Identification of Lbxgenes in extant Osteichthyes
To reconstruct the phylogeny of vertebrate Lbx genes we first attempted to identify the complete set of these genes in representatives of extant Osteichthyes. For this, BLAST-searches of sequence databases were carried out, using the known human and mouse Lbx1 and Lbx2, chicken Lbx1 and Lbx3, Xenopus laevis Lbx1 and zebrafish Lbx1 sequences as query sequences [1, 2, 4, 9, 14, 15, 18, 19]. To obtain outgroups for these phylogenetic analyses, we also searched the databases for lbx/ladybird sequences in invertebrate deuterostomes, including the cephalochordate Branchiostoma floridae (amphioxus), the urorchordates Oikopleura dioica and Ciona intestinalis, and the echinoderm Strongylocentrotus purpuratus (purple sea urchin); moreover we included sequences from various protostomes. Our search confirmed the presence of two distinct Lbx genes in placental mammals and marsupials (human, mouse, dog, cattle, opossum), one Lbx gene in the still incompletely sequenced platypus and Anole lizard genomes, and two Lbx genes in the chicken. Only one Lbx gene was found for the frog Xenopus tropicalis. On the other hand, besides the gene so far known as zebrafish lbx1 [14], two novel lbx genes were identified in this organism. Three Lbx genes were also identified in the teleosts Takifugu rubripes (fugu), Tetraodon nigroviridis, Gasterosteus aculeatus (stickleback), and two genes in Oryzias latipes (Medaka), while only one lbx/ladybird gene was retrieved for the invertebrate deuterostomes.
Phylogenetic analysis of osteichthyan Lbxprotein sequences
To determine the evolutionary relationship between osteichthyan Lbx genes, we first determined the phylogenetic relationship of Lbx proteins (Fig. 1). For this purpose, amino acid sequences were aligned and analysed, using maximum likelihood methods. We found that the vertebrate Lbx sequences were assigned to two distinct groups. The first group encompassed the known human, mouse, chicken and frog Lbx1 proteins. In addition, this group contained the two novel zebrafish Lbx proteins, encoded by the genes located on chromosomes 13 and 1, the fugu Lbx proteins whose genes are on scaffolds 52 and 62, the Tetraodon protein encoded by the Lbx gene on chromosome 18, the stickleback proteins with genes on groups VI and IX and the medaka protein encoded by the gene on chromosome 1.
The second group contained all of the mammalian Lbx2 proteins, the chicken protein currently known as Lbx3, the zebrafish protein so far named Lbx1 and encoded by the gene on chromosome 14, together with the Lbx sequences encoded by genes on fugu scaffold 70, Tetraodon chromosome 20, stickleback group IV and medaka scaffold 1066. The division into two distinct groups of Lbx proteins was supported by high bootstrap values. Our phylogenetic analysis shows significantly longer branch lengths for the amniote Lbx2 proteins, indicating that these Lbx2 genes have probably evolved at a quicker rate than the Lbx1 genes. No evidence was found for distinct Lbx3/4 proteins.
Comparison of genomic Lbxloci in Osteichthyes
It is generally held that in the lineage leading to jawed vertebrates, two rounds of whole genome duplication occurred, followed by a further genome duplication in the lineage leading to teleosts [20–23, 26]. Thus, theoretically, if no gene loss has occurred, four Lbx genes should be detectable in extant tetrapods and eight in teleost fish. However, our phylogenetic analyses suggest that only two Lbx genes were retained after the second vertebrate genome duplication and before the genome duplication in ray-finned fish. To confirm these results and to further analyse the orthology of the various vertebrate Lbx genes, we compared the organisation of genes associated with the vertebrate Lbx loci, reasoning that orthologous Lbx genes would share a similar chromosomal environment, while paralogous genes would exhibit a distinct arrangement of the locus (see [27–29] for examples).
Since the human genome information is the most accurate and complete, we began by recording the genes that according to the NCBI Map Viewer database are located in the environment of human LBX1 and 2 genes (Fig. 2). Subsequently, we looked for remnants of the LBX3 and 4 loci, initially by searching the human genome for paralogues of the genes co-localising with LBX1 and 2. We then identified and determined the arrangement of genes characteristic for each of these four paralogons in the genomes of other mammals, the chicken, the frog Xenopus tropicalis and the five teleosts (Fig. 3 and additional file 1).
