Novel Chlamydiae and Amoebophilus endosymbionts are prevalent in wild isolates of the model social amoeba Dictyostelium discoideum.

Amoebae interact with bacteria in multifaceted ways. Amoeba predation can serve as a selective pressure for the development of bacterial virulence traits. Bacteria may also adapt to life inside amoebae, resulting in symbiotic relationships. Indeed, particular lineages of obligate bacterial endosymbionts have been found in different amoebae. Here, we screened an extensive collection of Dictyostelium discoideum wild isolates for the presence of these bacterial symbionts using endosymbiont specific PCR primers. We find that these symbionts are surprisingly common, identified in 42% of screened isolates (N = 730). Members of the Chlamydiae phylum are particularly prevalent, occurring in 27% of the amoeba isolated. They are novel and phylogenetically distinct from other Chlamydiae. We also found Amoebophilus symbionts in 8% of screened isolates (N = 730). Antibiotic-cured amoebae behave similarly to their Chlamydiae or Amoebophilus-infected counterparts, suggesting that these endosymbionts do not significantly impact host fitness, at least in the laboratory. We found several natural isolates were co-infected with multiple endosymbionts, with no obvious fitness effect of co-infection under laboratory conditions. The high prevalence and novelty of amoeba endosymbiont clades in the model organism D. discoideum opens the door to future research on the significance and mechanisms of amoeba-symbiont interactions.

Introduction

As voracious microbial predators, free‐living amoebae are important shapers of their microbial communities (Clarholm, 1981). This predatory pressure can alter the presence and abundance of specific microbial constituents in the community (Ronn et al., 2002). Amoeba predation is also postulated to play an important role in microbial evolution, particularly in the evolution of bacterial virulence (Matz and Kjelleberg, 2005; Sun et al., 2018). Bacteria that evolve strategies to avoid or survive amoeba predation would be selected for in amoeba rich environments (Erken et al., 2013). Bacteria that are able to enter amoeba cells (via phagocytosis or other entry mechanisms) but avoid subsequent digestion gain access to an attractive intracellular niche. A diverse collection of intracellular bacterial symbionts of amoebae has been found, some of which appear pathogenic, neutral, or beneficial for their amoeba host (DiSalvo et al., 2015; Jeon, 1992; König et al., 2019; Maita et al., 2018; Shu et al., 2018; Taylor et al., 2012). Some of these symbionts belong to the same bacterial genera as important human pathogens such as Mycobacteria, Legionella, and Chlamydia (Drancourt, 2014; Tosetti et al., 2014; Paquet and Charette, 2016; Boamah et al., 2017; Cardenal‐Muñoz et al., 2018; Gomez‐Valero and Buchrieser, 2019).

Bacterial endosymbionts of amoebae were first discovered in the diverse, free‐living amoeba genus Acanthamoeba (Horn and Wagner, 2004). These amoebae are found in soil and freshwater environments and can protect themselves and their bacterial associates by forming cysts under environmentally unfavourable conditions (Rodríguez‐Zaragoza, 1994). Initially, specific identification of these endosymbionts was impeded by the inability to isolate them. Once molecular methods became available, it was clear that these bacteria were from particular lineages of Chlamydiae, Proteobacteria, Bacteroidetes, and Dependentiae (Amann et al., 1997; Fritsche et al., 1999; Horn and Wagner, 2004; Schmitz‐Esser et al., 2008; Delafont et al., 2015; Maita et al., 2018; König et al., 2019; Samba‐Louaka et al., 2019). Overall, a high proportion of sampled Acanthamoeba (25%–80%) are infected with endosymbionts; however, the broader distribution of symbiotic bacteria among other amoeba species and their prevalence in natural host populations is unclear (Schmitz‐Esser et al., 2008).

