Enterobacter hormaechei subsp. hoffmannii subsp. nov., Enterobacter hormaechei subsp. xiangfangensis comb. nov., Enterobacter roggenkampii sp. nov., and Enterobacter muelleri is a later heterotypic synonym of Enterobacter asburiae based on computational analysis of sequenced Enterobacter genomes. [version 2; peer review: 2 approved]

Background: The predominant species in clinical Enterobacter isolates is E. hormaechei. Many articles, clinicians, and GenBank submissions misname these strains as E. cloacae. The lack of sequenced type strains or named species/subspecies for some clades in the E. cloacae complex complicate the issue. Methods: The genomes of the type strains for Enterobacter hormaechei subsp. oharae, E. hormaechei subsp. steigerwaltii, and E. xiangfangensis, and two strains from Hoffmann clusters III and IV of the E. cloacae complex were sequenced. These genomes, the E. hormaechei subsp. hormaechei type strain, and other available Enterobacter type strains were analysed in conjunction with all extant Enterobacter genomes in NCBI’s RefSeq using Average Nucleotide Identity (ANI). Results: There were five recognizable subspecies of E. hormaechei: E. hormaechei subsp. hoffmannii subsp. nov., E. hormaechei subsp. xiangfangensis comb. nov., and the three previously known subspecies. One of the strains sequenced from the E. cloacae complex was not a novel E. hormaechei subspecies but rather a member of a clade of a novel species: E. roggenkampii sp. nov.. E. muelleri was determined to be a later heterotypic synonym of E. asburiae which should take precedence. Conclusion: The phylogeny of the Enterobacter genus, particularly the cloacae complex, was re-evaluated based on the type strain genome sequences and all other available Enterobacter genomes in RefSeq. Open Peer Review


Introduction
The name Enterobacter hormaechei was created for a taxon at the rank of species that had previously been called Enteric Group 75. O'Hara et al 1 . defined the type strain to be ATCC 49162 T from the 23 strains they studied. Twelve of the strains were shown to be closely related via DNA-DNA hybridization (DDH) and less closely related to other Enterobacter species. Numerous biochemical assays were performed on the 23 strains to characterize and differentiate the new species.
Hoffmann and Roggenkamp 2 investigated the genetic structure of the E. cloacae complex (the set of species included in this complex has varied over time) by a combination of sequencing of the three housekeeping genes hsp60, rpoB, and hemB; and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of ampC. They defined 12 genetic clusters (I-XII) based most exhaustively on the hsp60 sequencing. Three of the clusters (cluster III, 58 strains; cluster VI, 28 strains; and cluster VIII, 59 strains) accounted for 70% of the 206 strains studied. The authors noted that "Only 3% of our study strains clustered with the type strain of E. cloacae." (cluster XI), "We found that 3% of our study strains clustered around the E. hormaechei type strain." (cluster VII), and "Our clusters VI and VIII were closely related to E. hormaechei cluster VII. DDH studies are needed to verify whether these clusters form a common DNA relatedness group allowing emending and broadening of the species description of E. hormaechei.".
Hoffmann et al 3 . followed up with a characterization of clusters VI, VII, and VIII asserting based on DDH that these clusters were subspecies of the same species. Since cluster VII contained the type strain for E. hormaechei Hoffmann et al. named cluster VII E. hormaechei subsp. hormaechei, cluster VI E. hormaechei subsp. oharae, and cluster VIII E. hormaechei subsp. steigerwaltii. Forty-eight strains were characterized using 129 biochemical tests showing that there were phenotypic differences between the subspecies. Unfortunately the authors did not decide to include the other predominant cluster (III) in their analysis, nor did they validly publish these subspecies names. This was rectified recently in Validation List no. 172 4 .
Gu et al 5 . defined E. xiangfangensis using a phylogenetic tree based upon concatenated partial rpoB, atpD, gyrB and infB gene sequences from a novel isolate and existing type strains where E. xiangfangensis grouped closest to E. hormaechei.
Biochemical assays were performed and E. xiangfangensis strains were differentiable from the E. hormaechei type strain.
