General hypotheses on arthropod phylogeny are rapidly being altered by DNA sequence data 123. For instance, the Atelocerata concept held that hexapods and myriapods are united in one clade, but under the influence of molecular data (e.g. 4) this concept was replaced by the view that crustaceans and hexapods constitute a monophyletic group, which is known as Pancrustacea (e.g. 235).

Another recently proposed, but still highly debated viewpoint is the diphyletic origin of Hexapoda, which was initially raised by Nardi and co-workers in 2003 6. Based on four mitochondrial genes, they 6 observed that two species of Collembola (Tetrodontophora bielanensis and Gomphiocephalus hodgsoni) branched off before the other pancrustacean groups that were included in their study (Insecta and Crustacea), suggesting paraphyly of Hexapoda. Their thesis was that the six-legged body plan of Collembola and other hexapods evolved at least twice: once in the group of wingless hexapods and another time in the true insects.

The conclusions of Nardi et al. 6 resulted in a vivid scientific debate, and many studies have addressed the phylogenetic placement of Collembola since then. Some authors focused on mitochondrial sequences, others analyzed nuclear genes. Additional mitochondrial sequences confirmed that, due to the placement of Collembola separate from the other hexapods, Hexapoda are indeed not monophyletic 37. However, after thorough analyses exploring the effects of outgroup and gene choice, sequence handling and optimality criteria on inferred trees, Cameron and co-workers 8 concluded that the mitochondrial data as available at the time were inadequate to fully resolve hexapod relationships 8. Hassinin 9 arrived at a similar conclusion in a more recent study focusing on the effects of reverse strand-bias. Most recently, Carapelli and co-workers 10 reported new analyses on a very large dataset, consisting of no fewer than a hundred almost-complete mitochondrial genomes. These new analyses, which were based on a novel model of amino acid sequence evolution (MtPan), supported the non-monophyly of hexapod groups.

It has gradually become clear in pancrustacean phylogeny that nuclear and mitochondrial datasets tell different stories, and often result in different conclusions 10. Remarkably, studies that addressed the question using nuclear genomic data (ribosomal RNA and protein-encoding genes) indicate that the Collembola group between crustaceans and insects and that Hexapoda is monophyletic 251112131415161718. However, most of those studies included a relatively small number of loci 2, most likely because obtaining data on protein-encoding DNA sequences is not always straightforward for groups for which little genomic information is available. Here we try to fill this gap by re-evaluating the position of Collembola using a relatively large number of nuclear protein-encoding sequences that are, although all for ribosomal proteins, assumed to be distributed throughout the genome (see for example 19).

Several authors have shown that publicly available data can be useful when conducting a large-scale phylogenetic study (eg. 20), and that expressed sequence tags (ESTs) can be extremely valuable for phylogenetic purposes 2122. Here, we combine data from a recently finished EST sequencing project on the collembolan Folsomia candida 23, with data on 34 ecdysozoan species (Chelicerata, Hexapoda, Tardigrada, Nematoda and Crustacea) available in the public GenBank repository 24, and with data from a smaller EST dataset of the collembolan Orchesella cincta. We focus on ribosomal proteins to prevent the problem of analyzing paralogous genes (sensu 21).

In total, gene-sequences for 48 ribosomal proteins were obtained from the Folsomia candida EST dataset. This is almost two-thirds of the total set of 79 ribosomal proteins 19 found in the genome of Drosophila melanogaster. Four D. melanogaster ribosomal protein sequences (RpL15, RpL32, RpL36 and RpL39) showed high similarity with two, instead of one F. candida transcript cluster in the EST dataset. Comparison of the F. candida transcripts with those of D. melanogaster revealed insertions/deletions resulting in frame shifts in one of the two F. candida EST clusters for RpL15, RpL32 and RpL36. Transcripts with a frame-shift were discarded. Two highly diverse F. candida EST clusters (one consisting of six EST sequences and one singleton sequence) showed homology with D. melanogaster RpL39. The F. candida RpL39 singleton sequence was excluded from further analysis. The discarded RpL15, RpL32, RpL36 and RpL39 transcripts may stem from duplications in the F. candida genome (for example, in D. melanogaster nine ribosomal proteins are represented by two separate functional genes 19), or from constitutively expressed pseudogenes. This situation may be analogous to the apparent amplification of many mammalian ribosomal proteins; for instance, the human genome contains over 2000 ribosomal protein pseudogenes 25. Still, it seems that only one copy of each ribosomal protein is actually functional 2627.

