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4 nar Nucleic Acids Research Nucleic Acids Res Oxford University Press PMC8728215 8728215 8728215 34520557 10.1093/nar/gkab776 GTDB: an ongoing census of bacterial and archaeal diversity through a phylogenetically consistent, rank normalized and complete genome-based taxonomy Parks Donovan H 1 ✉ Chuvochina Maria 2 Rinke Christian 3 Mussig Aaron J 4 Chaumeil Pierre-Alain 5 Hugenholtz Philip 6 ✉ 1 The University of Queensland, School of Chemistry and Molecular Biosciences, Australian Centre for Ecogenomics, QLD 4072, Australia 2 The University of Queensland, School of Chemistry and Molecular Biosciences, Australian Centre for Ecogenomics, QLD 4072, Australia 3 The University of Queensland, School of Chemistry and Molecular Biosciences, Australian Centre for Ecogenomics, QLD 4072, Australia 4 The University of Queensland, School of Chemistry and Molecular Biosciences, Australian Centre for Ecogenomics, QLD 4072, Australia 5 The University of Queensland, School of Chemistry and Molecular Biosciences, Australian Centre for Ecogenomics, QLD 4072, Australia 6 The University of Queensland, School of Chemistry and Molecular Biosciences, Australian Centre for Ecogenomics, QLD 4072, Australia ✉ To whom correspondence should be addressed. Email: donovan.parks@gmail.com
✉ Correspondence may also be addressed to Philip Hugenholtz. Email: p.hugenholtz@uq.edu.au
14 9 2021 50 D1 D785 D785–D794 5 1 2022 © The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/4.0/ ), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract
The Genome Taxonomy Database (GTDB; https://gtdb.ecogenomic.org ) provides a phylogenetically consistent and rank normalized genome-based taxonomy for prokaryotic genomes sourced from the NCBI Assembly database. GTDB R06-RS202 spans 254 090 bacterial and 4316 archaeal genomes, a 270% increase since the introduction of the GTDB in November, 2017. These genomes are organized into 45 555 bacterial and 2339 archaeal species clusters which is a 200% increase since the integration of species clusters into the GTDB in June, 2019. Here, we explore prokaryotic diversity from the perspective of the GTDB and highlight the importance of metagenome-assembled genomes in expanding available genomic representation. We also discuss improvements to the GTDB website which allow tracking of taxonomic changes, easy assessment of genome assembly quality, and identification of genomes assembled from type material or used as species representatives. Methodological updates and policy changes made since the inception of the GTDB are then described along with the procedure used to update species clusters in the GTDB. claim_typeWe conclude with a discussion on the use of average nucleotide identities as a pragmatic approach for delineating prokaryotic species.
status released display-pdf yes is-olf no is-manuscript no is-preprint no is-journal-matter no is-scanned no is-retracted no Accepted 2021 Aug 28; Revised 2021 Aug 18; Received 2021 Jul 27; Collection date 2022 Jan 7.
INTRODUCTION
The Genome Taxonomy Database (GTDB) was developed to provide a phylogenetically consistent bacterial and archaeal taxonomy which can accommodate isolate genomes and the tens of thousands of metagenome-assembled genomes (MAGs) now being obtained from environmental and clinical samples ( 1–3 ). contributionmechanismssensescontent_tags↗In order to taxonomically organise this large and growing genome dataset, the GTDB uses relative evolutionary divergence (RED) to delineate higher-rank taxa and average nucleotide identity (ANI) to delineate species clusters ( 4 , 5 ). Use of these quantitative criteria for circumscribing taxa allows for automated classification of new genomes ( 6 ), ensures all genomes are classified from species to domain, and normalizes the definition of taxonomic ranks across the bacterial and archaeal domains.
GTDB builds upon a number of existing public resources in order to provide a taxonomic resource that reflects recently proposed taxa, changes in taxonomic opinion, and the wealth of publicly available genomes. The NCBI Taxonomy database ( 7 ) aids in the discovery of newly proposed taxa, provides initial species assignments and specifies co-identical strain identifiers for genomes in the NCBI Assembly database ( 8 ). We currently use the NCBI Assembly database as the sole genome repository for the GTDB as it is a member of the International Nucleotide Sequence Database Collaboration (INSDC; 9 ) which ensures it also contains genomes submitted to DDBJ ( 10 ) and EMBL-EBI ( 11 ). claim_relationsThe LPSN (List of Prokaryotic names with Standing in Nomenclature) database ( 12 ) is used to establish co-identical strain identifiers for the type strains of species and subspecies, the types of higher-rank taxa, and the nomenclatural status of newly proposed or reclassified taxa. The Living Tree Project ( 13 ) is used to classify 16S rRNA sequences and help resolve ambiguity in regard to the correct classification of genomes.
The latest version of the GTDB is R06-RS202 which was released in April, 2021. This is the sixth release (i.e. R06) of the GTDB since its inception in November 2017 and comprises genomes in the NCBI Assembly database as of September 2020, i.e. the release date of RefSeq 202 ( 14 ).
RESOURCE CONTENT
Growth of GTDB
The number of genomes in the GTDB has grown by over 270% since its inception in November 2017 (Figure 1A and B ; Supplementary Table S1 ). Perhaps surprisingly, bacterial isolates account for the majority of this growth rather than MAGs (or single amplified genomes; SAGs) despite recent metagenomic studies recovering tens of thousands of MAGs ( 2 , 3 , 15 ). This is a result of both the large numbers of genomes from human pathogens deposited in INSDC repositories ( Supplementary Table S2 ) and many metagenomic studies providing only a representative MAG per operational species cluster or no MAGs to an INSDC repository. Unfortunately, this means INSDC repositories are missing much of the strain diversity which has recently been discovered. Interestingly, this pattern is not observed for archaea where MAGs are responsible for the majority of additional genomes (Figure 1B ), perhaps reflecting both challenges in cultivating archaeal strains and the lack of archaeal human pathogens.
Figure 1. Growth of the GTDB since its inception in November 2017. ( A , B ) Number of bacterial and archaeal isolates, MAGs, and SAGs in the GTDB along with the total number of genomes. Archaea were introduced into the GTDB starting with R03-RS86 in August, 2018. ( C , D ) Percent growth in the number of bacterial and archaeal taxa in the GTDB. ( E , F ) Proportion of bacterial and archaeal taxa at each taxonomic rank in GTDB R06-RS202 comprised exclusively of environmental genomes (MAGs and/or SAGs), exclusively of isolates, or both isolate and environmental genomes. For comparison, the proportion of isolate and environmental genomes is shown in the right bar plot.
Growth in the number of genomes has coincided with a large increase in the number of bacterial (513%) and archaeal (459%) species in GTDB (Figure 1C and D ; Supplementary Table S1 ). claim_relationsImportantly, we implemented an ANI-based method for delineating species starting with R04-RS89 (June 2019) which allows all genomes to be classified at the rank of species and provides a uniform estimate of species-level diversity within the GTDB ( 5 ). As expected, growth in the number of higher-rank taxa has been more modest (Figure 1C and D ) though still appreciable, with the number of bacterial and archaeal genera increasing by 222% and 193%, respectively.
mechanismslimitationsconcepts↗concepts↗concept_edgescontent_tagsUnlike the number of bacterial genomes, MAGs account for the majority of taxonomic diversity covered by GTDB R06-RS202 (Figure 1E and F ; Supplementary Table S1 ). Over 50% of bacterial taxa, regardless of rank, consist exclusively of MAGs and/or SAGs despite MAGs (50 669 genomes) and SAGs (745 genomes) representing only 20.2% of the 254 090 bacterial genomes. Similarly, nearly 50% of all archaeal taxa, regardless of rank, consist exclusively of MAGs and/or SAGs with over 70% of archaeal species, genera, families, and orders lacking an available cultured representative (Figure 1F ).
