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Community analysis of microbial sharing rspb.royalsocietypublishing.org and specialization in a Costa Rican –plant–hemipteran symbiosis

Elizabeth G. Pringle1,2 and Corrie S. Moreau3 Research 1Department of Biology, Program in Ecology, Evolution, and Conservation Biology, University of Nevada, Cite this article: Pringle EG, Moreau CS. 2017 Reno, NV 89557, USA 2Michigan Society of Fellows, University of Michigan, Ann Arbor, MI 48109, USA Community analysis of microbial sharing and 3Department of Science and Education, Field Museum of Natural History, 1400 South Lake Shore Drive, specialization in a Costa Rican ant–plant– Chicago, IL 60605, USA hemipteran symbiosis. Proc. R. Soc. B 284: EGP, 0000-0002-4398-9272 20162770. http://dx.doi.org/10.1098/rspb.2016.2770 have long been renowned for their intimate mutualisms with tropho- bionts and plants and more recently appreciated for their widespread and diverse interactions with microbes. An open question in symbiosis research is the extent to which environmental influence, including the exchange of Received: 14 December 2016 microbes between interacting macroorganisms, affects the composition and Accepted: 17 January 2017 function of symbiotic microbial communities. Here we approached this ques- tion by investigating symbiosis within symbiosis. Ant–plant–hemipteran symbioses are hallmarks of tropical ecosystems that produce persistent close contact among the macroorganism partners, which then have substantial opportunity to exchange symbiotic microbes. We used metabarcoding and Subject Category: quantitative PCR to examine community structure of both and Ecology fungi in a Neotropical ant–plant–scale- symbiosis. Both phloem-feed- ing scale and honeydew-feeding ants make use of microbial Subject Areas: symbionts to subsist on phloem-derived diets of suboptimal nutritional qual- ecology, evolution, microbiology ity. Among the insects examined here, ants and pseudococcid scale insects had the most specialized bacterial symbionts, whereas ants appeared to consume or associate with more fungi than bacteria, and coccid Keywords: scale insects were associated with unusually diverse bacterial communities. Cordia alliodora, bacteria, fungi, Despite these differences, we also identified apparent sharing of microbes metabarcoding, myrmecophyte, scale insects among the macro-partners. How microbial exchanges affect the consumer- resource interactions that shape the evolution of ant–plant–hemipteran symbioses is an exciting question that awaits further research. Author for correspondence: Elizabeth G. Pringle e-mail: [email protected] 1. Introduction Mutualistic symbioses, i.e. mutually beneficial interactions where the partners live in prolonged physical contact, have been a major driver of the evolution of life on Earth, from the origin of eukaryotes to coral-reef diversity hotspots. Mutualistic symbioses are currently receiving unprecedented attention across biological subdisciplines as the use of high-throughput sequencing begins to reveal the ubiquitous and dynamic associations between microbes and macro- organisms. These associations provide vital functions for both partners, including increased metabolic capacity, nutrition and protection, but have so far been examined mostly as two-way host–microbe interactions. A central question in mutualism biology is how mutualistic interactions shape and are One contribution to a special feature ‘Ant shaped by their surrounding ecological communities [1]. interactions with their biotic environments’. Symbioses can be classified as ‘open’ or ‘closed’, where open symbioses have symbionts that can be gained or lost from the environment and are thus Electronic supplementary material is available typically subject to greater influence from the outside community than are online at https://dx.doi.org/10.6084/m9.fig- symbionts in closed systems [2]. Closed symbioses, by contrast, are usually transmitted vertically from parent to offspring and tend to show higher partner share.c.3677095. fidelity, which may lead more easily to coevolution between partners [2].

& 2017 The Author(s) Published by the Royal Society. All rights reserved. Downloaded from http://rspb.royalsocietypublishing.org/ on March 15, 2017

Although the distinction between open and closed symbioses that forms hollow domatia at stem nodes, where ant symbionts 2 provides a bird’s-eye view of mutualistic symbiosis in its nest and tend several of scale insects. In Costa Rica, rspb.royalsocietypublishing.org ecological context, the complete framework will probably Co. alliodora trees are commonly inhabited by both Azteca require ‘open’ and ‘closed’ at two ends of a continuum of spp. () and Cephalotes setulifer Emery (Myrmici- possible environmental interactions. What constitutes the nae) ants, either in separate trees or in different domatia on the space between requires investigation into how organisms’ same tree [19]. Whereas Azteca ants appear to be omnivorous evolutionary history and present ecology simultaneou- and to host very few specialized gut symbionts, Cephalotes is sly influence the extent of interaction between a given the quintessential cryptic herbivore, and this habit may be mutualistic symbiosis and its environment. facilitated by a core gut bacterial microbiome that is maintained Both coevolution and the key role of the ecological com- throughout the genus [10,12–14]. In Co. alliodora, however, munity in shaping a mutualistic symbiosis were first both Azteca and Cephalotes share a very similar environment demonstrated in a single pioneering experiment: Janzen [3] and diet: individual tree domatia can transition between B Soc. R. Proc. separated tropical acacia plants that provide housing and ant occupants over time, and both ants tend large numbers food for ant symbionts from those ant symbionts and of honeydew-producing scale insects in two subfamilies: showed that herbivore damage to plants increased, strongly Pseudococcidae : Pseudococcinae and Coccidae : Myzolecanii- reducing plant fitness. Ant–plant protective mutualisms, nae (E. G. Pringle 2007, personal observation). The vertically 284 including acacia-like ‘ant-plants’ (myrmecophytes) with transmitted intracellular symbionts of a major clade of Pseudo- hollow cavities (domatia) that host symbiotic ant defenders, coccinae species have been well studied and are unusual in that 20162770 : have been central to developing our understanding of mutu- the metabolic pathways are shared between two nested alism ever since [4]. The diversity of ant–plant interactions, bacteria—a within the cytoplasm of as well as the sheer abundance of ants that is maintained the Tremblaya—and the host insect by them, may have been driven primarily by the evolution genome [20,21]. To our knowledge, the microbial symbionts of protective-nutritional mutualism between ants and honey- of Myzolecanniinae remain virtually unstudied. Relatives in dew-producing hemipteran insects (e.g. scale insects and Coccidae : Coccinae were reported to have abundant fungal aphids) [5,6]. Hemipterans feed on plant sap and metabolize symbionts [9]. it into carbohydrate-rich honeydew, which allows honeydew- Using high-throughput sequencing of 16S rRNA from bac- feeding ants to take on primarily arboreal lifestyles as ‘cryptic teria and internal transcribed spacer (ITS) region rDNA from herbivores’ [6]. Indeed, the vast majority of myrmecophytes fungi, we tested the hypothesis that the persistent physical feed ants in part via the honeydew of hemipteran scale contact created by the symbiosis among the tree, ants and insects [7]. scale insects would influence the composition of the insects’ Although both ants and hemipterans can obtain their microbial associates and symbionts. For bacteria, we predicted required energy from plant phloem sap, several key nutri- that environmental influence would be strongest in Azteca, ents, and essential amino acids in particular, are in much weaker in Cephalotes and weakest for the coccoid scale insects, shorter supply [8]. Hemipterans were early and enduring based on the expected differences in localization, transmission models for studying how can obtain such key nutri- and partner fidelity of the associated bacteria. For fungi, we ents from microbes [9], but the broad relevance of bacteria predicted that Azteca ants would consistently associate with and fungi as potential sources of nutrition for ants, which domatia-derived fungi: these associations have been pre- are typically omnivorous, was recognized only recently viously reported from other Azteca-plant symbioses [22], and [10,11]. Most phloem-feeding hemipterans are associated we have observed black, fungal patches within the ant doma- with just one or a few highly specialized intracellular bac- tia. We had few predictions for the fungal associations of the teria that are vertically transmitted, i.e. ‘closed’ symbiosis, rest of the insect taxa, which have been very little studied. whereas ants are more typically associated with gut bacterial symbionts, i.e. ‘open’ symbiosis. Nevertheless, evidence to date suggests that these associations may provide good examples of the continuum of ecological interaction with 2. Material and methods symbiosis: some ants exhibit core microbiota that, though (a) Sample collection subject to dietary influence, vary little through evolutionary Samples were collected in June 2012 in the Area de Conservacio´n time [12–14], and hemipteran honeydew can contain pro- Guanacaste, Sector Santa Rosa, Costa Rica (108500 N, 858360 W). teins derived from their bacterial symbionts as well as even Two species of Azteca commonly occupy Co. alliodora trees at some of the bacteria themselves [15,16]. Microbial symbionts the site (Azteca pittieri and Azteca beltii; [23]), and they are difficult have in fact recently been suggested to mediate ant–hemi- to distinguish in the field. We therefore chose study trees based pteran mutualisms [17]. Because there is a potentially on which ant genera were present and subsequently identified strong feedback loop among plant chemistry, hemipteran Azteca to species level with molecular barcoding (electronic sup- honeydew, and the quantity and quality of ant defensive be- plementary material, text S1). We collected samples from five trees haviour [18], which may be modified by the insect- (approx. 9 cm average diameter at breast height) (electronic sup- associated microbes, microbial symbionts may also play plementary material, table S1). From each tree, we collected ants (Dolichoderinae: Azteca spp. and/or : Ce. setulifer), key roles in the eco-evolutionary outcomes of ant–plant hereafter Azteca and Ce. setulifer, scale insects (Coccidae: Myzoleca- protective mutualisms. niinae: Cryptostigma spp. and/or Pseudococcidae: Pseudococcinae: To investigate the extent of environmental influence on pat- Paraputo cf. larai), hereafter Cryptostigma and Paraputo, and environ- terns of microbial communities in an ant–plant–hemipteran mental samples from the ant domatia and tree leaves. Domatia symbiosis, we investigated the abundance and composition samples consisted of 2 mm2 scrapings of an interior domatium of bacteria and fungi in the myrmecophytic tree Cordia alliodora wall occupied by each ant species per tree; leaf samples consisted in Costa Rica. Cordia alliodora is a widespread Neotropical tree of approximately 4 cm2 per tree from each of two leaves from Downloaded from http://rspb.royalsocietypublishing.org/ on March 15, 2017 separate whorls. Stable-isotope samples were transferred to a free- (e) Sequencing of bacterial and fungal communities 3 zer within 1 h of collection and frozen for less than 12 h before

We first sequenced both 16S bacterial rRNA and ITS fungal rspb.royalsocietypublishing.org 8 they were dried for 48 h at 60 C. Microbial-survey samples rDNA amplicons using tag-encoded 454 FLX-titanium amplicon were stored in 100% ethanol until processing. pyrosequencing (Research and Testing Laboratory, Lubbock, TX, USA). 16S rRNA from bacteria was amplified in the V1–3 region using primers 28F and 519R [31]. ITS rDNA from fungi was amplified using fungal-specific primers ITS1-F and ITS4 [28]. (b) Stable isotopes Two blank negative controls from the PowerSoil DNA extraction To test whether there was a relationship between the trophic pos- kit produced no sequences. The 454 sequences were extracted ition of the insects and the abundance of their internal microbes, and processed in MOTHUR v. 1.36.0 [32], following the standard @15 we measured N in the ants and scale insects. Sets of approxi- operating procedure (accessed 2 February 2015; [33]) with ¼ mately 20 ant worker bodies (head and thorax) (n 6 Azteca and some modifications.

