Bacterial Distribution
After the raw data was processed and filtered through Qiime2, the dataset consisted of 1,003,775 sequences, with 481,514 sequences associated with the 24 ant samples, 470,669 sequences associated with the 24 fungal samples, and 51,592 sequences associated with the 7 soil samples. From these resulting sequences, 1,065 OTUs were detected, with 334 of these OTUs found within the ant samples, 775 OTUs found within the fungal samples, and 513 OTUs found the soil samples.
Regional Analysis of Ant, Fungus, and Soil Microbiome:
Initial multivariate analysis of the ant and fungal samples indicated that ants, fungus and soil samples formed visually distinct clusters (Fig. S1; stress = 0.184). Moreover, ants, fungus, and soil bacterial microbiomes were significantly different from one another (CCA; F = 3.1581, df = 2, p < 0.0001). The ant (H = 1.946), fungus garden (H = 1.947), and soil samples (H = 4.019) had significantly different Shannon’s Diversity Index values (F = 12.68, df = 2, p = 3.27 x 10-5), with the soil samples having a significantly higher average in comparison to the ant and fungus garden samples (Fig. S2). These results confirm that microbial communities of ants and fungus we were analyzing in this study are not contaminants originating from the surrounding soils. In all subsequent analyses, soils were removed since they were of no primary interest.
Ant Microbiome Composition
The two ant species were the primary driver of their microbiome (CCA; F = 3.8141, df = 1, p < 0.0001), whereas fungus garden clade (F = 0.8464, df = 2, p = 0.6684) and region (F = 0.6298, df = 1, p = 0.8807) did not appear to have significant roles in structuring the bacterial microbiome of ants. These findings were corroborated with NMDS plots showing that the ant samples formed distinct clusters based on species (Fig. 2a; stress = 0.190), and not region (Fig. 2b; stress = 0.190). Additionally, NMDS plots showed that T. septentrionalis had a less variable microbiome than M. turrifex, since T. septentrionalis ant samples were more clustered together while M. turrifex ant samples were more variable (Fig. 2a).
Ant microbiomes appeared to be structured by different bacterial taxa. The three most common bacterial taxa in the microbiome of T. septentrionalis ants were one strain of Solirubrobacter (29.409%), Luteimonas (15.8%), and an unknown member of Burkholderiaceae (9.209%) (Table 2a). In contrast, the three most common taxa in the microbiome of M. turrifex ants were Amycolatopsis (19.814%), an unknown member of Burkholderiaceae (14.633%), and an undescribed member of Microbacteriaceae (10.530%) (Table 2b). Additionally, Pseudonocardia strains consisted of 5.760% of the M. turrifex ant microbiome whereas it was nearly absent in (<1%) T. septentrionalis ants, (Table 2a,b). The indicator species analysis (ISA) of these two ant species also suggested different core microbiomes; with the Actinobacteria, Amycolatopsis (indicator value (IV) = 0.539, p = 0.0001) and Pseudonocardia (IV = 0.500, p = 0.001) strains were the top two significant contributors to overall variation in the M. turrifex ant microbiome whereas two strains of Solirubrobacter (IV = 0.894, p = 0.0001; IV = 0.816, p = 0.0001) were the top significant contributors to overall variation within the T. septentrionalis microbiome (Table 3a,b, Table S1). As a next step, we probed whether bacterial diversity in the classes identified as significant in the ISA were explained by 1) geographic region, 2) ant species or 3) fungal garden clade (Table S1). The ISA reported that four bacterial classes (Actinobacteria, Alphaproteobacteria, Bacteroidia, and Gammaproteobacteria) were important in describing the variation among ants. The Actinobacteria community of the ants was primarily explained by the ant species (CCA; Table 4a). Similarly, the Alphaproteobacteria and the Gammaproteobacteria communities of the ants were explained by ant species (CCA; Table 4a). However, the Bacteroidia community for the ants was not explained by any variable (RDA; Table 4a). The ISA found 15 significant OTUs within the Actinobacteria community that differed between the two ant species. OTUs of Pseudonocardia and Amycolatopsis associated with M. turrifex and OTUs of Naumannella, an undescribed Intrasporangiaceae, and Aeromicrobium within T. septentrionalis were important drivers in overall discrimination between the two ant associated Actinobacteria communities (Fig. 3a,b). Other significant OTUs driving the differentiation in the Alphaproteobacteria between the two ant species included a Sphingomonas OTU in M. turrifex and within the Gammaproteobacteria, a Lutimonas OTU in T. septentrionalis (Fig. S3a,b).