Lbxloci in humans
Human LBX1 has previously been mapped to chromosome 10, 1.7 Mb distant from NKX2.3 [2, 4]. LBX1 is tightly linked to the related Nk-type homeobox gene TLX1 (Fig. 2 and Additional file 1). This is followed by the Kazal-type serine peptidase inhibitor domain 1 gene KAZALD1, the LOXL4 gene encoding a Lysyl oxidase like protein and the SLIT1 gene encoding an axon guidance molecule. Moving in the opposite direction, LBX1 is linked to a Beta-transducin repeat containing gene (BTRC1, a F-box and WD repeat domain 11 type gene), the DNA polymerase lambda gene (POLL), the Deleted in a mouse model of Primary Ciliary Dyskinesia gene (DPCD), the F-box and WD repeat domain containing 4 gene (FBXW4), the Fibroblast growth factor 8 gene (FGF8), the Nucleophosmin 3 gene (NPM3), the Meningioma expressed antigen 5 gene (MGEA5), the gene encoding a Kv channel interacting protein 2 (KCNIP2), and further away, the gene encoding the Lim domain binding protein LDB1 and the NKX1.2 gene (Figs 2 &3).
Human LBX2 has been mapped to chromosome 2, and is linked to TLX2 ([2, 4]; Fig. 2). Neither of these genes are associated with other NK genes, suggesting that they have translocated from their original cluster [2, 3]. LBX2 and TLX2 are separated by the PCGF1 gene, which encodes a polycomb group ring finger. Facing away from PCGF1 and TLX2, LBX2 is linked to genes that encode Dynactin subunit 1 (DCTN1) and the Tetratricopeptide repeat protein 31 (TTC31). TLX2 on the other side is flanked by the DEAQ box 1 (DQX1) gene which encodes for an ATP-dependent RNA helicase, the gene encoding Ancient ubiquitous protein 1 (AUP1), the HTRA1 gene which encodes for a high temperature requiring serine protease, the LOXL3 gene, DOK1 which encodes for the Docking downstream of Tyrosine kinase 1 protein, and a gene encoding the uncharacterised protein NP620159.2. Thus, the human LBX1 and LBX2 loci are quite distinct (Fig. 2). However, they both harbour TLX genes, in line with the idea that Lbx and Tlx genes were linked in the original bilaterian cluster of Nk-type homebox genes [2, 3]. Moreover, both LBX loci include LOXL genes.
In humans, a further TLX gene exists 1.4 Mb distant from NKX2.5 on chromosome 5, thought to be a remnant of the third cluster of NK genes, which, after the second vertebrate genome duplication, lost its cognate LBX gene ([3]; Fig. 2, Additional file 1). Investigating the organisation of this former LBX3/TLX3 locus, we found that TLX3 is on one side linked to the gene that encodes for Ran-binding protein 17 (RANBP17, a member of the Exportin protein family), the gene encoding for the gamma-aminobutyric acid receptor (GABRP), a further KCNIP gene, KCNIP1, the Lymphocyte cytosolic protein gene LCP2, the Forkhead transcription factor gene FOXI1, the Dedicator of cytokinesis gene DOCK2, the coil-coil domain encoding CCDC99 gene, followed by SLIT3. On the other side, TLX3 is linked to NMP1, FGF18, FBXW11/BTRC2, and further away from the TLX3 gene, NKX2.5, MSX2 and DOK3. The order of NMP1, FGF18 and FBXW11/BTRC2 is reversed compared to the paralogous genes at the LBX1/TLX1 locus. However, it is remarkable that for both the LBX1/TLX1 and LBX3/TLX3 paralogons, the NPM and FGF genes are closely linked, separated by sequences of only 5 and 9 kb, respectively. Taken together, our data suggests that Kcnip, Fbxw11/Btrc2, Slit, Loxl, Dok and the closely associated Fgf-Npm genes are hallmarks of Lbx/Tlx loci.
To identify remnants of the fourth LBX locus, we searched the human genome for additional, linked KCNIP, BTRC/FBXW11, SLIT, LOXL, DOK and FGF-NPM sequences. No further homologues of BTRC/FBXW11 genes were identified. However, we found:
- FGF17 and NPM2 closely linked to DOK2 and about 1.8 Mb distant from LOXL2, NKX2.6 and NKX3.1 on chromosome 8, which also carries NKX6.3
- Linked KCNIP4-SLIT2 genes 7.7 Mb distant from NKX3.2 on chromosome 4
- KCNIP3 on the long arm of chromosome 2, however 20.9 Mb distant from and hence probably not a genuine part of the LBX2-TLX2 region.
