# A Novel Lineage of Endosymbiotic Actinomycetales: Genome Reduction and Acquisition of New Functions in Bifidobacteriaceae Associated With Termite Gut Flagellates

Joana Kästle Silva, Vincent Hervé, Undine S Mies, Katja Platt, Andreas Brune

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## To cite this version:

Joana Kästle Silva, Vincent Hervé, Undine S Mies, Katja Platt, Andreas Brune. A Novel Lineage of Endosymbiotic Actinomycetales: Genome Reduction and Acquisition of New Functions in Bifidobacteriaceae Associated With Termite Gut Flagellates. Environmental Microbiology, 2025, 27 (1), 10.1111/1462-2920.70010. hal-04908819

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Submitted on 23 Jan 2025

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BRIEF REPORT

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# A Novel Lineage of Endosymbiotic *Actinomycetales*: Genome Reduction and Acquisition of New Functions in *Bifidobacteriaceae* Associated With Termite Gut Flagellates

Joana Kästle Silva ID |

Vincent Hervé ID |

Undine S. Mies ID |

Katja Platt | Andreas Brune

Research Group Insect Gut Microbiology and Symbiosis, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany

**Correspondence:** Andreas Brune (brune@mpi-marburg.mpg.de)

**Received:** 16 August 2024 |

**Revised:** 29 October 2024 |

**Accepted:** 13 November 2024

**Funding:** This work was supported by Max-Planck-Gesellschaft.

**Keywords:** bifidobacteria | flagellates endosymbiosis | genome reduction | gut microbiota | horizontal gene transfer | termites

## ABSTRACT

Cellulolytic flagellates are essential for the symbiotic digestion of lignocellulose in the gut of lower termites. Most species are associated with host-specific consortia of bacterial symbionts from various phyla. 16S rRNA-based diversity studies and taxon-specific fluorescence in situ hybridization revealed a termite-specific clade of *Actinomycetales* that colonise the cytoplasm of *Trichonympha* spp. and other gut flagellates, representing the only known case of intracellular *Actinomycetota* in protists. Comparative analysis of eleven metagenome-assembled genomes from lower termites allowed us to describe them as new genera of *Bifidobacteriaceae*. Like the previously investigated *Candidatus* Ancillula trichonymphae, they ferment sugars via the bifido-bacterium shunt but, unlike their free-living relatives, experienced significant genome erosion. Additionally, they acquired new functions by horizontal gene transfer from other gut bacteria, including the capacity to produce hydrogen. Members of the genus *Ancillula* (average genome size 1.56 ± 0.2 Mbp) retained most pathways for the synthesis of amino acids, including a threonine/serine exporter, providing concrete evidence for the basis of the mutualistic relationship with their host. By contrast, *Opitulatrix* species (1.23 ± 0.1 Mbp) lost most of their biosynthetic capacities, indicating that an originally mutualistic symbiosis is on the decline.

## 1 | Introduction

Termites evolved from subsocial cockroaches about 150 million years ago (Chouvenc et al. 2021). With the help of their symbiotic gut microbiota, they digest wood and other lignocellulosic matter (Brune 2014), which lends them a key role in the carbon cycle and other ecosystem services in the tropics and subtropics (Jouquet et al. 2011; Griffiths et al. 2019). Except for the family Termitidae ('higher termites'), termites from all other families ('lower termites') harbour species-specific assemblages of cellulolytic flagellates that break down wood fibres into compounds that can be assimilated by the host (Brune 2014). The flagellates are themselves colonised by diverse prokaryotic consortia,

resulting in a multitiered network of symbiotic interactions (Ohkuma and Brune 2011; Hongoh and Ohkuma 2018).

Symbiotic bacteria are located on the surface of the flagellate cell (ectosymbionts) or colonise the cytoplasm or the nucleus of their flagellate host (endosymbionts) (Brune and Dietrich 2015). These associations are host-specific and may result in co-speciation between the partners (Noda et al. 2007; Ikeda-Ohtsubo and Brune 2009). The endosymbionts were recruited from a wide range of bacterial phyla, including *Bacteroidota* (Noda et al. 2006), *Spirochaetota* (Ohkuma et al. 2015), *Elusimicrobiota* (Stingl et al. 2005; Ohkuma et al. 2007; Ikeda-Ohtsubo et al. 2007), *Desulfobacterota* (Ikeda-Ohtsubo

<footnote>This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</footnote>

<footnote>© 2025 The Author(s). *Environmental Microbiology* published by John Wiley & Sons Ltd.</footnote>
et al. 2016), *Verrucomicrobiota* (Sato et al. 2014) and *Bacillota* (Takahashi et al. 2023), but also among *Archaea* (Hongoh and Ohkuma 2018; Kaneko et al. 2024).

The first and so far only case of endosymbiotic *Actinomycetota* in termite gut flagellates is a deep-branching clade of *Actinomycetales* that colonise certain *Trichonympha* species in dry-wood termites (Strassert et al. 2012). The draft genome of *Candidatus* Ancillula trichonymphae', which was assigned a sister position to *Bifidobacteriaceae*, revealed their capacity to ferment xylose, N-acetyl-glucosamine and glycerol 3-phosphate through the bifidobacterial pathway (Strassert et al. 2016). However, the diversity of the clade, the presence of related bacteria in other termite lineages, their full metabolic potential and the basis of a potentially mutualistic relationship between the endosymbionts and their flagellate hosts remained unresolved.

To fill this knowledge gap, we investigated the diversity and phylogenetic history of termite-associated *Bifidobacteriaceae* and the genomic changes involved in their adaptation to an intracellular lifestyle using culture-independent molecular and bioinformatic techniques. This includes phylogenomic and functional analyses comparing the metagenome-assembled genomes (MAGs) of termite-associated *Bifidobacteriaceae* to the genomes of their closest relatives, diversity analysis of 16S rRNA genes from cockroach and termite guts, including capillary-picked suspensions of termite gut flagellates, and the subcellular localization of the endosymbionts by fluorescence in situ hybridization (FISH). Finally, we classified the new lineages represented by the MAGs as new genera and species under the Code of Nomenclature of Prokaryotes Described from Sequence Data (SeqCode) (Hedlund et al. 2022).

## 2 | Results

### 2.1 | Phylogeny of Termite-Associated *Bifidobacteriaceae*

Among more than 2000 bacterial MAGs from all major termite lineages (Mies et al. 2024), we recovered 8 high-quality and 3 medium-quality genomes of termite-associated *Bifidobacteriaceae*, which were obtained exclusively from lower termites (Table S1). They represent a monophyletic clade in a well-supported sister position to the members of the family *Bifidobacteriaceae* (Figure 1). The relative evolutionary divergence (RED) values of the individual nodes indicate that the clade comprises four genus-level lineages that fall—albeit deep-branching—into the radiation of the family *Bifidobacteriaceae*. One lineage includes ‘Ca. Ancillula trichonymphae’, the endosymbiont of *Trichonympha paraspiralis* from *Incisitermes marginipennis* (Strassert et al. 2016), and numerous MAGs from a diverse range of kalotermitid, rhinotermitid and hodotermitid hosts. Each of the remaining lineages is represented by MAGs from only one or two termite species, but their host range increased when low-quality MAGs were included in the analysis (Figure A1). All lineages are recognised as separate genera also in the most recent version of GTDB (Parks et al. 2022) and were linked to the corresponding clades in the 16S rRNA-based analysis (see below). Therefore, we propose to classify the members of these lineages in the new genera *Ancillula*, *Opitulatrix*

(g\_JAITBJ01), *Servula* (g\_JAITFB01) and *Nutricula* (g\_JAISFQ01) (see Section 3.5).

### 2.2 | Genome Characteristics

Each of the new genera is represented by at least one high-quality genome (Figure 1). The genome sizes of all termite-associated genera are significantly smaller and their genomic GC content lower than those of other *Bifidobacteriaceae* (Figure 2). After completeness correction with a lineage-specific marker gene set, the smallest genomes and lowest GC contents (average ± SD) were found in the genus *Opitulatrix* (1.23 ± 0.1 Mbp, 33.6% ± 1.3%), whereas the values for *Ancillula* (1.56 ± 0.2 Mbp, 43.0% ± 5.0%) were in the same range as those for *Servula* (1.61 Mbp, 42.0%) and *Nutricula* (2.01 Mbp, 43.5%). The number of tRNA-coding genes and anticodons in the respective tRNAs of termite-associated *Bifidobacteriales* are only slightly lower than in the complete genomes of other *Bifidobacteriaceae* (Table S2), which corroborates the high completeness values of the MAGs. Although the recovery of rRNA genes in the MAGs was poor and their numbers have to be regarded with caution, there is no indication of the presence of more than one rRNA operon in termite-associated *Bifidobacteriaceae* (Table S2). The number of pseudogenes in the genomes of *Ancillula* (26 ± 19), *Nutricula* (17) and *Servula* (16) is higher than in the genomes of the genus *Opitulatrix* (10 ± 2) (Table S2).

