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.

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.

Ancestry analysis of locally adapted Crespa goats from southernmost Brazil.

Crespa goats are phenotypically similar to the Angora breed, and are traditionally reared in small, low-tech farms in southernmost Brazil. Whether they represent degenerated remnants of pure Angora goats or result from foreign breeds introduced during colonial times and recently mixed with commercial breeds is unknown. Since the degree of relatedness of Crespa in relation to other goats is completely unknown, we performed a comparative assessment of the genetic similarity between Crespa and foreign commercial breeds reared in the region (Angora, Alpine, Anglo-Nubian, Boer, and Saanen), particularly the Angora. We used 11 microsatellites to score alleles in 148 individuals and performed a Bayesian assignment test, which revealed six clusters (K = 6; Ln likelihood = -5047.6). In addition, a segment of the mitochondrial DNA (mtDNA) control region was sequenced to investigate the relatedness of Crespa goats to Portuguese autochthonous breeds (Algarvia, Bravia, Charnequeira, Serpentina, and Serrana). The origin of the Crespa breed could not be ascertained from the mtDNA, but it does not only descend from the Angora. It is probably related to other introduced and autochthonous Portuguese breeds, in particular the Algarvia. Therefore, our results indicate that this distinctive source of genetic diversity is partly a remnant of animals that were introduced during the colonial period. By recognizing it as genetically distinct, we provide further support for the protection of this particular gene pool.

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.

Ontogenetic allometry in the foot size of Oligoryzomys flavescens (Waterhouse, 1837) (Rodentia, Sigmodontinae).

Ontogenetic allometry is the study of how the size or shape of certain structures changes over the course of an animal's development. In this study, using Huxley's formula of allometric growth (1932), we assessed the changes in the rate of growth of the feet size of the sigmodontine rodent Oligoryzomys flavescens during its ontogeny and compared differences between males and females. We find evidence of a change of polarity during the ontogenetic development of the species, with the presence of positive allometry during pregnancy and negative allometry in adulthood. Moreover, we note the presence of sexual dimorphism in the size of the feet, in which males of the species have a higher rate of growth than females. This growth pattern is positively related to escape from predators in childhood in both sexes and, in adulthood, provides a higher encounter rate of females by males, due to the larger displacement of the latter. We suggest that both the forces of natural selection and sexual selection have acted to shape the evolution of foot size in this species.

Insights about the genetic diversity and population structure of an offshore group of common bottlenose dolphins (Tursiops truncatus) in the Mid-Atlantic.

Although the genus Tursiops has a worldwide distribution and is globally well-studied, some dolphin populations continue to face high risks of decline. Hence, it is necessary to assess the genetic diversity and structure of this genus to properly assess its conservation status and to implement appropriate management actions. In Brazil, genetic studies on this group remain rare, particularly for populations inhabiting offshore waters. Saint Peter and Saint Paul Archipelago (SPSPA) is a small group of islands located in the Mid- Atlantic Ridge, where recent studies of the Tursiops truncatus group indicate that individuals are resident throughout the year around the archipelago, exhibiting considerable site fidelity. A previous study with this group indicated that the individuals form an isolated population. To test this hypothesis, and describe the genetic diversity of SPSPA individuals, we assessed 12 microsatellite loci and a portion of the mitochondrial control region. Bayesian analysis revealed that SPSPA bottlenose dolphins form a unique population. In a phylogeographic perspective, we found that individuals from SPSPA shared mtDNA haplotypes with inshore and offshore individuals from North Atlantic, suggesting that they are not currently isolated from their conspecifics. Mirroring mtDNA findings, microsatellite analysis revealed that most of the pairs of individuals sampled seem to be unrelated (83.8%) and no indication of inbreeding, what would be expected if a small population such as SPSPA was reproductively isolated.

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.

DNA-based and geometric morphometric analysis to validate species designation: a case study of the subterranean rodent Ctenomys bicolor.

The genus Ctenomys (Rodentia: Ctenomyidae) shows several taxonomic inconsistencies. In this study, we used an integrative approach including DNA sequences, karyotypes, and geometric morphometrics to evaluate the taxonomic validity of a nominal species, Ctenomys bicolor, which was described based on only one specimen in 1912 by Miranda Ribeiro, and since then neglected. We sampled near the type locality assigned to this species and collected 10 specimens. A total of 820 base pairs of the cytochrome b gene were sequenced and analyzed together with nine other species and four morphotypes obtained from GenBank. Bayesian analyses showed that C. bicolor is monophyletic and related to the Bolivian-Matogrossense group, a clade that originated about 3 mya. We compared the cranial shape through morphometric geometrics of C. bicolor, including the specimen originally sampled in 1912, with other species representative of the same phylogenetic group (C. boliviensis and C. steinbachi). C. bicolor shows unique skull traits that distinguish it from all other currently known taxa. Our findings confirm that the specimen collected by Miranda Ribeiro is a valid species, and improve the knowledge about Ctenomys in the Amazon region.

