Using telomeric length measurements and methylation to understand the karyotype diversification of Ctenomys minutus (a small fossorial mammal).

The genus Ctenomys has been widely used in karyotype evolution studies due to the variation in their diploid numbers. Ctenomys minutus is characterized by intraspecific variation in diploid number (2n = 42, 46, 48, and 50), which makes it an interesting model to investigate genomic rearrangements mechanisms that could lead to different cytotypes in this species. Thereupon, it has been already shown that DNA methylation may participate in chromosome structure. Therefore, we aimed to investigate whether telomeres and global DNA methylation had a role in the genome rearrangements that led to this variation in C. minutus. We also realized an analysis for the presence of intrachromosomal telomeric repeats (ITRs) by fluorescence in situ hybridization. Our study demonstrated that neither telomere length nor DNA methylation had significant differences among the cytotypes. However, if only females were considered, there were significant differences for telomere length and methylation. Young individuals, regardless of their cytotypes, had the most methylated DNA. Regarding the ITRs, we found a signal on chromosome 1 in 2n = 50b. No evidence was found that telomere length or methylation could have influenced chromosomal rearrangements, although new cytotypes seem to have emerged within the distribution of parental cytotypes by the accumulation of different chromosomal rearrangements.

Diet, bite force and skull morphology in the generalist rodent morphotype.

For many vertebrate species, bite force plays an important functional role. Ecological characteristics of a species' niche, such as diet, are often associated with bite force. Previous evidence suggests a biomechanical trade-off between rodents specialized for gnawing, which feed mainly on seeds, and those specialized for chewing, which feed mainly on green vegetation. We tested the hypothesis that gnawers are stronger biters than chewers. We estimated bite force and measured skull and mandible shape and size in 63 genera of a major rodent radiation (the myomorph sigmodontines). Analysis of the influence of diet on bite force and morphology was made in a comparative framework. We then used phylogenetic path analysis to uncover the most probable causal relationships linking diet and bite force. Both granivores (gnawers) and herbivores (chewers) have a similar high bite force, leading us to reject the initial hypothesis. Path analysis reveals that bite force is more likely influenced by diet than the reverse causality. The absence of a trade-off between herbivores and granivores may be associated with the generalist nature of the myomorph condition seen in sigmodontine rodents. Both gnawing and chewing sigmodontines exhibit similar, intermediate phenotypes, at least compared to extreme gnawers (squirrels) and chewers (chinchillas). Only insectivorous rodents appear to be moving towards a different direction in the shape space, through some notable changes in morphology. In terms of diet, natural selection alters bite force through changes in size and shape, indicating that organisms adjust their bite force in tandem with changes in food items.

Evolution of dark colour in toucans (Ramphastidae): a case of molecular adaptation?

In the last decades, researchers have been able to determine the molecular basis of some phenotypes, to test for evidence of natural selection upon them, and to demonstrate that the same genes or genetic pathways can be associated with convergent traits. Colour traits are often subject to natural selection because even small changes in these traits can have a large effect on fitness via camouflage, sexual selection or other mechanisms. The melanocortin-1 receptor locus (MC1R) is frequently associated with intraspecific coat colour variation in vertebrates, but it has been far harder to demonstrate that this locus is involved in adaptive interspecific colour differences. Here, we investigate the contribution of the MC1R gene to the colour diversity found in toucans (Ramphastidae). We found divergent selection on MC1R in the clade represented by the genus Ramphastos and that this coincided with the evolution of darker plumage in members of this genus. Using phylogenetically corrected correlations, we show significant and specific relationships between the rate of nonsynonymous change in MC1R (dN) and plumage darkness across Ramphastidae, and also between the rate of functionally significant amino acid changes in MC1R and plumage darkness. Furthermore, three of the seven amino acid changes in MC1R that occurred in the ancestral Ramphastos branch are associated with melanism in other birds. Taken together, our results suggest that the dark colour of Ramphastos toucans was related to nonsynonymous substitutions in MC1R that may have been subject to positive selection or to a relaxation of selective pressure. These results also demonstrate a quantitative relationship between gene and phenotype evolution, representing an example of how MC1R molecular evolution may affect macroevolution of plumage phenotypes.

DNA metabarcoding diet analysis for species with parapatric vs sympatric distribution: a case study on subterranean rodents.

Closely related sympatric species commonly develop different ecological strategies to avoid competition. Ctenomys minutus and C. flamarioni are subterranean rodents parapatrically distributed in the southern Brazilian coastal plain, showing a narrow sympatric zone. To gain understanding on food preferences and possible competition for food resources, we evaluated their diet composition performing DNA metabarcoding analyzes of 67 C. minutus and 100 C. flamarioni scat samples, collected along the species geographical ranges. Thirteen plant families, mainly represented by Poaceae, Araliaceae, Asteraceae and Fabaceae, were identified in the diet of C. minutus. For C. flamarioni, 10 families were recovered, with a predominance of Poaceae, Araliaceae and Asteraceae. A significant correlation between diet composition and geographical distance was detected in C. minutus, whereas the diet of C. flamarioni was quite homogeneous throughout its geographical distribution. No significant differences were observed between males and females of each species. However, differences in diet composition between species were evident according to multivariate analysis. Our results suggest some level of diet partitioning between C. flamarioni and C. minutus in the sympatric region. While the first species is more specialized on few plant items, the second showed a more varied and heterogeneous diet pattern among individuals. These differences might have been developed to avoid competition in the region of co-occurrence. Resource availability in the environment also seems to influence food choices. Our data indicate that C. minutus and C. flamarioni are generalist species, but that some preference for Poaceae, Asteraceae and Araliaceae families can be suggested for both rodents.