### 2.3 | Energy Metabolism

Members of the genera *Ancillula* and *Opitulatrix* encode all enzymes of the fructose 6-phosphate phosphoketolase (F6PPK) pathway but lack phosphofructokinase (PFK) and fructose bisphosphate (FBP) aldolase, which are markers of the Embden-Meyerhoff (EM) pathway (Figure 3). The bifunctional xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp), the key enzyme of the F6PPK pathway, clusters with the homologues of other *Bifidobacteriaceae* (Figure A2). *Ancillula* can generate F6P from N-acetyl glucosamine and *Opitulatrix* from glucose and mannose via substrate-specific uptake and phosphorylation systems (Figures 3, 4 and A3). In addition, both genera encode an ABC transporter for xylose and arabinose and all enzymes for their metabolism via the pentose phosphate pathway (PPP). By contrast, the MAGs of *Nutricula* and *Servula* lack a homologue of Xfp but possess PFK and FBP aldolase, indicating that they metabolise hexoses via the EM pathway (Figure 3).

Members of the genera *Ancillula* and *Opitulatrix* also encode a glycerol 3-phosphate transporter (GlpT) of the organophosphate: inorganic phosphate antiporters (OPA) family, which allows the uptake of sugar phosphates from the host cell. The homologues from this family include only a few biochemically characterised enzymes, such as the phosphoglycerate transporter (PgpT) of *Salmonella typhimurium* and the hexose 6-phosphate transporters of *Escherichia coli* (UhpT) and *Chlamydia pneumoniae* (UhpC) (Figure A4); therefore, their exact substrate specificity remains uncertain. Except for *Nutricula*, termite-associated *Bifidobacteriaceae* also encode the enzymes required for the uptake and metabolism of hexuronic acids via the 2-keto-3-deoxy-phosphogluconate (KDPG) pathway (Figures 3 and 4).
<figure><img src="image_5.png" /><figcaption>**FIGURE 1** | Rank-normalized phylogeny of *Bifidobacteriaceae*, including type species of all genera validly published under the ICNP. Family-level lineages of *Actinomycetales* and selected orders of *Actinomycetia* were included for comparison. The maximum-likelihood tree is based on a concatenated alignment of 120 single-copy marker genes generated by GTDB-Tk and was inferred using IQ-TREE under the LG + F + I + G4 model of evolution. It was normalized using relative evolutionary divergence (RED) values determined with PhyloRank. Bullets on internal nodes indicate ultrafast bootstrap support (●, ≥ 99%; ○, ≥ 95%; 1000 replicates). The number of genomes in the collapsed clades and the type strains of novel genera (Ts) are indicated. The family of the host termite is indicated (H, Hodotermitidae; K, Kalotermitidae; R, Rhinotermitidae). For an expanded version of the tree that includes also low-quality MAGs, see Figure A1.</figcaption></figure>

All termite-associated *Bifidobacteriaceae* encode lactate dehydrogenase and a pyruvate-ferredoxin/flavodoxin oxidoreductase (PFOR), indicating that pyruvate can be reduced to lactate or oxidised to acetyl-CoA. In addition, members of *Ancillula* and *Nutricula* may produce acetyl-CoA via pyruvate-formate lyase. Almost all MAGs encode a bifunctional alcohol/acetaldehyde dehydrogenase (AdhE) that reduces acetyl-CoA to ethanol. This suggests that the main fermentation products are the same as those of other *Bifidobacteriaceae*, which include acetate, lactate, formate, and ethanol (de Vries and Stouthamer 1968; Palframan, Gibson, and Rastall 2003). The presence of PFOR and a [FeFe] hydrogenase of subgroup A1, which are absent from all other *Bifidobacteriaceae*, in members of *Ancillula* and *Opitulatrix* indicate the capacity to form H₂ as an additional fermentation

product (Figure 4). The phylogenies of the [FeFe] hydrogenase (Figure A5) and PFOR (Figure A6) suggest a clostridial origin. The PFOR sequences of *Ancillula* and *Opitulatrix* are most closely related to flavodoxin-dependent homologues, making it likely that they use flavodoxin rather than ferredoxin as a low-potential electron carrier.

Like all *Bifidobacteriaceae*, the termite-associated genera lack pyruvate dehydrogenase and have an incomplete TCA cycle (Figures 3 and 4). The general absence of biosynthetic pathways for cytochromes and quinones from all MAGs is consistent with the anaerobic metabolism characteristic for *Bifidobacteriaceae*. If ATP is generated exclusively via substrate-level phosphorylation, the F-type ATPase of
<figure><img src="image_6.png" /><figcaption>**FIGURE 2** | Genome size and genomic GC content of the order *Actinomycetales*, comparing the termite-associated genera with other members of *Bifidobacteriaceae*.</figcaption></figure>

*Bifidobacteriaceae* is not required for ATP synthesis but rather for the maintenance of the electrochemical membrane potential, as in other bacteria with an obligately fermentative metabolism (Buckel and Thauer 2013).

## 2.4 | Anabolic Pathways

While members of the genera *Ancillula* and *Nutricula* have retained the capacity for *de novo* biosynthesis of most proteinogenic amino acids, the corresponding pathways are mostly incomplete or absent among members of the genera *Opitulatrix* and *Servula* (Figure 5). Most termite-associated genera encode ABC transporters for methionine (MetQ) and aromatic amino acids (AroP) and an amino acid permease (AAP) with a broad substrate range. Although all MAGs of *Opitulatrix* and *Servula* lack the biosynthetic potential for branched-chain amino acids, the corresponding ABC transporter (LivGH) is encoded only by *Opitulatrix cubana* and *Servula enteritis*. Like other *Bifidobacteriaceae*, all genera lack asparagine synthetase (AsnAB) but (except for *Opitulatrix*) encode an unusual aspartyl-tRNA synthetase (AspS2) that loads an aspartyl residue onto tRNA<sup>Asn</sup>, where it is subsequently converted to an asparaginyl residue by aspartyl-tRNA amidotransferase (GatCAB) (Figure A7) (Min et al. 2002).

<figure><img src="image_7.png" /><figcaption>**FIGURE 3** | Key enzymes of major catabolic pathways encoded by MAGs from the termite-associated *Bifidobacteriaceae* and other family members (type species only). Selected representatives of other *Actinomycetales* were included for comparison. Genome size was estimated based on assembly size and completeness of the genomes. More details are shown in (Figures A2 and A3. ABC, ATP-binding cassette; OPA, organophosphate transporter, PTS, phosphotransferase system; Xfp, xylose-5-phosphate/fructose-6-phosphate phosphoketolase; Xpk, xylose-5-phosphate phosphoketolase.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**FIGURE 4** | Comparison of the metabolic pathways of the genera *Ancillula* and *Opitulatrix*. Pathways shown in black are present in both genera; the fructose-6-phosphate phosphoketolase (F6PPK) pathway (bifidobacteria shunt) is highlighted in red. Elements marked in green are present only in *Ancillula*; those marked in brown are present only in *Opitulatrix*. Dashed arrows indicate that a pathway is not present in all MAGs. Fermentation products are highlighted in bold letters, and products of biosynthetic pathways are shown in blue. Detailed biosynthetic pathways for amino acids and cofactors are shown in Figure A12. Non-standard abbreviations: AAP, amino acid permease; ABC, ATP-binding cassette transporter; Amt, ammonium transporter; AroP, aromatic amino acid transporter; Fd/Fld, ferredoxin/flavodoxin; GlcN, glucosamine; GlcNAc, N-acetyl-glucosamine; GlpT, glycerol-3-phosphate transporter; KDPG, 2-keto-3-deoxy-6-phosphogluconate; LivGH, branched-chain amino acid transporter; MetQ, methionine transporter; PLP, pyridoxal 5-phosphate; PPP, pentose phosphate pathway; PRPP, phosphoribosyl pyrophosphate; PTS, phosphotransferase system; SAM, S-adenosylmethionine; THF, tetrahydrofolate; ThrE, threonine/serine exporter; Xfp, xylose-5-phosphate/fructose-6-phosphate phosphoketolase.</figcaption></figure>

Almost all MAGs of *Ancillula* and *Opitulatrix* encode a thre-
onine/serine exporter (ThrE). It is most closely related to ho-
mologues from other *Bifidobacteriaceae* (Figure A8), indicating
that the gene was present already in the common ancestor of the
family but lost in *Servula* and *Nutricula*. Many MAGs encode
an ammonium transporter (Amt), glutamine synthetase and
a putative transaminase, indicating the capacity to assimilate
ammonia, whereas others must acquire combined nitrogen in
the form of amino acids (Figure 4). Genes encoding nitrogenase
(NifHDK) are absent from all MAGs.

The termite-associated genera cannot generally synthesise biotin,
riboflavin, and (unlike other *Bifidobacteriaceae*) also cobalamin
(Figure 5). *Ancillula glyptotermitis* and *A. incisitermitis* are excep-
tions, retaining the potential for the *de novo* synthesis of most B
vitamins (e.g., niacin, tetrahydrofolate, pantothenate, and thia-
mine). However, transport systems for biotin (BioY), riboflavin
(RibU) and folate (FolT) are absent from all genera but *Ancillula*.

