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ABSTRACT Arbuscular mycorrhizal fungi (AMF) have been symbionts of land plants for at least 450 Myr. It is known that some AMF host in their cytoplasm Gram-positive endobacteria called

bacterium-like organisms (BLOs), of unknown phylogenetic origin. In this study, an extensive inventory of 28 cultured AMF, from diverse evolutionary lineages and four continents, indicated

that most of the AMF species investigated possess BLOs. Analyzing the 16S ribosomal DNA (rDNA) as a phylogenetic marker revealed that BLO sequences from divergent lineages all clustered in a

well-supported monophyletic clade. Unexpectedly, the cell-walled BLOs were shown to likely represent a sister clade of the _Mycoplasmatales_ and _Entomoplasmatales_, within the

_Mollicutes_, whose members are lacking cell walls and show symbiotic or parasitic lifestyles. Perhaps BLOs maintained the Gram-positive trait whereas the sister groups lost it. The

intracellular location of BLOs was revealed by fluorescent _in situ_ hybridization (FISH), and confirmed by pyrosequencing. BLO DNA could only be amplified from AMF spores and not from spore

washings. As highly divergent BLO sequences were found within individual fungal spores, amplicon libraries derived from _Glomus etunicatum_ isolates from different geographic regions were

pyrosequenced; they revealed distinct sequence compositions in different isolates. Our results show a vertically inherited, monophyletic and globally distributed lineage of endobacteria

thriving in AMF cytoplasm. These bacteria split from their sister groups more than 400 Myr ago, colonizing their fungal hosts already before main AMF lineages separated. The BLO–AMF

symbiosis can, therefore, be dated back at least to the time when AMF formed the ancestral symbiosis with emergent land plants. SIMILAR CONTENT BEING VIEWED BY OTHERS COMPARATIVE GENOMICS OF

MOLLICUTES-RELATED ENDOBACTERIA SUPPORTS A LATE INVASION INTO MUCOROMYCOTA FUNGI Article Open access 18 September 2023 LARGE-SCALE GENOME SEQUENCING OF MYCORRHIZAL FUNGI PROVIDES INSIGHTS

INTO THE EARLY EVOLUTION OF SYMBIOTIC TRAITS Article Open access 12 October 2020 SYMBIOTIC BACTERIA OF THE GALL-INDUCING MITE _FRAGARIOCOPTES SETIGER_ (ERIOPHYOIDEA) AND PHYLOGENOMIC

RESOLUTION OF THE ERIOPHYOID POSITION AMONG ACARI Article Open access 09 March 2022 MAIN Symbiosis is central in the evolution of complexity, being crucial to the lifestyles of animals,

plants, fungi and also prokaryotes (Hoffmeister and Martin, 2003). Many functions could only be evolved because of the intimate interaction and often interdependence of different species, as

illustrated by endosymbioses, which gave rise to the eukaryotic organelles (Timmis et al., 2004). Scaling up to the ecological level, symbiotically living organisms have key roles in most

ecosystems (Moran et al., 2008). Bacterial endosymbionts are widespread in animals, in particular in insects in which they—as ubiquitous and heritable genetic components—offer excellent

models to investigate organelle evolution, genome reduction and horizontal gene transfer (Moran et al., 2008). In contrast, examples of endobacteria in fungi are limited to a few examples

(Bonfante and Anca, 2009); among them a _Burkholderia_ species living inside a strain of _Rhizopus_ is responsible for fungal pathogenicity, and hence showing the interdependence of the

bacterial–fungal alliance (Partida-Martinez and Hertweck, 2005). Arbuscular mycorrhizal fungi (AMF), which are analyzed in this study, are long known to possess endobacteria in their

cytoplasm. AMF belong to the _Glomeromycota_, a phylum known to be older than the _Ascomycota_ and _Basidiomycota_ (Schüßler et al., 2001), and form symbiotic associations with >80% of

land plants, in which both partners benefit from nutrient exchange. This association is commonly described as the result of co-evolution dating back to early Devonian times (Taylor et al.,

