Low STR variability in the threatened marsh deer, Blastocerus dichotomus, detected through amplicon sequencing in non-invasive samples.

Blastocerus dichotomus is the largest deer in South America. We have used 25 microsatellite markers detected and genotyped by Next Generation Sequencing to estimate the genetic variability of B. dichotomus in Argentina, where most of its populations are threatened. Primer design was based on the sequence of a shallow partial genome (15,967,456 reads; 16.66% genome coverage, mean depth 1.64) of a single individual. From the thousands of microsatellite loci found, even under high stringency selection, we chose and tested a set of 80 markers on 30 DNA samples extracted from tissue and feces from three Argentinean populations. Heterozygosity levels were low across all loci in all populations (H=0.31 to 0.40). Amplicon sequencing is a fast, easy, and affordable technique that can be very useful for the characterization of microsatellite marker sets for the conservation genetics of non-model organisms. This work is also one of the first ones to use amplicon sequencing in non-invasive samples and represents an important development for the study of threatened species.

Landscape genetics in the subterranean rodent Ctenomys "chasiquensis" associated with highly disturbed habitats from the southeastern Pampas region, Argentina.

Studies of genetic differentiation in fragmented environments help us to identify those landscape features that most affect gene flow and dispersal patterns. Particularly, the assessment of the relative significance of intrinsic biological and environmental factors affecting the genetic structure of populations becomes crucial. In this work, we assess the current dispersal patterns and population structure of Ctenomys "chasiquensis", a vulnerable and endemic subterranean rodent distributed on a small area in Central Argentina, using 9 polymorphic microsatellite loci. We use landscape genetics approaches to assess the relationship between genetic connectivity among populations and environmental attributes. Our analyses show that populations of C. "chasiquensis" are moderately to highly structured at a regional level. This pattern is most likely the outcome of substantial gene flow on the more homogeneous sand dune habitat of the Northwest of its distributional range, in conjunction with an important degree of isolation of eastern and southwestern populations, where the optimal habitat is surrounded by a highly fragmented landscape. Landscape genetics analysis suggests that habitat quality and longitude were the environmental factors most strongly associated with genetic differentiation/uniqueness of populations. In conclusion, our results indicate an important genetic structure in this species, even at a small spatial scale, suggesting that contemporary habitat fragmentation increases population differentiation.

Influence of environmental heterogeneity on the distribution and persistence of a subterranean rodent in a highly unstable landscape.

In this study we combine information from landscape characteristics, demographic inference and species distribution modelling to identify environmental factors that shape the genetic distribution of the fossorial rodent Ctenomys. We sequenced the mtDNA control region and amplified 12 microsatellites from 27 populations distributed across the Iberá wetland ecosystem. Hierarchical Bayesian modelling was used to construct phylogenies and estimate divergence times. We developed species distribution models to determine what climatic variables and soil parameters predicted species presence by comparing the current to the historic and predicted future distribution of the species. Finally, we explore the impact of environmental variables on the genetic structure of Ctenomys based on current and past species distributions. The variables that consistently correlated with the predicted distribution of the species and explained the observed genetic differentiation among populations included the distribution of well-drained sandy soils and temperature seasonality. A core region of stable suitable habitat was identified from the Last Interglacial, which is projected to remain stable into the future. This region is also the most genetically diverse and is currently under strong anthropogenic pressure. Results reveal complex demographic dynamics, which have been in constant change in both time and space, and are likely linked to the evolution of the Paraná River. We suggest that any alteration of soil properties (climatic or anthropic) may significantly impact the availability of suitable habitat and consequently the ability of individuals to disperse. The protection of this core stable habitat is of prime importance given the increasing levels of human disturbance across this wetland system and the threat of climate change.

Chromosome synapsis and recombination in simple and complex chromosomal heterozygotes of tuco-tuco (Ctenomys talarum: Rodentia: Ctenomyidae).

