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Page 6 of 18 Mueller et al. Microbiome Res Rep 2024;3:33 https://dx.doi.org/10.20517/mrr.2024.09

Figure 1. Phylogenetic analysis of Akkermansia genomes and evidence of speciation among seven major phylogroups. (A) ANI based on
whole-genome comparisons, as calculated using pyANI in Anvi’o. The heatmap displays levels of ANI similarity between pairwise
comparisons and grouping is set at a 95% similarity threshold. Red denotes a pairwise ANI of 95% or above. Major phylogroups are
highlighted and labeled to maintain consistency with previous publications [19-22] . Note the presence of the new AmV and AmVI
phylogroups; (B) Comparison of average ANI between Akkermansia phylogroups. Differences in average ANI below 95% were used to
denote new species among phylogroups and an average ANI between 95% and 98% was defined to assign new sub-phylogroups within
those new species (dotted lines on each subplot); (C) Phylogenetic analysis of core Akkermansia proteins. The predicted amino acid
sequences encoded by single-copy Akkermansia genes were aligned and concatenated using anvi-get-sequences-for-gene-clusters. anvi-
gen-phylogenomic-tree was used to generate a phylogenomic tree with FastTree. A. glycaniphila was used as an outgroup, and individual
strains are highlighted by the proposed species and subspecies. Numbers denote main branch lengths; (D) Comparison of average (red
line) genome sizes of high-quality complete genomes of representative members of Akkermansia phylogroups; (E) Phylogenetic grouping
of full-length 16S rRNA Akkermansia sequences is consistent with genomic ANI comparisons. Full-length 16S rRNA gene sequences were
extracted from genome sequences within Anvi’o. Clustal Omega was used to perform the alignment of those sequences, and the
resulting phylogenetic tree was used for visualization. A. glycaniphila was used as an outgroup, and individual strains have been
highlighted by phylogroup. ANI: Average nucleotide identity.

the commonly used threshold of 97%. At a higher species demarcating threshold of 98.65%, only
A. biwaensis and the AmVI phylogroup together would be considered a new Akkermansia species
[Figure 1E, Supplementary Figure 2 and Supplementary Table 3].