1995; Redecker et al., 2000; Bonfante and Genre, 2008). As for insect endosymbionts, the presence of endobacteria inside _AMF_ cytoplasm has long been documented by electron microscopy,

which has distinguished two bacterial morphotypes. The first, restricted to a phylogenetically relatively young AMF family (_Gigasporaceae_), is rod shaped, related to _Burkholderia_ and

described as an uncultured taxon, _Candidatus_ Glomeribacter gigasporarum (Bianciotto et al., 2003). A fungal line cured from these endobacteria showed that they confer an ecologically

relevant fitness to their fungal host (Lumini et al., 2007). The other bacterial type has been detected inside AMF spores and hyphae colonizing plant roots sampled in the field. It is

coccoid in shape and has been called ‘bacterium-like organism’ (BLO), as its identity is still obscure (MacDonald et al., 1982; Scannerini and Bonfante, 1991; Schüßler et al., 1994). When

BLOs were first detected, knowledge of AMF phylogeny was limited and the fungi were classified only by their morphological features. As a consequence, BLO attribution to specific AMF taxa is

uncertain and opens questions of whether BLOs in the fungal cytoplasm represent occasional re-infection by free-living bacteria, or whether they are a consistent feature. To unambiguously

identify the BLOs and assess their distribution in diverse members of the _Glomeromycota_, we analyzed AMF spores from 28 cultures, representing highly divergent lineages originating from

four continents. We used confocal microscopy, fluorescence _in situ_ hybridization (FISH) and electron microscopy to establish BLO localization in the AMF cytoplasm, together with sequencing

and phylogenetic analysis on BLOs from single spores or groups. We report that BLOs live only within the fungal cytoplasm and that divergent, but monophyletic bacterial lineages, co-exist

in an individual single spore. Unexpectedly, the cell-walled BLOs represent an old, formerly unknown bacterial group that likely falls within the clade harboring the cell wall-lacking

_Mollicutes_. This finding opens new questions about the evolution of _Mollicutes-_related bacteria, their biotrophic lifestyle, the complexity of interphylum interactions and the

symbiotic-genetic makeup of AMF. MATERIALS AND METHODS SPORE MANIPULATION AMF cultures were obtained from different culture collections (Supplementary Table S1), in which they had been

propagated for several generations either in pot soil cultures in the presence of a plant host or _in vitro_ on root organ cultures (Cranenbrouck et al., 2005). All steps requiring sterility

were performed under laminar flow, and plastic material was sterile and DNA free. Spores were cleaned as described in Schwarzott and Schüßler (2001) and surface sterilized with 3%

chloramine-T and 0.03% streptomycin. DNA EXTRACTION AND AMPLIFICATION DNA was extracted from spores (either single or groups of five) as described in Lumini et al. (2007) with 10 μl 5 ×

HotStarTaq PCR buffer (Qiagen, Milan, Italy), or following Schwarzott and Schüßler (2001). The bacterial 16S ribosomal DNA (rDNA) was amplified by PCR with Phusion High-Fidelity DNA

polymerase 2 × mastermix (Finnzymes, Espoo, Finland) and the general bacterial primers 16F27 (Bennasar et al., 1996) and 1495r (Bandi et al., 1994) nested with 16F27 and 1387R (Marchesi et

al., 1998). The final reaction mix contained 0.02 U μl−1 Phusion polymerase, 1 × Phusion HF Buffer with 1.5 mM MgCl2, 200 μM of each deoxynucleotide triphosphate and 0.5 μM of each primer.

