viruses Communication A Roadmap for Genome-Based Phage Taxonomy Dann Turner 1 , Andrew M. Kropinski 2,3 and Evelien M. Adriaenssens 4,* 1 Department of Applied Sciences, University of the West of England, Bristol BS16 1QY, UK; [email protected] 2 Department of Food Science, University of Guelph, Guelph, ON N1G 2W1, Canada; [email protected] 3 Department of Pathobiology, University of Guelph, Guelph, ON N1G 2W1, Canada 4 Quadram Institute Bioscience, Norwich Research Park, Norwich NR4 7UQ, UK * Correspondence: [email protected] Abstract: Bacteriophage (phage) taxonomy has been in flux since its inception over four decades ago. Genome sequencing has put pressure on the classification system and recent years have seen significant changes to phage taxonomy. Here, we reflect on the state of phage taxonomy and provide a roadmap for the future, including the abolition of the order Caudovirales and the families Myoviridae, Podoviridae, and Siphoviridae. Furthermore, we specify guidelines for the demarcation of species, genus, subfamily and family-level ranks of tailed phage taxonomy. Keywords: phage taxonomy; phage classification; Caudovirales; Myoviridae; Podoviridae; Siphoviridae; demarcation criteria 1. An Ongoing Revolution in Phage Taxonomy Historically, phages have been classified according to their morphology, dating from Citation: Turner, D.; Kropinski, A.M.; the time before the existence of PCR, sequencing or many of the molecular methods we Adriaenssens, E.M. A Roadmap for know today [1–3]. For tailed phages, the formal taxonomy was derived from the pioneering Genome-Based Phage Taxonomy. classification work of David Bradley (Memorial University, Canada) who classified them Viruses 2021, 13, 506. https:// into three morphotypes, A (contractile tail), B (long, non-contractile tail), C (short non- doi.org/10.3390/v13030506 contractile tail, based on electron microscopy, a system that was subsequently enhanced by Ackermann and Eisenstark (1974) [4,5]. In 1971, this system was formally adopted by the Academic Editor: Terje Dokland International Committee on Nomenclature of Viruses (ICNV) but not with the names we are familiar with today. The names Myoviridae, Podoviridae and Siphoviridae were formally Received: 8 February 2021 accepted by the International Committee on Taxonomy of Viruses (ICTV) in 1981 and Accepted: 16 March 2021 1984. The order Caudovirales, unifying all tailed phages, was proposed in 1998 by Hans- Published: 18 March 2021 Wolfgang Ackermann and approved by postal vote. Some of the other phage families have equally long histories with the families Inoviridae, Microviridae, Tectiviridae, Corticoviridae, Publisher’s Note: MDPI stays neutral Plasmaviridae, Leviviridae, and Cystoviridae all formalised by plenary session vote in 1978 (for with regard to jurisdictional claims in published maps and institutional affil- a history of taxonomy releases see https://talk.ictvonline.org/taxonomy/p/taxonomy_ iations. releases, accessed on 5 February 2021). This >40-year-old family-level classification system resulted in the classic textbook figures (Figure1) on phage taxonomy, easily represented by line drawings. As the age of genomics dawned in the early 2000s, the sequencing of phage genomes revealed a much higher genomic diversity than had previously been considered, especially Copyright: © 2021 by the authors. in bacteriophages belonging to the order Caudovirales, leading to the creation of the first Licensee MDPI, Basel, Switzerland. subfamilies within the existing three families Podoviridae [7], Myoviridae [8], and later on This article is an open access article distributed under the terms and Siphoviridae [9]. As the number of phage genomes in databases rose, it quickly became ap- conditions of the Creative Commons parent that these three families were not monophyletic and cohesive within a monophyletic Attribution (CC BY) license (https:// order. This paraphyly was illustrated by a number of tools and publications: The Phage creativecommons.org/licenses/by/ Proteomic Tree [10,11], the first phage genome relatedness network representation [12], a 4.0/). bipartite network of shared genes [13], an updated network of shared predicted proteins Viruses 2021, 13, 506. https://doi.org/10.3390/v13030506 https://www.mdpi.com/journal/viruses Viruses 2021, 13, 506 2 of 10 (vConTACT) [14,15], a composite tool combining gene homologies and gene order (GRAV- iTy) [16,17], a virus domain orthologous groups approach (VDOG) [18] and a concatenated protein phylogeny of members of the order Caudovirales (CCP77) [19]. Based on this ev- idence, the ICTV’s Bacterial and Archaeal Viruses Subcommittee started disentangling the web of overlapping and complementary groups of tailed phages by defining new, genome-based