Habitat and Host Species Drive the Structure of Bacterial Communities of Two Neotropical Trap-Jaw Odontomachus Ants : Habitat and Host Species Drive the Structure of Bacterial Communities of Two Neotropical Trap-Jaw Odontomachus Ants.

Ants have long been known for their associations with other taxa, including macroscopic fungi and symbiotic bacteria. Recently, many ant species have had the composition and function of their bacterial communities investigated. Due to its behavioral and ecological diversity, the subfamily Ponerinae deserves more attention regarding its associated microbiota. Here, we used the V4 region of the 16S rRNA gene to characterize the bacterial communities of Odontomachus chelifer (ground-nesting) and Odontomachus hastatus (arboreal), two ponerine trap-jaw species commonly found in the Brazilian savanna ("Cerrado") and Atlantic rainforest. We investigated habitat effects (O. chelifer in the Cerrado and the Atlantic rainforest) and species-specific effects (both species in the Atlantic rainforest) on the bacterial communities' structure (composition and abundance) in two different body parts: cuticle and gaster. Bacterial communities differed in all populations studied. Cuticular communities were more diverse, while gaster communities presented variants common to other ants, including Wolbachia and Candidatus Tokpelaia hoelldoblerii. Odontomachus chelifer populations presented different communities in both body parts, highlighting the influence of habitat type. In the Atlantic rainforest, the outcome depended on the body part targeted. Cuticular communities were similar between species, reinforcing the habitat effect on bacterial communities, which are mainly composed of environmentally acquired taxa. Gaster communities, however, differed between the two Odontomachus species, suggesting species-specific effects and selective filters. Unclassified Firmicutes and uncultured Rhizobiales variants are the main components accounting for the observed differences. Our study indicates that both host species and habitat act synergistically, but to different degrees, to shape the bacterial communities in these Odontomachus species.

Transcriptomic analysis of Malpighian tubules from the stingless bee Melipona scutellaris reveals thiamethoxam-induced damages.

The concern about pesticide exposure to neotropical bees has been increasing in the last few years, and knowledge gaps have been identified. Although stingless bees, (e.g.: Melipona scutellaris), are more diverse than honeybees and they stand out in the pollination of several valuable economical crops, toxicity assessments with stingless bees are still scarce. Nowadays new approaches in ecotoxicological studies, such as omic analysis, were pointed out as a strategy to reveal mechanisms of how bees deal with these stressors. To date, no molecular techniques have been applied for the evaluation of target and/or non-target organs in stingless bees, such as the Malpighian tubules (Mt). Therefore, in the present study, we evaluated the differentially expressed genes (DEGs) in the Mt of M. scutellaris after one and eight days of exposure to LC50/100 (0.000543 ng a.i./µL) of thiamethoxam (TMX). Through functional annotation analysis of four transcriptome libraries, the time course line approach revealed 237 DEGs (nine clusters) associated with carbon/energy metabolism and cellular processes (lysosomes, autophagy, and glycan degradation). The expression profiles of Mt were altered by TMX in processes, such as detoxification, excretion, tissue regeneration, oxidative stress, apoptosis, and DNA repair. Transcriptome analysis showed that cell metabolism in Mt was mainly affected after 8 days of exposure. Nine genes were selected from different clusters and validated by RT-qPCR. According to our findings, TMX promotes several types of damage in Mt cells at the molecular level. Therefore, interference of different cellular processes directly affects the health of M. scutellaris by compromising the function of Mt.

Evaluation of calcium hydroxide, calcium hypochlorite, peracetic acid, and potassium bicarbonate as citrus fruit sanitizers.