The chromosomal speciation hypothesis suggests that irregularities in synapsis, recombination, and segregation in heterozygotes for chromosome rearrangements may restrict gene flow between karyotypically distinct populations and promote speciation. Ctenomys talarum is a South American subterranean rodent inhabiting the coastal regions of Argentina, whose populations polymorphic for Robertsonian and tandem translocations seem to have a very restricted gene flow. To test if chromosomal differences are involved in isolation among its populations, we examined chromosome pairing, recombination, and meiotic silencing of unsynapsed chromatin in male meiosis of simple and complex translocation heterozygotes using immunolocalization of the MLH1 marking mature recombination nodules and phosphorylated histone γH2A.X marking unrepaired double-strand breaks. We observed small asynaptic areas labeled by γH2A.X in pericentromeric regions of the chromosomes involved in the trivalents and quadrivalents. We also observed a decrease of recombination frequency and a distalization of the crossover distribution in the heterozygotes and metacentric homozygotes compared to acrocentric homozygotes. We suggest that the asynapsis of the pericentromeric regions are unlikely to induce germ cell death and decrease fertility of the heterozygotes; however, suppressed recombination in pericentromeric areas of the multivalents may reduce gene flow between chromosomally different populations of the Talas tuco-tuco.

The evolution of a highly speciose group in a changing environment: are we witnessing speciation in the Iberá wetlands?

Delimiting species is very conflicting in the case of very young taxa that are in the process of diversification, and even more difficult if the species inhabit a heterogeneous environment. In this case, even population delimitation is controversial. The South American genus of subterranean rodents Ctenomys is highly speciose, with 62 species that appeared in the lapse of 3 Myr. Within the genus, the 'perrensi' group, formed by three named species and a group of forms of unknown taxonomic status, inhabits the Iberá wetland, in northern Argentina. Almost every locality shows a particular chromosomal complement. To understand the relationships and the evolutionary process among species and populations, we examined mitochondrial DNA sequences and microsatellite genotypes. We found an isolation-by-distance pattern with evidence of cluster-like behaviour of the system. The mitochondrial DNA network revealed two different groups, separated by one of the main rivers of the region. Clustering methods delimited 12 different populations and five metapopulation lineages that seem to be evolving independently. We found evidence of ancient migration among localities at the centre of the distribution but no signals of current migration among the 12 delimited clusters. Some of the genetic clusters found included localities with different chromosomal numbers, which points to the existence of gene flow despite chromosomal variation. The evolutionary future of these five lineages is controlled by the dynamics of their habitat: if stable, they may become distinct species; otherwise, they may collapse into a hybrid swarm, forming a single evolving metapopulation.

Signals of demographic expansion in Drosophila virilis.

BACKGROUND: The pattern of genetic variation within and among populations of a species is strongly affected by its phylogeographic history. Analyses based on putatively neutral markers provide data from which past events, such as population expansions and colonizations, can be inferred. Drosophila virilis is a cosmopolitan species belonging to the virilis group, where divergence times between different phylads go back to the early Miocene. We analysed mitochondrial DNA sequence variation among 35 Drosophila virilis strains covering the species' range in order to detect demographic events that could be used to understand the present characteristics of the species, as well as its differences from other members of the group. RESULTS: Drosophila virilis showed very low nucleotide diversity with haplotypes distributed in a star-like network, consistent with a recent world-wide exponential expansion possibly associated either with domestication or post-glacial colonization. All analyses point towards a rapid population expansion. Coalescence models support this interpretation. The central haplotype in the network, which could be interpreted as ancestral, is widely distributed and gives no information about the geographical origin of the population expansion. The species showed no geographic structure in the distribution of mitochondrial haplotypes, in contrast to results of a recent microsatellite-based analysis. CONCLUSION: The lack of geographic structure and the star-like topology depicted by the D. virilis haplotypes indicate a pattern of global demographic expansion, probably related to human movements, although this interpretation cannot be distinguished from a selective sweep in the mitochondrial DNA until nuclear sequence data become available. The particular behavioural traits of this species, including weak species-discrimination and intraspecific mate choice exercised by the females, can be understood from this perspective.

A simple method for domestic animal identification in Argentina using PCR-RFLP analysis of cytochrome b gene.

