Evolution of a novel subfamily of nuclear receptors with members that each contain two DNA binding domains

Background Nuclear receptors (NRs) are important transcriptional modulators in metazoans which regulate transcription through binding to the promoter region of their target gene by the DNA binding domain (DBD) and activation or repression of mRNA synthesis through co-regulators bound to the ligand binding domain (LBD). NRs typically have a single DBD with a LBD. Results Three nuclear receptors named 2DBD-NRs, were identified from the flatworm Schistosoma mansoni that each possess a novel set of two DBDs in tandem with a LBD. They represent a novel NR modular structure: A/B-DBD-DBD-hinge-LBD. The 2DBD-NRs form a new subfamily of NRs, VII. By database mining, 2DBD-NR genes from other flatworm species (Schmidtea mediterranea and Dugesia japonica), from Mollusks (Lottia gigantean) and from arthropods (Daphnia pulex) were also identified. All 2DBD-NRs possess a P-box sequence of CEACKK in the first DBD, which is unique to 2DBD-NRs, and a P-box sequence of CEGCKG in the second DBD. Phylogenetic analyses of both DBD and ligand binding domain sequences showed that 2DBD-NR genes originate from a common two DBD-containing ancestor gene. A single 2DBD-NR orthologue was found in Arthropoda, Platyhelminths and Mollusca. Subsequent 2DBD-NR gene evolution in Mollusks and Platyhelminths involved gene duplication. Chromosome localization of S. mansoni 2DBD-NR genes by Fluorescent in situ hybridization (FISH) suggests that 2DBD-NR genes duplicated on different chromosomes in the Platyhelminths. Dimerization of Sm2DBDα indicates that 2DBD-NRs may act as homodimers, suggesting either that two repeats of a half-site are necessary for each DBD of 2DBD-NRs to bind to its target gene, or that each 2DBD-NR can recognize multiple sites. Conclusion 2DBD-NRs share a common ancestor gene which possessed an extra DBD that likely resulted from a recombination event. After the split of the Arthropods, Mollusks and Platyhelminths, 2DBD-NR underwent a recent duplication in a common ancestor of Mollusks, while two rounds of duplication occurred in a common ancestor of the Platyhelminths. This demonstrates that certain NR gene underwent recent duplication in Prostostome lineages after the split of the Prostostomia and Deuterostomia.


Background
Nuclear receptors (NR) regulate homeostasis, differentiation, metamorphosis and reproduction in metazoans. Members of the nuclear receptor superfamily are characterized by a modular structure. Typical NRs contain an Nterminal A/B domain, a C domain (DNA binding domain, DBD), a D domain (hinge) and an E domain (ligand binding domain, LBD). The most conserved region in NRs is the DBD, which contains two zinc finger motifs (CI and CII). There is a conserved sequence element in the DBD, called the P-box, which confers target DNA binding specificity. Another moderately conserved region is the LBD [1][2][3]. Two highly conserved regions are present within the LBD. The first region is called the signature sequence of LBD (Tau, Tτ), from the C-terminus of helix 3 to the middle of helix 4 [1,4]. The second conserved region is helix 12 which contains the activation function core motif (AF2-AD) that is required for transcriptional activation and co-regulator recruitment. NRs regulate transcription through binding to the promoter region of their target gene by the DBD and activation or repression of mRNA synthesis through co-regulators bound to the LBD [5].
Recently, we isolated three partial cDNAs of nuclear receptors which contain two DBDs in the flatworm Schistosoma mansoni [6]. Typical NRs only have a single DBD with a LBD, unusual nuclear receptors are known only to have one DBD without a LBD [7][8][9][10] or to posses a LBD without a DBD [11,12]. To determine the modular structure of these novel nuclear receptors (that is, whether they contain a LBD), cDNAs encoding the entire open reading frame (ORF) of Sm2DBD-NRs were isolated. By data mining, additional 2DBD-NRs were identified in species of Mollusca, Arthropoda and other species of Platyhelminths. The phylogenetic relationship of 2DBD-NRs was constructed, the origin of 2DBD-NRs and their role in understanding metazoan phylogeny is discussed.