During analysis of the E. cloacae complex and E.(now Klebsiella 6 ) aerogenes strains looking at antimicrobial resistance patterns 7 , many of the Hoffmann et al. clusters were rediscovered using whole genome comparisons such as SNP analysis and average nucleotide identity (ANI). The clusters were identifiable by the hsp60 sequences deposited by the Hoffmann group. The three subspecies of E. hormaechei defined by Hoffmann et al. fell within the expected ANI range for bacterial species, being greater than 95% ANI between subspecies and greater than 98% ANI within a subspecies. Unexpectedly Hoffmann cluster III also met the ANI criteria to be an E. hormaechei subspecies. Further, genomes named E. xiangfangensis in GenBank fell within the E. hormaechei subsp. steigerwaltii cluster rather than a separate cluster. Moreover, most of the genomes in these clusters were mistakenly identified as E. cloacae when they were submitted to GenBank. To resolve the naming inconsistencies of these genomes the type strains for E. hormaechei subsp. steigerwaltii, E. hormaechei subsp. oharae, E. xiangfangensis, Hoffmann cluster III, and Hoffmann cluster IV were sequenced.
Tools for bacterial species assignment have changed over time 8,9 . Initially, morphology as viewed through a microscope and later aided by staining such as Gram staining 10 to distinguish cell wall differences was used. Biochemical assays and other methods to determine phenotype followed. Use of the genome started with DNA-DNA hybridization (DDH) where a 70% threshold for species followed later by a 79% threshold for subspecies were proposed. Widespread use of marker genes in particular the 16S rRNA gene made assays easier. A threshold of less than 97% identity for the 16S rRNA gene was used to determine a new species but values above 97% could not guarantee that isolates were the same species. The sequence of other less conserved marker genes such as hsp60 has also been used to differentiate species. More recently multiple marker genes are sequenced and a combined alignment is used. With the advent of inexpensive genome sequencing, computing ANI, which correlates very closely with DDH, has largely supplanted other methods. Studies have shown that an ANI threshold between 94-96.5% correlates well with existing species definitions and 97-98% for subspecies [11][12][13][14][15][16][17][18][19] . DDH has been shown to not only correlate with ANI but also with how many of the genes or what fraction of the genomes are shared in common so some ANI based tools take this measurement into account as well [17][18][19] . Most definitions of new species involve sequencing the genome and taking ANI and shared gene content into account in some fashion but many species definitions predate genome sequencing and some type strains have not been sequenced. There is no generally accepted method for reconciling older species definitions with genome comparisons but usually ANI and shared gene content form a basis for the analysis.
As Hoffmann 2,3 and others 20-26 discovered the predominant species in clinical Enterobacter isolates is E. hormaechei. Unfortunately many articles, clinicians, and GenBank submissions misname these strains as E. cloacae perhaps as a short hand for the E. cloacae complex and possibly due to the

Amendments from Version 1
This version of the paper addresses the referees' concerns. The paper is now in IMRAD format. E. asburiae subspecies are now discussed more fully. The "Candidatus" designation is discussed. PanOCT Average Nucleotide Identity (ANI) is compared to the Genome to Genome Distance Calculator (GGDC). A more thorough analysis of the outlier genomes is performed. The gene content differences between the E. hormaechei subspecies is made clearer. Author Thomas H. Clark's name has been corrected to "Thomas H. Clarke".

REVISED
E. hormaechei subspecies not being validly published until recently. Another issue was the lack of sequenced type strains or named species/subspecies for some clades. The definition of what species/subspecies make up the E. cloacae complex has been in flux 2,27,28 and even what species are in the genus Enterobacter 29-31 .
The E. cloacae complex was shown to have 18 clades (A-R) 7 , 12 of which corresponded to 11 of the 12 clusters defined previously by Hoffmann 2 . Hoffmann cluster X is E. nimipressuralis which has been reclassified as Lelliottia nimipressuralis 29 . Table 1 incorporates more recently sequenced genomes and published papers adding four clades (S-V) and incorporating the latest literature. For example, clade R (Hoffmann cluster IX) was recently defined to be E. bugandensis 31 .