Thermal cycling conditions were: 5 min initial denaturation at 99 °C; 30–40 cycles of 10 s denaturation at 98 °C, 30 s annealing at 60 °C and 1 min elongation at 72 °C; and a 10 min final

elongation. PCR products were TOPO cloned (Invitrogen, San Giuliano Milanese, Italy) and transformed into Top10 chemically competent _Escherichia coli_ according to the manufacturers

instructions. Colonies were screened for insert length by PCR. The PCR products were digested with the restriction endonucleases _Alu_I and _Rsa_I (Invitrogen) (1 U, 1 h at 37 °C) to produce

restriction fragment length polymorphism profiles. A modified heat-lysis protocol (Ganguly et al., 2005) was applied to extract plasmids. The plasmid inserts were sequenced from both ends

on an ABI 3730 48 capillary sequencer with 50 cm capillary length using BigDye v3.1 sequencing chemistry (Applied Biosystems, Darmstadt, Germany). BIOINFORMATIC ANALYSES Sequences were

assembled and curated using Seqassem (Sequentix, Klein Raden, Germany) and aligned with the SILVA 16S RNA database (Pruesse et al., 2007), version SSURef_release96, using ARB (Ludwig et al.,

2004). Phylogenetic trees were inferred with the PHYLIP package (Felsenstein, 1989), MrBayes (Huelsenbeck and Ronquist, 2001) and RaxML (Stamatakis et al., 2008). Only topologies are shown,

which are supported by at least two of the three phylogenetic analysis methods used (neighbor joining, maximum likelihood and Bayesian) with >50% bootstrap or >0.5 posterior

probability values. Others are collapsed to polytomies. Dashes instead of numbers indicate that the topology was not supported in the respective analysis (Figure 3). Pairwise distances

between sequences types were estimated with ClustalW (Larkin et al., 2007). PYROSEQUENCING A metagenomic approach was adopted for the 454-based experiments; as BLOs are so far uncultured,

the starting material was fungal spores. For the construction of amplicon libraries from endobacteria and bacteria associated with the surface of _Glomus versiforme_ Att 475–45, we used

three types of samples: (1) washed spores, (2) spores sonicated in 10 μl sterile DNA-free water for 2 min and washed three times afterwards and (3) the water remaining after the sonication.

Spores of _G. etunicatum_ culture Att 239–4 were decontaminated as in sample 2. Spores from _G. etunicatum_ isolates MUCL 47650, CA-OT126-3-2 and CA-OT-135-4-2 were collected from _in vitro_

culture and washed twice. Each sample combined five spores or the equivalent water and we used three biological replicates. DNA was extracted as in Schwarzott and Schüßler (2001), and

amplified by PCR with Phusion HF Mastermix (as described above) with modified versions of the primers 967F (5′-GCCTCCCTCGCGCCATCAGNNNNCRACGCGNAGAACCTTACC-3′) (Sogin et al., 2006) and 1495r

(5′-GCCTTGCCAGCCCGCTCAGCTACGGYTACCTTGTTACGAC-3′) (Bandi et al., 1994), chosen to construct tagged fusion primers (Supplementary Table S3, SI). Thermal cycling conditions were: 5 min initial

denaturation at 99 °C; 30 cycles of 10 s denaturation at 98 °C, 30 s annealing at 57 °C and 1 min elongation at 72 °C; and a 10 min final elongation. Each DNA extract was amplified with its

respectively tagged fusion primer in five independent PCRs. Products were pooled and purified with the AMPure kit (Agencourt, Beckman Coulter S.p.A, Milan, Italy), quantified with a NanoDrop

1000 spectrophotometer (Thermo Scientific, Euroclone S.p.A., Milan, Italy) and combined equimolar. Product quality was checked on an Agilent 2100 Bioanalyzer (Agilent Technologies,

Cernusco, Italy) and sent to BMR Genomic (Padova, Italy) for emulsion PCR and pyrosequencing on a GS FLX sequencer (Roche, Mannheim, Germany). Bacterial rDNA sequences from _G. etunicatum_

and _G. versiforme_ metagenomic DNA were quality trimmed (quality cutoff set to 20, omitting sequences <150 b), sorted to the respective sample through the tag sequences, and aligned and

clustered using the pyrosequencing pipeline provided by the ribosomal database project (Cole et al., 2009). Operational taxonomic units were classified with the ribosomal database project

classifier. In addition, a dereplicated subset of sequences (one sequences per sample per operational taxonomic unit recovered at the 3% sequence divergence level) was aligned with SINA (the

