Sequences homologous to human exon sequences were derived from filtering tblastn search results on 11 complete eukaryotic genomes. Because the most-criticized issue in resolving the Ecdysozoa-Coelomata problem seems to be the LBAE produced by the nematode species, we decided to rearrange homologous sequences in a series of nested datasets that gradually reduced LBAE. Aligned homologous sequences were arranged in eight datasets (D_i) and concatenated in their corresponding matrices (M_i) (see Materials and methods), such that as suffix i increases, datasets and matrices comprise a smaller number of homologous sequences showing more similar relative branch lengths (RBL) between C. elegans (L_Ce) and D. melanogaster (L_Dm) (Figure 1). RBL are relative human distances.

To quantify the effect on the RBL of C. elegans of concatenating alternative homologous sequences, maximum likelihood (ML) estimates of branch length were obtained using the star-like unrooted tree transformation for each dataset (see Materials and methods). Figure 2a shows that the RBL of C. elegans over D. melanogaster decreased by approximately 30% continuously from dataset D₁ to D₈. To test whether the gradual decrease in C. elegans branch length was enough to produce statistical confidence on equal evolutionary rates between the nematode and the arthropod sequences, relative rate tests using two outgroup schemes were assayed on concatenated sequences (see Materials and methods). Figure 2b shows that using Saccharomyces cerevisae as the unique outgroup species (OUG1), all the individual tests on the eight matrices failed to detect statistical deviations (at the 5% level family-wise) between sequences. Only when the phylogenetically weighted scheme of outgroup species (OUG2) was used did the relative rate test detect significant deviation of clock behavior from D₁ to D₅ datasets. We are therefore confident that the arthropod and nematode concatenated sequences of the M₆, M₇, and M₈ matrices meet the desired clock-like conditions to test the Coelomata and Ecdysozoa hypotheses and exclude any artifacts derived from a possible LBAE. This result supports previous work suggesting that the genetic distance between ingroup and outgroup modifies the power of the relative rate test 20.

To test whether concatenated matrices carry sufficient phylogenetic signal, the ML mapping method was used. The compound posterior probability point (P) for all the possible quartets of each M_i matrix could be placed, with almost equivalent values (approximately 33%), inside the corner areas of the equilateral triangle probability surface (see Additional data file 1). Thus, concatenated matrices derived from selecting a different number of homologous sequences contained sufficient phylogenetic signal to represent topologies as strictly bifurcating trees. Finally, using the Akaike information criterion (AIC) 21, the statistical test of the best-fit model of sequence evolution for each dataset was selected from six different alternatives (see Materials and methods). As all the models are not nested and share the same number of parameters, the best one was that with the greatest log likelihood result. The WAG amino-acid replacement matrix 22 adjusted for frequencies (+F), rate heterogeneity (+Γ) and invariable sites (+I) was the best evolutionary model chosen for all the datasets. Moreover, model-fit-data values followed the same inequality independently of the dataset (WAG 22 > VT 23 > BLOSUM62 24 > JTT 25 > PAM 26 > mtREV24 27), suggesting that the best models were those that consider more distantly related amino-acid sequences.

Distance and ML phylogenetic methods were used on all the datasets (see Materials and methods). Figure 3 shows phylogenetic reconstructions and statistical support for the two extreme conditions of the nested datasets. Whereas the M₁ matrix supported the Coelomata tree with the highest statistical confidence, M₈ showed the same result for the Ecdysozoa tree. Thus, by decreasing the RBL of C. elegans, the statistical support switched from the Coelomata to the Ecdysozoa hypothesis. Figure 4 shows that, whichever phylogenetic method was used, C. elegans bootstrap support between datasets and topologies changed in agreement with the gradual RBL decrement. Specifically, using M₁ and M₈ (the matrices showing the most extreme evolutionary rate conditions for C. elegans and D. melanogaster sequences - from a clock-absent to the most adjusted behavior), the statistical support moved from Coelomata to Ecdysozoa. The same occurred with M₂ and M₇. Alternatively, using M₃ and M₆, only one of the two distance and ML methods (Figure 4a,b) provided sufficient support (90% or more) to the hypothesis. Finally, using M₄ and M₅, only one distance method supported Coelomata and Ecdysozoa with confidence. Given that datasets differed principally in the RBL of C. elegans over D. melanogaster, the gradual change in topology strongly favors an LBAE between C. elegans and the more basal species. To test whether a paired-sites test 28 supports the bootstrap conclusions, Shimodaira-Hasegawa (SH) and expected-likelihood weight (ELW) tests were evaluated on the datasets (see Materials and methods).

