Integrating ANI and phylogenies for re-evaluation of Fusobacterium taxonomy and disease associations

Domain: biology
Published: 2026-03-10
Editorial outcome: minor_revision

Abstract
The genus Fusobacterium encompasses significant pathogens implicated in diseases spanning from infections to cancer. However, taxonomic ambiguities persist within the genus, particularly concerning Fusobacterium nucleatum (sensu lato). Through genus-wide average nucleotide identity (ANI) and phylogenetic analyses of 540 Fusobacterium genomes, we identify an ANI gap (93.38%--93.89%) for species delineation, leading to comprehensive taxonomic revisions that resolve these ambiguities. We further establish gyrB and rpoB as high-resolution taxonomic markers with phylogenies consistently supporting the revised taxonomy. Leveraging these markers, we develop B&B, a general strategy for precise species identification without whole-genomNe sequencing, and validate its accuracy in clinically relevant strains. Integrating the revised taxonom y with genomic/metagenomic toolkits demonstrate broad utilities, reinterpreting key colorectal cancer-associated species. This work establishes a unified taxonomic framework and enabTles standardised species classification for Fusobacterium isolates and microbiomes, highlightinAg the genetic divergence among Fusobacterium species and providing the taxonomic precision essential for advancing Fusobacterium-related research.

Introduction
The genus Fusobacterium has garnered broad attention due to its emerging roles in human diseases beyond infection, functioning as key components of the dysregulated microbiota1,2. In the recent decade, F. nucleatum (sensu lato) has been extensively studied as an oncobacterium in various malignancies, such as colorectal cancer and breast cancer, acting through multiple mechanisms3-9. Its presence has been implicated as potential diagnostic and therapeutic targets10,11.

However, the taxonomy within this genus has long been challenging and complex, particularly regarding F. nucleatum (sensu lato)12,13. Historically, F. nucleatum (sensu lato) has been classified into four subspecies, animalis, polymorphum, vincentNii, and nucleatum, with documented genotypic and phenotypic heterogeneity12,14-20. However, geno mic evidence including average nucleotide identity (ANI) and genome-to-genome distance analy ses has suggested that these subspecies are sufficiently divergent to be species; and accordingly, theyT were reclassified as F. animalis, F. polymorphum, F. vincentii, and F. nucleatum (sensu stricto)A by Kook et al.19. This revision has been further supported by studies with genome/marker-based approaches and/or biological species concept (BSC) strategies21-24. Recently, Zepeda-Rivera et al. proposed that subsp. animalis comprises two clades, Fna C1 and Fna C225. Subsequent works noted that Fna C1 represents a lineage distinct from F. animalis20,24. Most lately, Sivertsen et al.26, through ANI and BSC analyses, proposed that Fna C1 corresponds to F. watanabei, a species previously described by Tomida et al.27, while Fna C2 corresponds to F. animalis, Notably, the annotation of the Fna C1 lineage has evolved rapidly and fluctuated across studies. Most strains defined as Fna C1 were initially identified as F. nucleatum (sensu lato) subsp. animalis based on 16S rRNA gene profiling25. Zepeda-Rivera et al. designated them as Fna C1 after distinguishing them from the F. nucleatum (sensu lato) subsp. animalis 7_1 lineage based on pangenomic, ANI and phylogenetic analyses of F. nucleatum (sensu lato) genomes25. Meanwhile, Ma et al. assigned a strain (FNU) of this lineage as a novel subspecies (subsp. unique) based on pangenome‑based phylogeny28. A recent metagenomic study by Piccinno et al. regrouped Fna C1 with F. vincentii based on phylogenetic analysis of the species-level genome bins (SGBs) within F. nucleatum (sensu lato)29. Almost in parallel with the proposal reclassifying Fna C1 as F. watanabei26, Zepeda-Rivera et al. also observed, based on Genome Taxonomy Database (GTDB) analysis, that F. watanabei, Fna C1, subsp. unique (FNU) and the F. nucleatum_J group defined by GTDB may represent the same lineage13,30. These dynamic shifts in classification likely stem from differences in the datasets and methodologies emplNoyed across studies, highlighting the need to integrate genomic, phylogenetic, and marker-based appr oaches within a coherent framework. Recent studies have further highlighted their distinctive roles in disease pathogenesis12. For instance, these taxa are differentially distributed in CRC1T8,22,31,32 with Fna C2 (F. animalis) reported to dominate the CRC niche25. Similarly, it has been showAn that subsp. animalis (F. animalis), rather than the well-characterised subsp. nucleatum (F. nucleatum sensu stricto), is clinically relevant to inflammation in oral diseases23. Such studies have either supported the notion of reclassifying F. nucleatum (sensu lato) subspecies/clades as species or provided genomic evidence such as ANI values supporting that reclassification23,25. However, mixed use of nomenclature in literature, coupled with uncertain annotations risk raising ambiguity and have fueled ongoing debate12,13,26,33,34. More critically, this inconsistency often fails to adequately account for the underlying genetic and pathogenic diversity among these taxa, potentially obscuring precise correlations between species and their specific biological or clinical relevance. While experts in microbiological taxonomy and/or clinical microbiology may readily adopt properly updated classifications, a significant practical challenge remains in achieving consensus on consistent implementation across the broader research and clinical community (especially among those without the specialised expertise) now actively engaged in Fusobacterium studies. This challenge is particularly pressing given the expanding relevance of Fusobacterium in diverse diseases. Furthermore, the high level of similarity in 16S rRNA genes often limits their utility for species-level resolution within Fusobacterium22,25,35, particularly when partial regions such as V3-V4 are used, and may also lead to misclassification of non-nucleatum (sensu lato) taxa. Therefore, a timely clarification of Fusobacterium taxonomy, along with development of standardised, practical solutions to ensure consistent, accurate and reproducible species-level identification, both for Ncurrent applications and future research, is urgently needed.

Genome-based ANI has been widely recognised as a quantitative standard for defining bacterial species boundaries36,37. Phylogenomic aTpproaches have also been adopted to clarify taxonomic ambiguities in other genera38-41. AlthouAgh several studies have included ANI and/or phylogenetic analyses of Fusobacterium genomes13,22-26, genus-wide assessments remain scarce and a robust species boundary for the entire genus remains undefined. In addition, alternative genetic markers, including the RNA polymerase beta subunit gene rpoB and zinc protease gene znpA, have been proposed for species discrimination23-25,35. Dedicated profiling methods targeting these markers, including FrpoB-seq for rpoB V1 (Variable Region 1)22 and znpA amplicon sequencing23, have been developed for community analysis within the human microbiota, to overcome the low-resolution limitations of 16S rRNA gene sequencing. Similarly, primers targeting the DNA gyrase subunit B gene gyrB have been designed to differentiate the subspecies of F. nucleatum (sensu lato) as well as F. necrophorum42. However, the utility of gyrB has not been evaluated in a genus-wide context. Furthermore, the recent expansion of available Fusobacterium genome necessitates reassessment of the resolution of these markers and the establishment of high-resolution phylogenetic markers.

In this work, to address these gaps, we conduct a genus-wide study integrating large-scale whole genome ANI and phylogenetic analyses. This study has systematically established a unified taxonomic framework for Fusobacterium, resolved the taxonomic ambiguities, defined high-resolution genetic markers, and developed standardised species identification approaches applicable to both individual strains and complex microbiota.

Results
Genus-wide analysis revealed an ANI gap delineating species

To identify a unified species Tboundary within the Fusobacterium genus, we performed ANI and phylogenetic analysis on 540 genomes of Fusobacterium strains/isolates (Supplementary Data 1) collected from the NCBI RefSeq, a high-quality genome database. The frequency distribution of pairwise ANI values revealed a natural gap spanning 93.39% -- 93.94% (centred at 93.67%) (Fig. 1a and Supplementary Data 2), with complete discontinuity. To further characterise this discontinuity, we sorted all pairwise ANI values in descending order (Supplementary Fig. 1a) and calculated the first derivative (Δ) of the distribution to measure the rate of change in ANI, using an approach similar to that described by Passarelli‑Araujo et al.43. The maximum Δ among ANI values > 90%, defined as the ANI discontinuity (δ), precisely corresponded to this gap (Supplementary Fig. 1b), supporting the presence of a significant genetic break. Given that the bacterial species boundary is typically around 95% ANI36,37, we investigated whether this observed ANI gap could serve as a threshold for Fusobacterium species delineation. To anchor taxonomic references, we used the reference genomes (n =20) defined by the RefSeq, each representing a well-established species, to standardise and stabilise species definitions throughout this study. In the dendrogram derived from the ANI matrix, strains with pairwise ANI values above this the gap consistently formed discrete clades, each corresponding well to a specific species (Fig. 1b). The taxonomic anchors were uniquely distributed in individual clades. We then constructed a whole-genome phylogenetic tree using kSNP444 to evaluate species-level relationships and validate the ANI-based groupings (Fig. 1c). The whole-genome tree and the ANI-based dendrogram showed a normalised quartet score (NQS) of 0.904 (Fig. 2), indicating very stroNng concordance. Critically, the ANI-based grouping was corroborated by the whole genome-based phylogenetic tree, where strains within each ANI-defined group consistently clustered together within the same major clades parallel to each other (Figs. 1c and 2), confirming that the ANI gap reTpresents a standardised boundary for defining Fusobacterium species. Considering that recombiAnation might influence the topological accuracy of the whole genome-based phylogenetic tree, we additionally constructed trees based on single nucleotide polymorphism (SNP) loci occurring in at least 25%, 50%, and 75% of genomes (Supplementary Fig. 2). All these trees showed 0.96 -- 0.99 NQS to the primary tree, suggesting that the core phylogenetic relationships were robust against potential noise introduced by recombination. Finally, species names within each ANI-defined group were assigned based on the taxonomic anchor where available. Clades lacking a reference anchor either corresponded to unspecified species (previously recognised) or were designated as novel species. Thus, the 540 genomes were finally classified into 34 species (Table 1), six of which were newly proposed. The intra- and interspecies ANI distributions showed no overlap around this boundary (Supplementary Fig. 3).

Impact of genome number and completeness on ANI boundary definition

To assess the sufficiency of the genome sample size for capturing this boundary, we performed a rarefaction curve-like analysis. We randomly selected increased amounts of genomes with 100 replicates, and plotted the changes in the upper and lower limits of the ANI gap (Fig. 3a). Both limits reached plateau with over 350 genomes. Furthermore, considering that over half of the genomes analysed were draft assemblies, we evaluated the impact of genome completeness on the ANI gap range. Draft genomes exerted a shrinking effect on the gap width, as the gap range calculated using the draft genomes was narrower than that obtained from the complete genomes (Fig. 3b). Nevertheless, the above identified ANI gap based on both complete and draft genomes remNains a distinct boundary as it still spans an interval of over 0.5% ANI.

Taxonomic revisions resolved species designation ambiguities

The genome-based phylogeny aTnd the standardised ANI species boundary enabled a comprehensive review of the FusobacteriuAm taxonomy. Strikingly, we found that the species designations of 101 (18.6%) strains in RefSeq required correction, update, or redefinition (Supplementary Data 2 and Table 2). Corrections for 60 genomes were consistent with prior reports, validating our ANI-based species boundary. Crucially, our results confirmed that F. nucleatum (sensu lato) comprises multiple distinct species; specifically, the subspecies vincentii, polymorphum, nucleatum (sensu stricto), and the animalis clade 1 (Fna C1) and 2 (Fna C2) are separate species, consistent with previous observations19,22,23. In our analysis, Fna C2 was represented by the F. animalis anchor strain (KCOM 1325), whereas Fna C1 was clustered with the F. watanabei anchor (CCUG74246T). Our data also supported that the strain F. nucleatum (sensu lato) W1481 lacking subspecies designation belongs to F. simiae26 and validated the proposed reclassification of F. equinum into F. gonidiaformans45. Among the revisions proposed here, a group of species-unspecified isolates from bovine digital dermatitis form a new species, proposed here as Fusobacterium sp. bovis. Four strains misclassified as F. ulcerans or F. varium constitute another new species, proposed as F. heteroulcerans sp. nov. Notably, F. heteroulcerans was previously designated Fusobacterium sp. 81A6 to describe the F. ulcerans ATCC 49185 isolate 81A622. Our results further clarified that 81A6 is distinct from the original F. ulcerans ATCC 49185 strain. We also identified that five strains misclassed as F. varium, F. mortiferum or F. perfoetens actually represent four additional novel species. Meanwhile, six strains were corrected from F. periodonticum to F. pseudoperiodonticum and 14 unspecified species were assigned into estabNlished species, some of which were also in agreement with earlier reports20,24,26. Note that some of the RefSeq reference genomes used as taxonomic anchors might not be derived from type strains, as we employed a standardised, database-driven approach to maintain methodological consisteTncy. For instance, while KCOM 1325 was used as the F. animalis anchor, ATCC 51191 (type strain)A and 7_1 are commonly used for this species25. Our analysis confirmed their classification within the same species, indicating that the type strains or commonly used ones, though they might not be used here as anchors, remain important and reliable representative strains. In addition, given that several Fusobacterium species, such as F. perfoetens, exhibited distinct phylogenetic relationships to well-established species (Fig. 1c) and were observed to relate with non-Fusobacterium taxa in previous studies46,47, we performed an extended whole-genome phylogenetic analysis (Supplementary Fig. 4). This analysis included representative genomes (Supplementary Data 3) from other genera within the Fusobacteriaceae family, as well as F. naviforme, which has been proposed for reclassification as Zandiella naviformis45. Interestingly, our result indicated that although F. perfoetens, Fusobacterium sp. An877, sp. FSA-380-WT-3AF, sp. SB021 (F. sphaericum)48, sp. IOR10, sp. MFO224 and sp. An888 remained phylogenetically distinct from other genera, their degree of divergence from other Fusobacterium species appeared comparable to that observed in Cetobacterium. Meanwhile, Z. naviformis (F. naviforme) maintained a relatively close phylogenetic association with F. gonidiaformans. This evidence collectively suggested that certain highly divergent lineages may further warrant a taxonomic re-evaluation at the genus level.

Evaluation of gyrB and rpoB as high-resolution taxonomic markers

We then evaluated Fusobacterium taxonomic markers (Supplementary Data 4) using these species-curated genomes. Phylogenetic analysis of the 16SN rRNA gene (including all available copies within a genome) revealed inadequate species discrimin atory power, particularly for distinguishing F. animalis, F. polymorphum, Fusobacterium sp. SB 042, and F. hwasookii (Fig. 4a). The tree also did not efficiently cluster many species into distincTt clades. The 16S rRNA gene tree showed a NQS of 0.829 to whole-genome phylogeny, indicaAting moderately strong agreement. Accounting for its multi-copy nature, we calculated the maximum, minimum, and average 16S rRNA gene identities between each strain pair (Supplementary Data 5). The resulting interspecies and interspecies pairwise identity distributions showed extensive overlap, precluding reliable species boundary delineation at any threshold (Fig. 4b). In contrast, we demonstrated that rpoB, gyrB and the znpA genes are effective molecular markers for resolving phylogenetic relationships within Fusobacterium (Fig. 5a). We confirmed that rpoB provided high species-level resolution (NQS = 0.864), consistent with previous reports22,35. The znpA phylogeny also showed high discrimination power as previously documented23,35, but still exhibited moderately strong agreement with whole-genome phylogeny (NQS = 0.827), likely because it did not fully cluster F. watanabei and F. polymorphum into distinct clades. Additionally, znpA was found to be absent in F. necrophorum and F. gonidiaformans. Notably, we identified the housekeeping gene gyrB exhibiting strong discriminatory power for Fusobacterium species, with an NQS of 0.905 to whole-genome phylogeny. The concatenated rpoB+gyrB phylogeny also resolved species well (NQS = 0.894; Supplementary Fig. 5). A PHI test detected no significant recombination signals in all analysed genes except for rpoB (Supplementary Table 1). Nevertheless, the major phylogenetic structure of the rpoB tree remained highly informative for species-level clustering. To statistically compare marker performance, we calculated the nucleotide diversity (π) of all analysed genes and performed a bootstrap-based difference test. We also compared the distributions of NQS values obtained Nfrom bootstrap-resampled gene trees relative to whole-genome phylogeny. Given the draft nature of m any genomes and the natural absence of znpA in certain species, not every RefSeq genome con tained complete marker sequences; therefore, all comparisons and corresponding NQS calculationsT were performed only on the subset of genomes containing complete sequences for the markersA being analysed. Within the analysed subsets, rpoB, gyrB and znpA all exhibited higher nucleotide diversity than the 16S rRNA gene (Supplementary Fig. 6). The gyrB trees consistently yielded the highest NQS values, followed by rpoB and znpA, all of which were statistically higher than that of the 16S rRNA gene tree (Supplementary Fig. 7). However, the concatenated rpoB+gyrB phylogeny did not improve the NQS compared to gyrB alone (Supplementary Fig. 7). Notably, phylogenies of all three markers consistently supported the taxonomic revisions of Fusobacterium. However, for each of the three genes, defining a universal pairwise identity threshold to delineate species remain unsuccessful by persistent, albeit slight, overlap between intra- and interspecies identity distributions (Supplementary Data 5 and Fig. 5b). In addition, our previous study had identified 45 undescribed Fusobacterium species in the gut microbiota of CRC patients via FrpoB-seq 22. Phylogenetic analysis of rpoB V1 incorporating the FrpoB-seq-derived sequences revealed that the recently or newly defined F. watanabei, Fusobacterium sp. SB042, sp. SB021 (F. sphaericum), sp. An877, sp. An888 and sp. An425 matched the previously identified species22 Fusobacterium sp. B1_6, sp. B1_45, sp. B3_19, sp. B2_19, sp. B3_87 and sp. B3_12, respectively (Supplementary Fig. 8), supporting the notion that there are more Fusobacterium species to be explored. This phylogenetic analysis also assigned all species except Fusobacterium sp. PH-29 into previously established Fusobacterium lineages (Supplementary Fig. 8 and Table 1).

