Rheological and Biological Properties of Adhesive Skin Secretions from Eupsophus vertebralis (Anura: Alsodidae).

Skin secretions from Patagonian ground frogs, Eupsophus vertebralis, have previously been reported as a potent proteinaceous adhesive with potential biomedical applications. Here, we conducted a rheological analysis indicating the mechanical robustness of these secretions, with a storage modulus ranging from 1 to 10 Pa. In addition, antimicrobial and cytotoxicity assays were performed, revealing no antimicrobial activity against both the Gram-positive and Gram-negative bacteria. The cytotoxicity results were intriguing, as three samples showed no harm, and one exhibited a severe cytotoxic effect on the human cell line MG63. These properties, as indicated by these preliminary results, reinforce their potential for practical applications in the industrial and medical sectors.

Analysis of microbiota-host communication mediated by butyrate in Atlantic salmon.

Butyrate is a microbiota-produced metabolite, sensed by host short-chain fatty acid receptors FFAR2 (Gpr43), FFAR3 (Gpr41), HCAR2 (Gpr109A), and Histone deacetylase (HDAC) that promotes microbiota-host crosstalk. Butyrate influences energy uptake, developmental and immune response in mammals. This microbial metabolite is produced by around 79 anaerobic genera present in the mammalian gut, yet little is known about the role of butyrate in the host-microbiota interaction in salmonid fish. To further our knowledge of this interaction, we analyzed the intestinal microbiota and genome of Atlantic salmon (Salmo salar), searching for butyrate-producing genera and host butyrate receptors. We identified Firmicutes, Proteobacteria, and Actinobacteria as the main butyrate-producing bacteria in the salmon gut microbiota. In the Atlantic salmon genome, we identified an expansion of genes orthologous to FFAR2 and HCAR2 receptors, and class I and IIa HDACs that are sensitive to butyrate. In addition, we determined the expression levels of orthologous of HCAR2 in the gut, spleen, and head-kidney, and FFAR2 in RTgutGC cells. The effect of butyrate on the Atlantic salmon immune response was evaluated by analyzing the pro and anti-inflammatory cytokines response in vitro in SHK-1 cells by RT-qPCR. Butyrate decreased the expression of the pro-inflammatory cytokine IL-1ß and increased anti-inflammatory IL-10 and TGF-ß cytokines. Butyrate also reduced the expression of interferon-alpha, Mx, and PKR, and decreased the viral load at a higher concentration (4 mM) in cells treated with this molecule before the infection with Infectious Pancreatic Necrosis Virus (IPNV) by mechanisms independent of FFAR2, FFAR3 and HCAR2 expression that probably inhibit HDAC. Moreover, butyrate modified phosphorylation of cytoplasmic proteins in RTgutGC cells. Our data allow us to infer that Atlantic salmon have the ability to sense butyrate produced by their gut microbiota via different specific targets, through which butyrate modulates the immune response of pro and anti-inflammatory cytokines and the antiviral response.

Species limits and recent diversification of Cerradomys (Sigmodontinae: Oryzomyini) during the Pleistocene.

Cerradomys is a genus of the tribe Oryzomyini with eight species currently recognized, and a controversial taxonomy. These species are mainly distributed in the South America dry diagonal, but some species extend into Atlantic Forest, reaching the coastal sandy plains known as Restingas. This study aimed to address species limits and patterns of diversification of Cerradomys species. For this purpose, we performed cytogenetic and molecular analyses (phylogeny, coalescent species delimitation, barcoding, and divergence times estimation) using multiple mitochondrial and nuclear markers on a comprehensive sampling, representing all nominal taxa reported so far. Chromosomal information was a robust marker recognizing eight Cerradomys species. Reciprocal monophyly was recovered for all the species, except for C. subflavus. These results together with coalescent analyses recovered eight species as the most congruent species delimitation scenario for the genus (mean C tax : 0.72). Divergence time estimates revealed that Cerradomys' diversification occurred about 1.32 million years ago (Mya) during the Pleistocene. Although our results conservatively support the eight Cerradomys species described so far, different lines of evidence suggest that C. langguthi and C. subflavus could potentially be species-complexes. We discussed this scenario in the light of multiple evolutionary processes within and between species and populations, since Cerradomys comprises a species group with recent diversification affected by Pleistocene climatic changes and by the complex biogeographic history of South America dry diagonal. This work supports that the diversity of Cerradomys is underestimated and reiterates that interdisciplinary approaches are mandatory to identify small rodent species properly, and to unhide cryptic species.

