evidence suggests that marine iguana populations are not genetically diverse and may have recently expanded in the Gala´pagos archipelago [42]. Therefore, the unclear evolutionary history of this species makes it difficult to attribute the dearth of polymorphism in Amcr-UA to its nonclassical function. While rare, several truncated class I molecules are known to lack Tm/Cyt regions in humans and mice. For example, the mouse Q10 gene contains a 13 bp deletion in the exon encoding the Tm domain, causing a frame shift and the introduction of a premature termination signal downstream in the same exon. In addition, the remaining transcribed portion of the Tm domain has numerous polar amino acid residues that would likely prevent its insertion into the cell membrane. Therefore, it seems clear that the Q10 molecule is not involved in classical antigen presentation. It also possesses several other nonclassical features, including a lack of polymorphism and expression that is almost exclusive to mouse liver cells [43–45]. Several studies have shown that Q10 is likely secreted and can bind a wide array of non-self peptides in a similar manner to classical molecules, but its function is still not well understood [44,46]. While there is no phylogenetic similarity between Amcr-UA and the mouse Q10, the characteristics of the latter gene demonstrate that truncated, soluble, secreted class I molecules can exist which lack genetic variation and maintain protein binding capabilities. Numerous other classical and nonclassical class I molecules are known to exist in soluble form for secretion. For example, the human HLA-G locus possesses all of the coding features of a membrane-bound class I receptor, but is sometimes subjected to alternative transcription where the Tm/Cyt domains are deleted. This molecule is expressed specifically in placental tissue and is secreted during pregnancy [47]. The role of this gene is also not well understood, but is thought to be involved in inducing apoptosis in activated maternal CD8+ T cells [48]. Classical human class I genes (HLA-A, -B, and -C) are also known to exist in soluble forms and to play a role in cell death of activated T cells [49]. Nevertheless, a unique feature of the Amcr-UA sequence which distinguishes it from the Q10 and HLA-G loci, as well as other soluble class I molecules, is that there does not appear be any sign of a Tm/Cyt coding sequence in the genomic DNA, regardless of whether it is transcribed or not; but again, additional support for this conclusion must come from further collection of sequence data in the 39 region of the gene. Some truncated class Ib molecules, such as those in the mouse Qa-2 family, are not necessarily secreted, but are rather linked to the cell membrane through a glycosylphosphatidylinositol (GPI) anchor that is added to the carboxyl terminus of the protein during posttranslational modification [50,51]. Therefore, the apparent References 1. Klein J (1986) Natural history of the major histocompatibility complex. New York: Wiley. xv, 775 p. 2. Ploegh H, Watts C (1998) Antigen recognition. Curr Opin Immunol 10: 57–58. 3. Bjorkman PJ, Parham P (1990) Structure, function, and diversity of class I major histocompatibility complex molecules. Annu Rev Biochem 59: 253–288. 4. Potts WK, Wakeland EK (1990) Evolution of diversity at the major histocompatibility complex. Trends Ecol Evol 5: 181–187. 5. Brigl M, Brenner MB (2004) CD1: Antigen presentation and T cell function. Annual Rev Immunol 22: 817–890. 6. Braud VM, Allan DS, McMichael AJ (1999) Functions of nonclassical MHC and non-MHC-encoded class I molecules. Curr Opin Immunol 11: 100–108. 7. Hansen TH, Huang S, Arnold PL, Fremont DH (2007) Patterns of nonclassical MHC antigen presentation. Nat Immunol 8: 563–568. 8. Hughes AL, Nei M (1989) Evolution of the major histocompatibility complex: independent origin of nonclassical class I genes in different groups of mammals. Mol Biol Evol 6: 559–579. 