Genomic screening and molecular dynamics simulations of cyanovirin-N homologs from cyanobacteria phylum.

The phylum cyanobacteria are one of the most ancient groups of organisms on the planet and are well recognized due to its wide distribution, ecological role, and biotechnological potential. Cyanobacterial lectins are being extensively explored due to their antiviral activity, mainly because of their capacity of inhibiting HIV strains from infecting human cells by gp120 and gp41 binding. Cianovirin-N from Nostoc ellipsosporum was the first lectin isolated with this property. Since then, various homologs have been discovered and characterized. In this article, we present results of a genomic screening to find cyanovirin-N homologs (CVNH) in all cyanobacteria genomes available in the GenBank, resulting in 155 CVNH proteins with 63 presenting significant identity differences of cyanovirin-N. Homology modeling and molecular dynamics were employed to characterize 18 unexplored models and their functional capacity of binding to Manα(1-2)Man. Results presented here support the hypothesis of multiple ligand recognition for the CVNH family and may help to understand the function of these lectins for the producer cyanobacteria. Additionally, the theoretical results observed here justify carrying out experimental investigations that can expand the therapeutic potential of cyanobacterial lectins.

Current Knowledge on Microviridin from Cyanobacteria.

Cyanobacteria are a rich source of secondary metabolites with a vast biotechnological potential. These compounds have intrigued the scientific community due their uniqueness and diversity, which is guaranteed by a rich enzymatic apparatus. The ribosomally synthesized and post-translationally modified peptides (RiPPs) are among the most promising metabolite groups derived from cyanobacteria. They are interested in numerous biological and ecological processes, many of which are entirely unknown. Microviridins are among the most recognized class of ribosomal peptides formed by cyanobacteria. These oligopeptides are potent inhibitors of protease; thus, they can be used for drug development and the control of mosquitoes. They also play a key ecological role in the defense of cyanobacteria against microcrustaceans. The purpose of this review is to systematically identify the key characteristics of microviridins, including its chemical structure and biosynthesis, as well as its biotechnological and ecological significance.

Replication origin location might contribute to genetic variability in Trypanosoma cruzi.

BACKGROUND: DNA replication in trypanosomatids operates in a uniquely challenging environment, since most of their genomes are constitutively transcribed. Trypanosoma cruzi, the etiological agent of Chagas disease, presents high variability in both chromosomes size and copy number among strains, though the underlying mechanisms are unknown. RESULTS: Here we have mapped sites of DNA replication initiation across the T. cruzi genome using Marker Frequency Analysis, which has previously only been deployed in two related trypanosomatids. The putative origins identified in T. cruzi show a notable enrichment of GC content, a preferential position at subtelomeric regions, coinciding with genes transcribed towards the telomeres, and a pronounced enrichment within coding DNA sequences, most notably in genes from the Dispersed Gene Family 1 (DGF-1). CONCLUSIONS: These findings suggest a scenario where collisions between DNA replication and transcription are frequent, leading to increased genetic variability, as seen by the increase SNP levels at chromosome subtelomeres and in DGF-1 genes containing putative origins.

A new Piper nigrum cysteine proteinase inhibitor, PnCPI, with antifungal activity: molecular cloning, recombinant expression, functional analyses and molecular modeling.

MAIN CONCLUSION: A new Piper nigrum cysteine proteinase inhibitor, PnCPI, belonging to group I of phytocystatins, with inhibitory activity against papain and growth of Fusarium solani f. sp. piperis, was isolated and characterized. Previous studies (de Souza et al. 2011) have identified a partial cDNA sequence of putative cysteine proteinase inhibitor differentially expressed in roots of black pepper (P. nigrum L.) infected by F. solani f. sp. piperis. Here, we aimed to isolate the full-length cDNA and genomic sequences of the P. nigrum cysteine proteinase inhibitor gene, named PnCPI. Sequence analyses showed that the PnCPI gene encodes a deduced protein of 108 amino acid residues with a predicted molecular mass of 12.3 kDa and isoelectric point of 6.51. Besides the LARFAV-like sequence, common to all phytocystatins, PnCPI contains three conserved motifs of the superfamily cystatin: a glycine residue at the N-terminal region, the QxVxG reactive site more centrally positioned, and one tryptophan in the C-terminal region. PnCPI, belonging to group I of phytocystatins, showed high identity with cystatins isolated from several plant species. Sequence analyses also revealed no putative signal peptide at the N-terminal of PnCPI, as well as no introns within the genomic sequence corresponding to the PnCPI coding region. Molecular modeling showed the ability of PnCPI to interact with papain, while its inhibitory activity against this protease was confirmed after heterologous expression in Escherichia coli. The effects of heat treatments on the inhibitory activity of recombinant PnCPI, rPnCPI, were evaluated. In addition, rPnCPI exhibited in vitro activity against F. solani f. sp. piperis, revealing a new cystatin with the potential antifungal application. The identification of PnCPI as a functional cystatin able to inhibit the in vitro growth of F. solani f. sp. piperis indicates other factors contributing to in vivo susceptibility of black pepper to root rot disease.

