New karyotype information for Ctenomys (Rodentia: Ctenomyidae) from Midwest and Northern Brazil.

Its wide karyotypic variation characterizes the genus Ctenomys, and in Brazil, the genus is distributed in the country's southern, Midwest, and Northern regions. Recently, populations of Ctenomys have been found in the Midwest and northern Brazil, with two new lineages named C. sp. "xingu" and C. sp. "central". This work combines classical cytogenetic and molecular analyses to provide new chromosomal information on the boliviensis group distributed in Northern and Midwestern Brazil. This includes the validation of the karyotype of C. bicolor and C. nattereri, and the description of the karyotype of C. sp. "xingu" and C. sp. "central". We found three different karyotypes: 2n=40 for C. bicolor; 2n= 36 for C. nattereri, and specimens from a locality belonging to C. sp. "central"; 2n=34 for the lineage C. sp. "xingu" and specimens from a locality belonging to C. sp. "central". Furthermore, GTG-banding revealed homologous chromosomes between species/lineages and allowed the identification of the rearrangements that occurred, which proved the occurrence of fissions. Considering our results on the variation of 2n in the boliviensis group, we found two possibilities: the first, deduced by parsimony, is that 2n=36 appeared initially, and two fissions produced gave rise to 2n=40, and an independent fusion gave rise to 2n=34 from 2n=36; moreover, the second explanation is that all karyotypes arose independently.

Genomic Organization of Microsatellites and LINE-1-like Retrotransposons: Evolutionary Implications for Ctenomys minutus (Rodentia: Ctenomyidae) Cytotypes.

The Neotropical underground rodents of the genus Ctenomys (Rodentia: Ctenomyidae) comprise about 65 species, which harbor the most significant chromosomal variation among mammals (2n = 10 to 2n = 70). Among them, C. minutus stands out with 45 different cytotypes already identified, among which, seven parental ones, named A to G, are parapatrically distributed in the coastal plains of Southern Brazil. Looking for possible causes that led to such extensive karyotype diversification, we performed chromosomal mapping of different repetitive DNAs, including microsatellites and long interspersed element-1 (LINE-1) retrotransposons in the seven parental cytotypes. Although microsatellites were found mainly in the centromeric and telomeric regions of the chromosomes, different patterns occur for each cytotype, thus revealing specific features. Likewise, the LINE-1-like retrotransposons also showed a differential distribution for each cytotype, which may be linked to stochastic loss of LINE-1 in some populations. Here, microsatellite motifs (A)30, (C)30, (CA)15, (CAC)10, (CAG)10, (CGG)10, (GA)15, and (GAG)10 could be mapped to fusion of chromosomes 20/17, fission and inversion in the short arm of chromosome 2, fusion of chromosomes 23/19, and different combinations of centric and tandem fusions of chromosomes 22/24/16. These data provide evidence for a correlation between repetitive genomic content and localization of evolutionary breakpoints and highlight their direct impact in promoting chromosomal rearrangements.

Bridging macroecology and macroevolution in the radiation of sigmodontine rodents.

Investigations of phenotypic disparity across geography often ignore macroevolutionary processes. As a corollary, the random null expectations to which disparity is compared and interpreted may be unrealistic. We tackle this issue by representing, in geographical space, distinct processes of phenotypic evolution underlying ecological disparity. Under divergent natural selection, assemblages in a given region should have empirical disparity higher than expected under an evolutionarily oriented null model, whereas the opposite may indicate constraints on phenotypic evolution. We gathered phylogenies, biogeographic distributions, and data on the skull morphology of sigmodontine rodents to discover which regions of the Neotropics were more influenced by divergent, neutral, or constrained phenotypic evolution. We found that regions with higher disparity than expected by the evolutionary-oriented null model, in terms of both size and shape, were concentrated in the Atlantic Forest, suggesting a larger role for divergent natural selection there. Phenotypic disparity in the rest of South America, mainly the Amazon basin, northeastern Brazil, and Southern Andes, was constrained-lower than predicted by the evolutionary model. We also demonstrated equivalence between the disparity produced by randomization-based null models and constrained-evolution null models. Therefore, including evolutionary simulations into the null modeling framework used in ecophylogenetics can strengthen inferences on the processes underlying phenotypic evolution.

