On the Biological Diversity of Ant Alkaloids.

Ants have outstanding capacity to mediate inter- and intraspecific interactions by producing structurally diverse metabolites from numerous secretory glands. Since Murray Blum's pioneering studies dating from the 1950s, there has been a growing interest in arthropod toxins as natural products. Over a dozen different alkaloid classes have been reported from approximately 40 ant genera in five subfamilies, with peak diversity within the Myrmicinae tribe Solenopsidini. Most ant alkaloids function as venom, but some derive from other glands with alternative functions. They are used in defense (e.g., alarm, repellants) or offense (e.g., toxins) but also serve as antimicrobials and pheromones. We provide an overview of ant alkaloid diversity and function with an evolutionary perspective. We conclude that more directed integrative research is needed. We suggest that comparative phylogenetics will illuminate compound diversification, while molecular approaches will elucidate genetic origins. Biological context, informed by natural history, remains critical not only for research about focal species, but also to guide applied research.

The Evolution of Tyramides in Male Fungus-Growing Ants (Formicidae: Myrmicinae: Attini: Attina).

Ants use a variety of semiochemicals for essential activities and have been a source for many novel natural products. While ant taxa produce a wide variety of chemicals, the chemistry and ecology of male ants have remained understudied. Tyramides are a class of compounds that have been found only in males of the Myrmicinae ant subfamily. Tyramides found in the fire ant Solenopsis invicta are transferred to gynes during mating where they are converted to tyramine, leading to rapid reproductive development. To further understand the evolution of tyramide production in male ants, we determined the tyramide composition in males of 15 fungus-growing ant species (Formicidae: Myrmicinae: Attini: Attina) and a Megalomyrmex species (Formicidae: Myrmicinae: Solenopsidini). Thirteen tyramides were identified, four for the first time in natural sources, and their percent composition was mapped to the fungus-growing ant phylogeny.

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.

Chemically armed mercenary ants protect fungus-farming societies.

The ants are extraordinary in having evolved many lineages that exploit closely related ant societies as social parasites, but social parasitism by distantly related ants is rare. Here we document the interaction dynamics among a Sericomyrmex fungus-growing ant host, a permanently associated parasitic guest ant of the genus Megalomyrmex, and a raiding agro-predator of the genus Gnamptogenys. We show experimentally that the guest ants protect their host colonies against agro-predator raids using alkaloid venom that is much more potent than the biting defenses of the host ants. Relatively few guest ants are sufficient to kill raiders that invariably exterminate host nests without a cohabiting guest ant colony. We also show that the odor of guest ants discourages raider scouts from recruiting nestmates to host colonies. Our results imply that Sericomyrmex fungus-growers obtain a net benefit from their costly guest ants behaving as a functional soldier caste to meet lethal threats from agro-predator raiders. The fundamentally different life histories of the agro-predators and guest ants appear to facilitate their coexistence in a negative frequency-dependent manner. Because a guest ant colony is committed for life to a single host colony, the guests would harm their own interests by not defending the host that they continue to exploit. This conditional mutualism is analogous to chronic sickle cell anemia enhancing the resistance to malaria and to episodes in human history when mercenary city defenders offered either net benefits or imposed net costs, depending on the level of threat from invading armies.

Uncoupling Flight and Reproduction in Ants: Evolution of Ergatoid Queens in Two Lineages of Megalomyrmex (Hymenoptera: Formicidae).