¼ ¼ B Soc. R. Proc. n 6 Ce. setulifer) and entire scale insects (n 3 Cryptostigma and For ITS sequences, after running sff.multiple with minflows ¼ ¼ n 3 Paraputo) were ground with a mortar and pestle. Four 360, we trimmed all sequences at both ends to 250 total bases, dis- larval leaf-chewing herbivores, three spiders, and nine leaves carding shorter sequences. This trimming scheme produced a good were analysed for comparison. Analyses were performed at the match to a mock community that we sequenced for quality control UC Davis Stable Isotope Facility. We tested for differences in 15 (electronic supplementary material, text S1). We detected chimeras @ N among sample types using a generalized linear model 284 using the uchime algorithm implemented in MOTHUR.Operational because of the unbalanced sampling design and conducted taxonomic units (OTUs) were then determined by calculating pair- 20162770 : Tukey post hoc comparisons in the multcomp package in R wise distances between sequences and clustering using the average v. 3.3.1 [24,25]. neighbour method and a 0.03 distance. Although the 97% OTU cut-off may approximate species imprecisely in some taxa [34], it is currently the most common threshold for community analysis of both bacteria and fungi and allowed us to compare within and (c) DNA extraction between these hyperdiverse kingdoms. Representative OTUs Insects were surface sterilized prior to DNA extraction. Ant and phylotypes were classified using a recent UNITE database gasters and whole scale insects were dipped in 95% ethanol, (UNITEv6_sh_dynamic) [35]. We included singletons in the soaked for 1 min in 5% bleach and rinsed in sterile water. We analyses presented here; analyses excluding singletons were run clipped gasters (i.e. posterior abdomens, which contain the in parallel (see the electronic supplementary material). entire digestive tract apart from the mouth and oesophagus) Because our preliminary analyses of the 454 16S rRNA from ant bodies and pooled 1–3 gasters for each ant sample sequences indicated unusually high diversity among the bacteria ¼ ¼ (n 15 Azteca samples, n 15 Ce. setulifer samples). We dis- associated with our two Cryptostigma (Coccidae: Myzolecaniinae) sected the midgut from one additional Ce. setulifer ant and samples, we resequenced all of our samples for bacterial 16S extracted it separately. Ethanol was evaporated from the domatia rRNA, including five additional Cryptostigma spp. samples (elec- ¼ ¼ (n 10) and leaf (n 6) samples prior to extraction. DNA was tronic supplementary material, table S1) and 13 additional extracted using a modified version of the PowerSoil DNA Iso- Myzolecaniinae (electronic supplementary material, text S4), on lation Kit (MO BIO Laboratories, Carlsbad, CA, USA) (see [26]). an Illumina MiSeq platform (Argonne National Laboratory, Lemont, IL, USA), amplifying the V4 region using primers 515F and 806R [27]. Because these MiSeq 16S rRNA sequences produced very similar results and more total reads after filtering (d) Quantitative PCR to determine microbe copy (241 077 from MiSeq versus 201 246 from 454), here we focus on numbers the MiSeq results for 16S rRNA. MiSeq sequences were processed To estimate the abundance of bacteria and fungi in our samples, in MOTHUR v. 1.37.0 [32], following the standard operating pro- we performed real-time quantitative PCR (qPCR) of the bacterial cedure (accessed 1 April 2016; [36]). We again detected and 16S rRNA gene and fungal ITS rDNA gene (see also the elec- removed chimeras using the uchime algorithm. Sequences were tronic supplementary material, text S1). For 16S rRNA, we classified using the SILVA database (www.arb-silva.de). We used the universal bacterial primers 515F and 806R [27]. For removed sequences classified as Archaea, chloroplasts, or mito- ITS rDNA, we used ITS1-F and 5.8S primers [28,29]. All qPCRs chondria, and clustered sequences into OTUs using the were performed on a CFX Connect Real-Time System (Bio-Rad, dist.seqs and cluster commands. OTUs were subsequently classi- Hercules, CA, USA) using SsoAdvanced 2X SYBR green super- fied at 97% (see above). Sequences from four blank negative mix (Biorad) and 2 ml of DNA extract. For bacterial 16S rRNA, controls from the PowerSoil DNA extraction kit were analysed standard curves were generated from the 16S in parallel to test for possible reagent- or platform-derived rRNA gene. For fungal rDNA, standard curves were generated contamination. from the ITS rDNA gene of Pleurotus sp., obtained from store- bought oyster mushrooms (electronic supplementary material, text S1). To determine the number of gene copies per microgram (f) Analysis of bacterial and fungal communities of DNA, we measured the DNA concentration of each sample on To investigate the alpha diversity of our samples, we compared a Qubit (Life Technologies, Grand Island, NY, USA) (electronic composition based on phylotypes, conducted rarefaction analysis supplementary material, text S1). and calculated diversity. We calculated phylotypes from the We tested for differences among sample types in the abun- SILVA and UNITE database for bacteria and fungi, respectively. dance of 16S rRNA and ITS rDNA separately using the Rarefaction curves were calculated from data binned at the OTU nparcomp package in R v. 3.3.1 [25,30] because the sampling level with the rarefaction.single command in MOTHUR. To com- was unbalanced and the data exhibited a strong right skew. pare diversity, we calculated an inverse Simpson index for all We used two-sided tests on a multivariate t-distribution with replicates subsampled at 1100 sequences for bacteria and 902 a Satterthwaite approximation. We tested for a relationship sequences for fungi (see below) using the summary.single com- between the abundance of bacteria in our ant and scale-insect mand in MOTHUR. Because the distributions of diversity samples and their @15N signatures using a general linear model estimates were right-skewed, we assessed differences in diversity in R. among samples using the nparcomp package in R. Downloaded from http://rspb.royalsocietypublishing.org/ on March 15, 2017

Before beta-diversity analyses, the MOTHUR sub.sample com- 1.40 × 109 4 mand was used to subsample the data. Bacteria were 1.20 × 109 rspb.royalsocietypublishing.org subsampled at 1100 sequences, which excluded nine samples. Cephalotes Fungi were subsampled at 902 sequences, which excluded 1.00 × 109 three samples. Community composition was compared in two ways. First, we used nonmetric multidimensional scaling 8.00 × 108 (NMDS) to visualize differences in community composition 6.00 × 108 among all of our sample types. The vegan package in R v. 3.3.1 was used to calculate Bray–Curtis distances (vegdist function), 4.00 × 108 Coccidae and stable solutions to the first two NMDS axes were calculated Pseudococcidae 2.00 × 108 using the metaMDS function [25,37]. Second, we visualized the Azteca extent to which each ant shared bacterial and fungal OTUs 16S rRNA copies per m g DNA 0 0.5 1.5 2.5 3.5 4.5 5.5 6.5 with their scale insects and domatia environment using B Soc. R. Proc. ∂15 Venn diagrams. We searched for overlap among the Azteca, N Azteca domatia and Azteca-tended scale insects and among the Figure 1. Relationship between the abundance of bacteria in ants (posterior Ce. setulifer, Ce. setulifer domatia and Ce. setulifer-tended scale abdomens; red dots) and whole scale insects (blue dots) and their @15N insects (electronic supplementary material, figure S1). The mer- signatures. Error bars indicate s.e. Sample sizes are as follows: Azteca n ¼ 4; Cepha- ge.groups and venn commands in MOTHUR were used to 284 lotes n ¼ 5; Coccidae n ¼ 3; Pseudococcidae n ¼ 3. (Online version in colour.) visualize OTU overlap. 20162770 : (b) Alpha diversity of communities (g) Functional inference from inferred hemipteran (i) Bacteria There were 20 identifiable bacterial phyla among all of our metagenomes sample types, including ants, scale insects, domatia and To examine whether the two genera of hemipteran scale insects leaves, with , Actinobacteria, (Cryptostigma coccids and Paraputo pseudococcids) harboured and Verrucomicrobia representing nearly 92% of all 241 077 bacterial endosymbionts with similar predicted functional high-quality sequences in the MiSeq dataset. Nearly 7% of activity, we used the online Galaxy version of Phylogenetic sequences were not classified at the phylum level. Classifi- Investigation of Communities by Reconstruction of Unobserved cation by phylotype produced 50 classes, 90 orders and 203 States (PICRUSt, v. 1.0.0) to predict metagenome function from families. A class-level barplot indicated 15 abundant classes 16S rRNA sequences [38] (electronic supplementary material, text S1). To compare the relative abundance of predicted gene and particular abundance of and families from PICRUSt, we calculated the relative abundance of Gammaproteobacteria in all sample types (figure 2a). Surpris- each gene family per sample and tested for differences between ingly, at least 11 classes were present in high abundance in the Cryptostigma and Paraputo using t-tests with unequal variances Cryptostigma coccids, compared to only two classes in the and a Bonferroni correction for multiple comparisons. Paraputo pseudococcids (see §3d, below). The 16S rRNA sequences were binned at 97% similarity into 5069 OTUs, including 1555 non-singletons. 0.6% of these OTUs clustered with sequences from the blank negative controls 3. Results and represented potential reagent- or platform-derived contami- nants (electronic supplementary material, table S2). Rarefaction (a) Trophic position and microbial abundance analysis indicated that our sequence coverage was exhaustive in Among our insects, there was no relationship between the case of non-singletons (electronic supplementary material, bacterial abundance and @15N natural abundance (figure 1). figure S3b)—except in the case of leaves and Cephalotes domatia, Unexpectedly, the Paraputo pseudococcids, which feed on probably owing to low bacterial sequence counts and/or abun- tree phloem, exhibited @15N levels significantly higher than dance, respectively (electronic supplementary material, figure tree leaves and not significantly different than the Azteca S2a)—and the relative richness of the different sample types ants or the spiders (electronic supplementary material, was very similar when singletons were included (electronic figure S2a). By contrast, the Cryptostigma coccids, which supplementary material, figure S3a; see also the electronic sup- also feed on tree phloem, exhibited @15N levels similar to plementary material, figure S4a). Bacteria found in Azteca ants leaf-chewing herbivores and not significantly different than and Cryptostigma coccids exhibited higher OTU richness tree leaves. Cryptostigma coccids and Paraputo pseudococcids that was less stable across individual replicates than the bacteria contained similar abundances of bacteria, and both con- in Ce. setulifer ants or Paraputo pseudococcids (electronic tained low abundances of fungi (electronic supplementary supplementary material, figure S5). Indeed, Paraputo pseudo- material, figure S2b). Cephalotes setulifer ants exhibited much coccids exhibited the lowest bacterial diversity among our lower @15N than Azteca ants (figure 1). The posterior abdo- sample types, whereas Azteca domatia exhibited particularly mens of the Ce. setulifer ants contained the highest bacterial high diversity (electronic supplementary material, figure S6a). abundance of any of our samples (electronic supplementary The core microbiome of Ce. setulifer ants strongly mirrored pre- material, figure S2b), consistent with the indication that these vious reports of core bacterial species common to the genus bacteria play a role in Cephalotes nutrition [10]. By contrast, (electronic supplementary material, table S3; [10,12,13]). the posterior abdomens of Azteca ants contained very few bacteria but a higher abundance of fungi, suggesting that (ii) Fungi perhaps fungi are an important source of nutrition for There were 17 identifiable fungal classes among all of our these ants, as has been shown in other symbiotic ant–plant samples, with , Sordariomycetes, Agarico- mutualisms [11]. mycetes and Eurotiomycetes representing approximately Downloaded from http://rspb.royalsocietypublishing.org/ on March 15, 2017

(a)(bacteria bacterial class b) fungi fungal order 5 1.00 1.00

Verrucomicrobiae Xylariales rspb.royalsocietypublishing.org Verrucomicrobia_unclassified Trichosporonales Sphingobacteria Tremellales Proteobacteria_unclassified Sordariomycetes_unclassified 0.75 0.75 Planctomycetacia Saccharomycetales Opitutae Pleosporales Gammaproteobacteria Microbotryomycetes_unclassified Flavobacteria 0.50 0.50 Malasseziales Betaproteobacteria Hypocreales Bacteroidetes_unclassified Fungi_unclassified Bacteria_unclassified Dothideomycetes_unclassified Bacilli 0.25 0.25 Dothideales proportion of ITS rDNA sequences proportion of 16S rRNA sequences Alphaproteobacteria

Diaporthales B Soc. R. Proc. Actinobacteria Chaetothyriales Acidobacteria_Gp1 0 0 Ascomycota_unclassified Azteca leaf Azteca leaf Agaricomycetes_unclassified CephalotesAzteca dom Coccidae CephalotesAzteca dom Coccidae Cephalotes dom Agaricales Cephalotes dom Pseudococcidae Pseudococcidae 284

Figure 2. Taxonomic composition of (a) bacterial and (b) fungal communities for all sample types. Each bar corresponds to the pooled sequence counts for all 20162770 : replicates within a sample type. Coccidae (Myzolecaniinae: Cryptostigma) and Pseudococcidae (Pseudococcinae: Paraputo) are set apart in (a) for easier comparison of their distinct alpha diversity (see also the electronic supplementary material, figure S5). Note that nearly all of the sequences in the insects and approximately 50% of the sequences in the domatia that are classified by UNITE as ‘Ascomycota_unclassified’ provide good BLASTn hits to Chaetothyriales.