A general method for Fusobacterium species identification

We sought to develop a practical and accurate appNroach to overcome the limitations of the 16S rRNA gene-based method for Fusobacterium species identification. Given the absence of established identity thresholds for individual genetic markers, we investigated the feasibility of using a marker combination and considering only the best hit.T We selected gyrB and rpoB, which are ubiquitous in Fusobacterium. An ~1.5 kb gyrB variable regiAon (n.t. 240--1737; designated the gyrB V1 region) and the rpoB V1 region (n.t. 81--399)22 compatible for PCR with degenerate primers were assessed (Fig. 6 a and b). These regions exhibited nucleotide diversity comparable to their full-length genes (Supplementary Fig. 6) and no significant recombination signal was detected in rpoB V1 (Supplementary Table 1). Pan-Fusobacterium primers gyrB-219F and gyrB-1759R were designed for gyrB V1, whereas those for the latter were rpoB-55F and rpoB-422R, modified from fuso-rpoB-F2/R222. Analysis considering only the intra- and interspecies best hits of these regions from one genome against the counterparts in others, revealed that identity thresholds of 97% for gyrB V1 and 99% for rpoB V1 could accurately define species for nearly all genomes (Fig. 6 a and b; Supplementary Data 6). Only four genomes exhibited intraspecies gyrB V1 identities below 97% (96% --97%) and much lower interspecies identities (Fig. 6c). While the 97% gyrB V1 threshold failed to define their species, interestingly, three of them could be subsequently defined using the rpoB V1 region at the 99% threshold (Fig. 6c). The only exception was F. necrophorum KG35 (Fig. 6c). Therefore, we propose a B&B (gyrB & rpoB) strategy for Fusobacterium species identification (Fig. 6d). First, one can compare an amplified gyrB sequence (primer regions excluded) against its counterparts (Supplementary Data 4) from species-curated genomes, using a ≥97% best-hit identity threshold to assign species. If this proves inconclusive, on some occasions, the rpoB V1 can then be analysed with a ≥99% best-hit identity threshold. If both markers fail, probably in rare instances, genomic ANI and/or phylogenetic analysis is recommendeNd. The gyrB V1 is prioritised as the primary marker because rpoB V1 exhibited limited discriminat ory power between F. perfoetens and Fusobacterium sp. FSA-380-WT-3A. Moreover, if species assignment is successful using gyrB alone, rpoB analysis is unnecessary. We emphasise that Tthis workflow is specific to the B&B method for practical purposes and does not imply that gyrB iAs superior to rpoB for all taxonomic applications. To validate the performance of the B&B method, we applied it to nine Fusobacterium strains isolated from CRC tissues (sequenced in this study) and 36 public genomes from the NCBI GenBank database (Fig. 6e and Supplementary Data 7). These public genomes were excluded from the RefSeq database exclusively because of incorrect taxonomic assignments, not quality issues. They mostly represented medically relevant isolates, including 14 from the human gut (11 CRC-related, two IBD-related and one unspecified), 14 from the human oral cavity (5 CRC-related, one periodontal disease-related, and eight unspecified), two from human trans-tracheal site, one from human cervico-facial site, four from human unspecified sites, and one from dog mouth. Strikingly, the B&B species classifications for all 45 strains perfectly matched the reference assignments based on ANI values (Supplementary Data 7), whole-genome phylogeny (Supplementary Fig. 4) and previous curation20,24,26, confirming the classification accuracy of the method. One F. heteroulcerans strain (THCT13E1) was thus designated as the type strain of this newly proposed species. We further applied B&B to the genome of the recently established species F. paranimalis (type strain NCTC15132; Supplementary Data 7)26. The method correctly identified it as a novel species, consistent with whole-genome phylogeny (Supplementary Fig. 4). Interestingly, a few of its pairwise ANI values with RefSeq genomes (71.30%--93.51%) fell within the species gap described above. As its maximum ANI (93.51%) to any RefSeq genome remained below the midpoint (93.67%) and upper limit (93.94%) of the gap, this supported its novel species status aNnd suggested that inclusion of F. paranimalis would refine the gap boundary to 93.52%--93.94%. N ote that universal tails were incorporated into the primers to facilitate sequencing in this study, and in silico B&B analysis was applied to the public genomes. The gyrB and rpoB sequences from Tthe species-curated RefSeq genomes were used as a reference dataset (Supplementary Data 4).

Integration of the revised taxonomy with bioinformatics toolkits

To demonstrate the broader applicability of the revised Fusobacterium taxonomy, we integrated it with two representative bioinformatics toolkits, the Genome Taxonomy Database (GTDB)49 and MetaPhlAn50. GTDB is a widely utilised database for microbial taxonomy and genomic analyses based on genome phylogeny. We herein mapped the revised Fusobacterium taxonomy to GTDB assignments (Table 1) and particularly reassigned ambiguous GTDB taxonomic entities, such as F. nucleatum_J, F. nucleatum_D, F. priodonticum_D, F. periodonticum_B, Fusobacterium_A ulcerans_A and Fusobacterium_A varium_A, to defined species. Furthermore, given the importance of Fusobacterium in the human microbiota, which is commonly profiled via metagenomic approaches, we mapped the revised taxonomy to the SGBs used by MetaPhlAn (Fig. 7a, Table 1 and Supplementary Data 8), a state-of-the-art tool for species-level microbial profiling from metagenomic data. This mapping identified SGBs specifically matching 29 Fusobacterium species, including those reclassified from F. nucleatum (sensu lato). We found that all genomes of a given species exhibited > 92% ANIs to their species-specific SGB and lower ANIs to other SGBs, with very few exceptions observed only in F. canifelinum and F. nucleatum (sensu stricto) (Fig. 7b and Supplementary Data 8). Critically, this revised SGB annotation refined the interpretation of key CRC-associated species identified in previous large-scale metagenomic studies25,29, demonstrating that certain CRC-associated SGBs previously assigned to F. nNucleatum (sensu lato) actually correspond to distinct species (Fig. 7c).

Methods
Public bacterial genomic data

A total of 542 publicly available Fusobacterium genomes were retrieved from the NCBI RefSeq database as of 20 June 2025 (Supplementary Data 1) for genome-based taxonomic analysis. While the RefSeq has filtered out low-quality genomes with strict criteria (see https://www.ncbi.nlm.nih.gov/datasets/docs/v2/policies-annotation/genome-processing/genome_notes/), we further assessed genome quality using CheckM2 (version 1.1.0) 53. Based on this assessment, 540 genomes with >95% completeness and <5% contamination were retained for analysis. The reference genomes designated by the RefSeq (see https://www.ncbi.nlm.nih.gov/datasets/docs/v2/policies-annotation/genome-processing/reference-selection/) were selected as taxonomic anchors (Supplementary Data 1). Typically, one reference genome was available for each well-defined species. A set of representative genomes from other genera within the Fusobacteriaceae family were retrieved from RefSeq or GenBank database for extended genome-based taxonomic analysis (Supplementary Data 3). In addition, 36 genomes excluded from the RefSeq solely duNe to “unverified source organism” status (indicating incorrect taxonomic assignment rather than qu ality concerns) were obtained from the NCBI GenBank database (Supplementary Data 7) to validate the newly developed Fusobacterium species identification method. The genome of NCTCT15132 (Vestland19T), the type strain of the newly defined species F. paranimalis26, was also inAcluded from GenBank for this validation (Supplementary Data 7). All genomes acquired for validation, and the non-Fusobacterium genomes, were also subjected to quality assessment with CheckM2. All included genomes were derived from cultured strains/isolates and not assembled from metagenomic data.

Bacterial strains

F. pseudoperiodonticum THCT18E1, F. varium strains THCT4E2, THCT4E4, THCT23B1, THCT23E1 and THCT23E3, as well as F. heteroulcerans strains THCT1E1, THCT1E2 and THCT13E1, were previously isolated from CRC tissues22. They were cultivated anaerobically on Columbia agar supplemented with 5% defibrinated sheep blood (Comagal Microbial) at 37°C 22. Notably, THCT18E1 had been identified as F. periodonticum, whereas THCT1E1, THCT1E2 and THCT13E1 as F. varium based on16S rRNA genes22. They were reassigned in this study. The strain THCT13E1 was submitted to the China General Microbiological Culture Collection Center (CGMCC) and China Center for Type Culture Collection (CCTCC) with the deposition numbers CGMCC 46909 and CCTCC M 20253012, respectively.

Whole-genome sequencing of bacterial strains

Genome sequencing and assembly of bacterial strains were conducted at the Beijing Genomics Institute (BGI), Shenzhen. THCT18E1 was sequenced with the PacBio RSII platform (2639× coverage) combined with the Illumina HiSeq 4000 platform (469× coverage). PBdagcon (version 0.1; https://github.com/PacificBiosciences) was use d for subread correction. The genome was assembled with Celera Assembler v8.3 (http://wgs-assembler.sourceforge.net). GATK v1.6 (https://github.com/broadinstituteT/gatk) and the SOAP2 package54 were used for single-base correction. THCT1E1, THCT4E2, THACT4E4, THCT13E1 and THCT23E1 were sequenced with the PacBio Sequel II platform (1000× -- 3000× coverage) combined with the BGISEQ-500 platform (330× -- 450× coverage). Canu v1.5 was used for subread correction and genome assembly55. GATK v3.4 (https://www.broadinstitute.org/gatk/) was used to perform single-base corrections. THCT1E2, THCT23B1 and THCT23E3 were sequenced only with the BGISEQ-500 platform (310× -- 375× coverage), and the sequenced reads were assembled with SOAPdenovo v2.0456.

Whole-genome ANI analysis

ANI analysis of the genomes was performed using pyani (version 0.3.0-alpha) with the BLASTN method57. Pairwise ANI values using one as the query and the other as the subject and vice versa were visualised as a heatmap with the gplots package (version 3.3.0; https://github.com/talgalili/gplots) in R (version 4.4.2). A dendrogram was drawn with default parameters.

Whole-genome phylogenetic analysis

Phylogenetic trees inferred from genome sequences were constructed based on the parsimony method using the kSNP4 (version 4.1)44, with a kmer size of 23 as determined by its integrated tool Kchooser4. All other parameters were set to default. To assess the potential impact of recombination on phylogenetic inference, additional trees were built based on SNP loci occurring in at least 25%, 50%, and 75% of genomes using the ‘-min_frac’ option.

Nucleotide sequence comparison and phylogeneNtic analyses of makers

Nucleotide sequence comparison was conduc ted with BLASTN (BLAST+ version 2.9.0). Multiple sequence alignments were performed with MUSCLE v3.8.3158, based on which phylogenetic trees were constructed with the maximum-liTkelihood method and 1,000 bootstrap replicates using IQ-TREE (version 3.0.1)59. The ModelFinderA Plus (MFP) option60 was used to automatically select the best-fit nucleotide substitution model by minimising the Bayesian information criterion and the ultrafast bootstrap mode61 was applied. The resulting consensus trees were visualised in MEGA 5.262. The PHI test was performed to examine signals of recombination with PhiPack (version 1.1)63. Nucleotide diversity (π) was estimated with the pegas package (version 1.4)64 in R (version 4.4.2). Pan-Fusobacterium primers gyrB-219F and gyrB-1759R for gyrB were designed within the conserved regions identified through multiple sequence alignment. The gyrB-219F refers to a set of forward primers gyrB-219Fa (5`-WGATAATGGAAGAGGRATACC-3`), gyrB-219Fb (5`-NGATAAYGGDAGAGGDATTCC-3`), gyrB-219Fc (5`-RGACAATGGWAGAGGAATTCC-3`) and gyrB-219Fd (5`-WGATAAYGGAAGGGGWATTCC-3`) while the gyrB-1759R contains reverse primers gyrB-1759Ra (5`-CAAATAATTGGTCAGGATTCAT-3`), gyrB-1759Rb (5`-CCCANARYTGTTCNGGATTCAT-3`) and gyrB-1759Rc (5`-CCCAWARTTGTTCHGGGTTCAT-3`). Similarly, pan-Fusobacterium primers rpoB-55F and rpoB-422R were modified from fuso-rpoB-F2/R222. The rpoB-55F contains rpoB-55Fa (5`-GCCACATTTTCTTGAATTTCAATT-3`), rpoB-55Fb (5`-GCCTCATTTTYTDGARTTYCAATT-3`, same to the fuso-rpoB-F2) and rpoB-55Fc (5`- GCCTCAYTTCCTTGAATTYCAATT-3`), while the rpoB-422R contains rpoB-422Ra (5`- ACTACTCTTTCTGCTCCATTTAC-3`), rpoB-422Rb (5`- ACDACTCTTTCHGCHCCATTDAT-3`) and rpoB-422Rc (5`- ACTACTCTTTCWGCTCCGTTTAT-3`). The universal M13F (5`-CGCCAGGGTTTTCCCANGTCACGAC-3`) was added to the 5’ of each forward primer while M13R (5`-AGCGGATAACAAT TTCACACAGGA-3`) to the 5’ of each reverse primers, serving as sequencing primers of the amplicons. The sequences of 16S rRNA genes, rpoB, gyrB and znpA are provided in Supplementary DTata 4. Note that due to the draft nature of certain genomes, not all marker gene sequences were avaAilable for every RefSeq genome. Specifically, while gyrB sequences were available for all 540 genomes, rpoB sequences were available for 539 genomes, znpA for 436 genomes (also due to its natural absence in certain species), and at least one 16S rRNA gene copy for 414 genomes. Whole-genome phylogenetic trees were also constructed based on these genome subsets as described above.

Bacterial genomic DNA preparation

Genomic DNA was prepared from the bacterial strains with the TIANamp Bacteria DNA Kit (TIANGEN) according to the manufacturer’s instructions.

Polymerase chain reaction (PCR)

PCR was performed with Premix Taq™ (Ex Taq™ Version 2.0 plus dye; Takara) on a Mastercycler nexus gradient thermal cycler (Eppendorf) according to the manufacturer’s instructions. In each reaction, 10 ng of bacterial genomic DNA was used, and 30 cycles of 30 seconds at 98°C, 30 seconds at 50°C, and 90 seconds at 72°C were applied. PCR products were verified by agarose gel electrophoresis, and then sequenced by the Sanger method at Saiheng Biotech.