Changes to the GTDB Website
There have been a number of additional features and improvements to the GTDB website since its inception in November 2017, which we illustrate here using the Enterocloster bolteae genome GCF_002234575.2 (Figure 2 ). Each genome has an associated GTDB Genome page (Figure 2A – D ) indicating taxonomic, nomenclatural, and assembly quality information. A badge system is used to provide a quick assessment of overall genome quality and indicate that GCF_002234575.2 is a high-quality (HQ) isolate genome assembly which contains the 5S, 16S, and 23S rRNA genes along with genes for all 20 tRNAs (Figure 2A ). Additional assembly statistics such as CheckM completeness and contamination estimates ( 16 ), number of contigs, N50, genome size, protein count, and GC content are also provided on the Genome page (data not shown). A link to the NCBI Assembly page for GCF_002234575.2 is provided along with a link to the LPSN page for E. bolteae as this genome is recognized as being assembled from the type strain of the species according to LPSN (Figure 2B ). Both GTDB and NCBI classifications are provided to allow for easy comparison between these taxonomic resources which have identical classifications for GCF_002234575.2 (Figure 2C ). GCF_002234575.2 is annotated as being assembled from the strain ATCC BAA-613 at NCBI and considered to be assembled from the type strain of the species within the GTDB framework based on LPSN nomenclatural information for this species (Figure 2C ). This genome has also been selected as the GTDB representative of the E. bolteae species cluster which comprises 24 genomes in GTDB R06-RS202 (Figure 2C ).
Figure 2. Taxonomic, nomenclatural, and assembly quality information provided for individual genomes. ( A ) NCBI genome assembly accession and GTDB quality badges associated with this genome. Hovering over a tag provides information about the criteria used to establish the tag. ( B ) An external link is always provided to the NCBI Assembly page of the genome as all GTDB genomes are sourced from NCBI. A link is also provided to an LPSN species page when a genome is established to be assembled from the type strain of a species based on LPSN nomenclatural information. ( C ) GTDB and NCBI classifications for this genome along with its strain identifiers, nomenclatural status at GTDB (i.e. type strain of the species), and GTDB species representative status. GTDB taxa link to their corresponding position in the GTDB Taxonomy Tree (i.e. Figure 2F ) while NCBI taxa link to NCBI Taxonomy Browser pages. Each genome also links to a table indicating all genomes in the same GTDB species cluster. ( D ) GTDB classification of the genome in each GTDB release. GTDB taxa link to their corresponding Taxon History page (i.e. Figure 2E ). ( E ) GTDB Taxon History view for genomes classified as Enterocloster bolteae indicating this species was reclassified from Clostridium_M to Enterocloster in GTDB R95. Numbers in parenthesis indicate the number of genomes assigned to a taxon. The Not Present label indicates genomes that were not available at the time of a GTDB release or failed the GTDB quality-control criteria used for the release, and thus had no GTDB classification. ( F ) GTDB Taxonomy Tree which provides a hierarchical exploration of the GTDB taxonomy and indicates nomenclatural type information, genomes selected as GTDB representatives, and Latin names in the GTDB which remain to be validated. Genomes link to their corresponding GTDB Genome page (i.e. A–D).
We provide two avenues for exploring how GTDB classifications have changed between releases. At the bottom of each Genome page, the classification of the genome is provided for each GTDB release (Figure 2D ). For example, GCF_002234575.2 was reclassified from Clostridium_M bolteae to E. bolteae in GTDB R95 in accordance with a new taxonomic opinion ( 17 ). Changes in GTDB classifications and their relationship to the NCBI taxonomy can also be explored at the taxon level using the GTDB Taxon History tool (Figure 2E ). This visualization illustrates that E. bolteae was reclassified from Clostridium_M to Enterocloster in GTDB R05-RS95, that the number of genomes assigned to this species has steadily increased between releases, and that there are a number of incongruent classifications at NCBI including one genome assigned as Clostridium bolteae and another as Hungatella hathewayi .
The GTDB taxonomy can be explored hierarchically using the Taxonomy Tree tool (Figure 2F ) or as an alphabetically sortable table (not shown). The tree view provides an approximation of the reference phylogenetic tree where taxa within a group are listed alphabetically with Latin names listed ahead of alphanumeric placeholder names. Type species of genera and type strains of species and subspecies are highlighted by color-coding. For example, there are three genomes assembled from the type strain of E. bolteae with the genome selected to represent this species in GTDB marked with an asterisk (Figure 2F ) ( 5 ). GTDB includes over 400 Latin names that have been assigned by the GTDB curators but are still to be formally proposed (see Policy Changes ). These are identified in the Taxonomy Tree with hashes and can also be marked and downloaded from the table view.
Searching functionality on the GTDB website has been expanded to allow for searches that are restricted to the GTDB taxonomy, NCBI taxonomy, NCBI organism name, or NCBI genome ID. This is useful for identifying genomes assigned to a specific GTDB or NCBI taxon. For example, this can be used to identify the 35 Enterocloster clostridioformis genomes in GTDB R06-RS202 without retrieving the two additional genomes with this classification at NCBI which are considered misclassified E. bolteae genomes in the GTDB (Figure 2E ). An advanced search feature has been added to the GTDB website to allow specific subsets of genomes to be identified across all GTDB metadata fields. For example, this can be used to identify the 94 genomes in GTDB R06-RS202 which are classified as Enterocloster and have an estimated completeness >90%, estimated contamination <10%, and contain at least one 16S rRNA gene.
A primary goal of taxonomy is to aid in scientific communication. With this in mind, all GTDB web tools produce URLs which allow specific results to be communicated between researchers. The following URLs produce the Genome page, Taxonomy Tree view, Taxon History view, and described Advanced search results for E. bolteae (Figure 2 ):
Genome page: https://gtdb.ecogenomic.org/genomes?gid=GCF_002234575.2
Taxonomy Tree : https://gtdb.ecogenomic.org/tree?r=s__Enterocloster%20bolteae
Taxon History : https://gtdb.ecogenomic.org/taxon_history/?from=R89&to=NCBI&query=s__Enterocloster%20bolteae
Advanced Search : https://gtdb.ecogenomic.org/advanced?1=MX4yfmdfX0VudGVyb2Nsb3N0ZXI∼&3=Nn4xMn45MA∼∼&4=N34xMH4xMA∼∼&5=MTB.MTJ.MQ∼∼&exp=KDEmMyY0JjUp
RESOURCE METHODOLOGY AND POLICIES
Methodological updates
A number of methodological changes have occurred since the original description of the GTDB ( 4 ) including the addition of Archaea starting with GTDB R03-RS86 ( 18 ) and adoption of ANI-based genomic similarity criteria for delineating species in GTDB R04-RS89 ( 5 ), which is discussed in more detail below. The 120 bacterial and 122 archaeal marker genes used to infer the domain-specific GTDB reference trees have not changed but identification and alignment of these genes is performed using Pfam v33.1 instead of v27 ( 19 ) starting with R06-RS202. The archaeal tree was originally inferred with FastTree v2.1.10 ( 20 ) under the WAG + GAMMA model but has been inferred using IQ-TREE v1.6.9 ( 21 ) under the C10 + PMSF model as of GTDB R04-RS89 ( 18 ). The initial GTDB releases included in-house MAGs that at the time had not been deposited in an INSDC repository ( 1 , 4 ). However, starting with GTDB R05-RS95, the GTDB only incorporates genomes from the NCBI Assembly database ( 8 ) and explicitly excludes assemblies annotated as being from ‘large multi-isolate projects’ at NCBI as these comprise genomically well-represented species and would require substantial additional computational resources if considered. The methodology and reference data used for each release have been provided in the METHODS file starting with GTDB R04-RS89 ( https://data.gtdb.ecogenomic.org/ ).
