. Second, stable structural gene rearrangements are rare events because functional genomes must be maintained. Third, the great number of potential gene rearrangements makes it very unlikely that different lineages would independently adopt the same gene order or that any gene would move back to a previous location (Boore and Brown 1998).
Here we report the complete mitochondrial genome sequence of Spadella cephaloptera in which we identified three major striking and unique features: (1) the lack of both atp8 and atp6, (2) the absence of all tRNA genes, and (3) the smallest size among metazoan mtDNA genomes.
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Materials and Methods |
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Isolation of the Mitochondrial Genome
The ribosomal RNA 16S (rrnL) gene was the first mitochondrial sequence isolated in S. cephaloptera, using the following primers: 16SF (5'-CCTGTTTANCAAAAACAT-3') and 16SR (5'-GGTCCAACCAAAGATAGA-3'). We isolated the cytochrome b gene (cob) while randomly sequencing a cDNA library of S. cephaloptera (see Papillon et al. [2003]). Using rrnL and cob as anchor genes, we tried to amplify regions of mitochondrial genomes lying between these two genes with the Advantage 2 PCR Kit (CLONTECH) dedicated to long-range PCR. All combinations of genome organization were tested, and we were able to amplify a 2-kb region containing the NADH dehydrogenase subunit 6 (nad6) gene and a 10kb region, possibly spanning the almost complete mitochondrial genome. This last fragment was used as a template for the following PCR experiments. We amplified the cytochrome oxydase 1 (cox1) gene with universal degenerate primers COI5A2 (5'-TAATWGGTGGNTTYGGNA-3') and COI3A (5'-TCAGGRTGNCCRAARAAYCA-3'). This sequence and the rrnL-nad6-cob fragment were again used as anchor genes to isolate missing regions. Two 5-kb bands were obtained. Both fragments were cut with EcoR1 and resulting subfragments were cloned into pBC SK+ (Promega) and sequenced by "primer-walk."
Genome Assembling
After sequencing, mtDNA genome was assembled using MacVector version 7.1 program (Oxford Molecular Group). Protein-encoding genes and start codons were identified by Blast matching to other animal mtDNAs. Transfer RNA searching was done by hand and using the tRNA scan-SE Search Server (www.genetics.wustl.edu/eddy/tRNAscan-SE). Sequence of the mtDNA genome of S. cephaloptera has been submitted to GenBank under the accession number AY545549.
Alignments
We chose a broad representation of taxa from available complete mtDNA sequences for this study (table S1 in Supplementary Material online). Inferred amino acid sequences of 11 mitochondrial protein genes (cox1 to cox3, cob, nad1 to nad6, and nad4L) from 28 bilaterian ingroup and two outgroup taxa were concatenated and aligned using ClustalW in MacVector 7.1, and alignments were refined by eye. Phyla with long branches were constantly excluded from the analyses (notably the nematodes and the platyhelminths). Among ecdysozoans, except arthropods, only nematode mtDNA genomes are fully sequenced, but these sequences are problematic for phylogenetic reconstructions because of dramatically accelerated substitution rates (data not shown), even with the mtDNA genome of Trichinella, considered as the less derived of the nematodes phylum (Lavrov and Brown 2001). Thus, arthropods are the only ecdysozoan representatives. To avoid subjectivity in excluding unreliably aligned positions from phylogenetic analysis, the program Gblocks version 0.91b (Castresana 2000) was used with the least-stringent settings.
Phylogenetic Reconstruction
Sequence alignments were analyzed by Neighbor-Joining (NJ) (Gamma model of distances and sites pairwise deletion) and maximum-parsimony (MP) with MEGA version 2.1 (Kumar, Tamura, and Nei 1994). Confidence estimates included bootstrap analysis with 1,000 replicates. The full alignment comprised 2,511 amino acid positions, of which 1,671 were parsimony-informative. Maximum-likelihood (ML) analysis employed 10,000 quartet-puzzling steps, an mtREV24 model of substitution, and eight Gamma rate categories in Tree-Puzzle version 5.0 (Schmidt et al. 2002).
In another analysis, two set of trees were compared so that support for specific phylogenetic hypotheses could be assessed. We produced an exhaustive search of ML trees in ProtML (Molphy 2.3b3 [Jun Adachi and Masami Hasegawa 19921996]) using the mtREV24 model with various constraints. First, we tested hypotheses of Chaetognatha being a deuterostome, a protostome, a basal bilaterian, or between protostomes and deuterostomes. Then, four positions within protostomes were also assessed: chaetognaths included in the lophotrochozoans, in the ecdysozoans, as a third protostomian branch, or as a basal protostome. Two different approaches were applied in tree selection to obtain two sets of trees. First, we built consensus trees (PHYLIP version 3.5c [Felsenstein 1993]) for each phylogenetic hypothesis from the entire set of generated trees or from the first 1,000 top-ranking trees when more trees were generated. Second, we retained the best trees for each constraint using the Approximately Unbiased (AU), Kishino-Hasegawa (KH), and Shimodaira-Hasegawa (SH) tests (Shimodaira 2002, and references therein), as implemented in the CONSEL program (Shimodaira and Hasegawa 2001). Then, to chose among these phylogenetic hypotheses, the selected trees of each set ("consensus" or "best tree strategies") were compared using CONSEL, as described above to test whether the difference between the log-likelihood scores (LnL) of the ML trees obtained was statistically significant. The "best-tree" strategy was also conducted with a second substitution model, mtREV24-F, to test its influence on the results.