Results
All RefSeq genomes labelled as being in the genus Enterobacter were downloaded from NCBI RefSeq resulting in 1,249 genomes. A fast approximate ANI tool, called MASH 32 , was used to generate a pairwise ANI based distance matrix and average linkage hierarchical clustering was used to generate the tree shown in Figure 1. 1,216 genomes were assigned to 22 clades (A-V Table 1) in the E. cloacae complex (Supplemental Table 1) while 30 genomes were deemed to be outliers and not in the Enterobacter genus (best MASH matches in Supplemental Table 2) as well as 2 E. lignolyticus genomes and 1 E. timonensis genome deemed to be outside of the E. cloacae complex. Two species of Enterobacter: E. siamensis and E. tabaci do not have sequenced genomes and their type strains' 16S rRNA sequences while having full length matches at 98% and 99% respectively to some E. cloacae complex genomes did not have definitive matches to any particular clade. The type strains for E. asburiae and E. muelleri fall within the same clade (J -Hoffmann cluster I). All 78 genomes in this clade are above the 95% ANI species cut-off (Table 2) but using a 98% ANI subspecies cut-off produces 8 subclades of sizes 1, 1, 2, 2, 2 (E. muelleri), 3 (E. asburiae), 24, and 43. Thus E. muelleri 33 is a later heterotypic synonym of E. asburiae 34 which should take precedence. Whether the 8 subclades of E. asburiae should be treated as subspecies is beyond the scope of this paper but is revisited in the Discussion section.
Five clades (A-E) are above the 95% ANI cut-off to be considered the same species (Table 2). Almost all within-clade pairwise ANIs are greater than between-clade ANIs ( While we believe that ANI and other similar measures recently categorized as overall genome related index (OGRI) 35 should be used for species/subspecies determination, phenotypic differences due to gene content may play a role particularly for delineation of subspecies. To explore the gene content differences of the E. cloacae complex and the E. hormaechei subspecies in particular, the pan-genome of the 1,216 E. cloacae complex genomes was determined using PanOCT 36 . The pan-genome generates orthologous gene clusters that delineate which genes are in common between the clades and which genes differentiate the clades (Supplemental Table 3 and Supplemental Table 4). There were 2,966 genes in "common to all" of the clades (present in 90% of the genomes of each clade). The number of genes "specific to" a clade (present in 90% of the genomes of that clade and in less than 10% of genomes from any other clade) varied from 0 (L) to 465 (V). The number of genes "missing from" a clade (present in less than 10% of the genomes of that clade and present in at least 90% of the genomes of all other clades) varied from 0 (A,C,H,K,O) to 40 (U). The clades which represent named species and subspecies show no qualitative difference in gene content from clades with no named species (Supplemental Table 4). In particular, clade D which is the proposed E. hormaechei subsp. hoffmannii has more genes specific to it than 3 of the 4 recognized subspecies. The gene content numbers need to be looked at carefully since they depend on the number of genomes in a clade (T has 187 clade specific genes but this is based on a single genome which means it is really strain specific genes rather than species specific), the distance from other clades (V the most distant clade has 465 specific genes and also has only 3 genomes), and sampling bias such as if most genomes in a clade are from a clonal outbreak. Gene content analysis can also be confounded by misassembly or misannotation of draft genomes which is why we use RefSeq genomes which have passed a quality screen and are consistently annotated. Again we emphasize that ANI as our primary criterium appears to have less of these subjective issues to deal with.
Biochemical and other properties of the E. hormaechei subspp. clades have been previously published 3,5 except for clade D. These biochemical properties were used to differentiate between the subspecies but not between other species within the E. cloacae complex. With the availability of whole genome sequences and pan-genome analysis tools some of the observed phenotypic traits can be assigned to genetic features, such as the presence or absence of protein coding genes for known metabolic pathways. E. hormaechei subsp. hormaechei was previously distinguished from E. hormaechei subsp. oharae and E. hormaechei subsp. steigerwaltii by growth on dulcitol (a.k.a. galactitol) as the sole carbon source 3 . This phenotype can be explained by the presence of a gat operon 7,37 within all 7 of the hormaechei subsp. genomes while none of oharae, steigerwaltii, or hoffmannii genomes have the gat operon. In the same genomic location, between the D-galactarate dehydratase gene and the 16S rRNA methyltransferase gene, all of the steigerwaltii, oharae, and hoffmannii subspp. genomes have a related, but different operon, encoding for N-acetyl galactosamine metabolism (a.k.a., the aga operon) 7,38 . For xiangfangensis most (222 out of 255) of the genomes have the aga operon but 33 have the gat operon instead. Similarly, steigerwaltii isolates can be distinguished from hormaechei, oharae, xiangfangensis, and hoffmannii by their ability to grow on adonitol (a.k.a. ribitol) and D(+)-arabitol;  as a unit between core clusters (16694-5) and another 6 (15153-15156, 27141-2) occur between core clusters (17653-4). These clusters have no or vague annotation but are intriguing targets to provide functional phenotypic differences. which generated data that could be extracted into an all versus all ANI comparison (Supplemental Table 5). We used the GGRaSP 40 R package (version 1.0) which generated an ultramateric tree by using the R hclust function with average linkage from the distance matrix calculated by subtracting 100 from the MASH ANI results. The result was translated into Newick format with the APE 41 R package (Supplemental File 1) rendered with metadata annotated using the Interactive Tree of Life 42 into Figure 1.