Species limits and recent diversification of Cerradomys (Sigmodontinae: Oryzomyini) during the Pleistocene

Cerradomys is a genus of the tribe Oryzomyini with eight species currently recognized, and a controversial taxonomy. These species are mainly distributed in the South America dry diagonal, but some species extend into Atlantic Forest, reaching the coastal sandy plains known as Restingas. This study aimed to address species limits and patterns of diversification of Cerradomys species. For this purpose, we performed cytogenetic and molecular analyses (phylogeny, coalescent species delimitation, barcoding, and divergence times estimation) using multiple mitochondrial and nuclear markers on a comprehensive sampling, representing all nominal taxa reported so far. Chromosomal information was a robust marker recognizing eight Cerradomys species. Reciprocal monophyly was recovered for all the species, except for C. subflavus. These results together with coalescent analyses recovered eight species as the most congruent species delimitation scenario for the genus (mean Ctax: 0.72). Divergence time estimates revealed that Cerradomys’ diversification occurred about 1.32 million years ago (Mya) during the Pleistocene. Although our results conservatively support the eight Cerradomys species described so far, different lines of evidence suggest that C. langguthi and C. subflavus could potentially be species-complexes. We discussed this scenario in the light of multiple evolutionary processes within and between species and populations, since Cerradomys comprises a species group with recent diversification affected by Pleistocene climatic changes and by the complex biogeographic history of South America dry diagonal. This work supports that the diversity of Cerradomys is underestimated and reiterates that interdisciplinary approaches are mandatory to identify small rodent species properly, and to unhide cryptic species.

Blue mussels of the Mytilus edulis species complex from South America: The application of species delimitation models to DNA sequence variation.

Smooth-shelled blue mussels, Mytilus spp., have a worldwide antitropical distribution and are ecologically and economically important. Mussels of the Mytilus edulis species complex have been the focus of numerous taxonomic and biogeographical studies, in particular in the Northern hemisphere, but the taxonomic classification of mussels from South America remains unclear. The present study analysed 348 mussels from 20 sites in Argentina, Chile, Uruguay and the Falkland Islands on the Atlantic and Pacific coasts of South America. We sequenced two mitochondrial locus, Cytochrome c Oxidase subunit I (625 bp) and 16S rDNA (443 bp), and one nuclear gene, ribosomal 18S rDNA (1770 bp). Mitochondrial and nuclear loci were analysed separately and in combination using maximum likelihood and Bayesian inference methods to identify the combination of the most informative dataset and model. Species delimitation using five different models (GMYC single, bGMYC, PTP, bPTP and BPP) revealed that the Mytilus edulis complex in South America is represented by three species: native M. chilensis, M. edulis, and introduced Northern Hemisphere M. galloprovincialis. However, all models failed to delimit the putative species Mytilus platensis. In contrast, however, broad spatial scale genetic structure in South America using Geneland software to analyse COI sequence variation revealed a group of native mussels (putatively M. platensis) in central Argentina and the Falkland Islands. We discuss the scope of species delimitation methods and the use of nuclear and mitochondrial genetic data to the recognition of species within the Mytilus edulis complex at regional and global scales.

Phylogeographic analysis and species distribution modelling of the wood frog Batrachyla leptopus (Batrachylidae) reveal interglacial diversification in south western Patagonia.