Kudoa amazonica n. sp. (Myxozoa; Multivalvulida), a parasite of the esophageal musculature of the freshwater catfish, Hypophthalmus marginatus (Siluriformes: Pimelodidae), from a river of the Amazon region.

The class Myxozoa is a group of spore-producing eukaryote organisms that parasitize both freshwater and marine fish. The multivalvulide myxosporidian parasites of the genus Kudoa infect primarily the musculature of the fish host, some species are producing enzymes (proteases) capable of digesting muscle fibers. In the present study, 50 specimens of the freshwater catfish Hypophthalmus marginatus were collected from the Tocantins River in Pará, Brazil, and were analyzed. Overall, 68% of these specimens presented infections by Kudoa parasite in the esophageal musculature. The morphology of these parasite was examined under light microscopy and nucleotide sequences of the SSU rDNA gene were obtained for phylogenetic analyses. The species formed numerous whitish pseudocysts containing square spores with rounded extremities in the apical view, and four polar capsules of equal size. In the phylogenetic analyses, Kudoa amazonica n. sp. was characterized as a sister taxon of another freshwater species, Kudoa orbicularis. The combination of morphological, morphometric, and molecular data obtained in the present study provided a conclusive diagnosis of Kudoa amazonica n. sp., which is clearly distinct from all other Kudoa taxa described to date.

Effect of gluten diet on blood innate immune gene expressions and stool consistency in Spix's Saddleback Tamarin (Leontocebus fuscicollis) raised in captivity.

The callitrichids are non-human primates that feed on insects and plant matter in nature, but in captivity, they are fed mostly an artificial diet containing amounts of gluten, in their toxic forms in items such as wheat, barley and rye. The aim of this research was to estimate the blood ß-defensin and Toll like receptor 5 (TLR5) gene expressions and to analyze the stool consistency (firm, soft, diarrheic) in Leontocebus fuscicollis raised in captivity. Blood samples of animals under gluten-free and gluten diets were collected and their fecal output quality was periodically monitored and classified during the course of the study. Gene expression was evaluated using real-time PCR. The stool consistencies of individuals fed a gluten diet were most frequently soft or diarrheic, while it was mostly normal in individuals fed a gluten-free diet. ß-Defensin expression increased in individuals fed a gluten diet, but decreased after 15 days. Expression normalized between 30 and 45 days on a gluten-free diet. However, expression of the TLR5 gene did not change under a gluten diet. A gluten diet affects stool quality, and brings about an immediate increase in blood ß-defensin expression in the beginning but decreases after 15 days.

Optimization of Light Intensity and NaNO3 Concentration in Amazon Cyanobacteria Cultivation to Produce Biodiesel.

The objective of this study, for the first time, was to optimize Amazonian cyanobacterial culture conditions for improving cell productivity and lipid content, by analyzing the effect of light intensity and nitrogen concentration, for empirically evaluating biodiesel quality parameters. The strains Synechocystis sp. CACIAM05, Microcystis aeruginosa CACIAM08, Pantanalinema rosaneae CACIAM18, and Limnothrix sp. CACIAM25, were previously identified by morphological and molecular analysis (16S rRNA) and were selected based on their production of chlorophyll a and dry cell weight. Then, factorial planning (22) with central points was applied, with light intensity and NaNO3 concentration as independent variables. As response variables, cell productivity and lipid content were determined. Statistical analysis indicated that for all strains, the independent variables were statistically significant for cell productivity. Analysis of the fatty acid composition demonstrated diversity in the composition of the fatty acid profile from the experimental planning assays of each strain. The Biodiesel Analyzer software predicted the biodiesel quality parameters. CACIAM05 and CACIAM25 obtained better parameters with low levels of light intensity and NaNO3 concentration, whereas CACIAM08 and CACIAM18 obtained better parameters with low NaNO3 concentrations and high luminous intensity.