Integration of the revised taxonomy with GTDB classifications

GTDB taxonomic assignments and representative strain information for the genomes were retrieved via direct accession number mapping to the GTDB database49, as of 23 June 2025. These assignments were then mapped to the revised Fusobacterium species Ntaxonomy and summarised at the species level.

Integration of the revised taxonomy with Me taPhlAn SGBs

Genomes were aligned against the MetaPhlAn SGB database (vJan25_202503)50 with BLAST+ version 2.9.0. The best-matched SGB (higThest sequence identity and longest alignment coverage) of each genome, if available, was selected. AThe sequences of the selected SGBs were then extracted and subjected to ANI analysis against the genomes with pyani (version 0.3.0-alpha) as described above57. The mean ANI value between each SGB and all genomes assigned to a Fusobacterium species was calculated as a species-level ANI value. The ANI values were visualised as heatmaps.

Statistics

Frequency distributions of ANI values (bin width =0.01%) and nucleotide identity (bin width =0.05%) were analysed with GraphPad Prism (version 7). The ANI discontinuity was estimated following a method similar to Passarelli‑Araujo et al.43. Briefly, all pairwise ANI values were sorted in descending order. The first derivative (Δ) of ANI with respect to the rank position (j) was then calculated as the change in ANI between two consecutive ranks j and j + 1, divided by the rank difference: Δ = (ANI – ANI ) / (j+1-j) = j j+1 ANI – ANI . The ANI discontinuity (δ) was defined as the maximum Δ among ANI values > 90% (δ = j j+1 max{Δ|ANI ∈ [0.90, 1.00]}), representing a break or sharp change. A rarefaction curve-like analysis was performed to evaluate whether the current genome number was sufficient for ANI gap detection. Genomes were randomly subsampled at sizes of 10, 20, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500 and 540 using the “sample” function in R (version 4.4.2), with 100 replicates per size. The upper and lower limits of the ANI gap for each replicate were determined through frequency distribution analysis. Normalised quartet scores (NGS), which quantified the proportion of shared evolutionary relationships (quartets) between two trees, were calculated wNith ASTRAL v5.7.865 to assess overall topological concordance between the kSNP-derived tree and ANI-derived dendrogram or phylogenetic trees of individual markers. For each marker, we also computed the NQS for all trees generated during bootstrap resampling. The distributions of these bootstrap NQS values were then compared across markers using Kruskal-Wallis test followAed by Dunn's multiple comparisons test, as applicable. To compare nucleotide diversity (π) across markers, we employed a bootstrap-based difference test. For each marker, bootstrap resampling (1,000 iterations) was performed. Crucially, whereas the NQS comparison utilised bootstrapping to approximate the sampling distribution of the statistic itself, the π comparison employed bootstrapping to evaluate the uncertainty of a parameter estimate. Therefore, for each marker pair, we calculated the distribution of differences by subtracting the bootstrap π values of one marker from those of the other. Statistical significance was determined by examining whether the 95% confidence interval of this difference distribution contained zero. The corresponding two-sided p value was calculated as twice the proportion of bootstrap differences on the smaller side of zero, followed by Benjamini-Hochberg correction for multiple testing. All NQS and π comparisons were performed only on the subset of genomes that contained complete sequences for the markers being analysed. A two-sided p value <0.05 was considered statistically significant unless otherwise specified.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is another manuscript reassessing Fusobacterium taxonomy, with focus on the\nFusobacterium nucleatum group. The\nstudy is based mostly on publicly available genomes from GenBank that have already been included in multiple similar\nstudies and add little to the current understanding of the Fusobacterium taxonomy. It also fails to include several recent\npapers of major relevance to this subject.\nMajor comments:\nComment 1: Most of the wrongly annotated GenBank genomes flagged in this study have already been reported by others in\nprevious studies (https://doi.org/10.1038/s41586-024-07182-w, https://doi.org/10.1128/mbio.00941-25,\nhttps://doi.org/10.1038/s42003-024-06825-y, https://doi.org/10.1128/mbio.02989-24). In particular, all F. watanabei genomes\nerroneously annotated as F. animalis are provided by Sivertsen et al..\nComment 2: The authors focus on the elevation of the four previous subspecies of Fusobacterium nucleatum sensu lato into\nthe four separate species Fusobacterium nucleatum sensu stricto, Fusobacterium animalis, Fusobacterium vincentii and\nFusobacterium polymorphum. Taxonomically, these elevations are uncontroversial and in complete adherence to genome-\nbased taxonomic rules. They were proposed in 2017 and formally accepted in 2021.\nThe current taxonomy of the Fusobacterium nucleatum group has been confirmed in several recent studies, of which at least\nthree include the new “biological species concept”/gene flow network analyses in addition to the standard ANI-based\napproaches used in the present manuscript, thereby representing more contemporary, comprehensive and throughout\nanalyses (https://doi.org/10.1128/mbio.00941-25, https://doi.org/10.1038/s42003-024-06825-y,\nhttps://doi.org/10.1128/mbio.02989-24). The paper by Molteni et al. also assesses the taxonomy for the rest of the\nFusobacterium genus. None of these highly relevant papers have been discussed nor cited in the present manuscript. If ANI-\nbased methods should be further corroborated, methods like the first derivative of ordered ANI distance method\n(https://doi.org/10.1186/s13059-024-03443-z) could have been employed, as it has not been specifically used in\nfusobacterial datasets yet.\nStatements like “However, there are persistent taxonomic ambiguities within this genus, particularly regarding F. nucleatum\n(sensu lato)” and “However, a consensus on the taxonomy and nomenclature of these taxa has yet to be established.”\n(paragraphs on lines 48-75) are failing to describe an updated view on the literature.\nComment 3: In the introduction, the authors are specifically referring to Zepeda-Rivera et al. and their proposed division of\nFusobacterium animalis into clades Fna C1 and C2. This was a suggestion in clear violation of genome-based taxonomic\nrules. Soon after their publication, it was shown in two independent publications that Fna C1 was clearly a separate species\nfrom F. animalis (https://doi.org/10.1038/s42003-024-06825-y, https://doi.org/10.1128/mbio.02989-24). Later it was shown by\nSivertsen et al. that Fna C1 was simply a re-discovery and misclassification of F. watanabei\n(https://doi.org/10.1128/mbio.00941-25), and finally this was also acknowledged by Zepeda-Rivera et al.\n(https://doi.org/10.1080/19490976.2025.2514797), although no formal correction of their paper has been published.\nTherefore, if the authors want to mention Fna C1 and C2, it should be immediately followed by a sentence saying that this\nwas erroneous and that C1 was a rediscovery and misclassification of F. watanabei.\nFurther, there is a mention in lines 62-63 that in a study by Piccinno et al., Fna C1 was recently regrouped with F. vincentii in\na metagenomic study. We were not aware of this study, but this is just another example of people failing to keep their\ndatabases updated. If one does not have the correct genome reference in the reference database, many softwares will\nassign reads to the closest alternative species, which for F. watanabei often will be F. vincentii. However, Fna C1 is just a\nrediscovery and misclassification of F. watanabei as irrevocably established by Sivertsen et al. and there are no taxonomic\nambiguities.\nComment 4: Given the small number of available genomes for many Fusobacterium species and the narrow ANI-gap\ndescribed, it is not appropriate to call it a “robust” gap. However, this is not the major problem with so-called “genus-specific”\ncut-off values for species demarcations within a certain genus, representing an academic concept of little biological or\ntaxonomic relevance. What the authors do is simply to find the species with the largest intra-species ANI variability and the\nspecies with smallest inter-species ANI-differences and then defining the gap between these biologically unrelated\nmeasures as a genus specific gap. The authors fail to adequately define the utility of this measure. Each species has its own\nindependent intraspecies variability defined by the actual biological variation within that species, and its own distances to its\nown closest neighboring species and those are the only relevant demarcations for that species. A theoretical composite\nboundary based on all currently known species and currently available genomes within the entire genus has no biological\nmeaning and cannot be used for e.g. description of new species. Also, when more genomes become available (and they\nwill) and new Fusobacterial species are described (and they will) and some species are excluded from this genus (and they\nwill) - these boundaries will be obsolete. They are a snapshot of currently defined species and available genomes.\nComment 5: The recently proposed species Fusobacterium paranimalis has not been included in the analyses. Although it\nwas published outside of the IJSEM, it is a valid description according to IJSEM with a type-strain submitted to and accepted by\ntwo separate international strain collections (NCTC15132 and DSM119817) and a complete genome available in\nGenBank. It has also been included in the ENA/GenBank taxonomy. It is therefore likely to be formally accepted as a valid\npublication outside of the IJSEM.\nComment 6: The recently proposed species “Fusobacterium sphaericum” is included in the analyses. This is a proposed\nspecies from another publication by Zepeda-Rivera et al. It is not a valid description, since a type-strain has not been\nsubmitted to relevant microbial culture collections, and the name has not been adopted by ENA/GenBank. Also, it is highly\nquestionable that this is even a Fusobacterium, but Zepeda-Rivera et al. have not included relevant analyses to investigate\nthis (e.g. a POCP-analysis including neighboring genera). It has round cells (!), grows with large and waxy colonies, and\nshows low 16S rRNA (91-92%) and ANI homologies (70-72%) with other typical fusobacteria including F. nucleatum sensu\nstricto, the type species of the genus. It is most closely related to Fusobacterium perfoetens, representing another most likely\nmisclassified species belonging to a separate genus (see below). Based on these matters, we conclude that as it stands,\nthe description of F. sphaericum will not be accepted as a valid publication outside of the IJSEM. It should only be included in\nthe present manuscript if all these uncertainties are appropriately discussed and/or resolved.\nComment 7: While the taxonomy within the Fusobacterium nucleatum group is clear, there are other unresolved taxonomic\nmatters related to species included in the manuscript that are not addressed. Currently, there is growing evidence that the\nclade including “F. sphaericum”, F. perfoetens, and Fusobacterium naviforme (for some reason not included in this study)\nare not Fusobacteria, but belong to other genera. This is also the reason why GTDB as seen in your Table 1, has included a\nsuffix after the genus name in their genome-based taxonomy (Fusobacterium_B perfoetens). It indicates that this is probably\nnot a correct genus assignment. Fusobacterium naviforme is more closely related to Catonella morbi and has been\nproposed reclassified as Zandiella naviformis comb. nov. (https://doi.org/10.1007/s10482-023-01921-1), but this has not yet\nbeen formally accepted. Fusobacterium perfoetens is proposed as a non-fusobacterium in this paper\n(https://doi.org/10.1016/j.anaerobe.2014.06.007). In figure 1 in this paper (https://doi.org/10.1128/spectrum.00297-22), both\nF. perfoetens and F. naviforme cluster together with Cetobacterium and Leptotrichia outside of the Fusobacterium clade and\na similar finding is presented here in figure 1 (https://doi.org/10.1007/s10482-023-01921-1). As shown in your own Table 1, there are\nalso some Fusobacterium_A and Fusobacterium_C species in GTDB. For the community, it would be more valuable to include the\nnecessary phylogenetic analyses to appropriately address these unresolved matters, instead of\nreproducing taxonomy investigations within the Fusobacterium nucleatum group.\nComment 8: To underpin their claims, studies like this should include a phylogenetic tree based on whole genome data and\nnot solely a tree based on partial rpoB sequences. In all figures and tables, it should be clearly indicated which species that\nare not formally valid according to DSMZ or other authoritative bodies.\nComment 9: Line 315-316: Which community is this referring to? These matters are not controversial in the bacterial\ntaxonomy community or in the clinical microbiology community. Also, this is a political statement that we do not support.\nTaxonomy is a rule-based system developed for a purpose. It cannot be based on a perceived consensus in a vaguely\ndefined “community”. We divide bacteria into species because it is meaningful to cluster together isolates with the same\nbiological and pathogenic properties. When taxonomy is changed, it is because we acknowledge that the previous grouping\nhas been erroneous, and that we are grouping together organisms that should not be expected to share the same properties.\nTherefore, it is not rational to dispute changes like the elevation of former subspecies into separate species. Also, such\nopinions stand in direct conflict with what is written in the sentence before: “...and provides the taxonomic precision essential\nfor advancing mechanistic and clinical research on Fusobacterium pathogenesis”.\nOther comments:\nLines 66-67: What does this sentence mean? You just stated that “Recent studies have further highlighted their distinctive\nroles in disease pathogenesis” (Line 54). There is nothing in the present taxonomy preventing such investigations, although\npeople’s failure to adhere to or use contemporary taxonomy might definitely obscure such\ncorrelations.\nLine 67-68: This is not correct. It is true that the V3-V4 region commonly used for Illumina sequencing does not discriminate\nwell between some of these species, but other regions like e.g. V1-V3 or the full gene can be used to discriminate between\nmost Fusobacterium species, including the currently accepted species in the Fusobacterium nucleatum group.\nLine 69-70: This is not the case. There are several recent studies addressing this, but authors are not citing them.\nLines 127-129: As mentioned above, a range of recent studies have flagged these wrongly annotated genomes before, but\nthe authors are not citing them.\nLines 132-136: This has already been established by Sivertsen et al. (https://doi.org/10.1128/mbio.00941-25). This was the\nmain purpose of their study. They also provided the F. watanabei type strain whole genome used in the present study.\nLines 139-140: This has also been pointed out by others (10.1007/s10482-023-01921-1) and is acknowledged by DSMZ.\nLines 142-149: It is fine to conclude that some genomes are probably representatives of novel species. However, proposing\nnew names based on this is not very useful, since a valid description of novel species requires a type-strain submitted to at\nleast two microbial culture collections.\nLine 171-173: F. watanabei was not formerly Fna C1. F. watanabei was validly described in 2021 and Fna C1 is a\nmisconception from 2024.\nLine 187: rpoB- and gyrA primers are not universal. They are Pan-Fusobacterial.\nLines 202-205: rpoB is an excellent marker for Fusobacteria, but the authors are comparing a 320 bp segment of rpoB with a\n1500 bp segment of gyrB. That is not a very fair comparison, and a PCR providing a longer amplicon for rpoB is appropriate.\nThe failure to discriminate between F. perfoetens and Fusobacterium sp. FSA-380-WT-3A is not a strong argument for using\ngyrB over rpoB for Fusobacterium spp., since as commented above, the “F. perfoetens/sphaericum” clade most likely does\nnot consist of Fusobacterium species but representatives of a novel genus.\nLines 252-258: As mentioned above; other references have already resolved this (https://doi.org/10.1128/mbio.00941-25).\nLines 310-312: This paper only repeats what has already been confirmed by others and does not address any of the actual\nremaining issues within the taxonomy of the Fusobacterium genus, in particular removal of likely non-fusobacterial species\nas discussed in major comment 7.\nLine 312: what do the authors mean by “…for both individual strain and within microbiota”? Primers were not evaluated on\nmicrobiota samples and it cannot be known how efficient/specific they will be for amplifying fusobacterial DNA from a\ncomplex background of other DNA.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "Summary\nThe authors analyse 542 genomes of the genus Fusobacterium to better approach the taxonomy of the genus. They\nconducted ANI analyses, a whole genome phylogeny, plus individual gene phylogenies to get a sound idea of the\nphylogenetic relationships within the genus. This study has the potential to add some insights into the taxonomy of this\ngenus. However, there are some major and minor issues the authors want to address in a revised version.\nMajor comments\n1. To appreciate the quality of the genome assemblies, the authors want to run CheckM on all the genomes and report\ncompleteness and contamination; this info should be added to Supplementary Table 1. Ideally, for downstream analyses,\nthey just want to consider genomes with > 95% completeness and < 5% contamination. Also, the authors need to provide the\nBioProject of the newly sequenced genomes.\n2. Considering the concordance between the whole-genome phylogeny and the ANI-based grouping, a formal topology test\n(KH-test, SH-test, AU-test, RELL, etc) is desirable. So, to know if those strategies produce statistically significant different\ngroupings. Just from visually comparing the topologies, it is not possible to know whether or not they are statistically\ndifferent.