Publications

Mutualistic interactions typically involve the exchange of different commodities between species [1]. Nutritious secretions are produced by a number of insects and plants in exchange for services such as defense [2, 3]. These rewards are valuable metabolically and can be used to reinforce the behavior of symbiotic partners that can learn and remember them effectively [4, 5]. We show here novel effects of insect exocrine secretions produced by caterpillars in modulating the behavior of attendant ants in the food-for-defense interaction between lycaenid butterflies and ants [6]. Reward secretions from the dorsal nectary organ (DNO) of Narathura japonica caterpillars function to reduce the locomotory activities of their attendant ants, Pristomyrmex punctatus workers. Moreover, workers that feed from caterpillar secretions are significantly more likely to show aggressive responses to eversion of the tentacle organs of the caterpillars. Analysis of the neurogenic amines in the brains of workers that consumed caterpillar secretions showed a significant decrease in levels of dopamine compared with controls. Experimental treatments in which reserpine, a known inhibitor of dopamine in Drosophila, was fed to workers similarly reduced their locomotory activity. We conclude that DNO secretions of lycaenid caterpillars can manipulate attendant ant behavior by altering dopaminergic regulation and increasing partner fidelity. Unless manipulated ants also receive a net nutritional benefit from DNO secretions, this suggests that similar reward-for-defense interactions that have been traditionally considered to be mutualisms may in fact be parasitic in nature.

The biodiversity of tropical forests consists primarily of small organisms that are difficult to detect and characterize. Next-generation sequencing (NGS) methods can facilitate analyses of these arthropod and microbial communities, leading to a better understanding of existing diversity and factors influencing community assembly. The pitchers of carnivorous pitcher plants often house surprisingly discrete communities and provide ideal systems for analysis using an NGS approach. The plants digest insects in order to access essential nutrients while growing in poor soils; however, the pitchers are also home to communities of living organisms, called inquilines. Certain arthropods appear to have coevolved with their pitcher plant hosts and are not found in other environments. We used Illumina amplicon sequencing of 18S rDNA to characterize the eukaryotes in three species of Nepenthes (Nepenthaceae) pitcher plants – N. gracilis, N. rafflesiana and N. ampullaria – in each of three different parks in Singapore. The data reveal an unexpected diversity of eukaryotes, significant differences in community diversity among host species, variation in host specificity of inquilines and the presence of gregarine parasites. Counts of whole inquiline arthropods from the first collection year were roughly correlated with scaled 18S sequence abundances, indicating that amplicon sequencing is an effective means of gauging community structure. We barcoded a subset of the dipteran larvae using COI primers, and the resulting phylogenetic tree is mostly congruent with that found using the 18S locus, with the exception of one of five morphospecies. For many 18S and COI sequences, the best BLASTn matches showed low sequence identity, illustrating the need for better databases of Southeast Asian dipterans. Finally, networks of core arthropods and their host species were used to investigate degree of host specificity across multiple hosts, and this revealed significant specialization of certain arthropod fauna.

The Aphnaeinae (Lepidoptera: Lycaenidae) are a largely African subfamily of 278 described species that exhibit extraordinary life-history variation. The larvae of these butterflies typically form mutualistic associations with ants, and feed on a wide variety of plants, including 23 families in 19 orders. However, at least one species in each of 9 of the 17 genera is aphytophagous, parasitically feeding on the eggs, brood or regurgitations of ants. This diversity in diet and type of symbiotic association makes the phylogenetic relations of the Aphnaeinae of particular interest. A phylogenetic hypothesis for the Aphnaeinae was inferred from 4.4kb covering the mitochondrial marker COI and five nuclear markers (wg, H3, CAD, GAPDH and EF1) for each of 79 ingroup taxa representing 15 of the 17 currently recognized genera, as well as three outgroup taxa. Maximum Parsimony, Maximum Likelihood and Bayesian Inference analyses all support Heath's systematic revision of the clade based on morphological characters. Ancestral range inference suggests an African origin for the subfamily with a single dispersal into Asia. The common ancestor of the aphnaeines likely associated with myrmicine ants in the genus Crematogaster and plants of the order Fabales.