Anthropogenic Effects on Natural Mammalian Populations: Correlation Between Telomere Length and Coal Exposure.

The Candiota coal mine in Rio Grande do Sul (RS) is one of the largest in Brazil. Coal is a fossil fuel that causes environmental impacts from its extraction to combustion due to the release of different agents, such as polycyclic aromatic hydrocarbons (PAH) and heavy metals. Ctenomys torquatus are herbivorous and subterranean rodents that dig tunnels with their paws and teeth and can be exposed to coal through contaminated food. Exposure to pollutants can cause DNA damage and affect different tissues, inducing alterations in the population structure and genetic diversity. Our study aimed to evaluate the effect of exposure to coal and its derivatives on the C. torquatus population and to examine the relationship of coal exposure with variations in absolute telomere length (aTL), global DNA methylation and genotoxicity. Our study showed an inverse correlation between telomere length and coal exposure in addition to an increase in DNA damage. The results indicate that coal and its byproducts can contribute to the alteration of the C. torquatus population structure, as evidenced by a reduction in the number of adults.

Niche suitability affects development: skull asymmetry increases in less suitable areas.

For conservation purposes, it is important to take into account the suitability of a species to particular habitats; this information may predict the long-term survival of a species. In this sense, morphological measures of developmental stress, such as fluctuating asymmetry, can be proxies for an individual's performance in different regions. In this study, we conducted tests to determine whether areas with different levels of suitability for a species (generated by ecological niche models) were congruent with morphological markers that reflect environmental stress and morphological variance. We generated a Maxent niche model and compared the suitability assessments of several areas with the skull morphology data (fluctuating asymmetry and morphological disparity) of populations of the Atlantic forest endemic to Brazil rodent Akodon cursor. Our analyses showed a significant negative relationship between suitability levels and fluctuating asymmetry levels, which indicates that in less suitable areas, the individuals experience numerous disturbances during skull ontogeny. We have not found an association between morphological variance and environmental suitability. As expected, these results suggest that in environments with a lower suitability, developmental stress is increased. Such information is helpful in the understanding of the species evolution and in the selection of priority areas for the conservation of species.

Comparative assessment of genetic and morphological variation at an extensive hybrid zone between two wild cats in southern Brazil.

Increased attention towards the Neotropical cats Leopardus guttulus and L. geoffroyi was prompted after genetic studies identified the occurrence of extensive hybridization between them at their geographic contact zone in southern Brazil. This is a region where two biomes intersect, each of which is associated with one of the hybridizing species (Atlantic Forest with L. guttulus and Pampas with L. geoffroyi). In this study, we conducted in-depth analyses of multiple molecular markers aiming to characterize the magnitude and spatial structure of this hybrid zone. We also performed a morphological assessment of these species, aiming to test their phenotypic differentiation at the contact zone, as well as the correlation between morphological features and the admixture status of the individuals. We found strong evidence for extensive and complex hybridization, with at least 40% of the individuals sampled in Rio Grande do Sul state (southernmost Brazil) identified as hybrids resulting from post-F1 generations. Despite such a high level of hybridization, samples collected in this state still comprised two recognizable clusters (genetically and morphologically). Genetically pure individuals were sampled mainly in regions farther from the contact zone, while hybrids concentrated in a central region (exactly at the interface between the two biomes). The morphological data set also revealed a strong spatial structure, which was correlated with the molecular results but displayed an even more marked separation between the clusters. Hybrids often did not present intermediate body sizes and could not be clearly distinguished morphologically from the parental forms. This observation suggests that some selective pressure may be acting on the hybrids, limiting their dispersal away from the hybrid zone and perhaps favoring genomic combinations that maintain adaptive phenotypic features of one or the other parental species.

Molecular evidence for a recent demographic expansion in the puma (Puma concolor) (Mammalia, Felidae).