9. Nei M, Gu X, Sitnikova T (1997) Evolution by the birth-and-death process in multigene families of the vertebrate immune system. Proc Natl Acad Sci USA 94: 7799–7806. Marine Iguana Class I lack of a membrane-spanning domain in Amcr-UA doesn’t rule out its expression on the cell surface or its involvement in the primary T cell response. Overall, the Amcr-UA CDS showed the highest similarity with iguanine UB and Ameiva lizard sequences. In addition, phyloge- netic reconstruction supported the monophyly of all available squamate class I sequences. The basal position of Amcr-UA relative to all other squamate class I sequences suggests that this gene diverged very early in the evolution of this reptilian order. The most recent common ancestor of iguanines and the two groups represented by the Ameiva lizard (Family: Teiidae) and northern water snake (Suborder: Serpentes) is estimated to have existed between 179–206 million years ago [14], providing a minimum time for the split of Amcr-UA from class Ia genes in squamates. Outside of the squamate grouping, the tree topology is not well supported, suggesting that class I sequences are highly divergent among vertebrate groups. In summary, the Amcr-UA sequence possesses several charac- teristics of a functional, non-classical class I gene with a conserved protein structure. Additional work must be conducted to understand whether it is expressed at the level of the protein and what its position is in the genome relative to published classical loci [16]. While Amcr-UA is most closely related to the other published squamate sequences, it does not cluster with class Ia sequences from the same species, suggesting that it has long been on a separate evolutionary trajectory. Further characteriza- tion of UA-like sequences from other squamates will reveal whether this gene is part of a lineage that has maintained a non- classical function over the course of squamate evolution. Acknowledgments Maria Moreno and Stephen Dellaporta (Yale University) provided helpful advice for carrying out RACE protocol. Larry Buckley (Rochester Institute of Technology) contributed genomic DNA from four of the iguanine species used in this study (C. defensor, C. clarki, C. rileyi, and C. carinata). The C. cornuta specimen was provided by the Peabody Museum of Natural History with the help of Greg Watkins-Colwell. The Charles Darwin Research Station (Santa Cruz, Gala´pagos, Ecuador) and the Gala´pagos National Park provided logistical support for the collection and export of marine iguana blood and RNA. Cruz Marquez helped with marine iguana blood sampling. Special thanks to Ylenia Chiari and Hilary Miller for helpful comments on the manuscript. Author Contributions Conceived and designed the experiments: SG. Performed the experiments: SG. Analyzed the data: SG LdP. Contributed reagents/materials/analysis tools: AC. Wrote the paper: SG. 10. Rada C, Lorenzi R, Powis SJ, Vandenbogaerde J, Parham P, et al. (1990) Concerted evolution of class I genes in the major histocompatibility complex of murine rodents. Proc Natl Acad Sci USA 87: 2167–2171. 11. Hess CM, Edwards SV (2002) The evolution of the major histocompatibility complex in birds. Bioscience 52: 423–431. 12. Belov K, Deakin JE, Papenfuss AT, Baker ML, Melman SD, et al. (2006) Reconstructing an ancestral mammalian immune supercomplex from a marsupial major histocompatibility complex. PLoS Biol 4: 317–328. 13. Rest JS, Ast JC, Austin CC, Waddell PJ, Tibbetts EA, et al. (2003) Molecular systematics of primary reptilian lineages and the tuatara mitochondrial genome. Mol Phylogenet Evol 29: 289–297. 14. Vidal N, Hedges SB (2005) The phylogeny of squamate reptiles (lizards, snakes, and amphisbaenians) inferred from nine nuclear protein-coding genes. C R Biologies 328: 1000–1008. 15. Vitt LJ, Pianka ER, Cooper WE, Schwenk K (2003) History and the global ecology of squamate reptiles. Am Nat 162: 44–60. 16. Glaberman S, Caccone A (2008) Species-specific evolution of class I MHC genes in iguanas (Order: Squamata; Subfamily: Iguaninae). Immunogenetics 60: 371–382. PLoS ONE | www.plosone.org 10 August 2008 | Volume 3 | Issue 8 | e2859