50% of all 139 646 high-quality sequences. Unclassified that exhibited any considerable overlap were the Cryptostigma represented another approximately 28% of coccids and the Azteca domatia. The Cephalotes ants and the sequences, and nearly 18% of sequences were unclassified Paraputo pseudococcids grouped the farthest apart from the at the phylum level. Phylotypes at the order and family rest of the samples, consistent with a role for highly special- level produced 56 and 112 operational units, respectively. ized bacterial endosymbionts in these taxa. By contrast, An order-level barplot indicated 18 abundant orders and there was substantial NMDS overlap among all samples in particularly high abundance of Pleosporales and Chae- their fungal communities, with the sole exception of leaves. tothyriales in all sample types, and in ants and ant The leaves were composed of a very distinct community of domatia especially (figure 2b). endophytes and leaf pathogens (electronic supplementary The fungal ITS rDNA sequences were binned at 97% simi- material, table S4). larity into 1602 OTUs, which included 1398 non-singletons. The Venn diagrams of OTU overlap between a given ant Rarefaction analysis indicated that our sequence coverage taxon and its environment suggested that, despite the overall was exhaustive for all sample types, including for coccids, separation in bacterial communities indicated in the NMDS which had the fewest total sequences (electronic supplemen- analysis, there is leakage of individual bacterial OTUs tary material, figure S3c,d). Fungal OTUs were more variable among ants, their tended scale insects, and their domatia than bacteria among replicates within sample types (elec- (figure 4a; electronic supplementary material, table S5 and tronic supplementary material, figure S7). Leaves exhibited figure S4c,d). The fungal Venn diagrams, by contrast, particularly high fungal diversity compared to the rest of showed that despite extensive leakage between sample the samples (electronic supplementary material, figure S6b). types of the most common fungal OTUs, which was also Chaetothyriales comprised three of the five fungal OTUs suggested by the NMDS, these shared OTUs comprised a with the highest relative abundance. These three OTUs relatively small proportion of the overall OTU richness: produced BLAST hits at approximately 97% sequence iden- only 22% of all fungal OTUs were shared among samples tity to the so-called ‘domatia symbiont clade’ [39] for Azteca and 18% for Ce. setulifer (figure 4b; electronic sup- (E-values: e-97). They were present in the domatia and the plementary material, table S6). Many fungal OTUs were gasters of both Azteca and Ce. setulifer. The ninth most abun- unique to ants or their domatia, as well as, somewhat dant fungal OTU was also Chaetothyriales but produced a surprisingly, to the Paraputo pseudococcids tended by BLAST hit at 100% sequence identity to a previously Ce. setulifer ants. described domatia-carton-associated OTU from an ant- plant in Thailand [39]. This OTU was virtually absent from Azteca and Ce. setulifer gasters. (d) Bacterial diversity in Cryptostigma coccids The bacterial diversity in the Cryptostigma spp. coccids was unusually high for an insect in the Sternorrhyncha suborder (c) Beta diversity of communities of Hemiptera (figure 2a; electronic supplementary material, Beta-diversity analyses were conducted on 2026 bacterial figure S5c). To probe this unexpected result further, we OTUs and 1092 fungal OTUs after subsampling at 1100 and explored the diversity and function of these bacteria. In our 902 sequences per replicate, respectively. The NMDS ordina- dataset, the Paraputo pseudococcids exhibited much more tions revealed that the different sample types grouped much typical diversity for Sternorrhyncha—93.5% of all sequences more tightly by their communities of bacteria (stress ¼ 0.12) came from only two OTUs (figure 2a; electronic supplemen- than by their communities of fungi (stress ¼ 0.19) (figure 3; tary material, figure S5d). A search of the NCBI database electronic supplementary material, figures S4b and S8). In revealed good BLAST hits to known pseudococcid endosym- fact, in the bacterial communities, the only two samples bionts, Tremblaya princeps (E-value e-121; Betaproteobacteria Downloaded from http://rspb.royalsocietypublishing.org/ on March 15, 2017

(a) 6 Cephalotes

domatia rspb.royalsocietypublishing.org 1.0 (a)(b) Coccidae ants 0.5 680 314 domatia bacteria 61 0 64 6 16 17 73 8 57 scales 384 –0.5 325 99 84 NMDS axis 2 domatia –1.0 Azteca

Pseudococcidae (c) (d) B Soc. R. Proc. –1.5 102 161 32 20 6 fungi 12 33 –10 1 250 40 29 12 77 10 92 NMDS axis 1 284

(b) Figure 4. Venn diagrams of OTU overlap calculated separately for (a,c) Azteca 20162770 : and (b,d) Cephalotes microbial communities (see also the electronic sup- 0.6 domatia plementary material, tables S5 and S6). Ants are the top circles (red), 0.4 domatia are the lefthand circles (yellow) and scale insects (Cryptostigma coc- cids and Paraputo pseudococcids) are the righthand circles (blue). For both 0.2 bacteria (a,b) and fungi (c,d), the size of the circle is proportional to the 0 Pseudococcidae number of OTUs present in the sample type. (Online version in colour.)

NMDS axis 2 –0.2 Coccidae leaves and carbohydrate metabolism. Genes involved in the metab- –0.4 olism of secondary plant compounds were significantly more domatia abundant in the Cryptostigma coccids, whereas genes –0.6 involved in cellular and environmental processes were significantly more abundant in the Paraputo pseudococcids –1.0–0.5 0 0.5 (electronic supplementary material, figure S9 and table S8). NMDS axis 1 Figure 3. Nonmetric multidimensional scaling for (a) bacteria (stress ¼ 0.12) and (b) fungi (stress ¼ 0.19). Ellipses indicate 80% confidence intervals (CI). 4. Discussion Leaves are not included in (a) because no leaf samples included 1100 bac- terial sequences after chloroplast sequences were discarded. Note that the only In this study, we examined how the overlap in environment Cephalotes bacterial sample that falls outside of its 80% CI (halfway towards and natural history of the insects in an ant–plant– Azteca) was the sample where DNA was extracted from only the midgut. hemipteran symbiosis affected the community composition of their associated symbiotic microbes. Consistent with our predictions, Ce. setulifer ants and Paraputo pseudococcids in figure 2a) and a Gammaproteobacteria from Dysmicoccus exhibited distinct, apparently specialized bacterial commu- neobrevipes (E-value e-128; Gammaproteobacteria in nities that were comparatively closed to environmental figure 2a) [20,40] that presumably comes from the Sodalis- influence. In addition, based on the qPCR results, Azteca allied bacteria that have repeatedly replaced the symbiont ants consumed (or associated with) significantly more within Tremblaya [21]. A third OTU (OTU00031) that made than Ce. setulifer ants, which may provide the Azteca up 3.2% of sequences from our Paraputo pseudococcids ants with their required nitrogen in the absence of a special- (Proteobacteria_unclassified in figure 2a) did not produce ized bacterial microbiome. Counter to our predictions, the BLAST hits to pseudococcid symbionts but instead to Soda- Cryptostigma coccids exhibited an unexpectedly diverse bac- lis-like uncultured symbionts from stinkbugs and beetles. In terial community, and the Ce. setulifer ants were associated contrast to this low bacterial diversity in the Paraputo pseudo- with many of the same fungi as the Azteca ants. coccids, our six samples of Cryptostigma spp. coccids The bacterial communities of Azteca and Ce. setulifer pos- contained 1175 bacterial OTUs, of which 192 were present terior abdomens were very different despite the ants’ similar in 50% or more of our samples (89% of sequences) and 41 ecology (sharing the same individual trees and subsisting at were present in all six of our samples (31% of sequences) least in part on similar honeydew diets) and convergent (electronic supplementary material, table S7). adaptation to the Co. alliodora host tree (both A. pittieri and Despite the striking differences in the composition of Ce. setulifer are Co. alliodora specialists [41,42]). Substantial the Cryptostigma coccid and Paraputo pseudococcid bacterial evidence now suggests that Cephalotes ants have coevolved communities, the functional characteristics of the bacterial with their gut microbiome [10,12,13,43], and our Ce. setulifer metagenomes predicted to Level 2 KEGG orthologs by ants reflected these previously reported patterns in gut-bac- PICRUSt were remarkably similar (electronic supplementary teria alpha-diversity (electronic supplementary material, material, figure S9). NSTI values ranged from 0.03 to 0.12. table S3). The variance in bacterial diversity among Azteca Well represented gene categories included those of presumed replicates was also higher (electronic supplementary material, symbiotic importance, with prominent roles for amino acid figures S5a,b and S6a) and the overall abundance of bacteria Downloaded from http://rspb.royalsocietypublishing.org/ on March 15, 2017