Megalomyrmex Forel (Myrmicinae: Solenopsidini) consists of 44 species with diverse life history strategies. Most species are predatory and may also tend honeydew-producing insects. A morphologically derived group of species are social parasites that consume the brood and fungus garden within fungus-growing ant nests. The reproductive strategies of Megalomyrmex queens are somewhat aligned with these life-style patterns. Predatory species in the leoninus species group are large in body size and have ergatoid (i.e., permanently wingless) queens whereas the social parasitic species are smaller and typically have winged queens. We examined two ergatoid phenotypes of Megalomyrmex foreli Emery and Megalomyrmex wallacei Mann and compared them to winged species, one a social lestobiotic or "thief ant" parasite (Megalomyrmex mondabora Brandão) and the other a predator (Megalomyrmex modestus Emery). Megalomyrmex foreli colonies have a single queen with an enlarged gaster that is morphologically distinct from workers. Megalomyrmex wallacei colonies have several queens that are similar in body size to workers. Queens in both species showed a simplification of the thorax, but there was a dramatic difference in the number of ovarioles. Megalomyrmex foreli had 60-80 ovarioles compared to eight in M. wallacei and M. mondabora and M. modestus had 22-28. Along with flight loss in queens, there is an obligate shift to dependent colony founding (also called budding or fission) consequently influencing dispersal patterns. These constraints in life history traits may help explain the variation in nesting biology among Megalomyrmex species.

Bacterial symbiont sharing in Megalomyrmex social parasites and their fungus-growing ant hosts.

Bacterial symbionts are important fitness determinants of insects. Some hosts have independently acquired taxonomically related microbes to meet similar challenges, but whether distantly related hosts that live in tight symbiosis can maintain similar microbial communities has not been investigated. Varying degrees of nest sharing between Megalomyrmex social parasites (Solenopsidini) and their fungus-growing ant hosts (Attini) from the genera Cyphomyrmex, Trachymyrmex and Sericomyrmex allowed us to address this question, as both ant lineages rely on the same fungal diet, interact in varying intensities and are distantly related. We used tag-encoded FLX 454 pyrosequencing and diagnostic PCR to map bacterial symbiont diversity across the Megalomyrmex phylogenetic tree, which also contains free-living generalist predators. We show that social parasites and hosts share a subset of bacterial symbionts, primarily consisting of Entomoplasmatales, Bartonellaceae, Acinetobacter, Wolbachia and Pseudonocardia and that Entomoplasmatales and Bartonellaceae can co-infect specifically associated combinations of hosts and social parasites with identical 16S rRNA genotypes. We reconstructed in more detail the population-level infection dynamics for Entomoplasmatales and Bartonellaceae in Megalomyrmex symmetochus guest ants and their Sericomyrmex amabilis hosts. We further assessed the stability of the bacterial communities through a diet manipulation experiment and evaluated possible transmission modes in shared nests such as consumption of the same fungus garden food, eating of host brood by social parasites, trophallaxis and grooming interactions between the ants, or parallel acquisition from the same nest environment. Our results imply that cohabiting ant social parasites and hosts may obtain functional benefits from bacterial symbiont transfer even when they are not closely related.

Alkaloid venom weaponry of three Megalomyrmex thief ants and the behavioral response of Cyphomyrmex costatus host ants.

Social parasites exploit other societies by invading and stealing resources. Some enter protected nests using offensive chemical weaponry made from alkaloid-based venom. We characterized the venoms of three Megalomyrmex thief ant species (M. mondabora, M. mondaboroides, and M. silvestrii) that parasitize the fungus-growing ants, and developed an ethogram to describe host ant reactions to raiding M. mondaboroides and M. silvestrii parasites. We compared piperidine, pyrrolidine, and pyrolizidine venom alkaloid structures with synthetic samples from previous studies, and describe the novel stereochemistry of trans 2-hexyl-5-[8-oxononyl]-pyrrolidine (3) from M. mondabora. We showed that workers of Cyphomyrmex costatus, the host of M. mondaboroides and M. silvestrii, react to a sting by Megalomyrmex parasites mainly with submissive behavior, playing dead or retreating. Host submission also followed brief antennal contact. The behavior of C. costatus ants observed in this study was similar to that of Cyphomyrmex cornutus, host of M. mondabora, suggesting that the alkaloidal venoms with pyrrolidines from M. mondabora, piperidines from M. mondaboroides, and pyrolizidines from M. silvestrii may function similarly as appeasement and repellent allomones against host ants, despite their different chemical structure. With the use of these chemical weapons, the Megalomyrmex thief ants are met with little host resistance and easily exploit host colony resources.

