Gene NS1 NS2 PB1-F2 PB1 H5N1 H9N2 H5N1 H9N2 H5N1 H9N2 H5N1 H9N2 1.25 (0.75 1.93) 1.46 (1.07 2.24) 0.34 (0.24 0.52) 0.24 (0.15 0.35) 6.75 (5.74 9.88) 6.41 (5.52 7.92) 0.03 (0.02 0.05) 0.03 (0.02 0.05) 0.81 (0.41 1.52) 0.58 (0.38 0.86) 0.32 (0.22 0.50) 0.23 (0.13 0.36) 0.52 (0.40 0.76) 0.46 (0.34 0.75) 0.02 (0.02 0.04) 0.02 (0.01 0.04) Table 2. Estimation of selection intensity (v^ ) by independent and simultaneous estimation. Subtype a Independent v^ b, c Simultaneous v^ b aNumber of pairwise alignments of NS1 – NS2 overlaps is 10,569 and 8,745 for H5N1 and H9N2 subtypes, respectively; Number of pairwise alignments of PB1-F2 – PB1 overlaps is 16,112 and 33,720 for H5N1 and H9N2 subtypes, respectively. bMedian of v^ over all pairwise comparisons. Lower and upper quartiles are noted in parentheses. cValues of selection intensity in PB1-F2 and NS1 genes that appear as positive selection by independent estimation are bolded. doi:10.1371/journal.pone.0003996.t002 were excluded. In agreement with previous studies, PB1-F2 and NS1 genes appear to be under positive selection when gene overlap is not accounted for. However, by using our new method References 1. Barrell BG, Air GM, Hutchison CA 3rd (1976) Overlapping genes in bacteriophage phiX174. Nature 264: 34–41. 2. Smith RA, Parkinson JS (1980) Overlapping genes at the cheA locus of Escherichia coli. Proc Natl Acad Sci U S A 77: 5370–5374. 3. Montoya J, Gaines GL, Attardi G (1983) The pattern of transcription of the human mitochondrial rRNA genes reveals two overlapping transcription units. Cell 34: 151–159. 4. Jones CE, Fleming TM, Cowan DA, Littlechild JA, Piper PW (1995) The phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase genes from the thermophilic archaeon Sulfolobus solfataricus overlap by 8-bp. Isolation, sequencing of the genes and expression in Escherichia coli. Eur J Biochem 233: 800–808. 5. Makalowska I, Lin CF, Hernandez K (2007) Birth and death of gene overlaps in vertebrates. BMC Evol Biol 7: 193. 6. Lillo F, Krakauer DC (2007) A statistical analysis of the three-fold evolution of genomic compression through frame overlaps in prokaryotes. Biol Direct 2: 22. 7. Okamoto H, Imai M, Shimozaki M, Hoshi Y, Iizuka H, et al. (1986) Nucleotide sequence of a cloned hepatitis B virus genome, subtype ayr: comparison with genomes of the other three subtypes. J Gen Virol 67 (Pt 11): 2305–2314. 8. Johnson ZI, Chisholm SW (2004) Properties of overlapping genes are conserved across microbial genomes. Genome Res 14: 2268–2272. 9. Normark S, Bergstrom S, Edlund T, Grundstrom T, Jaurin B, et al. (1983) Overlapping genes. Annu Rev Genet 17: 499–525. 10. Cooper PR, Smilinich NJ, Day CD, Nowak NJ, Reid LH, et al. (1998) Divergently transcribed overlapping genes expressed in liver and kidney and located in the 11p15.5 imprinted domain. Genomics 49: 38–51. 11. Sakharkar KR, Sakharkar MK, Verma C, Chow VT (2005) Comparative study of overlapping genes in bacteria, with special reference to Rickettsia prowazekii and Rickettsia conorii. Int J Syst Evol Microbiol 55: 1205–1209. 12. Keese PK, Gibbs A (1992) Origins of genes: ‘‘big bang’’ or continuous creation? Proc Natl Acad Sci U S A 89: 9489–9493. 13. Krakauer DC (2000) Stability and evolution of overlapping genes. Evolution Int J Org Evolution 54: 731–739. 14. Miyata T, Yasunaga T (1978) Evolution of overlapping genes. Nature 272: 532–535. 15. Hughes AL, Westover K, da Silva J, O’Connor DH, Watkins DI (2001) Simultaneous positive and purifying selection on overlapping reading frames of the tat and vpr genes of simian immunodeficiency virus. J Virol 75: 7966–7972. 16. Hughes AL, Hughes MA (2005) Patterns of nucleotide difference in overlapping and non-overlapping reading frames of papillomavirus genomes. Virus Res 113: 81–88. 