Updating GTDB species clusters
method↗concepts↗concepts↗sensesconcept_edgescontent_tags↗Each GTDB species cluster is defined by a single representative genome and species assignments are established by considering the ANI and AF (alignment fraction) of genomes to these representatives as previously described ( 5 ). Here, we explain the methodology used to update these species clusters with each GTDB release. Species representatives are re-evaluated each release with an emphasis placed on retaining the same representative genomes for previously named species to preserve consistency between releases. However, the goal of stable representatives must be balanced with the utility of high-quality genomes as representatives, the need to incorporate changes in taxonomic opinion, and the need to correct genome classification or assembly errors.
Updating GTDB species clusters consists of four steps (Figure 3A ). Genomes in the current and previous releases are compared to identify new and updated genome assemblies, along with genomes that have been suppressed at NCBI indicating that they are no longer considered reliable (e.g. GCA_005039905.1). This is done by directly comparing the NCBI accession numbers (e.g. GCF_000267585.2) of the genomes in each release. Genomes of insufficient quality, as previously defined ( 5 ), are then identified and removed from further consideration. Next, species which contain multiple genomes identified as being assembled from the type strain of the species are examined. If any pair of these genomes has an ANI <99%, the genomes are manually inspected in order to establish which genome or genomes are most likely to represent the type strain of the species. limitationsEstablishing the provenance of type material remains an ongoing challenge and this decision is made by considering a number of factors including the classification of 16S rRNA genes against the Living Tree Project database ( 13 ), the type status of genomes at NCBI, previous GTDB classifications, consideration of all pairwise ANI values and literature review.
Figure 3. Updating species clusters with each GTDB release. ( A ) Workflow for updating GTDB species clusters with results for the most recent GTDB release, R06-RS202, given below each step. There were 90 368 new genomes in this release, 987 genomes where the assembly at NCBI was updated, and 158 genomes where the assembly was suppressed at NCBI and thus not used in this release. All genomes were subjected to quality control which resulted in 26 407 (9.3%) genomes being removed from consideration. There were 2,458 species where multiple genomes were identified as being assembled from the type strain of the species. Of these, 130 species had genomes that were sufficiently divergent to warrant manual inspection to establish the genome most likely to be from the type strain. The 31 910 representatives from the previous GTDB release, R05-RS95, were examined and 1131 (3.5%) updated to a new genome. In addition, 6 species defined in R05-RS95 were retired as the sole genome representing the species was suppressed at NCBI. ( B ) Illustrative example of a GTDB species cluster with previous and new genomes. Genomes are depicted by shapes and the distance between genomes scales with their ANI divergence. The large red circle indicates the ANI circumscription radii for assigning genomes to the current species clusters. The new/updated genome (blue triangle) will only replace the existing GTDB species representative (red circle) if the ANI between these genomes is sufficiently high and the new/updated genome is of sufficient quality (see Table 1 ). This decision is determined quantitatively using the balanced ANI score (see main text). ( C ) Updating the Macrococcus equipercicus species cluster from GTDB R05-RS95 to R06-RS202. The M. equipercicus genome assembly, GCF_004359525.1, was updated and found to be distinct from the previous assembly (ANI = 80.6%). Consequently, this genome formed a new species cluster and the genome GCF_004359515.1 was promoted to a species representative. GCF_004359525.2 is assembled from the type strain of M. equipercicus and GCF_004359515.1 assembled from the type strain of M. carouselicus indicating the M. equipercicus cluster in GTDB R05-RS95 actually represented the species M. carouselicus and was incorrectly classified as a result of the GCF_004359525.1 assembly being incorrect.
mechanismscontrolsconcepts↗concepts↗sensesconcept_edgescontent_tags↗GTDB representatives are updated according to two primary principles: (i) representatives should be assembled from the type strain of a species whenever possible, and (ii) representatives should only be replaced by equivalent assemblies of demonstrably higher quality (Figure 3B ). These two principles are quantitatively defined by the Balanced ANI Score (BAS) which is 0.5 × (ANI score) + 0.5 × (quality score), where the ANI score is 100 – 20 × (100 – ANI to current representative) and the quality score is defined by the criteria given in Table 1 . An existing representative is only replaced by a new or updated genome if it has a BAS ≥ 10 above the BAS of the current representative. This achieves the goal of taxonomic stability by requiring a new representative to be of increasingly higher quality (as defined by the quality score) the more dissimilar it is from the current representative (as defined by the ANI score).
Table 1. Criteria used to establish the quality score of genome assemblies
Criteria Score
Assembled from type strain of species 1 000 000
Effective type strain of species according to NCBI 100 000
NCBI representative of species 10 000
Assembled from type strain of subspecies 1000
Complete genome 100
CheckM quality estimate Completeness – 5 × contamination
MAG or SAG –100
Contig count –5 × (no. contigs/100)
Undetermined bases –5 × (no. undetermined bases/10 000)
Full length 16S rRNA gene 10
There are two special cases that must be handled when updating species representatives. The first is when the assembly of an existing representative is updated. Although atypical, it is possible the new assembly will be sufficiently divergent from the previous assembly (e.g. by removal of a substantial amount of contamination) that a new representative should be selected. This is determined by calculating the BAS between all genomes in the species cluster, including the new assembly, to the previous assembly of the representative genome. The selected representative is the genome with the highest BAS which is typically the new assembly. The second special case occurs when a GTDB representative becomes suppressed at NCBI indicating that the assembly is no longer reliable. In such cases, the representative is replaced with the genome in the species cluster with the highest BAS to the suppressed representative in order to provide stability to the set of GTDB representatives to the extent possible. If the species cluster does not contain any genomes other than the suppressed representative, the species cluster is retired and will no longer appear in the GTDB.
content_tagsGTDB species representatives have been largely stable since the introduction of ANI-based species clusters in RS04-RS89 with 96.7% of representatives being unchanged on average between releases (Table 2 ). This stability is particularly encouraging given the rapid growth in the number of GTDB species clusters (Figure 1C and D ; Table 2 ). Numerous factors account for the changes that have occurred illustrating the benefits and need to regularly update taxonomic frameworks. Between R05-RS95 and R06-RS202 there were 111 representatives with an updated genome assembly at NCBI of which two changed sufficiently that a new GTDB representative was selected for the species cluster. For example, this occurred for GCF_004359525.1 as it has an ANI of only 80.6% to its updated assembly GCF_004359525.2, but the original species cluster was still deemed valid as the representative could be replaced with GCF_004359515.1 which is 99.99% similar to GCF_004359525.1 (Figure 3C ). mechanismsconcepts↗concepts↗concept_edgescontent_tagsThis discrepancy can be explained by the fact that GCF_004359525.2 is assembled from the type strain of Macrococcus equipercicus while GCF_004359515.1 is assembled from the type strain of M. carouselicus indicating that prior to the GCF_004359525.1 assembly being updated, an M. carouselicus strain was erroneously considered as the type strain for M. equipercicus at NCBI and in the GTDB. While the exception, such large-scale reassignments and assembly errors do occur and we make a best effort to identify and handle all such cases. There were 158 genomes in R06-RS202 which were removed from the GTDB as they were suppressed at NCBI. Among these 158 genomes, were 20 GTDB representatives which resulted in 14 species having new representatives selected and six species being retired as they were singleton clusters. A relatively larger number of changes were the result of replacing a non-type representative with a genome assembled from the type strain of the species (312 cases) or subspecies (7 cases). However, the majority of changes (595 cases) were the result of replacing a non-type representative with a higher-quality genome assembly with sufficiently high ANI to the existing representative (i.e. they satisfied the BAS replacement criterion).