MASH
Based on the tree 30 genomes were deemed to be outliers and probably not in the Enterobacter genus as well as 2 E. lignolyticus genomes and 1 E. timonensis genome deemed to be outside of the E. cloacae complex. These 30 genomes were compared to all genome sequenced bacterial type strains from NCBI RefSeq (Supplemental Table 2) using MASH which confirmed that these genomes were likely misnamed as Enterobacter. The decision to leave E. lignolyticus and E. timonensis outside of the E. cloacae complex was based on two reasons: historically neither has been included in the complex, and there is a quantitative difference in the mean ANI values between genomes of these two species and genomes included in the 22 clades within the complex (last two rows of  Table 2), and all type strains from other genera closely related to Enterobacter and generated pairwise ANI values using PanOCT (Supplemental Table 7) to build both UPGMA and Neighbor-Joining trees (Supplemental Figure 2). This analysis supported our decision on what genomes are outliers. One anomaly arose from this analysis: the current type strain genome for Lelliottia nimipressuralis currently in GenBank (ASM187564v1) is the same species as the proposed E. roggenkampii (ASM172980v1) type strain. The type strain 16S sequence (Z96077) for Lelliottia nimipressuralis doesn't match this purported type strain genome sequence and this genome is an exact duplicate to the previously submitted Enterobacter sp. FB (ASM80579v1). The duplicate genomes are from the same submitter and the only reasonable conclusion is that this was a submission error for Lelliottia nimipressuralis. This has been reported to NCBI GenBank for resolution (Supplemental File 2).
From the all versus all MASH ANI comparison GGRaSP was used to generate average linkage clusters and the medoids of those clusters at both the 95% (species) and 98% (subspecies) levels. If type strains existed at the subspecies level those clusters were used (E. hormaechei and E. cloacae) otherwise species level clusters were used resulting in 22 clades (A-V). If a type strain genome sequence existed for a clade it was selected otherwise the medoid was selected as a proxy. The one exception for this was clade J where two different type strains existed: E. asburiae and E. muelleri where both were retained for the typing. These 23 representative genomes were used to "type" all 1,216 Enterobacter cloacae complex genomes (Supplemental Table 1). For typing the best MASH ANI match was used and resolved to either the species or subspecies level. As expected the typing was in complete agreement with the clades in the MASH ANI tree (Figure 1) The diverse 250 genome PanOCT run and the level 1 PanOCT batch runs used the PanOCT (version 3.27) command line:

panoct.pl -b results -t combined.blast -f genomes.list -g combined. att -P combined.fasta -S yes -L 1 -M Y -H Y -V Y -N Y -F 1.33 -G y -c 0,50,95,100 -T
The hierarchical PanOCT run of 1,216 genomes produced a matrix of orthologous gene clusters (Supplemental Table 3) where the rows are clusters and the columns are genomes with the cells containing the RefSeq IDs for the gene from the corresponding genome. This matrix was used to determine genes common to all, specific to, and missing from clades A-V. Individual PanOCT runs were also done for clade J, D, and M. Clade J to insure that PanOCT ANI values confirmed MASH ANI values that E. asburiae and E. muelleri are the same species which they did and these ANI values were used to determine the 8 subclades at 98% ANI using hierarchical clustering (hclust in R) average linkage. Clade D to confirm the MASH ANI values for E. hormaechei subsp. hoffmannii which they did. Clade M was done likewise to confirm E. roggenkampii which they did.