BACKGROUND: The evolutionary history of southern South American organisms has been strongly influenced by Pleistocene climate oscillations. Amphibians are good models to evaluate hypotheses about the influence of these climate cycles on population structure and diversification of the biota, because they are sensitive to environmental changes and have restricted dispersal capabilities. We test hypotheses regarding putative forest refugia and expansion events associated with past climatic changes in the wood frog Batrachyla leptopus distributed along ∼1,000 km of length including glaciated and non-glaciated areas in southwestern Patagonia. METHODS: Using three mitochondrial regions (D-loop, cyt b, and coI) and two nuclear loci (pomc and crybA1), we conducted multilocus phylogeographic analyses and species distribution modelling to gain insights of the evolutionary history of this species. Intraspecific genealogy was explored with maximum likelihood, Bayesian, and phylogenetic network approaches. Diversification time was assessed using molecular clock models in a Bayesian framework, and demographic scenarios were evaluated using approximate Bayesian computation (ABC) and extended Bayesian skyline plot (EBSP). Species distribution models (SDM) were reconstructed using climatic and geographic data. RESULTS: Population structure and genealogical analyses support the existence of four lineages distributed north to south, with moderate to high phylogenetic support (Bootstrap > 70%; BPP > 0.92). The diversification time of B. leptopus' populations began at ∼0.107 mya. The divergence between A and B lineages would have occurred by the late Pleistocene, approximately 0.068 mya, and divergence between C and D lineages was approximately 0.065 mya. The ABC simulations indicate that lineages coalesced at two different time periods, suggesting the presence of at least two glacial refugia and a postglacial colonization route that may have generated two southern lineages (p = 0.93, type I error: <0.094, type II error: 0.134). EBSP, mismatch distribution and neutrality indexes suggest sudden population expansion at ∼0.02 mya for all lineages. SDM infers fragmented distributions of B. leptopus associated with Pleistocene glaciations. Although the present populations of B. leptopus are found in zones affected by the last glacial maximum (∼0.023 mya), our analyses recover an older history of interglacial diversification (0.107-0.019 mya). In addition, we hypothesize two glacial refugia and three interglacial colonization routes, one of which gave rise to two expanding lineages in the south.

Comparative cytogenetics of the ground frogs Eupsophus emiliopugini Formas, 1989 and E. vertebralis Grandison, 1961 (Alsodidae) with comments on their inter- and intraspecific chromosome differentiation.

South American frogs of the genus Eupsophus Fitzinger, 1843 comprise 10 species. Two of them, Eupsophus vertebralis Grandison, 1961 and E. emiliopugini Formas, 1989 belong to the Eupsophus vertebralis group, exhibiting 2n = 28. Fundamental number differences between these species have been described using conventional chromosome staining of few specimens from only two localities. Here, classical techniques (Giemsa, C-banding, CMA3/DAPI banding, and Ag-NOR staining), and fluorescence in situ hybridization (FISH, with telomeric and 28S ribosomal probes), were applied on individuals of both species collected from 15 localities. We corroborate differences in fundamental numbers (FN) between E. vertebralis and E. emiliopugini through Giemsa staining and C-banding (FN = 54 and 56, respectively). No interstitial fluorescent signals, but clearly stained telomeric regions were detected by FISH using telomeric probe over spreads from both species. FISH with 28S rDNA probes and Ag-NOR staining confirmed the active nucleolus organizer regions signal on pair 5 for both species. Nevertheless, one E. emiliopugini individual from the Puyehue locality exhibited 28S ribosomal signals on pairs 4 and 5. Interestingly, only one chromosome of each pair showed Ag-NOR positive signals, showing a nucleolar dominance pattern. Chromosomal rearrangements, rRNA gene dosage control, mobile NORs elements, and/or species hybridization process could be involved in this interpopulation chromosomal variation.

Speciation in a biodiversity hotspot: Phylogenetic relationships, species delimitation, and divergence times of Patagonian ground frogs from the Eupsophus roseus group (Alsodidae).