The role of chromosomal rearrangements and geographical barriers in the divergence of lineages in a South American subterranean rodent (Rodentia: Ctenomyidae: Ctenomys minutus).

Identifying factors and the extent of their roles in the differentiation of populations is of great importance for understanding the evolutionary process in which a species is involved. Ctenomys minutus is a highly karyotype-polymorphic subterranean rodent, with diploid numbers ranging from 42 to 50 and autosomal arm numbers (ANs) ranging from 68 to 80, comprising a total of 45 karyotypes described so far. This species inhabits the southern Brazilian coastal plain, which has a complex geological history, with several potential geographical barriers acting on different time scales. We assessed the geographical genetic structure of C. minutus, examining 340 individuals over the entire distributional range and using information from chromosomal rearrangements, mitochondrial DNA (mtDNA) sequences and 14 microsatellite loci. The mtDNA results revealed seven main haplogroups, with the most recent common ancestors dating from the Pleistocene, whereas clustering methods defined 12 populations. Some boundaries of mtDNA haplogroups and population clusters can be associated with potential geographical barriers to gene flow. The isolation-by-distance pattern also has an important role in fine-scale genetic differentiation, which is strengthened by the narrowness of the coastal plain and by common features of subterranean rodents (that is, small fragmented populations and low dispersal rates), which limit gene flow among populations. A step-by-step mechanism of chromosomal evolution can be suggested for this species, mainly associated with the metapopulation structure, genetic drift and the geographical features of the southern Brazilian coastal plain. However, chromosomal variations have no or very little role in the diversification of C. minutus populations.

Genotoxicity biomonitoring in coal regions using wild rodent Ctenomys torquatus by Comet assay and micronucleus test.

Coal is a mixture of a variety of chemicals, especially hydrocarbons, which may give rise to polycyclic aromatic hydrocarbons (PAH). Many PAH compounds produce mutagenic and carcinogenic effects. The quality of mineral coal in Rio Grande do Sul (RS) is low and it is typically obtained by stripping operations; it represents approximately 87% of the Brazil reserves. This report concerns the application of the Comet assay to Ctenomys torquatus to detect the effects of coal, comparing the results with a micronucleus (MN) assay, both using peripheral blood. This study was performed over a 2-year period in an attempt to evaluate seasonal patterns. The wild rodent is fossorial, and its geographic distribution in RS coincides with the distribution of coal reserves. Three localitions were studied: two coal fields, Butiá (in a strip coal mine region) and Candiota (near a strip coal mine), and one control region, Pelotas (no coal). At the end of 2 years, 240 rodents had been analyzed. Our results showed that coal and derivatives induced DNA and chromosomal lesions in rodent cells that were demonstrated by Comet and MN assays. These tests also demonstrated quantitative differences between field exposures (Candiota > Butiá). The Comet assay was more sensitive and also showed a direct relationship between age and damage, and an inverse relationship between temperature and damage index.

Effects of chronic exposure to coal in wild rodents (Ctenomys torquatus) evaluated by multiple methods and tissues.

Rio Grande do Sul (RS) coal is low quality and typically obtained by strip mining. In a recent study concerning 2 years of biomonitoring in coal regions, we demonstrated the genotoxicity of coal and related products on blood cells of native rodents, from RS, Brazil. With the goal of studying the variations in the effects of RS coal on different tissues of the same rodent, we utilized, besides the single cell gel (SCG) and micronucleus (MN) assay on blood, histological analyses and SCG assay of bone marrow, spleen, kidney, liver and lung cells, and MN assay of bone marrow and spleen cells. In addition, to identify agents that can potentially influence the results, concentrations of several heavy metals were analyzed in livers and in soil, and the total concentration of hydrocarbons in the soil was determined. Rodents exposed to coal were captured at two different sites, Butiá and Candiota, in RS. Reference animals were obtained from Pelotas, where there is no coal mining. This report provides chemical and biological data from coal regions, indicating the possible association between Zn, Ni, Pb and hydrocarbons in the induction of DNA damage (e.g. single strand-breaks and alkali-labile sites) determined by the alkaline SCG assay in cells from Ctenomys torquatus. The results of the present SCG study indicate that coal and by-products not only induce DNA damage in blood cells, but also in other tissue cells, mainly liver, kidney and lung. Neither the MN assay nor histopathological observations showed significant differences; these analyses may be useful under circumstances where genotoxicity is higher. In conclusion we believe that the in vivo genotoxicity of coal can be biomonitored by the SCG assay, and our studies suggest that wild rodents, such as C. torquatus are useful for monitoring genotoxic damage by both methods, the SCG assay and the MN test.

Geographical variation of heterochromatin in Ctenomys flamarioni (Rodentia-Octodontidae) and its cytogenetic relationships with other species of the genus.

The first karyotype analysis of Ctenomys flamarioni, based on a sample of 46 specimens collected along the geographical range of this species, is reported. All specimens exhibited a diploid number of 48 but varied in autosomal arm number from a low of 50 to a high of 78. The amount of constitutive heterochromatin also varied with the species' geographical distribution. Comparison of G-band patterns showed a strong relationship between the karyotype of this species and those belonging to the Argentinian mendocinus group.