Discussion
The Introduction section reviews how the tools for defining a species have evolved. In a recent review of the genus Mycobacterium, the authors proposed that any newly defined bacterial species must have a genome sequence and an ANI comparison carried out against existing sequenced type strains to justify a novel species assignment 45 . ANI analysis should not be relied on in isolation for defining a species since historical or clinical phenotypic distinctions may be important for example in distinguishing between E. coli and Shigela which by ANI are the same species. However, genome sequencing appears to be outstripping the taxonomic definition of species within some genera. For the 22 clades of the E. cloacae complex identified here 9 do not have named type strains (7 if the two proposed here are adopted). For important pathogens where clinical practice may rely on proper classification the ability to name these clades/species and provide resources for identifying them could be pivotal. Unfortunately, the current established journal for validly publishing bacterial species' names, IJSEM, insists on phenotypic characterization and deposition of the type strain before naming is valid. This prevents computational based methods from moving quickly. Paradoxically almost all species identifying diagnostic tests are genotype not phenotype based so genotype is good enough for diagnosis but not species definition. Further, delineating what is acceptable to define as a new species is also genotype not phenotype based whether via DDH, marker genes, or more recently ANI. Worse there are no published standards for what defines the minimal set of phenotypic biochemical assays that must be performed. As the Mycobacterium review authors state: "The easy and affordable availability of reliable wholegenome sequences raises doubts about the real added value of investigating phenotypic traits when a new species is described. Actually, different taxonomists use their own panels of tests, often not standardized, to produce results of no use for colleagues and absolutely incomprehensible to the community of mycobacteriologists who have dismissed such approach since the '90s. For the genus Mycobacterium the major phenotypic traits that cannot be disregarded should include growth rate and pigmentation of colonies, while the classical investigation of biochemical activities is clearly obsolete.". If there were accepted standards for minimal phenotypic characterization then culture collection repositories could choose to provide the characterization as fee for service or even for free for type strains as an incentive for deposition. With the rapid growth in synthetic genomics capabilities one could argue that the deposition of a high quality complete genome might suffice rather than a culture.
We propose allowing "placeholder" species or subspecies names such as "E. cloacae complex clade S" in order to enable the most robust use of computational and genomic resources for clinical diagnosis. IJSEM currently recognizes provisional species names under the Candidatus designation 46 . Candidatus was designed for unculturable organisms where a type strain could not be maintained but phenotypic data is still required to be submitted. This is not a good fit for the case where genome sequences exist and species/subspecies are determined computationally because it was designed for environmental or unculturable samples with limited sequence data but at least some phenotypic or morphological data. We suggest that some similar designation be used for our proposed "placeholder" names. We do not want to computationally assign permanent names with a provisional status, but would rather have the name itself indicate it is provisional and to be replaced when someone does the hard work of depositing a type strain and any required minimal phenotypic information.
In the Results section we noted that the type strains for E. asburiae and E. muelleri fall within the same clade which could be separated into subspecies by ANI but we declined to do so. For E. hormaechei we did propose new subspecies but this was because subspecies for E. hormaechei had already been defined. We believe that there must be a cogent reason for delineating beyond the species level. We agree with Chun et al. 35 who state: "At this stage, we do not have sufficient data to provide a general guideline for defining subspecies using genome data. However, a good practice should involve the following criteria: (i) OGRIs between subspecies and other species should be lower than the species-level cutoff value, (ii) OGRIs between subspecies should be higher than the species-level cutoff, (iii) strains belonging to different subspecies should be genomically coherent and form distinguishable clades by OGRIs and phylogenomic treeing, (iv) subspecies should be differentiated by a sufficient number of phenotypes, and (v) there should be a sound rationale why subspecies should be created and separately recognized, such as showing different host specificity in the case of pathogens.". An overall genome related index (OGRI) is a computational measure of genome similarity or distance of which ANI is one such. Our ANI analysis possibly fullfill criteria i-iii although given how few strains are in most of the putative subspecies this does not seem robust and criteria iv-v are clearly not met. We only raised the subspecies issue for E. asburiae and E. muelleri because often in the past when two competing names exist for a species if the type strains can be separated into clear clades they become subspecies. Since the type strains fall into neither of the major clades for this species and certainly do not cleanly divide the species we did not feel this was appropriate.  In bacterial taxonomy, only type strains are valid, while strains belong to phylogenomically diverse clades are less important during defining a species. As there are limitations in the use of the GGDC tool to compare a large number of strains, it would have been more logical to estimate GGDC-dDDH values (only) for the type strains for which the ANI cutoff is ambiguous.
