evolutionary rates of synonymous and nonsynonymous substitu- tions at positions within the Xist gene are approximately equal. For the synonymous substitutions (Fig. 4a), the lengths of branches approximately correspond to the anticipated time estimates, thereby indicating the uniformity of evolutionary rates over time. Nonsynonymous substitutions (Fig. 4b) give characteristically short branches leading to Gglnx3 and Mdlnx3 (as compared to synonymous substitutions). This results from the action of stabilizing selection, which is typical for the majority of protein- coding genes. As Xist is not a protein-coding gene, this limitation is removed. Therefore, the branch leading to Xist genes is very long as compared with the other branches. If Xist were a protein-coding gene and displayed approximately the same evolutionary rate as Mdlnx3, the length of these branches would be approximately equal (the suggested position of Xist gene is denoted with arrow; Fig. 4b). However, the rest of the length of the branch leading to the last common ancestor of bovine and human (B1N in Fig. 4b) approximately equals the lengths of the branches leading to human and bovine (B2N in Fig. 4b). This indicates that the Xist gene has an ancient origin. The long branch B1N might result from an acceleration in the evolution of the Lnx3/Xist gene during the pseudogenization process. The fact that B1N is approximately 2 times longer (Table S4) in the Figure 4b (nonsynonymous sites) compared to BS (Fig. 4a) can probably be attributed to the nonsynonymous sites being far from equilibrium with the mutational pattern at the moment of pseudogenization, such that for a while the number of substitutions per site would have been higher for these sites than for the synonymous positions (which would have been already closer to mutational equilibrium). To test the reliability of the obtained results, we aligned nucleotide sequences of Gglnx3 and Mdlnx3 and all available mammalian Xist genes (Fig. S6). This alignment contained a much smaller number of sites (results not shown), however the results are similar to the results obtained for the four species alignment (Fig. S6). Similar estimates of branch lengths were obtained for different models of substitutions (Table S4) suggesting that the obtained results are reliable. In general, a short alignment and a lack of good descriptors of the pseudogenization process prevent precise time estimates; however, taking into account that divergence of bovine and human is assessed as 90–110 Mya (branch B2N in Fig. 4b) [8], we can assume that the time of Xist gene emergence might be approximately 2 times older than this estimate (in the assumption that the evolutionary rate of nonsynonymous substitutions at the branch B1N is 2 times faster than B2N and BS), i.e., 180–220 Mya. This estimate exceeds the assumed divergence time of marsupials and eutherians (130–180 Mya). Taking into account the inevitable errors in the estimation of evolutionary rates and branch lengths in short alignments, the data obtained suggest that the inactivation of the Lnx3 gene occurred during the divergence of marsupials and eutherians or in early eutherians. The Origin of Ftx and Enox(Jpx) genes Next we analysed the contribution of Uspl and Wave4(Wasf3) genes to the formation of the XIC, as this question has remained unresolved in previous studies. We conducted a comprehensive search for similarity between the chicken protein-coding genes Wave4(Wasf3) (ACC XM_420299.1), Rasl11c (ACC XM_420297.1), and Uspl (ACC NM_001031123.1) and the genes of the mammalian XIC. A more detailed analysis with less stringent parameters in the search programs detected several regions homologous to the chicken Wave4(Wasf3) gene in the human XIC. Exons 1 and 2 of Wave4(Wasf3) correspond to exons 2 and 3 of FTX (ACC AK057701.1), displaying 58 and 55% similarity, respectively. The A Dual Origin of the Xist Gene similarity is statistically significant in both cases (Table 4). Note that the exon–intron boundary between two homologous exons of Wave4(Wasf3) and FTX coincides (Fig. S2). Moreover, the consider- ably diverged regions homologous to Wave4(Wasf3) exons 3 and 4 are detectable at the same human loci (localized to Ftx 59 region). These data prompted us to search for homology to genes Uspl and Rasl11c. The considerably diverged remnants of the Rasl11c gene were found in a region between human ENOX(JPX) and XIST genes (Fig. S2). The most pronounced homology was for exon 3 of Rasl11c (63%), as described previously in [4]. The most likely explanation is that Enox(Jpx) is related to the chicken gene Uspl. As demonstrated previously, the main part of the Enox(Jpx) exons, except for exon 1, evolved from mobile elements of various classes [2]; moreover, different types of dispersed repeats gave rise to the exons of the gene in different mammalian species. When analyzing the human EST database, several alternatively spliced transcripts with common exons 1 and 2 were detected for the human ENOX(JPG) gene (data not shown). We found a statistically significant similarity (58%) between the putative ENOX(JPX) promoter region and exon 1 of chicken Uspl, between ENOX(JPX) exon 2 and Uspl exon 5, and between Uspl exon 7 and human EST BC071776, which is likely to be an alternatively spliced variant of the ENOX(JPX) gene (Table 4, Fig. S2). Thus, like Xist, Enox(Jpx) seems to have evolved from a protein-coding gene and a set of different transposable elements. Discussion The origin of the mammalian X-inactivation center In this study we analysed the origin and evolution of the group of linked genes from which the mammalian XIC originated. The tight linkage of Lnx3 (the precursor of Xist) with protein-coding genes Cdx4 and Chic1 (caudal type homeo box transcription factor 4; cysteine-rich hydrophobic domain 1), which flank Xist in eutherians, was detected in fish and, presumably, existed in the ancestor of vertebrates 450 Mya (Fig. 5). Unlike the evolutionary conserved syntenic group Cdx4, Chic1 and Lnx3, the other genes that contributed to the sequence of the Xic, Rasl11c, Uspl, and Wave4(Wasf3), are found within other linkage groups in fish (for details see Fig. 5 legends). Tight linkage of all the precursor genes of the XIC and the flanking conserved protein-coding genes is also detectable in amphibians [4,10,11]. In pipid frog (Xenopus tropicalis), the genes Cdx4, Chic1, Fip1l2, Lnx3, Rasl11c, Uspl, Wave4 (Wasf3), Slc16a2 (solute carrier family 16), and Rnf12 (ring finger protein 12) are detectable within the same scaffold (Fig. 5). Note that in an ancestral amphibian, the Fip1l2 gene was inserted into the conserved syntenic group Cdx4–Chic1–Lnx3, detectable in fish (Fig. 5). In chicken the order and orientation of the genes remains unchanged and they are integrated with genes which represent the main part of X conserved regions in mammals. Thus, it is likely that the formation of a proto XIC commenced about 450– 360 Mya in ancestral amphibians. An independent disruption of this linkage group took place in Prototheria and Methateria [10,11,12]. It is interesting, that these genes map to different regions of an autosome, chromosome 6 [11] in monotremes, while in marsupials they map to the X chromosome [12]. This linkage group remained in the eutherian lineage, where the five protein-coding genes of the proto XIC underwent pseudogenization about 180 Mya to give different sequences of the X-inactivation center [4]. The origin of the genes in the mammalian XIC Our comprehensive analysis together with previously published data [4] has enabled us to outline the origin of genes in the X- PLoS ONE | www.plosone.org 7 June 2008 | Volume 3 | Issue 6 | e2521