Fundamental Difficulties Prevent the Reconstruction of the Deep Phylogeny of Viruses

Abstract

The extension of virology beyond its traditional medical, veterinary, or agricultural applications, now called environmental virology, has shown that viruses are both the most numerous and diverse biological entities on Earth. In particular, virus isolations from unicellular eukaryotic hosts (heterotrophic and photosynthetic protozoans) revealed numerous viral types previously unexpected in terms of virion structure, gene content, or mode of replication. Complemented by large-scale metagenomic analyses, these discoveries have rekindled interest in the enigma of the origin of viruses, for which a description encompassing all their diversity remains not available. Several laboratories have repeatedly tackled the deep reconstruction of the evolutionary history of viruses, using various methods of molecular phylogeny applied to the few shared "core" genes detected in certain virus groups (e.g., the Nucleocytoviricota). Beyond the practical difficulties of establishing reliable homology relationships from extremely divergent sequences, I present here conceptual arguments highlighting several fundamental limitations plaguing the reconstruction of the deep evolutionary history of viruses, and even more the identification of their unique or multiple origin(s). These arguments also underline the risk of establishing premature high level viral taxonomic classifications. Those limitations are direct consequences of the random mechanisms governing the reductive/retrogressive evolution of all obligate intracellular parasites.

Keywords: Bamfordvirae; Nucleocytoviricota; Varidnaviria; obligate intracellular parasites; origin of viruses; phylogenetic reconstruction; reductive evolution.

Figure 1

Erroneous phylogenetic relationships between seven obligate intracellular parasitic bacteria. Top: The neighbor-joining tree was generated from 1014 conserved sites in the multiple alignment of their DNA polymerase alpha subunits using the JTT substitution matrix. The protein NCBI identifiers are indicated. In absence of free-living bacterial relatives, the tree erroneously suggests (with a strong statistical support) the existence of 3 separate “parasite families” emerging from 3 distinct evolutionary branches (Alpha, Beta, Gamma). Bottom: Using a different representation, the tree topology (inherent to the tree-building algorithm) can be interpreted as supporting the existence of an ancestral obligate parasite from which all three families of extant parasites derived. The true evolutionary history of these parasitic bacteria is shown in Figure 2.

Figure 2

A more realistic representation of the origin and evolution of the obligate intracellular parasitic bacteria depicted in Figure 1. The neighbor-joining tree was generated from 974 conserved sites in the multiple alignment of 16 DNA polymerase alpha subunits using the JTT substitution matrix. The protein NCBI identifiers are indicated. The red branches correspond to the 7 obligate intracellular parasites while free-living relatives are in black. The green branch corresponds to a distant bacterium from the Chloroflexi phylum, used as outgroup. This tree suggests (with strong statistical support) that the parasitic bacteria independently originated at least 5 times from within 5 lineages also containing free living members: once from within Actinobacteria and Betaproteobacteria, twice from Gammaproteobacteria, and once early in the Alphaproteobacteria class from which three members of the order Rickettsiales emerged. In each case, the switch to a parasitic lifestyle was associated to the loss of essential genes (reductive evolution) nowadays documented by direct comparative genomics. One exception, visible in the tree, is Protochlamydia amoebophila for which no free living relative could be found. P. amoebophila belongs to Chlamydiae, a phylum of highly diverse members all of which have—like viruses—an obligate intracellular lifestyle. In the absence of known free-living relatives, the origin of this bacterial phylum remains mysterious. Compared to Figure 1, this figure illustrates how the lack of known free-living relatives might suggest totally erroneous evolutionary scenarios. The DNA polymerase was used as a conserved protein present in all bacteria (parasitic of not). Its viral version is frequently used in global phylogenetic reconstructions of eukaryotic dsDNA viruses.

Figure 3

The “virus late” hypothesis: illustration of the intractable evolutionary scenarios resulting from random gene/function losses. A toy virus world is represented, starting from a hypothetical ancestral cell-like organism (level 0, w/o extant representative). Each box contains the abstract gene content inherited by a given virus (family) from its immediate ancestor. Red “genomes” indicate viruses with shared gene contents albeit possibly resulting from distinct evolutionary pathways. Random gene losses lead to very diverse overlaps of gene assortments (as in viruses) or to situations where no single “core gene” is shared by all virus family (here starting at level 3), as observed in actual viral genomes (in particular small ones). Individual genes recurring in multiple combinations (families) or ultimately remaining in the smallest genomes (level 6) are not more characteristic of the parasitic lifestyle than less ubiquitous ones. In addition, genes shared by more families than other (such as D) may not be better phylogenetic markers than others, as they could have been inherited from different ancestors (DE, DF, BD). Their polyphyly will not be detected if some of their above ancestors are extinct or unknown. This graph illustrates the difficulty of reconstructing the deep phylogeny of viruses beyond the immediate family level both due to the capacity of random gene losses enjoyed by obligate intracellular parasites and the lack of associated free-living organisms to be used as references.