Peptidoglycan biosynthesis is conserved in all lineages of
termite-associated *Bifidobacteriaceae* (Figure A9). An exception

is the pathway for diaminopimelic acid (L-DAP) production,
which is conserved only in the genus *Ancillula*, indicating a re-
quirement for lysine in all other lineages. The capacity to syn-
thesise murein is in agreement with the thick cell wall observed
in electron micrographs of *Ancillula trichonymphae* (Strassert
et al. 2012).

2.5 | Other Genomic Features

The annotation of the genes involved in DNA repair mecha-
nisms revealed that termite-associated *Bifidobacteriaceae*
lack many of the genes that in *Escherichia coli* are involved in
fixing DNA damage, particularly in mismatch repair (MMR)
pathways (Figure A10). However, numerous gene loci involved
in mismatch repair (*mutS*, *mutL*) and homologous recombina-
tion (*recABC*, *recD*, *recF*, *recG*, *recJ*, *recO*, *recR*) are generally
absent from *Bifidobacteriaceae* and other *Actinomycetales*.
Transposable elements, prophages, and CRISPR structures
and their associated *cas* genes were not detected in any of
the MAGs.
<figure><img src="image_9.png" /><figcaption>**FIGURE 5** | Capacities for biosynthesis and transport of amino acids and co-factors, and key genes for the assimilation of ammonia of termite-associated *Bifidobacteriaceae* and other family members (type species only). The full pathways and other details are shown in (Figures A7 and A12). Amt, ammonium transporter; AroP, aromatic amino acid transporter; AzlC, branched-chain amino acid transporter; BioY, biotin transporter; FolT, folate transporter; LysE, lysine exporter; LivGH, branched-chain amino acid transporter; MetQ, methionine transporter; ProV, glycine/proline transporter; RibU, riboflavin transporter; ThrE, threonine/serine exporter.</figcaption></figure>

A search for gene clusters involved in secondary metabo-
lite biosynthesis using automated annotation pipelines was
negative. We found no evidence for the biosynthesis of bacte-
riocins such as bifidins or lantibiotics that are produced by
other *Bifidobacteriaceae* (Cheikhyoussef et al. 2010; Lee, Li,
and O'Sullivan 2011). Extracellular structures like sortase-
dependent pili and tight adherence pili, which are essential for
host colonisation in the genus *Bifidobacterium* (Alessandri, van
Sinderen, and Ventura 2021), are not encoded by the termite-
associated genera. Likewise, there was no indication of a type IV
secretion system, which seems to be present in other bifidobac-
terial species (Guglielmetti et al. 2014).

The catalogue of carbohydrate-active enzymes (CAZymes) encoded by termite-associated *Bifidobacteriaceae* reveals the presence of several glycosyl hydrolases (GHs) and glycosyl transferases (Table S3). The majority of the GHs belong to the families GH13, GH1 and GH3 and are involved in glycogen metabolism. Only very few of the gene products were predicted to encode signal peptides that would indicate secretion into the extracellular space. GHs acting on polymers of plant cell walls were not detected.

2.6 | Diversity and Potential Flagellate Hosts

To assess the diversity of termite-associated *Bifidobacteriaceae*
and their potential association with specific flagellates, we com-
piled previously unpublished 16S rRNA gene sequences from

clone libraries and long-read amplicon libraries of termite and
cockroach gut microbiota obtained in our laboratory, includ-
ing those from numerous capillary-picked suspensions of ter-
mite gut flagellates (Table S4). Together with 16S rRNA genes
extracted from metagenomic libraries and MAGs from a wide
range of termites (Tables S1 and S2) (Hervé et al. 2020; Mies
et al. 2024), the dataset considerably expanded the known di-
versity of the termite-associated *Bifidobacteriaceae* (Figure 6).

Phylogenetic analysis revealed that all sequences of
*Bifidobacteriaceae* from termites formed a monophyletic group
(Figure 6). The largest clade comprised sequences of the genus
*Ancillula*, represented by the 16S rRNA genes obtained from
the MAGs that were assigned to this genus (Figures 1 and A1)
and related phylotypes from clone or amplicon libraries and
metagenomic dataset of other termites. One subclade con-
sists exclusively of phylotypes from flagellate suspensions of
*Trichonympha* spp. from dry-wood termites (Kalotermitidae)
or whole-gut samples of Kalotermitidae that harbour flagel-
lates of this genus. They include *Ancillula trichonymphae*, the
endosymbiont of *Trichonympha* spp. from *Incisitermes mar-
ginipennis* and *Incisitermes schwarzi* (Strassert et al. 2012),
and novel phylotypes from *Trichonympha* suspensions of
*I. tabogae* and *I. incisus*. The reads assigned to the genus
*Ancillula* dominate the amplicon libraries of capillary-picked
*Trichonympha* suspensions from the genus *Incisitermes*
(16%-81% of the bacterial reads) but are less abundant in
*Trichonympha* suspensions from *Glyptotermes* sp. 5 (0.38%)
(Table S5). The other subclades of *Ancillula* consist mostly of
<figure><img src="image_10.png" /><figcaption>**FIGURE 6** | 16S rRNA-based phylogeny of *Bifidobacteriaceae*, illustrating the relationship of the phylotypes recovered from whole-gut libraries of termites and cockroaches and capillary-picked suspensions of termite gut flagellates to the type species of all other genera. Accession numbers of novel sequences are shown in bold. The number of sequences in the collapsed clades and the type strains of novel genera (Ts) are indicated. The host species are indicated (termite family in parentheses: A, Archotermopsidae, H, Hodotermitidae; Hs, Hodotermopsidae, K, Kalotermitidae; R, Rhinotermitidae). Sequences from MAGs are marked in red. The maximum-likelihood tree is based on a curated alignment of near full-length 16S rRNA genes (1462 positions) and was generated using IQ-TREE under the SYM + R4 model of evolution. Bullets on internal nodes indicate ultrafast bootstrap support (●, ≥99%, ○, ≥95%, 1000 replicates). Other *Actinomycetales* were used as outgroups. Symbols created with **BioRender.com**.</figcaption></figure>

phylotypes from whole-gut samples of other termite species, including *Kalotermes italicus* and *Neotermes cubanus*. Here, a larger proportion of *Ancillula* reads (0.99%-3.10%) in whole-gut libraries of *K. italicus* than in the associated suspensions of *Joenia* spp. (0.18%-0.68%) suggest a contamination with bacterial symbionts of other gut flagellates, so that the identity of their host remains elusive.

The other clades comprised sequences that were assigned to the genera *Opitulatrix*, *Servula* and *Nutricula*, based on the 16S rRNA genes obtained from the corresponding MAGs (Figures 1 and A1) and related phylotypes from amplicon libraries and metagenomic datasets of other termites. In all cases, the termite-specific *Bifidobacteriaceae* account only for a small number of reads in the respective libraries (Table S5). None of these libraries was derived from capillary-picked *Trichonympha* suspensions or whole-gut samples of termites harbouring these flagellates, indicating that they colonise flagellates of

other genera. The genus *Servula* comprises a phylotype from a flagellate suspension of *Devescovina* (Cristamonadida) species from *Procryptotermes leewardensis* (0.04%), and the genus *Nutricula* was associated, albeit at very low abundance (0.14%), with another cristamonadid flagellate, *Joenia annectens*, from *Kalotermes italicus* (Table S5). Sequences of the genus *Opitulatrix* were linked to the phylogenomic dataset by MAGs from *Neotermes cubanus* and *Roisinitermes ebogensis* (0.06% in a whole-gut sample of *Neotermes cubanus*).

In addition, we recovered a novel genus-level lineage of uncultured *Bifidobacteriaceae* from whole-gut libraries of several cockroaches (Table S5). The phylotypes did not cluster with the termite-associated lineages but fell into the radiation of other *Bifidobacteriaceae*, with members of the genus *Aeriscardovia* as the closest relatives (Figure 6). They were represented in all cockroach samples investigated but made up only a small fraction (0.01%-1.1%) of the reads in the respective libraries (Table S5).
## 2.7 | Subcellular Localization

The intracellular location of *Ancillula trichonymphae* in the region is anterior to the nucleus in *Trichonympha* spp. of *Incisitermes schwarzi* and *I. marginipennis* has been documented already with FISH (Strassert et al. 2012). Using the same protocol, we assessed the subcellular localization patterns of other termite-associated *Bifidobacteriaceae* in fixed hindgut content of several kalotermitids using dual hybridization with general bacterial (Eub-338, fam-labelled) and taxon-specific (Act-490, cy3-labelled) probes. In the case of *Incisitermes incisus* and *Incisitermes tabogae*, we observed a large number of bacterial cells hybridising with the oligonucleotide probe Act-490 in *Trichonympha* spp. associated with the respective termite hosts (Figure 7), showing the same accumulation at the anterior pole of the host cell as previously documented for other *Incisitermes* spp. (Strassert et al. 2012). Simultaneous hybridization with a general bacterial probe (EUB338) indicated colonisation of the *Trichonympha* cells also by other bacterial symbionts. Although the target region of probe Act-490 is conserved among all representatives of termite-associated *Bifidobacteriaceae*, the fixed hindgut content of *Glyptotermes* species 5, *Kalotermes flavicollis* and *Kalotermes italicus* showed only cells hybridising with the bacterial probe.

