Heather D. Ishak

Bacterial Diversity in Solenopsis invicta and Solenopsis geminata Ant Colonies Characterized by 16S amplicon 454 Pyrosequencing

Social insects harbor diverse assemblages of bacterial microbes, which may play a crucial role in the success or failure of biological invasions. The invasive fire ant Solenopsis invicta (Formicidae, Hymenoptera) is a model system for understanding the dynamics of invasive social insects and their biological control. However, little is known about microbes as biotic factors influencing the success or failure of ant invasions. This pilot study is the first attempt to characterize and compare microbial communities associated with the introduced S. invicta and the native Solenopsis geminata in the USA. Using 16S amplicon 454 pyrosequencing, bacterial communities of workers, brood, and soil from nest walls were compared between neighboring S. invicta and S. geminata colonies at Brackenridge Field Laboratory, Austin, Texas, with the aim of identifying potential pathogenic, commensal, or mutualistic microbial associates. Two samples of S. geminata workers showed high counts of Spiroplasma bacteria, a known pathogen or mutualist of other insects. A subsequent analysis using PCR and sequencing confirmed the presence of Spiroplasma in additional colonies of both Solenopsis species. Wolbachia was found in one alate sample of S. geminata, while one brood sample of S. invicta had a high count of Lactococcus. As expected, ant samples from both species showed much lower microbial diversity than the surrounding soil. Both ant species had similar overall bacterial diversities, although little overlap in specific microbes. To properly characterize a single bacterial community associated with a Solenopsis ant sample, rarefaction analyses indicate that it is necessary to obtain 5,000–10,000 sequences. Overall, 16S amplicon 454 pyrosequencing appears to be a cost-effective approach to screen whole microbial diversity associated with invasive ant species.

Environment or kin: whence do bees obtain acidophilic bacteria?

As honey bee populations decline, interest in pathogenic and mutualistic relationships between bees and microorganisms has increased. Honey bees and bumble bees appear to have a simple intestinal bacterial fauna that includes acidophilic bacteria. Here, we explore the hypothesis that sweat bees can acquire acidophilic bacteria from the environment. To quantify bacterial communities associated with two species of North American and one species of Neotropical sweat bees, we conducted 16S rDNA amplicon 454 pyrosequencing of bacteria associated with the bees, their brood cells and their nests. Lactobacillus spp. were the most abundant bacteria in many, but not all, of the samples. To determine whether bee‐associated lactobacilli can also be found in the environment, we reconstructed the phylogenetic relationships of the genus Lactobacillus. Previously described groups that associate with Bombus and Apis appeared relatively specific to these genera. Close relatives of several bacteria that have been isolated from flowers, however, were isolated from bees. Additionally, all three sweat bee species associated with lactobacilli related to flower‐associated lactobacilli. These data suggest that there may be at least two different means by which bees acquire putative probiotics. Some lactobacilli appear specific to corbiculate apids, possibly because they are largely maternally inherited (vertically transmitted). Other lactobacilli, however, may be regularly acquired from environmental sources such as flowers. Sweat bee–associated lactobacilli were found to be abundant in the pollen and frass inside the nests of halictids, suggesting that they could play a role in suppressing the growth of moulds and other spoilage organisms.

Evolution of cold-tolerant fungal symbionts permits winter fungiculture by leafcutter ants at the northern frontier of a tropical ant-fungus symbiosis.

The obligate mutualism between leafcutter ants and their Attamyces fungi originated 8 to 12 million years ago in the tropics, but extends today also into temperate regions in South and North America. The northernmost leafcutter ant Atta texana sustains fungiculture during winter temperatures that would harm the cold-sensitive Attamyces cultivars of tropical leafcutter ants. Cold-tolerance of Attamyces cultivars increases with winter harshness along a south-to-north temperature gradient across the range of A. texana, indicating selection for cold-tolerant Attamyces variants along the temperature cline. Ecological niche modeling corroborates winter temperature as a key range-limiting factor impeding northward expansion of A. texana. The northernmost A. texana populations are able to sustain fungiculture throughout winter because of their cold-adapted fungi and because of seasonal, vertical garden relocation (maintaining gardens deep in the ground in winter to protect them from extreme cold, then moving gardens to warmer, shallow depths in spring). Although the origin of leafcutter fungiculture was an evolutionary breakthrough that revolutionized the food niche of tropical fungus-growing ants, the original adaptations of this host-microbe symbiosis to tropical temperatures and the dependence on cold-sensitive fungal symbionts eventually constrained expansion into temperate habitats. Evolution of cold-tolerant fungi within the symbiosis relaxed constraints on winter fungiculture at the northern frontier of the leafcutter ant distribution, thereby expanding the ecological niche of an obligate host–microbe symbiosis.