17. Narechania A, Terai M, Burk RD (2005) Overlapping reading frames in closely related human papillomaviruses result in modular rates of selection within E2. J Gen Virol 86: 1307–1313. Selection in Overlapping Genes for simultaneous estimation, these genes seem to be under weak purifying selection. As predicted by our simulation, the bias in the independent estimation is dependent on the degree of purifying selection acting on the overlapping gene, leading to higher bias in PB1-F2 compared to NS1. Discussion Overlapping genes are widespread in all taxa, but are particularly common in viruses [45]. The sequence interdepen- dence imposed by gene overlap adds complexity to almost any molecular evolutionary analysis. Here, we presented a new method for the estimation of selection intensities in overlapping genes. By simulation, we verified the accuracy of the method, tested its limitations, and compared the possible outcomes of estimating selection without accounting for gene overlap across different overlap types. We find that estimating selection as if the genes are independent of one another results in the false appearance of positive selection. Our model can be used to identify true functional genes, which are usually under negative or positive selection, from among hypothetical overlapping ORFs, which are mainly spurious. Author Contributions Conceived and designed the experiments: NS. Performed the experiments: NS. Analyzed the data: NS GL DG. Wrote the paper: NS GL DG. 18. Pavesi A (2006) Origin and evolution of overlapping genes in the family Microviridae. J Gen Virol 87: 1013–1017. 19. Pavesi A (2007) Pattern of nucleotide substitution in the overlapping nonstructural genes of influenza A virus and implication for the genetic diversity of the H5N1 subtype. Gene 402: 28–34. 20. Campitelli L, Ciccozzi M, Salemi M, Taglia F, Boros S, et al. (2006) H5N1 influenza virus evolution: a comparison of different epidemics in birds and humans (1997–2004). J Gen Virol 87: 955–960. 21. Suzuki Y (2006) Natural selection on the influenza virus genome. Mol Biol Evol 23: 1902–1911. 22. Zaaijer HL, van Hemert FJ, Koppelman MH, Lukashov VV (2007) Independent evolution of overlapping polymerase and surface protein genes of hepatitis B virus. J Gen Virol 88: 2137–2143. 23. Guyader S, Ducray DG (2002) Sequence analysis of Potato leafroll virus isolates reveals genetic stability, major evolutionary events and differential selection pressure between overlapping reading frame products. J Gen Virol 83: 1799–1807. 24. Holmes EC, Lipman DJ, Zamarin D, Yewdell JW (2006) Comment on ‘‘Large- scale sequence analysis of avian influenza isolates’’. Science 313: 1573; author reply 1573. 25. Obenauer JC, Denson J, Mehta PK, Su X, Mukatira S, et al. (2006) Large-scale sequence analysis of avian influenza isolates. Science 311: 1576–1580. 26. Li KS, Guan Y, Wang J, Smith GJ, Xu KM, et al. (2004) Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia. Nature 430: 209–213. 27. Rogozin IB, Spiridonov AN, Sorokin AV, Wolf YI, Jordan IK, et al. (2002) Purifying and directional selection in overlapping prokaryotic genes. Trends Genet 18: 228–232. 28. Hein J, Stovlbaek J (1995) A maximum-likelihood approach to analyzing nonoverlapping and overlapping reading frames. J Mol Evol 40: 181–189. 29. Li WH, Wu CI, Luo CC (1985) A new method for estimating synonymous and nonsynonymous rates of nucleotide substitution considering the relative likelihood of nucleotide and codon changes. Mol Biol Evol 2: 150–174. 30. Nei M, Gojobori T (1986) Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol Biol Evol 3: 418–426. 31. Pamilo P, Bianchi NO (1993) Evolution of the Zfx and Zfy genes: rates and interdependence between the genes. Mol Biol Evol 10: 271–281. 32. de Groot S, Mailund T, Hein J (2007) Comparative annotation of viral genomes with non-conserved gene structure. Bioinformatics 23: 1080–1089. 33. McCauley S, de Groot S, Mailund T, Hein J (2007) Annotation of selection strengths in viral genomes. Bioinformatics 23: 2978–2986. 34. McCauley S, Hein J (2006) Using hidden Markov models and observed evolution to annotate viral genomes. Bioinformatics 22: 1308–1316. PLoS ONE | www.plosone.org 6 December 2008 | Volume 3 | Issue 12 | e3996