Community analysis of microbial sharing and specialization in a Costa Rican ant-plant-hemipteran symbiosis

Abstract

Ants have long been renowned for their intimate mutualisms with trophobionts 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 microbes between interacting macroorganisms, affects the composition and function of symbiotic microbial communities. Here we approached this question 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 quantitative PCR to examine community structure of both bacteria and fungi in a Neotropical ant-plant-scale-insect symbiosis. Both phloem-feeding scale insects and honeydew-feeding ants make use of microbial symbionts to subsist on phloem-derived diets of suboptimal nutritional quality. Among the insects examined here, Cephalotes ants and pseudococcid scale insects had the most specialized bacterial symbionts, whereas Azteca ants appeared to consume or associate with more fungi than bacteria, and coccid scale insects were associated with unusually diverse bacterial communities. Despite these differences, we also identified apparent sharing of microbes 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.

Keywords: Cordia alliodora; bacteria; fungi; metabarcoding; myrmecophyte; scale insects.

Figure 1.

Relationship between the abundance of bacteria in ants (posterior abdomens; red dots) and whole scale insects (blue dots) and their ∂¹⁵N signatures. Error bars indicate s.e. Sample sizes are as follows: Azteca n = 4; Cephalotes n = 5; Coccidae n = 3; Pseudococcidae n = 3. (Online version in colour.)

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 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.

Figure 3.

Nonmetric multidimensional scaling for (a) bacteria (stress = 0.12) and (b) fungi (stress = 0.19). Ellipses indicate 80% confidence intervals (CI). Leaves are not included in (a) because no leaf samples included ≥1100 bacterial sequences after chloroplast sequences were discarded. Note that the only Cephalotes bacterial sample that falls outside of its 80% CI (halfway towards Azteca) was the sample where DNA was extracted from only the midgut.

Figure 4.

Venn diagrams of OTU overlap calculated separately for (a,c) Azteca and (b,d) Cephalotes microbial communities (see also the electronic supplementary material, tables S5 and S6). 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). For both bacteria (a,b) and fungi (c,d), the size of the circle is proportional to the number of OTUs present in the sample type. (Online version in colour.)