Proc. Natl. Acad. Sci. USA Vol. 88, pp. 5631-5634, July 1991 Evolution Phylogeny of viroids, viroidlike satellite RNAs, and the viroidlike domain of hepatitis 6 virus RNA (statistical geometry/quasispecies/RNA world/living fossils) SANTIAGO F. ELENA*, JOAQUIN DoPAZO*, RICARDO FLORESt, THEODOR 0. DIENER*, AND ANDR1S MOYA*§ *Departament de Gendtica i Servei de BioinformAtica, Universitat de Valdncia, Dr. Moliner 50, 46100 Burassot, Valdncia, Spain; tUnidad de Biologia Molecular y Celular de Plantas, Instituto de Agroquimica y Tecnologfa de Alimentos, Consejo Superior de Investigaciones Cientificas, 46010 Valdncia, Spain; and fCenter for Agricultural Biotechnology, and Department of Botany, University of Maryland, College Park, MD 20742, and Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705 Contributed by Theodor 0. Diener, March 21, 1991 ABSTRACT We report a phylogenetic study of viroids, assumed an intracellular mode of existence sometime after some plant satellite RNAs, and the viroidlike domain of human the evolution of cellular organisms. hepatitis 8 virus RNA. Our results support a monophyletic Implicit in this proposal is the possibility that all viroids and origin ofthese RNAs and are consistent with the hypothesis that viroidlike RNAs may have been derived from a common they may be "living fossils" of a precellular RNA world. ancestor. To obtain evidence for or against this proposition, Moreover, the viroidlike domain of human hepatitis 8 virus we have conducted a phylogenetic analysis of these small RNA appears closely related to the viroidlike satellite RNAs of pathogenic RNAs and report here that our results are con- plants, with which it shares some structural and functional sistent with a monophyletic origin of viroids and viroidlike properties. On the basis of our phylogenetic analysis, we satellite RNAs, as well as possibly of the viroidlike domain propose a taxonomic classification of these RNAs. of HDV RNA. Viroids, subviral pathogens of higher plants, are small (246- Phylogenetic Methods 375 nucleotide residues), unencapsidated, single-stranded, The nucleotide sequences of 15 viroids, 4 circular (viroidlike) circular RNAs characterized by highly base-paired, rodlike satellite RNAs, 2 linear satellite RNAs, and the viroidlike secondary structures (1). Viroids do not code for any pro- domain of HDV RNA were used (Table 1). teins, yet they replicate autonomously (without the assis- The first and most critical step in this work was the tance of helper viruses) in susceptible cells. Viroidlike sat- alignment of the sequences. It was carried out by means of a ellite RNAs resemble viroids, but they are found within the multiple sequence algorithm with hierarchical clustering (37, capsids of specific helper viruses required for their replica- 38), followed by minor adjustments of some stretches to tion (2). Human hepatitis 8 virus (HDV) RNA is a circular preserve previously described homologies (25, 39). RNA requiring hepatitis B virus as a helper virus for pack- Two methods of phylogenetic reconstruction were used: aging and transmission (3). HDV RNA contains a region, least squares (40) and maximum parsimony. In both cases the termed the viroidlike domain, with significant similarities bootstrap method (41, 42) was applied to evaluate the reli- with viroid and viroidlike satellite RNAs (4). Viroids, viroid- ability of the inferred trees. In the first case, 1000 replicates like satellite RNAs, and HDV RNA appear to replicate via were sampled from the original set of sequences, and the oligomeric RNA intermediates by some type of rolling-circle corresponding distance matrix was obtained. The number of mechanism (2-6). mutations between sequences was measured, correcting for Several hypotheses have been advanced to explain the multiple substitutions by using the formula d = -In(l - K)L evolution of viroids. It has been suggested, for example, that (43), K being the frequency of changes and L the homologous viroids may have originated from retroviruses or transpos- length of the sequences. Based on the distance matrix, a able elements by deletion of interior sequences (7), that they phylogenetic tree was obtained for each bootstrap replicate may represent escaped introns (8, 9), or that they have by means of the FITCH program of the PHYLIP package, evolved comparatively recently from hypothetical "anten- version 3.2 (44). Finally, a consensus tree was obtained by na" or "signal" RNAs, which eukaryotic cells are assumed using the CONSENSE program ofthe same package. It is worth to interchange (10). noting that the trees obtained were not significantly different With the demonstration that certain RNAs have catalytic whether gaps were excluded or treated as substitutions in the properties (11, 12), the idea that RNA preceded DNA as a estimation of d. carrier of genetic information has gained support. Most The bootstrap estimate (also based on 1000 replicates) of recent models for self-replicating precellular RNAs assume the tree obtained by using the second method was directly