\n3. Important details are missing about the phylogenetic analyses. First, many details about the whole genome phylogeny are\nneeded. For instance, kSNP4 estimates parsimony, maximum likelihood, and neighbour-joining trees; which method did the\nauthors select? Please state all the different parameters employed in the analysis. Importantly, given the distorting effects of\nrecombination, you want to run a phylogeny just on regions of the genome not affected by recombination. Did you use all the\nSNPs or just core SNPs? Second, re the individual gene markers, please state the DNA substitution model specified when\nrunning the gene phylogenies, maybe GTR+I+R?\n4. Finally, considering their top two markers (gyrB and rpoB), the authors want to make sure these genes are good\nphylogenetic markers. I would suggest the authors run topology tests (see Major comment 2) of these individual gene trees\n(and also a concatenated alignment of the two genes) versus the whole genome phylogeny and ensure that there are no\nsignificant differences. Also, they want to ensure that those two genes do not have signals of recombination. Lastly, please\nreport the nucleotide diversity of these two genes and how it compares to the nucleotide diversity of the other markers\npresented in Supplementary Data 3.\nMinor comments\nConsidering the title, if I were the authors, I would replace “genomic ANI” with just “ANI” — almost all ANI analyses involve\ngenome sequences\nLine 67: What do you mean by a high degree of homology? I guess you meant to say a high level of similarity or identity.\nLine 101:revise grammar.\ny-labels on Figure 1A and some other panels in other figures (for instance, Figure 5B) should be “Number of comparisons” or\njust “Frequency”\nLines 127-129: Previous studies using phylogenomic approaches have highlighted important cases of misclassification in\nother genera. See ref below\nhttps://pubmed.ncbi.nlm.nih.gov/31406982/\nhttps://pubmed.ncbi.nlm.nih.gov/39439205/\nhttps://pubmed.ncbi.nlm.nih.gov/34437546/\nhttps://pubmed.ncbi.nlm.nih.gov/30386709/\nLine 280: “The gyrB gene” reads better.", "reviewer_4": "In this manuscript, Bi et al provide quite a comprehensive genus-wide analysis that integrates ANI-based and phylogenetic\napproaches to clarify some longstanding issues that have arisen within Fusobacterium taxonomy and nomenclature. The y\nfurther introduce a practical dual-marker (gyrB and rpoB) identification method and demonstrate how it can be integrated\nwith GTDB and MetaPhlAn frameworks. The analyses are extensive, technically sound, and clearly the product of careful\ncomparative work across what has been a challenging genus. The effort to unify genomic, phylogenetic, and marker-based\napproaches into a coherent framework will represent a substantial step forward for reproducible species-level identification\nacross Fusobacterium. This reviewer particularly appreciates the authors’ effort to present a balanced, data-driven treatment\nof what is often a contentious subject. This reviewer also agrees that the novel markers outlined here could contribute to\novercoming challenges faced with traditional 16S rRNA sequencing efforts to elaborate on\nFusobacterium species relevant to human cancers.\nNevertheless, I do have concerns about some of the proposed updates, and along with a single major point, provide some\nconstructive feedback that may improve readability and decrease resistance within the field for adoption of the authors\nproposed taxonomy and nomenclature adjustments.\nMajor comment: Against a double meaning for F.n sensu lato.\nIt appears that the authors are proposing to change the nature of what Fusobacterium nucleatum sensu lato (Fn s.l.) means\n(at least as understood by this reviewer and by many colleagues working in this area). Several statements in the manuscript\nare ambiguous in this regard and warrant clarification, as they risk introducing additional significant confusion into an\nalready complex and historically inconsistent nomenclatural landscape. Specifically, the manuscript seems to redefine F.\nnucleatum sensu lato in a way that excludes F. animalis and F. watanabei, whereas in the broader literature, the term has\nconsistently referred to the entire F. nucleatum complex, (including these lineages).\nThese sentences include:\n- Lines 129–138, “the two F. nucleatum subsp. animalis clades (Fna C1 and Fna C2) constitute distinct species… which did\nnot justify the species status of F. nucleatum (sensu lato)”,\n- Lines 252–255, which describe the current subspecies/clade designations under “F. nucleatum (sensu lato)” as\n“taxonomically invalid”,\n- Table 2, which presents F. animalis and F. watanabei as reclassified species without reference to their origin within the F.\nnucleatum sensu lato complex.\nThis is in contrast to the current established usage of F. nucleatum sensu lato in the field (eg. Kook et al., 2017; Ma et al\n2023, Krieger et al., 2024; Zepeda-Rivera et al., 2025) encompasses the four canonical F. nucleatum subspecies\n(nucleatum, polymorphum, vincentii, animalis) and their derivative lineages, forming a coherent monophyletic group sharing\n~90–94 % ANI.\nIt is also important to note that the sensu lato designation functions not as a formal species rank but as a conceptual and\npractical umbrella, and is useful to preserve interpretive continuity across genomic, ecological, and clinical research where\nsubspecies resolution was unavailable.\nThis current framework is also relatively well established, as it provides an inclusive and pragmatic way to refer to the entire\nF. nucleatum complex by linking historical culture-based and 16S-based studies with more recent updated genome-resolved\nanalyses. It is also a default interpretive construct; even as individual lineages are refined to species rank.\nCould the authors please carefully consider and explicitly outline what the benefit to the field would be in creating a new\ninterpretation of F. nucleatum sensu lato that excludes F. animalis and F. watanabei, when there is already an established\nand widely understood F. nucleatum sensu lato framework that includes these lineages?\n- What is the practical or conceptual upside of redefining an existing and historically consistent umbrella term in this way? If\nthe intention is to describe or analyze a subset of species within the F. nucleatum complex, would it not be clearer and less\ndisruptive to the existing literature to introduce a new, unambiguous term for this more restricted grouping, rather than\naltering the meaning of F. nucleatum sensu lato?\n- Something like “F. nucleatum core group”?, or similar, where is unlikely to collide with the established framework. It would\nbe most beneficial if this could be then included in the figures, where the F.n sensu lato group and this new tighter group are\nall clearly highlighted in the main figures for future references. (so Fn sensu lato, Fn. core group, and Fn sensu stricto would\nall be in use, but distinguishable)\n- This reviewer would strongly advocate for such an alternative choice as currently it risks creating parallel usages of the\nsame term with different inclusivity. i.e: F.nucleatum sensu lato = the four canonical subspecies nucleatum, polymorphum,\nvincentii, and animalis and their derivative lineages (including F. watanabei)…….but also F.nucleatum sensu lato =\n(canonical subspecies nucleatum, polymorphum, vincentii).\n- Further, this redefinition raises another issue. F. animalis has long been considered a bona fide member of the F.\nnucleatum sensu lato and already included in this way within hundreds of studies linking Fusobacterium diversity to\ndisease. Reassigning F. animalis (and F. watanabei) outside of F. nucleatum sensu lato without a clear taxonomic or\ntranslational rationale risks severing continuity with a decade of literature that used F. nucleatum sensu lato precisely to\nunify these lineages under a single, interpretable construct.\nAt present, this reviewer cannot discern any clear benefit or added utility to the field in redefining F. nucleatum sensu lato in\nthis new way. Doing so may increase confusion among researchers, clinicians, and data curators who have relied on the\ninclusive sensu lato framework for consistent comparative and meta-analytical interpretation.\nIf the authors believe there is genuine conceptual or practical value in excluding F. animalis and F. watanabei from F.\nnucleatum sensu lato, could they please elaborate and articulate that rationale explicitly in the manuscript (what new clarity\ndoes this bring, and to whom?) Otherwise, I apologize, but the proposed redefinition appears to introduce new complexity\nwithout a tangible scientific or translational gain.\nMinor comments:\nLines 252–255 “Critically, our evidence supports the argument that the current subspecies/clade designations under ‘F.\nnucleatum’ (sensu lato) are taxonomically invalid..\nThe phrasing here could be perceived as a little aggressive, implying that prior taxonomic frameworks are incorrect or\ninvalid, which is inconsistent with taxonomic freedom and the evolving and interpretive nature of bacterial taxonomy noted\nearly in the discussion by the authors. Names and classifications reflect the data and consensus available at the time, but do\nnot become invalid with the emergence of new evidence.\nThe authors data clearly demonstrates that the historical F. nucleatum sensu lato subspecies exhibit genomic and\nphenotypic divergence consistent with species-level distinctions. However, rather than characterizing existing designations\nas “taxonomically invalid,” a more accurate and constructive framing might be that these designations may no longer\nadequately capture the observed diversity and that the authors’ analyses support recognition of these lineages as distinct\nspecies under a revised framework. This would keep the scientific strength of the findings but with a more balanced tone.\nLine 62: Notably, the status of Fna C1 remains inconclusive, as it was recently regrouped with F. vincentii in a metagenomic study\nSome additional context may be useful for this particularly unusual lineage that has quickly changed. The KCOM strains\nrepresenting the majority of the lineage Fna C1 (21 of 24) were original annotated as Fna/Fa isolates by the Korean\nCollection for Oral Microbiology using 16S rRNA profiling (partial) and released publicly between April 2020 and Feb 2021\nat NCBI. They remained annotated as such within the culture collection, until Zepeda et al noted that they were distinct from\nFna ATCC 51191 lineage, and they assigned them as Fna C1 and Fna C2. Ma et al also assigned a strain of this lineage as\nFNU (unique) around the same time based on their noted distinctions in a pangenome study. F. watanabei isolates were\nformally named after the efforts of Tomida et al, but remained disconnected from the other strains (and 13-08-02) due to a\nlack of whole genome information. The GTDB still refers to this lineage as F. nucleatum_J, although recent work by Tomida\ngroups and this work by Bi et al appear to verify that these different names all refer to a single lineage.\nIn this introduction, the authors note that Piccinno G et al determined this lineage to be clustered with Fnv. It may be useful to\nreaders for Bi et al to elaborate on why this lineage appears to fluctuate across studies, inherently based on the differences\nin the datasets and methods used (16s rRNA vs partial genome vs who genome vs SGBs of metagenomes)?\nLine 133: “As Fna C2 was represented by the F. animalis 134 anchor strain (KCOM 1325)”\nCould the authors elaborate on why KCOM 1325 was chosen as the anchor strain, and not ATCC 51191 or 7.1?\nLine 136: “the F. watanabei anchor (CCUG74246T) and herein reclassified into F. 136 watanabei.”\nIt appears authors of the FnaC1 study have made efforts with Tomida et al to provide clarity on where the\nFnaC1/F.nucleatum subspecies unique/F. watanabei/ 13-08-02 lineages reside with respect to one another -\ndoi/10.1128/mra.00132-25. Could Bi et al include this citation also.\nLine 89: developed standardised species identification approaches for strains and within microbiota.\n(is the ‘and”a misprint?)\nLine 140: “Severn strains”", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Answer: Thank you very much for this important comment. We have carefully\nexamined our data and revised our manuscript and Supplementary Data 1 accordingly.\nOur original intention was to illustrate how many annotations in RefSeq were\ncorrected specifically based on our integrated ANI & whole-genome phylogeny\napproach. We never intended to overlook previous studies. After careful examination,\nwe found that our corrections for 60 of the 101 genomes were consistent with the\nfindings in previously studies, strongly supporting the performance of our standardised\nANI-based species boundary. The remaining 41 corrections are newly proposed in our\nstudy and were not covered in previously studies.\nWe would like to clarify that the primary aim of our study was not simply to reassess\nand correct of the taxonomy of individual genomes. While we fully acknowledge and\nsupport the essential reclassifications presented in the earlier studies, our goal was to\nestablish a unified, data-driven taxonomic framework integrating genomic,\nphylogenetic, and marker-based approaches for reproducible species-level\nidentification across Fusobacterium. Our study provided a comprehensive, quantitative\nassessment of agreement among whole-genome phylogeny, ANI, and marker-based\nphylogenies, using the largest high-quality genome set to date for Fusobacterium. As a\nresult, we defined a genus-specific ANI boundary for species delineation, developed\nthe practical B&B method for species identification of isolates without requiring\ngenome sequencing, and refined the annotation of marker-based MetaPhlAn SGBs\nthrough large-scale analysis, which directly aids in improve metagenomic profiling\naccuracy. We wish to provide a foundation to improve taxonomic accuracy in future\nresearch, thereby helping to avoid the introduction of further ambiguity and\nmisclassification.\nThe taxonomic revisions we present were only one inherent outcome of our efforts.\nThe high consistency with previous studies in fact validated the performance of our\napproach, while our independent, large-scale analysis has confirmed and extended these\ncorrections.\nNevertheless, we sincerely apologise for the oversight of the significant work by\nSivertsen et al, which was published during the final stage of our manuscript\npreparation. We have revised the Introduction (lines 54-73), Results (lines 170-178),\nDiscussion (lines 341-343) and Supplementary Data 1 to properly credit all relevant\nstudies and to clarify our intention.\n2. Comment 2: The authors focus on the elevation of the four previous subspecies of\nFusobacterium nucleatum sensu lato into the four separate species Fusobacterium\nnucleatum sensu stricto, Fusobacterium animalis, Fusobacterium vincentii and\nFusobacterium polymorphum. Taxonomically, these elevations are uncontroversial and\nin complete adherence to genome-based taxonomic rules. They were proposed in 2017\nand formally accepted in 2021.\nThe current taxonomy of the Fusobacterium nucleatum group has been confirmed in\nseveral recent studies, of which at least three include the new “biological species\nconcept”/gene flow network analyses in addition to the standard ANI-based approaches\nused in the present manuscript, thereby representing more contemporary,\ncomprehensive and throughout analyses (https://doi.org/10.1128/mbio.00941-25,\nhttps://doi.org/10.1038/s42003-024-06825-y, https://doi.org/10.1128/mbio.02989-24).\nThe paper by Molteni et al. also assesses the taxonomy for the rest of the Fusobacterium\ngenus. None of these highly relevant papers have been discussed nor cited in the present\nmanuscript. If ANI-based methods should be further corroborated, methods like the first\nderivative of ordered ANI distance method (https://doi.org/10.1186/s13059-024-\n03443-z) could have been employed, as it has not been specifically used in\nfusobacterial datasets yet.\nStatements like “However, there are persistent taxonomic ambiguities within this genus,\nparticularly regarding F. nucleatum (sensu lato)” and “However, a consensus on the\ntaxonomy and nomenclature of these taxa has yet to be established.” (paragraphs on\nlines 48-75) are failing to describe an updated view on the literature.\nAnswer: We appreciate this comment and the constructive suggestion. We apologise\nfor any confusion caused. We wish to clarify that, as mentioned above, taxonomic\nreassessment including the established reclassification F. nucleatum sensu lato\nsubspecies was not the primary focus of our study. We consistently support and\nacknowledge the reclassification proposed by Kook et al. (Curr Microbiol, 2017), as\nwe have previously stated (Bi et al. Nat Commun, 2022). Our intention in mentioning\nthis historical issue was to highlight the complex and challenging nature of\nFusobacterium taxonomy. While experts in the field (particularly those with strong\nexpertise in microbiological taxonomy and/or clinical microbiology) are undoubtedly\nwell aware of and quickly adopt the updated taxonomy, a significant practical challenge\nremains in its consistent implementation across the broader research and clinical\ncommunities. Given the expanding relevance of Fusobacterium in diverse diseases\nbeyond infection, mixed usage in literatures and certain mis-leading annotations\ncontinue to create ambiguity. More critically, this inconsistency often fails to adequately\naccount for the underlying genetic and pathogenic diversity among species. Therefore,\nthe primary goal of our study was to perform a comprehensive phylogenetic and\ntaxonomic evaluation to develop standardised, practical solutions to ensure consistent\nand accurate species-level identification, both for current applications and future\nresearch. We have extensively revised the Introduction (lines 54-98), Results (lines 170-\n178) and Discussion (lines 341-343) to clarify these points and credit all relevant studies.