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1.  ITScan: a web-based analysis tool for Internal Transcribed Spacer (ITS) sequences 
BMC Research Notes  2014;7:857.
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.
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.
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
PMCID: PMC4258023  PMID: 25430816
Fungal biodiversity; Mycology; Pipeline; Web service
2.  A Brazilian Population of the Asexual Fungus-Growing Ant Mycocepurus smithii (Formicidae, Myrmicinae, Attini) Cultivates Fungal Symbionts with Gongylidia-Like Structures 
PLoS ONE  2014;9(8):e103800.
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.
PMCID: PMC4125159  PMID: 25101899
3.  The Mitochondrial Genome of the Leaf-Cutter Ant Atta laevigata: A Mitogenome with a Large Number of Intergenic Spacers 
PLoS ONE  2014;9(5):e97117.
In this paper we describe the nearly complete mitochondrial genome of the leaf-cutter ant Atta laevigata, assembled using transcriptomic libraries from Sanger and Illumina next generation sequencing (NGS), and PCR products. This mitogenome was found to be very large (18,729 bp), given the presence of 30 non-coding intergenic spacers (IGS) spanning 3,808 bp. A portion of the putative control region remained unsequenced. The gene content and organization correspond to that inferred for the ancestral pancrustacea, except for two tRNA gene rearrangements that have been described previously in other ants. The IGS were highly variable in length and dispersed through the mitogenome. This pattern was also found for the other hymenopterans in particular for the monophyletic Apocrita. These spacers with unknown function may be valuable for characterizing genome evolution and distinguishing closely related species and individuals. NGS provided better coverage than Sanger sequencing, especially for tRNA and ribosomal subunit genes, thus facilitating efforts to fill in sequence gaps. The results obtained showed that data from transcriptomic libraries contain valuable information for assembling mitogenomes. The present data also provide a source of molecular markers that will be very important for improving our understanding of genomic evolutionary processes and phylogenetic relationships among hymenopterans.
PMCID: PMC4020775  PMID: 24828084
4.  Asymmetric chromosome segregation in Xanthomonas citri ssp. citri 
MicrobiologyOpen  2013;3(1):29-41.
This study was intended to characterize the chromosome segregation process of Xanthomonas citri ssp. citri (Xac) by investigating the functionality of the ParB factor encoded on its chromosome, and its requirement for cell viability and virulence. Using TAP tagging we show that ParB is expressed in Xac. Disruption of parB increased the cell doubling time and precluded the ability of Xac to colonize the host citrus. Moreover, Xac mutant cells expressing only truncated forms of ParB exhibited the classical phenotype of aberrant chromosome organization, and seemed affected in cell division judged by their reduced growth rate and the propensity to form filaments. The ParB-GFP localization pattern in Xac was suggestive of an asymmetric mode of replicon partitioning, which together with the filamentation phenotype support the idea that Xac may control septum placement using mechanisms probably analogous to Caulobacter crescentus, and perhaps Vibrio cholerae, and Corynebacterium glutamicum. Xac exhibits asymmetric chromosome segregation, and the perturbation of this process leads to an inability to colonize the host plant.
PMCID: PMC3937727  PMID: 24339434
Cell division arrest; chromosome segregation; citrus canker.
5.  Cyatta abscondita: Taxonomy, Evolution, and Natural History of a New Fungus-Farming Ant Genus from Brazil 
PLoS ONE  2013;8(11):e80498.
Cyatta abscondita, a new genus and species of fungus-farming ant from Brazil, is described based on morphological study of more than 20 workers, two dealate gynes, one male, and two larvae. Ecological field data are summarized, including natural history, nest architecture, and foraging behavior. Phylogenetic analyses of DNA sequence data from four nuclear genes indicate that Cyatta abscondita is the distant sister taxon of the genus Kalathomyrmex, and that together they comprise the sister group of the remaining neoattine ants, an informal clade that includes the conspicuous and well-known leaf-cutter ants. Morphologically, Cyatta abscondita shares very few obvious character states with Kalathomyrmex. It does, however, possess a number of striking morphological features unique within the fungus-farming tribe Attini. It also shares morphological character states with taxa that span the ancestral node of the Attini. The morphology, behavior, and other biological characters of Cyatta abscondita are potentially informative about plesiomorphic character states within the fungus-farming ants and about the early evolution of ant agriculture.
PMCID: PMC3829880  PMID: 24260403
6.  Expressed sequence tags from Atta laevigata and identification of candidate genes for the control of pest leaf-cutting ants 
BMC Research Notes  2011;4:203.
Leafcutters are the highest evolved within Neotropical ants in the tribe Attini and model systems for studying caste formation, labor division and symbiosis with microorganisms. Some species of leafcutters are agricultural pests controlled by chemicals which affect other animals and accumulate in the environment. Aiming to provide genetic basis for the study of leafcutters and for the development of more specific and environmentally friendly methods for the control of pest leafcutters, we generated expressed sequence tag data from Atta laevigata, one of the pest ants with broad geographic distribution in South America.