was lower (electronic supplementary material, figure S2b) lymph-associated and intracellular fungi [9]. Even more sur- 7 than in Ce. setulifer, consistent with the hypothesis that the prisingly, our Cryptostigma insects had very diverse bacterial rspb.royalsocietypublishing.org Azteca gut bacteria represent a more transient and less func- associates, which, if accurate (electronic supplementary tionally important community than in Cephalotes [12]. material, text S4 and figure S10), has not been documented What the Co. alliodora-associated Azteca lack in a stable for any other insect in the Sternorrhyncha, the suborder of bacterial gut community, however, may be compensated for Hemiptera whose members include scale insects, whiteflies, by their consumption of (or association with) fungi, as indi- psyllids and aphids [47]. Two caveats, however, are: (i) that cated by the high abundance of fungal ITS sequences in we used adult female coccids in all cases, and it can be diffi- Azteca posterior abdomens (electronic supplementary cult to know whether these insects are still alive without material, figure S2b). Recent studies have found intimate co- careful dissection (P. J. Gullan 2016, personal communi- feeding relationships between Chaetothyriales fungi and cation); and (ii) we do not know where these symbionts are plant-ants, in which the ants fertilize the fungi and also con- located within the insect except that they were present after B Soc. R. Proc. sume it [11,44]. In other ant–plant symbioses, there has been surface sterilization. some evidence for distinct fungal communities associated Although the metagenome prediction analysis conducted with different ant species (e.g. [39]), although a recent with PiCRUST is a coarse tool that should be interpreted with study found evidence against codiversification between some caution, it too suggested possible symbiotic function of 284 Azteca and their associated Chaetothyriales [22]. Here we the Cryptostigma coccid bacteria. It is tempting to speculate found that Ce. setulifer and both Azteca species were associ- that if the Myzolecaniinae are associated with diverse 20162770 : ated with what appeared to be the same strains of bacterial symbionts, this unusual symbiosis could be related Chaetothyriales at the 97% OTU level, showing a potentially to the insects’ long, immobile period as adults. Immobility strong effect of the ants’ shared environment (host tree) on creates little if any chance to feed on different plant phloem the identity of their fungal associations (electronic sup- sieve-tubes over time, and this stationary lifestyle may require plementary material, text S2). Consistent with the the Myzolecaniinae to process many unusual and possibly suggestion that the thinner cell walls of domatia-associated toxic metabolites from the plant in its lifetime [48]. Indulging Chaetothyriales make them easier to digest than carton- in this speculation, we note that some of the few gene associated taxa [39,45], our ant posterior abdomens contained functions that were more abundant in the Cryptostigma coccids OTUs from the ‘domatia-symbiont clade’ [39] but not from a compared to in the Paraputo pseudococcids were those related carton-associated OTU. In all of our sample types, we also to the metabolism of plant secondary metabolites (electronic found abundant Pleosporales fungi, which, like Chaetothyr- supplementary material, figure S9). iales, is thought to be saprotrophic in other contexts; the This study represents a first step towards a holistic under- potential role of Pleosporales in ant–plant symbioses remains standing of how microbial symbionts are integrated with to be explored. ecological interactions among macroorganisms. The enor- Despite the distinct compositions of the insects’ symbio- mous diversity of microbes in this ant–plant–hemipteran tic bacterial communities, several bacterial OTUs appeared symbiosis (like, presumably, in most interactions among to be shared among the ants, their tended scales and/or macroorganisms) presents a challenge for determining the their domatia (figure 4a,b; electronic supplementary interactions of functional relevance, but such complexity material, table S5). Although the levels of sharing of diverse may be of profound importance to organismal ecology and OTUs supports this result overall (electronic supplementary evolution. Ants are emerging models for microbial study material, tables S5 and S6), individual cases of shared that also have wide-ranging effects on the functions of OTUs need to be verified because of the possibility for entire ecosystems, many of which are threatened by global cross-talk among multiplexed samples [46]. Two potential change. Elucidating how ants interact in a microbial world ecological pathways for microbes to be shared between is thus a necessary challenge. ants and scale insects are: (i) if the ants are ‘farming’ the scale insects for meat, or (ii) if these bacteria are passed Data accessibility. DNA sequences are available in GenBank (accessions: to the ants in low abundance via scale-insect honeydew, SRP095765). Code, OTU tables, and representative as has been shown for some gut-associated bacteria in sequences of 97% OTUs are deposited in Dryad (accession URL: http://dx.doi.org/10.5061/dryad.16830) [49]. aphids [15]. This kind of trophic microbial sharing could Authors’ contributions. E.G.P. and C.S.M. conceived the study; E.G.P. and have nutritional or other effects on the consumers in C.S.M. designed the experiments; E.G.P. performed the experiments ant–plant–hemipteran interactions [17]. and analysed the data. E.G.P. wrote the initial manuscript, and Some surprising results emerged from our investigation C.S.M. contributed to revising the manuscript. of the Co. alliodora scale insects. First, the Paraputo pseudococ- Competing interests. We declare we have no competing interests. cids exhibited unusually high @15N for herbivores (electronic Funding. E.G.P. was supported by the Michigan Society of Fellows and supplementary material, text S3). Another unexpected start-up funds from the University of Nevada, Reno. C.S.M. was result was that our Paraputo pseudococcids contained non- supported by the National Science Foundation (DEB-1442316 and IOS-1354193) and an anonymous donor. negligible quantities of fungi (electronic supplementary Acknowledgements. We thank Brian Wray for his invaluable assistance in material, figure S2b) and a surprisingly high diversity of the laboratory and Alexandra Westrich for the ant drawings. We are fungal OTUs (figure 4d; electronic supplementary material, grateful to Aileen Berastegui, Paul Dunlap, Penny Gullan, Timothy figure S7d). It is not clear how the pseudococcids acquire James, Takumasa Kondo, Manuela Ramalho and Benjamin Rubin these fungi or whether they play a functional role. Unlike for their analytical insights. The participants of both the 2015 the Paraputo pseudococcids, our Cryptostigma coccids con- -Microbe Symbioses GRC and the 2016 Ants Symposium in Munich provided helpful feedback. The molecular portion of this tained few fungi, suggesting that these insects have very research was completed in the Pritzker Laboratory for Molecular different microbial symbionts than their relatives in the Systematics and Evolution at the Field Museum of Natural History, Coccinae subfamily, which contain dense aggregations of Chicago, Illinois. Downloaded from http://rspb.royalsocietypublishing.org/ on March 15, 2017

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SUPPLEMENT

Text S1. Additional Material and Methods

(i) DNA barcoding of Azteca samples

To identify the Azteca samples to species level, we sequenced ~1 kb of Cytochrome Oxidase 1

(CO1) using methods described previously [1] and examined sequence similarity to identified specimens using BLASTn in the NCBI database. All sequences exhibited high similarity (98.9 ±

0.7%) to either A. pittieri or A. beltii (table S1). A subsequent examination of the community composition of the bacterial and fungal associates revealed no significant differences between the two Azteca species (analysis of dissimilarity: bacterial 16S rRNA: F1,11 = 1.00, P = 0.5; fungal ITS rDNA: F1,3 = 1.03, P = 0.4), so for the remainder of the analyses here we pooled these samples and refer to them jointly as Azteca.

(ii) Quantitative PCR to determine microbe copy numbers

The length of the 16S rRNA and ITS rDNA regions amplified via quantitative PCR were ~350 base pairs each. Negative controls were effective, indicating abundances orders of magnitude lower (3.8x102 compared to ~106 and 104 in bacteria and fungi, respectively). Reaction R2 values were 98.6 ± 0.26% for 16S rRNA and 99.0 ± 0.6%, respectively, for ITS rDNA.

To generate standard curves from store-bought oyster mushrooms, the Pleurotus ITS gene was cloned into plasmids using the TOPO® cloning kit (Invitrogen), verified by Sanger sequencing, and linearized with the restriction enzyme NcoI (New England BioLabs). All samples were run in triplicate, eight standards were run in triplicate with each plate, and the calculated starting quantities for each run were averaged prior to calculation of the number of gene copies per microliter of DNA solution. Supplement to Pringle and Moreau

When the DNA concentrations were measured for the 16S rRNA samples in November

2013, the Qubit did not register concentrations lower than 1 ng/µL; low-concentration readings were thus all standardized to 1 ng/µL (19/58 samples). When the DNA concentrations were measured for the ITS samples in October 2015, the Qubit registered concentrations as low as

0.057 ng/µL; four readings were too low to register and were excluded from subsequent analyses. The copy numbers per volume for 16S rRNA and ITS genes were then converted to the correct units and divided by the DNA concentration of the sample.

(iii) Verification of fungal analysis pipeline with fungal mock community

The mock fungal community was composed of six species of fungi using DNA extracted from pure cultures isolated from green coffee beans [see 2 for more details]. The dominant species in the mock community (Aspergillus niveoglaucus BLAST E-value e-124; 87% of the 5523 mock- community sequences) was mostly represented by two unique sequences (93% of 4830 A. cibarius sequences), with a pairwise distance of 0.012. In total, 0.1% of A. cibarius ITS sequences were misclassified as distinct OTUs at the 97% level.

(iv) Functional inference from inferred hemipteran metagenomes

To prepare the data, we reran classify.otu in mothur at 97% using the Greengenes taxonomy [3], rarefied the data to 1100 reads, and ran the make.biom command using closed-reference 97%

OTU picking (which resulted in the loss of only 10 OTUs, from ~600 to 590). Using PICRUSt, we predicted the metagenome of these closed-reference OTUs normalized by 16S rRNA gene copy number. Gene-family abundance was calculated based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) [4]. To assess the accuracy of gene-family predictions, we also calculated Supplement to Pringle and Moreau the Nearest Sequenced Taxon Index (NSTI), which determines the average divergence between the 16S rRNA reads in our dataset and those in the closest reference genomes.

References

1. Pringle E.G., Ramírez S.R., Bonebrake T.C., Gordon D.M., Dirzo R. 2012

Diversification and phylogeographic structure in widespread Azteca plant-ants from the northern

Neotropics. Molecular Ecology 21(14), 3576-3592. (doi:10.1111/j.1365-294X.2012.05618.x).

2. James, T. Y., J. A. Marino, I. Perfecto, and J. Vandermeer. 2016. Identification of putative coffee rust mycoparasites via single-molecule DNA sequencing of infected pustules. Applied and Environmental

Microbiology 82:631-639.

3. DeSantis T.Z., Hugenholtz P., Larsen N., Rojas M., Brodie E.L., Keller K., Huber T.,

Dalevi D., Hu P., Andersen G.L. 2006 Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Applied and Environmental Microbiology 72(7), 5069-

5072. (doi:10.1128/aem.03006-05).

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D114. (doi:10.1093/nar/gkr988).

Supplement to Pringle and Moreau

Text S2. Additional discussion of Chaetothyriales association with the two ant taxa

The presence of the same Chaetothyriales OTUs in both the C. alliodora domatia hosting Azteca, the protective mutualist, and those hosting Cephalotes, which does not protect the host plant

[1], contrasts with a study of the myrmecophyte Leonardoxa africana, where the protective ant species was found associated with Chaetothyriales, but the non-protective species was not [2].

Although the benefits of these ant-fungus symbioses do not seem to include nitrogen transfer to the plant, it remains possible that the fungi could degrade complex molecules beneficial to the plant [3]. If this were the case, the non-protective Cephalotes ants might not be the parasites of the C. alliodora mutualism that they first appear.

References

1. Tillberg C.V. 2004 Friend or foe? A behavioral and stable isotopic investigation of an ant-plant symbiosis. Oecologia 140(3), 506-515. (doi:10.1007/s00442-004-1601-8).

2. Defossez E., Selosse M.A., Dubois M.P., Mondolot L., Faccio A., Djieto-Lordon C.,

McKey D., Blatrix R. 2009 Ant-plants and fungi: a new threeway symbiosis. New Phytologist

182(4), 942-949. (doi:10.1111/j.1469-8137.2009.02793.x).

3. Defossez E., Djieto-Lordon C., McKey D., Selosse M.A., Blatrix R. 2011 Plant-ants feed their host plant, but above all a fungal symbiont to recycle nitrogen. Proceedings of the Royal

Society B-Biological Sciences 278(1710), 1419-1426. (doi:10.1098/rspb.2010.1884).

Supplement to Pringle and Moreau

Text S3. Discussion of unusual ∂15N signature in Paraputo pseudococcids

Sap-feeding herbivores are actually predicted to show even lower ∂15N than typical herbivores because their associated bacterial symbionts may preferentially recycle low ∂15N from plant- derived non-essential amino acids when synthesizing essential amino acids for the animal host

[1, 2]. Interestingly, unlike the bacterial symbionts predicted to perform such N recycling, the

Tremblaya/Gammaproteobacteria nested symbionts in the major clade of Pseudococcinae to which Paraputo belongs are missing the genes for two such recycling aminotransferases [3].

These genes are candidates for compensation from the host genome [3], but whether this would reduce the rate of recycling of lighter N isotopes is not clear.

References

1. Douglas A.E. 2006 Phloem-sap feeding by animals: problems and solutions. Journal of

Experimental Botany 57(4), 747-754. (doi:10.1093/jxb/erj067).

2. Cook S.C., Davidson D.W. 2006 Nutritional and functional biology of exudate-feeding ants. Entomologia Experimentalis Et Applicata 118(1), 1-10. (doi:10.1111/j.1570-

7458.2006.00374.x).

3. Husnik F., Nikoh N., Koga R., Ross L., Duncan R.P., Fujie M., Tanaka M., Satoh N.,

Bachtrog D., Wilson A.C.C., et al. 2013 Horizontal gene transfer from diverse bacteria to an insect genome enables a tripartite nested mealybug symbiosis. Cell 153(7), 1567-1578.

(doi:10.1016/j.cell.2013.05.040).

Supplement to Pringle and Moreau

Text S4. Confirmation of unusual bacterial diversity in Cryptostigma scales

We sought to confirm that our unusual finding of high bacterial diversity in the Cryptostigma scale insects was not a product of contamination in a few ways. First, after observing the unusually high diversity in two Cryptostigma replicates by means of 454 sequencing, we confirmed that these insects have high bacterial abundance by means of 16S rRNA qPCR (ESM, figure S2b). We also then re-sequenced the 16S rRNA of all of our samples on an Illumina

MiSeq platform, adding five additional Cryptostigma insects and 16 additional Myzolecaniinae

(not yet identified to genus level). All of the Myzolecaniinae showed the same pattern of high bacterial alpha diversity (ESM, figure S10). The consistency of these results, including distinct beta diversity between genera (bacterial communities of the Cryptostigma insects compared to the unidentified Myzolecaniinae; not shown) suggested that our result is not in error.