We developed a simple, quick assay in order to discriminate forensic samples among human, and common domestic and livestock species of the Pampean region, Argentina. A mitochondrial cytochrome b fragment amplified with universal primers was separately digested with three restriction enzymes (AluI, HaeIII, and HinfI) and the resulting fragments were resolved through electrophoresis in polyacrylamide gels. This PCR-RFLP method allowed us to identify the target species and worked on degraded samples. The assay was successfully applied in livestock robbery cases in Argentine, and may be useful when attempting a first assessment as to the specific status of a forensic evidence.

Demographic status and genetic tagging of endangered capercaillie in NW Spain.

Counting rare and elusive animals and evaluating their demographic status, are fundamental yet challenging aspects of population ecology and conservation biology. We set out to estimate population size (Nc), genetic effective population size (Ne gen), sex ratio, and movements based on genetic tagging for the threatened Cantabrian capercaillie. We used 9 microsatellite loci to genotype 134 droppings collected at 34 display areas during the breeding season. Using genetic capture-mark-recapture, we estimated 93 individuals (Nc, 95% CI: 70-116) in an area of about 500 km2, with sex ratio biased towards males (1∶1.6). Estimated Ne gen (35.5) was 38% of Nc, notably higher than the published average in wild populations. This capercaillie population is small and well within concern in terms of population viability. By genetic tagging, we detected mostly short movements; just a few males were recaptured between contiguous display areas. Non-invasive surveys of endangered populations have a great potential, yet adequate sample size and location are key to obtain reliable information on conservation status.

Studies of mitochondrial DNA, allozyme and morphometric variation in a house mouse hybrid zone.

An unusual chromosomal hybrid zone of the house mouse, Mus musculus domesticus, exists in Upper Valtellina, Northern Italy, consisting of four Robertsonian (Rb) races and the standard (all-acrocentric, or 2n = 40) race, all hybridizing freely within 10 km2. The hybrid zone in Valtellina provides an excellent opportunity to study the role of Rb fusions in reproductive isolation and speciation. This hybrid zone has already been well studied for the distribution of Rb fusions and the fertility of hybrids, but in order to understand the dynamics of the zone, a basic understanding of the origin and genetic similarity of the chromosomal races is necessary. This paper presents the results of three different methods of measuring genetic differentiation: multivariate analysis of morphological traits and analyses of allozyme variation and mitochondrial DNA sequences. The standard race is clearly distinguishable from the three Rb races by all three methods, but the Rb races are not distinguishable from one another. This provides strong evidence for our previous suggestions that the well-established Rb races in Valtellina are closely related, and that the standard race was introduced into the valley more recently from a distant source. The fact that the Rb races are indistinguishable is also consistent with our hypothesis that a within-village speciation event involving two of the races (Hauffe & Searle, 1992) was a recent occurrence. The low level of allozyme heterozygosity among the Rb races suggests that these populations are the products of at least one bottleneck. The present article substantially extends earlier studies and provides the first detailed morphological and molecular analysis of this complex hybrid zone.

Mitochondrial variability in the D-loop of four equine breeds shown by PCR-SSCP analysis

A fragment of 466 base pairs from a highly variable peripheral region of the mitochondrial D-loop of horses was amplified and analyzed by single stranded conformational polymorphism (SSCP). Fourteen distinct SSCP variants were detected in 100 horses belonging to four breeds (Arabian, ARB; Thoroughbred, TB; Argentinian Creole, ARC; and Peruvian Paso from Argentina, PPA). Each breed showed four to eight SSCP variants, many of which were shared between two or three of the studied breeds. Arabian horses were the most variable (eigth variants), with three variants unique to the breed. PPA and ARC showed two and one characteristic SSCP variants, respectively, while TB shared all its variants with at least one of the other breeds. An analysis based on the presence/absence of the variants revealed a closer relationship between PPA and TB, which was not completely unexpected considering the mixed ancestry of the PPA mares. The results also confirm the efficiency of SSCP to detect variability in horse mitochondrial DNA