Despite community level differences in taxa comprising the bacterial microbiomes of the two ant species, overall diversity of the two species was similar. Although M. turrifex was projected to have significantly more OTUs than T. septentrionalis (Fig. 2c), there was not a statistical difference in the average Shannon’s Diversity Index of bacteria between T. septentrionalis (H = 1.902) and M. turrifex (H = 1.998) ants (t = 0.70803, df = 22, p = 0.4864).
Fungal Microbiome Composition
Unlike ant bacterial microbiomes, geographic region appeared a greater role in structuring the bacterial microbiome of the fungus garden, though this was not statistically significant (CCA; F = 1.3466, df = 1, p = 0.1065) than fungal clade (F = 0.9482, df = 2, p = 0.5223) or host ant species (F = 1.0390, df = 1, p = 0.3347). The three most common taxa in the microbiome of East Texas fungus were Mesoplasma (57.891%) and two strains of Spiroplasma (21.857%, 5.020%) (Table 5a). The three most common taxa in the microbiome of Central Texas fungus were Mesoplasma (31.797%), Pseudomonas (8.750%), and Tyzzerella (4.880%) (Table 5b).
The ISA found that two OTUs defined East Texas fungus while 47 OTUs defined the Central Texas fungi; with most of these indicator OTUs found in the families Acetobacteraceae, Acidobacteriaceae, Burkholderiaceae, Cthoniobacteraceae, Enterobacteriaceae, Sphingobacteriaceae, and Weeksellaceae (Table 3c,d, Table S2). Except for a single undescribed OTU in the genus Entomoplasma, no members of the Entomoplasmataceae (the family of the most common taxa, Mesoplasma) were significant members of the fungus garden bacterial microbiomes (Tables 3,S2).
Since Mesoplasma was very common in the fungus garden samples, yet seemed to poorly characterize fungus garden microbiome structure (per the ISA), sequences of Mesoplasma were removed to examine for patterns among the other bacterial taxa. Without Mesoplasma, the fungus microbiome was significantly determined by region (CCA; F = 1.5921, df = 1, p = 0.0106), whereas fungal clade (F = 0.8532, df = 2, p = 0.7251) and host ant species (F = 1.0982, df = 1, p = 0.2832) were not significant factors in structuring fungus garden microbiomes. These findings were corroborated with an NMDS plot showing that the fungal samples formed clusters based around their region (Fig. 4, stress = 0.145).