Central American ants of the genus Megalomyrmex Forel (Hymenoptera: Formicidae): six new species and keys to workers and males.

Megalomyrmex Forel is a distinctive lineage of Neotropical ants, some of which are specialized parasites or predators of the fungus-growing ants Attini. Here we review and key the Central American fauna. Six new species are described from both female castes: M. brandaoi sp. n., M. fungiraptor sp. n., M. longinoi sp. n., M. milenae sp. n., M. megadrifti sp. n. and M. osadrifti sp. n. A worker-based key to all Central American species is presented, and all species are illustrated. Megalomyrmex drifti Kempf is redescribed and the first descriptions of queens for M. miri Brandão and M. foreli Emery are provided. New biological information, several new geographic records, and a discussion of the species-group schema of Brandão (1990) are presented. The male sex of Megalomyrmex is diagnosed at the genus-level and keyed to species for the Central American fauna, where known. The male of each species treated in the key is diagnosed, described, or redescribed. Males are known for fourteen out of twenty total Central American Megalomyrmex species. A distinct but unassociated male is described and keyed (M. male 01). The males of M. miri Brandão and M. wettereri Brandão are described for the first time, and the distinctness of these two species is confirmed. One potential synapomorphy of Megalomyrmex present in males and workers is the presence of a carina which posteriorly delimits the basalmost region of the petiolar dorsum. 

Histrionicotoxin alkaloids finally detected in an ant.

Workers of the ant Carebarella bicolor collected in Panama were found to have two major poison-frog alkaloids, cis- and trans-fused decahydroquinolines (DHQs) of the 269AB type, four minor 269AB isomers, two minor 269B isomers, and three isomers of DHQ 271D. For the first time in an ant, however, the DHQs were accompanied by six histrionicotoxins (HTXs), viz., 283A, 285A, 285B, 285C, 287A, and 287D. This co-occurrence of the HTX and DHQ alkaloids is the usual pattern seen in dendrobatid frogs. This finding contrasts with our earlier study, where workers of a Brazilian ant, Solenopsis (Diplorhoptrum) sp., were found to have a very similar DHQ complex but failed to show HTXs. Several new DHQ alkaloids of MW 271 (named in the frog as 271G) are reported from the above ants that have both m/z 202 and 204 as major fragment ions, unlike the spectrum seen for the poison-frog alkaloid 271D, which has only an m/z 204 base peak. Found also for the first time in skin extracts from the comparison frog Oophaga granulifera of Costa Rica is a trace DHQ of MW 273. It is coded as 273F in the frog; a different isomer is found in the ant.

Using an integrative taxonomic approach to delimit a sibling species, Mycetomoellerius mikromelanos sp. nov. (Formicidae: Attini: Attina).

The fungus-growing ant Mycetomoellerius (previously Trachymyrmex) zeteki (Weber 1940) has been the focus of a wide range of studies examining symbiotic partners, garden pathogens, mating frequencies, and genomics. This is in part due to the ease of collecting colonies from creek embankments and its high abundance in the Panama Canal region. The original description was based on samples collected on Barro Colorado Island (BCI), Panama. However, most subsequent studies have sampled populations on the mainland 15 km southeast of BCI. Herein we show that two sibling ant species live in sympatry on the mainland: Mycetomoellerius mikromelanos Cardenas, Schultz, & Adams and M. zeteki. This distinction was originally based on behavioral differences of workers in the field and on queen morphology (M. mikromelanos workers and queens are smaller and black while those of M. zeteki are larger and red). Authors frequently refer to either species as "M. cf. zeteki," indicating uncertainty about identity. We used an integrative taxonomic approach to resolve this, examining worker behavior, chemical profiles of worker volatiles, molecular markers, and morphology of all castes. For the latter, we used conventional taxonomic indicators from nine measurements, six extrapolated indices, and morphological characters. We document a new observation of a Diapriinae (Hymenoptera: Diapriidae) parasitoid wasp parasitizing M. zeteki. Finally, we discuss the importance of vouchering in dependable, accessible museum collections and provide a table of previously published papers to clarify the usage of the name T. zeteki. We found that most reports of M. zeteki or M. cf. zeteki-including a genome-actually refer to the new species M. mikromelanos.