SILVA web aligner, http://www.arb-silva.de/aligner/; Pruesse et al., 2007), imported and inserted into the ‘All-Species Living Tree’ alignment (Yarza et al., 2008) with ARB. This alignment

had been supplemented with the nearly full-length 16S rDNA sequence data of BLOs before use. MORPHOLOGICAL DETECTION OF BLOS The samples were observed with a confocal laser scanning

microscope (CLSM, Leica TCS SP2, Leica Microsystems Srl, Milan, Italy) exciting at 488 and 543 nm. A 40 × long-distance water immersion objective (HCX APO N.A. 0.80) and a 63 × water

immersion objective (HCX PL APO N.A. 1.20) were used. Images were taken sequentially at each excitation wavelength. Pseudocolors for emission wavelength were green for 500–530 nm

(fluorescein isothiocyanate and SYTO BC) and red for 550–580 nm (Cy3) and 605–635 nm (propidium iodide). To rapidly screen AMF spores for endobacteria, they were stained with the dye mixture

SYTO BC (1–5 μM; Molecular Probes, San Giuliano Milanese, Italy), known to penetrate intact living bacterial cells. Fungal nuclei and dead bacteria were counterstained with propidium iodide

(30–90 μM). Samples were incubated for 15 min at room temperature before spore crushing and observation. FISH EXPERIMENTS Before fixation, _G. versiforme_ Att 475–45 spores were vortexed

for 2 to 3 min to clean their surface, and washed three times with ultrapure water and once with phosphate-buffered saline. Spores were fixed in 3% formaldehyde buffered with

phosphate-buffered saline (Amann et al., 1990) incubated at 4 °C for 3 h or 6 h, washed three times in the saline, suspended in 50% ethanol in the saline, and stored at −20 °C until use.

Oligonucleotide probes were purchased from Sigma-Aldrich (Milan, Italy) and Thermo Fisher Scientific GmbH (Ulm, Germany), 5′-end labeled with Cy3 or fluorescein isothiocyanate. The

eubacterial probe EUB338I (Amann et al., 1990) was used as a general probe, and Apis2Pa (Moran et al., 2005), which targets the genus _Buchnera_, was used as a negative control to detect

nonspecific binding. Specific oligonucleotide probes were designed with ARB. They matched bacterial sequences amplified from _Glomus_ group B (_Glomerales_) and _Diversisporales_ and showed

a minimum of three centrally located mismatches to all other sequences in the ARB 16S database. The specificity of BLOgrBC (5′-GCCAATCCTACCCTTGTCA-3′) was tested empirically. Fungal spores

were immobilized on 10-well microscope slides (6 mm Ø each well, Structure Probe, West Chester, PA, USA) with a drop of 0.5% agarose (Sigma-Aldrich) and dehydrated in 50%, 75% and 100%

ethanol. Spores were crushed to allow penetration of the probes into the cytoplasm during hybridization, as described in Bertaux et al. (2005). The probes were hybridized at a stringency of

35% formamide (Sigma-Aldrich). Microscope slides were mounted with SlowFade Antifade kit component A (Molecular Probes). The hybridization to EUB338I and BLOgrBC was repeated 14 times,

corresponding to a total of 40 _G. versiforme_ spores. TRANSMISSION ELECTRON MICROSCOPY To allow fixative penetration into the spore, spore walls of surface-sterilized _G. versiforme_ Att

475–45 spores were incised with a fine syringe needle (U100 Insulin ACCU-FINE; Roche, Monza, Italy). These spores were immediately transferred to 50 mM sodium cacodylate buffer (pH 7)

containing in 2.5% (v/v) glutaraldehyde and then processed as described in Bianciotto et al. (2003) and Lumini et al. (2007). Ultrathin sections (70 nm) were cut with an ultramicrotome