## 3 | Discussion

Our study documents the diversity of termite-associated *Bifidobacteriaceae*, their distribution across a wide range of termite species, and their association with specific gut flagellates. Comparative genome analysis demonstrated the presence of four genus-level lineages that share many metabolic traits with *Bifidobacteriaceae* associated with mammalian hosts. However, their intracellular lifestyle led to strong genome erosion and the acquisition of new functions from other termite gut microbiota via horizontal gene transfer (HGT).

### 3.1 | Catabolism by Termite-Associated *Bifidobacteriaceae*

Members of the family *Bifidobacteriaceae* are distinguished from other *Actinomycetales* by the lack of pyruvate dehydrogenase,

an incomplete TCA cycle, and the absence of a respiratory metabolism (Genilloud 2017). The termite-associated genera are no exception. Like other *Bifidobacteriaceae*, the members of the genera *Ancillula* and *Opitulatrix* metabolise hexoses via the fructose 6-phosphate phosphoketolase (F6PPK) pathway (Scardovi 1965). This pathway, also known as 'bifidobacterium shunt' generates 0.5 ATP more per glucose than the Embden-Meyerhof (EM) pathway (Schramm, Klybas, and Racker 1958). The bifunctional phosphoketolase (Xfp) of *Bifidobacteriaceae*, which uses both xylulose 5-phosphate and fructose 6-phosphate as substrates (Grill, Crociani, and Ballongue 1995), forms a monophyletic group in the radiation of the monofunctional xylulose-5-phosphate phosphoketolase (Xpk) of *Actinomycetota* (Figure A2), which serves exclusively in the metabolism of pentoses (Meile et al. 2001; Xiong et al. 2015). The genera *Servula* and *Nutricula* are notable exceptions. They lack Xfp but possess phosphofructokinase and fructose biphosphate aldolase (Figure 3), suggesting that they retained the ancestral trait of *Actinomycetales* and metabolise hexoses via the EM pathway (Genilloud 2017).

While members of the termite-associated clade probably form the same fermentation products as other *Bifidobacteriaceae*, they differ in their capacity to produce hydrogen. In addition to pyruvate-formate lyase, which is present in most *Bifidobacteriaceae*, the termite-associated genera encode PFOR and an [FeFe] hydrogenase of subgroup A1 (Figure 3), which are probably coupled by flavodoxin and allow the generation of additional ATP by substrate-level phosphorylation, provided that the hydrogen partial pressure of the environment is low enough to release reducing equivalents as H₂ (Schuchmann, Chowdhury, and Müller 2018). Measurements of the intestinal H₂ partial pressure showed that *Kalotermes flavicollis* and *Incisitermes marginipennis* accumulate H₂ in the hindgut paunch colonised by flagellates, while *Neotermes* and other kalotermitids do not (Desai and Brune 2012).

Many host-associated *Bifidobacteriaceae* that colonise the gastrointestinal tract of mammals produce CAZymes that are secreted into the extracellular space and involved in the breakdown of complex polysaccharides (Pokusaeva, Fitzgerald, and van Sinderen 2011). However, the majority of GHs encoded by termite-associated *Bifidobacteriaceae* are α-glucosidases of the family GH13 (Table S3), which are common also in bifidobacterial species colonising the human gut (Milani et al. 2015)

<figure><img src="image_11.png" /><figcaption>**FIGURE 7** | Fluorescence in situ hybridization analysis of the hindgut content of *Incisitermes tabogae* (A) and *I. incisus* (B). Bacterial cells were hybridised with the general *Bacteria* probe EUB338 (Fam-labelled, green) and the probe ACT490 specific for the termite-associated lineages (Cy3-labelled, orange). Outlines of *Trichonympha* cells were drawn based on phase-contrast images of the same slides. Scale bars: 100 µm (A) and 50 µm (B).</figcaption></figure>
and involved in the breakdown of amylose and glycogen. We
found no evidence for extracellular xylanases, arabinofuranosidases or xylosidases that break down plant-derived glycans
in other bifidobacterial species (Kelly, Munoz-Munoz, and
van Sinderen 2021; Orihara et al. 2023). This is in agreement
with the hypothesis that endosymbiotic *Bifidobacteriaceae* use
monosaccharides, hexuronates, and sugar phosphates as energy
substrates.

3.2 | Genome Erosion

Termite-associated *Bifidobacteriaceae* have significantly smaller genomes and a lower genomic GC content than their gut-associated but free-living relatives. While the copy number of rRNA-specifying operons in the genomes of other *Bifidobacteriaceae* ranges between two and five (Bottacini et al. 2014), the termite-associated lineages seem to possess only one set of rRNA genes. Although rRNA genes are often excluded during the binning procedure, a low copy number is part of the genome streamlining common in intracellular bacteria and would be in agreement also with the low growth rates of intracellular symbionts. Genome erosion is most pronounced in the genus *Opitulatrix*, whose genome sizes and GC content are substantially lower than those of their free-living relatives (Figure 2), as observed also for other endosymbionts of termite gut flagellates (Mies et al. 2024). Intracellular symbionts are prone to harmful genetic drift, where increased mutation rates and the relaxed selection of non-essential traits lead to progressive gene loss (McCutcheon and Moran 2012). It is generally assumed that the primary cause of genome erosion is the loss of DNA repair mechanisms and the subsequent accumulation and fixation of point mutations (Hershberg and Petrov 2010). In intracellular *Mycoplasma* and *Spiroplasma* species, the decreased gene repertoire in the mismatch repair pathways has been linked to increased substitution rates and rapid genome evolution (Delaney et al. 2012; Gerth et al. 2021). However, the general absence of many genes involved in DNA repair also from other *Actinomycetales* (Figure A10) indicates that these are not gene losses related to an intracellular lifestyle. Rather, the presence of a non-canonical mismatch repair pathway in *Actinobacteria* and *Archaea* (Castañeda-García et al. 2017) indicates that actinobacteria might have further DNA repair mechanisms that remain elusive.

Despite the considerable genome reduction, the genera *Ancillula* and *Nutricula* have retained many biosynthetic pathways present also in host-associated but free-living *Bifidobacteriaceae* (Figures 3 and 5). By contrast, members of the genera *Opitulatrix* and *Servula* have lost the capacity for the *de novo* biosynthesis of most proteinogenic amino acids, which leaves them fully reliant on the acquisition of amino acids from the host environment (Figure 5).

3.3 | Acquisition of New Functions

Termite-associated *Bifidobacteriaceae* possess several func-
tions that are most likely acquired by horizontal gene transfer
from other gut bacteria. One example is PFOR, which is gen-
erally absent from *Actinomycetales*. The homologues encoded

by the termite-associated clade fall into the radiation of pyru-
vate-flavodoxin oxidoreductases from two groups of uncul-
tured *Lachnospiraceae* and *Acutalibacteraceae* (*Clostridia*)
that comprise many representatives from termite guts, includ-
ing other endosymbionts of flagellates, such as members of
the genus *Trichonympha* (Figure A6) (Takahashi et al. 2023;
Mies et al. 2024). This is in accordance with the phylogeny of
the accompanying subgroup A1 [FeFe] hydrogenase, in which
*Acutalibacteraceae* homologues from the termite gut also share
the phylogenetic neighbourhood with sequences from *Ancillula*
and *Opitulatrix* (Figure A5). In that context, it is important that
also the folate transporter (FolT) found in the genus *Ancillula*
has been acquired by lateral gene transfer from other gut mi-
crobiota. Notably, phylogenetic analysis places it among homo-
logues in the family *Oscillospirales*, with *Acutalibacteraceae*
again as close relatives (Figure A11).

Another example is the sugar-phosphate transporter (OPA)
present in *Ancillula* and *Opitulatrix*, which is phylogenetically
embedded among homologues from MAGs of *Lactobacillaceae*
that were also obtained from termite guts (Figure A4). Although
its exact substrate specificity is uncertain, any OPA would
provide the capacity to utilise sugar phosphates, which are in
ample supply in the cytoplasm of the host. OPAs are consid-
ered a hallmark of an intracellular lifestyle (Jeckelmann and
Erni 2020). Their independent acquisition also by members
of the genus *Endomicrobiellum* and putatively endosymbiotic
*Alphaproteobacteria* and *Verrucomicrobiae* documents conver-
gent evolution among unrelated intracellular symbionts of ter-
mite gut flagellates (Mies et al. 2024).