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.

Penial morphology in three species of Brazilian tuco-tucos, Ctenomys torquatus, C. minutus, and C. flamarioni (Rodentia: Ctenomyidae).

The present study analyses the glans penis and baculum morphology of three Brazilian tuco-tucos, Ctenomys torquatus Lichtenstein, 1830, Ctenomys minutus Nehring, 1887 and Ctenomys flamarioni Travi, 1981, in order to identify possible variations and understand some more about this taxonomically complex group. We used fixed penis from 15 previously listed adult specimens. For a more detailed baculum analysis, the penis underwent dissection and diaphanisation, whereas to analyse the glans penis surface we used Scanning Electron Microscopy (SEM). Results showed striking differences in baculum morphology among the three species. While C. minutus have a particular V-shaped proximal baculum tip, C. flamarioni baculum is thin throughout the shaft with rounded proximal and distal tips. Ctenomys torquatus have a shorter and larger baculum, similar to what has previously been described for the species. Glans penis surface microstructure analyses also revealed inter-specific differences, with penial spines varying in shape, size and, especially density. Although C. torquatus has a relatively small penis, it has the largest penial spine density, which suggests a more complex penial ornamentation in this species.

Molecular evolution of the melanocortin 1-receptor pigmentation gene in rodents.

Adaptive variation in the melanocortin 1-receptor gene (MC1R), a key locus in melanogenesis, has been identified in some species of rodents. However, in others, MC1R has no causative role in pigmentation phenotypes despite their coat color variation. In this study, we characterized the rates and patterns of MC1R nucleotide and amino acid sequence evolution and, particularly, selective pressures in the separated domains of the protein using a comparative analysis of 43 species representing three major lineages of rodents with variable coat colors. We found high amino acid variation (44% of sites) throughout the protein. Most substitutions were observed in extracellular and transmembrane domains; the intracellular segment was conserved across species. Pairwise non-synonymous substitutions did not vary significantly in different domains among the rodent lineages - i.e., variation was not associated with phylogenetic distance. Phylogeny-based likelihood analysis suggested that purifying selection has mostly shaped the evolutionary course of MC1R. However, a high proportion of sites (27%) were under relaxation of functional constraints (ω = 0.38), and four sites (3, 14, 26, and 251) clearly evolved under positive selection (ω ≅ 2.9). Thus, our data indicate a high proportion of sites evolving under relaxed evolutionary constraints, which might indicate the evolvability of the system in the generation of adaptive changes in specific taxa in rodent lineages.

Identification of the e allele at the Extension locus (MC1R) in Brazilian Creole sheep and its role in wool color variation.

The melanocortin 1 receptor (MC1R) gene has been described as responsible for the black color in some breeds of sheep, but little is known about its function in many colored breeds, particularly those with a wide range of pigmentation phenotypes. The Brazilian Creole is a local breed of sheep from southern Brazil that has a wide variety of wool colors. We examined the MC1R gene (Extension locus) to search for the e allele and determine its role in controlling wool color variation in this breed. One hundred and twenty-five animals, covering the most common Creole sheep phenotypes (black, brown, dark gray, light gray, and white), were sequenced to detect the mutations p.M73K and p.D121N. Besides these two mutations, three other synonymous sites (429, 600, and 725) were found. The dominant allele (E(D): p.73K, and p.121N) was found only in colored animals, whereas the recessive allele (E⁺: p.73M, and p.121D) was homozygous only in white individuals. We concluded that MC1R is involved in the control of wool color in Brazilian Creole sheep, particularly the dark phenotypes, although a second gene may be involved in the expression of the white phenotype in this breed.

Permanent Genetic Resources added to the Molecular Ecology Resources Database 1 February 2010-31 March 2010.