The GGDC tool shows E. xiangfangensis and E. hormaechei are in fact two different species (DDH estimate (GLM-based): 59.80% [56.9 -62.5%]). Indeed, there is only a 12% probability that these two type strain can be classified as subspecies of a either species. Extension of this estimate to clade level indicates clade A-D and E are two different species and represents E. xiangfangensis and E. hormaechei, respectively. Just because several strains of clade A-D were historically identified as E. hormaechei subspecies, there is no reason to ignore the recently validated E. xiangfangensis species.
Currently, phenotypic identification remains the gold standard for identification of microorganisms in standard diagnostic laboratories and provides the bulk of the data for taxonomic classification. WGS is presently largely done in research laboratories or as pilot endeavors in specialized diagnostic laboratories. As genotype-phenotype correlations are at present incomplete, current classification schemes would give phenotypic data priority.
group ΔTm values of up to 2.7. By evaluating the DNA relatedness among the clusters, we found that clusters VI and VIII are closely related (mean ΔTm value = 2.2), while a relatively longer distance for E. hormaechei cluster VII from the members of clusters VI and VIII was indicated by the mean ΔTm value of 4.0. However, all three genetic clusters could still be assigned to the same species (14). They could be genetically distinguished from the other species of the E. cloacae complex, which had ΔTm values of 5.6 to 10.3 (Table 2).". Unfortunately they did not report DNA-DNA relatedness values but only ΔTm values. They did cite previous work which gave DNA-DNA relatedness: "Davin-Regli et al. (4) reported an outbreak with an "E. cloacae strain with the E. hormaechei genotype" but an aberrant biotype. The strain exhibited all of the characteristics of E. hormaechei and was 80% related to the type strain in DNA-DNA reassociation experiments but was positive for growth on Dsorbitol and α-D-melibiose. Obviously, this outbreak was caused by a strain of genetic cluster VI. Hence, these studies are in agreement with our observation that genetic clusters VI and VIII belong to the species E. hormaechei (4,6).". We agree that by ANI and dDDH that E. hormaechei subsp. hormaechei is borderline at best to be grouped as the same species as the other E. hormaechei subspecies but Drs. Chakraborty and Doijad cannot have it both ways. Hoffman et al showed phenotypic data supporting there grouping of the subspecies and delineation from other subspecies as well as genotypic support using marker genes which has since been used in clinical papers to differentiate the subspecies from each other and other species. Certainly one could propose making these separate species but the bar for undoing historical precedent is much higher than arguing that the ANI or dDDH values are borderline.
Drs. Chakraborty and Doijad state: "Currently, phenotypic identification remains the gold standard for identification of microorganisms in standard diagnostic laboratories and provides the bulk of the data for taxonomic classification. WGS is presently largely done in research laboratories or as pilot endeavors in specialized diagnostic laboratories. As genotype-phenotype correlations are at present incomplete, current classification schemes would give phenotypic data priority.". Not being clinicians we are not sure if this is true but based on our reading of the literature if it is true it is likely to not be true in the near future. We are not against phenotypic characterization if it is economical and reliable. We look forward to a robust discussion of the pros and cons of phenotyic versus genotypic diagnostic methods. Regardless, assignment of species and species delineation has long been genotype based since DDH is a genotypic measure as well as marker genes and OGRI. We are not against phenotypic characterization of type strains although one could argue that this only really makes sense if a clade of strains of the same species is characterized to evaluate variability. We reached out to DSMZ to inquire about phenotypic characterization services which they are willing to provide at some level on a case by case basis but they could not tell us what minimal characterization is necessary for a type strain. Perhaps Drs. Chakraborty and Doijad could intervene on our behalf with DSMZ and have the appropriate characterization performed and placed in the DSMZ supported "The Bacterial Diversity Metadatabase" (BacDive). This could be the first step towards some form of phenotypic characterization standard for type strains. The bulk of the analysis is based on a single tool viz. MASH-based ANI and is supplemented by the panOCT tool developed by the authors. The authors should consider the use of additional software tools to determine the overall genome-related index (OGRI). The final paragraph on biochemical properties is inadequate and could lead to confusion of phenotypes and undo the very purpose of the proposed classification scheme. Thus the gat operon is not exclusive to E. hormaechei subspecies hormaechei as stated, but is also present for e.g. in type strain E. bugandensis EB-247 T .