These endosymbionts interact with their amoeba hosts in varied ways. Some inhabit host‐derived vacuoles, while others persist in the cytoplasm. A Neochlamydia symbiont can protect its host against the detrimental consequences of Legionella bacterial infection (Ishida et al., 2014; Maita et al., 2018; König et al., 2019) and other strains have been shown to improve the growth rate and motility of their hosts (Okude et al., 2012). Other known amoeba endosymbionts are from the less‐studied genera Amoebophilus and Procabacter whose effects on their hosts are largely unknown. Genome sequencing of the amoeba symbiont Candidatus Amoebophilus asiaticus found no evidence that it could provide novel pathways to supplement host nutrition, suggesting a parasitic lifestyle, but other fitness effects have not been tested (Schmitz‐Esser et al., 2010).

Another amoeba that has been used as a model to study host‐bacteria interactions is the social amoeba, Dictyostelium discoideum. This amoeba, distantly related to Acanthamoeba, is terrestrial, living and feeding as single cells in forest soils while the bacterial food supply is plentiful. When food becomes scarce, amoebae begin a social cycle, which entails thousands of cells aggregating and forming a slug to crawl to the surface of the soil. The cycle culminates in the formation of a fruiting body, made of a ball of spores (sorus) sitting on top of a stalk, which can then be dispersed to more favourable conditions (Kessin, 2001). In many cases, bacteria are cleared from D. discoideum cells during the social cycle through innate immune‐like mechanisms (Chen et al., 2007). However, vegetative amoebae can be infected with human pathogens such as Mycobacterium, Bordetella, and Legionella for experimental study (Bozzaro and Eichinger, 2011; Taylor‐Mulneix et al., 2017).

Recently, a variety of bacterial symbionts, both transient and persistent, have been discovered to colonize D. discoideum (DiSalvo et al., 2015; Brock et al., 2018; Haselkorn et al., 2018; Sallinger et al., 2021). Three different newly named Paraburkholderia species, P. agricolaris, P. hayleyella, and P. bonniea, are persistent bacterial symbionts that intracellularly infect vegetative amoebae and spore cells (Brock et al., 2011, 2020; Haselkorn et al., 2018; Shu et al., 2018; Khojandi et al., 2019). While these Paraburkholderia themselves do not nourish D. discoideum, they confer farming to their hosts by allowing food bacteria to survive in the fruiting body. These food bacteria can then be re‐seeded in new environments after spore dispersal, which benefits hosts in food‐scarce conditions (Brock et al., 2011; Haselkorn et al., 2018; Khojandi et al., 2019). In natural populations, an average of 25% of D. discoideum are infected with Paraburkholderia (Haselkorn et al., 2018). Other bacteria, some edible and others not, have the ability to transiently associate with D. discoideum even in the absence of Paraburkholderia co‐infections (Brock et al., 2018). Thus, natural D. discoideum fruiting bodies host a small‐scale microbiome, representing a simple model system to study the mechanisms of microbiome formation (Dinh et al., 2018; Farinholt et al., 2019). However, all previous sampling of natural D. discoideum associates has relied on isolating bacterial colonies from nutrient media plates, which would miss any bacterial species incapable of growing under these conditions, such as obligate amoeba symbionts.

Since obligate endosymbionts are common in other free‐living amoebae, we endeavoured to identify them in D. discoideum. Here, we screened a frozen stock collection of 730 natural D. discoideum isolates. We first used a 16S rRNA gene amplification strategy with ‘universal’ primers and direct sequencing to detect and identify bacteria within D. discoideum spores in a single population. In addition to detecting expected Paraburkholderia symbionts, we identified many Chlamydiae and Amoebophilus sequences, and a few Procabacter sequences in screened samples. To determine the extent of Chlamydiae and Amoebophilus symbionts in our collection we then used symbiont‐specific PCR screening. From each of these prevalent bacterial lineages, we found genetically distinct novel species and were able to visualize representative endosymbiont cells within spores via electron microscopy. We also used antibiotics to cure natural hosts of Chlamydiae and Amoebophilus and compared host fitness to uncured counterparts. Finally, we looked at the fitness effects of co‐infections since co‐infections occur in nature.