A novel NR modular structure: A/B-DBD-DBD-hinge-LBD
cDNAs of three S. mansoni 2DBD-NRs (Sm2DBDα, 5144 bp, Sm2DBDβ, 5525 bp and Sm2DBDγ, 6374 bp) were isolated. Each cDNA encodes a large protein: Sm2DBDα, 1527 amino acids; Sm2DBDβ, 1523 amino acids and Sm2DBDγ, 1816 amino acids (Fig. 1A). Each protein exhibits a modular structure characteristic of the nuclear receptor superfamily with a divergent N terminal A/B domain, a hinge region and a less well conserved LBD. Remarkably, each possesses two DBDs in tandem (Fig.  1A). Thus the A/B-DBD-DBD-hinge-LBD organization represents a novel NR modular structure. All the members of this novel group have been placed in a new subfamily, NR VII.

Identification of 2DBD-NR in other organisms
By an extensive search of whole genomic sequence (WGS) databases extracted from NCBI, three 2DBD-NRs were found in the freshwater turbellarian Schmidtea mediterranea, two were found in the mollusk Lottia gigantean (owl limpet) and one was found in the crustacean Daphnia pulex (water flea) ( Fig. 2 and additional file 1). No 2DBD-NR or any sequence encoding a P-box sequence of CEACKK, which is unique to the first DBD of 2DBD-NR (see below and Fig. 3), was found in sponge (Reniera sp), cnidarian (Hydra magnipapillata and Nematostella vectensis), arthropod insects, sea urchin or vertebrate sequence data bases. One additional 2DBD-NR was found in the turbellarian Dugesia japonica in the NCBI EST database ( Fig. 2 and additional file 1).

Sequence analysis and phylogenetic tree construction
Alignment of the deduced DBD sequences showed that all 2DBD-NRs possess a P-box sequence, CEACKK, in the first DBD, and the P-box sequence, CEGCKG, in the second DBD (Fig. 2). A blast search against all available databases showed that the P-Box sequence CEACKK is not present in any other NR. This unique P-box present in the first DBD of 2DBD-NRs suggests a novel target DNA specificity may exist for the first DBD. The P-box sequence of second DBD, CEGCKG followed by the amino acid sequence FFRR (CEGCKGFFRR) is identical to that of most members in NR subfamily I (NR I) suggesting that 2DBD-NRs may have a close functional or evolutionary relationship with receptors in NR subfamily I.
Both Maximum Likelihood method and Bayesian inference analysis show that the first DBD of 2DBD-NRs belongs to one monophyletic group and the second DBD belongs to a separate monophyletic group (Fig. 3). The results suggest that 2DBD-NRs originated from a common ancestor gene. Both the first and the second DBD are sister Functional domains of 2DBD-Nuclear receptors and sequence alignment of the LBD domain Figure 1 Functional domains of 2DBD-Nuclear receptors and sequence alignment of the LBD domain. A. Schematic representation of functional domains of 2DBD-NRs isolated from the fluke Schistosoma mansoni. hRORα (human RAR-related orphan receptor, NM_002943) as an example that shows the 'typical' modular structure of nuclear receptors, which contain an A/B domain, a C domain (DNA binding domain), a D domain (hinge region) and an E domain (ligand binding domain). Three Sm2DBD NRs (Sm2DBDα, Sm2DBDβ and Sm2DBDγ) exhibit a novel modular structure with an AB domain, two tandem DNA binding domains (C1 and C2), a D domain and an E domain. Sm2DBDα possesses an F domain at the C-terminal end of the E domain. The size of each domain in amino acids is indicated. B. Alignment of sequences from Helices 3-12 of the LBD domain of three S. mansoni 2DBD-NRs with that of members in NR subfamily I. Helices described in [64] are boxed, the signature sequence of the LBD (Tτ) is boxed with dash line. The autonomous activation domain (AF2-AD) is indicated and the conserved glutamic acids are shown in bold. Numbers at the end of each line indicate residue positions in the original sequence, amino acids of Sm2DBDα 1371-1527 and hRARα 453-462 are not shown in the alignment. Dark shaded areas show conserved residues in the signature sequence of the LBD. The accession numbers of the aligned human nuclear receptors can be found in additional file 2.  groups to members of NR subfamily I, suggesting that the common ancestral gene of 2DBD-NRs was close to a common ancestral gene of NR subfamily I genes and the extra DBD was gained by a recombination event (Fig. 3). Another phylogenetic tree was constructed employing LBD sequences, the same result was obtained (Fig. 4)