\nWe sincerely thank the reviewer for the valuable and very constructive suggestion\nregarding the first derivative analysis of ordered ANI values. We have performed this\nanalysis (Supplementary Fig. 1) using a similar approach described in Passarelli‑Araujo\net al. (https://doi.org/10.1186/s13059-024-03443-z). We noticed that Passarelli‑Araujo\net al. used Mash distance (Ondov et al. Genome Biol, 2016), which correlates strongly\nwith ANI, to assess genetic discontinuity. Their method is certainly applicable to ANI,\nbut our implementation is slightly different. Whereas Passarelli‑Araujo et al. analysed\nthe Mash identities against a representative genome, we included all pairwise ANI\nvalues in our analysis, which we consider a more stringent approach. As detailed in the\nrevised Methods, we have sorted all pairwise ANI values in descending order and\ncalculated the first derivative (Δ) of the ANI with respect to the rank position. The ANI\ndiscontinuity (δ) was defined as the maximum Δ among ANI values > 90% (δ =\nmax{Δ|ANI ∈ [0.90, 1.00]}), which represented a break or sharp change. Notably, The\nANI discontinuity (δ) occurred precisely at the ANI gap we previously described,\nsupporting that this gap represents significant genetic discontinuity within\nFusobacterium. We have revised the Methods (lines 548-554) and Results (lines 126-\n131) accordingly and added a Supplementary Fig. 1.\nMoreover, we emphasise that our taxonomic analysis did not rely solely on ANI.\nWe systematically evaluated the consistency between ANI-based grouping and whole-\ngenome based phylogenic clustering at species level (Fig. 2). Furthermore, we have\nnow also quantitatively assessed the topological agreement between the ANI matrix-\nbased dendrogram and the whole-genome phylogeny by calculating the normalised\nquartet score (Fig. 2).\n3. Comment 3: In the introduction, the authors are specifically referring to Zepeda-\nRivera et al. and their proposed division of Fusobacterium animalis into clades Fna C1\nand C2. This was a suggestion in clear violation of genome-based taxonomic rules.\nSoon after their publication, it was shown in two independent publications that Fna C1\nwas clearly a separate species from F. animalis (https://doi.org/10.1038/s42003-024-\n06825-y, https://doi.org/10.1128/mbio.02989-24). Later it was shown by Sivertsen et\nal. that Fna C1 was simply a re-discovery and misclassification of F. watanabei\n(https://doi.org/10.1128/mbio.00941-25), and finally this was also acknowledged by\nZepeda-Rivera et al. (https://doi.org/10.1080/19490976.2025.2514797), although no\nformal correction of their paper has been published. Therefore, if the authors want to\nmention Fna C1 and C2, it should be immediately followed by a sentence saying that\nthis was erroneous and that C1 was a rediscovery and misclassification of F. watanabei.\nFurther, there is a mention in lines 62-63 that in a study by Piccinno et al., Fna C1 was\nrecently regrouped with F. vincentii in a metagenomic study. We were not aware of this\nstudy, but this is just another example of people failing to keep their databases updated.\nIf one does not have the correct genome reference in the reference database, many\nsoftwares will assign reads to the closest alternative species, which for F. watanabei\noften will be F. vincentii. However, Fna C1 is just a rediscovery and misclassification\nof F. watanabei as irrevocably established by Sivertsen et al. and there are no taxonomic\nambiguities.\nAnswer: We understand the reviewer’s concern regarding the proposal of Fna C1 and\nC2 by Zepeda-Rivera et al. As stated above, in our revision, we have acknowledged\nthat that C1 corresponds to F. watanabei, as established by Sivertsen et al and extended\nthe context regarding the shifting history of the annotation of Fna C1 (lines 56-73). Our\nindependent, large-scale analysis provides strong and consistent support for this\nconclusion.\nRegarding the mention of Piccinno et al work, we have revised the text to accurately\nreflect the context (lines 68-70). We noticed that they had attempted to clarify the\ndesignation of Fna C1 through phylogenetic analysis of F. nucleatum sensu lato SGBs.\nHowever, their analysis was mostly confined to F. nucleatum sensu lato rather than\nencompassing the entire genus and not genome-wide. We believe this limited scope\nprevented them from obtaining a global phylogenetic view essential for correct\nassignment of this taxon.\n4. Comment 4: Given the small number of available genomes for many Fusobacterium\nspecies and the narrow ANI-gap described, it is not appropriate to call it a “robust” gap.\nHowever, this is not the major problem with so-called “genus-specific” cut-off values\nfor species demarcations within a certain genus, representing an academic concept of\nlittle biological or taxonomic relevance. What the authors do is simply to find the\nspecies with the largest intra-species ANI variability and the species with smallest inter-\nspecies ANI-differences and then defining the gap between these biologically unrelated\nmeasures as a genus specific gap. The authors fail to adequately define the utility of this\nmeasure. Each species has its own independent intraspecies variability defined by the\nactual biological variation within that species, and its own distances to its own closest\nneighboring species and those are the only relevant demarcations for that species. A\ntheoretical composite boundary based on all currently known species and currently\navailable genomes within the entire genus has no biological meaning and cannot be\nused for e.g. description of new species. Also, when more genomes become available\n(and they will) and new Fusobacterial species are described (and they will) and some\nspecies are excluded from this genus (and they will) - these boundaries will be obsolete.\nThey are a snapshot of currently defined species and available genomes.\nAnswer: Thanks for this very insightful comment. We have revised our wording\naccordingly, throughout manuscript. Our original rationale for describing the gap as\n\"robust\" was that species clustering based on this interval is fully consistent with the\nwhole-genome phylogeny and clearly discriminates all established species. From a\npractical perspective, we therefore considered it suitable as a universal boundary\napplicable across the genus.\nWe agree with the reviewer that each species possesses its own independent\nintraspecies variability, defined by its actual biological diversity. In fact, since ANI has\nbeen widely adopted for species classification due to its accuracy and practical utility,\nwe performed systematic, data-driven analyses to link this gap to biological and\ntaxonomic relevance. As described in our manuscript, the ANI gap was initially\nidentified through distribution analysis near the commonly recognised species\nthreshold without prior taxonomic assessment. Then we showed that the gap was highly\nconsistent with a genetic discontinuity revealed by the first derivative analysis\n(Supplementary Fig. 1) suggested the by the reviewer. We subsequently evaluated its\nutility as a species boundary by correlating ANI-based groupings with the whole\ngenome phylogeny (Fig. 2). Furthermore, while the current gap is a shared interval\nderived from genus-wide analysis, many species, particular those with relatively large\nnumbers of genomes available consistently exhibited intraspecies and interspecies\nboundaries that closely align with the upper and lower limits of this gap, respectively\n(Supplementary Fig. 3).\nWe also agree with the reviewer’s point regarding the dynamic nature of this\nboundary. In fact, we do not expect the ANI gap boundaries to be fixed and they should\nbe iteratively updated with new genomic data, as now discussed in our revised\nmanuscript (lines 368-375, 412-414). From a historical perspective, and as illustrated\nin our rarefaction curve-like analysis, the current observed discontinuity may\neventually converge as technology advances and more genomes become available.\nWhile the specific thresholds defined in this current study may not capture every\nbiological variation, our framework, based on exhaustive whole-genome analysis and\nvalidation, offers a reliable, updatable solution for achieving precise species\nclassification, moving beyond reliance on imperial thresholds that might introduce\narbitrary results. It is also noteworthy that, compared to many proposed single-\nbreakpoint species boundaries, a gap spanning >0.5% (corresponding to discontinuity\nΔ > 0.5%) represented a relatively wide interval. In the work by Passarelli‑Araujo et al.\nmentioned in your Major comment 2, many species exhibited lower Δ values (less than\n0.5%). Thus, this relatively broad range reflected a significant genetic discontinuity\nacross species and moreover provides a practical buffer zone for future convergence\nand for handling borderline cases in real-world applications (this scenario will certainly\noccur as the reviewer correctly noted). Such future convergence does not invalidate our\nstudy but rather indicates an evolving understanding of evolutionary patterns as more\nspecies are characterised.\n5. Comment 5: The recently proposed species Fusobacterium paranimalis has not been\nincluded in the analyses. Although it was published outside of the IJSEM, it is a valid\ndescription according to IJSEM with a type-strain submitted to and accepted by two\nseparate international strain collections (NCTC15132 and DSM119817) and a complete\ngenome available in GenBank. It has also been included in the ENA/GenBank\ntaxonomy. It is therefore likely to be formally accepted as a valid publication outside\nof the IJSEM.\nAnswer: Thank you very much for highlighting the recent proposal of Fusobacterium\nparanimalis. As mentioned above, we were not aware of this publication during the\nfinal stage of manuscript preparation, and again we sincerely apologise for this\noversight.\nWe agree that F. paranimalis represents a significant taxonomic update and should\nbe considered in our analysis. As stated in our manuscript, our primary taxonomic\nframework was strictly based on genomes from the RefSeq database to ensure\nconsistent quality control. As the F. paranimalis genome was not yet part of RefSeq, to\nmaintain methodological consistency, it was not yet included in the RefSeq-based\nanalyses presented in the manuscript.\nNevertheless, recognising its importance, we have conducted a complete set of\nsupplemental analyses on its type strain (Vestland19T) genome. Importantly, it served\nas an excellent validation case for our newly developed B&B method, which correctly\nidentified it as a novel species, with no match with the defined thresholds in the current\nreference set. Our whole-genome phylogenetic analysis confirmed that F. paranimalis\noccupied a distinct phylogenetic position in the whole genome-based tree (see\nSupplementary Fig. 4). Interestingly, a few of its pairwise ANI values with RefSeq\ngenomes (71.30%--93.51%) fell within the species gap, while all values remained\nbelow the gap's upper limit and midpoint. This did not contradict our findings but rather\nprovides a practical example of how our framework handled such borderline cases. As\nits maximum ANI to any RefSeq genome is still below the gap's midpoint, it should be\nclassified as a novel species. This case appropriately revised the lower boundary of the\ngap. We have explicitly pointed out that the gap derived from the RefSeq dataset could\nbe revised when considering the inclusion of the F. paranimalis genome. These results\nhave been added to the manuscript (lines 297-305, 368-375, 447-451, Fig. 6e,\nSupplementary Fig. 4 and Supplementary Data 7).\nThis example perfectly aligns with the point the reviewer raised and our discussion\nin your Major comment 4. We are truly grateful for this valuable suggestion, which has\nundoubtedly strengthened our work.\n6. Comment 6: The recently proposed species “Fusobacterium sphaericum” is included\nin the analyses. This is a proposed species from another publication by Zepeda-Rivera\net al. It is not a valid description, since a type-strain has not been submitted to relevant\nmicrobial culture collections, and the name has not been adopted by ENA/GenBank.\nAlso, it is highly questionable that this is even a Fusobacterium, but Zepeda-Rivera et\nal. have not included relevant analyses to investigate this (e.g. a POCP-analysis\nincluding neighboring genera). It has round cells (!), grows with large and waxy\ncolonies, and shows low 16S rRNA (91-92%) and ANI homologies (70-72%) with other\ntypical fusobacteria including F. nucleatum sensu stricto, the type species of the genus.\nIt is most closely related to Fusobacterium perfoetens, representing another most likely\nmisclassified species belonging to a separate genus (see below). Based on these matters,\nwe conclude that as it stands, the description of F. sphaericum will not be accepted as\na valid publication outside of the IJSEM. It should only be included in the present\nmanuscript if all these uncertainties are appropriately discussed and/or resolved.\nAnswer: We thank the reviewer for this thoughtful and highly informative comment\nregarding Fusobacterium sphaericum. In fact, we were also aware of the observations\ndocumented in the Zepeda-Rivera et al. paper and therefore understand your concerns.\nOur decision to include its genome in our analyses was based on the following\nconsiderations. It is a RefSeq genome under the genus Fusobacterium and met our\ninclusion criteria. To maintain methodological consistency and ensure an unbiased\nscreening process, we applied the same filtering rules to all genomes before any\ntaxonomic re-evaluation. We consider that this is essential for the reproducibility of our\nworkflow. In addition, the species name was formally proposed in a peer-review paper.\nAt the time of our analysis, there was no published note or studies clarifying or\nquestioning its classification at the genus level. Crucially, even if it belongs to other\ngenus, its inclusion did not represent a flaw of our study. On the contrary, our analyses,\nparticularly the whole genome phylogenetic analysis along with neighboring non-\nFusobacterium genera in Supplementary Fig. 4, provided evidence relevant to its\nplacement and offered data for future taxonomic discussion (see our answer to your\nMajor comment 7 below).\nWe agree that the points raised by the reviewer are significant. Our results also\nsupport the need for its future taxonomic re-evaluation at the genus level. We have\nadded a dedicated part regarding these uncertainties in the Discussion. To maintain a\nbalanced expression, we have now primarily used its objective NCBI identifier\n(Fusobacterium sp. SB021), and mentioned the proposed name F. sphaericum only\nparenthetically, throughout the manuscript, table, figures and relevant supplementary\nmaterials. Furthermore, we would like to emphasise that our study primarily focused\non establishing a species-level classification framework. In all our analyses, this\noutlying species did not distort the well-supported clustering of validated\nFusobacterium species, confirming that our species-level conclusions remain focused\nand undisturbed.\n7. Comment 7: While the taxonomy within the Fusobacterium nucleatum group is clear,\nthere are other unresolved taxonomic matters related to species included in the\nmanuscript that are not addressed. Currently, there is growing evidence that the\nclade including “F. sphaericum”, F. perfoetens, and Fusobacterium naviforme (for some\nreason not included in this study) are not Fusobacteria, but belong to other genera. This\nis also the reason why GTDB as seen in your Table 1, has included a suffix after the\ngenus name in their genome-based taxonomy (Fusobacterium_B perfoetens). It\nindicates that this is probably not a correct genus assignment. Fusobacterium naviforme\nis more closely related to Catonella morbi and has been proposed reclassified as\nZandiella naviformis comb. nov. (https://doi.org/10.1007/s10482-023-01921-1), but\nthis has not yet been formally accepted. Fusobacterium perfoetens is proposed as a non-\nfusobacterium in this paper (https://doi.org/10.1016/j.anaerobe.2014.06.007). In figure\n1 in this paper (https://doi.org/10.1128/spectrum.00297-22), both F. perfoetens and F.\nnaviforme cluster together with Cetobacterium and Leptotrichia outside of the\nFusobacterium clade and a similar finding is presented here in figure 1\n(https://doi.org/10.1007/s10482-023-01921-1). As shown in your own Table 1, there are\nalso some Fusobacterium_A and Fusobacterium_C species in GTDB. For the\ncommunity, it would be more valuable to include the necessary phylogenetic analyses\nto appropriately address these unresolved matters, instead of reproducing taxonomy\ninvestigations within the Fusobacterium nucleatum group.\nAnswer: We appreciate this valuable and constructive suggestion. we have conducted\nan extended whole-genome phylogenetic analysis (supplementary Fig. 4), which\nincluded representative genomes from other genera within the Fusobacteriaceae family\nas well as F. naviforme (Zandiella naviformis).\nWe initially excluded F. naviforme from our analysis due to the lack of a RefSeq\ngenome and its proposed reclassification (https://doi.org/10.1007/s10482-023-01921-1)\nas mentioned by the reviewer. We have included the genome of its ATCC 25832 strain\nin the extended analysis.\nOur result (supplementary Fig. 4) indicated that F. perfoetens and F. sphaericum were\nphylogenetically distinct from well-established Fusobacterium species, but did not\nshow that F. perfoetens and F. naviforme clustered closely together with Cetobacterium\nand Leptotrichia, respectively. We noticed that the analysis in the studies mentioned by\nthe reviewer were based on core gene or 16S rRNA gene phylogenies, while our whole\ngenome-based analysis offered a comprehensive, potentially more resolved view.\nBesides, the Fig. 1 in Bista et al. mentioned by the reviewer contains a topology-only\ntree without providing distance information. In Gupta, et al. They just stated “The\nspecies Fusobacterium perfoetens was found to branch earlier than the other three main\nsubclades of Fusobacterium, which are marked in this tree.” In our analysis, although\nF. perfoetens and Cetobacterium have close ancestral nodes, they were also distinctly\nrelated. But the divergence degree of F. perfoetens (as well as F. sphaericum and several\nunspecified species) from other Fusobacterium species appeared comparable to that\nobserved in Cetobacterium. As for F. naviforme, interestingly, we found that its ANI\nvalues to F. gonidiformans were 76%~81%, higher than the reported ~67% to Catonella\nmorbi. And, it also showed relatively close phylogenetic relationship to F.