(Ultracut; Reichert and Jung, Vienna, Austria) counterstained with uranyl acetate and lead citrate and observed with a transmission electron microscope (Philips CM10, Philips Medical

Systems, Eindhoven, The Netherlands). In all, 10 individual spores were examined. RESULTS BLOS ARE PRESENT IN DIVERSE AMF FAMILIES Spores from 28 cultures, including _in vitro-_grown root

organ cultures and pot cultures, were studied. They represent 16 phylogenetically characterized species belonging to diverse _Glomeromycota_ lineages and originating from four continents

(Figure 1; Supplementary Table S1, SI). After staining with the fluorescent dyes SYTO BC and propidium iodide, the BLOs were observed within the spore cytoplasm as green fluorescent spots

approximately 500 nm in diameter, whereas fungal nuclei appeared red (Figure 2a). BLOs were consistently found in 19 of the cultures and were associated with 11 of the 16 AMF species tested,

being present in members of the _Ambisporaceae_ and _Geosiphonaceae_ (_Archaeosporales_, an ancient AMF lineage), _Glomus_ group A and _Glomus_ group B (corresponding to two families in the

_Glomerales_), and _Diversisporaceae_, _Gigasporaceae_ and _Acaulosporaceae_ (_Diversisporales_) (Figure 1; Supplementary Table S1, SI). BLOS ARE RELATED TO THE _MOLLICUTES_ Bacterial 16S

rDNA was amplified from surface-sterilized or _in vitro_-cultured AMF spores using general bacterial primers. A set of BLO-specific primers was then developed, based on the sequence data

obtained. In total, 107 distinct sequence types were obtained from 17 of the 28 cultures, including members of the _Glomerales_ (_G. caledonium_, _G. mosseae_, _G. claroideum_ and _G.

etunicatum_), _Diversisporales_ (_Gigaspora margarita_, _Scutellospora gilmorei_ and _G. versiforme_) and the _Archaeosporales_ (_Ambispora appendicula_, _A. fennica_ and _Geosiphon

pyriformis_). BLO 16S rDNA could not be amplified from an _Acaulospora_ sp. and _Kuklospora colombiana_ (_Acaulosporaceae, Diversisporales_), notwithstanding the morphological evidence of

bacterial structures by fluorescent staining. All sequences clustered in a well-supported monophyletic clade that was not closely related to any described bacterial group. BLOs therefore

represent a new, undescribed bacterial higher taxon that, although there is a distinct cell wall (Figure 2b), forms a subclade within the wall-less _Mollicutes_ (Ludwig et al., 2008) (Figure

3) likely being a sister clade to the _Entomoplasmatales_ and _Mycoplasmatales_. HIGHLY DIVERGENT BLO SEQUENCES EXIST WITHIN A SINGLE AMF SPORE Within the monophyletic BLO clade (Figure 3),

highly variable 16S rDNA BLO sequences were found, showing up to 20% intrasporal sequence divergence even within single AMF spores. BLO sequences from an AMF species formed from one (_A.

fennica_) up to six divergent subclades (_G. claroideum_). The branches in the basal part of the tree included sequence types from the _Archaeosporales_, _Diversisporales_ and _Glomerales_,

and their topological relationships could not be resolved. In contrast, the more terminal part of the tree was better resolved. It only contained bacterial sequences from _Glomerales_ and

_Diversisporales_ (Supplementary Figure S1, SI). Here, BLO sequences stemming from closely related fungi clustered together, indicating co-evolution with their hosts. BLOS ARE LOCATED WITHIN

THE AMF CYTOPLASM To unambiguously show that BLOs are located in the cytoplasm of glomeromycotan fungi, _G. versiforme_ (Att 475–45) was selected because of easy production and handling of

spores, low degree of cytoplasm autofluorescence and its long-standing use as a model fungus in arbuscular mycorrhizal research (e.g., Harrison and Buuren, 1995). The 16S rDNA sequences

obtained in our work allowed us to develop specific probes for FISH. The new BLO-specific probe BLOgrBC colocalized with the widely used general bacterial probe EUB338I within the _G.