The puma is an iconic predator that ranges throughout the Americas, occupying diverse habitats. Previous phylogeographic analyses have revealed that it exhibits moderate levels of genetic structure across its range, with few of the classically recognized subspecies being supported as distinct demographic units. Moreover, most of the species' molecular diversity was found to be in South America. To further investigate the phylogeographic structure and demographic history of pumas we analyzed mtDNA sequences from 186 individuals sampled throughout their range, with emphasis on South America. Our objectives were to refine the phylogeographic assessment within South America and to investigate the demographic history of pumas using a coalescent approach. Our results extend previous phylogeographic findings, reassessing the delimitation of historical population units in South America and demonstrating that this species experienced a considerable demographic expansion in the Holocene, ca. 8,000 years ago. Our analyses indicate that this expansion occurred in South America, prior to the hypothesized re-colonization of North America, which was therefore inferred to be even more recent. The estimated demographic history supports the interpretation that pumas suffered a severe demographic decline in the Late Pleistocene throughout their distribution, followed by population expansion and re-colonization of the range, initiating from South America.

Human impact in naturally patched small populations: genetic structure and conservation of the burrowing rodent, tuco-tuco (Ctenomys lami).

Isolated or semi-isolated small populations are commonly found among species, due to a naturally patchy occupancy of suitable habitats or also as a result of habitat alterations. These populations are subject to an increased risk of local extinction because they are more vulnerable to demographic, genetic, and environmental stochasticity. Considering that natural areas have been becoming progressively more fragmented and smaller, understanding the genetic structure and evolutionary dynamics of small populations is critical. Ctenomys lami has 26 karyotypes distributed in a small area (936 km(2)) continually modified by human actions. We assessed the genetic geographical structure of this species, examining 178 specimens sampled on a fine scale, using information from chromosomal variability, mitochondrial DNA control region and cytochrome c oxidase subunit I sequences, and 14 microsatellite loci. The observed isolation-by-distance pattern and a clinal genetic variation suggest a stepping-stone population model. The results did not indicate genetic structuring associated with distinct karyotypes. However, mitochondrial and nuclear molecular markers demonstrated the existence of 2 demes, which are not completely isolated but are probably reinforced by a geographical barrier. The vulnerability of C. lami is greater than previously supposed, and our data support the designation of one Evolutionary Significant Unit and one Management Unit, and also the inclusion of this species' conservation status as vulnerable.

Sequence variation in the melanocortin-1 receptor (MC1R) pigmentation gene and its role in the cryptic coloration of two South American sand lizards.

In reptiles, dorsal body darkness often varies with substrate color or temperature environment, and is generally presumed to be an adaptation for crypsis or thermoregulation. However, the genetic basis of pigmentation is poorly known in this group. In this study we analyzed the coding region of the melanocortin-1-receptor (MC1R) gene, and therefore its role underlying the dorsal color variation in two sympatric species of sand lizards (Liolaemus) that inhabit the southeastern coast of South America: L. occipitalis and L. arambarensis. The first is light-colored and occupies aeolic pale sand dunes, while the second is brownish and lives in a darker sandy habitat. We sequenced 630 base pairs of MC1R in both species. In total, 12 nucleotide polymorphisms were observed, and four amino acid replacement sites, but none of them could be associated with a color pattern. Comparative analysis indicated that these taxa are monomorphic for amino acid sites that were previously identified as functionally important in other reptiles. Thus, our results indicate that MC1R is not involved in the pigmentation pattern observed in Liolaemus lizards. Therefore, structural differences in other genes, such as ASIP, or variation in regulatory regions of MC1R may be responsible for this variation. Alternatively, the phenotypic differences observed might be a consequence of non-genetic factors, such as thermoregulatory mechanisms.

A hybrid zone of the genus Ctenomys: A case study in southern Brazil.