Table 2. Updated GTDB species representatives and cluster assignments between releases
R04-RS89 to R05-RS95
R05-RS95 to R06-RS202
Species representative
24 706 to 31 910 31 910 to 47 894
Unchanged 23 957 (96.97%) 30 773 (96.44%)
Changed 743 (3.01%) 1131 (3.54%)
New species
7218 (29.2% increase) 16 004 (50.2% increase)
Genome cluster assignment
145 896 to 194 600 194 600 to 258 406
Same cluster 145 566 (99.77%) 193 668 (99.52%)
Different cluster 172 (0.12%) 774 (0.40%)
Suppressed 158 (0.11%) 158 (0.08%)
After updating previous GTDB representatives, representatives for species with new effectively or validly published names must be selected and all remaining genomes are assigned to these species clusters or de novo species clusters using ANI to delineate species as previously described ( 5 ). Only 0.26% of genomes on average are assigned to a different species cluster between releases (Table 2 ). This is despite the assignment of genomes to species clusters needing to be determined de novo each release in order to account for new and modified GTDB species representatives. Such reassignments are the direct result of allowing representatives to change between releases but as illustrated above there are situations where new representatives must be selected (e.g. replacing GCF_004359525.1). We consider this small amount of reassignment acceptable as it allows genomes assembled from type strains and high-quality genome assemblies to become species representatives.
Policy changes
The GTDB is an evolving resource that aims to balance continuity between releases with changes in classification methodology and taxonomic opinion to best serve the user community, which has necessitated a number of policy changes over time. As of R06-RS202, Latin names are no longer proposed by GTDB curators unless there is an associated publication with taxon descriptions ( 22 , 23 ). This change reflects our efforts to follow the International Code of Nomenclature of Prokaryotes ( 24 ) for determining correct names, including the proposal of Candidatus names. We currently discover new effectively published names by consulting the NCBI taxonomy and LPSN, receiving feedback from the community, and our own best efforts to read the literature. controlsEffectively published Latin names above the rank of genus without designated type material, either a sequenced type strain or MAG, will no longer be incorporated into GTDB, and those that do will only be introduced when the associated type genome is present in GTDB. This change is necessary as establishing the correct interior node in the reference tree for taxa without type material can be ambiguous, particularly when the addition of new genomes or alternative inference methods results in the named taxon becoming polyphyletic in later releases. Latin names in preprints are also no longer incorporated into the GTDB even if their type genomes are already in the database as names can change between preprint and peer-reviewed publication ( 25 , 26 ). mechanismscontrolssensescontent_tagsFinally, to preserve name continuity, we do not replace alphanumeric placeholder names with other placeholder names, even when names (mostly introduced in early releases) are unwieldy, e.g. CG2-30-70-394 which is used as a genus, family, order, class and phylum name. Placeholder names for new taxa in each release are now manually selected from a longer list of unique identifiers to avoid unwieldy names: NCBI organism name, NCBI infraspecific identifier, NCBI WGS identifier, or NCBI assembly identifier. We had previously aimed to release an update of the GTDB every six months and have averaged a new release every seven months. However, starting with R06-RS202 we plan to release updates every nine months in order to accommodate development of the GTDB between releases.
AVERAGE NUCLEOTIDE IDENTITY FOR DELINEATING SPECIES
Observations on the discreteness of ANI-based species in GTDB
research_questioncontent_tagscontent_tags↗An ANI threshold of 95–96% is widely considered the ‘gold standard’ for quantitatively delineating species ( 27–30 ) and it has recently been proposed that a genetic discontinuity exists between 83% and 95% ANI which would further support the use of ANI for defining species boundaries ( 31 , 32 ). This genetic discontinuity has also been observed between genomes recovered from specific environments such as the human gut and soil ( 33 ). However, when genome data are compared globally, i.e. not taking strain cohabitation into account, it is not uncommon for pairs of strains to have ANI values between 83% and 95% ( 5 , 34 ).
This issue is of interest as the GTDB uses a species definition based on the ANI to genomes selected as species representatives ( 5 ). Here we re-evaluated the evidence for a genetic discontinuity as the number of species and genomes within the GTDB has increased substantially since our original study. object_of_studymechanismsconcepts↗concepts↗concepts↗concepts↗claim_relationsconcept_edgesconcept_edgescontent_tagscontent_tagscontent_tags↗We demonstrate the lack of such a discontinuity by considering the ANI between closest pairs of representative genomes from GTDB species within the same genus (Figure 4A ). If a meaningful discontinuity existed, we would expect to find few such pairs with an ANI between 83% and 95% supporting the idea of discrete, well-separated species boundaries. In contrast, we find nearly equal numbers of pairs with ANI values between 78% and 95%. The addition of non-representative genomes can only increase the likelihood of there being genomes with ANI values between 83% and 95% to a species representative and thus a genetic continuum between species. controlscontent_tagsThis lack of a genetic discontinuity also persists when considering only genomes assembled from the type strain of a species, indicating that this result is independent of the methodology used to select GTDB species representatives (Figure 4B ). The above analyses do not speak to the possibility of discrete groups of strains defined by a large genetic discontinuity existing within specific environments as suggested by Olm et al. ( 33 ). However, further work is required to establish this discontinuity and specify how an environment-specific species definition might be used in practice.
Figure 4. Use of genomic similarity for delineating species. ( A ) ANI values between 36 781 GTDB species representatives and their closest representative within the same genus. ( B ) Same plot as in A but restricted to the 9,687 species where the GTDB representative genome is assembled from the type strain. ( C ) Pairwise ANI between the 24 Enterocloster bolteae and 34 E. clostridioformis genomes in GTDB RS06-RS202. ( D ) Pairwise ANI between the 14 Bradyrhizobium elkanii and 8 B. pachyrhizi genomes in GTDB RS06-RS202. ( E ) ANI between genomes and their closest genome in a different, intrageneric species cluster (108 503 total pairs). ( F ) ANI between the closest pairs in plot E for each of the 35 147 species considered.
While the above analysis indicates that there is no global genetic discontinuity between 83% and 95% ANI in the GTDB, it remains unclear as to whether strains generally form discrete, ANI-based species clusters. concepts↗concepts↗concept_edgesFor example, the pairwise ANI values between Enterocloster bolteae and E. clostridioformis genomes form discrete clusters and have a bimodal distribution with a discontinuity between 92% and 96% ANI (Figure 4C ). In contrast, pairwise ANI values between Bradyrhizobium elkanii and B. pachyrhizi genomes display a genetic continuum (Figure 4D ) and can be described as fuzzy species ( 35 ). The discreteness of GTDB species clusters was assessed by considering the ANI between each genome and its closest genome in a different, intrageneric species cluster (see Methods). Discrete species will contain few or ideally no genomes with a high ANI to genomes from a different species. Disregarding GTDB species clusters delineated at an ANI >95% indicated that only 6.0% of the closest interspecific genome pairs have an ANI ≥ 95% and 80.8% of pairs are well-separated as indicated by having an ANI ≤94% (Figure 4E ). mechanismslimitationssensescontent_tags↗content_tagscontent_tags↗In terms of species, 2.2% can be described as fuzzy as they have one or more genomes with an interspecific ANI value ≥95% and 89.5% can be described as discrete as they contain no genomes with an interspecific ANI value >94% (Figure 4F ). Encouragingly, this indicates that GTDB species clusters are largely discrete, but this result should be interpreted with care as most bacterial and archaeal species lack genomic representation based on even conservative estimates of the number of prokaryotic species in nature ( 36 ).
phenomenonmechanismslimitationscontent_tagscontent_tagsThe use of ANI to delineate species despite the lack of clear evidence for discrete species boundaries in the GTDB dataset is a pragmatic approach for organizing the rapidly growing biodiversity being discovered with metagenomic approaches ( 2 , 3 , 15 ). Leaving this diversity unclassified would severely limit the ability to effectively communicate scientific results as exemplified by 28.5% of genomes at NCBI lacking a species classification and 77.0% of GTDB R06-RS202 species clusters having an alphanumeric placeholder name. mechanismsclaim_relationscontent_tagscontent_tags↗Doolittle and Papke ( 37 ) best articulate the rationale for a pragmatic species definition: ‘What we want from a species definition is a set of easily applied and stable rules by which to decide when two organisms are similar enough in their genomic and/or phenotypic properties to be given the same name. The needs for such a guide to taxonomic practice in medicine, biotechnology and defense are obvious, and even arbitrary rules to satisfy them would be better than no rules at all.’