Of the four most diverse insect orders, Lepidoptera contains remarkably few predatory and parasitic species. Although species with these habits have evolved multiple times in moths and butterflies, they have rarely been associated with diversification. The wholly aphytophagous subfamily Miletinae (Lycaenidae) is an exception, consisting of nearly 190 species distributed primarily throughout the Old World tropics and subtropics. Most miletines eat Hemiptera, although some consume ant brood or are fed by ant trophallaxis. A well-resolved phylogeny inferred using 4915 bp from seven markers sampled from representatives of all genera and nearly one-third the described species was used to examine the biogeography and evolution of biotic associations in this group. Biogeographic analyses indicate that Miletinae likely diverged from an African ancestor near the start of the Eocene, and four lineages dispersed between Africa and Asia. Phylogenetic constraint in prey selection is apparent at two levels: related miletine species are more likely to feed on related Hemiptera, and related miletines are more likely to associate with related ants, either directly by eating the ants, or indirectly by eating hemipteran prey that are attended by those ants. These results suggest that adaptations for host ant location by ovipositing female miletines may have been retained from phytophagous ancestors that associated with ants mutualistically.

We isolated and characterized a total of 22 microsatellite loci for the leafcutter ant, Acromyrmex lundii. The loci were screened for 24 individuals from southern Brazil and Uruguay. The number of alleles per locus ranged from 5 to 20, the observed heterozygosity ranged from 0.417 to 0.917, and the probability of identity values ranged from 0.011 to 0.38. These genetic markers will be useful for understanding the population and conservation biology of the leafcutter ant A. lundii and closely related species, and will provide novel insights into the evolutionary biology of social parasitism and leafcutter ant mating systems.

Obligate social parasites, or inquilines, exploit the colonies of free-living social species and evolved at least 80 times in ants alone. Most species of the highly specialized inquiline social parasites are rare, only known from one or very few, geographically isolated populations, and the sexual offspring of most inquiline species mates inside the maternal colony. Therefore, inquiline populations are believed to be small and genetically homogeneous due to inbreeding. To comparatively study the genetic diversity of the socially parasitic fungus-growing ant, Mycocepurus castrator, and its only known host species, Mycocepurus goeldii, and to infer the parasite's conservation status, we developed 21 microsatellite markers for the host species, M. goeldii, and evaluated whether these markers cross-amplify in the social parasite, M. castrator. We isolated and characterized a total of 21 microsatellite loci for M. goeldii. The loci were screened for 24 individuals from geographically distant and genetically divergent populations in Brazil. The number of alleles per locus ranged from 18 to 4, the observed heterozygosity ranged from 0.25 to 0.636, and the probability of identity values ranged from 0.011 to 0.146. Preliminary analyses show that these markers cross amplify in the closely related social parasite species M. castrator. These newly developed loci provide tools for studying the genetic diversity and the evolution of social parasitism in the Mycocepurus host-parasite system.

The butterfly family Pieridae comprises approximately 1000 described species placed in 85 genera, but the higher classification has not yet been settled. We used molecular data from eight gene regions (one mitochondrial and seven nuclear protein-coding genes) comprising a total of similar to 6700bp from 96 taxa to infer a well-supported phylogenetic hypothesis for the family. Based on this hypothesis, we revise the higher classification for all pierid genera. We resurrect the tribe Teracolini stat. rev. in the subfamily Pierinae to include the genera Teracolus, Pinacopteryx, Gideona, Ixias, Eronia, Colotis and most likely Calopieris. We transfer Hebomoia to the tribe Anthocharidini and assign the previously unplaced genera Belenois and Dixeia to the subtribe Aporiina. Three lineages near the base of Pierinae (Leptosia, Elodina and Nepheronia + Pareronia) remain unplaced. For each of these, we describe and delineate new tribes: Elodinini Braby tribus nova, Leptosiaini Braby tribus nova and Nepheroniini Braby tribus nova. The proposed higher classification is based on well-supported monophyletic groups and is likely to remain stable even with the addition of more data.

Inquiline social parasitic ant species exploit colonies of other ant species mainly by producing sexual offspring that are raised by the host. Ant social parasites and their hosts are often close relatives (Emery's rule), and two main hypotheses compete to explain the parasites' evolutionary origins: (1) the interspecific hypothesis proposes an allopatric speciation scenario for the parasite, whereas (2) the intraspecific hypothesis postulates that the parasite evolves directly from its host in sympatry [1-10]. Evidence in support of the intraspecific hypothesis has been accumulating for ants [3, 5, 7, 9-12], but sympatric speciation remains controversial as a general speciation mechanism for inquiline parasites. Here we use molecular phylogenetics to assess whether the socially parasitic fungus-growing ant Mycocepurus castrator speciated from its host Mycocepurus goeldii in sympatry. Based on differing patterns of relationship in mitochondrial and individual nuclear genes, we conclude that host and parasite occupy a temporal window in which lineage sorting has taken place in the mitochondrial genes but not yet in the nuclear alleles. We infer that the host originated first and that the parasite originated subsequently from a subset of the host species' populations, providing empirical support for the hypothesis that inquiline parasites can evolve reproductive isolation while living sympatrically with their hosts.