Protein-protein interaction
NRs can regulate transcription as a homodimer or as a heterodimer with retinoid × receptor (RXR), another nuclear receptor. To determine whether 2DBD-NRs may form dimers and to begin to define the quarternary structure of 2DBD-NRs, the interaction of Sm2DBDα, SmRXR1 and SmRXR2 was evaluated in a yeast two hybrid system.
Yeast transformed with pSV40/p53 (positive control), pSV40/plamin C (negative control), pGBK-Sm2DBDα-C-F/pACT-SmRXR1, pAS-SmRXR1-C-F/pGAD-Sm2DBDα, pGBK-Sm2DBDα-C-F/pACT-SmRXR2, pAS-SmRXR2/ pGAD-Sm2DBDα and pGBK-Sm2DBDα-C-F/pGAD-Sm2DBDα grew on SD/-trp-leu plate as expected ( Fig. 5Aa). If Sm2DBDα interacts with SmRXR1, SmRXR2 or with itself, the Gal4 binding domain fusion partner bound to the Gal1 UAS element will interact with the Gal4 activation domain to drive transcription of the reporter gene. Yeast co-transformed with pGBK-Sm2DBDα-C-F/pGAD-Sm2DBDα grew on SD/-trp-his-leu plus 3 mM 3-AT, indicating that Sm2DBDα can act as a homodimer. Yeast cotransformed with pGBK-Sm2DBDα-C-F/pACT-SmRXR1, pGBK-Sm2DBDα-C-F/pACT-SmRXR2 or pAS-SmRXR1-C-F/pGAD-Sm2DBDα and pAS-SmRXR2/pGAD-Sm2DBDα did not grow on SD/-trp-his-leu plus 3 mM 3-AT plates, indicating that Sm2DBDα did not interact with SmRXR1 nor SmRXR2 (Fig. 5A). The positive control plasmids, pSV40/p53, grew on SD/-trp-his-leu plus 3 mM 3-AT plates while the negative control plasmids, pSV40/plamin C did not (Fig. 5A). The results show that Sm2DBDα can interact as a homodimer, but can not interact with Alignment of the deduced peptide sequences of DNA binding domains of 2DBD-NRs Figure 2 Alignment of the deduced peptide sequences of DNA binding domains of 2DBD-NRs. Each DBD is boxed with a solid line, P-box sequences (bold letters) are boxed with a dashed line. Stars identify the conserved cysteine residues that comprise the zinc finger of each DBD. Dashes indicate gaps in the sequence. The deduced amino acid sequence between DBDs for the NR from species other than S. mansoni is indicated with dots as we could not differentiate in the in silico analysis, exon sequence from intron sequence. All 2DBDs possess a P-box sequence of CEACKK in the first DBD and a P-box sequence of CEGCKG in the second DBD. Sm: flatworm Schistosoma mansoni, Se: flatworm Schmidtea mediterranea, Dj: flatworm Dugesia japonica, Lg: mollusk Lottia gigantea, Dp: arthropod Daphnia pulex.