\ngonidiaformans.\nNevertheless, our data clearly aligned with the reviewer’s point regarding the\ntaxonomic placement of F. perfoetens, F. sphaericum, F. naviforme and several other\nunspecified species. Our analysis could contribute genome-scale evidence to this\nongoing discussion. However, the limited number of available high-quality genomes\nfor these taxa and non-Fusobacterium currently precludes the establishment of a robust,\ngenome-based genus-level boundary. Therefore, our study remains primarily focused\non establishing a species-level classification framework.\nWe have added these results and expanded the Discussion accordingly (lines 188-\n297, 358-367, 414-416, 441-444; Supplementary Fig. 4; Supplementary Data 3).\nIn line with this, we agree that a future comprehensive taxonomic evaluation at the\ngenus level will be essential as more genomes become available.\n8. Comment 8: To underpin their claims, studies like this should include a phylogenetic\ntree based on whole genome data and not solely a tree based on partial rpoB sequences.\nIn all figures and tables, it should be clearly indicated which species that are not\nformally valid according to DSMZ or other authoritative bodies.\nAnswer: We would like to clarify that we had indeed constructed a phylogenetic tree\nbased on whole genome data (Fig. 1C) and have additionally constructed multiples trees\nfor further analysis (Supplementary Figs. 2 and 4). In fact, the taxonomic framework\nand conclusions of our study were primarily founded on the whole-genome phylogeny\nand phylogeny of full-length marker genes, not on trees based on partial gene sequences.\nIn our analysis, the whole-genome phylogenetic tree was constructed from genome-\nwide SNPs and served as the key reference for evaluating species-level relationships\nand validating the ANI-based groupings. As for the phylogenetic analysis of marker\ngenes (gyrB, rpoB, znpA and 16S rRNA gene), we use all their full-length sequences,\nnot partial regions. The phylogeny of the partial region of rpoB (the V1 region) was\nanalysed only for comparative purposes and lineage classification in relation to our\nearlier study that employed FrpoB-seq (Bi et al. Nat Commun, 2022), and those results\nare mostly confined to the Supplementary Fig. 8.\nConcerning the annotation of species validity, we agree that indicating the formal\nrecognition status of species (e.g., according to DSMZ) is important. We have found\nthat adding such annotations to every figure and table significantly impacted layout\nclarity and readability. As a constructive alternative, we have added this information in\nTable 1.\n9. Comment 9: Line 315-316: Which community is this referring to? These matters are\nnot controversial in the bacterial taxonomy community or in the clinical microbiology\ncommunity. Also, this is a political statement that we do not support. Taxonomy is a\nrule-based system developed for a purpose. It cannot be based on a perceived consensus\nin a vaguely defined “community”. We divide bacteria into species because it is\nmeaningful to cluster together isolates with the same biological and pathogenic\nproperties. When taxonomy is changed, it is because we acknowledge that the previous\ngrouping has been erroneous, and that we are grouping together organisms that should\nnot be expected to share the same properties. Therefore, it is not rational to dispute\nchanges like the elevation of former subspecies into separate species. Also, such\nopinions stand in direct conflict with what is written in the sentence before: “...and\nprovides the taxonomic precision essential for advancing mechanistic and clinical\nresearch on Fusobacterium pathogenesis”.\nAnswer: Thank you for this important comment! It seems we failed to express our\nintended meaning clearly in the original manuscript, which led to a misunderstanding.\nWe sincerely apologise for the confusion caused.\nAs explained in our answer to your Major comment 2, the updated taxonomy, of\ncourse, could be well-understood and adopted within community specialised in\nbacterial taxonomy and/or clinical microbiology. Our use of the term “community”\nmainly referred to the much broader and expanding cross-disciplinary research\ncommunity now actively engaged in Fusobacterium (particular F. nucleatum) studies\ndue to its close links to diverse diseases (e.g., CRC). This includes researchers\n(scientists and medical professionals) from fields such as oncology, gastroenterology,\nimmunology, and cell biology, who may not have deep expertise in microbial\ntaxonomy/clinical microbiology.\nTo be absolutely clear, we do not suggest that taxonomic revisions should be based\non community consensus. We fully support the established taxonomic revisions. Our\ngenomic analyses robustly affirm these updates. Our point is that there remains a\npractical challenge in its consistent application across the broad community. The mixed\nnomenclature in literature (even now) and databases can create confusion, potentially\nobscuring the precise correlation between species and their specific biological or\nclinical relevance and even misguiding mechanistic studies. More critically, this\ninconsistency often fails to adequately account for the underlying genetic and\npathogenic diversity among species.\nThus, the “consensus” is on the consistent adoption and use of updated taxonomy\nwithin this expansive research landscape. The primary goal of our study is to bridge the\ngap between taxonomic consensus and its consistent application, by providing a clear\nframework and practical tools that enable the accurate and consistent application of the\nrevised taxonomy across all research contexts involving Fusobacterium. Therefore, our\nstatement that our work “provides the taxonomic precision essential for advancing\nmechanistic and clinical research” is entirely aligned with this goal. We have revised\nthe relevant sentences (lines 426-428) and the Introduction (lines 87-92) to clarify our\nintent as described above.\nOther comments:\n1. Lines 66-67: What does this sentence mean? You just stated that “Recent studies have\nfurther highlighted their distinctive roles in disease pathogenesis” (Line 54). There is\nnothing in the present taxonomy preventing such investigations, although people’s\nfailure to adhere to or use contemporary taxonomy might definitely obscure such\ncorrelations.\nAnswer: We thank the reviewer for this comment, which helps us clarify our intended\nmeaning. The reviewer is absolutely correct that the updated taxonomy itself does not\nprevent investigations into genetic and pathogenic diversity. Our point was not a\ncritique of the taxonomic rules, but an observation about the practical consequences of\nusing a single, broad name (e.g. the F. nucleatum) in rapidly expanding, cross-\ndisciplinary fields as mentioned above. Our intended meaning was that persistent mixed\nuse of outdated, ambiguous, or inconsistent nomenclature in the literature can obscure\nthe underlying genetic and pathogenic differences among its members. As the reviewer\nnoted, this is often a result of researchers, especially those from the broader community,\nnot yet aware of and adopting the updated taxonomy. We wish to highlight a key\nchallenge that if studies only report associations with \"F. nucleatum\" without species-\nlevel resolution, it becomes difficult to determine whether a reported disease link,\nmechanism, or biomarker is attributable to the overall F. nucleatum sensu lato or its\nspecific members. This lack of resolution can indeed lead to ambiguous clinical\ninterpretations and hinder mechanistic research. We have revised the sentence (lines 83-\n87).\n2. Line 67-68: This is not correct. It is true that the V3-V4 region commonly used for\nIllumina sequencing does not discriminate well between some of these species, but\nother regions like e.g. V1-V3 or the full gene can be used to discriminate between\nmost Fusobacterium species, including the currently accepted species in the Fusobacterium\nnucleatum group.\nAnswer: Thanks for pointing that out. We have revised the text to be more precise (line\n94). We agree that the full-length 16S rRNA gene or V1-V3 region can discriminate\nFusobacterium species. It should be noted, however, that even the full-length 16S rRNA\ngene may not provide sufficient resolution in some cases, as documented in previous\nstudies (e.g Kim, et al. J Clin Microbiol. 2009) and observed in our analysis.\n3. Line 69-70: This is not the case. There are several recent studies addressing this, but\nauthors are not citing them.\nAnswer: We have revised the text while revising the Introduction as mentioned above\n(lines 73-76, 84-87, 95-98).\n4. Lines 127-129: As mentioned above, a range of recent studies have flagged these\nwrongly annotated genomes before, but the authors are not citing them.\nAnswer: Thank you for reiterating this point regarding this text. As detailed in our\nanswer to your Major Comment 1, we have made revisions accordingly (lines 170-178;\nSupplementary Data 1).\n5. Lines 132-136: This has already been established by Sivertsen et al.\n(https://doi.org/10.1128/mbio.00941-25). This was the main purpose of their study.\nThey also provided the F. watanabei type strain whole genome used in the present study.\nAnswer: We acknowledge our oversight again of this very recent work (we were at the\nfinal stage of our manuscript preparation and did not read it in time. Apologise!). As\ndetailed in our response to your Major comment 1, we have revised the manuscript\naccordingly (lines 171-176, 56-73).\n6. Lines 139-140: This has also been pointed out by others (10.1007/s10482-023-\n01921-1) and is acknowledged by DSMZ.\nAnswer: Thank you very much for the comment! We have also corrected this while\nrevising the manuscript in response to your Major Comment 1 (lines 176-178).\n7. Lines 142-149: It is fine to conclude that some genomes are probably representatives\nof novel species. However, proposing new names based on this is not very useful, since\na valid description of novel species requires a type-strain submitted to at least two\nmicrobial culture collections.\nAnswer: Thank you for this thoughtful suggestion. We agree and have exercised\ncaution regarding this matter. As noted in the manuscript, among the six novel species\nwe had already used the provisional identifier “sp.” for four of them rather than\nproposing formal names. For the “F. bovis” with numerous available genomes, we have\nrevised the designation to Fusobacterium sp. bovis, as we do not hold the physical\nstrains. Regarding “F. heteroulcerans”, we had previously isolated three strains from\ncolorectal cancer tissues, which were sequenced in this study. One strain (THCT13E1)\nhas been designated as the type strain and deposited in two official culture collections\nin China, the China General Microbiological Culture Collection Center (CGMCC) and\nChina Center for Type Culture Collection (CCTCC) under the deposition numbers\nCGMCC 46909 and CCTCC M 20253012, respectively. The CGMCC and CCTCC are\nculture collections of the National Microbial Resource Center of China and also\ninternational depositary authorities under the Budapest Treaty. The CCTCC is also a\nmember of World Federation for Culture Collections. Due to current national biosafety\nand biosecurity regulations, we are advised that overseas deposition (e.g., to ATCC) is\nnot presently feasible. However, upon publication of this study, the both collections will\nmake this microbial resource publicly available, according to their policies. We have\nrevised the manuscript accordingly (Table 1 and all figures mentioning the species;\nlines 179-180, 297-298, 459-462).\n8. Lines 171-173: F. watanabei was not formerly Fna C1. F. watanabei was validly\ndescribed in 2021 and Fna C1 is a misconception from 2024.\nAnswer: Sorry for causing this confusion. Our intended meaning was “including the\nformerly misclassified Fna C1”. We have revised the sentence (line 250).\n9. Line 187: rpoB- and gyrA primers are not universal. They are Pan-Fusobacterial.\nAnswer: Thank you for pointing it out. Our intended meaning was that the primers are\nuniversal within Fusobacterium. We now realise that this term was potentially\nmisleading and apologise for any confusion caused. We have used the more accurate\nterm “degenerate” or “pan-Fusobacterium primers” throughout the text (lines 263, 265,\n498, 506), as suggested by the reviewer.\n10. Lines 202-205: rpoB is an excellent marker for Fusobacteria, but the authors are\ncomparing a 320 bp segment of rpoB with a 1500 bp segment of gyrB. That is not a\nvery fair comparison, and a PCR providing a longer amplicon for rpoB is appropriate.\nThe failure to discriminate between F. perfoetens and Fusobacterium sp. FSA-380-WT-\n3A is not a strong argument for using gyrB over rpoB for Fusobacterium spp., since as\ncommented above, the “F. perfoetens/sphaericum” clade most likely does not consist\nof Fusobacterium species but representatives of a novel genus.\nAnswer: We wish to clarify that our choice of gyrB (V1 region) over the rpoB (V1\nregion) was a practical design consideration only specific to our B&B method, not a\ngeneral statement on their taxonomic value. As demonstrated in our study, both full-\nlength gyrB and rpoB are excellent markers for broad phylogenetic purposes and can\nbe used individually or in combination.\nThe B&B method was developed for the practical classification of Fusobacterium\nspecies based on these two genes. The selection of target regions during its design was\nguided by the following considerations to balance accuracy, robustness, and cost-\neffectiveness for processing large numbers of isolates. 1) The chosen regions were\nflanked by conserved sequences, allowing the design of generalised primers that can\nsuccessfully amplify a target from a Fusobacterium isolate without prior knowledge of\nits sequence. 2) The selected regions were kept as short as feasible to reduce the\nnumbers of Sanger sequencing reactions required, which would be particularly cost-\neffective when handling a large amount of isolates. 3) We required a region where clear\nnucleotide identity thresholds for species discrimination could be established. The\n1500 bp segment of gyrB was chosen because a shorter, equally informative region could\nnot be identified within this gene. We employed the rpoB V1 region not only because it\nmet our requirement, but also its utility been assessed in our previous work (Bi et al. Nat\nCommun, 2022). Furthermore, our analysis detected no significant recombination\nsignal within this region (Supplementary Table 1), and its nucleotide diversity (π) was\ncomparable to that of the full-length gene (Supplementary Fig. 6).\nNaturally, using partial gene region would reduce the resolving power. Therefore, we\nevaluated how to reduce this limitation through a combined-use scheme, as detailed in\nFig. 6C, which illustrated the rational of B&B designing rather than comparing the\nperformance of the two regions. Regarding the specific case of F. perfoetens and\nFusobacterium sp. FSA-380-WT-3A, although the rpoB V1 failed to distinguish them,\nwithin the B&B framework, the gyrB region provided sufficient resolution, making the\nanalysis of rpoB V1 unnecessary in this instance. If F. perfoetens is indeed\nreclassified into another genus of Fusobacteriaceae in future studies, it would not\ninvalidate our method but suggest the potential to extend the B&B method’s\napplicability across the family.\nThus, this only reflects one specific, practical workflow within the B&B method, and\ndoes not imply that gyrB is superior to rpoB for all taxonomic applications. We have\nclarified this point in the revised manuscript (lines 283-285).\n11. Lines 252-258: As mentioned above; other references have already resolved this\n(https://doi.org/10.1128/mbio.00941-25).\nAnswer: We acknowledge this. As detailed in our earlier responses, we have\nincorporated these findings into our revised manuscript and have cited them accordingly.\nWe apologise again for the initial oversight.\n12. Lines 310-312: This paper only repeats what has already been confirmed by others\nand does not address any of the actual remaining issues within the taxonomy of the\nFusobacterium genus, in particular removal of likely non-fusobacterial species as\ndiscussed in major comment 7.\nAnswer: We thank the reviewer for this comment. As explained above, our goal was to\nestablish a unified, data-driven taxonomic framework integrating genomic,\nphylogenetic, and marker-based approaches for reproducible species-level\nidentification across Fusobacterium. We have carefully conducted a comprehensive,\nquantitative assessment of agreement among whole-genome phylogeny, ANI, and\nmarker-based phylogenies, using a large high-quality genome set for Fusobacterium,\nand designed practical solutions to solidify taxonomic accuracy and prevent future\nambiguity. Thus, our work certainly did not repeat what has already been confirmed.\nOn the contrary, we wish to address a next practical challenge, that is how to implement\nthe accurate Fusobacterium taxonomy consistently across diverse research and clinical\nsettings. Regarding the point on potentially non-fusobacterial species, as stated in our\ndetailed responses to you Major comments 6 & 7, rather than simply removing them,\nwe have now constructed an extended whole-genome phylogenic tree that included\nthese uncertain species alongside neighboring non-Fusobacterium genera (see\nSupplementary Fig. 4). This provided a broader phylogenetic context for evaluating\ntheir placement within the Fusobacteriaceae family. We have revised the text to better\nsummarise our study (lines 73-76, 95-98, 421-428).\n13. Line 312: what do the authors mean by “…for both individual strain and within\nmicrobiota”? Primers were not evaluated on microbiota samples and it cannot be known\nhow efficient/specific they will be for amplifying fusobacterial DNA from a complex\nbackground of other DNA.