Genomic screening of new putative antiviral lectins from Amazonian cyanobacteria based on a bioinformatics approach.

Lectins are proteins of nonimmune origin, which are capable of recognizing and binding to glycoconjugate moieties. Some of them can block the interaction of viral glycoproteins to the host cell receptors acting as antiviral agents. Although cyanobacterial lectins have presented broad biotechnological potential, little research has been directed to Amazonian Cyanobacterial diversity. In order to identify new antiviral lectins, we performed genomic analysis in seven cyanobacterial strains from Coleção Amazônica de Cianobactérias e Microalgas (CACIAM). We found 75 unique CDS presenting one or more lectin domains. Since almost all were annotated as hypothetical proteins, we used homology modeling and molecular dynamics simulations to evaluate the structural and functional properties of three CDS that were more similar to known antiviral lectins. Nostoc sp. CACIAM 19 as well as Tolypothrix sp. CACIAM 22 strains presented cyanovirin-N homologues whose function was confirmed by binding free energy calculations. Asn, Glu, Thr, Lys, Leu, and Gly, which were described as binding residues for cyanovirin, were also observed on those structures. As for other known cyanovirins, those residues in both our models also made favorable interactions with dimannose. Finally, Alkalinema sp. CACIAM 70d presented one CDS, which was identified as a seven-bladed beta-propeller structure with binding sites predicted for sialic acid and N-acetylglucosamine. Despite its singular structure, our analysis suggested this molecule as a new putative antiviral lectin. Overall, the identification and the characterization of new lectins and their homologues are a promising area in antiviral research, and Amazonian cyanobacteria present biotechnological potential to be explored in this regard.

Anti-dengue virus activity of scytovirin and evaluation of point mutation effects by molecular dynamics and binding free energy calculations.

The absence of a specific treatment against DENV has led to intensive research into developing strategies for curing the infection. One lectin with high antiviral activity is scytovirin, which was isolated from the cyanobacterium Scytonema varium and has proven activity against HIV and Zaire Ebola Virus. To achieve the results presented here, we tested the affinity of full-length scytovirin, SD1 and SD2 separately, and six SD1 mutants for DENV glycoprotein E carbohydrate by Molecular Dynamics (MD) simulations and binding free energy calculations. It was possible to identify the key residues for protein-ligand interaction such as Glu10, Ala11, Pro17, Ans18, Arg30, Thr41, Ser42 and Arg43, which also has importance action against HIV. All binding free energy calculations showed negative values to ΔGbind of protein-DENV carbohydrate complexation. Additionally, these results are similar to the values of scytovirin and HIV gp120 carbohydrate complexation (-32.20 kcal/mol). Furthermore, we found that SD1 individually has more affinity to the carbohydrate and the Asn9, Glu10, Asn18, Arg30 and Arg43 demonstrated an important role in this matter. We also found that mutant G48R has better affinity (-34.10 kcal/mol) for the DENV carbohydrate than the wild type protein (-27.15 kcal/mol).

In silico analysis of the cyanobacterial lectin scytovirin: new insights into binding properties.

Scytovirin is a lectin isolated from the cyanobacterium Scytonema varium that has shown activity against HIV, SARS coronavirus and Zaire Ebola virus. Its 95 amino acids are divided into two structural domains (SD), the first spanning amino acids 1-48 (SD1) and the second 49-95 (SD2). Interestingly, the domains are nearly identical but differ in their affinities for carbohydrates. With the aim of enhancing understanding of the binding properties of scytovirin, we performed molecular dynamics (MD) simulations of scytovirin complexed with Man4. We set up three systems: (i) Man4 bound to both domains (SD1 + SD2) using the full-length protein; (ii) Man4 bound to an incomplete protein, containing only SD1 and (iii) Man4 bound to an incomplete protein containing only SD2. Contrary to other reports, binding free energy results suggest that Man4 can bind simultaneously to SD1 and SD2 binding regions, but SD1 individually has the best values of energy and the best affinity for Man4. Decomposition of the binding free energy showed that the residues that interact with Man4 were different in the three systems, suggesting that the binding mechanism of Man4 varies between full-length protein, SD1 and SD2. The results presented here may help to formulate strategies to use scytovirin and promote mutagenesis studies to improve the antiviral activity of scytovirin.