As described in the methods section, the remaining 48 ribosomal protein sequences were used to retrieve ribosomal protein sequence information on 32 additional ecdysozoan species. In addition, ribosomal protein sequences of D. melanogaster, Apis mellifera and Caenorhabditis elegans were retrieved from the Ribosomal Protein Gene-database (RPG; 28). The number of usable (partial) ribosomal protein gene sequences that were obtained per species ranged from two (4% of the 48 genes: Amblyomma variegatum) to 48 (100% of the 48 genes: D. melanogaster and Apis mellifera) (Table 1). Redundancy for a given ribosomal protein gene in a given species was often low, and many gene sequences were represented by one or a few EST sequences only. It should be mentioned that due to this rather low sequence coverage the dataset is most probably not free from sequencing errors. Furthermore, none of the 48 ribosomal proteins that were included in the dataset were observed in all of the 36 species investigated (Table 1 and Additional file 1). In summary, for each ribosomal protein gene information was available for 25% to 86% of the taxa.

We calculated "similarity" values between the amino acid sequences of F. candida and three well-represented species (C. elegans (most-distant outgroup), Daphnia magna and A. mellifera). These values were mapped onto a ternary graph (Figure 1). Almost all points cluster in the lower region of the ternary graph, showing that for almost all genes the distance between F. candida and C. elegans is greater than the distance between F. candida and A. mellifera. The graph also shows that most genes of F. candida are more "similar" to A. mellifera, while some have more in common with D. magna.

The individual alignments were concatenated and phylogenetic analyses were conducted to investigate the position of Collembola. Two species were excluded from the analyses (Table 1). The final alignment had a length of 5034 inferred amino acids, representing in total 15,102 nucleotides. Information was available for 66% of the amino acid positions.

Likelihood mapping was applied to obtain estimates of phylogenetic signal. The concatenated dataset contained more phylogenetic signal (89% fully resolved quartets) than each of the independent ribosomal protein alignments (9–72% fully resolved quartets; data not shown).

The trees obtained by the different tree-reconstruction algorithms were highly comparable (Figure 2). In all reconstructions (MP, ML and Bayesian), Chelicerata and Pancrustacea each formed a monophyletic group, with relatively high support (Bayesian posterior probabilities both 100%). The two branchiopods included in this study (D. magna and Artemia franciscana) grouped together, and remained separate from the other crustaceans (Malacostraca).

The relationships within the Insecta were weakly resolved; however, Diptera was recovered as a monophyletic clade, as were Lepidoptera and Coleoptera (Figure 2). However, the Hemiptera were resolved as a paraphyletic group. Homalodisca coagulata grouped with Locusta migratoria (Orthoptera), rather than with the other hemipterans Acyrthosypon pisum and Toxoptera citricida. The highly supported, but obviously incorrect, positioning of Homalodisca coagulata does not seem to be an artefact of the method that allowed for missing data, since all three Hemiptera, as well as Locusta migratoria, were represented by a large number of ribosomal protein gene sequences (32 to 47). The incorrect placement of H. coagulata could be a consequence of the inability of ribosomal protein genes to resolve more recent evolutionary splits, which may be a trade-off of their suitability for deeper phylogenies.

Hexapoda was clearly monophyletic: Both collembolans (F. candida and O. cincta) grouped together and formed the sister-group to the Insecta in all analyses conducted (ML bootstrap RtRev+G+F = 91%, ML bootstrap Wag+G+F = 86%, MP bootstrap = 78%, Bayesian posterior probabilities = 100%).

Several C. elegans and D. melanogaster ribosomal proteins are duplicated (see also RPG database). MP analyses of a second concatenated dataset that contained D. melanogaster homologs for ribosomal protein RpS5, RpS15A, RpS19, RpS28, RpL34 and RpL10A resulted in a similar topology (data not shown).

In this study we reassessed the position of Collembola, using (partial) genes for 48 nuclear encoded proteins. The main result of our study is clear evidence of monophyly of Hexapoda. All phylogenetic reconstruction methods employed in this study support this hypothesis (Figure 2). Based on our nuclear dataset we conclude that the six-legged body plan, as found among insects and Collembola, evolved only once in the course of evolution. This is in contrast to results obtained using large mitochondrial datasets 36910 that by and large suggest that the characteristic hexapod body plan was acquired in parallel by Collembola and insects due to convergent evolution, rather than by descent.