\nAnswer: We would like to clarify that the primers of the B&B method were designed\nand applied to pure isolates only, not for use on complex microbiota samples. The value\nof our work for improving Fusobacterium identification within microbiota studies lies\nin the flexible integration and refinement of existing bioinformatics tools, rather than\nin PCR-based detection from mixed samples as demonstrated in our study. For instance,\nwith comprehensive comparison analysis, we systematically evaluated, curated and\nrevised the species-level annotations of Fusobacterium SGBs in the MetaPhlAn\ndatabase. While MetaPhlAn is powerful for differentiating taxonomic units, certain\nspecies labels were ambiguous or incorrect in its results. Our work provided a reference\nessential for resolving these issues. Furthermore, as noted in the Discussion, all\ngenomes with revised species annotations in this study, as well as future genomes\nclassified using our framework, could serve as high-accuracy reference for\nmetagenomic profiling. Thus, our contribution is to provide a resolved taxonomic\nframework and validated reference data that can be flexibly integrated into existing\nbioinformatics tools. We believe this offers a practical solution to accurate species-level\nidentification of Fusobacterium in metagenomic studies, without the need to develop\nentirely new dedicated tools.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nAnswer: Thank you very much for the careful review. We have provided detailed\nresponses to the specific comments and questions raised in the reviewer reports.\nReviewer #3 (Remarks to the Author):\nSummary\nThe authors analyse 542 genomes of the genus Fusobacterium to better approach the\ntaxonomy of the genus. They conducted ANI analyses, a whole genome phylogeny, plus\nindividual gene phylogenies to get a sound idea of the phylogenetic relationships within\nthe genus. This study has the potential to add some insights into the taxonomy of this\ngenus. However, there are some major and minor issues the authors want to address in\na revised version.\nMajor comments\n1. To appreciate the quality of the genome assemblies, the authors want to run CheckM\non all the genomes and report completeness and contamination; this info should be\nadded to Supplementary Table 1. Ideally, for downstream analyses, they just want to\nconsider genomes with > 95% completeness and < 5% contamination. Also, the authors\nneed to provide the BioProject of the newly sequenced genomes.\nAnswer: Thank you for your thoughtful suggestion! We have assessed the quality of\nall genomes in our study using CheckM2 (Chklovski, et al. Nature Method, 2023), the\nlatest version of CheckM and applied the suggested inclusion criteria (> 95%\ncompleteness and < 5% contamination) (lines 436-438). The genome completeness and\ncontamination data have now been added to Supplementary Data 1 (for the RefSeq\ngenomes), Supplementary Data 7 (for newly sequenced and the GenBank genomes)\nand Supplementary Data 3 (for the representative genomes from other genera within\nFusobacteriaceae). Two RefSeq genomes failed to meet the quality thresholds and were\ntherefore excluded from subsequent analyses. We have re-executed the entire analysis\nworkflow with this updated RefSeq dataset and revised all relevant Results, Methods,\nfigures, and Supplementary Data accordingly. Importantly, all major conclusions of our\nstudy remain fully supported.\nPlease note that the BioProject accession number (PRJNA704481) of the newly\nsequenced genomes was already provided in the Data and resource availability section\nof the original manuscript and the related GenBank accession numbers of individual\ngenomes are also listed in Supplementary Data 7.\n2. Considering the concordance between the whole-genome phylogeny and the ANI-\nbased grouping, a formal topology test (KH-test, SH-test, AU-test, RELL, etc) is\ndesirable. So, to know if those strategies produce statistically significant different\ngroupings. Just from visually comparing the topologies, it is not possible to know\nwhether or not they are statistically different.\nAnswer: Thank you for very much for the constructive and insightful suggestion! We\nfully agree that a formal quantitative assessment of the concordance between the whole-\ngenome phylogeny and ANI-based grouping is crucial and would significantly\nstrengthen our study.\nWe have given careful consideration to the topology tests including KH-test, SH-test,\nAU-test or RELL. We recognise that these methods are powerful tools for comparing\nphylogenetic trees inferred from the same dataset under different models or parameters\nto choose the best-fitting tree for this dataset. In our case, however, we were comparing\ngroupings derived from fundamentally different data types, namely, a whole genome-\nbased alignment (used for phylogeny) and a genomic ANI matrix (used for clustering).\nThese data captured different evolutionary signals (substitution history vs. genome-\nwide identity), and thus their topologies and branch lengths are not expected to be\nstatistically equivalent in a branch-by-branch manner. For this reason, we considered\nthat direct application of these topology tests would not be methodologically\nappropriate here.\nAlternatively, to evaluate the topological concordance between the two approaches,\nwe employed ASTRAL (v5.7.8) to calculate a normalised quartet score (NQS), which\nquantified the proportion of shared evolutionary relationships (quartets) between two\ntrees, making it ideal for comparing trees built from different data sources. The analysis\nyielded an NQS of 0.904 between the whole-genome tree and the ANI-based\ndendrogram, indicating very strong concordance. We have included the result in Fig. 2\nand expanded the Method section accordingly (lines 559-562).\nSimilarly, we have also calculated the NQS values between the whole-genome tree\nand the phylogenic trees based on the 16S rRNA gene, rpoB, gyrB, and znpA, as detailed\nin our response to your Major comment 4. Notably, ASTRAL is particularly suitable\nhere, given its compatibility with multi-copy genes like the 16S rRNA gene.\n3. Important details are missing about the phylogenetic analyses. First, many details\nabout the whole genome phylogeny are needed. For instance, kSNP4 estimates\nparsimony, maximum likelihood, and neighbour-joining trees; which method did the\nauthors select? Please state all the different parameters employed in the analysis.\nImportantly, given the distorting effects of recombination, you want to run a phylogeny\njust on regions of the genome not affected by recombination. Did you use all the\nSNPs or just core SNPs? Second, re the individual gene markers, please state the DNA\nsubstitution model specified when running the gene phylogenies, maybe GTR+I+R?\nAnswer: We sincerely thank the reviewer for raising these crucial points regarding\nphylogenetic methodology.\nThe whole-genome tree was inferred using the parsimony method in kSNP4, which\nis recommended by the kSNP4 paper (Hall et al. Mol Biol Evol, 2023), as a simulation\nstudy (Hall et al. Cladistics, 2015) have shown that kSNP parsimony trees are the most\naccurate. All other parameters were at default setting.\nIndeed, recombination could influence topological accuracy. We understand that\nexcluding recombination regions is ideal for inferring a robust tree. However, in our\ncase, over half of the studied genomes were draft assemblies, which were fragmented\nand contained missing data, making large-scale, genome-wide recombination detection\nand filtering technically unreliable. To mitigate this concern, we have attempted to\nconstruct a tree using core SNPs via the ‘-core’ parameter in kSNP4, as suggested by\nthe reviewer. However, this approach failed, very likely due to the high nucleotide\ndiversity across species and the prevalence of draft genomes, which prevented the\nidentification of a universal set of core SNPs sufficient for tree reconstruction. As an\nalternative, we have constructed multiple trees (Supplementary Fig. 2) based on SNP\nloci occurring in at least 25%, 50%, and 75% of genomes (using the ‘-min_frac’ option).\nRemarkably, all resulting trees showed 0.96 – 0.99 (>0.998 if considering the species\nlevel) NQS to the primary tree (Supplementary Fig. 2). This high concordance indicated\nthat the core phylogenetic relationships were robust against potential noise introduced\nby recombination. We fully acknowledge not filtering for recombination as a limitation\nof the kSNP4-based phylogeny in this study and have explicitly point it out in the\nDiscussion. Importantly, the species-level clustering resolved by the kSNP4 tree was\nalso consistently supported by the ANI matrix and phylogenies derived from the high-\nresolution marker genes rpoB, gyrB, and znpA. Therefore, we consider that the major\ntaxonomic conclusions regarding species delineation remain robust despite this\nlimitation.\nAs for individual gene markers, we have re-inferred all gene phylogenies with IQ-\nTREE based on the updated genome dataset. The ModelFinder Plus (MFP) option was\nemployed to automatically select the best-fit nucleotide substitution model for each\ngene by minimising the Bayesian information criterion (BIC). The specific model\nselected for each marker is now clearly specified in the corresponding figure legends.\nWe have now provided a more comprehensive description in the Methods section\nand revised the Results, Discussion and figures accordingly (lines 483-488, 492-496,\n146-150, 408-412; Figs. 4 & 5 and Supplementary Fig. 2).\n4. Finally, considering their top two markers (gyrB and rpoB), the authors want to make\nsure these genes are good phylogenetic markers. I would suggest the authors run\ntopology tests (see Major comment 2) of these individual gene trees (and also a\nconcatenated alignment of the two genes) versus the whole genome phylogeny and\nensure that there are no significant differences. Also, they want to ensure that those two\ngenes do not have signals of recombination. Lastly, please report the nucleotide\ndiversity of these two genes and how it compares to the nucleotide diversity of the other\nmarkers presented in Supplementary Data 3.\nAnswer: This is also a very constructive and insightful suggestion, thanks!\nAs mentioned above, we have calculated the NQS between each individual gene tree\n(and a concatenated rpoB+gyrB tree, see Supplementary Fig. 5) and the whole genome\nphylogeny for topological concordance evaluation. The result indicated that gyrB (NQS\n= 0.905) and rpoB (NQS = 0.864) trees exhibited high topological agreement. Moreover,\nto statistically compare the performance across markers, we leveraged the distributions\nof NQS values generated from bootstrap replicates of each gene tree (see Statistics part\nin the Methods). A Kruskal-Wallis test followed by Dunn’s post-hoc tests revealed that\nboth gyrB and rpoB trees exhibited higher NQS than the 16S rRNA gene tree (both p <\n0.05; see Supplementary Fig. 7), further supporting their superior resolution.\nWe have examined the signals of recombination in all analysed genes with the PHI\ntest in PhiPack (version 1.1). No significant recombination signal was detected in these\ngenes except rpoB (Supplementary Fig. 1). A recent study has also reported\nrecombination in the rpoB gene of Fusobacterium (Molteni et al. Commun Biol, 2024).\nWe therefore acknowledged in the Discussion that rpoB tree topology should be\ninterpreted with caution regarding evolutionary trajectory assessment. Nevertheless, its\nhigh NQS confirmed that its major phylogenetic structure remains highly informative\nfor species-level clustering. In addition, we did not detect significant recombination\nsignal in the V1 region of rpoB employed by the B&B method.\nWe have also calculated the nucleotide diversity (π) of all analysed genes (Figs. 4&5\nand Supplementary Fig. 6). To compare nucleotide diversity (π) across markers, we\nemployed a bootstrap-based difference test. For each marker, bootstrap resampling\n(1,000 iterations) was performed (see Statistics part in the Methods). Crucially, whereas\nthe NQS comparison utilised bootstrapping to approximate the sampling distribution of\nthe statistic itself, the π comparison employed bootstrapping to evaluate the uncertainty\nof a parameter estimate. Therefore, for each maker pair, we calculated the distribution\nof differences by subtracting the bootstrap π values of one maker from those of the other.\nStatistical significance was determined by examining whether the 95% confidence\ninterval of this difference distribution contained zero. The corresponding two-tailed p\nvalue was calculated as twice the proportion of bootstrap differences on the smaller side\nof zero, followed by Benjamini-Hochberg correction for multiple testing. The\ncomparison results are provided in Supplementary Fig. 6.\nNote that due to the draft nature of certain genomes, not all marker gene sequences\nwere available for every genome in the RefSeq dataset. Specifically, while gyrB\nsequences were available for all genomes, rpoB sequences were available for 539\ngenomes, znpA for 436 genomes (also due to its natural absence in certain species), and\nat least one 16S rRNA gene copy for 414 genomes. Therefore, to ensure fair\ncomparisons, all NQS and π comparisons were performed only on the subset of\ngenomes that contained complete sequences for the markers being analysed.\nWe have added these results and methodological details in the manuscript (lines 496-\n498, 517-522, 559-577, 213-215, 220-243, 387-388; and the figures and table\nmentioned above).\nMinor comments\n1. Considering the title, if I were the authors, I would replace “genomic ANI” with just\n“ANI” — almost all ANI analyses involve genome sequences.\nAnswer: That is a thoughtful suggestion. We have done that (line 1). Thanks!\n2. Line 67: What do you mean by a high degree of homology? I guess you meant to say\na high level of similarity or identity.\nAnswer: Yes. We meant to say a high level of similarity or identity. We have revised\nour expression as “high level of similarity” (lines 92-93). Thanks for pointing that out!\n3. Line 101: revise grammar.\nAnswer: Corrected (line 134). Sorry for the error.\n4. y-labels on Figure 1A and some other panels in other figures (for instance, Figure\n5B) should be “Number of comparisons” or just “Frequency”\nAnswer: Indeed, that is the meaning we meant to deliver. We have revised the y-labels\nas “Frequency” in all relevant Figures.\n5. Lines 127-129: Previous studies using phylogenomic approaches have highlighted\nimportant cases of misclassification in other genera. See ref below\nhttps://pubmed.ncbi.nlm.nih.gov/31406982/\nhttps://pubmed.ncbi.nlm.nih.gov/39439205/\nhttps://pubmed.ncbi.nlm.nih.gov/34437546/&#x00A0;\nhttps://pubmed.ncbi.nlm.nih.gov/30386709/\nAnswer: Thank you for this very kind and helpful information! We have cited these\nimportant works in the Introduction of the revised manuscript (lines 100-102), to\nhighlight the importance of the phylogenomic approaches in taxonomic studies. Their\ninsights have also informed our methodological approach and the interpretation of our\nphylogenetic results.\nLine 280: “The gyrB gene” reads better.\nAnswer: Revised as suggested (line 385). Thanks!\nReviewer #4 (Remarks to the Author):\nIn this manuscript, Bi et al provide quite a comprehensive genus-wide analysis that\nintegrates ANI-based and phylogenetic approaches to clarify some longstanding issues\nthat have arisen within Fusobacterium taxonomy and nomenclature. They further\nintroduce a practical dual-marker (gyrB and rpoB) identification method and\ndemonstrate how it can be integrated with GTDB and MetaPhlAn frameworks. The\nanalyses are extensive, technically sound, and clearly the product of careful\ncomparative work across what has been a challenging genus. The effort to unify\ngenomic, phylogenetic, and marker-based approaches into a coherent framework will\nrepresent a substantial step forward for reproducible species-level identification across\nFusobacterium. This reviewer particularly appreciates the authors’ effort to present a\nbalanced, data-driven treatment of what is often a contentious subject. This reviewer\nalso agrees that the novel markers outlined here could contribute to overcoming\nchallenges faced with traditional 16S rRNA sequencing efforts to elaborate on\nFusobacterium species relevant to human cancers.\nNevertheless, I do have concerns about some of the proposed updates, and along with\na single major point, provide some constructive feedback that may improve readability\nand decrease resistance within the field for adoption of the authors proposed taxonomy\nand nomenclature adjustments.\nMajor comment: Against a double meaning for F.n sensu lato.\nIt appears that the authors are proposing to change the nature of what Fusobacterium\nnucleatum sensu lato (Fn s.l.) means (at least as understood by this reviewer and by\nmany colleagues working in this area). Several statements in the manuscript are\nambiguous in this regard and warrant clarification, as they risk introducing additional\nsignificant confusion into an already complex and historically inconsistent\nnomenclatural landscape. Specifically, the manuscript seems to redefine F. nucleatum\nsensu lato in a way that excludes F. animalis and F. watanabei, whereas in the broader\nliterature, the term has consistently referred to the entire F. nucleatum complex,\n(including these lineages).\nThese sentences include:\n- Lines 129–138, “the two F. nucleatum subsp. animalis clades (Fna C1 and Fna C2)\nconstitute distinct species… which did not justify the species status of F. nucleatum\n(sensu lato)”,\n- Lines 252–255, which describe the current subspecies/clade designations under “F.\nnucleatum (sensu lato)” as “taxonomically invalid”,\n- Table 2, which presents F. animalis and F. watanabei as reclassified species without\nreference to their origin within the F. nucleatum sensu lato complex.\nThis is in contrast to the current established usage of F. nucleatum sensu lato in the field\n(eg. Kook et al., 2017; Ma et al 2023, Krieger et al., 2024; Zepeda-Rivera et al., 2025)\nencompasses the four canonical F. nucleatum subspecies (nucleatum, polymorphum,\nvincentii, animalis) and their derivative lineages, forming a coherent monophyletic\ngroup sharing ~90–94 % ANI.\nIt is also important to note that the sensu lato designation functions not as a formal\nspecies rank but as a conceptual and practical umbrella, and is useful to preserve\ninterpretive continuity across genomic, ecological, and clinical research where\nsubspecies resolution was unavailable.