The alsodid ground frogs of the Eupsophus genus are divided into two groups, the roseus (2n = 30) and vertebralis (2n = 28), which are distributed throughout the temperate Nothofagus forests of South America. Currently, the roseus group is composed by four species, while the vertebralis group consists of two. Phylogenetic relationships and species delimitation within each group are controversial. In fact, previous analyses considered that the roseus group was composed of between four to nine species. In this work, we evaluated phylogenetic relationships, diversification times, and species delimitation within the roseus group using a multi-locus dataset. For this purpose, mitochondrial (D-loop, Cyt b, and COI) and nuclear (POMC and CRYBA1) partial sequences from 164 individuals were amplified, representing all species. Maximum Likelihood (ML) and Bayesian approaches were used to reconstruct phylogenetic relationships. Species tree was estimated using BEAST and singular value decomposition scores for species quartets (SVDquartets). Species limits were evaluated with six coalescent approaches. Diversification times were estimated using mitochondrial and nuclear rates with LogNormal relaxed clock in BEAST. Nine well-supported monophyletic lineages were recovered in Bayesian, ML, and SVDquartets, including eight named species and a lineage composed by specimens from the Villarrica population (Bootstrap:>70, PP:> 0.99). Single-locus species delimitation analyses overestimated the species number in E. migueli, E. calcaratus, and E. roseus lineages, while multi-locus analyses recovered as species the nine lineages observed in phylogenetic analyses (Ctax = 0.69). It is hypothesized that Eupsophus diversification occurred during Mid-Pleistocene (0.42-0.14 Mya), with most species having originated after the Last Southern Patagonian Glaciation (0.18 Mya). Our results revitalize the hypothesis that the E. roseus group is composed of eight species and support the Villarrica lineage as a new putative species.

Mitochondrial genomes of the South American frogs Eupsophus vertebralis and E. emiliopugini (Neobatrachia: Alsodidae) and their phylogenetic relationships.

We report the sequencing and compare the mitochondrial genomes of the South American ground frogs Eupsophus vertebralis and E. emiliopugini and reconstruct phylogenetic relationships among Eupsophus species. These genomes consist of 16,156 and 16,711 bp in length, respectively and contain 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes (tRNA), and partial non-coding D-loop region. Both genomes share 94.5% identity with 879 variable sites. A phylogenetic analysis with other available mitogenomes recovered both species as the sister clade of Alsodes gargola. Sequences from D-loop, CO1, and Cyt b, amplified and sequenced with primers developed from the mitochondrial genomes, allowed us to reconstruct phylogenetic relationships among Eupsophus species. Since our report represents the first mitogenomes for the genus Eupsophus, we expect these data will be valuable for further studies on conservation genetics and on the evolution of Patagonian amphibians.

The ancestral chromosomes of Dromiciops gliroides (Microbiotheridae), and its bearings on the karyotypic evolution of American marsupials.

BACKGROUND: The low-numbered 14-chromosome karyotype of marsupials has falsified the fusion hypothesis claiming ancestrality from a 22-chromosome karyotype. Since the 14-chromosome condition of the relict Dromiciops gliroides is reminecent of ancestrality, its interstitial traces of past putative fusions and heterochromatin banding patterns were studied and added to available marsupials' cytogenetic data. Fluorescent in situ hybridization (FISH) and self-genomic in situ hybridization (self-GISH) were used to detect telomeric and repetitive sequences, respectively. These were complemented with C-, fluorescent banding, and centromere immunodetection over mitotic spreads. The presence of interstitial telomeric sequences (ITS) and diploid numbers were reconstructed and mapped onto the marsupial phylogenetic tree. RESULTS: No interstitial, fluorescent signals, but clearly stained telomeric regions were detected by FISH and self-GISH. Heterochromatin distribution was sparse in the telomeric/subtelomeric regions of large submetacentric chromosomes. Large AT-rich blocks were detected in the long arm of four submetacentrics and CG-rich block in the telomeric regions of all chromosomes. The ancestral reconstructions both ITS presence and diploid numbers suggested that ITS are unrelated to fusion events. CONCLUSION: Although the lack of interstitial signals in D. gliroides' karyotype does not prove absence of past fusions, our data suggests its non-rearranged plesiomorphic condition.

Filling phylogenetic gaps and the biogeographic relationships of the Octodontidae (Mammalia: Hystricognathi).

