J Mol Evol (1995) 40:362-371 jou..ao MOLECULAR [EVOLUTION © Springer-Veflag New YorkInc. 1995 Evolutionary Analysis of the Picornavirus Family Maria Jesds Rodrigo, 1 Joaquln Dopazo 2 1 Instituto de Biologfa Molecular y Celular de Plantas, CSIC-UPV, Camino deVera s/n, 46022 Valencia, Spain 2 Centro Nacional de Biotecnologfa, CSIC. Universidad Aut6noma. 28049 Madrid, Spain Received: 3 August 1993 / Accepted: 1 June 1994 Abstract. An exhaustive evolutionary analysis of the picornavirus family has been carried out using the amino acid sequences of several proteins of the viruses includ- ing: the capsid proteins (1D, 1B, and 1C) situated at the 5' end of the genome and responsible for the serotype of the viruses, and the viral polymerase (3D), located at the 3' end of the genome. The evolutionary relationships found among the viruses studied support the new classi- fication, recently suggested, in contrast to the classical one, and the existence of a new genus for the picorna- virus family. In the new taxonomic organization, five genera form the picornavirus family: (1) aphthoviruses, (2) cardioviruses, (3) hepatoviruses (previously classi- fied as enteroviruses), (4) renteroviruses (which mainly constitute a combination of the previous genera rhinovi- rus and enterovirus), and (5) a new genus, with a new and unique representative: the echovirus 22. Our analysis also allowed us, for the first time, to propose the most probable sequence of speciation events to have given rise to the current picornavirus family. The bootstrap procedure was used to check the reli- ability of the phylogenetic trees obtained. The applica- tion of the method of the statistical geometry in distance space to internal branches of the tree revealed a high degree of evolutionary "noise," which makes the reso- lution of some internal branching points difficult. Key words: Picornavirus -- Phylogeny -- Neighbor- joining method -- Least-squares method -- Bootstrap -- Variance of branch length -- Statistical geometry in dis- tance space -- Split decomposition Correspondence to: J. Dopazo Introduction The picornavirus family comprises a wide range of hu- man and animal pathogens. Classically, they have been divided, on the basis of their physicochemical properties, into four genera: enteroviruses, rhinoviruses, cardiovi- ruses, and aphthoviruses (Cooper et al. 1978). The pi- cornavirus family shows remarkable antigenic diversity. To date, more than 230 different serotypes have been reported (Rueckert 1985). However, this variability is not homogeneously distributed among genera. More than 120 serotypes have been described in the rhinovirus ge- nus, and approximately 70 in the enterovirus genus, which is composed of, among others, polioviruses, cox- sackieviruses, echoviruses, enteroviruses, and hepatovi- ruses. On the other hand, the aphthovirus genus displays seven serotypes and the cardiovirus genus is composed of a series of viruses that are considered to belong to a unique serotype. As has recently been suggested by Palmemberg (1989; Stanway 1990), the classical division of this fam- ily in genera is not completely supported by the available genetic data. The proposal for the new classification in- cluded the addition of a new, separate genus for hepato- viruses as well as the combination of rhinoviruses and enteroviruses into a unique genus, now called renterovi- rus (Porter 1993). On the other hand, the cardio- and aphthovirus genera remained unchanged. These conclu- sions were derived from a phylogenetic tree inferred by the UPGMA method (Sneath and Sokal 1973)--that is, assuming that a molecular clock exists for the evolution of the picornavirus family (Palmemberg 1989; Rico- Hesse et al. 1987; Stanway 1990). However, no indica- tion as to whether a molecular clock held or not was