\nThis current framework is also relatively well established, as it provides an inclusive\nand pragmatic way to refer to the entire F. nucleatum complex by linking historical\nculture-based and 16S-based studies with more recent updated genome-resolved\nanalyses. It is also a default interpretive construct; even as individual lineages are\nrefined to species rank.\nCould the authors please carefully consider and explicitly outline what the benefit to\nthe field would be in creating a new interpretation of F. nucleatum sensu lato that\nexcludes F. animalis and F. watanabei, when there is already an established and widely\nunderstood F. nucleatum sensu lato framework that includes these lineages?\n- What is the practical or conceptual upside of redefining an existing and historically\nconsistent umbrella term in this way? If the intention is to describe or analyze a subset\nof species within the F. nucleatum complex, would it not be clearer and less disruptive\nto the existing literature to introduce a new, unambiguous term for this more restricted\ngrouping, rather than altering the meaning of F. nucleatum sensu lato?\n- Something like “F. nucleatum core group”?, or similar, where is unlikely to collide\nwith the established framework. It would be most beneficial if this could be then\nincluded in the figures, where the F.n sensu lato group and this new tighter group are\nall clearly highlighted in the main figures for future references. (so Fn sensu lato, Fn.\ncore group, and Fn sensu stricto would all be in use, but distinguishable)\n- This reviewer would strongly advocate for such an alternative choice as currently it\nrisks creating parallel usages of the same term with different inclusivity. i.e: F.\nnucleatum sensu lato = the four canonical subspecies nucleatum, polymorphum,\nvincentii, and animalis and their derivative lineages (including F. watanabei)…….but\nalso F.nucleatum sensu lato = (canonical subspecies nucleatum, polymorphum,\nvincentii).\n- Further, this redefinition raises another issue. F. animalis has long been considered a\nbona fide member of the F. nucleatum sensu lato and already included in this way within\nhundreds of studies linking Fusobacterium diversity to disease. Reassigning F. animalis\n(and F. watanabei) outside of F. nucleatum sensu lato without a clear taxonomic or\ntranslational rationale risks severing continuity with a decade of literature that used F.\nnucleatum sensu lato precisely to unify these lineages under a single, interpretable\nconstruct.\nAt present, this reviewer cannot discern any clear benefit or added utility to the field in\nredefining F. nucleatum sensu lato in this new way. Doing so may increase confusion\namong researchers, clinicians, and data curators who have relied on the inclusive sensu\nlato framework for consistent comparative and meta-analytical interpretation.\nIf the authors believe there is genuine conceptual or practical value in excluding F.\nanimalis and F. watanabei from F. nucleatum sensu lato, could they please elaborate\nand articulate that rationale explicitly in the manuscript (what new clarity does this\nbring, and to whom?) Otherwise, I apologize, but the proposed redefinition appears to\nintroduce new complexity without a tangible scientific or translational gain.\nAnswer: We sincerely thank the reviewer for their appreciation of our work and for this\nexceptionally thorough, careful, and thoughtful comment!!! Clearly, our phrasing in the\nmanuscript caused an unintended and significant misunderstanding, and we deeply\napologise for this.\nWe would like to clarify that we fully acknowledge and consistently support the\nwidely-recognised concept of F. nucleatum sensu lato and had absolutely no intention\nto redefine it. We have comprehensively revised the entire manuscript to eliminate this\nconfusion (e.g. lines 171-174 and Table 2). We have clearly stated that F. nucleatum\nsensu lato includes the four canonical subspecies nucleatum, polymorphum, vincentii,\nand animalis and their derivative lineage F. watanabei. Also, as our own results confirm,\nthere is no conceptual or practical value in excluding F. animalis and F. watanabei from\nF. nucleatum sensu lato. We are truly sorry that our original phrasing and presentation\ninadvertently created ambiguity.\nWe are profoundly grateful to the reviewer for catching this critical issue, which\nallows us to improve the clarity of our manuscript. Beyond correcting this specific point,\nall the reviewer’s comprehensive perspective and insightful suggestions have also\nstrengthened the overall impact of our study. Their guidance greatly helped us address\nconcerns from other reviewers more effectively!\nMinor comments:\n1. Lines 252–255 “Critically, our evidence supports the argument that the current\nsubspecies/clade designations under ‘F. nucleatum’ (sensu lato) are taxonomically\ninvalid.\nThe phrasing here could be perceived as a little aggressive, implying that prior\ntaxonomic frameworks are incorrect or invalid, which is inconsistent with taxonomic\nfreedom and the evolving and interpretive nature of bacterial taxonomy noted early in\nthe discussion by the authors. Names and classifications reflect the data and consensus\navailable at the time, but do not become invalid with the emergence of new evidence.\nThe authors data clearly demonstrates that the historical F. nucleatum sensu lato\nsubspecies exhibit genomic and phenotypic divergence consistent with species-level\ndistinctions. However, rather than characterizing existing designations as\n“taxonomically invalid,” a more accurate and constructive framing might be that these\ndesignations may no longer adequately capture the observed diversity and that the\nauthors’ analyses support recognition of these lineages as distinct species under a\nrevised framework. This would keep the scientific strength of the findings but with a\nmore balanced tone.\nAnswer: We agree with the reviewer. Thank you so much for this kind and thoughtful\nsuggestion! We have realised that the expression was unnecessarily strong and did not\nacknowledge their historical context and utility. We have revised the phrasing in the\nmanuscript to adopt a more accurate and constructive tone as suggested (lines 340-344):\n2. Line 62: Notably, the status of Fna C1 remains inconclusive, as it was recently\nregrouped with F. vincentii in a metagenomic study\nSome additional context may be useful for this particularly unusual lineage that has\nquickly changed. The KCOM strains representing the majority of the lineage Fna C1\n(21 of 24) were original annotated as Fna/Fa isolates by the Korean Collection for Oral\nMicrobiology using 16S rRNA profiling (partial) and released publicly between April\n2020 and Feb 2021 at NCBI. They remained annotated as such within the culture\ncollection, until Zepeda et al noted that they were distinct from Fna ATCC 51191\nlineage, and they assigned them as Fna C1 and Fna C2. Ma et al also assigned a strain\nof this lineage as FNU (unique) around the same time based on their noted distinctions\nin a pangenome study. F. watanabei isolates were formally named after the efforts of\nTomida et al, but remained disconnected from the other strains (and 13-08-02) due to a\nlack of whole genome information. The GTDB still refers to this lineage as F.\nnucleatum_J, although recent work by Tomida groups and this work by Bi et al appear\nto verify that these different names all refer to a single lineage.\nIn this introduction, the authors note that Piccinno G et al determined this lineage to be\nclustered with Fnv. It may be useful to readers for Bi et al to elaborate on why this\nlineage appears to fluctuate across studies, inherently based on the differences in the\ndatasets and methods used (16s rRNA vs partial genome vs whole genome vs SGBs of\nmetagenomes)?\nAnswer: Thank you very much for this very constructive comment and for providing\nsuch a detailed context regarding Fna C1. This information is particularly useful for\nour revision and for keeping us maintaining a balanced tone! We have revised the text\nto include more details (lines 56-76). It is indeed true that these studies utiliesed\ndifferent datasets and methods and mostly confined to a context only including F.\nnucleatum sensu lato. For instance, Piccinno G et al actually had attempted to clarify\nthe designation of Fna C1 through phylogenetic analysis of SGBs. However, their\nanalysis was confined to F. nucleatum sensu lato rather than encompassing the entire\ngenus. We believe this limited scope prevented them from obtaining a global\nphylogenetic view essential for correct assignment of this taxon.\n3. Line 133: “As Fna C2 was represented by the F. animalis 134 anchor strain (KCOM\n1325)”\nCould the authors elaborate on why KCOM 1325 was chosen as the anchor strain, and\nnot ATCC 51191 or 7.1?\nAnswer: Thank you for raising this point. Our choice of KCOM 1325 was based on a\nstandardised, database-driven approach to ensure methodological consistency and\nreproducibility across the genus-wide analysis. As mentioned in the Methods, we used\nthe RefSeq-defined reference genome for each well-established species as its\ntaxonomic anchor; and KCOM 1325 is formally assigned as the reference genome for\nF. animalis. The criteria for RefSeq reference genome selection are detailed in\nhttps://www.ncbi.nlm.nih.gov/datasets/docs/v2/policies-annotation/genome-\nprocessing/reference-selection/. By doing so, we intended to provide a consistent and\nobjective basis for species assignment.\nWe fully acknowledge the importance of other key F. animalis strains, such as ATCC\n51191 and 7.1, which have been previously characterised and the ATCC 51191 is also\nthe type strain of F. animalis. Our genomic analysis indicated that these strains form a\ncoherent cluster along with KCOM 1325, confirming their classification within the\nsame species. We have revised the manuscript to highlight ATCC 51191 and 7.1 as\nimportant and reliable representative strains of F. animalis, while maintaining KCOM\n1325 as the reference anchor for our analytical pipeline (lines 188-194).\n4. Line 136: “the F. watanabei anchor (CCUG74246T) and herein reclassified into F.\n136 watanabei.”\nIt appears authors of the Fna C1 study have made efforts with Tomida et al to provide\nclarity on where the Fna C1/F.nucleatum subspecies unique/F. watanabei/ 13-08-02\nlineages reside with respect to one another - doi/10.1128/mra.00132-25. Could Bi et al\ninclude this citation also.\nAnswer: Thank you very much for this valuable suggestion. We have now included this\ncitation and revised the text, particularly in the Introduction, to provide a more\ncomprehensive review of this matter (lines 56-76). This revision is also related to our\nresponse to your Minor comment 2.\n5. Line 89: developed standardised species identification approaches for strains and\nwithin microbiota.\n(is the ‘and”a misprint?)\nAnswer: Thank you for your careful reading and sorry for causing the confusion. Our\nintended meaning is that we have developed standardised approaches for species\nidentification of individual strains, while the integration of the revised taxonomy with\nexisting tools could enable precise profiling of Fusobacterium species within complex\nmicrobiota. We have revised the sentence to read more clearly (line 118).\n6. Line 140: “Severn strains”\nAnswer: Sorry for the typo. It should be “seven strains”. Please note that due to the\nexclusion of two genomes following CheckM2 quality assessment (as detailed in\nresponse to Reviewer #3’s Major comment 1), the number has now been updated from\nseven to six in the revised manuscript (line 186).\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nAnswer: Thank you very much for the careful review. We have provided detailed\nresponses to the specific comments and questions raised in the reviewer reports.\nReviewer #1 (Remarks to the Author):\nThe manuscript has been updated with regards to the most recent literature, and the\nauthors have appropriately addressed unresolved matters in the Fuosbacterium genus\noutside of the Fusobacterium nucleatum group. However, despite our criticism of the\nconcept of a universal genus-wide species boundary this has been kept as a major part\nof the manuscript. We still don’t think it is a good idea to contribute legitimizing in\nscientific literature a concept that is principally and practically of little value and\nsuggest that the emphasis on this concept should be reduced. This could also help\nshorten the manuscript.\nLines 102-103: …,genus-wide assessments remain scarce and a robust, universal\nspecies boundary for the entire genus remains undefined.\nWe do not understand why the authors keep insisting that this is a problem, when they\napparently agree, based on their response to our former comment 4, that each species\nhas its own independent intraspecies variability defined by the actual biological\nvariation within that species, and its own distances to its own closest neighboring\nspecies. Those are the only relevant demarcations for that species. A genus-wide\nspecies boundary will never have priority over standard requirements for describing\nnew species but will need to be adjusted when new species are described.\nLines 331-332: This study established a unified taxonomic framework for the genus\nFusobacterium by defining a standardised ANI boundary for species delineation…\nPlease refer to our comment above. You cannot claim this when each species has its\nown boundaries regardless of such “genus-wide” boundaries and that in the occasion\nthat a new species is described it will be described based on current recommendations\nfrom the IJSEM regardless of any deviation from your “genus-wide” boundaries in\nwhich case they will need to be adjusted to encompass the new valid species. When\nyour write in your response that you “do not expect the ANI gap boundaries to be\nfixed and they should be iteratively updated with new genomic data, as now discussed\nin our revised manuscript”, how can you claim this to be a taxonomic framework for\nthe genus? Your genus specific boundary is simply an observation based on currently\ndescribed species of little biological or practical relevance.\nAnswer: Thank you for the thoughtful comments. ANI is an objective and widely\nused genetic similarity measure in bacterial taxonomy, including many previous\nstudies on Fusobacterium. One of its key strengths is its ability to reveal clear genetic\ndiscontinuities that can serve as robust species boundaries. In this study, we identified\na natural ANI gap within the genus Fusobacterium through distribution and\nfirst‑derivative analyses, without any prior taxonomic assumption. Importantly, we\nfurther showed that groupings defined by this gap were fully concordant with\nwhole‑genome phylogeny and clearly discriminated all currently recognised species.\nThis consistency indicates that the observed gap reflects a speciation‑associated\ngenetic discontinuity inherent to the genus. As shown in Supplementary Fig. 3, this\ngap represents a shared region between intra- and interspecies ANI distributions\nacross all established species. Critically, species with relatively large numbers of\ngenomes available consistently exhibited intraspecies and interspecies boundaries that\nclosely align with the upper and lower limits of this gap, respectively, suggesting a\nconverging trend. Thus, this gap can serve as a practical and a “standardised”\nreference for species delineation within Fusobacterium. In contrast, if we were to\ndefine species-specific thresholds individually, a newly sequenced genome with an\nunknown closest neighbour would require multiple rounds of comparisons, and new\nstandards would need to be repeatedly established for each novel species.\nWe also would like to clarify that the dynamic nature we refer to lies in the upper\nand lower limits of the ANI gap themselves. The currently defined limits were based\non the present genome set. Large-scale genomic analyses usually are a key approach\nto infer such limits, and we have additionally assessed their stability using a\nrarefaction curve-like analysis. Future refinement of these limits does not invalidate\nthe existence of a genetic discontinuity; rather, it improves its precision. Moreover,\nfuture updates to the gap limits will be driven by the emergence of borderline cases,\nnot merely by the description of additional new species that fall within the existing\ngap.\nOur genus‑wide approach does not contradict the fact that each species has its own\nindependent intraspecies variability, nor do we claim that the ANI gap defines the\ndegree of intraspecies diversity. Rather, we emphasise that a genus-wide perspective\nis essential, particularly in light of the historical instability in annotating lineages such\nas Fna C1, which largely stemmed from studies confined to F. nucleatum sensu lato.\nIn line with the journal’s guidelines, we have removed the term “universal” and\nsome unnecessary use of “standardised”, throughout the manuscript to avoid any\nover‑interpretation of our findings.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers who\nco-review manuscripts.\nAnswer: Thank you very much for the careful review.\nReviewer #3 (Remarks to the Author):\nThe authors have done a good job addressing my concerns. I have nothing else to add;\ncongratulations on their work.\nAnswer: Thank you very much for your appreciation and the help in improving our\nmanuscript.\nReviewer #4 (Remarks to the Author):\nThis revised manuscript by Bi et al shows a high level of care and professionalism in\naddressing complex and evolving issues in Fusobacterium taxonomy and\nnomenclature. The update text has clearly distinguished historical usage from current\nspecies assignments, (updates in Table 1 and 2 are quite useful) integrated more recent\ngenome-based studies and techniques appropriately and explicitly acknowledged\nareas where taxonomic boundaries remain unresolved across Fusobacterium lineages\n(and may be subject to further updates in the future). My previous comments have\nbeen addressed, and I am broadly supportive of this manuscript, I believe it will be a\nvaluable and clarifying addition to the field. Great work!\nAnswer: Thank you very much for your appreciation and the help in improving our\nmanuscript."}]

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