Endemic to South America, octodontid rodents are remarkable by being the only mammal taxa where allotetraploidy has been documented. The taxon's extensive morpho-physiological radiation associated to niche shifts has allowed testing phylogeographic hypotheses. Using maximum likelihood and Bayesian inference analyses, applied to all nominal species of octodontids, phylogenetic reconstructions based on sequences of 12S rRNA and growth hormone receptor gene are presented. Species boundaries were determined by coalescent analyses and divergence times among taxa were estimated based on mutation rates. Two main clades associated to the Andean orogenesis were recognized. The essentially western clade comprises genera Aconaemys, Octodon, Spalacopus, and Octodontomys whereas the eastern one included genera Octomys, Pipanacoctomys, Salinoctomys, and Tympanoctomys. Genetic relationships, coalescent analyses, and genetic distance supported the specific status given to Octodon pacificus and that given to Pipanacoctomys aureus as a species of Tympanoctomys. However, these analyses failed to recognize Salinoctomys loschalchalerosorum as a valid taxon considering its position within the diversity of Tympanoctomys barrerae. Although the origin of genome duplication remains contentious, the coincidence of the basal clade split with distinctive modes of karyotypic evolution across the Andes emphasizes the role of physiographic barriers and westerlies in shaping different edaphological conditions, selective grounds, and concomitantly distinct adaptations within the octodontids.

Meiotic behavior and H3K4m distribution in B chromosomes of Characidium gomesi (Characiformes, Crenuchidae).

Characidium gomesi Travasso, 1956 specimens from the Pardo River have up to four heterochromatic supernumerary chromosomes, derived from the sex chromosomes. To access the meiotic behavior and distribution of an active chromatin marker, males and females of Characidium gomesi with two or three B chromosomes were analyzed. Mitotic chromosomes were characterized using C-banding and FISH with B chromosome probes. Meiocytes were subjected to immunofluorescence-FISH assay using anti-SYCP3, anti-H3K4m, and B chromosomes probes. Molecular homology of supernumeraries was confirmed by FISH and by its bivalent conformation in individuals with two of these chromosomes. In individuals with three Bs, these elements formed a bivalent and a univalent. Supernumerary and sex chromosomes exhibited H3K4m signals during pachytene contrasting with their heterochromatic and asynaptic nature, which suggest a more structural role than functional of this histone modification. The implications of this result are discussed in light of the homology, meiotic nuclear organization, and meiotic silencing of unsynapsed chomatin.

Homomorphic sex chromosomes and the intriguing Y chromosome of Ctenomys rodent species (Rodentia, Ctenomyidae).

Unlike the X chromosome, the mammalian Y chromosome undergoes evolutionary decay resulting in small size. This sex chromosomal heteromorphism, observed in most species of the fossorial rodent Ctenomys, contrasts with the medium-sized, homomorphic acrocentric sex chromosomes of closely related C. maulinus and C. sp. To characterize the sequence composition of these chromosomes, fluorescent banding, self-genomic in situ hybridization, and fluorescent in situ hybridization with an X painting probe were performed on mitotic and meiotic plates. High molecular homology between the sex chromosomes was detected on mitotic material as well as on meiotic plates immunodetected with anti-SYCP3 and anti-γH2AX. The Y chromosome is euchromatic, poor in repetitive sequences and differs from the X by the loss of a block of pericentromeric chromatin. Inferred from the G-banding pattern, an inversion and the concomitant prevention of recombination in a large asynaptic region seems to be crucial for meiotic X chromosome inactivation. These peculiar findings together with the homomorphism of Ctenomys sex chromosomes are discussed in the light of the regular purge that counteracts Muller's ratchet and the probable mechanisms accounting for their origin and molecular homology.

First cytogenetic information for Drymoreomys albimaculatus (Rodentia, Cricetidae), a recently described genus from Brazilian Atlantic Forest.

The recently described taxon Drymoreomys albimaculatus is endemic to the Brazilian Atlantic Forest and its biology and genetics are still poorly known. Herein, we present, for the first time, the karyotype of the species using classical and molecular cytogenetics, which showed 2n=62, FN=62, and interstitial telomeric signals at the sex chromosomes. Nuclear and mitochondrial DNA sequences from the two karyotyped individuals verify the taxonomic identity as the recently described Drymoreomys albimaculatus and confirm the relationship of the species with other Oryzomyini. Additionally, external morphological information is provided.