Supplement to Pringle and Moreau

Supplementary Figures and Tables

Figure S1. Ant associations with coccid and pseudococcid scale insects. Composition of (a) coccid soft scale insects and (b) pseudococcid mealybugs tended by Azteca (N = 24) and

Cephalotes (N = 9) ants per domatium. Asterisks (*) indicate significant differences by Wilcoxon signed rank tests (P < 0.002).

(a) (b) 14 * *

12 8

10 6 8

6 4

4 2 2 Adult scale insects (Coccidae)

0 Adult mealybugs (Pseudococcidae) 0

Azteca Cephalotes Azteca Cephalotes

Supplement to Pringle and Moreau

Figure S2. Stable isotope and qPCR results. (a) Trophic position of the insect samples estimated from natural abundance of ∂15N compared to that of leaf-chewing herbivores, spiders

(predators), and plant material (leaf). Different letters indicate significant differences by a generalized linear model and Tukey post-hoc comparisons (P < 0.05). (b) Bacterial and fungal abundance estimated from qPCR of 16S rRNA and ITS rDNA, respectively. Different letters indicate significant differences by nonparametric multiple comparisons (P < 0.05). Multiple comparisons were conducted separately for 16S rRNA (letters a-c) and ITS rDNA (letters d-g).

Note that the high 16S abundance estimate in leaf samples is due to amplification of chloroplast

DNA; these sequences were discarded in the MiSeq dataset before community analyses.

a (a) a 6 a ab ab 4 b

15 ab

2

0

Azteca Leaf Cephalotes Coccidae Predators Pseudococcidae herbivores

b (b) b 1.5+e09 1+e09

d

abc bc 5+e08 c ac a ef e 0

16S ITS 16S ITS 16S ITS 16S ITS 16S ITS 16S ITS 16S ITS

Azteca Cephalotes Coccidae Pseudococcidae Azteca Cephalotes Leaf domatium domatium Supplement to Pringle and Moreau

Figure S3. Alpha diversity rarefaction curves. Rarefaction curves for each sample type from

(a) all bacterial MiSeq sequences including singleton OTUs; (b) bacterial MiSeq sequences without singletons; (c) all fungal 454 sequences including singleton OTUs; (d) fungal 454 sequences without singletons. Curves were generated using the rarefaction.single command in mothur, sampling every 10 sequences from the total binned sequences for all replicates within each sample type.

(a) (b) 800 1500 600 TUs TUs

1000 Azteca

Cephalotes 400 Coccidae

Bacterial O Pseudococcidae Bacterial O 500

Azteca domatium 200 Cephalotes domatium Leaf 0 0

0e+00 2e+04 4e+04 6e+04 8e+04 1e+05 0e+00 2e+04 4e+04 6e+04 8e+04 1e+05 Number of Sequences (Illumina MiSeq) Number of Sequences (Illumina MiSeq)

(c) (d) 1000 800 TUs TUs 600 600 400 Fungal O Fungal O 200 200 0 0

0 5000 10000 15000 20000 25000 30000 0 5000 10000 15000 20000 25000 30000 Number of Sequences (454 pyrosequencing) Number of Sequences (454 pyrosequencing)

Supplement to Pringle and Moreau

Figure S4. Analyses of the 16S rRNA sequences derived from 454 sequencing. (a) Alpha diversity of bacterial OTUs, defined at 97% similarity. Bars represent SE. (b) Non-metric multidimensional scaling plots of bacterial beta diversity based on shared OTUs. Ellipses represent 95% confidence intervals. This plot represents 1136 total OTUs at 97% similarity, including singletons. (c,d) Venn diagrams of OTU overlap for (c) Azteca and (d) Cephalotes bacterial communities. Ants are the top circles (red), domatia are the lefthand circles (yellow), and scale insects (Cryptostigma coccids and Paraputo pseudococcids) are the righthand circles

(blue).

Supplement to Pringle and Moreau

Figure S5. Alpha diversity of bacterial OTUs. Barplots of 97% 16S rRNA bacterial OTUs from the MiSeq platform for all insect replicates. All plots represent core OTUs present in ≥50% of replicates for better visualization.

(a) Cephalotes setulifer

1.00 OTU (97%)

1: Xanthomonadaceae_

14: Rhizobiales_

0.75 12: Alcaligenaceae_

9: Betaproteobacteria_

15: Betaproteobacteria_

3: Opitutus_

0.50 4: Gammaproteobacteria_ 13: Rhizobiales_

16: Alcaligenaceae_

22: Betaproteobacteria_

7: Comamonadaceae 0.25 25:

28: Bacteroidetes_ Proportion of 16S rRNA sequences 46: Flavobacteriaceae_

0.00

Ceph_T165_433_1Ceph_T165_523Ceph_T2_532Ceph_T2_539_1Ceph_T2_539_2Ceph_T4_425_1Ceph_T4_425_2Ceph_T4_497Ceph_T7_429_1Ceph_T7_429_2Ceph_T7_516Ceph_T8_431_1Ceph_T8_431_2Ceph_T8_510

(b) Azteca spp. OTU (97%)

1.00 5: Wolbachia

23: 31: Enterobacteriaceae_ 98: Cloacibacterium_

34: Acinetobacter guillouiae 0.75 68: Comamonadaceae_

113: Acidovorax_ 97: Streptococcus infantis 111: Candidatus_Phlomobacter_ 38: Enterobacteriaceae_ 0.50 118: Lactobacillus 128: Acetobacteraceae_ 121: Actinomycetales_ 129: Actinomycetales_ 0.25 135: Bacillus cereus_ 162: Aquabacterium_

Proportion of 16S rRNA sequences 164: Gammaproteobacteria_ 145: Staphylococcus_ 176: Pseudomonas

0.00 94: Sphingomonadaceae

Az_T103_529Az_T103_538_1Az_T103_538_2Az_T165_526Az_T165_537_1Az_T165_537_2Az_T4_423Az_T4_424Az_T4_494Az_T6_430_1Az_T6_430_2Az_T6_519Az_T7_412Az_T7_432Az_T7_513

Supplement to Pringle and Moreau

(c) Cryptostigma spp.

OTU (97%)

1.00 20: Actinomycetales_ 29: Luteimonas_

40: Betaproteobacteria_ 33: Niabella_

21: Rhizobiales_ 184: Verrucomicrobium spinosum

36: Chitinophagaceae_ 39: Actinomycetales_

0.75 24: Parapedobacter_ 62: Rhizobiales_

79: Nitrosomonadaceae_ 160: Olivibacter_

140: Pseudofulvimonas gallinarii 37: Actinomycetales_

78: Verrucomicrobia_ 100: Actinomycetales_

0.50 89: Betaproteobacteria_ 108: Phyllobacteriaceae_ 65: Dermacoccus_ 70: Xanthomonadaceae_

27: Actinomycetales_ 90: JG30-KF-CM45_

95: Sphingomonadales_ 175: Luteolibacter_

91: Bacteroidetes_ 270: Isophaeraceae_ 0.25 109: Chitinophagaceae_ 60: Actinomycetales_

Proportion of 16S rRNA sequences 181: Bacteroidetes_ 86: Steroidobacter_

211: Ellin6075_ 103: Devosia_

191: Devosia_ 193: Isophaeraceae_ 0.00 38: Enterobacteriaceae_ 32: Sphingobacteriaceae_

Coc_T103_530 Coc_T165_527 Coc_TG16_Z192 Coc_TG28_Z206_1Coc_TG28_Z206_2Coc_TG57_Z241_3

(d) Paraputo sp.

OTU (97%) 1.00 2: Gammaproteobacteria (Sodalis-allied)

6: Tremblaya

31: Uncultured Gammaproteobacteria

0.75

0.50

0.25 Proportion of 16S rRNA sequences

0.00

PsCoc_T165_524 PsCoc_T2_533 PsCoc_T6_521 PsCoc_T7_514 PsCoc_T7_517 PsCoc_T8_511

Supplement to Pringle and Moreau

Figure S6. Simpson diversity indices for bacteria and fungi. Diversity as calculated by the inverse Simpson metric among sample types after subsampling in (a) bacteria and (b) fungi.

Error bars indicate SE. Different letters indicate P < 0.05 by nonparametric multiple comparisons implemented in the nparcomp package in R. In (b), Coccidae lacks an error bar and was not included in statistical analyses because there was only one replicate with ≥ 902 sequences that was retained after subsampling.

(a) c 50 16S rRNA (bacteria) 45 40 35 ac 30 ab 25 20 15 ab 10 a 5 b 0 Diversity (inverse Simpson) Azteca CephalotesCoccidae domatiumdomatium PseudococcidaeAzteca (b) Cephalotes 40 ITS rDNA (fungi) b 35 30 25 20 15

10 ab ab ab 5 a a 0 Diversity (inverse Simpson) Azteca Leaf CephalotesCoccidae domatiumdomatium PseudococcidaeAzteca Cephalotes Supplement to Pringle and Moreau

Figure S7. Alpha diversity of fungal OTUs. Barplots of 97% ITS rDNA fungal OTUs from the 454 platform for all insect replicates. All plots represent core OTUs present in ≥50% of replicates for better visualization.

(a) Cephalotes setulifer

1.00

OTU (97%)

Otu0001: Pleosporales sp. 0.75 Otu0002: Chaetothyriales sp. Otu0003: Chaetothyriales sp. Otu0004: Chaetothyriales sp. Otu0008: Ascomycota sp. 0.50 Otu0018: Chaetothyriales sp. Otu0019: Cladosporiaceae sp. Otu0057: Mycosphaerella yunnanensis Otu0080: Fungi_ 0.25 Otu0107: Leptosphaeria sp. Otu0296: Mycosphaerellaceae_ Proportion of ITS rDNA sequences

0.00

Ceph_T165_433_1Ceph_T2_532Ceph_T2_539Ceph_T4_425Ceph_T4_425Ceph_T4_497Ceph_T8_431_1Ceph_T8_431_2

(b) Azteca spp.

1.00

0.75

OTU (97%)

Otu0003: Chaetothyriales sp.

0.50 Otu0004: Chaetothyriales sp. Otu0019: Cladosporiaceae sp. Otu0285: Fungi_

0.25 Proportion of ITS rDNA sequences

0.00

Az_T103_538_1 Az_T103_538_2 Az_T4_423 Az_T4_424 Az_T4_494

Supplement to Pringle and Moreau

(c) Cryptostigma spp.

1.00

0.75 OTU (97%) Otu0002: Chaetothyriales sp. Otu0032: Ascomycota sp. Otu0037: Ascomycota sp. 0.50 Otu0038: Ascomycota sp. Otu0049: Ascomycota sp. Otu0063: Sordariomycetes sp. Otu0202: Basidiomycota sp. 0.25 Proportion of ITS rDNA sequences

0.00

Coc_T103_530 Coc_T165_527

(d) Paraputo sp.

OTU (97%) 1.00 Otu0001 Otu0002: Chaetothyriales sp. Otu0003: Chaetothyriales sp. Otu0004: Chaetothyriales sp. 0.75 Otu0005: Alternaria sp. Otu0007: Pleosporales sp. Otu0008: Ascomycota sp. Otu0013: Ascomycota sp.

0.50 Otu0014: Agaricomycetes sp. Otu0019: Cladosporiaceae sp. Otu0021: Saccharomycetales sp. Otu0030: Ascomycota sp. Otu0042: Ascomycota sp. 0.25 Otu0049: Ascomycota sp.

Proportion of ITS rDNA sequences Otu0063: Sordariomycetes sp. Otu0108: Ascomycota sp. Otu0152: Ascomycota sp. 0.00 Otu0350: Fungi_

PsCoc_T165_524 PsCoc_T2_533 PsCoc_T4_492 PsCoc_T7_514 PsCoc_T7_517

Supplement to Pringle and Moreau

Figure S8. Beta diversity NMDS plots excluding singletons. Nonmetric multidimensional scaling for (a) bacteria and (b) fungi after exclusion of singleton OTUs. Ellipses indicate 80% confidence intervals (CI). Compare to Figure 3, where singletons are included.