versiforme_ cytoplasm (Figure 4) in which it labeled coccoid structures (Figure 4, compare with Figure 2a). The FISH signal was not detected in any free-living bacteria or those associated

with the spore surface (data not shown). FISH also indicated that the structures stained by SYTO BC were indeed bacteria. Electron microscopy of the same _G. versiforme_ culture confirmed

the presence of coccoid bacteria in the fungal cytoplasm. They were 250–500 nm in size, possessed a homogeneous, Gram-positive type cell wall and, unlike _Candidatus_ Glomeribacter, were not

surrounded by a fungal membrane (Figure 2b). To confirm the cytoplasmic localization of the BLOs we used the 454 GS-FLX technology (Margulies et al., 2005) to sequence 16S rDNA amplicon

libraries. The metagenomic DNA was derived from either (1) five washed spores, (2) five sonicated and washed spores or (3) the water in which the five spores were held during sonication,

with three biological replicates each. A total of 1125 high-quality sequences were obtained. Sequences detected from the sonicated water were considered as derived from spore

surface-associated bacteria. The results showed that sonication did not remove all surface bacteria but the percentage of non-BLO sequences associated with the surface of sonicated spores

was lower than for spores only washed (Figure 5; Supplementary Figure S2, SI). They belonged to bacterial groups already reported as associated with AMF spores (reviewed in Bonfante and

Anca, 2009) and in studies of soil biodiversity (Roesch et al., 2007; Elshahed et al., 2008; Kielak et al., 2008). In contrast, BLO sequences were exclusively obtained from washed and

sonicated spores but never from sonicated water. This points to their presence inside fungal spores and more importantly shows that BLOs were not detected as free-living bacteria. Sequences

related to those of BLOs have been reported in some recent studies. With the exception of one that was shown to be associated with _Gigaspora margarita_ (Long et al., 2009), all the other

sequences are annotated as environmental and have been retrieved in studies on soil bacterial diversity, from tree and grassland environments (Dunbar et al., 2002; Elshahed et al., 2008;

Hansel et al., 2008; Lesaulnier et al., 2008; Cruz-Martinez et al., 2009). Most likely, these sequences originated from BLO-harboring AMF present in the samples, either as spores or, hyphae

in the soil or in root fragments. BLO RDNA SEQUENCE VARIABILITY: ANOTHER SOURCE OF GENETIC VARIABILITY IN ARBUSCULAR MYCORRHIZAL FUNGI The model AMF _Glomus_ sp. ‘intraradices-like’

DAOM197198, whose genome is currently under sequencing, has been indicated to contain multiple, polymorphic genomes (Hijri and Sanders, 2005; Martin et al., 2008). In contrast, the

mitochondrial genome of this fungus and its close relatives (Lee and Young, 2009) including the mt-rDNA (Börstler et al., 2008), is nearly invariable. For isolates of _G. intraradices_ and

the species represented by _Glomus_ sp. DAOM197198, exceptionally variable nuclear rDNA regions were reported with up to >20% intrasporal sequence divergence in the internal transcribed

spacer region (Stockinger et al., 2009). A similarly high internal transcribed spacer variability was recently also reported for AMF from other phylogenetic clades (Stockinger et al., 2010).