Results

Obligate endosymbionts are common in wild‐collected clones of Dictyostelium discoideum

The vast majority of our 730 D. discoideum isolates were collected from the Eastern half of the United States (Fig. 1A) and had previously been PCR‐screened for Paraburkholderia (Haselkorn et al., 2018; Supporting Information Table S1). This included 16 populations with seven or more individuals collected. Our largest collection from Virginia consists of about 200 individuals collected in 2000 and in 2014. Across all locations we found over 41% of samples harboured at least one obligate endosymbiont. Specifically, 117 (~16%) were infected with Amoebophilus and 198 (~27%) with Chlamydiae. Sixteen (2.1%) were co‐infected with both Amoebophilus and Chlamydiae (Fig. 1B). Two isolates (both from Virginia) were infected with an obligate bacterium identified as belonging to the genus Procabacter.

Fig. 1

Unculturable symbiont prevalence in natural D. discoideum populations. A. Pie charts indicate Chlamydiae, Amoebophilus, and Chlamydiae‐Amoebophilus co‐infection patterns in respective populations of D. discoideum isolates from across the Eastern United States. Procabacter, due to its low prevalence, is not included. Numbers next to pie charts indicate the number of isolates for each population. B. Of all symbiont‐positive amoebae isolates, several are co‐infected with more than one symbiont species, represented by the Venn diagram indicating the number of isolates infected with Paraburkholderia, Amoebophilus, and Chlamydiae.

Associations among Chlamydiae, Amoebophilus, and Paraburkholderia D. discoideum symbionts are inconsistent across populations

Our earlier culture‐based screening of this D. discoideum natural collection found that 25% of isolates were infected with Paraburkholderia symbionts (Haselkorn et al., 2018; Brock et al., 2020). For this new round of sequence‐based screening, we identified multiple instances of Paraburkholderia‐Amoebophilus and Paraburkholderia‐Chlamydiae co‐infections in addition to the Amoebophilus‐Chlamydiae co‐infections from above. Specifically, 42 isolates (6%) were co‐infected with Paraburkholderia and Amoebophilus, 25 (3.5%) with Paraburkholderia and Chlamydiae, and 9 (1%) were co‐infected with all three symbiont types (Fig. 1B). Association of the different endosymbionts was tested in our three highest sampled populations: Texas‐Houston Arboretum, Virginia Mountain Lake 2000, and Virginia Mountain Lake 2014. Pairwise fisher exact tests showed a small positive association between Chlamydiae and Paraburkholderia in the Texas population (P = 0.0132, 15 observed with 10 expected) but not the Virginia populations. There was a positive association between Paraburkholderia and Amoebophilus in the Virginia 2000 collection (P = 0.000277, 43 observed with 30 expected) and a negative association between Chlamydiae and Amoebophilus in the Virginia 2014 collection (P = 0.000024, none observed with 10 expected). Positive associations between Paraburkholderia and other bacteria may be consistent with the farming phenotype (Brock et al., 2011, DiSalvo et al., 2015), although this was not seen consistently across all populations. Variable associations across populations may suggest environmental‐context dependent effects.

The Procabacter, Amoebophilus, and Chlamydiae endosymbionts of D. discoideum are novel and phylogenetically distinct from known symbionts

For Procabacter, Amoebophilus, and the three most prevalent Chlamydiae strains (those occurring in more than one individual), we sequenced the full length 16S rRNA for our D. discoideum endosymbionts to better determine their evolutionary relationships. In all cases, the 16S rRNA haplotypes were novel and highly diverged, exhibiting a range of 6%–12% sequence difference from other sequences currently in GenBank or SILVA (Supporting Information Table S2). For the Procabacter and Amoebophilus endosymbionts, only a single haplotype was found for each. Our phylogenetic analysis shows that the Procabacter endosymbiont of D. discoideum is sister to all of the Procabacter endosymbionts of Acanthamoeba (Fig. 2A). While it is most closely related to this genus, forming a clade with 100% bootstrap support, the 16S rRNA sequence is only a 93% match. Although this Procabacter endosymbiont is not common in these D. discoideum populations, its phylogenetic placement represents an important link for understanding the evolution and adaptation of Procabacter to amoeba hosts.

