Table 3. Uncorrected pairwise distances (p-distance) between Amcr-UA and similar sequences from other iguanine species. 1 Ctde 2 Ctcl 3 Cyco 4 Cyca 5 Cyri 6 Cosu 7 Amcr-UA 1 0.04546 0.04789 0.04693 0.04791 0.04671 0.05366 2 0.04435 0.05722 0.05822 0.05430 0.05694 0.06577 3 0.03226 0.03629 0.01975 0.02073 0.03846 0.04924 4 0.02823 0.03226 0.00403 0.00988 0.03552 0.04633 5 0.03226 0.03629 0.00806 0.00403 0.03850 0.05120 6 0.04435 0.06048 0.03226 0.02823 0.03226 0.02650 Distances were calculated separately for exon 3 (above the diagonal) and intron 3 (below the diagonal). See methods for Iguaninae taxa abbreviations. Pairwise values between Ctcl and Cosu are italicized because they deviate from the otherwise typical pattern of higher divergence in intron 3 versus exon 3. doi:10.1371/journal.pone.0002859.t003 The higher divergence in intron 3 compared to exon 3 among the iguanine UA sequences suggests that this gene is conserved and under some degree of purifying selection, or at least is not evolving neutrally as would be expected for a nonfunctional pseudogene. However, other physiological and molecular data, including expression at the protein level, must be collected in order to confirm the functionality of Amcr-UA. But for the purpose of discussion, we will proceed with the assumption that this gene is functional in order to explore its characteristics and relationship with other vertebrate class I genes. Despite the absence of a Tm/Cyt region, the Amcr-UA sequence shares many of the conserved peptide-binding residues that are suggestive of classical class I function (Figure 3). However, the comparison of UA exon 3 sequences between Iguaninae taxa shows that there is little adaptive divergence in the antigen-binding region between species, suggesting that this gene is under purifying selection, rather than balancing-selection, and has a conserved function. Thus, it is possible that the product of this gene is involved in antigen binding, but not in the classical sense. Although Kaufman et al. [24] interpreted the presence of certain conserved residues at peptide-binding sites as preliminary evidence of classical function, many of these amino acid character states are maintained in nonclassical genes such as the human HLA-E, -F, and -G loci, as well as the mouse H2-M3 gene. None of these loci differ from the classical mammalian type by more than three out of the nine residues [36]. Thus, it is not at all unprecedented that Amcr-UA displays several nonclassical features while still showing signs of peptide-binding ability. Since a lack of polymorphism is an important criterion for describing nonclassical loci, sequence data was collected from exon 3 and intron 3 of Amcr-UA for three marine iguana populations. The near absence of exon 3 variability in these samples seems to support the pattern of purifying selection indicating a conserved function. However, the simultaneous lack of diversity in the adjacent intron indicates either strong linkage to the conserved exon or perhaps that an insufficient amount of time has passed for a large number of substitutions to accumulate in the intron. The latter pattern would not be surprising since mitochondrial DNA Marine Iguana Class I 7 0.04839 0.05645 0.02823 0.02419 0.02823 0.01210 Figure 5. (a) Nucleotide and (b) amino-acid alignments of iguanine exon 3 (a-2 domain) sequences. The lower-case letters and shaded boxes represent specific codon positions that show variation in amino acid residues between species. Species abbreviations are described in the Materials and methods section. doi:10.1371/journal.pone.0002859.g005 PLoS ONE | www.plosone.org 9 August 2008 | Volume 3 | Issue 8 | e2859