Male fire ant neurotransmitter precursors trigger reproductive development in females after mating.

Social insect queens have evolved mechanisms to prevent competition from their sexual daughters. For Solenopsis invicta, the fire ant, queens have evolved a primer pheromone that retards reproductive development in their winged reproductive daughters. If these daughters are removed from the influence of the queen, it takes about a week to start reproductive development; however, it starts almost immediately after mating. This dichotomy has been unsuccessfully investigated for several decades. Here we show that male fire ants produce tyramides, derivatives of the biogenic amine tyramine, in their reproductive system. Males transfer tyramides to winged females during mating, where the now newly mated queens enzymatically convert tyramides to tyramine. Tyramine floods the hemolymph, rapidly activating physiological processes associated with reproductive development. Tyramides have been found only in the large Myrmicinae ant sub-family (6,800 species), We suggest that the complex inhibition/disinhibition of reproductive development described here will be applicable to other members of this ant sub-family.

Venom Function of a New Species of Megalomyrmex Forel, 1885 (Hymenoptera: Formicidae).

Alkaloids are important metabolites found across a variety of organisms with diverse ecological functions. Of particular interest are alkaloids found in ants, organisms well known for dominating the ecosystems they dwell in. Within ants, alkaloids are found in venom and function as potent weapons against heterospecific species. However, research is often limited to pest species or species with parasitic lifestyles and thus fails to address the broader ecological function of ant venom alkaloids. Here we describe a new species of free-living Megalomyrmex ant: Megalomyrmex peetersi sp. n. In addition, we identify its singular venom alkaloid (trans-2-butyl-5-heptylpyrrolidine) and elucidate the antibiotic and insecticidal functions of its venom. Our results show that Megalomyrmex peetersi sp. n. venom is an effective antibiotic and insecticide. These results are comparable to venom alkaloids found in other ant species, such as Solenopsis invicta. This research provides great insight into venom alkaloid function, and it is the first study to explore these ideas in the Megalomyrmex system.

The emerging field of venom-microbiomics for exploring venom as a microenvironment, and the corresponding Initiative for Venom Associated Microbes and Parasites (iVAMP).

Venom is a known source of novel antimicrobial natural products. The substantial, increasing number of these discoveries have unintentionally culminated in the misconception that venom and venom-producing glands are largely sterile environments. Culture-dependent and -independent studies on the microbial communities in venom microenvironments reveal the presence of archaea, algae, bacteria, fungi, protozoa, and viruses. Venom-centric microbiome studies are relatively sparse to date with the adaptive advantages that venom-associated microbes might offer to their hosts, or that hosts might provide to venom-associated microbes, remaining largely unknown. We highlight the potential for the discovery of venom microbiomes within the adaptive landscape of venom systems. The considerable number of convergently evolved venomous animals, juxtaposed with the comparatively few known studies to identify microbial communities in venom, provides new possibilities for both biodiversity and therapeutic discoveries. We present an evidence-based argument for integrating microbiology as part of venomics (i.e., venom-microbiomics) and introduce iVAMP, the Initiative for Venom Associated Microbes and Parasites (https://ivamp-consortium.github.io/), as a growing collaborative consortium. We express commitment to the diversity, inclusion and scientific collaboration among researchers interested in this emerging subdiscipline through expansion of the iVAMP consortium.

Evolution of ant-cultivar specialization and cultivar switching in Apterostigma fungus-growing ants.