Correlation between gut microbiota and host phylogeny could reflect codiversification over shared evolutionary history or a selective environment that is more similar in related hosts. These alternatives imply substantial differences in the relationship between host and symbiont, but can they be distinguished based on patterns in the community data themselves? We explored patterns of phylogenetic correlation in the distribution of gut bacteria among species of turtle ants (genus Cephalotes), which host a dense gut microbial community. We used 16S rRNA pyrosequencing from 25 Cephalotes species to show that their gut community is remarkably stable, from the colony to the genus level. Despite this overall similarity, the existing differences among species' microbiota significantly correlated with host phylogeny. We introduced a novel analytical technique to test whether these phylogenetic correlations are derived from recent bacterial evolution, as would be expected in the case of codiversification, or from broader shifts more likely to reflect environmental filters imposed by factors such as diet or habitat. We also tested this technique on a published data set of ape microbiota, confirming earlier results while revealing previously undescribed patterns of phylogenetic correlation. Our results indicated a high degree of partner fidelity in the Cephalotes microbiota, suggesting that vertical transmission of the entire community could play an important role in the evolution and maintenance of the association. As additional comparative microbiota data become available, the techniques presented here can be used to explore trends in the evolution of host-associated microbial communities.

Eusociality is taxonomically rare, yet associated with great ecological success. Surprisingly, studies of environmental conditions favouring eusociality are often contradictory. Harsh conditions associated with increasing altitude and latitude seem to favour increased sociality in bumblebees and ants, but the reverse pattern is found in halictid bees and polistine wasps. Here, we compare the life histories and distributions of populations of 176 species of Hymenoptera from the Swiss Alps. We show that differences in altitudinal distributions and development times among social forms can explain these contrasting patterns: highly social taxa develop more quickly than intermediate social taxa, and are thus able to complete the reproductive cycle in shorter seasons at higher elevations. This dual impact of altitude and development time on sociality illustrates that ecological constraints can elicit dynamic shifts in behaviour, and helps explain the complex distribution of sociality across ecological gradients.

We isolated and characterized a total of 22 microsatellite loci for the leafcutter ant, Acromyrmex lundii. The loci were screened for 24 individuals from southern Brazil and Uruguay. The number of alleles per locus ranged from 5 to 20, the observed heterozygosity ranged from 0.417 to 0.917, and the probability of identity values ranged from 0.011 to 0.38. These genetic markers will be useful for understanding the population and conservation biology of the leafcutter ant A. lundii and closely related species, and will provide novel insights into the evolutionary biology of social parasitism and leafcutter ant mating systems.

Obligate social parasites, or inquilines, exploit the colonies of free-living social species and evolved at least 80 times in ants alone. Most species of the highly specialized inquiline social parasites are rare, only known from one or very few, geographically isolated populations, and the sexual offspring of most inquiline species mates inside the maternal colony. Therefore, inquiline populations are believed to be small and genetically homogeneous due to inbreeding. To comparatively study the genetic diversity of the socially parasitic fungus-growing ant, Mycocepurus castrator, and its only known host species, Mycocepurus goeldii, and to infer the parasite's conservation status, we developed 21 microsatellite markers for the host species, M. goeldii, and evaluated whether these markers cross-amplify in the social parasite, M. castrator. We isolated and characterized a total of 21 microsatellite loci for M. goeldii. The loci were screened for 24 individuals from geographically distant and genetically divergent populations in Brazil. The number of alleles per locus ranged from 18 to 4, the observed heterozygosity ranged from 0.25 to 0.636, and the probability of identity values ranged from 0.011 to 0.146. Preliminary analyses show that these markers cross amplify in the closely related social parasite species M. castrator. These newly developed loci provide tools for studying the genetic diversity and the evolution of social parasitism in the Mycocepurus host-parasite system.