CONCLUDING REMARKS AND FUTURE PLANS
GTDB is in active development and there are a number of initiatives underway to make this resource more comprehensive and accurate. As MAGs are not always submitted to INSDC repositories, we are exploring incorporation of additional repositories into the GTDB such as MGnify ( 38 ). The quality of MAGs is an ongoing concern and we are evaluating methods for decontaminating MAGs ( 39 ) and identifying and removing MAGs that may contribute to phylogenetic instability ( 40 ). mechanismscontent_tagscontent_tagscontent_tags↗Related to MAG quality, we expect to change the AF criteria used for assigning genomes to GTDB species clusters from 0.65 to 0.5 starting in the next GTDB release in order to better accommodate the growing number of incomplete MAGs contained in the GTDB which can cause the AF between MAGs to be artificially low. There is also an ongoing need to evaluate best practices in terms of tree inference methods, which has been initially explored for the archaeal tree and resulted in the adoption of IQ-TREE for this domain. We envisage that further evaluations will result in modifications to the bacterial and archaeal marker sets in future GTDB releases. Feedback is essential for the GTDB to best serve the community and we encourage suggestions and comments on either the GTDB Forum ( https://forum.gtdb.ecogenomic.org/ ) or directly to the GTDB development team ( https://gtdb.ecogenomic.org/about ).
METHODS
ANI and AF values were calculated with FastANI v1.3 ( 31 ). The discreteness of GTDB species clusters was assessed by identifying the closest genome to a genome within a different, intrageneric species cluster. The 35 147 species in genera with multiple species and with an ANI circumscription radius of 95% were considered in this analysis (species with an ANI radius >95% are exceptional cases in the GTDB and result from another species being within 95% ANI that was retained in the GTDB to preserve historical classifications). limitationscontent_tags↗In order to reduce computational requirements, it was assumed that if the closest GTDB representative genome for species A was found to be species B, that the closest genome for any genome in species A will be in species B. Furthermore, for the 87 species containing >150 genomes only a randomly selected subset of 150 genomes was considered though these were compared to all genomes in species B regardless of the number of genomes in this species.
DATA AVAILABILITY
The GTDB can be accessed at https://gtdb.ecogenomic.org/ and data files for each GTDB release are available from https://data.gtdb.ecogenomic.org/ . Genomes comprising the GTDB are obtained from the NCBI Assembly database ( www.ncbi.nlm.nih.gov/assembly ). GTDB species clusters are updated using the Python code at https://github.com/Ecogenomics/gtdb-species-clusters which is made available under the GNU GPL v3.0.
Supplementary Material
gkab776_Supplemental_File Click here for additional data file.
ACKNOWLEDGEMENTS
We thank Brian Kemish for systems engineering support, and members of the Australian Centre for Ecogenomics for beta-testing new features. We also thank the user community for feedback and highlighting errors in the taxonomy.
Contributor Information
Donovan H Parks, The University of Queensland, School of Chemistry and Molecular Biosciences, Australian Centre for Ecogenomics, QLD 4072, Australia.
Maria Chuvochina, The University of Queensland, School of Chemistry and Molecular Biosciences, Australian Centre for Ecogenomics, QLD 4072, Australia.
Christian Rinke, The University of Queensland, School of Chemistry and Molecular Biosciences, Australian Centre for Ecogenomics, QLD 4072, Australia.
Aaron J Mussig, The University of Queensland, School of Chemistry and Molecular Biosciences, Australian Centre for Ecogenomics, QLD 4072, Australia.
Pierre-Alain Chaumeil, The University of Queensland, School of Chemistry and Molecular Biosciences, Australian Centre for Ecogenomics, QLD 4072, Australia.
Philip Hugenholtz, The University of Queensland, School of Chemistry and Molecular Biosciences, Australian Centre for Ecogenomics, QLD 4072, Australia.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
FUNDING
GTDB is supported by Australian Research Council Laureate Fellowship [FL150100038 to P.H.]; Australian Research Council Future Fellowship [FT170100213 to C.R.]; strategic funding from the University of Queensland. Funding for open access charge: Australian Research Council Laureate Fellowship [FL150100038 to P.H.].
Conflict of interest statement . None declared.
Relations on this page carry a quotation for the claim. Where a relation was separately evidenced — and so tested against the vocabulary for its type — the row says so; 7 of 7 relations here carry that mark. The rest are the tagger’s reading of the sentence quoted beside them.