The first DBD
The second DBD Maximum Likelihood phylogenetic tree produced using sequences of NR ligand binding domain Figure 4 Maximum Likelihood phylogenetic tree produced using sequences of NR ligand binding domain. Phylogenetic relationship was examined by the Maximum Likelihood (ML) method as described for Fig. 3. Support values for the tree were obtained by bootstrapping a 100 replicates and are indicated above each branch. Branches under the threshold value of 46 (this value was set to support the subfamily IV as a monophyletic group) were shown as polytomies. Bayesian inference with the same methods as in Fig. 3, by running 3 million generations. The PPs are shown below each branch or after the ML bootstrapping value separated by a slash. Star indicates the node obtained form by Bayesian inference which was different from that obtained by the ML method. The accession number of each sequence used for phylogenetic analysis can be found in additiona1 file 2 and 3. SmRXR1 nor SmRXR2. As a strong dimer interface is known to be located in the LBD [23,24], GST pull-down experiments were performed (Fig. 5B). The results verified that the LBDs of Sm2DBDα can form a homodimer in vitro.
Nuclear receptors act on target genes by recognizing and binding to specific DNA core motifs in the promoter region of target genes via their P-box motif located in the DBD. The DNA core motif is a typical consensus hexameric sequence AGGTCA called a half-site. When NRs bind to the half-site as a dimer, two P-boxes and a repeat of half-site, with different orientations and spacings between the half sites, are required. 2DBD-NR can interact as a homodimer indicating that four P-boxes may be involved in DNA binding, thus a novel mechanism of DNA binding, which requires two independent pairs of half-site repeats, or four half-site repeats, each with unknown orientations and spacing, are predicted to exist to allow 2DBD-NR to bind to target DNA cis-elements. The protein of the first DBD, second DBD and the first DBD with second DBD were tested for their ability to bind to a direct repeat, an everted repeat and palindromes of the half-site AGGTCA with 1-6 nucleotide spacings by electrphoretic mobility shift assay (EMSAs). No binding compared to controls was observed (data not shown). However, as the flanking sequence of the AGGTCA motif and the spacing between half sites also determines the protein binding to the DNA element, further experiments will be performed using different sets of templates and by determining DNA binding sites using a PCR/EMSA-based approach.
If the traditional phylogenetic scheme is correct, 2DBD-NRs originated in a common ancestor of the Bilateria, because 2DBD-NRs were found in both Acoelomates (flatworms) and Coelomates (mollusks and arthropods) (Fig.  6A). The 2DBD-NR was lost in Pseudocoelomates (nema-todes) after the split of Pseudocoelomates and Coelomates (Protostomes and Deuterostomes). As 2DBD-NRs have not been found in Deuterostomes, they were lost in the Deuterostome lineage after the split of Protostomes and Deuterostomes (Fig. 6A).
If the molecular phylogeny hypothesis is correct, there were two possibilities for 2DBD-NR origin. The 2DBD-NR might originate in a common ancestor of Protostomes, since 2DBD-NRs were identified both in Lophotrochozoans (Platyhelminths and Molluscs) and in Ecdysozoans (Crustaceans) (Fig. 6B). The other possibility is that 2DBD-NR might originate in a common ancestor of the Bilateria and was lost in the Deuterostome lineage after the split of Protostomes and Deuterostomes. 2DBD-NR is absent in nematodes suggesting that this gene was lost after the split of the nematodes and arthropods. In arthropods, no 2DBD-NR was found in insects suggesting 2DBD-NR was lost after the split of insects and crustaceans (Fig. 6B).
The phylogeny of the Platyhelminths has itself been under debate (eg. [34][35][36][37]). The Platyhelminths have always played a central role in hypotheses concerning metazoan phylogeny and evolution. Recently, many platyhelminth flatworms, previously regarded in the traditional phylogeny as basal bilaterians (Fig. 6A), are now placed within the lophotrochozoan protostomates (Fig. 6B). Furthermore, the Acoelomorpha (Aceola + Nerertoderdermatida) are no longer considered part of the Platyhelminths but are still considered basal bilaterians [34][35][36][37][38]. Certainly, further studies on nuclear receptor evolution in these taxa can contribute to our understanding of the evolution of the Metazoa and Bilateria, especially as nuclear receptors have been identified in sponges [39], a group that is hypothesized to have given rise to the hypothetical metazoan ancestor [40].