the existence of primitive RNA enzymes with properties that implemented in the program DNABOOT of the PHYLIP pack- are derived from extant self-splicing introns (13, 14). Because age. In this case the estimate of the branch lengths from the one viroid, all known viroidlike satellite RNAs, and the inferred tree was obtained with the FITCH program, using the viroidlike domain of HDV RNA are self-cleaving (3, 15, 16), previously obtained consensus topology. it is equally plausible to consider these RNAs as relics of the As a method for testing the tree-likeness of the data (i.e., RNA world, thus leading to an alternative hypothesis for the their possible common origin), we have employed the method evolution of viroids and viroidlike satellite RNAs (17). In this ofstatistical geometry in distance space (45, 46). This method view, one must suppose that these RNAs have evolved from is especially useful for the analysis of "old" relationships that "free-living" molecules and that they, like introns, have have suffered to a large extent from ulterior randomizations, The publication costs of this article were defrayed in part by page charge Abbreviations: HDV, hepatitis 8 virus; ASBVd, avocado sunblotch payment. This article must therefore be hereby marked "advertisement" viroid; see Table 1 for other abbreviations. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 5631 Downloaded by guest on September 27, 2021 5632 Evolution: Elena et al. Proc. Natl. Acad. Sci. USA 88 (1991) Table 1. Viroids and satellite RNAs examined 100 substitutions Abbre- Size, ASBVd RNA viation nt Ref. Avocado sunblotch viroid ASBVd 247 18 Potato spindle tuber viroid PSTVd 359 19 vLTSV Tomato apical stunt viroid TASVd 360 7 Tomato planta macho viroid TPMVd 360 7 ASSVd Citrus exocortis viroid CEVd 371 20 Chrysanthemum stunt viroid CSVd 356 21 GIBVd Columnea latent viroid CLVd 370 22 HLVd Coconut cadang-cadang viroid CCCVd 246 23 Hop stunt viroid HSVd 297 24 Hop latent viroid HLVd 256 25 Apple scar skin viroid ASSVd 330 26 Grapevine yellow speckle viroid GYSVd 367 27 T CEVd Grapevine 1B viroid G1BVd 363 28 TASVd Australian grapevine viroid AGVd 369 29 TPMVd Coconut tinangaja viroid CTiVd 254 30 CSVd sArMV Velvet tobacco mottle virus* vVTMoV 366 31 Solanum nodiflorum mottle FIG. 1. Consensus phylogenetic tree obtained for the RNA sequences listed in Table 1 (see text). ***, Group monophyletic in all virus* vSNMV 378 31 of the replicates; **, monophyletic in more than 99% of the repli- Lucerne transient streak virus* vLTSV 324 32 cates; *, in more than 95%; +, in more than 90%; and -, in more than Subterranean clover mottle virus* vSCMoV 332 33 80%. The remaining groups appeared as monophyletic in less than Tobacco ringspot virust sTRSV 359 34 80% of 1000 replicates. ASBVd has been taken as outgroup. Groups Arabis mosaic virust sArMV 300 35 are considered from ASBVd to the left of the figure within the viroid Hepatitis 8 virus RNAt vHDV 368 36 family, and from ASBVd to the right within the satellite family (including the viroidlike domain of HDV RNA). For example, nt, Nucleotides. sTRSV and sArMV (satellite family) or CCCVd, CTiVd, and HLVd *Circular (viroidlike) satellite RNA of virus listed. (viroid family) conform to two well-defined monophyletic groups in tLinear satellite RNA of virus listed. all of the bootstrap replicates. tViroidlike domain as indicated in ref. 4. HDV RNA appears closely associated in the tree with the as is the case with the present sequences. The method is subgroup formed by most of the plant viroidlike satellite based on the statistical analysis of the six distances defined RNAs. In fact, despite its mammalian host, HDV RNA is for four sequences in all the possible quartets that can be closer to this subgroup than is the plant viroidlike satellite formed with all the sequences. RNA of Lucerne transient streak virus (Fig. 1). Within viroids themselves the high frequency of appear- RESULTS ance of groups in the consensus tree supports the four groups described above. The length of the branch that separates trees the were The phylogenetic relating different RNAs that ASBVd from all other sequences clearly shows that ASBVd two were obtained by the phylogenetic procedures similar. constitutes an independent group. It has been suggested that, The inferred evolutionary process is shown in Fig. 1. The on the basis of its intermediate biological properties, ASBVd following groups can be distinguished: could represent an evolutionary link between typical viroids Linear satellite RNAs (sTRSV, sArMV) and satellite RNAs (17). The location of ASBVd in the tree Circular (viroidlike) satellite RNAs (vLTSV, vSCMoV, supports this hypothesis. vSNMV, vVTMoV), together with the viroidlike domain of Application of the method of statistical geometry in dis- HDV RNA tance space to the whole set of sequences resulted in a value Avocado sunblotch viroid (ASBVd) of x/y = 0.18, where x/y stands for the rate of deviation from Typical viroids, which can be subdivided into tree-likeness to the true branch length (Table 2).