Fig. 2

Unculturable endosymbionts of Dictyostelium are related to unculturable endosymbionts of other amoebae. 16S rRNA phylogenies of Procobacter (A) Amoebophilus (B) and Chlamydiae (C) endosymbionts constructed using the Maximum Likelihood method in Mega7, with 1000 bootstrap replicates. Family names, where applicable are delineated in bold italics. All sequences are full length 16S rRNA except for the Chlamydiae haplotypes 4–8.

A similar phylogenetic pattern is seen for the Amoebophilus symbiont of D. discoideum (Fig. 2B). This symbiont groups most closely with the putative Amoebophilus symbiont from genome assembly of the recently sequenced social amoeba Coremiostelium polycephalum, suggesting that there may be some host specificity of Amoebophilus among social amoebae, although sampling of additional amoeba species would be necessary to elucidate this pattern. This Dictyostelid symbiont clade is sister to the Amoebophilus asiaticus endosymbionts of Acanthamoeba (Horn et al., 2001), and is 6% diverged at the 16S rRNA gene (Supporting Information Table S2). The next most closely related bacteria to the Amoebophilus genus are the insect facultative endosymbionts of the Cardinium genus (Santos‐Garcia et al., 2014). These bacteria infect up to 7% of all insect species and have diverse effects on their hosts ranging from reproductive parasitism to nutritional supplementation and defence against parasites. Comparative genome sequencing of this additional Amoebophilus species could lend insight into the process of symbionts adapting to diverse hosts and highlight potential amoeba species‐specific adaptations.

There are eight distinct and novel haplotypes of D. discoideum endosymbionts in the Chlamydiae. For the three most prevalent haplotypes 1, 2, and 3 (22%, 4%, and 1% population prevalence respectively), we sequenced the full 16S rRNA gene to reconstruct a more resolved phylogeny which included their nearest neighbours from the SILVA database and representatives from many Chlamydiae families (Fig. 2C). There are currently six formally named and eight proposed Chlamydiae families (Taylor‐Brown et al., 2015; Pillonel et al., 2018), and several other recently discovered novel clades from marine sediments (Dharamshi et al., 2020). The D. discoideum Chlamydiae endosymbionts do not appear to be closely related (<97% sequence match) to any of these groups, and some may represent novel families. Greater than 10% sequence divergence at the full length 16S rRNA gene has been proposed as family level difference (Everett et al., 1999), although full genome sequences may be necessary to make this assertion.

Haplotypes 1, 2, and 4 form their own well‐supported clade, grouping with the Rhabdochlamydiaceae and Ca. Arenachlamydiaceae, sister to the Simkaniaceae (Fig. 2C). Many of the internal nodes on this phylogeny have low bootstrap support, making more detailed phylogenetic inferences impossible. This D. discoideum endosymbiont clade, however, does have only a 91% identity to its closest phylogenetic neighbour and may represent a new genus or family of Chlamydiae. These are, to the best of our knowledge, novel, as yet unnamed, Chlamydiae, though they may be commonly found in the soil. Detailed searches for this most common Chlamydiae haplotype 1 in the IMNGS database (Lagkouvardos et al., 2016), which queries the sequence read archives from NCBI, returned 13 matches (>99% sequence identity), mostly as ‘uncultured bacteria’ from soil metagenomic studies (Supporting Information Table S3). One such study was in the Northeast United States (Massachusetts), in a region close to where we sampled D. discoideum, and it is likely that amoeba endosymbionts are being sequenced as part of these metagenomes.

Our other Chlamydiae haplotypes are scattered throughout the phylogeny. Haplotypes 3 and 5 form a well‐supported clade and may be sister to Rhabdochlamydiaceae/Ca. Arenachlamydiaceae/Simkaniaceae families. These haplotypes have a greater than 11% nucleotide difference from their nearest neighbours on the phylogeny (and in the SILVA and NCBI databases; Supporting Information Table S2) and may represent a novel family as well. Haplotypes 6, 7, and 8 fall in different places in the phylogeny and with long branch lengths separating them from other taxa, although these haplotypes are based on only 220 bp of sequence information so their taxonomic identity is less clear. Finally, while the newly discovered marine Chlamydiae lineages (Dharamshi et al., 2020) were not included in this phylogeny because they were not full‐length 16S rRNA sequences, when we aligned our Chlamydiae haplotypes with their partial 16S rRNA sequences, the closest match had only an 89% sequence identity.