Xanthomonas citri (X. citri) is a quarentenary plant pathogen and the causal agent of the citrus canker. X. citri forms biofilms and remains fixed on the surface of plant tissues, especially on leaves and fruits. Considering this, all the citrus fruits have to be sanitized before they can be commercialized. NaOCl is the main sanitizer used to decontaminate fruits in the world. Due to its toxicity, treatment with NaOCl is no longer accepted by some Europe Union countries. Therefore, the aim of this work was to evaluate potassium bicarbonate (KHCO3), calcium hydroxide (Ca(OH)2), calcium hypochlorite (Ca(OCl)2) and peracetic acid (CH3CO3H) as alternatives to NaOCl for the sanitization of citrus fruit. By monitoring cell respiration and bacterial growth, we determined that peracetic acid and calcium hypochlorite exhibit bactericidal action against X. citri. Time-response growth curves and membrane integrity analyses showed that peracetic acid and calcium hypochlorite target the bacterial cytoplasmatic membrane, which is probably responsible for cell death in the first minutes of contact. The simulation of the sanitization process of citrus fruit in packinghouses showed that only peracetic acid exhibited a performance comparable to NaOCl. Among the tested compounds, peracetic acid constitutes an efficient and safer alternative to NaOCl.

Three phylogenetically distinct and culturable diazotrophs are perennial symbionts of leaf-cutting ants.

The obligate mutualistic basidiomycete fungus, Leucocoprinus gongylophorus, mediates nutrition of leaf-cutting ants with carbons from vegetal matter. In addition, diazotrophic Enterobacteriales in the fungus garden and intestinal Rhizobiales supposedly mediate assimilation of atmospheric nitrogen, and Entomoplasmatales in the genus Mesoplasma, as well as other yet unidentified strains, supposedly mediate ant assimilation of other compounds from vegetal matter, such as citrate, fructose, and amino acids. Together, these nutritional partners would support the production of high yields of leafcutter biomass. In the present investigation, we propose that three phylogenetically distinct and culturable diazotrophs in the genera Ralstonia, Methylobacterium, and Pseudomonas integrate this symbiotic nutrition network, facilitating ant nutrition on nitrogen. Strains in these genera were often isolated and directly sequenced in 16S rRNA libraries from the ant abdomen, together with the nondiazotrophs Acinetobacter and Brachybacterium. These five isolates were underrepresented in libraries, suggesting that none of them is dominant in vivo. Libraries have been dominated by four uncultured Rhizobiales strains in the genera Liberibacter, Terasakiella, and Bartonella and, only in Acromyrmex ants, by the Entomoplasmatales in the genus Mesoplasma. Acromyrmex also presented small amounts of two other uncultured Entomoplasmatales strains, Entomoplasma and Spiroplasma. The absence of Entomoplasmatales in Atta workers implicates that the association with these bacteria is not mandatory for ant biomass production. Most of the strains that we detected in South American ants were genetically similar with strains previously described in association with leafcutters from Central and North America, indicating wide geographic dispersion, and suggesting fixed ecological services.

Marine associated microbial consortium applied to RBBR textile dye detoxification and decolorization: Combined approach and metatranscriptomic analysis.

The combination of different microorganisms and their metabolisms makes the use of microbial consortia in bioremediation processes a useful approach. In this sense, this study aimed at structuring and selecting a marine microbial consortium for Remazol Brilliant Blue R (RBBR) detoxification and decolorization. Experimental design was applied to improve the culture conditions, and metatranscriptomic analysis to understand the enzymatic pathways. A promising consortium composed of Mucor racemosus CBMAI 847, Marasmiellus sp. CBMAI 1062, Bacillus subtilis CBMAI 707, and Dietzia maris CBMAI 705 was selected. This consortium showed 52% of detoxification and 86% of decolorization in the validation assays after seven days of incubation in the presence of 500 ppm of RBBR. Reduction in RBBR color and toxicity were achieved by biosorption and microbial metabolisms. Metatranscriptomic data indicate that the consortium was able to decolorize and breakdown the RBBR molecule using a coordinated action of oxidases, oxygenases, and hydrolases. Epoxide hydrolases and glyoxalases expression could be associated with the decrease in toxicity. The efficiency of this marine microbial consortium suggests their use in bioremediation processes of textile effluents.