Microbiomes of ant castes implicate new microbial roles in the fungus-growing ant Trachymyrmex septentrionalis

Fungus-growing ants employ several defenses against diseases, including disease-suppressing microbial biofilms on their integument and in fungal gardens. Here, we compare the phenology of microbiomes in natural nests of the temperate fungus-growing ant Trachymyrmex septentrionalis using culture-dependent isolations and culture-independent 16S-amplicon 454-sequencing. 454-sequencing revealed diverse actinobacteria associated with ants, including most prominently Solirubrobacter (12.2–30.9% of sequence reads), Pseudonocardia (3.5–42.0%), and Microlunatus (0.4–10.8%). Bacterial abundances remained relatively constant in monthly surveys throughout the annual active period (late winter to late summer), except Pseudonocardia abundance declined in females during the reproductive phase. Pseudonocardia species found on ants are phylogenetically different from those in gardens and soil, indicating ecological separation of these Pseudonocardia types. Because the pathogen Escovopsis is not known to infect gardens of T. septentrionalis, the ant-associated microbes do not seem to function in Escovopsis suppression, but could protect against ant diseases, help in nest sanitation, or serve unknown functions.

Cryptic sexual populations account for genetic diversity and ecological success in a widely distributed, asexual fungus-growing ant

Sex and recombination are central processes in life generating genetic diversity. Organisms that rely on asexual propagation risk extinction due to the loss of genetic diversity and the inability to adapt to changing environmental conditions. The fungus-growing ant species Mycocepurus smithii was thought to be obligately asexual because only parthenogenetic populations have been collected from widely separated geographic localities. Nonetheless, M. smithii is ecologically successful, with the most extensive distribution and the highest population densities of any fungus-growing ant. Here we report that M. smithii actually consists of a mosaic of asexual and sexual populations that are nonrandomly distributed geographically. The sexual populations cluster along the Rio Amazonas and the Rio Negro and appear to be the source of independently evolved and widely distributed asexual lineages, or clones. Either apomixis or automixis with central fusion and low recombination rates is inferred to be the cytogenetic mechanism underlying parthenogenesis in M. smithii. Males appear to be entirely absent from asexual populations, but their existence in sexual populations is indicated by the presence of sperm in the reproductive tracts of queens. A phylogenetic analysis of the genus suggests that M. smithii is monophyletic, rendering a hybrid origin of asexuality unlikely. Instead, a mitochondrial phylogeny of sexual and asexual populations suggests multiple independent origins of asexual reproduction, and a divergence-dating analysis indicates that M. smithii evolved 0.5–1.65 million years ago. Understanding the evolutionary origin and maintenance of asexual reproduction in this species contributes to a general understanding of the adaptive significance of sex.

Placement of attine ant-associated Pseudonocardia in a global Pseudonocardia phylogeny (Pseudonocardiaceae, Actinomycetales): a test of two symbiont-association models

We reconstruct the phylogenetic relationships within the bacterial genus Pseudonocardia to evaluate two models explaining how and why Pseudonocardia bacteria colonize the microbial communities on the integument of fungus-gardening ant species (Attini, Formicidae). The traditional Coevolution-Codivergence model views the integument-colonizing Pseudonocardia as mutualistic microbes that are largely vertically transmitted between ant generations and that supply antibiotics that specifically suppress the garden pathogen Escovopsis. The more recent Acquisition model views Pseudonocardia as part of a larger integumental microbe community that frequently colonizes the ant integument from environmental sources (e.g., soil, plant material). Under this latter model, ant-associated Pseudonocardia may have diverse ecological roles on the ant integument (possibly ranging from pathogenic, to commensal, to mutualistic) and are not necessarily related to Escovopsis suppression. We test distinct predictions of these two models regarding the phylogenetic proximity of ant-associated and environmental Pseudonocardia. We amassed 16S-rRNA gene sequence information for 87 attine-associated and 238 environmental Pseudonocardia, aligned the sequences with the help of RNA secondary structure modeling, and reconstructed phylogenetic relationships using a maximum-likelihood approach. We present 16S-rRNA secondary structure models of representative Pseudonocardia species to improve sequence alignments and identify sequencing errors. Our phylogenetic analyses reveal close affinities and even identical sequence matches between environmental Pseudonocardia and ant-associated Pseudonocardia, as well as nesting of environmental Pseudonocardia in subgroups that were previously thought to be specialized to associate only with attine ants. The great majority of ant-associated Pseudonocardia are closely related to autotrophic Pseudonocardia and are placed in a large subgroup of Pseudonocardia that is known essentially only from cultured isolates (rather than cloned 16S sequences). The preponderance of the known ant-associated Pseudonocardia in this latter clade of culturable lineages may not necessarily reflect abundance of these Pseudonocardia types on the ants, but isolation biases when screening for Pseudonocardia (e.g., preferential isolation of autotrophic Pseudonocardia with minimum-nutrient media). The accumulated phylogenetic patterns and the possibility of isolation biases in previous work further erode support for the traditional Coevolution-Codivergence model and calls for continued revision of our understanding how and why Pseudonocardia colonize the microbial communities on the integument of fungus-gardening ant species.

Co-evolutionary patterns and diversification of ant-fungus associations in the asexual fungus-farming ant Mycocepurus smithii in Panama.