| facet | tag | field | conf. | whose words |
|---|---|---|---|---|
| object_of_study | A SPECIES BOUNDARY WHOSE JUSTIFICATION WAS TESTED, DISPROVED AND KEPT ON PURPOSE - the 95-96% average nucleotide identity threshold, defended in the literature by a claimed genetic discontinuity that this paper looks for in its own 254,090 genomes and does not find | object_of_study | the paper's words | |
| research_question | whether prokaryotic species are discrete enough for a similarity threshold to be a natural boundary, and how to keep a genome-based taxonomy stable while it grows by 270% and its ranks are set by two continuous statistics | research_question | the paper's words | |
| contribution | a rank-normalised genome taxonomy in which higher ranks are delineated by relative evolutionary divergence and species by ANI, with a published policy for names, a quantitative rule for replacing type genomes, and a measurement showing the species threshold has no natural discontinuity behind it | contribution | the paper's words | |
| claim_type | method | claim_type | the paper's words | |
| phenomenon | prokaryotic diversity as a continuum, most of it never cultured and most of it unnamed, being cut into named classes so that it can be talked about at all | phenomenon | the paper's words | |
| method | system-or-tool-building also in: system-or-tool-building | method | the paper's words | |
| mechanisms | THE JUSTIFICATION FOR THE SPECIES THRESHOLD WAS TESTED AND FAILED: `An ANI threshold of 95-96% is widely considered the `GOLD STANDARD` ... and it has recently been proposed that a genetic discontinuity exists between 83% and 95% ANI which would further support the use of ANI for defining species boundaries.` Re-tested here: `If a meaningful discontinuity existed, we would expect to find few such pairs with an ANI between 83% and 95% ... In contrast, we find nearly equal numbers of pairs with ANI values between 78% and 95%.` The gap the line was supposed to sit inside is not there | mechanisms[0] | 0.95 | the tagger's words |
| mechanisms | AND THE THRESHOLD IS KEPT, WITH THE REASON WRITTEN DOWN: `The use of ANI to delineate species despite the lack of clear evidence for discrete species boundaries in the GTDB dataset is a pragmatic approach for organizing the rapidly growing biodiversity ... Leaving this diversity unclassified would severely limit the ability to effectively communicate scientific results.` Read against simonneau2018 in the same corpus - `THIS DEFINITION IS, THEREFORE, NO LONGER ARBITRARY, BUT BASED ON A SCIENTIFIC APPROACH`. One field says it has removed the arbitrariness; this one measures for a natural boundary, does not find it, and keeps the rule anyway | mechanisms[1] | 0.95 | the tagger's words |
| mechanisms | AND THE ARGUMENT FOR DOING SO IS QUOTED FROM SOMEBODY ELSE, IN FULL: `What we want from a species definition is a set of easily applied and stable rules by which to decide when two organisms are similar enough ... to be given the same name. The needs for such a guide to taxonomic practice in medicine, biotechnology and defense are obvious, and even arbitrary rules to satisfy them would be better than no rules at all.` The clearest statement in the corpus that a definition can be justified by its usefulness rather than by nature - and it is a quotation, not this paper's own sentence | mechanisms[2] | 0.95 | the tagger's words |
| mechanisms | AND THE RANKS ARE NORMALISED BY A CONTINUOUS STATISTIC: `the GTDB uses RELATIVE EVOLUTIONARY DIVERGENCE (RED) to delineate higher-rank taxa and average nucleotide identity (ANI) to delineate species clusters. Use of these quantitative criteria for circumscribing taxa allows for automated classification ..., ensures all genomes are classified from species to domain, and normalizes the definition of taxonomic ranks across the bacterial and archaeal domains.` A genus in one phylum and a genus in another are made comparable by a number - which is the thing traditional taxonomy could never guarantee | mechanisms[3] | 0.95 | the tagger's words |
| mechanisms | AND STABILITY IS ENFORCED BY A SCORE WITH AN EXPLICIT ASYMMETRY: `representatives should only be replaced by equivalent assemblies of demonstrably higher quality ... quantitatively defined by the BALANCED ANI SCORE (BAS) which is 0.5 x (ANI score) + 0.5 x (quality score) ... An existing representative is only replaced ... IF IT HAS A BAS >= 10 ABOVE the BAS of the current representative. This achieves the goal of taxonomic stability by requiring A NEW REPRESENTATIVE TO BE OF INCREASINGLY HIGHER QUALITY THE MORE DISSIMILAR IT IS from the current representative.` The cost of changing what a name points at rises with how much the pointing would move | mechanisms[4] | 0.95 | the tagger's words |
| mechanisms | AND A NAME CAN TURN OUT TO HAVE POINTED AT THE WRONG ORGANISM: `GCF_004359525.2 is assembled from the type strain of Macrococcus equipercicus WHILE GCF_004359515.1 is assembled from the type strain of M. carouselicus, indicating that prior to the ... assembly being updated, an M. carouselicus strain was erroneously considered as the type strain for M. equipercicus at NCBI and in the GTDB.` For at least one release a species cluster carried one name and contained the other organism, and the correction came from an assembly update rather than from biology | mechanisms[5] | 0.95 | the tagger's words |
| mechanisms | AND MOST OF THE TAXONOMY HAS NO NAME AND NO ORGANISM: `28.5% of genomes at NCBI lacking a species classification and 77.0% of GTDB R06-RS202 species clusters having an alphanumeric placeholder name`; and `over 70% of archaeal species, genera, families, and orders lacking an available cultured representative`. Three quarters of the species in a species database are named by a string like CG2-30-70-394, and half the taxa exist only as assembled sequence | mechanisms[6] | 0.95 | the tagger's words |
| mechanisms | AND THE NAMING POLICY IS FIVE RULES ABOUT WHO MAY NAME WHAT: Latin names are `no longer proposed by GTDB curators unless there is an associated publication with taxon descriptions`; names above genus `without designated type material ... will no longer be incorporated`; `Latin names in preprints are also no longer incorporated ... as names can change between preprint and peer-reviewed publication`; and placeholder names are never swapped for other placeholder names, `EVEN WHEN [THEY] ARE UNWIELDY, e.g. CG2-30-70-394 which is used as A genus, family, order, class and phylum name`. Continuity of reference beats readability, explicitly | mechanisms[7] | 0.95 | the tagger's words |
| mechanisms | AND A DEFINITIONAL PARAMETER IS ABOUT TO MOVE BECAUSE OF DATA QUALITY: `we expect to change the AF criteria used for assigning genomes to GTDB species clusters from 0.65 to 0.5 starting in the next GTDB release in order to better accommodate the growing number of incomplete MAGs contained in the GTDB which can cause the AF between MAGs to be artificially low.` The membership rule is loosened because the evidence got worse, not because the organisms changed - and the sentence says so | mechanisms[8] | 0.95 | the tagger's words |
| mechanisms | AND DISCRETENESS IS MEASURED RATHER THAN ASSUMED: `2.2% can be described as fuzzy as they have one or more genomes with an interspecific ANI value >=95% AND 89.5% can be described as discrete as they contain no genomes with an interspecific ANI value >94% ... but this result should be interpreted with care as most bacterial and archaeal species lack genomic representation.` The clusters are mostly clean; the boundary between them is not a gap in nature - both are true, and the paper keeps both | mechanisms[9] | 0.95 | the tagger's words |
| limitations | THE SPECIES DEFINITION IS ADMITTED TO HAVE NO NATURAL BOUNDARY BEHIND IT: `The use of ANI to delineate species despite the lack of clear evidence for discrete species boundaries in the GTDB dataset is a pragmatic approach.` This is a stronger disclosure than most definitional papers make - the authors ran the test that would have vindicated their own threshold and published the negative result | limitations[0] | 0.95 | the tagger's words |
| limitations | AND THE DISCRETENESS RESULT IS BOUNDED BY WHAT HAS BEEN SEQUENCED: `this result should be interpreted with care as most bacterial and archaeal species lack genomic representation based on even conservative estimates of the number of prokaryotic species in nature.` The clusters look discrete partly because the space between them has not been sampled | limitations[1] | 0.95 | the tagger's words |
| limitations | AND HALF THE TAXA HAVE NEVER BEEN GROWN: `over 70% of archaeal species, genera, families, and orders lacking an available cultured representative`, and over 50% of bacterial taxa `consist exclusively of MAGs and/or SAGs` although those are only 20.2% of the genomes. A taxonomy of organisms nobody has in a flask | limitations[2] | 0.95 | the tagger's words |