Fungus-growing insects host a distinctive microbiota apparently adapted to the fungiculture environment.

Some lineages of ants, termites, and beetles independently evolved a symbiotic association with lignocellulolytic fungi cultivated for food, in a lifestyle known as fungiculture. Fungus-growing insects' symbiosis also hosts a bacterial community thought to integrate their physiology. Similarities in taxonomic composition support the microbiota of fungus-growing insects as convergent, despite differences in fungus-rearing by these insects. Here, by comparing fungus-growing insects to several hosts ranging diverse dietary patterns, we investigate whether the microbiota taxonomic and functional profiles are characteristic of the fungiculture environment. Compared to other hosts, the microbiota associated with fungus-growing insects presents a distinctive taxonomic profile, dominated by Gammaproteobacteria at class level and by Pseudomonas at genera level. Even with a functional profile presenting similarities with the gut microbiota of herbivorous and omnivorous hosts, some differentially abundant features codified by the microbiota of fungus-growing insects suggest these communities occupying microhabitats that are characteristic of fungiculture. These features include metabolic pathways involved in lignocellulose breakdown, detoxification of plant secondary metabolites, metabolism of simple sugars, fungal cell wall deconstruction, biofilm formation, antimicrobials biosynthesis, and metabolism of diverse nutrients. Our results suggest that the microbiota could be functionally adapted to the fungiculture environment, codifying metabolic pathways potentially relevant to the fungus-growing insects' ecosystems functioning.

Symbiotic bacterial communities in rainforest fungus-farming ants: evidence for species and colony specificity.

Animals may host diverse bacterial communities that can markedly affect their behavioral physiology, ecology, and vulnerability to disease. Fungus-farming ants represent a classical example of mutualism that depends on symbiotic microorganisms. Unraveling the bacterial communities associated with fungus-farming ants is essential to understand the role of these microorganisms in the ant-fungus symbiosis. The bacterial community structure of five species of fungus-farmers (non-leaf-cutters; genera Mycocepurus, Mycetarotes, Mycetophylax, and Sericomyrmex) from three different environments in the Brazilian Atlantic rainforest (lowland forest, restinga forest, and sand dunes) was characterized with amplicon-based Illumina sequencing of 16 S ribosomal RNA gene. Possible differences in bacterial communities between ants internal to the nest (on the fungus garden) and external foragers were also investigated. Our results on the richness and diversity of associated bacteria provide novel evidence that these communities are host- and colony-specific in fungus-farming ants. Indeed, the bacterial communities associated with external foragers differ among the five species, and among colonies of the same species. Furthermore, bacterial communities from internal ants vs. foragers do not differ or differ only slightly within each ant species. This study highlights the importance of describing ant-associated bacterial communities to better understand this host-bacterial interaction in the social environment of insect colonies and provides the foundation for future studies on the ecological and evolutionary processes that drive the success of fungus-farming ants.

Pyrene degradation by marine-derived ascomycete: process optimization, toxicity, and metabolic analyses.

Marine-derived fungi are relevant genetic resources for bioremediation of saline environments/processes. Among the five fungi recovered from marine sponges able to degrade pyrene (Py) and benzo[a]pyrene (BaP), Tolypocladium sp. strain CBMAI 1346 and Xylaria sp. CBMAI 1464 presented the best removal rates of Py and BaP, respectively. Since the decrease in BaP was related to mycelial adsorption, a combined strategy was applied for the investigation of Py degradation by the fungus Tolypocladium sp. CBMAI 1346. The selected fungus was able to degrade about 95% of Py after 7 days of incubation (optimized conditions), generating metabolites different from the ones found before optimization. Metabolites and transcriptomic data revealed that the degradation occurred mainly by the cytochrome P450 pathway. Putative monooxygenases and dioxygenases found in the transcriptome may play an important role. After 21 days of degradation, no toxicity was found in the optimized culture conditions. The findings from the present study highlight the potential of marine-derived fungi to degrade environmental pollutants and convey innovative information related to the metabolism of pyrene.