Supplement to Pringle and Moreau

Figure S9. PICRUSt results for scale insects. Predicted relative abundance of KEGG ortholog groups from the PICRUSt analysis for Paraputo spp. (Pseudococcidae:

Pseudococcinae) mealybugs (light blue) and Cryptostigma spp. (Coccidae: Myzolecaniinae) soft scale insects (dark blue). Error bars indicate SD. Asterisks (*) indicate statistically significant differences by t-test after a Bonferroni correction for multiple comparisons (P < 0.0012). See also electronic supplementary material, table S4.

Cell Communication Cellular Processes Cell Growth and Death Cell Motility * Transport and Catabolism Membrane Transport Environmental * Signal Transduction Information Processing * Signaling Molecules and Interaction Folding, Sorting and Degradation Genetic Replication and Repair Information Processing Transcription Translation Cancers Cardiovascular Diseases Human Diseases Immune System Diseases Infectious Diseases Metabolic Diseases Neurodegenerative Diseases Amino Acid Metabolism Biosynthesis of Other Secondary Metabolites Carbohydrate Metabolism Energy Metabolism Enzyme Families Metabolism Glycan Biosynthesis and Metabolism Lipid Metabolism Metabolism of Cofactors and Vitamins Metabolism of Other Amino Acids Metabolism of Terpenoids and Polyketides * Nucleotide Metabolism Xenobiotics Biodegradation and Metabolism Circulatory System Digestive System Endocrine System Pseudococcids Environmental Adaptation Organismal Systems Coccids Excretory System Immune System Nervous System * Sensory System Cellular Processes and Signaling * Genetic Information Processing Unclassified Metabolism Poorly Characterized 0.0 0.05 0.10 0.15 0.20 Relative Abundance of Gene Function Supplement to Pringle and Moreau

Figure S10. Alpha diversity of bacteria in Myzolecaniinae. Taxonomic composition of the bacteria found in 19 surface-sterilized Coccidae:Myzolecaniinae replicates.

1.00 Bacterial Class

Actinobacteria Acidobacteria_Gp3

Gammaproteobacteria Planctomycetacia

Alphaproteobacteria Bacteroidia

0.75 Sphingobacteria Clostridia

Betaproteobacteria Opitutae

Proteobacteria_unclassified Negativicutes

Bacteroidetes_unclassified TM7_class_incertae_sedis

0.50 Verrucomicrobia_unclassified Acidobacteria_Gp1

Verrucomicrobiae

Bacteria_unclassified

Bacilli

0.25 Acidobacteria_Gp4

Proportion of 16S rRNA sequences Proportion of 16S rRNA Chloroflexi_unclassified

Deltaproteobacteria

Flavobacteria

0.00 Acidobacteria_Gp6

Supplement to Pringle and Moreau

Table S1. Trees and insects sampled in this study.

Tree Date Ant species N Scale insect N collected G_SI/AG Tree 4 Jun-12 Azteca pittieri 3 Coccidae--sequencing (16S & ITS) failed 0 G_SI/AG Tree 4 Jun-12 Cephalotes setulifer 3 Paraputo sp. (Pseudococcidae) 1 G_SI/AG Tree 7 Jun-12 Azteca pittieri 3 Paraputo sp. (Pseudococcidae) 1 G_SI/AG Tree 7 Jun-12 Cephalotes setulifer 3 Paraputo sp. (Pseudococcidae) 1 G165.3 Jun-12 Azteca beltii 3 Cryptostigma sp. (Coccidae) 1 G165.3 Jun-12 Cephalotes setulifer 3 Paraputo sp. (Pseudococcidae) 1 G103 Jun-12 Azteca beltii 3 Cryptostigma reticulolaminae (Coccidae) 1 AvC 6 Jun-12 Azteca pittieri 3 Paraputo sp. (Pseudococcidae) 1 AvC 8 Jun-12 Cephalotes setulifer 3 Paraputo sp. (Pseudococcidae) 1 G_CarbTree2 Jun-12 Cephalotes setulifer 3 + midgut Paraputo sp. (Pseudococcidae) 1 Azteca sp.--not G16 Feb-07 included Cryptostigma sp. (Coccidae) 1 Azteca sp.--not G28 Feb-07 included Cryptostigma sp. (Coccidae) 2 Azteca sp.--not G57 Feb-07 included Cryptostigma sp. (Coccidae) 2

Gray rows indicate coccid samples that were added to the study for the MiSeq sequencing of 16S rRNA

after unusual diversity in Coccidae was detected in the 454 sequencing of 16S rRNA.

Supplement to Pringle and Moreau

Table S2. Potential contaminants. Bacterial 16S rRNA OTUs from MiSeq that form 97% clusters with sequences from blank controls and/or samples from another study that were part of the same sequencing run.

No. No. N in data sequences Top BLAST BLAST OTU sequences in set (of BLAST taxonomy in blanks hit score dataset 74) (N = 4) Otu00005 14207 64 1 Wolbachia sp. GQ275145 e-126 Otu00019 2774 40 N/A* Blochmannia GU226303 e-120 Otu00023 2185 32 N/A* Blochmannia CP010049 e-124 Otu00034 1340 39 128 Acinetobacter sp. KY213422 e-128 Otu00052 678 36 140 Mycobacterium sp. KU961789 e-128 Otu00054 656 35 N/A* Blochmannia GU226314 e-128 Otu00087 359 19 48 Stenotrophomonas sp. KY067436 e-128 Otu00097 307 38 82 Streptococcus infantis KY218827 e-127 Otu00145 180 31 254 Staphylococcus aureus KY218901 e-128 Otu00153 165 21 22 Paracoccus sp. KU845477 e-128 Otu00176 134 25 30 Pseudomonas sp. KU986688 e-128 Methylobacterium Otu00207 89 13 218 LT594351 e-128 extorquens Otu00226 76 24 28 Kocuria sp. KY119373 e-128 Otu00264 63 18 N/A* Blochmannia GU226308 e-118 Otu00265 63 18 282 Neisseria sp. KX454108 e-128 Otu00321 42 17 27 Streptococcus vestibularis KX373563 e-127 Otu00341 38 17 68 Uncultured LC198555 e-129 Otu00359 33 15 182 Finegoldia sp. KX367833 e-127 Otu00360 33 15 47 Uncultured KX993583 e-129 Otu00432 20 11 13922 Pseudomonas fluorescens CP017951 e-128 Otu00555 12 7 111 Uncultured KU886661 e-129 Otu00561 11 7 35 Uncultured KX997772 e-128 Otu00618 9 7 25 Uncultured KX437318 e-127 Otu00608 9 8 1 Porphyromonas sp. KU506606 e-128 Otu00641 8 7 346 Sphingobium sp. KU174187 e-128 Otu01127 3 1 2 Lactobacillus gasseri KY123806 e-128 Otu01301 2 1 180 Bacteroides uniformis KT006902 e-126 Otu01465 2 1 65 Methylobacillus sp. KC439358 e-128 Otu04341 1 1 415 Bacteroides vulgatus JN084208 e-128 Otu03040 1 1 380 raoultii KX506724 e-128

*N/A = Sequences found in high abundance in other samples that were part of the same sequencing run.

Supplement to Pringle and Moreau

Table S3. Core bacterial microbiome of Cephalotes setulifer. These 97% OTUs were present in at least 50% of replicates and with at least 100 sequences.

Top BLAST BLAST OTU Taxonomy (SILVA) hit score Host in top BLAST hit Reference Hu et al. 2014 Molec Otu00001 Xanthomonadaceae KF730303 e-128 Cephalotes varians Ecol Otu00003 Opitutus sp. KM066004 e-128 Cephalotes varians Lin et al. 2016 Hu et al. 2014 Molec Otu00004 Gammaproteobacteria KF730304 e-123 Cephalotes varians Ecol Otu00007 KP904433 e-116 N/A Hu et al. 2014 Molec Otu00009 Burkholderiales KF730294 e-125 Cephalotes varians Ecol Otu00012 Alcaligenaceae KT258895 e-128 Cephalotes rohweri Lin et al. 2016 Hu et al. 2014 Molec Otu00013 Rhizobiales KF730299 e-128 Cephalotes varians Ecol Hu et al. 2014 Molec Otu00014 Alphaproteobacteria KF730289 e-121 Cephalotes varians Ecol Otu00015 Burkholderiales KX242540 e-126 Cephalotes varians Lin et al. 2016 Hu et al. 2014 Molec Otu00016 Alcaligenaceae KF730291 e-126 Cephalotes varians Ecol Hu et al. 2014 Molec Otu00022 Betaproteobacteria KF730300 e-120 Cephalotes varians Ecol Cephalotes Russell et al. 2009 Otu00025 Xanthomonadaceae FJ477594 e-123 unimaculatus PNAS Russell et al. 2009 Otu00028 Bacteroidetes FJ477649 e-103 Procryptocerus batesi PNAS Otu00046 Myroides sp. JX990230 e-120 Cephalotes varians Kautz et al. 2013 AEM

Supplement to Pringle and Moreau

Table S4. Leaf fungal alpha diversity. Fungal OTUs represented by at least 50 sequences among all replicates of C. alliodora leaves.

UNITE classification

Gro Top BLAST BLAST Putativ Phylum Class Order Family Genus Species up hit score e role Otu Chaetothyrial e-97 Ascomycota( Ascomycota_unc Ascomycota_uncla Ascomycota_unclassifi Ascomycota_unclassified(92) 0004 es sp. 96) lassified(92) ssified(92) ed(92) Otu Phoma sp. e-125 endop Ascomycota( Dothideomycete Pleosporales(100) Pleosporales_family_In Pleosporales_family_Incertae 0011 hyte 100) s(100) certae_sedis(100) _sedis_unclassified(72) Otu Phaeophleos e-97 Ascomycota( Dothideomycete Capnodiales(100) Mycosphaerellaceae(1 Mycosphaerellaceae_unclassi 0012 pora 100) s(100) 00) fied(100) Otu Septoria e-126 endop Ascomycota( Dothideomycete Capnodiales(100) Mycosphaerellaceae(1 Mycosphaerellaceae_unclassi 0016 escalloniae hyte 100) s(100) 00) fied(100) Otu Passalora e-126 Ascomycota( Dothideomycete Capnodiales(100) Mycosphaerellaceae(1 Mycosphaerellaceae_unclassi 0020 microsora 100) s(100) 00) fied(98) Otu Allophoma e-126 Ascomycota( Dothideomycete Pleosporales(99) Pleosporales_family_In Phoma(92) 0023 tropica 100) s(99) certae_sedis(99) Otu Setophoma e-124 endop Ascomycota( Dothideomycete Dothideomycetes_ Dothideomycetes_uncl Dothideomycetes_unclassifie 0024 chromolaena hyte 100) s(99) unclassified(85) assified(85) d(85) Otu Capnobotryell e-40 Fungi_unclas Fungi_unclassifi Fungi_unclassified( Fungi_unclassified(62) Fungi_unclassified(62) 0025 a sp. sified(62) ed(62) 62) Otu Unidentified e-46 Ascomycota( Ascomycota_unc Ascomycota_uncla Ascomycota_unclassifi Ascomycota_unclassified(95) 0040 leaf- 95) lassified(95) ssified(95) ed(95) associated from Panama Otu Pleosporales e-103 Ascomycota( Sordariomycetes Xylariales(100) Diatrypaceae(100) Libertella(100) Libertella_sp_EX 0044 sp. 100) (100) P0557F(100) Otu Bullera sp. e-126 Basidiomycot Tremellomycetes Tremellales(100) Tremellales_family_Inc unclassified_Tremellales(100) Tremellales_sp( 0045 a(100) (100) ertae_sedis(100) 100) Otu Stagonospora e-125 Ascomycota( Dothideomycete Pleosporales(100) Phaeosphaeriaceae(10 Phaeosphaeriaceae_unclassif 0046 sp. 100) s(100) 0) ied(100) Otu Diatractium e-41 pathog Fungi_unclas Fungi_unclassifi Fungi_unclassified( Fungi_unclassified(100 Fungi_unclassified(100) 0047 sp. en sified(100) ed(100) 100) ) Otu Phaeonectriell e-49 Ascomycota( Ascomycota_unc Ascomycota_uncla Ascomycota_unclassifi Ascomycota_unclassified(94) 0050 a lignicola 94) lassified(94) ssified(94) ed(94) Otu Sporobolomy e-126 endop Ascomycota( Ascomycota_unc Ascomycota_uncla Ascomycota_unclassifi Ascomycota_unclassified(100 0051 ces sp. hyte 100) lassified(100) ssified(100) ed(100) ) Otu Peltaster e-40 Ascomycota( Ascomycota_unc Ascomycota_uncla Ascomycota_unclassifi Ascomycota_unclassified(100 0053 fructicola 100) lassified(100) ssified(100) ed(100) ) Otu Polychaeton e-45 Ascomycota( Ascomycota_unc Ascomycota_uncla Ascomycota_unclassifi Ascomycota_unclassified(62) 0055 citri 62) lassified(62) ssified(62) ed(62) Otu Chaetothyrial e-99 Ascomycota( Eurotiomycetes( Chaetothyriales(91 Chaetothyriales_unclas Chaetothyriales_unclassified( 0056 es sp. 93) 92) ) sified(91) 91) Otu Ascomycota e-125 Ascomycota( Ascomycota_unc Ascomycota_uncla Ascomycota_unclassifi Ascomycota_unclassified(52) 0061 sp. 52) lassified(52) ssified(52) ed(52) Supplement to Pringle and Moreau