The duplication of 2DBD-NRs was deduced from phylogenetic analysis employing the first and the second DBD sequences as a unit. Maximum Likelihood method and Bayesian inference were performed and the same result was obtained (Fig. 7A). Since the results show that there is only one Arthropod, Mollusk and Platyhelminth 2DBD-NR orthologue, the duplication of the ancient 2DBD-NR first occurred after the split of Arthropods, Mollusks and Platyhelminths (Fig. 7B). The absence of a 2DBD-NR in the insect and nematode lineages supports this hypothesis, since it is likely that there was less of a chance for all earlier duplicated genes to be lost in all the animals in which no 2DBD-NR was found. The analysis also showed that 2DBD-NRs underwent two rounds of duplication in a common ancestor of the Platyhelminths that eventually gave rise to three genes. The 2DBD-NRα and 2DBD-NRβ are considered the most recently duplicated, since the orthologue of 2DBD-NRγ is found in Crustacea and Mollusca (Fig. 7A, B). Interestingly, a single gene is found in the crustacean, one duplication event occurred in mollusks and two duplications occurred in the Platyhelminths.
S. mansoni bacterial artificial chromosome (BAC) clones containing Sm2DBDα (BAC: CHOR-18I10) were identified by screening the S. mansoni CHOR-1 BAC library. BAC clones containing Sm2DBDβ (BAC: SmBAC1 54O21) and Sm2DBDγ (BAC: SmBAC1 18F9) were iden-Protein-protein interaction of Sm2DBDα Figure 5 Protein-protein interaction of Sm2DBDα. A. Yeast two hybrid assays show that S. mansoni 2DBD-NR (Sm2DBDα) can act as a homodimer but not as a heterodimer with SmRXR1 and SmRXR2. Yeast AH109 was transformed with 1 μg of the following co-transformats: 1) pGBK-Sm2DBDα-C-F/pACT-SmRXR1, 2) pGAD-Sm2DBDα/pAs-SmRXR1-C-F, 3) pGBK-Sm2DBDα-C-F/pGAD-Sm2DBDα, 4) negative control plasmid pSV40/plamin C, 5) positive control plasmid pSV40/p53, 6) pGBK-Sm2DBDα-C-F/pACT-SmRXR2, 7) pGAD-Sm2DBDα/pAS-SmRXR2. Transformed yeast were plated on SD/-trp-leu and SD/-trp-his-leu-ade plus 3 mM 3-amino-1,2,4-triazole (3-AT). The results show that Sm2DBDα can form a homodimer but not a heterodimer with SmRXRs. B. GST pull down verified that the S. mansoni 2DBDα-E-F domain, in which the dimer interface is located, can form a homodimer in vitro. Duplication of 2DBD-NRs. A. Maximum Likelihood phylogenetic tree derived from sequences in the first and the second DBD of 2DBD-NRs. Phylogenetic relationship was examined by the Maximum Likelihood method as described for Fig. 3. Support values for the tree were obtained by bootstrapping a 100 replicates and are indicated above each branch. Branches under the threshold value of 45 were shown as polytomies. Bayesian inference with the same methods as in Fig. 3 running 5 million generations. The PPs are shown below each branch or after the ML bootstrapping value separated by a slash. B. Figure shows that the common ancestor gene of 2DBD-NRs underwent duplication after the split of the Arthropods, Molluscs and Platyhelminths. In Mollusks, the orthologue gene (γ) underwent a duplication giving rise to the α/β gene. In Platyhelminths, the orthologue gene (γ) underwent a duplication giving rise to a new gene, this new gene underwent a recent duplication to give birth to the two present genes (α and β genes). All three genes are present in the flatworm S. mansoni and the planarian S. mediterranea suggesting that the two rounds of 2DBD-NR duplication occurred in a common ancestor of the Platyhelminths. A tified by blast searching databases of S. mansoni BAC ends and verified by PCR. Sm2DBDα, Sm2DBDβ and Sm2DBDγ were localized on chromosomes by FISH using BAC DNA as the probe (Fig. 8). The results show that Sm2DBDα localized to chromosome 1. Sm2DBDβ is located on sex chromosomes Z and W and chromosome 3 indicating that there are two copies of Sm2DBDβ. However, there are other possibilities. It could be a fourth gene. However, we do not think that it is a fourth gene as we should have found it in our search of the genome sequence. It might be a pseudogene or a second copy of the 2DBDβ gene. We favor a second copy of the genes as the probe hybridizes to the euchromatic region of the Z and W. However, we cannot rule out that it is an artifact due to hybridization of a repetitive sequence in the BAC clone. Sm2DBDγ is localized on chromosome 4. In figures 9a and 9c a repeat sequence in the BAC clones that commonly hybridizes to the W chromosome is shown as well. The results suggest that 2DBD-NRs duplicated among different chromosomes in a common ancestor of the Platyhelminths.
Analysis of the NR superfamily, mainly in Drosophila and vertebrates supports the hypothesis that evolution of nuclear receptors occurred by two serial rounds of duplication [41][42][43][44][45]. The duplication of 2DBD-NRs suggests that certain NR genes have undergone recent duplication in invertebrates after the divergence of various clades within the Bilateria. Our previous study of S. mansoni NRs supports this hypothesis [6]. NRs in insects seem to have undergone extensive gene loss. For example, a recently identified estrogen receptor in the mollusk Aplysia californica [46] and two thyroid hormone receptors in the Platyhelminth S. mansoni [6,47] are missing in the insect genera Drosophila and Anopheles. To address the importance of gene duplication in NR evolution, more invertebrates NR complements await to be analyzed.