A previous report suggested that LBX2/TLX2 may once have belonged to the NKX2.6-NKX3.1 region on chromosome 8 [3]. However, the presence of LOXL and DOK genes at both the LBX2 locus and the NKX2.6-NKX3 locus is not consistent with this idea. KCNIP4-SLIT2 were found linked to the Prominin gene PROM1, so far not associated with LBX/TLX loci. However, as KCNIP3 is also linked to PROM2, this suggests that Prom genes may once have belonged to Lbx/Tlx containing regions. Taken together, these observations suggest that the LBX2/TLX2 region may have originated from the NKX3.2 containing cluster on chromosome 4, while FGF17-NPM2 linked to LOXL2 and DOK2 represent the remnants of the fourth LBX/TLX region associated with NKX2.6 and 3.1 on chromosome 8.
Lbxloci in other mammals
Investigating the arrangement of genes at Lbx loci in additional placental mammals (mouse, dog and cattle), a marsupial (opossum) and a monotreme (platypus), we found that the loci are arranged in the same fashion as in humans (Fig. 3, Additional file 1 and data not shown). The exception is the platypus Lbx2/Tlx2 locus, whose existence could not be confirmed as genes linked to this paralogon in other mammals were found on short, unlinked and poorly characterised DNA fragments. Moreover, in mouse and Monodelphis, Prom2-Kcnip3 are not on the same chromosome as the Lbx2 locus, supporting the idea that these genes secondarily intercalated into the Lbx2 carrying chromosome in the lineage leading to humans.
Lbxloci in the chicken
As the information on the chicken genome is still fragmentary, the localisation of chicken Lbx genes could not be determined with certainty. However, chicken chromosome 6 contains a region that is syntenic with the LBX1 region on Human chromosome 10, encompassing Kazald1, Tlx1, Btrc, Poll, Dpcd, Fbxw4, Fgf8, Nmp3, Mgea5 and Kcnip2 in the same order as the human genes (Fig. 3). Loxl4, Slit1 and further genes located in the wider environment of the mammalian Lbx1 locus were also identified, with gene groups displaying a similar arrangement in the chicken and in mammals (Additional file 1). Furthermore, we identified sequences 300 bp upstream and 2.2 kb downstream of the two Lbx1 exons, which are conserved between the mammalian Lbx1 loci and the putative chicken Lbx1 site; in the chicken these regions flank a region that has not been fully sequenced yet (data not shown). Thus, it is likely that this area on chromosome 6 indeed harbours the Lbx1 gene. As will become relevant below, in the chicken the Btrc1 gene is separated from the Poll gene by the Slc2a15 gene (our name) encoding a solute carrier (Fig. 3).
The Lbx2-type gene so far named Lbx3 [18] is situated on a short contig not assigned to a chromosome (Fig. 3). However, Aup1, Htra2, Loxl3, the gene encoding NP620159.2, together with a further Mgea gene, were found on the so far poorly-characterised contig 242 assigned to chromosome 4; all genes are arranged in the same order as the corresponding genes at the Lbx2/Tlx2 containing region of mammalian chromosomes (Fig. 3; Additional file 1). Moreover, separated by sequence gaps, these genes are linked to the Dctn1 gene, which is downstream of Lbx2 in mammals. Significantly, this chromosome also carries genes associated with the NKX3.2 locus in humans, adding weight to the hypothesis that Lbx2 genes were once associated with this particular NK cluster.
Tlx3 was found on chicken chromosome 13 in a region syntenic with the TLX3 containing region of human chromosome 5 and grouped with Fbxw11/Btrc2, Fgf18, Npm1, Ranbp17, Gabrp, Kcnip1, Foxi1, Lcp2, Dock2, Ccdc99 and Slit3 in the same fashion as mammalian Tlx3. In agreement with this being the third Lbx/Tlx paralogon, other members of the NK cluster associated with this paralogon are also found on chick chromosome 13 (Additional file 1).
Finally, a representative of Fgf17 could not be identified, but Npm2, Dok2 and Loxl2 were found on chromosome 22, in a region syntenic to the FGF17-NPM2 region on human chromosome 8 and linked to Nkx2.8 and 6.3 (Fig. 3 and Additional file 1). Notably, this region is also linked to Prom2, a gene in mammals associated with Kcnip3 (Kcnip3 was not found in the chicken). This finding supports the idea that Kcnip3-Prom2 are not an original component of the Lbx2/Tlx2 paralagon but instead probably belong to the fourth Lbx/Tlx paralagon.