Henneguya paraensis n. sp. (Myxozoa; Myxosporea), a new gill parasite of the Amazonian fish Cichla temensis (Teleostei: Cichlidae): morphological and molecular aspects.

The present study describes light microscopy, transmission electron microscopy, and molecular analyses of a myxosporid found parasitizing the gill region of the teleost fish Cichla temensis, collected from the Tocantins River, near Cametá, Pará State, Brazil. The prevalence of infection was 60 %. The spore-containing cysts that were located in the gill lamellae were oval and whitish. The spores had a mean length of 42.3 ± 0.65 µm; fusiform body, 12.8 ± 0.42-µm long and 8.6 ± 0.32-µm wide; each of the two valves exhibited a tapering tail of 29.5 ± 0.73 µm length. The spores had two polar capsules, 7.4 ± 0.16-µm long by 2.6 ± 0.08-µm wide, containing a polar filament with 5-7 twists. The spores differ from the species previously described, and phylogenetic analysis based on spore morphology and molecular aspects indicated that the fish parasite Henneguya sp. has a strong trend to form clades mainly based on the environment and host order/family. Thus, we conclude that the species belongs to the family Myxobolidae, genus Henneguya, which comprises a new species: Henneguya paraensis n. sp.

Splenic architecture disruption and parasite-induced splenocyte activation and anergy in Plasmodium falciparum-infected Saimiri sciureus monkeys.

BACKGROUND: The understanding of the mechanisms of immunity in malaria is crucial for the rational development of interventions such as vaccines. During blood stage infection, the spleen is considered to play critical roles in both immunity and immunopathology of Plasmodium falciparum infections. METHODS: Saimiri sciureus monkeys were inoculated with blood stages of P. falciparum (FUP strain) and spleens removed during acute disease (days 7 and 13 of infection) and during convalescence (15 days after start of chloroquine treatment). Cytokine (IFNγ, TNFα, IL2, IL6, IL10, and IL12) responses of splenocytes stimulated with P. falciparum-parasitized red blood cells were assessed by real-time PCR using specific Saimiri primers, and histological changes were evaluated using haematoxylin-eosin and Giemsa-stained slides. RESULTS: Early during infection (day 7, 1-2% parasitaemia), spleens showed disruption of germinal centre architecture with heavy B-cell activation (centroblasts), and splenocytes showed increased expression of IFNγ, IL6 and IL12 upon in vitro stimuli by P. falciparum-parasitized red blood cells (pRBC). Conversely, 15 days after treatment of blood stage infection with chloroquine, splenocytes showed spontaneous in vitro expression of TNFα, IL2, IL6, IL10, and IL12, but not IFNγ, and stimulation with P. falciparum pRBC blocked the expression of all these cytokines. During the acute phase of infection, splenic disarray with disorganized germinal centres was observed. During convalescence, spleens of the chloroquine-treated animals showed white pulp hyperplasia with extensive lymphocyte activation and persistency of heavily haemozoin-laden macrophages throughout the red pulp. CONCLUSIONS: Inability to eliminate haemozoin is likely involved in the persistent lymphocyte activation and in the anergic responses of Saimiri splenocytes to P. falciparum pRBC, with important negative impact in immune responses and implications for the design of malaria vaccine.

Potaspora aequidens n. sp. (Microsporidia, Tetramicridae), a parasite infecting the freshwater fish Aequidens plagiozonatus (Teleostei, Cichlidae) from Brazil.

Morphological and molecular procedures were used to describe a new species of microsporidian that infects the muscles of the sub-opercular region and the caudal fins of the freshwater Aequidens plagiozonatus in Brazil. This microsporidian forms whitish xenomas containing variable number of spores, reaching up to ~0.4 mm in diameter. The mature spores, pyriformin shape, with slightly round ends, measured 3.4 ± 0.5 µm long and 1.9 ± 0.3 µm wide (n = 50) and showed characteristics typical of Microsporidia. The average thickness of the spore wall was 100 (96-108) nm (n = 50), and the spore wall was composed of two layers, a thin, electron-dense exospore and a thick electron-transparent endospore. The exospore was surrounded by a thin, irregular layer of granular material. The anchoring disc was mushroom-like, located in the apical region of the spore in an eccentric position relative to the spore axis, rendering bilateral asymmetry to the spore. The anterior part of the polar filament (PF) (manubrium) measured approximately 125 (122-128) nm thick (n = 30), and the angle of tilt between the anterior PF and the spore axis was ~45°; the posterior part was packed in 8-9 coils. Phylogenetic analysis showed a strongly supported clade containing family Spragueidae Weissenberg, 1976, family Tetramicridae Matthews and Matthews, 1980, Microsporidium sp. RBS1, and Kabatana spp. In conclusion, the available morphological, ultrastructural, and molecular data shows that this microsporidian is a new species belonging to group 4, classified as Potaspora aequidens n. sp. This is the second species described in the genus Potaspora.