We describe variation at microsatellite loci and the chromosomal polymorphisms of a hybrid population, and hybridizing populations of Ctenomys minutus (the minor tuco-tuco) from the coastal plain of Rio Grande do Sul, southern Brazil. Cytogenetic analysis and a survey of six microsatellite loci included 101 specimens of C. minutus from the parental populations (2n/AN = 42/74 and 48a/76) and their contact zone. Cytogenetic analysis recorded 26 different karyotypes exhibited by 50 individuals from the hybrid population. Of the 26 karyotypes, only 14% presented a parental-like configuration, and none had the combinations of 2n and AN expected for an F1 hybrid. The remaining karyotypes were alternative hybrid forms, with 2n varying from 42 to 46 and AN from 68 to 80. These results suggest chromosomal rearrangements are only of minor significance in the establishment of reproductive barriers for this species.

Evaluation of genetic variability in the collared peccary Pecari tajacu and the white-lipped peccary Tayassu pecari by microsatellite markers.

In this study, the microsatellite technique was used to evaluate the genetic variability in populations of collared and white-lipped peccaries kept in captivity. Six primers developed for domestic pigs were used and amplified in both species. They revealed the presence of five polymorphic loci and one monomorphic locus. The polymorphic loci included 4 of the 16 alleles in collared peccaries, and 3 of the 10 alleles in the white-lipped peccaries. Polymorphic information content (PIC) in both species and all the loci was highly informative. The probability of paternity exclusion (PEC), if one of the parents is known, was almost as high in white-lipped peccaries (95.53%) as in the collared (99,48%). The Fst values for collared (0.042) and white-lipped (0.1387) peccaries showed that both populations are not structured. The Fis values for all loci, except ACTG2 in white-lipped peccaries (-0.0275) and in both species (0.1985 to 0.9284 in collared peccaries and 0.3621 to 0.4754 in the white-lipped), revealed a high level of homozygosis, probably caused by inbreeding. Data on heterologous amplification and genetic variability in collared and white-lipped peccaries are presented for the first time.

Karyotypic and molecular polymorphisms in Ctenomys torquatus (Rodentia: Ctenomyidae): taxonomic considerations.

The rodent genus Ctenomys (tuco-tucos) comprises more than 60 described species, and shows extraordinary inter- and intraspecific karyotypic variation. The most widely distributed species of Ctenomys in Brazil is C. torquatus. Although several cytogenetic studies have been done, the karyotypic variability of this species is still poorly known. In this paper we report two new diploid numbers for C. torquatus: 2n = 40 and 2n = 42, both showing AN = 72. The new distribution limits of C. torquatus here reported include localities in the southern, central and western parts of Rio Grande do Sul (RS) State in southern Brazil. The phylogenetic relationship between C. torquatus from Alegrete, RS, and Ctenomys sp. from Corrientes, Argentina, is described by means of mtDNA cytochrome b analysis. Although both entities share similar karyotypes and sperm morphology, these two species are not phylogenetically close.

New karyotypes and some considerations about the chromosomal diversification of Ctenomys minutus (Rodentia: Ctenomyidae) on the coastal plain of the Brazilian state of Rio Grande do Sul.

The dominant mammals occupying the subterranean niche in South America are rodents of the genus Ctenomys, which form a large group of 56 species with chromosome numbers ranging from 2n = 10 to 70. In southern Brazil, Ctenomys minutus is the species with the widest geographic distribution, inhabiting sandy fields and dunes extending from Jaguaruna beach in the state of Santa Catarina to the town of São José do Norte in the state of Rio Grande do Sul. Eleven karyotypes (2n = 42; 2n = 46a; 2n = 46b; 2n = 47a; 2n = 47b; 2n = 48a; 2n = 48b; 2n = 49a; 2n = 49b; 2n = 50a and 2n = 50b) were described for this species and zones of hybridization are also known. A sample of 51 C. minutus specimens was collected from five sampling sites about 20km apart along the coastal plain of Rio Grande do Sul between the municipalities of Tavares (31 degrees 23'S 51 degrees 09'W) and São José do Norte (31 degrees 52'S 51 degrees 54'W). We were able to extend the known geographic distribution of C. minutus by 90 km, from Tavares southwards to São José do Norte. During our study we found five karyotypes (2n = 46b, 47b, 48b, 49b and 50b), four of which (2n = 47b, 48b, 49b and 50b) have not previously been described for this species.