Developmentally Regulated Expression
Quantitative real-time RT-PCR was performed to evaluate mRNA expression of Sm2DBDα, Sm2DBDβ and Sm2DBDγ. Normalized gene expression (ΔΔCT) [48] was standardized to the relative quantities of S. mansoni αtubulin. Sm2DBDα was detected in secondary sporocysts, cercariae, 21-day schistosomules, 28-day schistosomules, female and male worms. Sm2DBDβ was expressed relatively high in eggs, secondary sporocysts, cercariae and male stages. Sm2DBDγ was only detected in cercariae and 28-day worms. The results indicate that the three genes are developmentally regulated and thus have a role in different development stages (Fig. 9). It is of note that Sm2DBDγ, the putative ancestral gene is only expressed in 2 of the developmental stages studied and that both Sm2DBDα and Sm2DBDβ show sex-specific gene expression.

Conclusion
A protein modular structure containing an AB domain, two DNA binding domains in tandem, a hinge region and a ligand binding domain (A/B-DBD-DBD-hinge-LBD) represents a novel NR modular structure, and is named 2DBD-NR. 2DBD-NRs were identified from mollusks, arthropods (crustaceans) and flatworms. 2DBD-NRs may act as homodimers. 2DBD-NRs share a common ancestor gene which possessed an extra DBD that likely resulted from a recombination event. 2DBD-NRs were found in flatworms, mollusks and arthropods whose phylogeny is still under debate [30,31,33] (Fig. 6A and 6B). Further studies of 2DBD-NR gene subfamily may contribute to our understanding of gene duplication as an evolutionary force and to the phylogeny of the Metazoa. The conserved zinc finger motifs in each of the two DBDs are the most readily recognized features of 2DBD-NRs. The P-box sequences in the first DBD and the second DBD give members of the 2DBD-NR their unique feature. This feature makes 2DBD-NRs an interesting gene subfamily for studies of metazoan phylogeny.