Lbx loci in Xenopus tropicalis
In the frog, both Lbx1 and the Kazald1, Tlx1, Btrc, Poll, Dpcd, Fbxw4, Fgf8, Nmp3, Mgea5 and Kcnip2 genes typical for the amniote Lbx1 locus were found on scaffold 238 (Fig. 3 and Additional file 1). As in the chicken, the Slc2a15 gene was located between Btrc and Poll, suggesting that the solute carrier gene was present in the original Lbx1 locus and was lost from this location in the lineage leading to mammals.
A Tlx2 gene was found on the scaffold 2223, but this scaffold does not contain any further genes associated with Lbx/Tlx paralagons (Fig. 3 and Additional files). Similarly, an isolated Pcgf1 gene was found on scaffold 47. Searches for Loxl3 and Aup genes were unsuccessful. Thus, the existence of a Lbx2/Tlx2 locus could not be determined. However, linked Tlx3, Gabrp, Kcnip1, Foxi, Dock2 and Ccdc99 genes were found on scaffold 313, suggesting that a Lbx3/Tlx3 locus exists in frogs (Fig. 3).
As in chicken, we did not find a Xenopus tropicalis ortholog of Fgf17, but we detected Npm2 and Dok2 on scaffold 34, linked to each other and arranged in the same order as the corresponding genes surrounding human and chicken Npm2. Notably, as in amniotes, these genes are also linked to Nkx6.3 (Additional file 1). Moreover, scaffold 30 contains linked Loxl2, Nkx3.1, Nkx2.6, Prom2 and Kcnip3 genes, which in amniotes, with the exception of Prom2-Kcnip3 that have translocated to a different chromosome in placental mammals, are linked to Fgf17, Npm2, Dok2 and Nkx6.3 (Additional file 1). Taken together, this suggests that all of these genes belong to the same Nkx3.1, 2.6 and 6.3 containing cluster.
Lbxloci in teleost fish
For teleosts, we expected to find chromosomal arrangements corresponding to that of tetrapods, if, in line with our analysis of Lbx proteins, only two Lbx bearing loci were present prior to the genome duplication in the ray-finned fish lineage. Alternatively, if in teleosts Lbx3 and Lbx4 genes were maintained, they should be embedded in distinct loci, with Lbx3 next to Tlx3, Kcnip1, Npm1, Fgf18 and Btrc2 and/or Lbx4 near to Fgf17 and Npm2.
We found for the two novel Lbx1-type teleost genes that the organisation of their gene loci fell into two classes. The genes on zebrafish chromosome 13, Tetraodon scaffolds 8483 and 13770, fugu scaffold 52 and stickleback group VI are surrounded by Tlx1 (sequence incomplete for Tetraodon), Slc2a15, Fbxw4 and Fgf8, thus bearing the hallmarks of the tetrapod Lbx1/Tlx1 locus (Fig. 3 and additional files). The same arrangement of genes was found on medaka chromosome 15, with a sequence gap at the place where the lbx gene is expected to be. Significantly, in all of these teleosts the order of genes is identical to that of the equivalent tetrapod genes, with the exception of an inversion between the Tlx1 and Fgf8 region (Figure 3). The Lbx genes on zebrafish chromosome 1, Tetraodon chromosome 18 (sequence incomplete), fugu scaffold 62, stickleback group IX and medaka chromosome 1 are linked to a second Slc2a15 gene, and to Poll, Dpcd and a second Fgf8-type gene (there is a sequence gap at the position of the Fgf8 gene in Tetraodon), thus also bearing hallmarks of the tetrapod Lbx1/Tlx1 locus (Fig. 3 and additional files). Both stickleback and medaka loci are accompanied by Kcnip2 and Mgea5 genes; for stickleback group VI and medaka chromosome 15, these genes are also associated with Npm3 and Slit1, while linked Npm3-Mgea5 genes were found on zebrafish chromosome 13. Moreover, all of the teleost Lbx1-type loci are associated with gene groups found in the wider environment of Lbx1 loci in amniotes (Additional file 1). Taken together, these findings support the idea that all of these fish have two Lbx1/Tlx1 paralogons, although in many cases there has been reciprocal gene loss following the teleost-specific whole genome duplication