Unexpectedly, a very high variability (up to 20% intrasporal sequence divergence) was found between BLO 16S rDNA sequences attributed to a single fungal culture and even to single spores in

the present study, although not all these variations could be recovered from each culture. To understand whether this was due to insufficient sampling density, amplicon libraries of _G.

etunicatum_ spores were 454 GS-FLX pyrosequenced. _G. etunicatum_ was chosen because four cultures were available for comparison. Three of these were single spore isolates established as

root organ cultures (CA-OT-126-3-2, CA-OT-135-4-2 and MUCL47650), and one was propagated in pot (Att 239–4) (Figure 1; Supplementary Table S1, SI). Each library was again generated from five

spores, with three biological replicates each, leading to 2827 sequences. To define phylotypes, all sequences with ⩽3% divergence were clustered. Only one of the resulting 20 BLO phylotypes

was present in all four _G. etunicatum_ cultures, one was exclusively detected in the biological replicates of MUCL47650 and Att 239–4, one only in the biological replicates of

CA-OT-135-4-2, whereas several other phylotypes could be attributed to both CA-OT-126-3-2 and CA-OT-135-4-2 isolates, both originating from California (Table S2, SI). Thus, pyrosequencing

confirmed that different phylotype compositions do indeed exist between isolates of the same AMF species, and highlighted that the BLO sequences represent the vast majority (89.8%) of the

bacterial sequences recovered from cleaned spores. When the _in vitro_ cultures, expected to be free of contaminating bacteria, were considered, BLO sequences represented even 93.6%.

Although the BLO phylotypes were present throughout all biological replicates of a sample, the remaining non-BLO sequences were highly diverse and variable in between replicates, indicating

contaminants acquired during sample preparation. However, the percentage values have to be interpreted with care, as sequences may be artificially replicated during pyrosequencing

(Gomez-Alvarez et al., 2009). DISCUSSION Mycorrhizas, the symbioses established by most land plants and soil fungi, are often described as the result of tripartite interactions, as many

bacteria are known to be associated as free-living helper microbes (Frey-Klett et al., 2007). In contrast, existence of true endobacteria in mycorrhizal fungi has been convincingly shown

only for the betaproteobacterium _Candidatus_ Glomeribacter gigasporarum, whose presence is restricted to members of the family _Gigasporaceae_ (Bonfante and Anca, 2009). In this study we

show that the Gram positive endobacteria (the BLOs) live in the cytoplasm of many AMF lineages, and that their occurrence is not a sporadic event. BLOs are present in _Glomus_ group A and B

of the _Glomerales_, in _Diversisporales_ and in _Archeosporales_, covering diverse evolutionary lineages in the AMF. Interestingly, they were not detected in _Glomus_ sp.

‘intraradices-like’ DAOM197198, _G. intraradices_ and _G. clarum,_ which are all closely related and belong to the subclade _Glomus_ group Ab (Schwarzott et al., 2001). The AMF-BLO sequences

cluster within those of the _Mollicutes_, despite the presence of a distinct cell wall and represent a new bacterial higher taxon. The AMF cultures that tested positive for BLOs originated

from four continents and represent diverse families, including more basal evolutionary lineages. This indicates that BLOs already colonized AMF before the split of those early diverging AMF

lineages. As a consequence, and from the phylogenetic tree topology, the BLOs must have been living in AMF for more than 400 Myr. Because many of the AMF cultures studied here were

maintained in _in vitro_ root organ cultures, we also obtained evidence that BLOs are vertically transmitted, similar to many endobacteria living in insects (Baumann & Moran, 1997) and

_Candidatus_ Glomeribacter gigasporarum inside AMF of the _Gigasporaceae._ Theory predicts that vertically transmitted symbionts, for example, in insects, should offer some benefit to the

host as they are maintained by it (Brownlie and Johnson, 2009). Even if, at the moment, we have no clue to BLO function, we can infer that BLOs likely confer fitness on their fungal hosts.

Electron microscopy shows that BLOs are directly embedded in the fungal cytoplasm, without a surrounding host membrane, indicating a high level of compatibility or an ancient interaction.