Phylogenetic patterns of ant-fungus associations indicate that farming strategies, not only a superior fungal cultivar, explain the ecological success of leafcutter ants.

To elucidate fungicultural specializations contributing to ecological dominance of leafcutter ants, we estimate the phylogeny of fungi cultivated by fungus-growing (attine) ants, including fungal cultivars from (i) the entire leafcutter range from southern South America to southern North America, (ii) all higher-attine ant lineages (leafcutting genera Atta, Acromyrmex; nonleafcutting genera Trachymyrmex, Sericomyrmex) and (iii) all lower-attine lineages. Higher-attine fungi form two clades, Clade-A fungi (Leucocoprinus gongylophorus, formerly Attamyces) previously thought to be cultivated only by leafcutter ants, and a sister clade, Clade-B fungi, previously thought to be cultivated only by Trachymyrmex and Sericomyrmex ants. Contradicting this traditional view, we find that (i) leafcutter ants are not specialized to cultivate only Clade-A fungi because some leafcutter species ranging across South America cultivate Clade-B fungi; (ii) Trachymyrmex ants are not specialized to cultivate only Clade-B fungi because some Trachymyrmex species cultivate Clade-A fungi and other Trachymyrmex species cultivate fungi known so far only from lower-attine ants; (iii) in some locations, single higher-attine ant species or closely related cryptic species cultivate both Clade-A and Clade-B fungi; and (iv) ant-fungus co-evolution among higher-attine mutualisms is therefore less specialized than previously thought. Sympatric leafcutter ants can be ecologically dominant when cultivating either Clade-A or Clade-B fungi, sustaining with either cultivar-type huge nests that command large foraging territories; conversely, sympatric Trachymyrmex ants cultivating either Clade-A or Clade-B fungi can be locally abundant without achieving the ecological dominance of leafcutter ants. Ecological dominance of leafcutter ants therefore does not depend primarily on specialized fungiculture of L. gongylophorus (Clade-A), but must derive from ant-fungus synergisms and unique ant adaptations.

De novo transcriptome assembly: a new laccase multigene family from the marine-derived basidiomycete Peniophora sp. CBMAI 1063.

Laccases are multicopper oxidases that are able to catalyze reactions involving a range of substrates, including phenols and amines, and this ability is related to the existence of different laccases. Basidiomycetes usually have more than one gene for laccase, but until now, this feature has not been demonstrated in a marine-derived fungus. Peniophora sp. CBMAI 1063 is a basidiomycete fungus isolated from a marine sponge that exhibits the ability to secrete significant amounts of laccase in saline conditions. In the present study, we identified laccase sequences from the transcriptome of Peniophora sp. CBMAI 1063 and used them to perform different molecular in silico analyses. The results revealed the presence of at least eight putative genes, which may encode ten different laccases with peptide lengths ranging from 482 to 588 aa and molecular weights ranging from 53.5 to 64.4 kDa. These laccases seem to perform extracellular activities, with the exception of one that may represent an intracellular laccase. The 10 predicted laccases expressed by Peniophora sp. CBMAI 1063 in laccase-induced media showed different patterns of N-glycosylation and isoelectric points and are divided into two classes based on the residue associated with the regulation of the redox potential of the enzyme. None of the predicted laccases showed more than 61% similarity to other fungal laccases. Based on the differences among the laccases expressed by Peniophora sp. CBMAI 1063, this marine-derived basidiomycete represents a valuable resource with strong potential for biotechnological exploitation.

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.

Rediscovery of the enigmatic fungus-farming ant "Mycetosoritis" asper Mayr (Hymenoptera: Formicidae): Implications for taxonomy, phylogeny, and the evolution of agriculture in ants.