Convergent evolution is also evidenced by the acquisi-
tion of a hexuronate transporter (ExuT) and all enzymes
of the 2-keto-3-deoxy-phosphogluconate (KDPG) pathway
(Suvorova et al. 2011) by termite-associated *Bifidobacteriaceae*
(Figure 4). The capacity to metabolise hexuronic acids is a
common trait of many intracellular symbionts of termite gut
flagellates, including *Azobacteroides pseudotrichonymphae*
(*Bacteroidota*) (Hongoh et al. 2008), *Ancillula trichonymphae*
(Strassert et al. 2016), and *Endomicrobiellum trichonym-
phae* (*Elusimicrobiota*) (Zheng, Dietrich, and Brune 2017).
Comparative analysis of a wide range of MAGs from termite
guts revealed that the KDPG pathway is present in all mem-
bers of these endosymbiotic lineages and was independently
acquired from other gut bacteria, possibly as an adaptation to
the products of hemicellulose digestion provided by their flag-
ellate host (Mies et al. 2024).

3.4 | Interaction With Flagellate Host

While the acquisition of new metabolic functions by the en-
dosymbionts of termite gut flagellates provides access to
substrates provided by the host environment, such as sugar
phosphates and hexuronates, the basis for a mutualistic in-
teraction is unclear. Although the biosynthetic capacities of
termite gut flagellates are unknown, it is generally assumed
that the symbionts provide their hosts with important nutri-
ents such as cofactors and amino acids, which are scarce in
the lignocellulose-rich diet of termites. However, the termite
gut itself is more nutrient-rich, and an export of essential
nutrients from the symbiont to the host remains to be documented. Moreover, functional traits that are potentially beneficial for the host are rarely common among all members of an endosymbiotic lineage.

It is therefore remarkable that the threonine/serine exporter (ThrE) of *Actinomycetales* is conserved in the genera *Ancillula* and *Opitulatrix* (Figures 4 and 5). ThrE is an active transporter that was first discovered in *Corynebacterium glutamicum*, where it serves to avoid the intracellular accumulation of threonine and structurally related solutes (Simic, Sahm, and Eggeling 2001). Homologues are present in most *Bifidobacteriaceae* and many other members of the order *Actinomycetales* (Figure A8). ThrE is the first amino acid exporter identified in the genome of a flagellate endosymbiont and may provide the basis for a nutritional symbiosis at least between members of the genus *Ancillula* and their flagellate hosts. In the case of the genus *Opitulatrix*, the loss of the capacity for threonine and serine production, together with a much lower symbiont load (Table S5), suggests that formerly mutualistic symbionts have lost their beneficial role due to progressive genome erosion. They may eventually disappear from their flagellate hosts because they no longer provide a selective advantage. A similar case exists in certain *Endomicrobiellum* lineages, which compensated for severe gene losses in energy metabolism with the acquisition of an ATP/ADP antiporter (Mies et al. 2024). However, direct evidence for an exchange of metabolites between gut flagellates and their bacterial symbionts is so far lacking.

Another potentially beneficial role of termite-associated *Bifidobacteriaceae* is the supplementation of essential cofactors required by their flagellate hosts. *Bifidobacteriaceae* colonising the mammalian gut are avid producers of various B vitamins (LeBlanc et al. 2013), and the provision of B vitamins is a common trait among intracellular symbionts of insect tissues (Blow et al. 2020; Michalik et al. 2023). Nothing is known about the biosynthetic capacities of termite gut flagellates, but since most termite-associated *Bifidobacteriaceae* have lost a significant amount of their biosynthetic potential for vitamin production (Figure 5), the provision of B vitamins is an unlikely basis for the symbiosis.

*Bifidobacteriaceae* are also common in the intestines of honeybees, bumblebees and wasps (Kopecný et al. 2010). The bifidobacterial species in these insects represent a different lineage (Bifidobacterium+Bombiscardovia) from those found in cockroaches and termites (Figure 6), suggesting that these symbionts were acquired independently at least three times by different host lineages. *Bifidobacterium* spp. that colonise the guts of honey bees have been attributed to protective functions against bacterial pathogens (Forsgren et al. 2010; Vásquez et al. 2012). Similar to the bifidobacterial lineages found in mammals, they also influence the production of developmental hormones in the host insect (Kešnerová et al. 2017) and may play a role in hemi-cellulose degradation by producing various carbohydrate-active enzymes (Bottacini et al. 2012; Kwong and Moran 2016). The loss of these functions in termite-associated *Bifidobacteriaceae* is most likely an adaptation to their intracellular environment within the flagellate host.

<table><caption>**TABLE 1** | New taxa of termite-associated *Bifidobacteriaceae* proposed under SeqCode and the designated taxonomic types. The full protologues are in appendix (Text B).</caption><thead><tr><th>New or emended taxa</th><th>Taxonomic type (genome acc. no.)</th></tr></thead><tbody><tr><td><strong>Family</strong></td><td></td></tr><tr><td><em>Bifidobacteriaceae</em><br>Stackebrandt, Rainey, and Ward-Rainey 1997 emend. Kästle Silva and Brune</td><td><em>Bifidobacterium bifidum</em></td></tr><tr><td><strong>Genus</strong></td><td></td></tr><tr><td><em>Ancillula</em> gen. nov. Strassert and Brune</td><td><em>Ancillula trichonymphae</em><br>sp. nov.</td></tr><tr><td><em>Nutricula</em> gen. nov. Kästle Silva and Brune</td><td><em>Nutricula glyptotermitis</em><br>sp. nov.</td></tr><tr><td><em>Opitulatrix</em> gen. nov. Kästle Silva and Brune</td><td><em>Opitulatrix roisinitermitis</em><br>sp. nov.</td></tr><tr><td><em>Servula</em> gen. nov. Kästle Silva and Brune</td><td><em>Servula neotermitis</em><br>sp. nov.</td></tr><tr><td><strong>Species</strong></td><td></td></tr><tr><td><em>Ancillula trichonymphae</em> sp. nov. Strassert and Brune</td><td>GCA_000485475.1</td></tr><tr><td><em>Ancillula hodotermitis</em> sp. nov. Kästle Silva and Brune</td><td>GCA_031262545.1</td></tr><tr><td><em>Ancillula glyptotermitis</em> sp. nov. Kästle Silva and Brune</td><td>GCA_031268675.1</td></tr><tr><td><em>Ancillula kalotermitis</em> sp. nov. Kästle Silva and Brune</td><td>GCA_031273495.1</td></tr><tr><td><em>Ancillula prorhinotermitis</em> sp. nov. Kästle Silva and Brune</td><td>GCA_031280795.1</td></tr><tr><td><em>Nutricula glyptotermitis</em> sp. nov. Kästle Silva and Brune</td><td>GCA_031263495.1</td></tr><tr><td><em>Opitulatrix roisinitermitis</em> sp. nov. Kästle Silva and Brune</td><td>GCA_031288355.1</td></tr><tr><td><em>Opitulatrix cubana</em> sp. nov. Kästle Silva and Brune</td><td>GCA_031283665.1</td></tr><tr><td><em>Servula neotermitis</em> sp. nov. Kästle Silva and Brune</td><td>GCA_031281695.1</td></tr></tbody></table>

## 3.5 | Taxonomy

The delineation of the family *Bifidobacteriaceae* is based on phylogenetic criteria and supported by biochemical characteristics such as the presence of fructose-6-phosphate phosphoketolase activity (Biavati 2015; Stackebrandt, Rainey, and Ward-Rainey 1997). Based on 16S rRNA gene phylogenies reconstructed with distance-based methods, where *Bifidobacteriaceae* occupied a sister position to *Actinomycetales*, the family was
classified in the order *Bifidobacteriales* (Stackebrandt, Rainey, and Ward-Rainey 1997; Zhi, Li, and Stckebrandt 2009). However, phylogenomic analyses using concatenated gene trees place *Bifidobacteriaceae* within the radiation of *Actinomycetales* (GTDB, Figure 1), rendering the latter polyphyletic. Therefore, we propose to transfer the family *Bifidobacteriaceae* to the order *Actinomycetales* and to delete the now vacant order *Bifidobacteriales* from the class *Actinomycetia* under the rules of the *International Code of Nomenclature of Prokaryotes* (ICNP, Oren et al. 2023). An emended description of the family *Bifidobacteriaceae* is given below.

The current GTDB taxonomy (release 220) recognises the ge-
nomes of termite-associated *Bifidobacteriaceae* as four genus-
level lineages of the family *Bifidobacteriaceae*, which is in
agreement with our phylogenomic analysis and the RED values
of the internal nodes (Figure 1). Further support for their in-
clusion in the family include the synapomorphies shared with
other family members, such as loss of pyruvate dehydrogenase
and aerobic metabolism, and the metabolism of hexoses via the
fructose-6-phosphate phosphoketolase pathway.