Environmental Chlamydiae appear to be diverse and ubiquitous and many Chlamydiae endosymbionts associate with a wide range of protozoa and other organisms (Corsaro and Venditti, 2006; Coulon et al., 2012; Corsaro et al., 2013). The incredible genetic diversity within the phylum Chlamydiae suggests ancient interactions with eukaryotes, with estimates that the association is 700 million years old (Greub and Raoult, 2003). The diversity of the phylum Chlamydiae is much higher than the currently characterized and named families would indicate. Using novel methods which search various 16S rRNA genome databases, metagenomes, and sequence read archives, it is estimated from the vast DNA sequence diversity detected that there are anywhere from 1157 to 1483 different families (Collingro et al., 2020). This type of screening, however, does not identify the hosts, so insight into adaptation to particular hosts is limited. Five of the eight unique D. discoideum haplotypes fall into two divergent and well‐supported D. discoideum‐specific phylogenetic clades, potentially representing two novel Chlamydiae families and suggesting ongoing evolution within the D. discoideum host. The separation of these novel D. discoideum Chlamydiae lineages from the other amoeba lineages suggests a long‐standing association and possible co‐adaptation.

Many Chlamydiae and Amoebophilus endosymbiont haplotypes are widely distributed across D. discoideum populations

While the average overall prevalence of infection of Chlamydiae endosymbionts was 27%, it varied among populations, ranging from 0% to 75% (Supporting Information Table S1). Haplotype 1 represented 79.8% of all D. discoideum Chlamydiae infections, while haplotype 2 was 13.6% and haplotype 3 was 3.5%. Haplotypes 4–8 were represented only once. Haplotype 1 was found in 12 populations, haplotype 2 was found in eight different populations, and haplotype 3 was found in three populations. The diversity of Chlamydiae strains was the highest in the Virginia Mountain Lake collection from 2000, which had six of the eight haplotypes (1–6), and the second highest diversity of Chlamydiae haplotypes was found in the Texas Houston Arboretum collection (1, 2, 3 and 8). Interestingly, haplotype 1 was the only haplotype circulating in the most recent 2014 Virginia Mountain Lake collection. While haplotype diversity was lower in this collection, Chlamydiae prevalence (for all haplotypes) in the region increased from 20% in 2000 to 38% in 2014.

The one strain of Amoebophilus found to infect D. discoideum was distributed across nine populations, although at low prevalence, often with just a single individual infected in many locations. This endosymbiont was generally less prevalent than Chlamydiae, with the exception of the 2000 Virginia Mountain Lake population, where it infected 37% of the population. Amoebophilus prevalence in this region, however, decreased to 10% in 2014. Amoebophilus was also relatively prevalent in the Texas Houston Arboretum population, infecting 23% of the population.

Obligate endosymbionts can be visualized within reproductive host spore cells

Transmission electron microscopy (TEM) revealed bacterial cells inside developed spores from representative Amoebophilus, Chlamydiae, and Procabacter host isolates (Fig. 3). Symbiont cells were detectable in over 50% of the spores of these symbiont hosts in the TEM cross sections, suggesting a high infection prevalence. Each symbiont appeared to have gram‐negative type cell walls but displayed distinct morphologies within spore cells. Amoebophilus endosymbionts were rod‐like, measured 0.3–0.5 μm in width and 0.5–1.3 μm in length, and appeared to be distributed throughout the cytoplasm within host membranes, similar to previous observations of Amoebophilus symbionts in Acanthamoeba sp. (Horn et al., 2001; Schmitz‐Esser et al., 2010). Procabacter endosymbionts were rod shaped, measured 0.25–0.5 μm in width and 0.75–1.5 μm in length, and in many cases were surrounded by a host membrane, as seen for the Acanthamoeba spp. Procabacter in some cells (Heinz et al., 2007), but not others (Fritsche et al., 2002). Chlamydiae endosymbionts appeared as dense wrinkled spheres measuring 0.3–0.6 μm that were distributed throughout the cytoplasm. The wrinkled morphology of Chlamydiae cells in D. discoideum spores is somewhat reminiscent of the spiny structure of Chlamydia‐like symbionts in Acanthamoeba, Naegleria, and Hartmannella host species (Horn et al., 2000; Corsaro and Greub, 2006; Casson et al., 2008). However, these morphological observations may be artefacts of (or exaggerated by) sample preparation prior to TEM and may be better resolved using cryo‐electron tomography (Huang et al., 2010; Santarella‐Mellwig et al., 2013).