Almost all of the more than 200 species of fungus-growing ants (Formicidae: Attini) cultivate litter-decomposing fungi in the family Lepiotaceae (Basidiomycota: Agaricales). The single exception to this rule is a subgroup of ant species within the lower attine genus Apterostigma, which cultivate pterulaceous fungi distantly related to the Lepiotaceae. Comparison of cultivar and ant phylogenies suggests that a switch from lepiotaceous to pterulaceous fungiculture occurred only once in the history of the fungus-growing ants. This unique switch occurred after the origin of the genus Apterostigma, such that the basal Apterostigma lineages retained the ancestral attine condition of lepiotaceous fungiculture, and none of the Apterostigma lineages in the monophyletic group of pterulaceous fungiculturists are known to have reverted back to lepiotaceous fungiculture. The origin of pterulaceous fungiculture in attine ants may have involved a unique transition from the ancestral cultivation of litter-decomposing lepiotaceous fungi to the cultivation of wood-decomposing pterulaceous fungi. Phylogenetic analyses further indicate that distantly related Apterostigma ant species sometimes cultivate the same cultivar lineage, indicating evolutionarily frequent, and possibly ongoing, exchanges of fungal cultivars between Apterostigma ant species. The pterulaceous cultivars form two sister clades, and different Apterostigma ant lineages are invariably associated with, and thus specialized on, only one of the two cultivar clades. However, within clades Apterostigma ant species are able to switch between fungi. This pattern of broad specialization by attine ants on defined cultivar clades, coupled with flexible switching between fungi within cultivar clades, is also found in other attine lineages and appears to be a general phenomenon of fungicultural evolution in all fungus-growing ants.

Extended parental care in communal social groups.

Recent developments in social insect research have challenged the need for close kinship as a prerequisite for the evolution of stable group living. In a model communal bee species, Lasioglossum (Chilalictus) hemichalceum, previous allozyme work indicated that groups of cooperating adult females are not relatives. Yet at any given time, not all group members perform the risky task of foraging. We previously hypothesized that tolerance for non-foragers was a component of extended parental care, previously known only for kin based social systems. DNA microsatellites were used to study colony genetic structure in order to test this hypothesis. Microsatellite polymorphism was substantial (He = 0.775). Overall intracolony relatedness, mainly of immatures, was low but significant in nine, late season nests (r = 0.136 +/- 0.023), indicating that broods contain five to six unrelated sib ships. Detailed analyses of kinship between pairs of individuals revealed that most pairs were unrelated and most related pairs were siblings. Mothers are absent for 89-91% of the developing immature females, and 97% of developing males. Alternatively, 46% of adult females had neither sibs nor offspring in their nests. These findings indicate that the extended parental care model applies broadly to both kin based and nonkin based social systems in the Hymenoptera.

Inter-island movements and population differentiation in a pelagic seabird.

We used mark-resight data and amplified fragment length polymorphism (AFLP) markers to assess movements and gene flow between Central Pacific breeding colonies of the great frigatebird, Fregata minor. Of 715 adult frigatebirds marked on Tern Island and Johnston Atoll, 21.3% were resighted at other frigatebird colonies at least 582 km away. Mark-resight data indicated regular movement of males and females between Tern Island and Johnston Atoll (873 km apart), and less frequent movements to other islands; no birds marked on Tern or Johnston were seen on Christmas Island, but one was seen in the Philippines, 7627 km from where it was marked. Despite the regular occurrence of interisland movements, Bayesian analyses of AFLP data showed significant genetic differentiation between Tern Island and Johnston Atoll, and more pronounced differentiation between these two islands and the more distant Christmas Island. The AFLP profiles of three birds breeding on Tern Island fell within the profile-cluster typical for Christmas Island birds, both in a nonmetric multidimensional scaling analysis and in a population assignment test, suggesting dispersal events from Christmas Island to Tern Island. Several factors could explain the persistence of genetic structure despite frequent movements between colonies: many movements occurred during the nonbreeding season, many breeding-season movements did not involve mate-acquisition behaviours and individuals that do disperse may be selected against, as suggested by morphometric differences between colonies. The persistence of genetic structure among breeding colonies despite significant interisland movements suggests limits to the effectiveness of migration as a homogenizing force in this broadly distributed, extremely mobile species.