The presence of both high-quality genomes and 16S rRNA gene
sequences for most lineages allows us to propose new taxa
under the Code of Nomenclature of Prokaryotes Described
from Sequence Data (SeqCode) (Hedlund et al. 2022; Whitman
et al. 2022). The names of the new genera and species are listed
in Table 1, along with the designated nomenclatural types.
Moreover, we propose to remove the order of *Bifidobacteriales*
and transfer the family *Bifidobacteriaceae* to the order
*Actinomycetales*. The protologues including etymologies and
the full description of all taxa can be found in appendix (Text B).

**3.5.1 | Emended Description of Bifidobacteriaceae**
**Stackebrandt, Rainey, and Ward-Rainey 1997**

Family defined by phylogenomic analysis. Members form a
monophyletic group that shows a relative evolutionary divergence
(RED) similar to that of the neighbouring families. Contains the
genera 'Ancillula' Strassert and Brune, *Aeriscardovia* Simpson
et al. 2004, *Alloscardovia* Huys et al. 2007, *Bifidobacterium* Orla-
Jensen 1924, *Bombiscardovia* Killer et al. 2010, *Galliscardovia*
Pechar et al. 2017, *Gardnerella* Greenwood and Pickett 1980,
*Metascardovia* Okamoto et al. 2007, *Neoscardovia* García-Aljaro
et al. 2012, 'Nutricula' Kästle Silva and Brune, 'Opitulatrix'
Kästle Silva and Brune, *Parascardovia* Jian and Dong 2002,
*Pseudoscardovia* Killer et al. 2013, *Scardovia* Jian and Dong
2002, and 'Servula' Kästle Silva and Brune.

Type genus: *Bifidobacterium* Orla-Jensen 1924 (Approved Lists 1980).

Parent taxon: *Actinomycetales* Buchanan 1917 (Approved Lists 1980).

**3.6 | Conclusion**

Termite-associated *Bifidobacteriaceae* are the first represen-
tatives of *Actinomycetota* that occur intracellularly in protists.

They are found exclusively in lower termites but are highly abun-
dant only in the genus *Incisitermes*, where members of the genus
*Ancillula* colonise gut flagellates of the genus *Trichonympha*.
Substantial genome erosion in all genus-level lineages strongly
suggests that also the other genera are intracellular symbionts,
but their specific hosts remain elusive. A genus-level lineage of
putatively free-living *Bifidobacteriaceae* detected in cockroach
guts is only distantly related. The conservation of numerous
biosynthetic pathways in the genus *Ancillula* and a threonine/
serine exporter in most lineages provides the first direct evi-
dence for the long-standing hypothesis that the intracellular
symbionts of termite gut flagellates provide their hosts with
amino acids. In other genera, however, many of the metabolic
capacities were lost, suggesting that the originally mutualistic
symbiosis is on its decline. Our study strongly supports the hy-
pothesis that the evolution of flagellate symbionts is influenced
by both progressive genome erosion and the acquisition of new
functions. These new functions not only compensate for gene
losses but also provide convergent adaptations to the intra-
cellular lifestyle through horizontal gene transfer from other
endosymbionts.

**4 | Experimental Procedures**

**4.1 | Annotation of Metagenome-Assembled Genomes**

The MAGs of termite-associated *Bifidobacteriaceae* were ob-
tained in the course of a metagenomic study of numerous lower
and higher termites (Mies et al. 2024). For a detailed list of MAGs
and reference genomes included in the present study, see Table S2.
Completeness and contamination of MAGs were assessed using
CheckM (Parks et al. 2015; Chklovski et al. 2023). The absence
of four single-copy genes in the lineage-specific marker set of
CheckM v.1. from all genomes of *Bifidobacteriaceae*, including
the circularised genomes of all type species (Table S6), caused a
considerable bias in the completeness values. The situation did
not improve when CheckM v.2 was used, which is based on a
reference-independent machine-learning algorithm. Therefore,
we removed the offending genes from the set of marker genes
used by CheckM v.1. MAGs with > 90% completeness and < 5%
contamination were considered high-quality (HQ) genomes,
those with > 50% completeness and < 10% contamination
medium-quality (MQ) genomes.

**4.2 | Genome Annotation and Phylogenomic Analysis**

The GTDB reference database (r.220) (Parks et al. 2022) and
the accompanying GTDB-Toolkit (v. 2.4) (Chaumeil et al. 2022)
standard pipeline for the classification of MAGs and genomes
were used to align and classify our MAGs together with refer-
ence *Actinomycetales* and *Bifidobacteriaceae* genomes. The tool-
kit uses both the average nucleotide identity (ANI) and the RED
scores to infer taxonomic classification. The multiple sequence
alignment (MSA) output based on 120 bacterial marker genes
was used to reconstruct a maximum-likelihood genome tree in
IQ-TREE (Minh et al. 2020). Trees were inferred using the best
model of evolution recommended by Model Finder and using
UFBoot (Hoang et al. 2018) and the Shimodaira–Hasegawa approximate-likelihood ratio test (SH-aLRT) (Guindon et al. 2010) for node support assessment.

Genes were predicted and annotated using Prokka (Seemann 2014). Ghost koala (Kegg) (Kanehisa, Sato, and Morishima 2016) was used for automated MAGs annotation, and SignalP 6.0 (Teufel et al. 2022) software was used for signal peptide prediction in the identification of secreted proteins and transport systems. Since the termite-associated *Bifidobacteriaceae* represent a novel lineage poorly represented in public databases, the annotation of the MAGs was refined by Hidden Markov Model (HMM) searching in HMMER version 3.3 (Finn, Clements, and Eddy 2011) using hmmsearch (Eddy 2011) and HMMs from Pfam (release 33.1) (El-Gebali et al. 2019) and TIGRFAM (Haft, Selengut, and White 2003) (release 15.0) databases against a database containing annotated termite gut MAGs as well as curated sequences from SwissProt (The UniProt Consortium 2023). Results were imported for visualisation into iTOL (v.3) (Letunic and Bork 2016). Identification of pseudogenes is based on the automatic annotation of NCBI PGAP (Li et al. 2020). Clustered regularly interspaced palindromic repeats (CRISPR) arrays and their associated cas proteins were searched for in the MAGs using CRISPRCasFinder (Couvin et al. 2018). Transposable elements were predicted in the MAGs using Issaga (Varani et al. 2011).

Additionally, antiSMASH 7.1 (Blin et al. 2023) was used for the annotation and analysis of secondary metabolite biosynthetic gene clusters. For the annotation and classification of carbohydrate-active enzymes, the MAGs were submitted to dbCAN3 (Zheng et al. 2023). Hydrogenases were classified with the HydDB database (Søndergaard, Pedersen, and Greening 2016).

To create the protein phylogenies, we gathered amino acid sequences from the termite-associated *Bifidobacteriaceae* MAGs. Homologues were then extracted from a custom database containing termite gut MAGs and non-redundant high-quality SWISS-PROT entries using BlastP (v. 2.6.0). These sequences were aligned using MAFFT (v. 7.427) and used to build a tree with IQ-TREE, using the best model of evolution recommended by Model Finder (Kalyaanamoorthy et al. 2017; Minh et al. 2020). Node support was assessed using SH-aLRT (Guindon et al. 2010) and ultrafast bootstrap analysis (UFBoot; Hoang et al. 2018). The alignment and consensus tree were then imported into Arb software (v. 6.1) (Ludwig et al. 2004), where we made manual improvements. If needed, we exported the alignment to generate a second tree using the same settings.

## 4.3 | 16S rRNA-Based Diversity

Full-length 16S rRNA gene sequences were extracted from gut metagenomes of lower and higher termites (Table S1) and MAGs of termite-associated *Bifidobacteriaceae* (Table S2) using Barrnap (v. 0.9) (Seemann 2013). Together with sequences previously obtained from clone libraries and long-read amplicon libraries (Table S4), they were imported into the ARB-SILVA database (v. 138.1) (Quast et al. 2013) using the ARB software package (v. 7.02) (Ludwig et al. 2004), aligned with SINA (v1.2.12) (Pruesse, Peplies, and Glöckner 2012), and placed into the phylogenetic framework of Silva v.138.1. The overall alignment was

manually curated using the alignment editor integrated into ARB (Protasov et al. 2023). The improved alignment was exported and a maximum-likelihood tree was inferred with IQ-TREE using the substitution model suggested by ModelFinder. Node support was assessed with SH-aLRT and UFBoot.

## 4.4 | Fluorescence In Situ Hybridization

*Incisitermes incisus* and *Incisitermes tabogae* (Gualedoupe), *Glyptotermes* species 5 (Cameroon), and *Kalotermes italicus* (Italy) were collected in the field. *Kalotermes flavicollis* was obtained from the Bundesanstalt für Materialforschung (BAM) in Berlin, Germany. The FISH followed a previously published protocol, using gut contents of live specimens fixed with paraformaldehyde (Strassert et al. 2012). Termite-associated *Bifidobacteriaceae* and other bacteria were visualised by double hybridization with the previously established oligonucleotide probe ACT490 (5'-AGCCGGTCCTTTCTTGTTAG-3'; Strassert et al. 2012) and a general *Bacteria*-specific probe (EUB338); non-specific probe binding was checked by additional hybridizations using a nonsense probe (NON338EUB; Wallner, Amann, and Beisker 1993).