The analysis of the expressed sequence tags allowed us to characterize 2,006 unique sequences in Atta laevigata. Sixteen of these genes had a high number of transcripts and are likely positively selected for high level of gene expression, being responsible for three basic biological functions: energy conservation through redox reactions in mitochondria; cytoskeleton and muscle structuring; regulation of gene expression and metabolism. Based on leafcutters lifestyle and reports of genes involved in key processes of other social insects, we identified 146 sequences potential targets for controlling pest leafcutters. The targets are responsible for antixenobiosis, development and longevity, immunity, resistance to pathogens, pheromone function, cell signaling, behavior, polysaccharide metabolism and arginine kynase activity.
The generation and analysis of expressed sequence tags from Atta laevigata have provided important genetic basis for future studies on the biology of leaf-cutting ants and may contribute to the development of a more specific and environmentally friendly method for the control of agricultural pest leafcutters.
PMCID: PMC3132717  PMID: 21682882
7.  Phylogenetic relationships in genus Arachis based on ITS and 5.8S rDNA sequences 
BMC Plant Biology  2010;10:255.
The genus Arachis comprises 80 species and it is subdivided into nine taxonomic sections (Arachis, Caulorrhizae, Erectoides, Extranervosae, Heteranthae, Procumbentes, Rhizomatosae, Trierectoides, and Triseminatae). This genus is naturally confined to South America and most of its species are native to Brazil. In order to provide a better understanding of the evolution of the genus, we reconstructed the phylogeny of 45 species using the variation observed on nucleotide sequences in internal transcribed spacer regions (ITS1 and ITS2) and 5.8 S of nuclear ribosomal DNA.
Intraspecific variation was detected, but in general it was not enough to place accessions of the same species in different clades. Our data support the view that Arachis is a monophyletic group and suggested Heteranthae as the most primitive section of genus Arachis. The results confirmed the circumscriptions of some sections (Caulorrhizae, Extranervosae), but raised questions about others. Sections Erectoides, Trierectoides and Procumbentes were not well defined, while sections Arachis and Rhizomatosae seem to include species that could be moved to different sections. The division of section Arachis into A and B genome species was also observed in the phylogenetic tree and these two groups of species may not have a monophyletic origin. The 2n = 2x = 18 species of section Arachis (A. praecox, A. palustris and A. decora) were all placed in the same clade, indicating they are closely related to each other, and their genomes are more related to B genome than to the A genome. Data also allowed insights on the origin of tetraploid A. glabrata, suggesting rhizome appeared twice within the genus and raising questions about the placement of that species in section Rhizomatosae.
The main clades established in this study in general agreed with many other studies that have used other types of evidences and sets of species, being some of them included in our study and some not. Thus, the relationships established can be a useful framework for future systematic reviews of genus Arachis and for the selection of species to pre-breeding programs.
PMCID: PMC3095334  PMID: 21092103
8.  Thelytokous Parthenogenesis in the Fungus-Gardening Ant Mycocepurus smithii (Hymenoptera: Formicidae) 
PLoS ONE  2009;4(8):e6781.
The general prevalence of sexual reproduction over asexual reproduction among organisms testifies to the evolutionary benefits of recombination, such as accelerated adaptation to changing environments and elimination of deleterious mutations. Documented instances of asexual reproduction in groups otherwise dominated by sexual reproduction challenge evolutionary biologists to understand the special circumstances that might confer an advantage to asexual reproductive strategies. Here we report one such instance of asexual reproduction in the ants. We present evidence for obligate thelytoky in the asexual fungus-gardening ant, Mycocepurus smithii, in which queens produce female offspring from unfertilized eggs, workers are sterile, and males appear to be completely absent. Obligate thelytoky is implicated by reproductive physiology of queens, lack of males, absence of mating behavior, and natural history observations. An obligate thelytoky hypothesis is further supported by the absence of evidence indicating sexual reproduction or genetic recombination across the species' extensive distribution range (Mexico-Argentina). Potential conflicting evidence for sexual reproduction in this species derives from three Mycocepurus males reported in the literature, previously regarded as possible males of M. smithii. However, we show here that these specimens represent males of the congeneric species M. obsoletus, and not males of M. smithii. Mycocepurus smithii is unique among ants and among eusocial Hymenoptera, in that males seem to be completely absent and only queens (and not workers) produce diploid offspring via thelytoky. Because colonies consisting only of females can be propagated consecutively in the laboratory, M. smithii could be an adequate study organism a) to test hypotheses of the population-genetic advantages and disadvantages of asexual reproduction in a social organism and b) inform kin conflict theory.
For a Portuguese translation of the abstract, please see Abstract S1.
PMCID: PMC2728836  PMID: 19707513
9.  Metabolism of Plant Polysaccharides by Leucoagaricus gongylophorus, the Symbiotic Fungus of the Leaf-Cutting Ant Atta sexdens L. 
Applied and Environmental Microbiology  1998;64(12):4820-4822.
Atta sexdens L. ants feed on the fungus they cultivate on cut leaves inside their nests. The fungus, Leucoagaricus gongylophorus, metabolizes plant polysaccharides, such as xylan, starch, pectin, and cellulose, mediating assimilation of these compounds by the ants. This metabolic integration may be an important part of the ant-fungus symbiosis, and it involves primarily xylan and starch, both of which support rapid fungal growth. Cellulose seems to be less important for symbiont nutrition, since it is poorly degraded and assimilated by the fungus. Pectin is rapidly degraded but slowly assimilated by L. gongylophorus, and its degradation may occur so that the fungus can more easily access other polysaccharides in the leaves.
PMCID: PMC90928  PMID: 9835568

Results 1-9 (9)