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound? Partly

If applicable, is the statistical analysis and its interpretation appropriate? Not applicable
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Partly
Competing Interests: No competing interests were disclosed.
We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above.

Author Response 26 Jun 2018
Granger Sutton, J Craig Venter Institute, Rockville, USA We thank Dr. Pallen for the thoughtful review and respond to issues below. "I don't see the need for the separate Introduction and Background sections. According to the guidelines for authors, papers in this journal should follow the usual IMRAD format, so I think that the two sections should simply become sub-sections of the Introduction, perhaps with brief explanatory headers." We removed the Background and Conclusion section headings to conform to the IMRAD format. "I am not sure why the authors abdicate responsibility for determining whether "8 subclades of E. asburiae should be treated as subspecies". Why not roll their approach out to cover these lineages too?" We now address this in the Discussion section. "The authors discuss the concept of "placeholder" species and subspecies in the Discussion, but fail to mention the "Candidatus" designation, which is recognised by the current bacterial taxonomy apparatchiks: http://ijs.microbiologyresearch.org/content/journal/ijsem/10.1099/00207713-45-1-186 https://en.wikipedia.org/wiki/Candidatus They should include some discussion of this designation that includes a recognition of its major shortcoming in requiring phenotypic data in addition to genome sequence." We thank Dr. Pallen for pointing this out to us and have included this in the Discussion section. We thank Dr. Chakraborty for the thoughtful review and respond to issues below. "The bulk of the analysis is based on a single tool viz. MASH-based ANI and is supplemented by the panOCT tool developed by the authors. The authors should consider the use of additional software tools to determine the overall genome-related index (OGRI)." and "For such closely related clades, multi-tool-based analysis of taxonomy are helpful to reassure the claims. To support the species/subspecies distinction, particularly for those closely related clades, the use of widely used taxonomic tools such as the digital DNA-DNA hybridization tool, GGDC should be employed to strengthen the claims." We have included the comparison of GGDC to MASH and PanOCT ANI in the Methods section. "Clade A-E represent the five subspecies of E. hormaechei. The average nucleotide identity (ANI) for the clades A-D and E are at the borderline ANI-species definition." This is certainly true but is also true of the already existing E. hormaechei subspecies: clade B E. hormaechei ssp. steigerwaltii, clade C E. hormaechei ssp. oharae, and clade E E. hormaechei ssp. hormaechei. While in the absence of previous taxonomic assignments one might choose to be reluctant to combine clades B, C, and E into a single species based on ANI because they have already been grouped as a species the borderline ANI values are not strong enough to argue for changing this. Given this adding clades A and D to E. hormaechei is strongly confirmed by the ANI values between clades A, B, C, and D.
