Molecular Evolution of the Plant Virus Family Bromoviridae Based on RNA 3-Encoded Proteins Francisco M. Codon˜er, 1 Jose´ M. Cuevas, 2 Jesu´s A. Sa´nchez-Navarro, 1 Vicente Palla´s, 1 Santiago F. Elena 1 1 Instituto de Biologı´a Molecular y Celular de Plantas, CSIC-UPV, Avenida de los naranjos s/n, 46022 Valencia, Spain 2 Department of Zoology, University of Oxford, Oxford OX1 3PS, UK Received: 26 January 2005 / Accepted: 26 May 2005 [Reviewing Editors: Dr. Martin Kreitman and Dr. James Bull] Abstract. We have carried out an evolutionary study of the two proteins encoded by the RNA 3 from members of the plant virus family Bromoviridae. Using maximum likelihood methods, we have in- ferred the patterns of amino acid substitution that better explain the diversification of this viral family. The results indicate that the molecular evolution of this family was rather complex, with each protein evolving at different rates and according to different patterns of amino acid substitution. These differences include different amino acid equilibrium frequencies, heterogeneity in substitution rates among sites, and covariation among sites. Despite these differences, the model of protein evolution that better fits both pro- teins is one specifically proposed for the evolution of globular proteins. We also found evidence for coevolution between domains of these two proteins. Finally, our analyses suggest that the molecular clock hypothesis does not hold, since different lineages evolved at different rates. The implications of these results for the taxonomy of this important family of plant viruses are discussed. Key words: Bromoviridae — Maximum likelihood — Plant viruses — Protein evolution — Viral evo- lution — Virus taxonomy Introduction The family Bromoviridae constitutes one of the most important families of plant viruses. A large number of its members are distributed worldwide, and several are responsible for major disease epidemics in crop plants. Their host range covers more than 1000 spe- cies, including herbaceous plants, shrubs, and trees. Most of the type members of the different genera, such as cucumber mosaic virus (CMV), brome mo- saic virus (BMV), and alfalfa mosaic virus (AMV), constitute excellent model systems in plant virology. The genome of the Bromoviridae is approximately 8 kb long and organized into three linear, positive sense ssRNAs. The genomic RNAs 1 and 2 each encode a single large ORF (the replicase proteins P1 and P2), and in some genera, RNA 2 also encodes a second ORF that is apparently translated from a subgenomic RNA (sgRNA). RNA 3 is bicistronic and has two ORFs encoding the movement protein (MP) and the coat protein (CP), the latter being translated from a sgRNA. Besides all these common features, the gen- era Ilarvirus and Alfamovirus share the phenomenon of genome activation (Bol et al. 1971). Genome activation is the binding of CP to specific sites near the 3¢ end of the RNA to initiate infection. The present taxonomy of the Bromoviridae divides the family into five genera (Table 1), named after their most representative member. Genera are dis- tinguished by common biological properties of their members. The Ilarvirus genus, which includes more members than the other genera, has been subdivided into seven groups according to their antigenic Correspondence to: Santiago F. Elena; email: sfelena@ibmcp.upv. es J Mol Evol (2005) 61:697–705 DOI: 10.1007/s00239-005-0021-7