Comet assay using mullet (Mugil sp.) and sea catfish (Netuma sp.) erythrocytes for the detection of genotoxic pollutants in aquatic environment.

The development of comet assay for aquatic organisms is of particular relevance in light of the importance of coastal fisheries to several countries around the world. Two of the most common fish species native to southern Brazil are the gray mullet (Mugil sp.) and sea catfish (Netuma sp.) for which we have produced a standardized comet assay using whole erythrocytes taken from samples of these fish. We investigated the potential of the comet assay for monitoring genotoxicity in mullet and sea catfish and made a preliminary investigation of the baseline levels of DNA damage in the erythrocytes of samples of these fish from non-polluted areas as well as assessing the in vitro sensitivity of erythrocyte exposed to 2, 4 and 8 x 10(-5) M of methyl methanesulfonate (MMS) for 1, 2, 6 and 24h at 25 and 37 degrees C. Our results show that there was an increase in baseline DNA damage at higher temperatures and that the amount of MMS-induced DNA damage also increased at higher temperatures and that there was a clear dose/time response to treatment with MMS. To assess the possibility of using fish for environmental biomonitoring we also used the comet assay to investigate the in vitro genotoxic effect of MMS on whole blood cells from human donors and found a clear concentration-related effect at all exposure times, findings which agree with those of other workers. This study demonstrates the potential application of the comet assay to erythrocytes of mullets and sea catfish. However, these findings also suggest that temperature could alter both baseline DNA damage in untreated animals and in vitro cell sensitivity towards genotoxic pollutants.

Genetic and demographic structure in a population of Ctenomys lami (Rodentia-Ctenomyidae).

The variability at eight microsatellite loci was analyzed in 88 individuals of Ctenomys lami, a species of tuco-tucos. Initially considered as three subpopulations, the estimated Fst values ranged from 0.0007 to 0.0589. Additionally, a Bayesian approach presented probabilities of 0.9933 for the existence of only one population. A Fis value of 0.19 could indicate an incipient structure. Sexual dimorphism was observed. The reproductive period was short and a seasonal pattern was observed with sex ratio of 1:1 during all seasons. Presence of recent bottleneck is described suggesting further research in this population to assess more complete and detailed data.

Genotoxicity biomonitoring in regions exposed to vehicle emissions using the comet assay and the micronucleus test in native rodent Ctenomys minutus.

Exposure to motor vehicle emissions represents an important concern for possible long-term health effects. The present report describes: 1) the application and verification of the alkaline comet assay in Ctenomys minutus to detect the possible genotoxicity of automobile emissions; 2) a comparison of the comet assay results with peripheral blood micronucleus (MN) assay results performed in the same animals; and 3) the identification of agents involved in the responses and in the seasonal variation of the effects. Ctenomys minutus (Octodontidae-Rodentia) were captured in two different fields from both sides of RS/030, a highway on the coastal plain of the Brazilian state of Rio Grande do Sul. Reference animals were obtained from a nearby field that was about 3 km distant from any road. By the end of this study, 123 rodents (73 females and 50 males) were live-trapped. Our results indicate that there was an increase in cells with DNA damage for C. minutus environmentally exposed to automobile emissions, as demonstrated by the alkaline comet assay, but there was no increase in micronucleated cells. The alkaline comet assay showed age and gender differences in the response. The comet assay results suggest that adult females are the principal population affected by air pollutants from vehicle emissions. Chemical data were also collected from areas exposed to automobile exhaust and these indicated that elevated levels of hydrocarbons, metals, and NO(2) were associated with the elevated levels of damaged cells observed in the wild rodent C. minutus. Our results agree with previous data on engine and fuel components, where weak increases in damage for native rodents exposed to emissions have been observed. Other larger, controlled studies are needed to better understand how the metabolism of C. minutus affects its response to emission exposure.