We report the rediscovery of the exceedingly rarely collected and enigmatic fungus-farming ant species Mycetosoritis asper. Since the description of the type specimen in 1887, only four additional specimens are known to have been added to the world's insect collections. Its biology is entirely unknown and its phylogenetic position within the fungus-farming ants has remained puzzling due to its aberrant morphology. In 2014 we excavated and collected twenty-one colonies of M. asper in the Floresta Nacional de Chapecó in Santa Catarina, Brazil. We describe here for the first time the male and larva of the species and complement the previous descriptions of both the queen and the worker. We describe, also for the first time, M. asper biology, nest architecture, and colony demographics, and identify its fungal cultivar. Molecular phylogenetic analyses indicate that both M. asper and M. clorindae are members of the genus Cyphomyrmex, which we show to be paraphyletic as currently defined. More precisely, M. asper is a member of the Cyphomyrmex strigatus group, which we also show to be paraphyletic with respect to the genus Mycetophylax. Based on these results, and in the interest of taxonomic stability, we transfer the species M. asper, M. clorindae, and all members of the C. strigatus group to the genus Mycetophylax, the oldest available name for this clade. Based on ITS sequence data, Mycetophylax asper practices lower agriculture, cultivating a fungal species that belongs to lower-attine fungal Clade 2, subclade F.

Protein depletion using the arabinose promoter in Xanthomonas citri subsp. citri.

Xanthomonas citri subsp. citri (X. citri) is a plant pathogen and the etiological agent of citrus canker, a severe disease that affects all the commercially important citrus varieties, and has worldwide distribution. Citrus canker cannot be healed, and the best method known to control the spread of X. citri in the orchards is the eradication of symptomatic and asymptomatic plants in the field. However, in the state of São Paulo, Brazil, the main orange producing area in the world, control is evolving to an integrated management system (IMS) in which growers have to use less susceptible plants, windshields to prevent bacterial spread out and sprays of cupric bactericidal formulations. Our group has recently proposed alternative methods to control citrus canker, which are based on the use of chemical compounds able to disrupt vital cellular processes of X. citri. An important step in this approach is the genetic and biochemical characterization of genes/proteins that are the possible targets to be perturbed, a task not always simple when the gene/protein under investigation is essential for the organism. Here, we describe vectors carrying the arabinose promoter that enable controllable protein expression in X. citri. These vectors were used as complementation tools for the clean deletion of parB in X. citri, a widespread and conserved gene involved in the essential process of bacterial chromosome segregation. Overexpression or depletion of ParB led to increased cell size, which is probably a resultant of delayed chromosome segregation with subsequent retard of cell division. However, ParB is not essential in X. citri, and in its absence the bacterium was fully competent to colonize the host citrus and cause disease. The arabinose expression vectors described here are valuable tools for protein expression, and especially, to assist in the deletion of essential genes in X. citri.

Bacterial microbiomes from vertically transmitted fungal inocula of the leaf-cutting ant Atta texana.

Microbiome surveys provide clues for the functional roles of symbiotic microbial communities and their hosts. In this study, we elucidated bacterial microbiomes associated with the vertically transmitted fungal inocula (pellets) used by foundress queens of the leaf-cutting ant Atta texana as starter-cultures for new gardens. As reference microbiomes, we also surveyed bacterial microbiomes of foundress queens, gardens and brood of incipient nests. Pseudomonas, Acinetobacter, Propionibacterium and Corynebacterium were consistently present in high abundance in microbiomes. Some pellet and ant samples contained abundant bacteria from an Entomoplasmatales-clade, and a separate PCR-based survey of Entomoplasmatales bacteria in eight attine ant-genera from Brazil placed these bacteria in a monophyletic clade within the bacterial genus Mesoplasma. The attine ant-Mesoplasma association parallels a similar association between a closely related, monophyletic Entomoplasmatales-clade and army ants. Of thirteen A. texana nests surveyed, three nests with exceptionally high Mesoplasma abundance died, whereas the other nests survived. It is unclear whether Mesoplasma was the primary cause of mortality, or Mesoplasma became abundant in moribund nests for non-pathogenic reasons. However, the consistent and geographically widespread presence of Mesoplasma suggests an important functional role in the association with attine ants.