Isolation of 2DBD-NR cDNAs in the Platyhelminth S. mansoni
cDNAs encoding the entire open reading frame (ORF) of three S. mansoni 2DBD-NRs (Sm2DBDα, Sm2DBDβ and Sm2DBDγ) were isolated by a PCR strategy using a S. mansoni female worm cDNA library (pBluescript SK (+/-) phagemid) pool as template DNA [6,22]. The PCR primers for one end (either 5' or 3' end) were designed according to a fragment encoding the previously identified DBD region of these genes [6]. The primer for the other end (either 5' or 3' end) was a vector universal primer (M13-Rev and T3, or M13-For and T7 primers). PCR products were separated on 1.2% agarose gels, ligated into pCR2.1 TOPO vector (Invitrogen) and sequenced. After the correct fragments were identified, the cDNA sequence containing the 5' UTR, ORF and 3' UTR were obtained by PCR. The cDNAs were shown to be related to a single mRNA species by sequencing the PCR products obtained from single-stranded cDNA using primers located within the 5'UTR and 3'UTR of each gene.

Data mining
Whole genomic sequences (WGS) were extracted from the GenBank public ftp site [49] (up to October 2005) and imported into StarBlast program (DNASTAR) to build a local database which was screened by tblastn using the sequence of the first and second DBD of Sm2DBDα as the query. Any sequence that contained a zinc finger structure of the DBD (Cys-X2-Cys-X13-Cys-X2-Cys or Cys-X5-Cys-X9-Cys-X2-Cys) was retained. Sequence walking was carried out to assemble the contigs. Website databases of GenBank (nr, EST_human, EST_mouse and EST_other databases) [50], European Bioinformatics Institute [51] and Swiss-Prot [52] were also mined by tblasn or blastp using the same query sequence as above.

Phylogenetic tree construction
Phylogenetic trees were constructed from deduced sequences of the DBD and the LBD, respectively. Sequences were aligned with ClustalW [53]. Phylogenetic analysis of the data set was carried out using the Maximum Likelihood method under Jones-Taylor-Thornton (JTT) substitution model [54] with a gamma distribution of rates between sites (eight categories, parameter alpha, estimated by the program) using PHYML (v2.4.4)) [55]. Support values for the tree were obtained by bootstrapping a 100 replicates. The same data set was also tested by Bayesian inference using MrBAYES v3.1.1 with a mixed amino acid replacement model + invgamma rates (Huelsenbeck and Ronquist, 2001). The trees were started randomly; four simultaneous Markov chains were run for 5 million generations for the DBD data set and 3 million generations for the LBD data set, respectively. The trees were sampled every 100 generations. Bayesian posterior probabilities (PPs) were calculated using a Markov chain Monte Carlo (MCMC) sampling approach implemented in MrBAYES v3.1.1, with a burn-in value setting at 12,500 for DBD data set and 7,500 generations for the LBD data set, respectively.

BAC clone screening and localization of 2DBD-NRs on chromosomes of S. mansoni
S. mansoni BAC clones containing Sm2DBD-NRs were identified by screening the S. mansoni CHOR-1 BAC library with methods previously described [59] or by blast searching databases of S. mansoni BAC end sequences in TIGR [60] and verified by PCR. Fluorescent in situ hybridization (FISH) was performed on S. mansoni sporocyst metaphase chromosome spreads with BAC DNAs that each contained one of the three S. mansoni 2DBD-NRs (Sm2DBDα, Sm2DBDβ and Sm2DBDγ). FISH was performed using techniques previously described [61,62].