Figure 5 shows the assessment of paired-sites tests for the two competing trees on all the datasets. Paired-sites tests supporting topologies (p > 0.05) changed almost gradually on datasets. Figure 5a and 5b show that the SH test is more conservative than the ELW 29. Using matrices M₁ and M₂, both tests strongly rejected the Ecdysozoa hypothesis, whereas M₆, M₇, and M₈ rejected the Coelomata tree. Interestingly, datasets between them did not reject any topology with sufficient statistical evidence. We can conclude that by decreasing the RBL of C. elegans over D. melanogaster by around 13% (Figure 2a) the LBAE favoring the Coelomata hypothesis disappears and we can confirm that under strict conditions of clock-like behavior, the Coelomata hypothesis was strongly rejected by paired-sites tests and bootstrap support.

To test if the shortness of the evolutionary distances between C. elegans and D. melanogaster resulting from the above filtering method biased topology over the common ancestry of arthropods and nematodes, we searched for chordate, arthropod, and nematode sequences showing clock-like behavior between them. To increase the probability of finding sequences to fit the criteria, we focused on sequences from the most closely related chordate to the molting species, that is, the ascidian Ciona intestinalis. Only 14 exon sequences met the above criteria. A relative rate test showed that the probability of a perfect clock-like behavior was p = 0.515 for C. elegans and D. melanogaster, p = 0.308 for C. intestinalis and D. melanogaster and p = 0.712 for C. intestinalis and C. elegans. The ML mapping method showed that the concatenation of all the 810 characters carried sufficient phylogenetic signal in the matrix to represent a strictly bifurcating tree (see Additional data file 2). Despite the reduced number of characters, phylogenetic analysis showed significant support for the Ecdysozoa hypothesis. Using distance and ML methods, bootstrap values reached 97%. Moreover, the Ecdysozoa hypothesis was accepted with a probability of p = 1.00 and p = 0.997 when SH and ELW paired-sites tests, respectively, were performed. Conversely, the Coelomata hypothesis was rejected at p = 0.006 and p = 0.0023, respectively.

In order to discard a probable biased selection of exon sequences favoring the Ecdysozoa hypothesis, two additional matrices were built by removing from the original dataset (D₁) the exons in which the C. elegans sequences evolved at a faster rate. Figure 6 shows that by removing the fastest 15% of total exon sequences the reliability of the Coelomata hypothesis is reduced from 100% to 78%. Moreover, when the fastest 30% of all exons were removed, the topology changes to Ecdysozoa with 90% confidence level. The change in topology in parallel with the reduction of the C. elegans branch length points to the LBAE as the main obstacle to obtaining the true phylogenetic relationship between chordates, arthropods and nematodes. We conclude that the Ecdysozoa hypothesis does not depend on adjusting a particular set of homologous exon sequences to clock-like behavior.

Complete genome sequences from Plasmodium falciparum 41, Arabidopsis thaliana 42, Oryza sativa 43, Saccharomyces cerevisae 44, Caenorhabditis elegans 45, Anopheles gambiae 46, Drosophila melanogaster 47, Ciona intestinalis 48, Fugu rubripes 49, Mus musculus 50 and Homo sapiens 51 were downloaded and formatted to run local BLAST 52. Amino-acid sequences corresponding to all the gene exons in a sample of 18 human chromosome including 6-18, 20-22, X and Y (approximately 14,000 genes and 140,000 exons), were obtained from the Ensembl database project 53. Human paralogous exons were excluded by running local blastp 52 on a human exon database built ad hoc. Only the best of those sequences, with more than a single hit with a fraction of aligned and conserved amino-acid sequence ≥ 95% and ≥ 90% respectively, were retained to find homologous sequences in the other eukaryotic species (threshold values based on a previous human paralogous study 54). We used tblastn 52 that searches a query amino-acid sequence on the six translation frames of the target sequence to search for homology in the complete genome databases of the species mentioned above. Exons less than 22 amino acids were removed from the analysis. Each best hit of tblastn was filtered by means of a threshold e-value (≤ 1e-03) and a threshold proportion of the query over the subject sequence length (≥ 75%). Only those exons that pass through all the species filter conditions were selected as the final dataset of human exon homologous sequences. All the exon homologous sequences were aligned using Clustal W 55 with default parameters. The total number of homologous sequences, derived from 18 human chromosomes, corresponds to 1,192 exons selected from 610 known genes, adding up to more than 55,500 amino-acid characters.