The ISA reported that five bacterial classes (Acidobacteriia, Actinobacteria, Alphaproteobacteria, Bacteroidia, and Gammaproteobacteria) were important in describing the variation among the fungus gardens (Table 3 and S2). As with the ants, the following analyses probed whether variation in the OTUs in these classes were explained by 1) geographic region, 2) host ant species or 3) fungal clade. The Acidobacteria community of the fungus gardens was explained by both fungal clade and the region (RDA; Table 4b). The Actinobacteria community of fungus gardens was significantly explained by the host ant species, but the fungal clade may play a role in determining community variation, though the test was not statistically significant (CCA; Table 4b), but low enough to warrant concern about a Type II error. The Alphaproteobacteria community of fungus gardens was explained by host ant species and by fungal clade (CCA; Table 4b). The Gammaproteobacterial community of fungus gardens was explained by ant species (CCA; Table 4b). The Bacteroidia community for fungus gardens was not explained by any variable (RDA; Table 4b). The ISAs within the Acidobacteriia community found that OTUs of Granulicella, Edaphorbacter, and 2 undescribed OTUs from the Acidobacteria subgroup 2 being significant to Central Texas fungal samples (Fig. 5a). Similarly, regional differences among the Actinobacteria were driven by five OTUs, with Nocardioides, Mycobacterium, and Corynebacterium in Central Texas fungus gardens and Intrasporangiaceae and Naumannella significant for East Texas fungal samples (Fig. 5b). Nine OTUs within Alphaproteobacteria were found to drive the differentiation of this community based on region, with some of these significant OTUs for the Central Texas fungal samples being associated with Caulobacteracea, Acetobacteraceae, Xanthobacteraceae, and Micropepsaceae (Fig. 5c). 4 OTUs within Gammaproteobacteria were found to drive regional community differentation, with OTUs within Ideonella, Enhydrobacter, and the Burkholderia-Caballeronia-Paraburkholderia complex significant for Central Texas fungal samples (Fig. 5d). Additionally, OTUs within Acidobacteriia and Alphaproteobacteria were found to significantly drive community differences of the three fungal lineages, but this significance was due to these driver OTUs being found primarily within the Clade B3 samples, which was relatively undersampled (n=3) (Fig. S4a,b).
Comparing extrapolated rarefaction curves for fungus garden microbiomes (Mesoplasma excluded) show that Central Texas fungi contain significantly more OTUs in comparison to East Texas fungi (Fig. 6a); the average Shannon’s Diversity Index for Central Texas (H = 3.155) fungus was greater than that of East Texas (H = 2.151) fungus (t = -2.1604, df = 13.524, p = 0.049). Rarefaction curves of fungus garden microbiome based on host ant species showed no significant differences (Fig. 6b). Rarefaction curves based on fungal clade showed significant differences; with Clade B4 fungus having more expected OTUs than Clade B5 fungus (Fig. 6c). The similarity between the rarefaction curves of fungus grown by the different ant host species was also reflected in the Shannon’s Diversity Index values for fungus grown by T. septentrionalis (H = 2.430) and M. turrifex (H = 2.459) (t = 0.5936, df = 21.908, p = 0.9532) (Fig. 6d). However, there was not a statistical difference in the average Shannon’s Diversity Index between Clade B3 (H = 3.033), Clade B4 (H = 2.581), and Clade B5 (H = 2.205) fungal samples (F = 0.608, df = 2, p = 0.554) (Fig. 6e).
Comparing extrapolated rarefaction curves for fungus garden microbiomes (Mesoplasma included) show that Central Texas fungi contain significantly more OTUs in comparison to East Texas fungi (Fig. S5a); the average Shannon’s Diversity Index for Central Texas (H = 3.058) fungus was greater than that of East Texas (H = 1.489) fungus (t = -2.6417, df = 22, p = 0.0149). Rarefaction curves of fungus garden microbiome based on host ant species showed no significant differences (Fig. S5b). Rarefaction curves based on fungal clade showed significant differences; with Clade B4 fungus having more expected OTUs than Clade B5 fungus (Fig. S5c). The similarity between the rarefaction curves of fungus grown by the different ant host species was also reflected in the Shannon’s Diversity Index values for fungus grown by T. septentrionalis (H = 1.675) and M. turrifex (H = 2.268) (t = 0.97297, df = 22, p = 0.3411) (Fig. S5d). However, there was not a statistical difference in the average Shannon’s Diversity Index between Clade B3 (H = 3.056), Clade B4 (H = 2.191), and Clade B5 (H = 1.659) fungal samples (F = 1.188, df = 2, p = 0.325) (Fig. S5e). In summary, Mesoplasma did not appear to influence overall diversity patterns.