"In view of the fact that data is based mainly on draft genomes, the utility of supportive assignments based on the total numbers of unique genes must be considered carefully." We have noted this concern in the results section. Gene content is not a primary consideration in our proposed new species designation but rather a possible reason to delineate at the subspecies level. In our experience most recent draft genome sequences are of high quality and the RefSeq genomes we used are screened by NCBI to meet certain quality requirements. Draft genome breaks tend to be at and due to repetitive elements such as transposons which would not affect the representation of most genes. We also try to take this into account by using a 90% rather than a 100% threshold. "ANI values can vary when using different calculation tools as for e.g. with JSpecies and ANI calculator. The use of MASH algorithm leads to minor variation in ANI values and makes the borderline species definitions presented here difficult to interpret." ANI values for the newly proposed type strains were backed up by PanOCT ANI and now by GGDC and are not borderline except as consistent with previous taxonomy. "To confirm separation of E. timonensis and E. lignolyticus from the genus Enterobacter, comparison with members of the closest genera (for e.g., Klebsiella, Citrobacter etc.) should be added." We have added this analysis to the Methods section. "Finally, biochemical and fermentation characteristics are key indicators for phenotypic characterization of isolates in diagnostic laboratories." As the paper mentions we are not opposed to the biochemical characterization of type strains but need a standard that can be implemented by culture collections so that computationalists can acquire this data. The DSMZ for instance supports doing some of this characterization but does not claim it to be standard. In addition, DSMZ supports storing this characterization data in "The Bacterial Diversity Metadatabase" (BacDive) such as for the E. bugandensis type strain (https://bacdive.dsmz.de/strain/132404). What is interesting is that most biochemical characterization is not used to define a species in current practice. Researchers no longer collect phenotypic features and cluster based on a feature vector. Rather, genotypic characteristics are captured such as 16S or hsp60 or rpoB or WGS which are used to define a cluster of strains and then phenotypic characterization of those strains is performed and used as part of the species definition no matter how divergent those features may be. Computational taxonomy provides a structure by which strains can be clustered, named, referenced, discussed and compared to related clades. Biologists should follow up on clinically or otherwise interesting clades. We are not sure whether Dr. Chakraborty is arguing for historical consistency in what characterization is minimally required for a type strain or is arguing that there is little or no value in computational taxonomy without phenotypic characterization because it is required for clinical diagnosis. We would disagree with both since with the advent of whole genome sequences (or even DDH) phenotype is not needed to define species and clinical diagnosis can be done with molecular markers. "The final paragraph on biochemical properties is inadequate and could lead to confusion of phenotypes and undo the very purpose of the proposed classification scheme. Thus the gat operon is not exclusive to E. hormaechei subspecies hormaechei as stated, but is also present for e.g. in type strain E. bugandensis EB-247T." We apologize for being unclear. We were summarizing what is already in the literature for distinguishing E. hormaechei subspecies from each other. We have been more precise and clarified this issue in the Results section.
Enterobacter huaxiensis and Enterobacter chuandaensis https://protectus.mimecast.com/s/gvM8CL9no8s8XxuqHnU5?domain=ijs.microbiologyresearch.org Enterobacter chengduensis https://www.ncbi.nlm.nih.gov/pubmed/30302649 Enterobacter sichuanensis https://ijs.microbiologyresearch.org/content/journal/ijsem/10.1099/ijsem.0.003089#tab5 The most recent of these papers references this paper but makes no real attempt to use what is in it. As pointed out in the paper we gave temporary names to species clades using NCBI's preferred genomospecies format and these temporary names should be updated. Below are the four type strains from the papers plus two strains from the NCBI taxonomy. Columns 5 and 6 are MASH ANI best hits to the type and proxy type strains from our paper. So we would like genomosp. T to become chuandaensis, genomosp. L to become chengduensis, and genomosp. N to become sichuanensis. Enterobacter huaxiensis appears to be a novel species with no corresponding genomosp. clade.

Version 1
Author Response 27 Jun 2018 Granger Sutton, J Craig Venter Institute, Rockville, USA We thank Florian Plaza Onate for pointing this out. To confirm this observation we started with the PanOCT run of the 250 most diverse genomes including the outlier genomes. We selected all clusters which were present in more than 151 genomes which would include all core clusters and many others. We extracted the medoid fasta sequences for these 3833 clusters. We then used our LOCUST tool to search for and extract homologous sequences from the three Enterobacter mori strains (LMG25796, 80072117, ECC1766). For LMG25796, 208 genes were missing and 328 were short. For 80072117, 95 genes were missing and 331 were short. For ECC1766, 72 genes were missing and 332 were short. For default LOCUST parameters, short genes are ones missing more than 5bp from either end of a Blast match so some short genes can be due to divergence from the medoid sequence rather than genome incompleteness. For missing genes, a small fragment may be present but was not significant enough to be found by Blast using LOCUST's blast parameters. Regardless of these caveats, it is clear that LMG25796 is the most incomplete of the three strains and for analyses needing more complete genomes should be handled with caution. However LMG25796 is the type strain and has full length genes for 3297 of the 3833 genes we selected which is more than enough for Average Nucleotide Identity calculations.