To arrange homologous sequences in different datasets, pairwise distances between sequences were extracted using the PROTDIST program (Kimura option) of the PHYLIP package 56. Distances between C. elegans, D. melanogaster and H. sapiens were transformed into branch lengths in a star-like unrooted tree (l_a = (d_ab + d_ac - d_bc)/2, where l_a is the length of the branch leading to a and d_ab, d_ac, d_bc are the distances between a and b, a and c, and b and c, respectively). It is important to emphasize that we are not considering that the phylogenetic relationships of C. elegans, D. melanogaster and H. sapiens is a star topology. We used this exact equation for determining the branch lengths of the three species, because the unique way to arrange three species in a phylogenetic tree is a star topology. We consider C. elegans, D. melanogaster and H. sapiens to be members of the ingroup and P. falciparum, A. thaliana, O. sativa and S. cerevisae as the outgroup species at the moment to root the phylogenetic tree. Homologous exon sequences were arranged in eight datasets according to their pertinence to more inclusive areas surrounding the straight line representing identical relative branch lengths (RBLs) of C. elegans (L_Ce = l_Ce/l_Hs) and D. melanogaster (L_Dm = l_Dm/l_Hs). The D_i dataset clusters all the homologous exon alignments where L_Dm - δ_i ≤ L_Ce ≤ L_Dm + δ_i, where i is an integer ranging from 2 to 7 and δ_i = 5.0, 3.0,2.5,2.0,15,1.0,0.5. The D₁ dataset contains all the exon homologous sequences without the constraints of evolutionary rates. Exons with negative or undefined normalized distances (l_Hs = 0) were excluded from the analysis. All the aligned homologous exon sequences of the D_i dataset were concatenated in the M_i matrix. Three additional matrices were derived from D₁: two by removing exons containing L_Ce ≥ and L_Ce ≥ , and the last one by adjusting the sequences of C. intestinalis, D. melanogaster and C. elegans to clock-like behavior.

The relative rate test was performed at the 5% statistical level by means of the RRTree program 57 using outgroups with one (S. cerevisae; OUG1) or more species (S. cerevisae, A. thaliana, O. sativa and P. falciparum; OUG2). In the latter case, an explicit weighted phylogenetic scheme was chosen (1/2 S. cerevisae, ((1/8 A. thaliana, 1/8 O. sativa), 1/4 P. falciparum)). Given that three ingroups were set for all analyses (the chordates H. sapiens, M. musculus, F. rubripes, and C. intestinalis; the arthropods Anopheles gambiae and Drosophila melanogaster; and the nematode C. elegans), the threshold value was corrected for multiple testing to 5/3 = 1.7%. TREE-PUZZLE 58 was used to evaluate six alternative evolutionary models adjusted for frequencies (+F), site rate variation (+Γ distribution with two rates) and a proportion of invariable sites (+I), to estimate the amount of evolutionary information of datasets by the likelihood-mapping method 59, to derive the maximum likelihood (ML) trees using the quartet-puzzling algorithm, to set the ML pairwise sequence distances, and to test alternative topologies using SH 60 and ELW 29 tests. The PROML (JTT+f) program of the PHYLIP package 56 was used to estimate ML trees derived from the stepwise addition algorithm. Distance methods of phylogenetic reconstruction were performed using PROTDIST (JTT, Kimura options), NEIGHBOR (neighbor-joining (NJ) 61) and least squares (LS) 62 algorithms, and CONSENSE (50% majority-consensus rule option) programs on 100 bootstrap replications using PHYLIP.