Fig. 3

Unculturable symbionts have distinct morphologies in infected spores. Transmission electron micrographs of spores from the indicated representative uninfected and infected D. discoideum isolates. Images on the left show single D. discoideum spores with arrows pointing to individual endosymbionts followed by a close‐up image on the right of individual endosymbionts from the same spore.

Chlamydiae‐like organisms typically have two or more morphotypes that correspond to distinct developmental stages. Elementary bodies are the infectious form that can be transmitted extracellularly from cell to cell. Once internalized, elementary bodies differentiate into reticulate bodies that typically replicate within inclusion vesicles, although some environmental Chlamydiae can be found directly in the cytosol (Horn, 2008; Benamar et al., 2017; Bou Khalil et al., 2017; Bayramova et al., 2018; Nylund et al., 2018; Collingro et al., 2020). However, in D. discoideum host spores, we only visualize one morphology that does not appear to be in an inclusion but is rather distributed throughout the cytosol. It is unclear what morphotype this represents and whether other morphotypes exist in D. discoideum prior to spore formation. It is possible that additional Chlamydiae developmental stages occur in vegetative amoebae and that only one form is favoured within metabolically inert host spores. Future studies using fluorescent in situ hybridization would lend insight into infection dynamics during different stages of the amoeba lifecycle.

Amoebophilus and Chlamydiae can be eliminated from host isolates without significantly impacting host fitness

We did not observe any obvious defects in the growth and development of hosts infected by Chlamydiae or Amoebophilus. To better assess the impact of these endosymbionts on their hosts, we measured the reproductive fitness of representative host isolates before and after endosymbiont curing (Fig. 4). To eliminate endosymbionts from host isolates, we selected four Chlamydiae positive and four Amoebophilus positive isolates and cultured each for two social cycles on antibiotic saturated plates. After antibiotic exposure and resumption of development on normal media, we confirmed stable loss of endosymbionts via endosymbiont‐specific PCR screening (Fig. 4A). We next cultured 1 × 105 spores from antibiotic‐treated and untreated D. discoideum counterparts on nutrient media with Klebsiella pneumoniae food bacteria. Following culmination of fruiting body development after a week of incubation, we harvested and measured total spore contents (Fig. 4B). We found no significant differences in spore productivity according to endosymbiont status (one‐way analysis of variance: F(5,42) = 1.106, P = 0.372). Thus, symbiont infections in these natural host isolates have no significant impact on host spore productivity under these conditions. This does not, however, rule out the possibility that these symbionts may impart significant impacts on their hosts which may be life‐stage, environmental, or genotypically context‐dependent (Taylor‐Brown et al., 2015). We could not assess the fitness impact of Chlamydiae or Amoebophilus in new host amoebae as our attempts to establish infections in new hosts have been thus far unsuccessful.

Fig. 4

Elimination of Amoebophilus and Chlamydiae from natural hosts with antibiotics does not impact host reproductive fitness. Four uninfected, Amoebophilus‐infected, and Chlamydiae‐infected representative host isolates were treated with rifampicin to eliminate symbionts (A) and reproductive fitness (B) was assessed. Symbiont presence is indicated by amplification with Amoebophilus‐specific or Chlamydiae‐specific PCR and DNA gel electrophoresis (with Eukaryote specific gene amplification serving as an internal control) (A). Reproductive fitness was assessed by quantifying total spore productivity of D. discoideum cultures after completion of one social cycle (B). Individual D. discoideum isolates are represented by point colour.

