ABSTRACT
With the exponential increase in the number of bacterial taxa with genome sequence data, a new standardized method to assign species designations is needed that is consistent with classically obtained taxonomic analyses. This is particularly acute for unculturable, obligate intracellular bacteria with which classically defined methods, like DNA-DNA hybridization, cannot be used, such as those in the Rickettsiales. In this study, we generated nucleotide-based core genome alignments for a wide range of genera with classically defined species, as well as those within the Rickettsiales. We created a workflow that uses the length, sequence identity, and phylogenetic relationships inferred from core genome alignments to assign genus and species designations that recapitulate classically obtained results. Using this method, most classically defined bacterial genera have a core genome alignment that is ≥10% of the average input genome length. Both Anaplasma and Neorickettsia fail to meet this criterion, indicating that the taxonomy of these genera should be reexamined. Consistently, genomes from organisms with the same species epithet have ≥96.8% identity of their core genome alignments. Additionally, these core genome alignments can be used to generate phylogenomic trees to identify monophyletic clades that define species and neighbor-network trees to assess recombination across different taxa. By these criteria, Wolbachia organisms are delineated into species different from the currently used supergroup designations, while Rickettsia organisms are delineated into 9 distinct species, compared to the current 27 species. By using core genome alignments to assign taxonomic designations, we aim to provide a high-resolution, robust method to guide bacterial nomenclature that is aligned with classically obtained results.
IMPORTANCE With the increasing availability of genome sequences, we sought to develop and apply a robust, portable, and high-resolution method for the assignment of genera and species designations that can recapitulate classically defined taxonomic designations. Using cutoffs derived from the lengths and sequence identities of core genome alignments along with phylogenetic analyses, we sought to evaluate or reevaluate genus- and species-level designations for diverse taxa, with an emphasis on the order Rickettsiales, where species designations have been applied inconsistently. Our results indicate that the Rickettsia genus has an overabundance of species designations, that the current Anaplasma and Neorickettsia genus designations are both too broad and need to be divided, and that there are clear demarcations of Wolbachia species that do not align precisely with the existing supergroup designations.
INTRODUCTION
While acknowledging the disdain that some scientists have for taxonomy, Stephen Jay Gould frequently highlighted in his writings how classifications arising from a good taxonomy both reflect and direct our thinking, stating, “the way we order reflects the way we think. Historical changes in classification are the fossilized indicators of conceptual revolutions” (1). Historically, bacterial species delimitation relied on phenotypic, morphologic, and chemotaxonomic characterizations (2–4). The 1960s saw the introduction of molecular techniques in bacterial species delimitation through the use of GC content (5), DNA-DNA hybridization (DDH) (6), and 16S rRNA sequencing (7, 8). Currently, databases like SILVA (9) and Greengenes (10) use 16S rRNA sequencing to identify bacteria. However, 16S rRNA sequencing often fails to separate closely related taxa, and its utility for species-level identification is questionable (10–12). Multilocus sequence analysis (MLSA) has also been used to delineate species (13), as has the phylogenetic analysis of both rRNA and protein-coding genes (3, 14, 15). Nongenomic mass spectrometry-based approaches, in which expressed proteins and peptides are characterized, provide complementary data to phenotypic and genomic species delimitations (16, 17) and are used in clinical microbiology laboratories. However, DDH remains the “gold standard” of defining bacterial species (18, 19), despite the intensive labor involved and its inability to be applied to nonculturable organisms. A new genome-based bacterial species definition is attractive, given the increasing availability of bacterial genomes, rapid sequencing improvements with decreasing sequencing costs, and data standards and databases that enable data sharing.
Average nucleotide identity (ANI) and digital DDH (dDDH) were developed as genomic-era tools that allow for bacterial species classification with a high correlation to results obtained using wet lab DDH, while bypassing the associated difficulties (20–22). For ANI calculations, the genome of the query organism is split into 1-kbp fragments, which are then searched against the whole genome of a reference organism. The average sequence identity of all matches having >60% overall sequence identity over >70% of their length is defined as the ANI between the two organisms (18). dDDH uses the sequence similarity of conserved regions between two genomes of interest (23) to calculate genome-to-genome distances. These distances are converted to a dDDH value, which is intended to be analogous to DDH values obtained using traditional laboratory methods (23). There are three formulas for calculating dDDH values between two genomes using either (i) the length of all matches divided by the total genome length, (ii) the sum of all identities found in matches divided by the overall match length, and (iii) the sum of all identities found in matches divided by the total genome length, with the second formula being recommended for assigning species designations for draft genomes (24, 25). However, neither ANI nor dDDH reports the total length of fragments that match the reference genome, and problems arise when only a small number of fragments are unknowingly used. Additionally, neither method generates an alignment that can be used for complementary phylogenetic analyses, instead relying solely