Taken together, our results show that AMF-BLOs are pan-global, vertically inherited, monophyletic, ancient and, so far, uncultured and most likely _Mollicutes_-related endobacteria. Although

the 16S phylogenies place BLOs with _Mollicutes_ rather than with _Firmicutes_, the exact placement of the BLO clade should currently be treated with caution. The relationship between

_Mollicutes,_ which belong to the _Tenericutes,_ and the _Firmicutes_ is in fact not clearly solved (Ludwig et al., 2008; Battistuzzi and Hedges, 2009). To unambiguously resolve the

relationships within these phyla and then attribute BLOs to one of them, data from other genes will be required. Nevertheless, the finding that the BLOs, _Entomoplasmatales_ and

_Mycoplasmatales_ likely share a common ancestor raises questions about the biology and evolution of the _Mollicutes_. A common characteristic is their endosymbiotic or parasitic lifestyle

(Johansson and Pettersson, 2002). The intracellular status of the AMF-BLOs supports the concept that the ancestors of _Mollicutes_ had already evolved mechanisms to exploit intracellular or

symbiotic niches, as showed by the present-day species in the genera _Phytoplasma_ and _Mycoplasma_, which are widespread parasites of animals and plants. Intriguingly, the BLO cell wall,

absent from other members of the _Mollicutes_, indicates that BLOs maintained a trait that sister clades have lost, after the divergence of _Mollicutes_ from their _Firmicute-_related

ancestors, between 600 and 2000 Myr (Maniloff, 2002; Battistuzzi and Hedges, 2009). The presence of a cell wall and the phylogenetic branching of the BLOs support the concept of an ancestral

association between BLOs and AMF. Because AMF themselves have been involved in symbioses with plants since Devonian times (Taylor et al., 1995; Redecker et al., 2000), our findings open new

questions on the complexity and age of multiple and symbiotic inter-phylum interactions. Lastly, in the broader context of eukaryotic cell evolution, the plant-symbiont AMF have been unique

up to now; they maintain an extraordinarily high polymorphism in their genomes (Martin et al., 2008), as reflected by intrasporal nuclear rDNA variability (Stockinger et al., 2009, 2010),

likely resulting in a population of heterokaryotic nuclei. The variability of BLO rDNA sequences now indicates a similar variability for the AMF-associated endobacterial population, even

within one fungal spore. BLOs seem to be widespread not only within the AMF maintained in culture, as investigated in this study, but also in environmental samples, as is emerging from

ultrastructural observations (e.g., Ligrone et al., 2007). For this reason, their pan-global distribution and genetic variability, apparently associated with avoidance of a bottleneck, add a

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241–250. Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank T Pawlowska (USA), C Walker (GB), the Genebank at NIAS (Japan), S Cranenbrouck and S Declerck

from GINCO (Belgium), as well as all original collectors for providing AMF cultures. We thank V Bianciotto and E Lumini for useful comments and A Faccio for TEM preparation. The research

leading to these results received funding from the European Community's Sixth Framework Programme (_FP6/2005–2009_) under grant agreement no. MEST-CT-2005-021016 (TRACEAM), from

Compagnia di San Paolo, Torino and from Converging technology Project (BIOBIT, CIPE) to PB. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Plant Biology, University of Turin and

IPP-CNR, Turin, Italy Maria Naumann & Paola Bonfante * Department of Biology, Inst. Genetics, University of Munich (LMU), Planegg-Martinsried, Germany Maria Naumann & Arthur

Schüßler Authors * Maria Naumann View author publications You can also search for this author inPubMed Google Scholar * Arthur Schüßler View author publications You can also search for this

author inPubMed Google Scholar * Paola Bonfante View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Paola Bonfante.

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Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Naumann, M., Schüßler, A. & Bonfante, P. The obligate endobacteria of arbuscular mycorrhizal fungi are ancient heritable

components related to the _Mollicutes_. _ISME J_ 4, 862–871 (2010). https://doi.org/10.1038/ismej.2010.21 Download citation * Received: 23 November 2009 * Revised: 28 January 2010 *

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initiative KEYWORDS * arbuscular mycorrhizal fungi * endobacteria * interphylum interactions * _Mollicutes_ * pyrosequencing * vertical transmission