UNITE classification Gro Top BLAST BLAST Putativ Phylum Class Order Family Genus Species up hit score e role Otu Chaetothyrial e-99 Ascomycota( Ascomycota_unc Ascomycota_uncla Ascomycota_unclassifi Ascomycota_unclassified(85) 0066 es sp. 86) lassified(85) ssified(85) ed(85) Otu Bullera e-120 Basidiomycot Tremellomycetes Tremellales(99) Tremellales_family_Inc Bullera(99) Bullera_sakaerat 0073 sakaeratica a(99) (99) ertae_sedis(99) ica(99)

We conducted BLASTn using the FASTA sequences selected from the get.oturep command in mothur. Phylum-species classifications are based on classify.otu output in comparison to the UNITE database. Numbers in parentheses indicate Wang bootstrap values.

Supplement to Pringle and Moreau

Supplementary Table S5. Bacterial OTUs at the center of venn diagrams in Figure 4a,b.

Rows highlighted in gray clustered at 97% with sequences derived from blanks and/or samples from another study that were part of the same sequencing run. Supplementary Table S5. Characteristics of 97% bacterial 16S rRNA OTUs shared among ants, their domatia, and their scale insects (see Figure 4). Rows highlighted in gray clustered at 97% with sequences derived from blanks.

Number of sequences non-subsampled data, although the venn diagrams were constructed using subsampled data Cephalotes setulifer Ceph_dom Scale_Ceph N (of 4) - all no.sequences N (of 15) no.sequences N (of 4) no.sequences pseudococcids total.seq BLAST taxonomy Top BLAST hit BLAST score Otu00005 106 11 244 4 50 4 14207 Wolbachia GQ275145 e-126 Otu00006 7 4 2 1 6502 4 13879 Tremblaya princeps AF476083 e-126 Otu00019 167 9 122 3 5 2 2774 Blochmannia GU226303.1 e-120 Uncultured (cf. Otu00031 41 7 34 3 581 4 1583 Sodalis) AB821285.1 e-126 Otu00094 8 4 6 2 7 1 338 Uncultured KY125653.1 e-128 Otu00098 12 5 6 3 3 2 306 Uncultured KY124865.1 e-128 Otu00113 15 2 11 3 6 3 240 Uncultured KY124681 e-128 Otu00118 20 5 27 4 4 3 231 Uncultured KU505387.1 e-126 Uncultured, from Otu00121 22 5 15 3 3 2 228 Polyrachis robsoni KC137133 e-98 Acetobacteraceae, from Camponotus Otu00128 12 4 18 3 4 1 205 chromaiodes KU289115.1 e-127 Otu00129 17 4 18 3 2 2 203 Uncultured KJ424427.1 e-128 Staphylococcus Otu00145 5 4 12 2 2 1 180 aureus KY197819.1 e-128 Otu00163 6 2 5 2 1 1 150 KY206743.1 e-128 Uncultured, from Otu00216 3 1 5 3 1 1 79 Polyrachis robsoni KC137133.1 e-98 Bacteroidetes, from Otu00247 6 2 8 3 1 1 70 Coccoidea DQ133549.1 e-115 Blochmannia from Camponotus Otu00264 13 3 8 3 1 1 63 (Tanaemyrmex) GU226308 e-118 Planococcus Otu00384 1 1 2 2 1 1 28 plakortidis CP016539.2 e-128

Azteca spp. Azteca_dom Scale_Azteca N (of 4) - 2 coccids and 2 no.sequences N (of 15) no.sequences N (of 5) no.sequences pseudococcids total.seq Top BLAST taxonomy Top BLAST hit BLAST score Otu00005 13003 15 56 5 34 3 14207 Wolbachia GQ275145 e-126 Otu00018 15 5 799 5 91 3 2995 Shinella zoogloeoides LC177122.1 e-128 Otu00019 1620 15 557 4 17 3 2774 Blochmannia GU226303.1 e-120 Uncultured, from Otu00020 25 8 41 4 18 3 2593 attine ants LN563671 e-121 Uncultured, from Otu00021 7 3 797 5 205 3 2504 attine ants LN567209.1 e-123 Otu00023 1918 14 14 1 3 2 2185 Blochmannia CP010049.1 e-124 Otu00024 19 3 1005 5 293 2 2118 KX417294.1 e-125 Otu00027 2 2 839 5 9 1 1913 Actinotalea suaedae NR_134690.1 e-120 Otu00029 8 4 936 5 110 3 1756 Uncultured KP808096.1 e-128 Otu00030 14 6 705 5 168 2 1696 Xenophilus KU534350.1 e-128 Uncultured (cf. Otu00031 413 15 19 3 333 2 1583 Sodalis) AB821285.1 e-126 Uncultured, from Otu00033 3 2 553 5 160 2 1357 attine ants LN560252.1 e-120 Otu00035 6 2 518 5 58 3 1312 Uncultured KU125548.1 e-118 Otu00036 22 2 213 5 9 2 1220 Uncultured KY125135.1 e-126 Otu00040 5 2 399 5 41 3 1052 Uncultured KX635859.1 e-126 Amycolatopsis Otu00041 3 2 49 3 4 1 998 keratiniphila LT629789.1 e-128 Uncultured (cf. Otu00044 2 1 44 5 1 1 959 Mesorhizobium) KU505784.1 e-128 Uncultured, from Otu00045 3 1 667 5 124 2 896 attine ants LN565014.1 e-120 Uncultured (cf. Otu00048 8 4 238 5 13 2 721 Bosea) KY012472.1 e-128 Otu00052 33 8 331 5 5 3 678 Mycobacterium sp. KU961789.1 e-128 Blochmannia from Otu00054 450 15 14 5 5 2 656 Camponotus rufipes GU226314.1 e-128 Otu00055 17 7 280 5 75 2 638 Paracoccus sanguinis KT944225.1 e-128 Uncultured, from Otu00057 3 2 439 5 46 2 610 attine ants LN565172.1 e-128 Otu00059 19 7 316 5 25 2 567 Leifsonia sp. KX354861.1 e-128 Otu00061 9 1 367 5 97 2 535 Uncultured DQ116010.1 e-121 Otu00063 1 1 385 5 95 1 527 Uncultured KP801633.1 e-123

Supplement to Pringle and Moreau

Azteca spp. Azteca_dom Scale_Azteca N (of 4) - 2 coccids and 2 no.sequences N (of 15) no.sequences N (of 5) no.sequences pseudococcids total.seq Top BLAST taxonomy Top BLAST hit BLAST score Sphingomonas Otu00064 12 4 16 5 2 2 522 melonis LC191557.1 e-128 Otu00065 5 1 291 4 13 1 518 Uncultured KU571922.1 e-127 Otu00068 201 15 140 5 18 2 492 Ottowia sp. KX832990.1 e-128 Otu00069 4 2 409 5 35 3 488 Uncultured KX993233.1 e-126 Otu00072 2 2 163 5 9 2 447 Nocardioides sp. KP003999.1 e-126 Brachybacterium Otu00073 17 8 345 5 10 1 445 rhamnosum KU147424.1 e-128 Otu00074 16 6 76 4 16 2 444 Gordonia sp. KX459809.1 e-128 Otu00075 3 1 270 5 75 2 437 Uncultured KC439360.1 e-120 Otu00080 2 1 290 4 51 2 394 Uncultured HM276949.1 e-120 Otu00081 1 1 292 4 49 1 372 Uncultured KT360403.1 e-115 Otu00084 5 2 70 5 6 2 363 Uncultured KY012653.1 e-125 Uncultured, from Otu00085 2 1 183 5 23 2 361 attine ants LN563492.1 e-128 Otu00086 3 2 90 5 3 2 360 Steroidobacter LC016942.1 e-128 Stenotrophomonas Otu00087 69 5 111 1 20 1 359 pavanii KY067436.1 e-128 Otu00102 21 6 124 5 8 3 281 Aeromicrobium sp. KX056207.1 e-125 Otu00110 2 2 98 5 100 3 245 Cytophagaceae sp. KC252932 e-120 Otu00113 197 15 4 2 2 1 240 Uncultured KY124681 e-128 actinobacterium, Otu00114 8 4 75 5 26 2 236 from bees HM109806.1 e-121 Actinomyces Otu00120 2 1 32 4 2 1 229 urogenitalis KU726628.1 e-108 Otu00125 4 2 94 5 3 2 217 Uncultured AJ576407.1 e-126 Otu00127 2 2 104 3 4 2 207 Nocardia sp. KP326339.1 e-120 Acetobacteraceae, from Camponotus Otu00128 163 14 1 1 1 1 205 chromaiodes KU289115.1 e-127 Otu00130 2 1 90 5 19 2 202 Alererythrobacter sp. KX585254.1 e-128 Otu00131 3 2 147 5 23 1 202 Uncultured KY012724.1 e-125 Otu00134 3 1 107 4 32 2 194 Uncultured JQ077065.1 e-115 Otu00138 5 3 35 5 33 1 188 Shinella sp. KY054581.1 e-128 Otu00143 1 1 30 3 10 2 181 Flavobacterium sp. GU726988 e-126 Otu00144 2 1 156 3 8 1 181 Brevibacteriaceae GU797885.1 e-123 Otu00146 3 3 94 5 7 2 179 Uncultured JQ337640 e-126

Otu00151 4 2 143 4 8 1 172 Brevibacterium album KX168130.1 e-125 Otu00153 13 8 4 2 1 1 165 Paracoccus sp. KU845477 e-128 Uncultured Otu00187 2 1 29 3 3 2 113 actinobacterium JF981099.1 e-126 Otu00194 10 2 53 3 2 2 102 Acinetobacter sp. JQ316376.1 e-125 Otu00198 1 1 35 1 56 1 98 Uncultured KT910254 e-126 Otu00214 1 1 69 5 10 1 84 Uncultured KT360403.1 e-111 Otu00216 57 12 1 1 1 1 79 Uncultured KC137133.1 e-98 Otu00235 3 1 33 4 1 1 73 Nocardioides sp. KX119426.1 e-128 Acinetobacter sp., Otu00251 37 4 29 3 1 1 67 palm weevil gut KF115254.1 e-108 Otu00252 3 1 19 3 37 1 67 Niabella sp. JX133200 e-121 Otu00272 2 1 45 4 6 2 60 Uncultured KP913731.1 e-115 Otu00273 8 3 33 2 13 1 58 Uncultured KR851176.1 e-121 Otu00296 3 1 37 2 1 1 49 Uncultured KX634250.1 e-121 Otu00298 2 1 10 1 22 1 48 Uncultured KT928555.1 e-128 Otu00299 2 1 34 1 5 1 48 Uncultured KC632842.1 e-128 Uncultured, from Otu00307 1 1 34 3 2 1 47 attine ants LN560175.1 e-128 Otu00330 12 3 15 5 2 1 40 Uncultured KU966499 e-128 Otu00479 2 1 4 4 6 1 16 Uncultured KU104751.1 e-128

Supplement to Pringle and Moreau

Supplementary Table S6. Fungal OTUs at the center of venn diagrams in Figure 4c,d.