### Author Contributions

**Joana Kästle Silva:** conceptualization, methodology, data curation, investigation, validation, formal analysis, supervision, visualization, writing – original draft, project administration, writing – review and editing.
**Vincent Hervé:** data curation, formal analysis, resources, writing – review and editing.
**Undine S. Mies:** data curation, resources.
**Katja Platt:** resources, writing – review and editing, methodology.
**Andreas Brune:** writing – original draft, writing – review and editing, visualization, supervision, resources, funding acquisition, conceptualization, methodology, validation, data curation, project administration, formal analysis, investigation.

### Acknowledgements

This work was supported by the Max Planck Society (MPG), JMKS and USM received scholarships from the International Max Planck Research School 'Principles of Microbial Life: From molecules to cells, from cells to interactions'. We thank Rudy Plarre (Bundesanstalt für Materialforschung und -prüfung, Berlin), Jan Šobotník (Czech University of Life Sciences, Prague) and Mario Marini (University of Bologna) for providing termites, Sebastian Treitli for the hydrogenase reference alignment, and Jakob Wenning for his contributions during a student project. Open Access funding enabled and organized by Projekt DEAL.

### Conflicts of Interest

The authors declare no conflicts of interest.

### Data Availability Statement

The metagenomic datasets used in this study are available in the NCBI Sequence Read Archive (SRA) (Table S1). The GenBank accession numbers of the MAGs are listed in Table S2. For the GenBank accession numbers of the 16S rRNA gene sequences obtained in this study, see Tables S1, S2 and S4. The Supporting information, including tables, can be accessed using the following DOI: 10.5281/zenodo.14186872.

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## Supporting Information

Additional supporting information can be found online in the Supporting Information section.
Appendix A

<figure><img src="image_12.png" /><figcaption>**FIGURE A1** | Phylogenomic tree of *Bifidobacteriaceae*, including the names of all genera validly published under the ICNP and low-quality (LQ) MAGs from termite guts. The maximum-likelihood tree is based on an alignment of 120 single-copy marker genes generated by GTDB-Tk and was inferred with IQ-TREE under the LG + F + I + G4 model of evolution. Numbers at internal nodes indicate ultrafast bootstrap support (in percent, 1000 replicates). MAGs with 16S rRNA genes are in red typeface, type strains of new species are printed in bold. The host families are indicated in parentheses (A, Archotermopsidae, H, Hodotermitidae; Hs, Hodotermopsidae, K, Kalotermitidae; R, Rhinotermitidae; T, Termitidae).</figcaption></figure>
<figure><img src="image_13.png" /><figcaption>**FIGURE A2** | Phylogeny of the bifunctional phosphoketolase (Xfp) of termite-associated *Bifidobacteriaceae* (TAB) and other family members, and the homologous monofunctional phosphoketolases (Xpk) of *Actinomycetota* and other bacterial phyla. Biochemically characterised homologues (SWISS-PROT) are highlighted. The maximum-likelihood tree is based on a curated alignment of 490 amino acid positions and was inferred under the LG + G4 model of evolution. Bootstrap values were omitted for clarity.</figcaption></figure>
A

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

B

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

**FIGURE A3** | PTS systems for N-acetyl-glucosamine (A) and glucose/mannose (B) and their conversion to fructose 6-phosphate in termite-associated *Bifidobacteriaceae*.
<figure><img src="image_18.png" /><figcaption>**FIGURE A4** | Phylogeny of the organophosphate antiporter (OPA) of termite-associated *Bifidobacteriaceae* (TAB) and their closest relatives from public reference databases. Biochemically characterised homologues (SWISS-PROT) are highlighted. The maximum-likelihood tree is based on a curated alignment of 428 amino acid positions and was inferred under the LG + F + R9 model of evolution. Bootstrap values were omitted for clarity.</figcaption></figure>
<figure><img src="image_19.png" /></figure>

FIGURE A5 | Phylogeny of Group A [FeFe] hydrogenases in the genus *Ancillula* and *Opitulatrix* and their homologues in public databases. Homologues from MAGs of termite gut microbiota are marked in bold. The maximum-likelihood tree is based on a curated alignment of 281 amino acid positions and was inferred under the LG+R8 model of evolution. Numbers at internal nodes indicate ultrafast bootstrap support (1000 replicates).
<figure><img src="image_20.png" /><figcaption>**FIGURE A6** | Phylogeny of PFOR in termite-associated *Bifidobacteriaceae* and their homologues from public databases. The maximum-likelihood tree is based on a curated alignment of 323 amino acid positions and was inferred under the LG + R5 model of evolution. Numbers at internal nodes indicate ultrafast bootstrap support (1000 replicates).</figcaption></figure>
<figure><img src="image_21.png" /><figcaption>**FIGURE A7** | Enzymes of asparagine biosynthesis encoded by termite-associated *Bifidobacteriaceae* and other family members. Abbreviations: AsnA, aspartate-ammonia ligase; AsnB, asparagine synthetase B; AspS, aspartate-tRNA ligase; GatABC, aspartyl/glutamyl-tRNA(Asn/Gln) amidotransferase.</figcaption></figure>
<figure><img src="image_22.png" /><figcaption>**FIGURE A8** | Phylogeny of the threonine/serine exporter (ThrE) of termite-associated *Bifidobacteriaceae* and its homologues in public databases. The maximum-likelihood tree is based on a curated alignment of 259 amino acid positions and was inferred under the LG + F + R6 model of evolution. Numbers at internal nodes indicate ultrafast bootstrap support (1000 replicates).</figcaption></figure>
<table><thead><tr><th></th><th>MurJ</th><th>Ddl</th><th>MurF</th><th>MurG</th><th>Mpl</th><th>MurA</th><th>Alr</th><th>MraY</th><th>MurB</th><th>FTSW</th></tr></thead><tbody><tr><td>Nutricula glyptotermitis (Gx485_bin43)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Servula neotermitis (Nm470_bin85)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Opitulatrix cubana (Ncb351_bin81)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Opitulatrix roisinitermitis (Roe453_bin66)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Opitulatrix roisinitermitis (Roe453_bin58)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Opitulatrix roisinitermitis (Roe453_bin28)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Ancillula hodotermitis (Hm464_bin179)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Ancillula prorhinotermitis (Pc512_bin108)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Ancillula kalotermitis (Kf353_bin77)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Ancillula glyptotermitis (Gsp477_bin129)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Ancillula trichonymphae (Im94_bin94)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Ancillula trichonymphae (ImTpAt1)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Aeriscardovia sp903789535</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Aeriscardovia aeriphila</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Pseudoscardovia suis</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Pseudoscardovia radai</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Parascardovia denticolens</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Scardovia inopinata</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Alloscardovia macacae</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Alloscardovia theropithecii</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Alloscardovia omnicolens</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Galliscardovia tissieri</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Galliscardovia ingluviei</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bifidobacterium indicum</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bifidobacterium asteroides</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bifidobacterium bombi</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bifidobacterium vaginale</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bifidobacterium minimum</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bifidobacterium magnum</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bifidobacterium animalis</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bifidobacterium ruminantium</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bifidobacterium adolescentis</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bifidobacterium bifidum</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bifidobacterium merycicum</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bifidobacterium angulatum</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bifidobacterium rousetti</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bifidobacterium scaligerum</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bifidobacterium saguini</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

**FIGURE A9** | Enzymes of murein biosynthesis encoded by termite-associated *Bifidobacteriaceae* and other family members. Abbreviations: MurJ, Lipid II flippase; Ddl, D-alanine-D-alanine ligase; MurF, UDP-N-acetylmuramoyl-tripeptide-D-alanyl-D-alanine ligase; Mpl, UDP-N-acetylmuramate-L-alanyl-gamma-D-glutamyl-meso-2,6-diaminoheptandioate ligase; MurA, UDP-N-acetylglucosamine 1-carboxyvinyltransferase; Alr, Alanine racemase; MraY, phospho-N-acetylmuramoyl pentapeptide transferase; MurB, UDP-N-acetylenolpyruvoylglucosamine reductase; FTSW, peptidoglycan glycosyltransferase.
<figure><img src="image_23.png" /><figcaption>**FIGURE A10** | Elements of different DNA repair mechanisms encoded by *Bifidobacteriaceae* and other *Actinomycetales*.</figcaption></figure>
<figure><img src="image_24.png" /><figcaption>**FIGURE A11** | Phylogeny of the folate transporter (FoIT) encoded by members of the genus *Ancillula* and its homologues from public databases. The maximum-likelihood tree is based on a curated alignment of 324 amino acid positions and was inferred under the LG+G4 model of evolution. Numbers at internal nodes indicate ultrafast bootstrap support (1000 replicates).</figcaption></figure>
A

<figure><img src="image_25.png" /></figure>

B

<figure><img src="image_26.png" /></figure>

**FIGURE A12** | Expanded biosynthetic pathways for amino acid (A) and cofactor (B) biosynthesis in the *Ancillula* and *Opitulatrix* MAGs. Genes in black are encoded by both genera. Genes in purple are present only in *Ancillula*, and genes in grey are absent in both genera.
# Appendix B

## Protologues for New Prokaryotic Taxa

Etymology and description of the new taxa proposed under SeqCode.

