Virus Evolution, 2015, 1(1): vev019 doi: 10.1093/ve/vev019 Research article Tobamoviruses have probably co-diverged with their eudicotyledonous hosts for at least 110 million years Downloaded from https://academic.oup.com/ve/article/1/1/vev019/2568707 by guest on 28 September 2021 Adrian J. Gibbs,1,* Jeffrey Wood,2 Fernando Garcia-Arenal,3,† Kazusato Ohshima,4 and John S. Armstrong1 1Emeritus Faculty, Australian National University, Canberra, ACT 2601, Australia, 2Statistical Consulting Unit, Australian National University, Canberra, ACT 2601, Australia, 3Centro de Biotecnologı´a y Genomica de Plantas (UPM-INIA) and E.T.S.I. Agronomos, Campus de Montegancedo, Universidad Polite´cnica de Madrid, Madrid, Spain and 4Laboratory of Plant Virology, Department of Applied Biological Sciences, Faculty of Agriculture, Saga University, 1-banchi, Honjo-machi, Saga 840-8502, Japan *Corresponding author: E-mail: [email protected] †http://orcid.org/0000-0002-5327-3200 Abstract A phylogeny has been calculated by maximum likelihood comparisons of the concatenated consensus protein sequences of 29 tobamoviruses shown to be non-recombinant. This phylogeny has statistically significant support throughout, including its basal branches. The viruses form eight lineages that are congruent with the taxonomy of the hosts from which each was first isolated and, with the exception of three of the twenty-nine species, all fall into three clusters that have either asterid or rosid or caryophyllid hosts (i.e. the major subdivisions of eudicotyledonous plants). A modified Mantel permutation test showed that the patristic distances of virus and host phylogenies are significantly correlated, especially when the three anomalously placed viruses are removed. When the internal branches of the virus phylogeny were collapsed the congru- ence decreased. The simplest explanation of this congruence of the virus and host phylogenies is that most tobamovirus lineages have co-diverged with their primary plant hosts for more than 110 million years, and only the brassica-infecting lineage originated from a major host switch from asterids to rosids. Their co-divergence seems to have been ‘fuzzy’ rather than ‘strict’, permitting viruses to switch hosts within major host clades. Our conclusions support those of a coalesence analysis of tobamovirus sequences, that used proxy node dating, but not a similar analysis of nucleotide sequences from dated samples, which concluded that the tobamoviruses originated only 100 thousand years ago. Key words: tobamovirus evolution; plant evolution; co-divergence; phylogenetic congruence. 1. Introduction few hundred or thousand years, but doubts about these interpre- Knowledge of a pathogen’s origin(s) and its rate of evolution pro- tations (Firth et al. 2010; Sharp and Simmonds 2011; Ducheˆne, vides a crucial framework for understanding its biology. Holmes, and Ho 2014), and clues of much more ancient origins of However, for most viruses, this information can only be inferred some viruses, are forcing a re-think. Much of the doubt has arisen indirectly, as viruses leave no fossils and so existing populations from evidence of viruses or virogenes integrated in the genomes provide the only certain information of them. Many recent re- of plants (Bejarano et al. 1996; Geering et al. 2005; Chiba et al. ports of statistical analyses of the gene sequences of viruses col- 2011; James et al. 2011; Lefeuvre et al. 2011; Lyttle, Orlovich, and lected on different dates conclude that extant viruses have large Guy 2011; Kondo et al. 2013; Filloux et al. 2015) and animals mutation rates and must therefore have originated in the past (Gifford et al. 2008; Katzourakis et al. 2009; Belyi, Levine, and VC The Author 2015. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 1 2|Virus Evolution, 2015, Vol. 1, No. 1 Skalka 2010a, b; Gilbert and Feschotte 2010; Horie et al. 2010; In this article, we report experiments to resolve the contrast- Taylor et al. 2010, 2011; Worobey et al. 2010; Han and Worobey ing conclusions obtained by coalescence methods. We used dif- 2012), where the taxonomy of the integrated genes and their ferent methods and obtained a tobamovirus phylogeny, that ‘hosts’ indicates an ancient origin. Attempts to establish ancient was better supported, especially its internal (basal) branches, origins by phylogenetic evidence of co-divergence of host and vi- and checked its congruence with the hosts’ phylogeny. Our aim rus (e.g. Wu et al. 2008) have proven to be more controversial (De was to provide independent support, or otherwise, for co- Vienne et al. 2013). In the tests reported here, we used new