Supplementary Table S6. Characteristics of 97% fungal ITS rDNA OTUs shared among ants, their domatia, and their scale insects (see Figure 4).

Cephalotes setulifer Ceph_dom Scale_Ceph BLAST taxonomy Top BLAST hit BLAST score N (of 4) - all no.sequences N (of 8) no.sequences N (of 5) no.sequences pseudococcids total.seq Otu0001 103 4 15310 4 474 2 22348 Pleosporales sp. KP890523.1 e-123 Otu0002 2844 8 1198 3 16286 4 21997 Chaetothyriales sp. HQ634652.1 e-95 Otu0003 4621 8 3031 4 1876 4 13379 Chaetothyriales sp. HQ634653.1 e-97 Otu0004 8153 7 148 3 48 3 10158 Chaetothyriales sp. HQ634653.1 e-97 Otu0008 327 5 3087 2 19 4 4007 Ascomycota sp. KF221635.1 e-77 Otu0013 98 1 73 2 1508 2 2320 Uncultured KF221635.1 e-75 Otu0015 412 3 1437 1 27 2 1901 Fusarium oxysporum KX009445.1 e-126 Otu0018 184 5 280 4 853 1 1487 Chaetothyriales sp. HQ634652.1 e-93 Otu0112 63 2 4 1 8 1 81 Ascomycota sp. KF638538.1 e-123 Otu0129 2 1 4 1 28 1 54 Fungal sp. KX247443.1 e-126 Otu0244 2 1 12 1 3 2 18 Chaetothyriales sp. HQ634652.1 e-93 Otu0488 1 1 2 1 1 1 7 Cyberlindnera jadinii KY103054.1 e-122

Azteca spp. Azteca_dom Scale_Azteca N (of 3) - 2 coccids and 1 no.sequences N (of 5) no.sequences N (of 5) no.sequences pseudococcid total.seq BLAST taxonomy Top BLAST hit BLAST score Otu0002 258 1 188 4 30 3 21997 Chaetothyriales sp. HQ634652.1 e-95 Otu0003 1044 3 41 3 2661 2 13379 Chaetothyriales sp. HQ634653.1 e-97 Otu0004 1208 3 177 3 216 2 10158 Chaetothyriales sp. HQ634653.1 e-97 Otu0005 5062 1 2 1 17 1 5728 Alternaria sp. JQ346907.1 e-123 Otu0014 1928 1 2 2 2 1 2012 Waitea circinata EU693449.1 e-124 Otu0065 4 2 192 3 4 3 240 Uncultured KF221635.1 e-79

Supplement to Pringle and Moreau

Table S7. The Cryptostigma spp. (Coccidae: Myzolecaniinae) core microbiome (41 OTUs

present in 100% of the six replicates). Identification based on comparison to the SILVA database.

OTU Phylum Class Order Family Genus Chitinophagacea Chitinophaga(100 Otu00008 Bacteroidetes(100) Sphingobacteria(100) (100) e(100) ) Rhodocyclaceae( Otu00018 Proteobacteria(100) Betaproteobacteria(56) Rhodocyclales(56) 56) Shinella(56) Tsukamurellacea Otu00020 Actinobacteria(100) Actinobacteria(100) Actinomycetales(100) e(85) Tsukamurella(85) Otu00021 Proteobacteria(100) Alphaproteobacteria(100) Rhizobiales(85) unclassified(52) unclassified(52) Sphingobacteriac Parapedobacter(9 Otu00024 Bacteroidetes(100) Sphingobacteria(100) Sphingobacteriales(100) eae(100) 7) Xanthomonadac Otu00029 Proteobacteria(100) Gammaproteobacteria(100) Xanthomonadales(100) eae(100) Luteimonas(72) Comamonadace Otu00030 Proteobacteria(100) Betaproteobacteria(100) Burkholderiales(100) ae(100) unclassified(93) Chitinophagacea Otu00033 Bacteroidetes(100) Sphingobacteria(100) Sphingobacteriales(100) e(100) unclassified(100) Cytophagaceae( Leadbetterella(10 Otu00035 Bacteroidetes(100) Sphingobacteria(100) Sphingobacteriales(100) 100) 0) Chitinophagacea Otu00036 Bacteroidetes(100) Sphingobacteria(100) Sphingobacteriales(100) e(100) Flavitalea(100) Otu00037 Actinobacteria(100) Actinobacteria(100) Actinomycetales(100) unclassified(100) unclassified(100) Otu00039 Actinobacteria(100) Actinobacteria(100) Actinomycetales(100) unclassified(100) unclassified(100) Otu00040 Proteobacteria(100) Betaproteobacteria(100) unclassified(74) unclassified(74) unclassified(74) Bradyrhizobiacea Otu00048 Proteobacteria(100) Alphaproteobacteria(100) Rhizobiales(100) e(100) Bosea(100) Chitinophagacea Otu00051 Bacteroidetes(100) Sphingobacteria(100) Sphingobacteriales(100) e(100) Niabella(80) Mycobacteriacea Mycobacterium(1 Otu00052 Actinobacteria(100) Actinobacteria(100) Actinomycetales(100) e(100) 00) Rhodobacterace Otu00055 Proteobacteria(100) Alphaproteobacteria(100) Rhodobacterales(100) ae(100) Paracoccus(90) Otu00057 Actinobacteria(100) Actinobacteria(100) Actinomycetales(100) unclassified(84) unclassified(84) Microbacteriacea Otu00059 Actinobacteria(100) Actinobacteria(100) Actinomycetales(100) e(100) Leucobacter(64) Otu00060 Actinobacteria(100) Actinobacteria(100) Actinomycetales(100) unclassified(89) unclassified(89) Rhodobacterace Otu00061 Proteobacteria(100) Alphaproteobacteria(100) Rhodobacterales(99) ae(99) unclassified(99) Xanthomonadac Otu00070 Proteobacteria(100) Gammaproteobacteria(100) Xanthomonadales(100) eae(100) Xanthomonas(52) Nocardioidaceae Otu00072 Actinobacteria(100) Actinobacteria(100) Actinomycetales(100) (100) Nocardioides(81) Phyllobacteriace Aquamicrobium(9 Otu00076 Proteobacteria(100) Alphaproteobacteria(100) Rhizobiales(100) ae(100) 6) Chitinophagacea Otu00085 Bacteroidetes(100) Sphingobacteria(100) Sphingobacteriales(100) e(100) unclassified(100) Sinobacteraceae Steroidobacter(10 Otu00086 Proteobacteria(100) Gammaproteobacteria(100) Xanthomonadales(100) (100) 0) Otu00090 unclassified(100) unclassified(100) unclassified(100) unclassified(100) unclassified(100) Otu00091 Bacteroidetes(100) unclassified(100) unclassified(100) unclassified(100) unclassified(100) Chitinophagacea Otu00096 Bacteroidetes(100) Sphingobacteria(100) Sphingobacteriales(100) e(100) unclassified(51) Nocardioidaceae Aeromicrobium(82 Otu00102 Actinobacteria(100) Actinobacteria(100) Actinomycetales(100) (97) ) Hyphomicrobiace Otu00103 Proteobacteria(100) Alphaproteobacteria(100) Rhizobiales(100) ae(98) Devosia(98) Phyllobacteriace Otu00108 Proteobacteria(100) Alphaproteobacteria(100) Rhizobiales(93) ae(84) unclassified(55) Nocardiaceae(84 Smaragdicoccus( Otu00114 Actinobacteria(100) Actinobacteria(100) Actinomycetales(100) ) 84) Nakamurellaceae Otu00125 Actinobacteria(100) Actinobacteria(100) Actinomycetales(100) (100) Saxeibacter(82) Supplement to Pringle and Moreau

Otu00134 Bacteroidetes(100) Sphingobacteria(85) Sphingobacteriales(85) unclassified(84) unclassified(84) Phyllobacteriace Otu00146 Proteobacteria(100) Alphaproteobacteria(100) Rhizobiales(100) ae(65) unclassified(64) Otu00148 Chloroflexi(93) unclassified(90) unclassified(90) unclassified(90) unclassified(90) Otu00150 Chloroflexi(82) unclassified(82) unclassified(82) unclassified(82) unclassified(82) Sphingobacteriac Otu00157 Bacteroidetes(100) Sphingobacteria(100) Sphingobacteriales(100) eae(99) (57) Phyllobacteriace Otu00180 Proteobacteria(100) Alphaproteobacteria(100) Rhizobiales(91) ae(67) unclassified(67) Otu00208 Actinobacteria(100) Actinobacteria(100) Acidimicrobiales(100) Lamiaceae(99) Lamia(99)

Numbers in parentheses indicate bootstrap values calculated in mothur classify.seqs,

method=wang.

Supplement to Pringle and Moreau

Table S8. Statistics of PICRUSt results for coccid and pseudococcid scale insects. Relative

abundance of gene categories predicted by KEGG-2 orthologs compared between the bacterial

communities of Pseudococcidae and Coccidae (see also figure S9).

KEGG-2 Ortholog Categories t statistic p.value Higher abundance: Cell Communication -0.03 0.9753 Cell Growth and Death 3.92 0.0043 Coccids Cell Motility -6.28 0.0003* Pseudococcids Transport and Catabolism 5.31 0.0014 Membrane Transport -5.36 0.0005* Pseudococcids Signal Transduction -6.14 0.0001* Pseudococcids Signaling Molecules and Interaction 2.35 0.0429 Folding, Sorting, and Degradation 0.85 0.4304 Replication and Repair 1.81 0.1278 Transcription -3.78 0.0037 Pseudococcids Translation -0.52 0.6171 Cancers 2.37 0.0614 Cardiovascular Diseases 3.90 0.0039 Coccids Immune System Diseases -1.54 0.1823 Infectious Diseases -4.16 0.0020 Pseudococcids Metabolic Diseases -1.16 0.2740 Neurodegenerative Diseases 3.36 0.0119 Amino Acid Metabolism 3.65 0.0045 Coccids Biosynthesis of Other Secondary Metabolites 3.41 0.0078 Carbohydrate Metabolism 1.83 0.1144 Energy Metabolism 2.43 0.0541 Enzyme Families -0.77 0.4599 Glycan Biosynthesis and Metabolism -3.30 0.0136 Lipid Metabolism 3.38 0.0070 Metabolism of Cofactors and Vitamins 2.04 0.0959 Metabolism of Other Amino Acids 1.82 0.1061 Metabolism of Terpenoids and Polyketides 5.18 0.0008* Coccids Nucleotide Metabolism 1.52 0.1874 Xenobiotics Biodegradation and Metabolism 2.71 0.0242 Circulatory System 3.08 0.0189 Digestive System 3.86 0.0032 Coccids Endocrine System 1.90 0.0886 Environmental Adaptation 0.29 0.7798 Excretory System 4.03 0.0032 Coccids Immune System -0.27 0.7939 Nervous System 5.76 0.0006* Coccids Sensory System -0.03 0.9753 Cellular Processes and Signaling -5.37 0.0006* Pseudococcids Genetic Information Processing 0.77 0.4594 Metabolism -1.43 0.2121 Poorly Characterized -1.49 0.1915

Bold indicates P < 0.005; asterisks (*) indicates significance by comparison to Bonferroni

correction (0.05/41 = 0.0012).