Testing the New Animal Phylogeny: First Use of Combined Large-Subunit and Small-Subunit rRNA Gene Sequences to Classify the Protostomes

Abstract

Although the small-subunit ribosomal RNA (SSU rRNA) gene is widely used in the molecular systematics, few large-subunit (LSU) rRNA gene sequences are known from protostome animals, and the value of the LSU gene for invertebrate systematics has not been explored. The goal of this study is to test whether combined LSU and SSU rRNA gene sequences support the division of protostomes into Ecdysozoa (molting forms) and Lophotrochozoa, as was proposed by Aguinaldo et al. (1997) (Nature 387:489) based on SSU rRNA sequences alone. Nearly complete LSU gene sequences were obtained, and combined LSU ϩ SSU sequences were assembled, for 15 distantly related protostome taxa plus five deuterostome outgroups. When the aligned LSU ϩ SSU sequences were analyzed by tree-building methods (minimum evolution analysis of LogDet-transformed distances, maximum likelihood, and maximum parsimony) and by spectral analysis of LogDet distances, both Ecdysozoa and Lophotrochozoa were indeed strongly supported (e.g., bootstrap values Ͼ90%), with higher support than from the SSU sequences alone. Furthermore, with the LogDet-based methods, the LSU ϩ SSU sequences resolved some accepted subgroups within Ecdysozoa and Lophotrochozoa (e.g., the polychaete sequence grouped with the echiuran, and the annelid sequences grouped with the mollusc and lophophorates)-subgroups that SSU-based studies do not reveal. Also, the mollusc sequence grouped with the sequences from lophophorates (brachiopod and phoronid). Like SSU sequences, our LSU ϩ SSU sequences contradict older hypotheses that grouped annelids with arthropods as Articulata, that said flatworms and nematodes were basal bilateralians, and considered lophophorates, nemerteans, and chaetognaths to be deuterostomes. The position of chaetognaths within protostomes remains uncertain: our chaetognath sequence associated with that of an onychophoran, but this was unstable and probably artifactual. Finally, the benefits of combining LSU with SSU sequences for phylogenetic analyses are discussed: LSU adds signal, it can be used at lower taxonomic levels, and its core region is easy to align across distant taxa-but its base frequencies tend to be nonstationary across such taxa. We conclude that molecular systematists should use combined LSU ϩ SSU rRNA genes rather than SSU alone.

Molecular evidence that phoronids are a subtaxon of brachiopods (Brachiopoda: Phoronata) and that genetic divergence of metazoan phyla began long before the early Cambrian

“…In these trees, the expected relationships of deuterostome, ecdysozoan and polychaete were found (i.e. as in Mallatt and Winchell 2002), suggesting that the alignment itself was not suspect. Similar results were obtained when the other more distant outgroups (polychaete, nemertean, priapulan) were used as sole outgroup in turn or in various combinations.…”

“…Newly determined SSU regions were amplified in three overlapping fragments as described by Cohen et al (2004). The sequenced LSU region was also obtained in three overlapping fragments, using amplification and sequencing primers from published descriptions (Hillis and Dixon 1991;Mallatt and Winchell 2002), but renumbered according to their forward (5 0 ) or reverse (3 0 ) positions in the articulate brachiopod Terebratalia transversa LSU sequence (GenBank accession AF342802), as follows: forward primers F700, F1033, F1483, F1818, F2118; reverse primers R1007, R1039, R1460, R1797, R2105, R2743. Satisfactory amplification was generally obtained with an annealing temperature of 50 1C, but occasionally required a few initial cycles at 45 1C followed by continued amplification at 55 1C.…”

“…(1) Quasi-complete, nuclear-encoded, ribosomal small subunit rDNA (18S or SSU rDNA), because it is widely used to resolve the high-level relationships of metazoans and has been used in several attempts to place brachiopods and phoronids in relation to other phyla (Halanych et al 1995;Gawthrop 1996, 1997;Cohen et al 1998a, b;Cohen 2000;Mallatt and Winchell 2002). However, this sequence alone is unable to resolve the inter-relationships of most lophotrochozoan, protostome phyla with strong support (e.g.…”

Multigene phylogenies of clonal Spumella-like strains, a cryptic heterotrophic nanoflagellate, isolated from different geographical regions

“…The reasoning for such multigene phylogenies is that a larger number of characters improves phylogenetic accuracy and resolution when the number of alignable and informative nucleotides in single-gene analyses is too limited or the rates of sequence evolution are too different (Poe & Swofford, 1999). This approach has been successful in minimizing difficulties based on single protein-coding gene evolution in plants (Bowe et al, 2000;Karol et al, 2001;Mallatt & Winchell, 2002), animals (Mallatt & Winchell, 2002), various groups of algae (Gontcharov et al, 2004;HoefEmden et al, 2002;Nozaki et al, 2000) and the radiation of eukaryotes (Baldauf et al, 2000;Parfrey et al, 2006). The combination of seven different genes seems to minimize any potential bias in phylogenetic reconstruction of Spumellalike strains that is caused by single genes in single taxa.…”

The phylogeny of the Schistosomatidae based on three genes with emphasis on the interrelationships of Schistosoma Weinland, 1858

“…Olson et al 2001 ;Olson & Littlewood, 2002 ;Olson et al manuscript submitted). However, rather than relying on partial lsrDNA alone, there is growing evidence that combining the complete sequences of both genes adds stability and resolution at a number of levels within and between metazoan taxa (Mallatt & Winchell, 2002 ;Medina et al 2001), including platyhelminths (Lockyer, Olson & Littlewood, 2003). Additionally, almost complete mitochondrial COI was sequenced in order to provide greater resolution among more closely related taxa.…”