This article documents the addition of 228 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Anser cygnoides, Apodemus flavicollis, Athene noctua, Cercis canadensis, Glis glis, Gubernatrix cristata, Haliotis tuberculata, Helianthus maximiliani, Laricobius nigrinus, Laricobius rubidus, Neoheligmonella granjoni, Nephrops norvegicus, Oenanthe javanica, Paramuricea clavata, Pyrrhura orcesi and Samanea saman. These loci were cross-tested on the following species: Apodemus sylvaticus, Laricobius laticollis and Laricobius osakensis (a proposed new species currently being described).

Mitochondrial and nuclear DNA analyses reveal population differentiation in Brazilian Creole sheep.

Using ND5 sequences from mtDNA and 10 nuclear markers, we investigated the genetic differentiation of two South American Creole sheep phenotypes that historically have been bred in different biomes in southern Brazil. In total, 18 unique mtDNA haplotypes were detected, none of which was shared between the two phenotypes. Bayesian analysis also indicated two different groups (k = 2). Thus, these varieties are supported as being genotypically distinct. This situation could have resulted either from geographical isolation, associated with differences in the cultural habits of sheep farmers and in the way that flocks were managed, or more likely, from the introduction of different stocks four centuries ago.

Inter-species hybridization among Neotropical cats of the genus Leopardus, and evidence for an introgressive hybrid zone between L. geoffroyi and L. tigrinus in southern Brazil.

Natural hybrid zones between distinct species have been reported for many taxa, but so far, few examples involve carnivores or Neotropical mammals in general. In this study, we employed mitochondrial DNA (mtDNA) sequences and nine microsatellite loci to identify and characterize a hybrid zone between two Neotropical felids, Leopardus geoffroyi and L. tigrinus, both of which are well-established species having diverged from each other c. 1 million years ago. These two felids are mostly allopatric throughout their ranges in South America, with a narrow contact zone that includes southern Brazil. We present strong evidence for the occurrence of hybridization between these species and identify at least 14 individuals (most of them originating from the geographical contact zone) exhibiting signs of interspecific genomic introgression. The genetic structure of Brazilian L. tigrinus populations seems to be affected by this introgression process, showing a gradient of differentiation from L. geoffroyi correlated with distance from the contact zone. We also corroborate and extend previous findings of hybridization between L. tigrinus and a third related felid, L. colocolo, leading to an unusual situation for a mammal, in which the former species contains introgressed mtDNA lineages from two distinct taxa in addition to its own.

Genetic diversity in the Pantaneiro horse breed assessed using microsatellite DNA markers.

The genetic variability for a sample of 227 animals from three populations of Pantaneiro horses was estimated using data from 10 microsatellite loci. The number of alleles and the proportion of heterozygosity indicated high variability. A total of 91 alleles were found, with a significantly high mean number of alleles. The mean polymorphic information content was 0.7 and the paternity exclusion probability was 99.3%. The inbreeding coefficient (F(IS)) was low for the three populations: Ipiranga (F(IS) = 0.147), Nova Esperança (F(IS) = 0.094) and Promissão (F(IS) = 0.108). Genetic differentiation among all three populations was low (F(ST) = 0.008 to 0.064). Three methods were used to test for a recent bottleneck effect. The graphical method and the Wilcoxon test using the stepwise mutation model showed no bottleneck pattern for any of the populations. The test by two-phase mutation model showed genetic signatures of bottleneck for Ipiranga and Promissão. When we consider standard deviation value for Nova Esperança, the M-statistic detected a bottleneck pattern, but this result could be explained by a sample size effect. Therefore, there is no immediate cause for concern regarding loss of variation within the breed.

Genetic diversity in the Pantaneiro horse breed assessed using microsatellite DNA markers

The genetic variability for a sample of 227 animals from three populations of Pantaneiro horses was estimated using data from 10 microsatellite loci. The number of alleles and the proportion of heterozygosity indicated high variability. A total of 91 alleles were found, with a significantly high mean number of alleles. The mean polymorphic information content was 0.7 and the paternity exclusion probability was 99.3%. The inbreeding coefficient (Fis) was low for the three populations: Ipiranga (FIS = 0.147), Nova Esperança (Fis = 0.094) and Promissão (Fis = 0.108). Genetic differentiation among all three populations was low (FST = 0.008 to 0.064). Three methods were used to test for a recent bottleneck effect. The graphical method and the Wilcoxon test using the stepwise mutation model showed no bottleneck pattern for any of the populations. The test by two-phase mutation model showed genetic signatures of bottleneck for Ipiranga and Promissão. When we consider standard deviation value for Nova Esperança, the M-statistic detected a bottleneck pattern, but this result could be explained by a sample size effect. Therefore, there is no immediate cause for concern regarding loss of variation within the breed.