| limitations | AND THE DISCRETENESS ANALYSIS TAKES TWO SHORTCUTS, BOTH NAMED: `it was assumed that if the closest GTDB representative genome for species A was found to be species B, that the closest genome for any genome in species A will be in species B. Furthermore, for the 87 species containing >150 genomes only a randomly selected subset of 150 genomes was considered.` Declared in the Methods, and both would move the fuzzy/discrete split in the same direction - toward discreteness | limitations[3] | 0.95 | the tagger's words |
| limitations | AND THE TYPE MATERIAL ITSELF IS NOT ALWAYS KNOWABLE: `Establishing the provenance of type material remains an ongoing challenge and this decision is made by considering a number of factors including the classification of 16S rRNA genes ..., the type status of genomes at NCBI, previous GTDB classifications, consideration of all pairwise ANI values and literature review.` One of the inputs to the decision is the database's own previous answer | limitations[4] | 0.95 | the tagger's words |
| controls | repeating the discontinuity analysis using only genomes assembled from type strains eliminates: the objection that the missing discontinuity is an artefact of how GTDB picks species representatives · outcome: held | controls[0] | 0.95 | the tagger's words |
| controls | requiring a Balanced ANI Score at least 10 above the incumbent before a species representative may be replaced eliminates: a name drifting to a different genome every release as new assemblies arrive · outcome: held | controls[1] | 0.95 | the tagger's words |
| controls | refusing Latin names without an associated publication, without designated type material, or published only as a preprint eliminates: names that cannot be anchored to a genome, and names that change between preprint and publication · outcome: held | controls[2] | 0.95 | the tagger's words |
| controls | never replacing an alphanumeric placeholder with another placeholder, however unwieldy eliminates: breaking the link between a published result and the taxon it named · outcome: held | controls[3] | 0.95 | the tagger's words |
| concepts | Each GTDB species cluster also in: Each GTDB species cluster | concepts[0] | 0.95 | the paper's words |
| concepts | a single representative genome also in: a single representative genome | concepts[1] | 0.95 | the paper's words |
| concepts | two primary principles also in: two primary principles | concepts[2] | 0.95 | the paper's words |
| concepts | the Balanced ANI Score (BAS) also in: the Balanced ANI Score (BAS) | concepts[3] | 0.95 | the paper's words |
| concepts | Enterocloster bolteae also in: Enterocloster bolteae | concepts[4] | 0.95 | the paper's words |
| concepts | Bradyrhizobium elkanii also in: Bradyrhizobium elkanii | concepts[5] | 0.95 | the paper's words |
| concepts | a meaningful discontinuity also in: a meaningful discontinuity | concepts[6] | 0.95 | the paper's words |
| concepts | nearly equal numbers of pairs with ANI values between 78% and 95% | concepts[7] | 0.95 | the paper's words |
| concepts | Macrococcus equipercicus also in: Macrococcus equipercicus | concepts[8] | 0.95 | the paper's words |
| concepts | M. carouselicus also in: M. carouselicus | concepts[9] | 0.95 | the paper's words |
| concepts | bacterial taxa also in: bacterial taxa | concepts[10] | 0.95 | the paper's words |
| concepts | MAGs and/or SAGs also in: MAGs and/or SAGs | concepts[11] | 0.95 | the paper's words |
| concepts | The addition of non-representative genomes | concepts[12] | 0.95 | the paper's words |
| concepts | a genetic continuum between species also in: a genetic continuum between species | concepts[13] | 0.95 | the paper's words |
| senses | `species` (GTDB sense) - a cluster around a single representative genome, entered by ANI and alignment fraction to that representative; NOT a claim about a discontinuity in nature | senses[0] | 0.95 | the tagger's words |
| senses | `rank normalized` - higher ranks set by relative evolutionary divergence so that a genus in one phylum is comparable with a genus in another | senses[1] | 0.95 | the tagger's words |
| senses | `fuzzy species` - a species with one or more genomes at >=95% ANI to a genome in another species; 2.2% of those examined. The complement, `discrete`, is 89.5% | senses[2] | 0.95 | the tagger's words |
| senses | `placeholder name` - an alphanumeric string standing in for a Latin name, carried by 77.0% of GTDB species clusters and never replaced by another placeholder | senses[3] | 0.95 | the tagger's words |
| senses | `Balanced ANI Score` - `0.5 x (ANI score) + 0.5 x (quality score)`, the rule deciding when the genome a species name points at may be swapped | senses[4] | 0.95 | the tagger's words |
| claim_relations | extends → the earlier GTDB papers that introduced relative evolutionary divergence for higher ranks and ANI-based species clusters, whose criteria this release applies and re-tests | claim_relations[0] | 0.95 | the tagger's words |
| claim_relations | critiques → the claim that a genetic discontinuity exists between 83% and 95% ANI, which is the main argument for treating the species threshold as a natural boundary | claim_relations[1] | 0.95 | the tagger's words |
| claim_relations | extends → LPSN, the NCBI Taxonomy and the Living Tree Project, which supply type material, nomenclatural status and 16S classification to this taxonomy | claim_relations[2] | 0.95 | the tagger's words |
| claim_relations | extends → Doolittle and Papke's argument for a pragmatic species definition, quoted in full as the rationale for keeping a threshold with no natural boundary behind it | claim_relations[3] | 0.95 | the tagger's words |
| concept_edges | Each GTDB species cluster —operationalised-as→ a single representative genome the relation itself is separately evidenced · strength of the relation: 0.95 · note: 'Each GTDB species cluster is defined by a single representative genome and species assignments are established by considering the ANI and AF (alignment fraction) of genomes to these representatives.' A definition by exemplar - and it is why the paper needs a rule for replacing representatives at all: change the exemplar and the class moves. Elsewhere in the paper, only '0.26% of genomes on average are assigned to a different species cluster between releases' · the relation stated: a species is whatever falls within an ANI and alignment-fraction radius of one chosen genome, so the class is defined by an exemplar rather than by a property | concept_edges[0] | 0.95 | the tagger's words |
| concept_edges | two primary principles —operationalised-as→ the Balanced ANI Score (BAS) the relation itself is separately evidenced · strength of the relation: 0.95 · note: 'GTDB representatives are updated according to two primary principles: (i) representatives should be assembled from the type strain ... and (ii) representatives should only be replaced by equivalent assemblies of demonstrably higher quality. These two principles are quantitatively defined by the Balanced ANI Score (BAS) WHICH IS 0.5 x (ANI SCORE) + 0.5 x (QUALITY SCORE).' A qualitative preference turned into an arithmetic one - and the quality score behind it encodes an ordinal ranking as powers of ten, so the sum is only meaningful because one term dominates · the relation stated: prefer the type strain, and replace only for demonstrably higher quality - expressed as a weighted sum with a fixed replacement margin | concept_edges[1] | 0.95 | the tagger's words |
| concept_edges | a meaningful discontinuity —contrasted-with→ nearly equal numbers of pairs with ANI values between 78% and 95% the relation itself is separately evidenced · strength of the relation: 0.95 · note: 'If a meaningful discontinuity existed, we would expect to find few such pairs with an ANI between 83% and 95% supporting the idea of discrete, well-separated species boundaries. In contrast, we find nearly equal numbers of pairs with ANI values between 78% and 95%.' A prediction stated before the test and a negative result published against the authors' own threshold - and the result 'persists when considering only genomes assembled from the type strain', so it is not an artefact of how representatives were chosen · the relation stated: the discontinuity that would justify the species threshold predicts few pairs in the 83-95% band; the data show a flat distribution across 78-95% | concept_edges[2] | 0.95 | the tagger's words |
| concept_edges | The addition of non-representative genomes —causes→ a genetic continuum between species direction on the measure: increases · the relation itself is separately evidenced · strength of the relation: 0.9 · note: 'The addition of non-representative genomes can only increase the likelihood of there being genomes with ANI values between 83% and 95% to a species representative and thus a genetic continuum between species.' A boundary claim that gets weaker with every new observation, by construction - the opposite of how thresholds usually behave as evidence accumulates · the relation stated: every additional strain can only fill in the gap the threshold was supposed to sit in, so the evidence for a natural boundary weakens as the database grows