Ecological opportunity, defined as access to new resources free from competitors, is thought to be a catalyst for the process of adaptive radiation. Much of what we know about ecological opportunity, and the larger process of adaptive radiation, is derived from vertebrate diversification on islands. Here, we examine lineage diversification in the turtle ants (Cephalotes), a species-rich group of ants that has diversified throughout the Neotropics. We show that crown group turtle ants originated during the Eocene (around 46 mya), coincident with global warming and the origin of many other clades. We also show a marked lineage-wide slowdown in diversification rates in the Miocene. Contrasting this overall pattern, a species group associated with the young and seasonally harsh Chacoan biogeographic region underwent a recent burst of diversification. Subsequent analyses also indicated that there is significant phylogenetic clustering within the Chacoan region and that speciation rates are highest there. Together, these findings suggest that recent ecological opportunity, from successful colonization of novel habitat, may have facilitated renewed turtle ant diversification. Our findings highlight a central role of ecological opportunity within a successful continental radiation.

The butterfly family Pieridae comprises approximately 1000 described species placed in 85 genera, but the higher classification has not yet been settled. We used molecular data from eight gene regions (one mitochondrial and seven nuclear protein-coding genes) comprising a total of similar to 6700bp from 96 taxa to infer a well-supported phylogenetic hypothesis for the family. Based on this hypothesis, we revise the higher classification for all pierid genera. We resurrect the tribe Teracolini stat. rev. in the subfamily Pierinae to include the genera Teracolus, Pinacopteryx, Gideona, Ixias, Eronia, Colotis and most likely Calopieris. We transfer Hebomoia to the tribe Anthocharidini and assign the previously unplaced genera Belenois and Dixeia to the subtribe Aporiina. Three lineages near the base of Pierinae (Leptosia, Elodina and Nepheronia + Pareronia) remain unplaced. For each of these, we describe and delineate new tribes: Elodinini Braby tribus nova, Leptosiaini Braby tribus nova and Nepheroniini Braby tribus nova. The proposed higher classification is based on well-supported monophyletic groups and is likely to remain stable even with the addition of more data.

Inquiline social parasitic ant species exploit colonies of other ant species mainly by producing sexual offspring that are raised by the host. Ant social parasites and their hosts are often close relatives (Emery's rule), and two main hypotheses compete to explain the parasites' evolutionary origins: (1) the interspecific hypothesis proposes an allopatric speciation scenario for the parasite, whereas (2) the intraspecific hypothesis postulates that the parasite evolves directly from its host in sympatry [1-10]. Evidence in support of the intraspecific hypothesis has been accumulating for ants [3, 5, 7, 9-12], but sympatric speciation remains controversial as a general speciation mechanism for inquiline parasites. Here we use molecular phylogenetics to assess whether the socially parasitic fungus-growing ant Mycocepurus castrator speciated from its host Mycocepurus goeldii in sympatry. Based on differing patterns of relationship in mitochondrial and individual nuclear genes, we conclude that host and parasite occupy a temporal window in which lineage sorting has taken place in the mitochondrial genes but not yet in the nuclear alleles. We infer that the host originated first and that the parasite originated subsequently from a subset of the host species' populations, providing empirical support for the hypothesis that inquiline parasites can evolve reproductive isolation while living sympatrically with their hosts.

Correlation between gut microbiota and host phylogeny could reflect codiversification over shared evolutionary history or a selective environment that is more similar in related hosts. These alternatives imply substantial differences in the relationship between host and symbiont, but can they be distinguished based on patterns in the community data themselves? We explored patterns of phylogenetic correlation in the distribution of gut bacteria among species of turtle ants (genus Cephalotes), which host a dense gut microbial community. We used 16S rRNA pyrosequencing from 25 Cephalotes species to show that their gut community is remarkably stable, from the colony to the genus level. Despite this overall similarity, the existing differences among species' microbiota significantly correlated with host phylogeny. We introduced a novel analytical technique to test whether these phylogenetic correlations are derived from recent bacterial evolution, as would be expected in the case of codiversification, or from broader shifts more likely to reflect environmental filters imposed by factors such as diet or habitat. We also tested this technique on a published data set of ape microbiota, confirming earlier results while revealing previously undescribed patterns of phylogenetic correlation. Our results indicated a high degree of partner fidelity in the Cephalotes microbiota, suggesting that vertical transmission of the entire community could play an important role in the evolution and maintenance of the association. As additional comparative microbiota data become available, the techniques presented here can be used to explore trends in the evolution of host-associated microbial communities.