Morphological aspects of Henneguya aequidens n. sp. (Myxozoa: Myxobolidae) in Aequidens plagiozonatus Kullander, 1984 (Teleostei: Cichlidae) in the Amazon region, Brazil.

A new species of Myxosporea, Henneguya aequidens sp. n. (Myxozoa: Myxobolidae), was described based on its ultrastructural features. This is a parasite of the freshwater fish Aequidens plagiozonatus, in the Peixe-boi River, Pará, Brazil. This parasite was found in the gills, in the form of whitish ellipsoid cysts with mature spores inside them. The average spore body was 15 ± 0.9 µm in length (n = 30) and 6 ± 0.8 µm in width (n = 30), and the tail measured 27 ± 0.5 µm in length (n = 15). The spores showed typical features of the genus Henneguya with two valves of equal size and two symmetrical polar capsules of 3 ± 0.3 µm in length and 2 ± 0.3 µm in width. Each polar capsule had a polar filament forming a helix from the apical region to the polar caps, with four to six turns. Based on the ultrastructural differences in morphology of these spores, the location of the parasite, and its host specificity, this parasite was described as a new species.

Complete nucleotide sequences of two blaKPC-2-bearing IncN Plasmids isolated from sequence type 442 Klebsiella pneumoniae clinical strains four years apart.

We sequenced the oldest blaKPC-2-bearing plasmid isolated in Brazil and another plasmid also carried by a Klebsiella pneumoniae strain of sequence type 442 (ST442), isolated 52 months later. Both plasmids present an IncN backbone and few acquired regions. Because the 2005 plasmid presented deletions and a truncated gene within Tn4401b compared to the 2009 plasmid, we can thus infer that IncN blaKPC-2-bearing plasmids pFCF1305 and pFCF3SP had a common ancestor circulating in Brazil prior to May 2005.

Structural and functional analysis of Cyanovirin-N homologs: Carbohydrate binding affinities and antiviral potential of cyanobacterial peptides.

Cyanobacteria, a group of photosynthetic prokaryotes, can sinthesize several substances due to their secondary metabolism, with notable properties, such as Cyanovirin-N(CVN), a carbohydrate-binding lectin, that exhibits antiviral activity against several pathogens, due to its ability to bind viral surface carbohydrates such as mannose, thus interfering with the viral entry on the cell. CVN has been described in several cyanobacterial strains and shows biotechnological potential for the development of drugs of pharmaceutical interest. This study focuses on the genomic exploration and characterization of Cyanovirin-N homologs to assess the conservation of carbohydrate-binding affinity within the group. The analysis of their antiviral properties was carried out using bioinformatics tools to study protein models through an in silico pipeline, following the steps of genomic prospection on public databases, homology modeling, docking, molecular dynamics and energetic analysis. Mannose served as the reference ligand, and the lectins' binding affinity with mannose was assessed across Cyanovirin-N homologs. Genomic mining identified 33 cyanobacterial lectin sequences, which underwent structural and functional characterization. The results obtained from this work indicate strong carbohydrate affinity on several homologs, pointing to the conservation of antiviral properties alongside the group. However, this affinity was not uniformly distributed among sequences, exhibiting significant heterogeneity in binding site residues, suggesting potential multi-ligand binding capabilities on the Cyanovirin-N homologs group. Studies focused on the properties involved in these molecules and the investigation of the genetic diversity of Cyanovirin-N homologs could provide valuable insights into the discovery of new drug candidates, harvesting the potential of bioinformatics for large-scale functional and structural analysis.

A new species of Mesocoelium (Digenea: Mesocoeliidae) found in Rhinella marina (Amphibia: Bufonidae) from Brazilian Amazonia.