Discrepancies between pancrustacean relationships as revealed by either nuclear or mitochondrial datasets seem almost universal. It is of major importance to focus on the causes of these discrepancies, and whether or not one of the two types of markers is superior. Elaborate discussions on the 'pros and cons' of one or both of the two different markers, and the possible approaches on how to correct for ambiguous signals are given in several recent papers 8929303132. Comparative studies that contrast nuclear and mitochondrial datasets suggest that nuclear markers are preferred in deep arthropodan molecular phylogenetics, as mitochondrial genes tend to be more substitutionally biased and evolve (in general) in a much faster way 30.

Already in 1999 Curole and Kocher 33 stated in a review paper that the value of mitochondrial genes in deep-level phylogeny is debatable and that "controversial" mitochondrial DNA (mtDNA) results should be verified with nuclear encoded genes. This was also the final conclusion of Springer and co-workers 34. These authors compared the usability of nuclear and mitochondrial encoded genes in inferring deep-level mammalian phylogenies. The authors report that nuclear encoded genes (exons) outperform mitochondrial markers in resolving deep splits. Springer and co-workers suggest that the reason for this dissimilarity in resolving-power might be found, among others, in the rate of nucleotide substitution 34.

Still, although the nuclear protein-encoding sequences in the study of Springer et al. 34 outperformed the mitochondrial genes, mtDNA-based studies are not necessarily useless for deep phylogeny. They are only problematic if mitochondrial genomes evolve at such a rate that saturation of substitutions makes actual phylogenetic signals from deeper nodes hazy 35. Otherwise, analyses using appropriate models should still be able to retrieve a plausible tree 35. In a recent study, Kjer and Honeycutt 35 used an approach that included all data found in mitochondrial genomes (including for instance 3^rd codon positions, but excluding the control region). After applying a site-specific rate model, these authors retrieved a phylogenetic tree of mammals that was in accordance with recent nuclear DNA based phylogenies 35.

When investigating cheliceratan relationships Jones et al. 36 arrived at a comparable conclusion. These authors state that mtDNA can be applied in molecular phylogenetics, but only when an appropriate substitution model (e.g. to correct for strand-bias) is used. These authors state as a final remark that earlier mtDNA studies that focused on deep-phylogenetic questions should be thoroughly re-evaluated 36. However, such models of mitochondrial sequence evolution might first need to be developed before Collembola can be placed with certainty in the arthropod phylogenetic tree. As mentioned before, Carapelli and co-workers 10 investigated an innovative pancrustacean-model of mitochondrial protein change. This model significantly aided the tree building, but did not yield a monophyletic Hexapoda 10.

An advantage of ribosomal protein genes is that the sequences of different species can be relatively easily homologized due to their conserved nature. However, there are also disadvantages. Although ribosomal protein genes are distributed all over the genome, they definitely do not evolve independently. Coevolving sites are known to exist in ribosomal proteins 37. For example, amino acid residues that are near tRNA binding sites in the ribosome appear to evolve in a related manner 37.

It has to be mentioned that we included only two Collembola in our analyses. Preferably, more springtail species, and maybe even more importantly, proturans and diplurans, should be included. Those latter basal hexapod groups were excluded from the current analysis as they lack available (EST) data. While earlier work suggests that proturan and dipluran genes might be fairly divergent from other arthropods 11, this and other papers (e.g. 2122) suggest that it should be relatively easy to obtain phylogenetically relevant sequence information on those groups by EST sequencing.

Another intriguing result of this study is the non-monophyly of the crustaceans. The branchiopods D. magna and A. franciscana clustered with the hexapods rather than with the other crustaceans in the malacostracan group. This is in accordance with studies by Regier and co-workers 5 and Mallat and Giribet 12, which suggests that the hexapod lineage evolved from within the crustaceans 38. The observed close relationship between hexapods and branchiopods, in combination with some other characteristics, made Glenner and co-workers 38 suggest that branchiopod groups colonized terrestrial ecosystems as insects.

As a final remark we would like to point out that this study shows that Collembola occupy a crucial position. Obtaining additional (EST) sequence information on Collembola, as well as other basal hexapods (Protura, Diplura and Microcoryphia) will definitely result in a better understanding of the phylogenetic origin of insects.

The phylogenetic efforts presented here clearly show that Collembola is a sister group of Insecta (Figure 2). Our results reinforce the discrepancy between results obtained using mitochondrial and nuclear datasets. It seems of major importance to unravel the underlying causes of the disagreements observed, or otherwise focus on nuclear encoded genes.

MJTNT, DR and NMvS designed the study. MJTNT conducted the bioinformatic and phylogenetic analyses and wrote the manuscript. JM generated Orchesella cincta EST data. DR, NMvS and JM commented on the manuscript. All authors read and approved the last version of the manuscript.