Shared Escovopsis parasites between leaf-cutting and non-leaf-cutting ants in the higher attine fungus-growing ant symbiosis.

Fungus-gardening (attine) ants grow fungus for food in protected gardens, which contain beneficial, auxiliary microbes, but also microbes harmful to gardens. Among these potentially pathogenic microorganisms, the most consistently isolated are fungi in the genus Escovopsis, which are thought to co-evolve with ants and their cultivar in a tripartite model. To test clade-to-clade correspondence between Escovopsis and ants in the higher attine symbiosis (including leaf-cutting and non-leaf-cutting ants), we amassed a geographically comprehensive collection of Escovopsis from Mexico to southern Brazil, and reconstructed the corresponding Escovopsis phylogeny. Contrary to previous analyses reporting phylogenetic divergence between Escovopsis from leafcutters and Trachymyrmex ants (non-leafcutter), we found no evidence for such specialization; rather, gardens from leafcutters and non-leafcutters genera can sometimes be infected by closely related strains of Escovopsis, suggesting switches at higher phylogenetic levels than previously reported within the higher attine symbiosis. Analyses identified rare Escovopsis strains that might represent biogeographically restricted endemic species. Phylogenetic patterns correspond to morphological variation of vesicle type (hyphal structures supporting spore-bearing cells), separating Escovopsis with phylogenetically derived cylindrical vesicles from ancestral Escovopsis with globose vesicles. The new phylogenetic insights provide an improved basis for future taxonomic and ecological studies of Escovopsis.

The most relictual fungus-farming ant species cultivates the most recently evolved and highly domesticated fungal symbiont species.

Fungus-farming (attine) ant agriculture is made up of five known agricultural systems characterized by remarkable symbiont fidelity in which five phylogenetic groups of ants faithfully cultivate five phylogenetic groups of fungi. Here we describe the first case of a lower-attine ant cultivating a higher-attine fungus based on our discovery of a Brazilian population of the relictual fungus-farming ant Apterostigma megacephala, known previously from four stray specimens from Peru and Colombia. We find that A. megacephala is the sole surviving representative of an ancient lineage that diverged ∼39 million years ago, very early in the ∼55-million-year evolution of fungus-farming ants. Contrary to all previously known patterns of ant-fungus symbiont fidelity, A. megacephala cultivates Leucoagaricus gongylophorus, a highly domesticated fungal cultivar that originated only 2-8 million years ago in the gardens of the highly derived and recently evolved (∼12 million years ago) leaf-cutting ants. Because no other lower fungus-farming ant is known to cultivate any of the higher-attine fungi, let alone the leaf-cutter fungus, A. megacephala may provide important clues about the biological mechanisms constraining the otherwise seemingly obligate ant-fungus associations that characterize attine ant agriculture.

Leaf-cutter ant fungus gardens are biphasic mixed microbial bioreactors that convert plant biomass to polyols with biotechnological applications.

Leaf-cutter ants use plant matter to culture the obligate mutualistic basidiomycete Leucoagaricus gongylophorus. This fungus mediates ant nutrition on plant resources. Furthermore, other microbes living in the fungus garden might also contribute to plant digestion. The fungus garden comprises a young sector with recently incorporated leaf fragments and an old sector with partially digested plant matter. Here, we show that the young and old sectors of the grass-cutter Atta bisphaerica fungus garden operate as a biphasic solid-state mixed fermenting system. An initial plant digestion phase occurred in the young sector in the fungus garden periphery, with prevailing hemicellulose and starch degradation into arabinose, mannose, xylose, and glucose. These products support fast microbial growth but were mostly converted into four polyols. Three polyols, mannitol, arabitol, and inositol, were secreted by L. gongylophorus, and a fourth polyol, sorbitol, was likely secreted by another, unidentified, microbe. A second plant digestion phase occurred in the old sector, located in the fungus garden core, comprising stocks of microbial biomass growing slowly on monosaccharides and polyols. This biphasic operation was efficient in mediating symbiotic nutrition on plant matter: the microbes, accounting for 4% of the fungus garden biomass, converted plant matter biomass into monosaccharides and polyols, which were completely consumed by the resident ants and microbes. However, when consumption was inhibited through laboratory manipulation, most of the plant polysaccharides were degraded, products rapidly accumulated, and yields could be preferentially switched between polyols and monosaccharides. This feature might be useful in biotechnology.

