Imbroglios of viral taxonomy: genetic exchange and failings of phenetic approaches

Abstract

The practice of classifying organisms into hierarchical groups originated with Aristotle and was codified into nearly immutable biological law by Linnaeus. The heart of taxonomy is the biological species, which forms the foundation for higher levels of classification. Whereas species have long been established among sexual eukaryotes, achieving a meaningful species concept for prokaryotes has been an onerous task and has proven exceedingly difficult for describing viruses and bacteriophages. Moreover, the assembly of viral "species" into higher-order taxonomic groupings has been even more tenuous, since these groupings were based initially on limited numbers of morphological features and more recently on overall genomic similarities. The wealth of nucleotide sequence information that catalyzed a revolution in the taxonomy of free-living organisms necessitates a reevaluation of the concept of viral species, genera, families, and higher levels of classification. Just as microbiologists discarded dubious morphological traits in favor of more accurate molecular yardsticks of evolutionary change, virologists can gain new insight into viral evolution through the rigorous analyses afforded by the molecular phylogenetics of viral genes. For bacteriophages, such dissections of genomic sequences reveal fundamental flaws in the Linnaean paradigm that necessitate a new view of viral evolution, classification, and taxonomy.

FIG. 1.

Conflicts between morphological classification and genetic relatedness in dsDNA-tailed bacteriophages. Although bacteriophages HK97 (70) and L5 (59) share very similar morphologies, including a long flexible tail, there are no genes or encoded proteins that are detectable as close homologues. In contrast, bacteriophage P22 (132) has substantial numbers of genes in common with HK97; genetic differences lead to different tail morphologies, which led to classification into two distinct families. Proteins shared between HK97 and P22 exhibit BLASTP (4, 5) E values between 10⁻¹⁶ and 10⁻¹⁰¹; BLASTP E values for comparisons with L5 proteins failed to reveal matches with significance values lower than 10⁰.

FIG. 2.

Evidence for gene exchange via homologous recombination among P2-related bacteriophages Wφ, φD, HK111, and HK241, which was initially detected by significant lack of homoplasy among certain gene sequences (99). Here phylogenetically informative sites (listed at the center of the figure) were extracted from sequences encoding the structural genes from four bacteriophages sufficiently closely related that multiple substitutions have not likely occluded phylogenetic relationships. A recombination event is evident between bases 2008 and 2472 of the aligned sequences and is denoted by the gap in the alignment of informative sites. Nucleotide positions supporting the significantly most parsimonious phylogeny on either side of the recombination join point are noted with asterisks; different phylogenies are robustly supported by the 5′ and 3′ portions of the sequence. ✽, P < 0.05 by Felsenstein's S test (41); ✽✽, P < 0.05 by both Felsenstein's S test and C test (41).

FIG. 3.

Mosaicism of gene cassettes among dsDNA-tailed bacteriophages. Genes in cassettes bearing homologous genes are coordinately colored; striped genes indicate that protein products perform analogous functions. The sequences for bacteriophages N15 (112), λ (116), HK97 (70), and Mu (94) were obtained from public databases; the SfV sequence (3) was kindly provided by N. Verma prior to publication.

FIG. 4.

Evidence for homologous recombination and mosaicism of gene cassettes among ssDNA filamentous bacteriophages. Genes in cassettes bearing homologous genes are coordinately colored; orthologous gene cassettes with various degrees of similarity are represented by various shades of the same color, whereas nonorthologous replacements are denoted by differently colored cassettes. The darker regions in genes I and IV of phages IKe and I2-2 indicate homologous recombination events with M13/fd-like sequences first noted by Peeters et al. (106). The sequences of bacteriophages fd (8), M13 (140), Ike (106), and I2-2 (123) were obtained from the public database; the φYP01 sequence represents an inferred prophage in the Yersinia pestis genome (104) beginning at gene YP02274 and continuing through downstream genes.

FIG. 5.

Problems arising in using phenetic methods for bacteriophage taxonomy. (A) Relationships inferred from overall similarity (e.g., number of shared genes, DNA sequence similarity, DNA-DNA hybridization, proteomic overlap) are here depicted in a Venn (141) diagram. These data can be misleading in two ways (see the text). (B) A phylogeny is drawn for all taxa in part A by using the robust phenetic approach UPGMA (122, 127). (C) Relationships are contingent upon the taxa included in the analysis; here, the elimination of taxon “Y” eliminates the connection between taxon “Z” and the remaining taxa.