meth- divergence. We inferred phylogenies by maximum likelihood ods to re-examine the evidence for and against co-divergence for (ML), to contrast with those obtained by Bayes coalescence and tobacco mosaic virus (TMV) and its relatives in the genus mentioned above, and we used simple procedures to minimize Tobamovirus. ‘mutational noise’ at the tips of the tree, and thereby increase Over the years, several suggestions have been made about statistical support for the internal branches of the phylogeny; when and where tobamoviruses arose, each reflecting the data the concatenated amino acid sequences of all three tobamovi- and methods available at the time. Holmes (1951) was perhaps rus genes were compared rather than individual gene se- the first to comment, albeit indirectly. He noted that the quences, and the consensus sequences for tobamovirus species Downloaded from https://academic.oup.com/ve/article/1/1/vev019/2568707 by guest on 28 September 2021 Nicotiana spp. and other solanaceous plants, that responded to were used rather than sequences from individual isolates. infection by TMV in a hypersensitive manner, are all natives of Central and South America, whereas Nicotiana species that are most susceptible to systemic TMV infection are mostly found in 2. Materials and methods North America, southern South America, and Australia. Holmes 2.1 Sequences argued that these differences resulted from the selection of TMV-resistant plant populations in Central and South America, Sequences were edited using BioEdit (Hall 1999) and MSWord and hence that this region was probably the ‘original habitat of Gene sequences were sorted, checked, grouped, and duplicates tobacco-mosaic virus in prehistoric times’. removed using the neighbour-joining (NJ) facility in ClustalX When the idea of a molecular clock was devised (Zuckerkandl (Jeanmougin et al. 1998), and were then aligned using MAFFT and Pauling 1965), it was applied first to the amino acid se- (Katoh and Standley 2013) with its ‘iterative refinement using quences of the coat proteins (CPs) of the different tobamoviruses local pairwise alignment’ option. Wasabi mosaic virus se- knownatthattime(Gibbs 1980). Their sequence relationships, quences were so close to the youcai mosaic virus population, represented as a Darwinian tree, were found to correlate with they were treated as such. The Accession Codes of the 185 se- their serological relationships. If those viral proteins had evolved quences in the resulting alignment are given in Supplementary at much the same rate as similar proteins of cellular organisms Data Table 1; a few tobamoviruses are represented by single se- (i.e. cf. 1% per million years), then the differences between them quences, and the 67 for TMV are the most for a single species. suggested that tobamoviruses had origins as ancient as their The genomic sequences were separated into their constituent plant hosts, which first appeared as fossils more than 100 million ORFs (i.e. the separate genes for the replicase, movement, and years ago. This timing has since been supported by several lines CPs, including any overlapping regions of each gene). They were of circumstantial evidence (Gibbs 1999; Gibbs et al. 2008, 2010, aligned using the encoded amino acids as guide by the 2011), notably the possibility of co-divergence with their long- TranslatorX server (Abascal, Zardoya, and Telford 2010; http:// term eudicotyledonous hosts, because the relationships of the translatorx.co.uk) with its MAFFT option. The ORFs were ana- genomes of different tobamovirus species reflects the relation- lysed both separately, and in combinations, but mostly as the ships of their hosts (Lartey, Voss, and Melcher 1996; Gibbs 1999), complete concatenated ORFs (concats) of each genome. The lat- and as the relevant host clades diverged at least 100 million years ter were tested for the presence of phylogenetic anomalies us- ago (Magallon and Castillo 2009; Magallon, Hilu, and Quandt ing the full suite of options in RDP4 (Martin and Rybicki 2000). 2013), it suggests that the tobamoviruses did too. Sites in the aligned protein sequences with more than five gaps There have however been two recent reports of estimates of were removed using POSORT, a DOS command line program the age of the tobamoviruses obtained by Bayesian MCMC coa- available at http://192.55.98.146/_resources/e-texts/README- lescence analyses of viral gene nucleotide sequences, and these POSORT.pdf. Consensus protein sequences were derived for have produced conclusions about the age of the genus that dif- each of the species using the ‘Create Consensus Sequence’ func- fer by three