families. At the time of writing, three new families of myoviruses have Viruses 2021, 13, 506 been officially ratified Ackermannviridae [20], Chaseviridae [21], Herelleviridae [22,23]; two2 of 9 for the siphoviruses, Demerecviridae [21], and Drexlerviridae [21], and one of podoviruses, Autographiviridae [21]. Figure 1. Line drawing of bacteriophage morphotypes, adapted from Ackermann, 2005 [6]. Figure 1. Line drawingIf we of look bacteriophage beyond the morphotypes, traditional tailedadapted bacteriophages, from Ackermann, we 2005 are [6] observing. a similar increase in genomic diversity in other phage clades, but interestingly, these expansions are mainlyAs driven the age by of metagenome-derived genomics dawned in information.the early 2000s, Using the sequencing a combination of phage of sequencing, genomes isolationrevealed and a much imaging higher methods, genomic a new diversity major lineage than had of non-tailed previously dsDNA been considered, phages was iden-espe- tifiedcially inin marinebacteriophages bacteria, belonging named Autolykiviridae to the order Caudovirales[24]. Similarly,, leading isolation to the of acreation new ssDNA of the phagefirst subfamilies and description within of the the existing new family threeFinnlakeviridae families Podoviridaelinks non-tailed [7], Myoviridae icosahedral [8], and ssDNA later andon Siphoviridae dsDNA phages [9]. As together the number [25,26 of]. phage Major genomes lineages in of databases presumed rose, novel it quickly dsDNA became tailed phagesapparent have that also these been three inferred families and were isolated not basedmonophyletic on metagenomic/viromic and cohesive within assemblies, a mono- includingphyletic order. the crAssphage This paraphyly lineage was [27 illustrated–29], Lak megaphage by a number [30 of], andtools multiple and publications: other lineages The ofPhage “huge Proteomic phages” Tree [31]. [10,11], the first phage genome relatedness network representation [12],For a bipartite the filamentous, network of ssDNA shared phages, genes [13] the, familyan updatedInoviridae networkhas beenof shared split predicted into two families,proteins (vConTACT)Inoviridae and [14,15]Plectroviridae, a compositewhich tool are combining grouped together gene homologies in the order andTubulavi- gene or- ralesder (GRAViTy)[21], with a [16,17] potential, a virus further domain increase orthologous with five groups new families approach based (VDOG) on the [18] analysis and a ofconcatenated cryptic inoviruses protein phylogeny from bacterial of members genome of datasets the order [32 Caudovirales]. In a similar (CCP77) vein, [19] many. Based ad- ditionalon this evidence, subfamilies the have ICTV’s been Bacterial proposed and in Archaeal the ssDNA Viruses family SubcommitteeMicroviridae started, beyond disen- the existingtangling subfamilies the web of overlappingBullavirinae [and9] and complementaryGokushovirinae groupsbased of on tailed the detection phages by in defining virome data,new, i.e.genome the subfamilies-based famil “Alpavirinae”ies. At the time [33], of “Pichovirinae” writing, three [ 34new], “Stokavirinae” families of myoviruses [35], and “Aravirinae”have been officially [35]. Recently, ratified computationalAckermannviridae approaches [20], Chaseviridae identified [21] a massive, Herelleviridae expansion [22,23] in; thetwo number for the of siphoviruses, ssRNA phage Demerecviridae genomes of the [21]Leviviridae, and Drexlerviridaefamily, first with[21], 158and [ 36one] then of podo- with aviruses, further Autographiviridae 1k complete and [21] 15k.partial genomes [37]. If we look beyond the traditional tailed bacteriophages, we are observing a similar increase in genomic diversity in other phage clades, but interestingly, these expansions are mainly driven by metagenome-derived information. Using a combination of sequenc- ing, isolation and imaging methods, a new major lineage of non-tailed dsDNA phages was identified in marine bacteria, named Autolykiviridae [24]. Similarly, isolation of a new ssDNA phage and description of the new family Finnlakeviridae links non-tailed icosahe- dral ssDNA and dsDNA phages together [25,26]. Major lineages of presumed novel dsDNA tailed phages have also been inferred and isolated based on metagenomic/viromic assemblies, including the crAssphage lineage [27–29], Lak megaphage [30], and multiple other lineages of “huge phages” [31]. Viruses 2021, 13, 506 3 of 10 Across all the different lineages of bacteriophages it has become clear that fundamental changes to classification are required in order to address this increasing genomic diversity. 2. The Next Steps for Tailed Phage Taxonomy Within phage taxonomy, the most pressing issue remains the paraphyly of the tailed phage families, because they