Amoebophilus and Chlamydiae infections do not alter the fitness of hosts during exposure to Paraburkholderia

Since co‐infections with unculturable symbionts and Paraburkholderia occur in the wild, we wanted to determine whether these co‐infections would result in a different fitness outcome than Paraburkholderia infections alone. We tested the fitness impact of Chlamydiae and Amoebophilus during Paraburkholderia exposure using the same strategy as above, but with the addition of 5% by volume of the indicated Paraburkholderia strain to food bacteria prior to plating (Fig. 5). These Paraburkholderia symbionts range from highly detrimental (P. hayleyella strain 11), to intermediately detrimental (P. agricolaris strains 159 and 70), to neutral (P. bonniea strain 859) under the culturing conditions used for this assay (Haselkorn et al., 2018; Shu et al., 2018; Khojandi et al., 2019; Miller et al., 2020). In line with previous observations, Paraburkholderia strains differentially influence spore productivity for both Amoebophilus (F(3,60) = 36.87, P = <0.001) and Chlamydiae (F(3,60) = 28.52, P = <0.001) originating host lines (Fig. 5). However, as with single unculturable symbiont infections, we found that the presence or absence of Amoebophilus or Chlamydiae had no significant impact on spore productivity during exposure to P. hayleyella‐11 (F(5,42) = 1.171, P = 0.339), P. agricolaris‐159 (F(5,42) = 0.985, P = 0.438), P. agricolaris‐70 (F(5,42) = 1.095, P = 0.377), or P. bonniea‐859 (F(5,42) = 0.661, P = 0.655) (Fig. 5). Thus, under these conditions, the fitness impact of Paraburkholderia symbiosis on host spore productivity does not appear to be altered (for better or worse) by co‐infections with obligate endosymbionts. This laboratory finding is consistent with the random associations among symbionts found more often than not in our populations. In different environments, however, other microbe–microbe interactions may have an effect that favours certain combinations of symbionts (Rock et al., 2018).

Fig. 5

Amoebophilus and Chlamydiae infections do not impact the reproductive fitness of hosts co‐infected with Paraburkholderia symbionts. Reproductive fitness was quantified for four untreated and antibiotic‐cured Amoebophilus‐infected (A) and Chlamydiae‐infected isolates (B) following social cycle completion after exposure to the indicated Paraburkholderia symbiont strains. Individual D. discoideum isolates are represented by point colour.

Conclusion

In a large survey across the eastern United States, we have identified the presence and prevalence of novel strains of obligate intracellular symbionts from within the Chlamydiae, Amoebophilus, and Procabacter lineages in natural populations of the social amoeba Dictyostelium discoideum. We have demonstrated that these symbionts do not affect amoeba fitness under standard laboratory conditions. High, but variable, prevalences in natural populations and the observation of some co‐infection rates that deviate from those expected for random distributions suggest that these symbionts may have environmental context‐dependent effects. The presence of these novel bacterial strains that are highly diverged from known amoeba endosymbionts provides an opportunity to study host–symbiont interactions in the well‐studied D. discoideum model organism, enabling explorations into the evolutionary history of these widespread obligate symbionts that are associated with a large variety of hosts.

Appendix S1. Supporting information

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Table S1 Table of Screened Table lists each D. discoideum isolate by identification number and location of collection. PCR amplification of Dictyostelium, Paraburkholderia, Amoebophilus, or Chlamydiae specific DNA is indicated with a 1 (for positive amplification) or 0 (for negative amplification). Samples used for fitness assays are highlighted in yellow.

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Table S2 SILVA Database Classification of Symbiont Haplotypes. Percent sequence identity to closest hit in the database for the five full length 16S rRNA sequence haplotypes and their least common ancestor taxonomic classification.

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Table S3 IMNGS database > 99% matches for Chlamydiae Endosymbiont of Includes metagenome sample type and collection location.

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Table S4 PCR Primers Used for this Study. PCR primer names, sequences, and associated references.

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