Partner fidelity through vertical symbiont transmission is thought to be the primary mechanism stabilizing cooperation in the mutualism between fungus‐farming (attine) ants and their cultivated fungal symbionts. An alternate or additional mechanism could be adaptive partner or symbiont choice mediating horizontal cultivar transmission or de novo domestication of free‐living fungi. Using microsatellite genotyping for the attine ant Mycocepurus smithii and ITS rDNA sequencing for fungal cultivars, we provide the first detailed population genetic analysis of local ant–fungus associations to test for the relative importance of vertical vs. horizontal transmission in a single attine species. M. smithii is the only known asexual attine ant, and it is furthermore exceptional because it cultivates a far greater cultivar diversity than any other attine ant. Cultivar switching could permit the ants to re‐acquire cultivars after garden loss, to purge inferior cultivars that are locally mal‐adapted or that accumulated deleterious mutations under long‐term asexuality. Compared to other attine ants, symbiont choice and local adaptation of ant–fungus combinations may play a more important role than partner‐fidelity feedback in the co‐evolutionary process of M. smithii and its fungal symbionts.

Biogeography of Mutualistic Fungi Cultivated by Leafcutter Ants

Leafcutter ants propagate co‐evolving fungi for food. The nearly 50 species of leafcutter ants (Atta, Acromyrmex) range from Argentina to the United States, with the greatest species diversity in southern South America. We elucidate the biogeography of fungi cultivated by leafcutter ants using DNA sequence and microsatellite‐marker analyses of 474 cultivars collected across the leafcutter range. Fungal cultivars belong to two clades (Clade‐A and Clade‐B). The dominant and widespread Clade‐A cultivars form three genotype clusters, with their relative prevalence corresponding to southern South America, northern South America, Central and North America. Admixture between Clade‐A populations supports genetic exchange within a single species, Leucocoprinus gongylophorus. Some leafcutter species that cut grass as fungicultural substrate are specialized to cultivate Clade‐B fungi, whereas leafcutters preferring dicot plants appear specialized on Clade‐A fungi. Cultivar sharing between sympatric leafcutter species occurs frequently such that cultivars of Atta are not distinct from those of Acromyrmex. Leafcutters specialized on Clade‐B fungi occur only in South America. Diversity of Clade‐A fungi is greatest in South America, but minimal in Central and North America. Maximum cultivar diversity in South America is predicted by the Kusnezov–Fowler hypothesis that leafcutter ants originated in subtropical South America and only dicot‐specialized leafcutter ants migrated out of South America, but the cultivar diversity becomes also compatible with a recently proposed hypothesis of a Central American origin by postulating that leafcutter ants acquired novel cultivars many times from other nonleafcutter fungus‐growing ants during their migrations from Central America across South America. We evaluate these biogeographic hypotheses in the light of estimated dates for the origins of leafcutter ants and their cultivars.

Characterization of 14 microsatellite loci in a tropical palm, Attalea phalerata (Arecaceae)

UNLABELLED PREMISE OF THE STUDY We developed microsatellite primers for the widely distributed tropical palm Attalea phalerata for studies on the dispersal and spatial genetic structure of palm populations. • METHODS AND RESULTS Fourteen di-, tri-, and tetra-nucleotide microsatellite primer pairs were identified. The number of alleles in the population tested ranged between 3 and 25, with a mean of 12.1. Ten microsatellite loci exhibited no significant deviations from Hardy-Weinberg Equilibrium or presence of null alleles, and their combined probability of exclusion was 0.998. • CONCLUSIONS These microsatellite loci will be useful in parentage analysis and population genetics studies of Attalea phalerata.

Nuclear populations of the multinucleate fungus of leafcutter ants can be dekaryotized and recombined to manipulate growth of nutritive hyphal nodules harvested by the ants

ABSTRACT We dekaryotized the multinucleate fungus Leucocoprinus gongylophorus, a symbiotic fungus cultivated vegetatively by leafcutter ants as their food. To track genetic changes resulting from dekaryotization (elimination of some nuclei from the multinuclear population), we developed two multiplex microsatellite fingerprinting panels (15 loci total), then characterized the allele profiles of 129 accessions generated by dekaryotization treatment. Genotype profiles of the 129 accessions confirmed allele loss expected by dekaryotization of the multinucleate fungus. We found no evidence for haploid and single-nucleus strains among the 129 accessions. Microscopy of fluorescently stained dekaryotized accessions revealed great variation in nuclei number between cells of the same vegetative mycelium, with cells containing typically between 3 and 15 nuclei/cell (average = 9.4 nuclei/cell; mode = 8). We distinguish four mycelial morphotypes among the dekaryotized accessions; some of these morphotypes had lost the full competence to produce gongylidia (nutritive hyphal-tip swellings consumed by leafcutter ants as food). In mycelial growth confrontations between different gongylidia-incompetent accessions, allele profiles suggest exchange of nuclei between dekaryotized accessions, restoring full gongylidia competence in some of these strains. The restoration of gongylidia competence after genetic exchange between dekaryotized strains suggests the hypothesis that complementary nuclei interact, or nuclear and cytoplasmic factors interact, to promote or enable gongylidia competence.