For phylogenetic analysis of gene arrangements, two different methods were used to construct pairwise distance matrices. The first is based on minimum-breakpoint analysis (Blanchette, Kunisawa, and Sankoff 1999). The program is given a set of gene orders, and it finds the tree and the ancestral gene orders that relate them. The optimization criterion used is the minimal number of breakpoints. The second method is used in the GRIMM program (Tesler 2002), which computes the minimum possible number of rearrangement steps and determines a possible scenario taking this number of steps. This method takes only into account gene inversions. Pairwise distance matrices obtained with these two methods (figure S2 in Supplementary Material online) were used to construct phylogenetic trees with the NJ algorithm in MEGA version 2.1.
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Results and Discussion |
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The loss of atp8 can be observed not only in S. cephaloptera but also in a few other species of mollusks, nematodes, and platyhelminths. In these last organisms, it is not clear whether atp8 (1) moved to the nucleus, (2) became dispensable entirely, or (3) had its function coopted by one of the other ATPase subunits (Boore 1999). Unlike the atp8 loss, the absence of atp6 in S. cephaloptera is unique among metazoan mtDNAs.
Chaetognaths are the first metazoans that could possess an mtDNA genome with a complete absence of tRNA genes. Total lack of tRNA genes has previously been reported only for some protozoans (Plasmodium), and some tRNA genes are lacking in green alga (Pedinomonas) and angiosperm plants (Arabidopsis) (Burger, Gray, and Lang 2003). The metazoans closest to this situation are the cnidarians, for instance, Metridium senile (Beagley, Okimoto, and Wolstenholme 1998) and Acropora tenuis (Van Oppen et al. 2002), where only Tryptophan and Methionine tRNAs are observed and the other necessary tRNAs are imported nuclear products (Boore 1999). As in cnidarians, paucity of mtRNA genes in S. cephaloptera is probably a derived condition rather than a conserved primitive state for multicellular animals.
Finally, given the very peculiar features of the S. cephaloptera mtDNA genome, the existence of other mtDNA molecules containing the lacking genes cannot be excluded (linear concatemers or supplementary circular mtDNA), as it has been reported in few unrelated organisms (for review see Burger, Gray, and Lang [2003]).
Despite this very unusual organization, mitochondrial data analyses based on deduced proteins sequences allowed us to investigate the phylogenetic position of chaetognaths. The search strategies for inferring phylogenetic trees included Neighbor-Joining (NJ), maximum parsimony (MP), and maximum likelihood (ML). In each analysis, deuterostome and protostome monophylies are highly supported (fig. 2). Discrepancies among these analyses are in the deuterostome relationships and the monophyly of lophotrochozoans. First, B. lanceolatum, P. marinus, and E. burgeri change phylogenetic positions, depending on the methods used. Second, lophotrochozoans monophyly is supported in the NJ and ML trees (support values 99% and 88%, respectively) but not in MP analyses (39%). Nevertheless, the three methods yielded unambiguously inclusion of Chaetognatha within the protostomes (100% branch-support values in each tree) either among (in ML tree [fig. 2] or outside the lophotrochozoans (in MP and NJ tree [figure S1a and b in Supplementary Material online]). Tree topology presented in figure 2 also indicates that chaetognaths are distant from arthropods. This hypothesis is supported by a recent study in which a specific tissue marker showed that chaetognaths are excluded from ecdysozoans (Haase et al. 2001).
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It should be noticed that the inclusion of Chaetognatha within protostomes, based on mitochondrial data, is conflicting with previous conclusions, based on Hox genes (Papillon et al. 2003). This could imply that the proposed basal bilaterian feature conserved (a mosaic Hox gene that has retained characteristics of both central and posterior classes of Hox genes) could rather be a derived feature of the phylum.
The way to divide the bilaterians is traditionally based on two characters: the origin of the coelom and the fate of the blastopore. In the new phylogeny (Adoutte et al. 2000), several phyla initially described as deuterostomes have been placed into protostomes. This is, for instance, the case for brachiopods (de Rosa 2001; Cohen 1998; Stechmann and Schlegel 1999) and phoronids (Helfenbein and Boore 2004), even though in these species, the coelomic cavities form by enterocoely and the mouth does not arise from the blastopore, like in deuterostomes. Valentine (1997), supported by Peterson and Eernisse (2001), previously advocated that traditional characters placing the lophophorates into the deuterostomes are plesiomorphies of bilaterians. Chaetognaths exhibit such plesiomorphies: a complete gut with a mouth not arising from the blastopore and coelomic cavities forming by enterocoely. Moreover, chaetognaths do not display any of the typical ecdysozoan and lophotrochozoan synapomorphies (possession of a molting habit and presence of lophophores or trochophore larvae, respectively). As Chaetognatha clearly exhibit plesiomorphies and lack synapomorphies, they cannot be accurately placed among the current protostomes, but this could bring them closer to the common ancestor, excluded from lophotrochozoan and ecdysozoan groups. Another embryological character traditionally used to link Chaetognatha with deuterostomes is the radial cleavage of the egg. However, recent cell fate analysis indicates that the four-cell embryo displays similarities with classic spiralians (Shimotori and Goto 2001), supporting the conclusion of the work presented here. Nevertheless, more precise phylogenetic investigations on arrow worms will need further sequencing efforts, for example, EST programs, to isolate new nuclear molecular markers.
The acceptance that Chaetognatha are genealogically allied to undoubted protostomes strengthens the phylogenetic validity of some morphological characters such as the presence of a ventral nerve cord and chitinous structures but stresses that ontological criteria (i.e., embryological location of mouth and anus and mode of coelom formation) that traditionally define the deuterostome and protostome lineages can be misleading and raises once more the question of their relevance to define a bilaterian phylogeny.
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Acknowledgements |
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Footnotes |
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