Audit 2026-09-28: DOUBTFUL - read against its full span and kept, but the span says the addition can only increase the LIKELIHOOD of such genomes; causes is loose. | concept_edges[3] | 0.95 | the tagger's words |
| concept_edges | Enterocloster bolteae —contrasted-with→ Bradyrhizobium elkanii the relation itself is separately evidenced · strength of the relation: 0.95 · note: 'the pairwise ANI values between ENTEROCLOSTER BOLTEAE and E. clostridioformis genomes FORM DISCRETE CLUSTERS and have a BIMODAL DISTRIBUTION with a discontinuity between 92% and 96% ANI. IN CONTRAST, pairwise ANI values between BRADYRHIZOBIUM ELKANII and B. pachyrhizi genomes DISPLAY A GENETIC CONTINUUM and can be described as FUZZY SPECIES.' One threshold, two kinds of object - and the paper measures how often each occurs rather than choosing between them · the relation stated: the same ANI rule produces a clean bimodal separation in one genus and an unbroken continuum in another - the species concept behaves differently by clade also in: Enterocloster bolteae · Bradyrhizobium elkanii | concept_edges[4] | 0.95 | the tagger's words |
| concept_edges | Macrococcus equipercicus —distinct-from→ M. carouselicus the relation itself is separately evidenced · strength of the relation: 0.95 · note: 'GCF_004359525.2 is assembled from the type strain of Macrococcus equipercicus while GCF_004359515.1 is assembled from the type strain of M. CAROUSELICUS, indicating that ... an M. carouselicus strain was erroneously considered as the type strain for M. equipercicus at NCBI and in the GTDB.' The distinction was always real; the pointer was wrong - which is a different failure from a definition being wrong, and only visible because the assembly was updated · the relation stated: two species whose type strains were confused in the source database, so a cluster carried one name and contained the other until an assembly was corrected also in: Macrococcus equipercicus · M. carouselicus | concept_edges[5] | 0.95 | the tagger's words |
| concept_edges | MAGs and/or SAGs —part-of→ bacterial taxa the relation itself is separately evidenced · strength of the relation: 0.9 · note: 'Over 50% of bacterial taxa, regardless of rank, consist exclusively of MAGs and/or SAGs despite MAGs (50 669 genomes) and SAGs (745 genomes) representing only 20.2% of the 254 090 bacterial genomes.' The taxa and the genomes are differently distributed: uncultured sequence is a minority of the data and a majority of the classes, because isolate genomes pile up inside a few well-sampled pathogens · the relation stated: over half of bacterial taxa at every rank contain nothing but assembled sequence, although such genomes are only a fifth of the database also in: MAGs and/or SAGs · bacterial taxa | concept_edges[6] | 0.95 | the tagger's words |
| content_tags | THE JUSTIFICATION FOR THE SPECIES THRESHOLD WAS TESTED AND FAILED: `If a meaningful discontinuity existed, we would expect to find few such pairs ... In contrast, we find nearly equal numbers of pairs with ANI values between 78% and 95%` | content_tags.findings[0] | 0.95 | the tagger's words |
| content_tags | AND THE THRESHOLD IS KEPT ON PURPOSE: `despite the lack of clear evidence for discrete species boundaries ... is a pragmatic approach`, because `Leaving this diversity unclassified would severely limit the ability to effectively communicate scientific results` | content_tags.findings[1] | 0.95 | the tagger's words |
| content_tags | AND THE ARGUMENT IS QUOTED, NOT ASSERTED: `even arbitrary rules to satisfy them would be better than no rules at all` - Doolittle and Papke, quoted in full | content_tags.findings[2] | 0.95 | the tagger's words |
| content_tags | AND THE RESULT SURVIVES THE OBVIOUS OBJECTION: the missing discontinuity `also persists when considering only genomes assembled from the type strain of a species` | content_tags.findings[3] | 0.95 | the tagger's words |
| content_tags | AND MORE DATA MAKES IT WORSE: `The addition of non-representative genomes can only increase the likelihood ... and thus a genetic continuum between species` | content_tags.findings[4] | 0.95 | the tagger's words |
| content_tags | over 50% of bacterial taxa consist only of MAGs or SAGs, and over 70% of archaeal species, genera, families and orders lack a cultured representative | content_tags.findings[5] | 0.95 | the tagger's words |
| content_tags | AND A CLUSTER ONCE CARRIED THE WRONG NAME: `an M. carouselicus strain was erroneously considered as the type strain for M. equipercicus at NCBI and in the GTDB` | content_tags.findings[6] | 0.95 | the tagger's words |
| content_tags | AND A MEMBERSHIP PARAMETER IS ABOUT TO BE LOOSENED FOR DATA QUALITY: the alignment fraction moves `from 0.65 to 0.5 ... to better accommodate the growing number of incomplete MAGs` | content_tags.findings[7] | 0.95 | the tagger's words |
| content_tags | AND NAME CONTINUITY BEATS READABILITY: placeholders are never swapped `EVEN WHEN [THEY] ARE UNWIELDY, e.g. CG2-30-70-394 which is used as A genus, family, order, class and phylum name` | content_tags.findings[8] | 0.95 | the tagger's words |
| content_tags | `Each GTDB species cluster is defined by a single representative genome`, entered by ANI and alignment fraction to that genome - a definition by exemplar also in: species | content_tags.definitions[0] | 0.95 | the tagger's words |
| content_tags | a species with one or more genomes at `an interspecific ANI value >=95%` - 2.2% of those measured, against 89.5% `DISCRETE` also in: fuzzy species | content_tags.definitions[2] | 0.95 | the tagger's words |
| content_tags | `0.5 x (ANI score) + 0.5 x (quality score)`, replacement only at `BAS >= 10` above the incumbent also in: the Balanced ANI Score | content_tags.definitions[3] | 0.95 | the tagger's words |
| content_tags | Doolittle and Papke: `a set of easily applied and stable rules by which to decide when two organisms are similar enough ... to be given the same name` also in: a pragmatic species definition | content_tags.definitions[4] | 0.95 | the tagger's words |
| content_tags | instead, `nearly equal numbers of pairs with ANI values between 78% and 95%` | content_tags.quantities[0] | 0.95 | the tagger's words |
| content_tags | and over 50% of bacterial taxa `consist exclusively of MAGs and/or SAGs` | content_tags.quantities[1] | 0.95 | the tagger's words |
| content_tags | `this result should be interpreted with care as most bacterial and archaeal species lack genomic representation` | content_tags.quantities[2] | 0.95 | the tagger's words |
| content_tags | achieved by requiring `A BAS >= 10 ABOVE the BAS of the current representative` | content_tags.quantities[3] | 0.95 | the tagger's words |
| content_tags | `to better accommodate the growing number of incomplete MAGs ... which can cause the AF between MAGs to be artificially low` | content_tags.quantities[4] | 0.95 | the tagger's words |
| content_tags | the prediction is stated first - `we would expect to find few such pairs` - and the observation contradicts it. The control is run and reported (`persists when considering only genomes assembled from the type strain`), and the direction of future error is named (`The addition of non-representative genomes can only increase the likelihood`). This is what a threshold paper looks like when it tests its own foundation | content_tags.statistics[0] | 0.95 | the tagger's words |
| content_tags | `it was assumed that if the closest GTDB representative genome for species A was found to be species B, that the closest genome for any genome in species A will be in species B`, and for 87 large species only 150 genomes were sampled. Declared, and both would make the fuzzy fraction look smaller than it is | content_tags.statistics[1] | 0.95 | the tagger's words |
| content_tags | `89.5% can be described as discrete` sits beside `most bacterial and archaeal species lack genomic representation`. The clusters look separated partly because the space between them is empty of data | content_tags.statistics[2] | 0.95 | the tagger's words |
| content_tags | the continuous quantity a species boundary is cut from - `widely considered the `GOLD STANDARD` for quantitatively delineating species`, and shown here to have no gap at the cut also in: average nucleotide identity (ANI) | content_tags.variables[0] | 0.95 | the tagger's words |
| content_tags | the statistic that makes ranks comparable across clades - it `normalizes the definition of taxonomic ranks across the bacterial and archaeal domains` | content_tags.variables[1] | 0.95 | the tagger's words |
| content_tags | the second membership criterion, and the one about to move from 0.65 to 0.5 because incomplete MAGs depress it also in: AF | content_tags.variables[2] | 0.95 | the tagger's words |
Tags that could not be placed in the text:
What was deliberately not tagged, and why, is recorded per paper by the tagger. Where a paper has no such record, this page says so rather than implying that nothing was declined.