Mesocoelium lanfrediae sp. nov. (Digenea: Mesocoeliidae) inhabits the small intestine of Rhinella marina (Amphibia: Bufonidae) and is described here, with illustrations provided by light, scanning electron microscopy and molecular approachs. M. lanfrediae sp. nov. presents the typical characteristics of the genus, but is morphometrically and morphologically different from the species described previously. The main diagnostic characteristics of M. lanfrediae sp. nov. are (i) seven pairs of regularly-distributed spherical papillae on the oral sucker, (ii) ventral sucker outlined by four pairs of papillae distributed in a uniform pattern and interspersed with numerous spines, which are larger at the posterior margin and (iii) small, rounded tegumentary papillae around the opening of the oral sucker, which are morphologically different from those of the oral sucker itself, some of which are randomly disposed in the ventrolateral tegumentary region of the anterior third of the body. Addionally, based on SSU rDNA, a phylogenetic analysis including Brachycoeliidae and Mesocoeliidae taxa available on GenBank established the close relationship between M. lanfrediae sp. nov. and Mesocoelium sp.

Critical points of Brazil nuts: a beginning for food safety, quality control and Amazon sustainability.

BACKGROUND: One difficulty of self-sustainability is the quality assurance of native products. This research was designed to study the risks and critical control points in the collection, handling and marketing of Brazil nuts from native forests and urban fairs in the Brazilian Amazon by characterisation of morphological aspects of fungi and posterior identification by molecular biology and determination of aflatoxins by high-performance liquid chromatography. RESULTS: Several corrective actions to improve product quality were found to be necessary in both sites. Growth of fungi was observed in 95% of fragments of Brazil nuts from both sites during the between-harvest period. Aflatoxin levels indicated that, although fungal growth was observed in both sites, only Brazil nuts from the native forest showed a high risk to human health (total aflatoxin level of 471.69 µg kg(-1)). CONCLUSION: This study has shown the main issues related to the process design of Brazil nuts, supporting the necessity for research on new strategies to improve the quality of nuts. Also, the habit of eating Brazil nuts stored throughout the year may represent a risk to farmers.

Description of Rhabdias breviensis n. sp. (Rhabditoidea: Rhabdiasidae) in two Neotropical frog species.

Nematodes of the genus Rhabdias Stiles & Hassall, 1905 (Rhabditoidea: Rhabdiasidae) have a dioecious free-living stage and a hermaphroditic stage that parasitises the lungs of amphibians and reptiles. Approximately 94 species of Rhabdias have been described. Because the similar morphological characteristics such as the labial structures, the location of the vulva and the shape of the tail of Rhabdias spp. hinder their identification, molecular biology techniques and scanning electron microscopy have been employed to diagnose species of this genus. This study describes Rhabdias breviensis n. sp., parasitic in the lungs of two Neotropical frog species Leptodactylus petersii Steindachner and Leptodactylus macrosternum Miranda-Ribeiro. The description of this species integrates classical taxonomy, scanning electron microscopy and a molecular analysis of the mitochondrial COI gene. The new species differs from all other Rhabdias species parasitic in Neotropical hosts in certain morphometric parameters, the position of the vulva, the host group and the cephalic characters.

Performance of intron 7 of the ß-fibrinogen gene for phylogenetic analysis: An example using gladiator frogs, Boana Gray, 1825 (Anura, Hylidae, Cophomantinae).

Boana, the third largest genus of Hylinae, has cryptic morphological species. The potential applicability of b-fibrinogen intron 7 - FGBI7 is explored to propose a robust phylogeny of Boana. The phylogenetic potential of FGBI7 was evaluated using maximum parsimony, MrBayes, and maximum likelihood analysis. Comparison of polymorphic sites and topologies obtained with concatenated analysis of FGBI7 and other nuclear genes (CXCR4, CXCR4, RHO, SIAH1, TYR, and 28S) allowed evaluation of the phylogenetic signal of FGBI7. Mean evolutionary rates were calculated using the sequences of the mitochondrial genes ND1 and CYTB available for Boana in GenBank. Dating of Boana and some of its groups was performed using the RelTime method with secondary calibration. FGBI7 analysis revealed high values at informative sites for parsimony. The absolute values of the mean evolutionary rate were higher for mitochondrial genes than for FGBI7. Dating of congruent Boana groups for ND1, CYTB, and FGBI7 revealed closer values between mitochondrial genes and slightly different values from those of FGBI7. Divergence times of basal groups tended to be overestimated when mtDNA was used and were more accurate when nDNA was used. Although there is evidence of phylogenetic potential arising from concatenation of specific genes, FGBI7 provides well-resolved independent gene trees. These results lead to a paradigm for linking data in phylogenomics that focuses on the uniqueness of species histories and ignores the multiplicities of individual gene histories.