### Bifidobacteriaceae Stackebrandt, Rainey, and Ward-Rainey 1997 emend. Kästle Silva and Brune

Family defined by phylogenomic analysis. Members form a monophyletic group that shows a relative evolutionary divergence (RED) similar to that of the neighbouring families. Contains the genera *Ancillula* Strassert and Brune, *Aeriscardovia* Simpson et al. 2004, *Alloscardovia* Huys et al. 2007, *Bifidobacterium* Orla-Jensen 1924, *Bombiscardovia* Killer et al. 2010, *Galliscardovia* Pechar et al. 2017, *Gardnerella* Greenwood and Pickett 1980, *Metascardovia* Okamoto et al. 2007, *Neoscardovia* García-Aljaro et al. 2012, *Nutricula* Kästle Silva and Brune, *Opitulatrix* Kästle Silva and Brune, *Parascardovia* Jian and Dong 2002, *Pseudoscardovia* Killer et al. 2013, *Scardovia* Jian and Dong 2002, and *Servula* Kästle Silva and Brune.

### Ancillula gen. nov. Strassert and Brune

**Etymology:** An.cil'lu.la. L. fem. dim. n. *Ancillula*, a little female servant.

**Synonym:** 'Candidatus Ancillula' Strassert et al. 2012 (not validly published).

**Description:** Genus defined by phylogenomic analysis of metagenome-assembled genomes and single-cell amplified genomes. Members of the genus form a monophyletic group that shows a relative evolutionary divergence (RED) similar to that of the neighbouring genera.

**Type species:** *Ancillula trichonymphae* sp. nov.

### Ancillula trichonymphae sp. nov. Strassert and Brune

**Etymology:** tri.cho.nym'phae. N.L. gen. fem. n. *trichonymphae*, of *Trichonympha*, a genus of termite gut flagellates.

**Synonym:** 'Candidatus Ancillula trichonymphae' Strassert et al. 2012 (not validly published).

**Description:** Short, slightly pleomorphic rods (approximately 1.2 mm in length and 0.3 mm in diameter) with a typical Gram-positive cell wall. Colonise the cytoplasm of a lineage of closely related flagellates (Trichonympha Cluster II) in the hindgut of termites. SSU rRNA genes form a cluster of host-specific phylotypes.

**Type genome:** ImTpAt1<sup>ST</sup>, GCA_000485475.1 (genome); JQ993633 (16S rRNA).

**Additional genomes:** GCA_031276145.1.

### Ancillula hodotermitis sp. nov. Kästle Silva and Brune

**Etymology:** ho.do.ter'mi.tis. N.L. gen. n. *hodotermitis*, of *Hodotermes*, a genus of termites.

**Description:** The species comprises one metagenome-assembled genome. The species will include all bacteria with more than 95% average nucleotide identity (ANI) to the type genome. The GC content of the type genome is 34.5% and the estimated genome size is 1.51 Mbp.

**Type genome:** Hm464_bin179, GCA_031262545.1 (genome), OR396865 (16S rRNA).

### Ancillula glyptotermitis sp. nov. Kästle Silva and Brune

**Etymology:** glyp.to.ter'mi.tis. N.L. gen. n. *glyptotermitis*, of *Glyptotermes*, a genus of termites.

**Description:** The species comprises one metagenome-assembled genome. The species will include all bacteria with more than 95% average nucleotide identity (ANI) to the type genome. The GC content of the type genome is 37.92% and the estimated genome size is 1.80 Mbp.

**Type genome:** Gsp477_bin129 from the lower termite *Glyptotermes*.

**GenBank accession number:** GCA_031268675.1 (genome), OR396855 (16S rRNA).

### Ancillula kalotermitis sp. nov. Kästle Silva and Brune

**Etymology:** ka.lo.ter'mi.tis. N.L. gen. n. *kalotermitis*, of *Kalotermes*, a genus of termites.

**Description:** The species comprises one metagenome-assembled genome. The species will include all bacteria with more than 95% average nucleotide identity (ANI) to the type genome. The GC content of the type genome is 49.22% and the estimated genome size is 1.07 Mbp.

**Type genome:** Kf353_bin77 from the lower termite *Kalotermes flavicollis*.

**GenBank accession number:** GCA_031273495.1 (genome), OR396858 (16S rRNA).

### Ancillula prorhinotermitis sp. nov. Kästle Silva and Brune

**Etymology:** pro.rhi.no.ter'mi.tis. N.L. gen. n. *prorhinotermitis*, of *Prorhinotermes*, a genus of termites.

**Description:** The species comprises one metagenome-assembled genome. The species will include all bacteria with more than 95% average nucleotide identity (ANI) to the type genome. The GC content of the type genome is 46.36% and the estimated genome size is 0.673 Mbp.

**Type genome:** Pc512_bin108 from the lower termite *Prorhinotermes canalifrons*.

**GenBank accession number:** GCA_031280795.1 (genome), PP623112 (16S rRNA).

### Opitulatrix gen. nov. Kästle Silva and Brune

**Etymology:** O.pi.tu.la'trix. N.L. fem. n. *Opitulatrix*, a female helper, maid.

**Description:** Genus defined by phylogenomic analysis of metagenome-assembled genomes. Members of the genus form a monophyletic group that shows a relative evolutionary divergence (RED) similar to that of the neighbouring genera.

**Type species:** *Opitulatrix roisinitermitis* sp. nov.

### Opitulatrix roisinitermitis sp. nov. Kästle Silva and Brune

**Etymology:** roi.si.ni.termi.tis. N.L. gen. n. *roisinitermitis*, of *Roisinitermes*, a genus of termites.

**Description:** The species comprises only metagenome-assembled genomes. The species includes all bacteria with more than 95% average nucleotide identity (ANI) to the type genome. The GC content of the type genome is 33.45% and the estimated genome size is 0.98 Mbp.

**Type genome:** Roe453_bin28 from the lower termite *Roisinitermes ebogoensis*.

**GenBank accession number:** GCA_031288355.1 (genome); OR396864 (16S rRNA).

**Additional genomes:** GCA_031288055.1, GCA_031288095.1.

### Opitulatrix cubana sp. nov. Kästle Silva and Brune

**Etymology:** cu.ba'na. N.L. fem. adj. cubana, of Cuba, Cuban, referring to the geographic origin of the host.

**Description:** The species comprises one metagenome-assembled genomes. The species will include all bacteria with more than 95% average nucleotide identity (ANI) to the type genome. The GC content of the type genome is 35.44% and the estimated genome size is 0.768 Mbp.

**Type genome:** Ncb351_bin81 from the lower termite *Neotermes cubanus*.
**GenBank accession number:** GCA_031283665.1 (genome); OR396859 (16S rRNA).

**Nutricula gen. nov. Kästle Silva and Brune**

*Etymology:* Nu.tri'cu.la. L. fem. n. *nutricula*, a wet nurse.

**Description:** Genus defined by phylogenomic analysis of metagenome-assembled genomes. The single genome shows a relative evolutionary divergence (RED) similar to that of the neighbouring genera.

**Type species:** *Nutricula glyptotermitis* sp. nov.

**Nutricula glyptotermitis sp. nov. Kästle Silva and Brune**

*Etymology:* glyp.to.ter'mi.tis. N.L. gen. n. *glyptotermitis*, of *Glyptotermes*, a genus of termites.

**Description:** The species comprises one metagenome-assembled genome. The species will include all bacteria with more than 95% average nucleotide identity (ANI) to the type genome. The GC content of the type genome is 43.55% and the estimated genome size is 1.54 Mbp.

**Type genome:** Gx485_bin43 from the lower termite *Glyptotermes* sp.

**GenBank accession number:** GCA_031263495.1 (genome); OR396856 (16S rRNA).

**Servula gen. nov. Kästle Silva and Brune**

*Etymology:* Ser'vu.la. L. fem. dim. n. *servula*, a young servant girl.

**Description:** Genus defined by phylogenomic analysis of metagenome-assembled genomes. The single genome shows a relative evolutionary divergence (RED) similar to that of the neighbouring genera.

**Type species:** *Servula neotermitis* sp. nov.

**Servula neotermitis sp. nov. Kästle Silva and Brune**

*Etymology:* ne.o.termi.tis. N.L. gen. n. *neotermitis*, of *Neotermes*, a genus of termites.

**Description:** The species comprises one metagenome-assembled genome. The species will include all bacteria with more than 95% average nucleotide identity (ANI) to the type genome. The GC content of the type genome is 42.03% and the estimated genome size is 1.10 Mbp.

**Type genome:** Nm470_bin85 from the lower termite *Neotermes meruensis*.

**GenBank accession number:** GCA_031281695.1 (genome); OR396861 (16S rRNA).