For the third of the teleost Lbx genes, namely the gene currently known as zebrafish Lbx1 on chromosome 14 and the Lbx genes on Tetraodon chromosome 20, fugu scaffold 70, stickleback group IV and medaka scaffold 1066, we found an environment reminiscent of the mammalian Lbx2 locus, with the Lbx genes being separated from a Tlx gene by Pcgf1 (Fig. 3). The exception is the medaka scaffold 1066, which is a short fragment that only contains the Lbx and Pcgf genes. Different from that of mammals, an Fgf24-Npm4 gene set was found between the Lbx and Tlx genes. In the wider environment of the Lbx-Pcgf1-Fgf24-Npm4-Tlx gene set, we found two types of arrangement (Additional file 1). On zebrafish chromosome 14, the genes are associated with Aup1 and Loxl3, reminiscent of the mammalian Lbx2/Tlx2 paralagon. The genes on fugu scaffold 70, Tetraodon chromosome 20 and stickleback group IV, are associated with Nanos1, Limch1, Phox2b, Tmem33, Bbs7, Anxa5, Fgfbp, Prom1, Tapt1, Ldb2 (Lbx1 side) and Adad1, Spata5, Ankrd50, Leprot1, Srp72, Arl9, Hop, Sec24B (Tlx side); the ultracontigs 115 and 117 and chromosome 10 of medaka harbour the same genes in the same order. Notably, the genes surrounding the Lbx-Pcgf-Fgf-Npm-Tlx group in fugu, Tetraodon, stickleback and medaka are also found on zebrafish chromsome 14, while Aup1 is found on stickleback group IV and medaka chromosome 10 (Additional file 1), i.e. all of these genes were located in the wider environment of the Lbx-Pcgf-Fgf-Npm-Tlx locus. Taken together, our data suggest that, despite substantial gene rearrangements, these chromosomal regions in teleosts are Lbx2/Tlx2 paralogons. Interestingly, the genes closely associated with fugu, Tetraodon, stickleback and medaka Lbx2, including the Fgfbp-Prom1-Tapt1-Ldb2-Anxa5 genes, are linked with the Nkx3.2 locus on human chromosome 4 and chicken chromosome 4, consistent with our proposal that the mammalian Lbx2 genes were originally part of the Nkx3.2 cluster (additional file 1).
Searching for remnants of the second teleost Lbx2 paralagon, we found Loxl3, a further Nanos1 gene and Ttc31 on Tetraodon chromosome 10, fugu scaffold 338, stickleback group XV and medaka chromosome 22. Dok1-NP620159.2-Sema4f genes were found on Tetraodon scaffold 7074, fugu scaffold 186, stickleback group VII and medaka chromosome 18; in stickleback and medaka these genes were also linked to Kcnip4-Gba3-Gpr125, which in tetrapods co-localise with Nkx3.2 (Additional file 1; the scaffolds were too short to determine whether this linkage also exists for fugu and Tetraodon). Finally, at a distance from the second Lbx1 locus on zebrafish chromosome 1, Tetraodon chromosome 18, fugu scaffold 17, stickleback group IX and medaka chromosome 1, we found Slit2 and a second group of Fgfbp-Prom1-Tapt1-Anxa5 genes linked to Fbxw7, which in humans are all associated with the LBX2 locus. Thus, it seems that the second teleost Lbx2 paralagon has dispersed, with parts having translocated to one of the Lbx1 carrying chromosomes.
For Tlx3 genes, two types of arrangements were identified (Additional file 1). On zebrafish chromosome 14, fugu scaffold 160, Tetraodon chromosome 1, stickleback group IV and medaka chromosome 10, i.e. inserted into the Lbx2 containing chromosome, Tlx3 is on one side linked to Kcnip1, and on the other linked to the Hrh2 gene encoding a Histamine receptor, Dock2 and, slightly more distant, Fgf18 and Fbxw11. On zebrafish chromosome 10, fugu scaffold 6, Tetraodon chromosome 7, stickleback group VII and medaka chromosome 14, Hrh2 is linked with Kncip1, Npm1 and in the case of zebrafish, Tlx3b, Fgf18 and Fbxw11, suggesting that remnants of the two Tlx3 loci created by the teleost-specific genome duplication still exist.