ITScan: a web-based analysis tool for Internal Transcribed Spacer (ITS) sequences.

BACKGROUND: Studies on fungal diversity and ecology aim to identify fungi and to investigate their interactions with each other and with the environment. DNA sequence-based tools are essential for these studies because they can speed up the identification process and access greater fungal diversity than traditional methods. The nucleotide sequence encoding for the internal transcribed spacer (ITS) of the nuclear ribosomal RNA has recently been proposed as a standard marker for molecular identification of fungi and evaluation of fungal diversity. However, the analysis of large sets of ITS sequences involves many programs and steps, which makes this task intensive and laborious. FINDINGS: We developed the web-based pipeline ITScan, which automates the analysis of fungal ITS sequences generated either by Sanger or Next Generation Sequencing (NGS) platforms. Validation was performed using datasets containing ca. 2,000 to 40,000 sequences each. CONCLUSIONS: ITScan is an online and user-friendly automated pipeline for fungal diversity analysis and identification based on ITS sequences. It speeds up a process which would otherwise be repetitive and time-consuming for users. The ITScan tool and documentation are available at http://evol.rc.unesp.br:8083/itscan.

A social parasite evolved reproductive isolation from its fungus-growing ant host in sympatry.

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.

A Brazilian population of the asexual fungus-growing ant Mycocepurus smithii (Formicidae, Myrmicinae, Attini) cultivates fungal symbionts with gongylidia-like structures.

Attine ants cultivate fungi as their most important food source and in turn the fungus is nourished, protected against harmful microorganisms, and dispersed by the ants. This symbiosis evolved approximately 50-60 million years ago in the late Paleocene or early Eocene, and since its origin attine ants have acquired a variety of fungal mutualists in the Leucocoprineae and the distantly related Pterulaceae. The most specialized symbiotic interaction is referred to as "higher agriculture" and includes leafcutter ant agriculture in which the ants cultivate the single species Leucoagaricus gongylophorus. Higher agriculture fungal cultivars are characterized by specialized hyphal tip swellings, so-called gongylidia, which are considered a unique, derived morphological adaptation of higher attine fungi thought to be absent in lower attine fungi. Rare reports of gongylidia-like structures in fungus gardens of lower attines exist, but it was never tested whether these represent rare switches of lower attines to L. gonglyphorus cultivars or whether lower attine cultivars occasionally produce gongylidia. Here we describe the occurrence of gongylidia-like structures in fungus gardens of the asexual lower attine ant Mycocepurus smithii. To test whether M. smithii cultivates leafcutter ant fungi or whether lower attine cultivars produce gongylidia, we identified the M. smithii fungus utilizing molecular and morphological methods. Results shows that the gongylidia-like structures of M. smithii gardens are morphologically similar to gongylidia of higher attine fungus gardens and can only be distinguished by their slightly smaller size. A molecular phylogenetic analysis of the fungal ITS sequence indicates that the gongylidia-bearing M. smithii cultivar belongs to the so-called "Clade 1"of lower Attini cultivars. Given that M. smithii is capable of cultivating a morphologically and genetically diverse array of fungal symbionts, we discuss whether asexuality of the ant host maybe correlated with low partner fidelity and active symbiont choice between fungus and ant mutualists.