Eusociality is taxonomically rare, yet associated with great ecological success. Surprisingly, studies of environmental conditions favouring eusociality are often contradictory. Harsh conditions associated with increasing altitude and latitude seem to favour increased sociality in bumblebees and ants, but the reverse pattern is found in halictid bees and polistine wasps. Here, we compare the life histories and distributions of populations of 176 species of Hymenoptera from the Swiss Alps. We show that differences in altitudinal distributions and development times among social forms can explain these contrasting patterns: highly social taxa develop more quickly than intermediate social taxa, and are thus able to complete the reproductive cycle in shorter seasons at higher elevations. This dual impact of altitude and development time on sociality illustrates that ecological constraints can elicit dynamic shifts in behaviour, and helps explain the complex distribution of sociality across ecological gradients.

In the last two decades, the widespread application of genetic and genomic approaches has revealed a bacterial world astonishing in its ubiquity and diversity. This review examines how a growing knowledge of the vast range of animal-bacterial interactions, whether in shared ecosystems or intimate symbioses, is fundamentally altering our understanding of animal biology. Specifically, we highlight recent technological and intellectual advances that have changed our thinking about five questions: how have bacteria facilitated the origin and evolution of animals; how do animals and bacteria affect each other's genomes; how does normal animal development depend on bacterial partners; how is homeostasis maintained between animals and their symbionts; and how can ecological approaches deepen our understanding of the multiple levels of animal-bacterial interaction. As answers to these fundamental questions emerge, all biologists will be challenged to broaden their appreciation of these interactions and to include investigations of the relationships between and among bacteria and their animal partners as we seek a better understanding of the natural world.

The African lycaenid butterfly, Anthene usamba, is an obligate myrmecophile of the acacia ant, Crematogaster mimosae. Female butterflies use the presence of C.mimosae as an oviposition cue. The eggs are laid on the foliage and young branches of the host plant, Acacia drepanolobium. Larvae shelter in the swollen thorns (domatia) of the host tree, where they live in close association with the acacia ants, and each larva occupies a domatium singly. Anthene usamba are tended by ants that feed from the dorsal nectary organ at regular intervals. Larvae also possess tentacle organs flanking the dorsal nectary organ and appear to signal to ants by everting these structures. Larvae were observed to spend most of their time within the domatia. Stable isotope analysis of matched host plantantbutterfly samples revealed that Anthene usamba are 15N enriched relative to the ants with which they associate. These data, based on the increase in 15N through trophic levels, indicate that the caterpillars of these butterflies are aphytophagous and either exploit the ant brood of C.mimosae within the domatia, or are fed mouth to mouth by adult workers via trophallaxis. This is the first documented case of aphytophagy in African Anthene. Pupation occurs inside the domatium and the imago emerges and departs via the hole chewed by the larva. The adult females remain closely associated with their natal patch of trees, whereas males disperse more widely across the acacia savannah. Females prefer to oviposit on trees with the specific host ant, C.mimosae, an aggressive obligate mutualist, and avoid neighbouring trees with other ant species. Adult butterflies are active during most months of the year, and there are at least two to three generations each year. Observations made over a 5-year period indicate that a number of different lycaenid species utilize ant-acacias in East Africa, and these observations are summarized, together with comparisons from the literature.(c) 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109, 302312.

. 1.Standardised transect counts of butterflies in old-growth rainforests in different biogeographical regions are lacking. Such data are needed to mitigate the influence of methodological and environmental factors within and between sites and, ultimately, to discriminate between long-term trends and short-term stochastic changes in abundance and community composition. 2.We compared butterfly assemblages using standardised Pollard Walks in the understory of closed-canopy lowland tropical rainforests across three biogeographical regions: Barro Colorado Island (BCI), Panama; Khao Chong (KHC), Thailand; and Wanang (WAN), Papua New Guinea. 3.The length and duration of transects, their spatial autocorrelation, and number of surveys per year represented important methodological factors that strongly influenced estimates of butterfly abundance. Of these, the effect of spatial autocorrelation was most difficult to mitigate across study sites. 4.Butterfly abundance and faunal composition were best explained by air temperature, elevation, rainfall, wind velocity, and human disturbance at BCI and KHC. In the absence of weather data at WAN, duration of transects and number of forest gaps accounted for most of the explained variance, which was rather low in all cases (<33%). 5.Adequate monitoring of the abundance of common butterflies was achieved at the 50ha BCI plot, with three observers walking each of 10 transects of 500m for 30min each, during each of four surveys per year. These data may be standardised further after removing outliers of temperature and rainfall. Practical procedures are suggested to implement global monitoring of rainforest butterflies with Pollard Walks.