Finally, searching for additional Btrc, Prom, Loxl, Slit, Dok, Kcnip, and Fgf-Npm genes as indicators of other possible Lbx/Tlx paralogons, we identified linked Npm2-Dok2-Fgf17 genes on zebrafish chromosome 8, linked Fgf17-Loxl2-Kcnip3 genes on stickleback group XIII, and linked Kcnip3-Loxl2-Dok2 genes on Tetraodon chromosome 12 and medaka chromosome 9. A second set of linked Kcnip3-Loxl2-Dok2 genes was identified on zebrafish chromosome 10 and a second set of Loxl2 and Kcnip3 genes in a conserved environment but split between two chromosomes were also identified for the other four fish species. Notably, additional genes were also identified that co-localise with Loxl2, Dok2, Kcnip3, Fgf17 and Npm2 genes both in teleosts and tetrapods, including orthologs of Nkx2.6 (Additional file 1 and data not shown), supporting the idea that all these genes once belonged to the same locus, which in tetrapods still encompasses Nkx 2.6, 3.1 and 6.3.
Phylogenetic relationship of signature genes for Lbx/Tlxparalogons
Characterising Lbx/Tlx loci in tetrapods, we found evidence for three distinct paralogons, with the possibility of Fgf17-Npm2 representing the remnant of the fourth. In teleosts, the arrangement of Lbx1/Tlx1 and Tlx3 genes closely follows the pattern observed in tetrapods. However, the putative teleost Lbx2/Tlx2 paralogon (genes currently named Lbx1 and Tlx3a in zebrafish) includes additional Fgf-Npm genes. Moreover, all of the teleosts except zebrafish show a somewhat divergent organisation of genes in the chromosomal regions surrounding the Lbx2/Tlx2 loci. Furthermore, the duplicate Lbx2 paralogon and the two Fgf17-Npm2 paralogons are poorly preserved in teleosts. Therefore, to further confirm the evolutionary relationship of the Lbx loci and our assignment of genes to specific paralogons, we carried out a comprehensive phylogenetic analysis of protein sequences encoded by the genes in the various Lbx/Tlx regions. For this, we BLAST-searched sequence databases for related sequences and built phylogenetic trees which included various invertebrate outgroups (Additional file 2).
Phylogenetic relationship of genes co-localising with several Lbxparalogons
Btrc/Fbxw11, Mgea, Kazald, Ldb and Prom genes occurred at two, Tlx, Loxl, Slit and Dok genes at three and Kcnip, and the linked Fgf-Npm genes at four putative Lbx loci in tetrapods. If our assignment of Lbx/Tlx paralogons is correct, then the genes that we have assigned to particular paralogons should group together. In addition, if the fundamental lay-out of Lbx/Tlx paralogons was established in vertebrates before the divergence of lobe-finned and ray-finned fish and the additional genome duplication in the ray-finned fish lineage, then the teleost protein sequences should group with the tetrapod proteins. If, however, distinct Lbx/Tlx loci were maintained in the lineage leading to lobe-finned fish/tetrapods and ray-finned fish/teleosts, then our phylogenetic analysis should reveal additional groups of genes.
With the exception of Dok2 sequences, which seem to have become highly diverged, all of our analyses supported our assignment of the different Lbx/Tlx paralogons (discussed in detail in Additional file 2). Notably, our analyses of Ldb and Prom1 genes also support our model that the tetrapod Lbx2 loci was originally located in the Nkx3.2 containing cluster: the Ldb2 sequences, which are associated with Lbx2 in teleosts and with the now Lbx-less locus carrying Nkx3.2 in tetrapods group together, as do the Prom1 genes which are linked to Ldb2-Tapt-Anxa5 genes and hence, to the current plus the dispersed Lbx2 and Nkx3.2 loci (Fig 3). In addition, our analyses of Prom2, Tlx, Kcnip, Fgf and Npn genes shows that orthologues associated with the Lbx1 and Lbx4 paralogons are more closely related to each other than to orthologues associated with the Lbx2 and Lbx3 paralogons and vice versa. This is most informative for the genes where four paralogues are still linked to potential Lbx loci in extant vertebrates (Fgf, Npm and Kcnip genes). In these cases, we found that Kcnip1 and Kcnip4 genes are more closely related to each other than to Kcnip2 and 3. Similarly, Fgf8 sequences are closely related to Fgf17 sequences, and Fgf24 sequences are closely related to Fgf18. Finally, the Npm3-Npm2 and Npm4-Npm1 sequences are more closely related to each other, respectively (Additional file 2). Taken together, this suggests that the Lbx1/4 paralogons and the Lbx2/3 paralagons arose from different ancestral Lbx loci generated during the first vertebrate genome duplication.