Genetic data are currently providing a large amount of new information on past distribution of species and are contributing to a new vision of Pleistocene ice ages. Nonetheless, an increasing number of studies on the 'time dependency' of mutation rates suggest that date assessments for evolutionary events of the Pleistocene might be overestimated. We analysed mitochondrial (mt) DNA (COI) sequence variation in 225 Parnassius mnemosyne individuals sampled across central and eastern Europe in order to assess (i) the existence of genetic signatures of Pleistocene climate shifts; and (ii) the timescale of demographic and evolutionary events. Our analyses reveal a phylogeographical pattern markedly influenced by the Pleistocene/Holocene climate shifts. Eastern Alpine and Balkan populations display comparatively high mtDNA diversity, suggesting multiple glacial refugia. On the other hand, three widely distributed and spatially segregated lineages occupy most of northern and eastern Europe, indicating postglacial recolonization from different refugial areas. We show that a conventional 'phylogenetic' substitution rate cannot account for the present distribution of genetic variation in this species, and we combine phylogeographical pattern and palaeoecological information in order to determine a suitable intraspecific rate through a Bayesian coalescent approach. We argue that our calibrated 'time-dependent' rate (0.096 substitutions/ million years), offers the most convincing time frame for the evolutionary events inferred from sequence data. When scaled by the new rate, estimates of divergence between Balkan and Alpine lineages point to c. 19 000 years before present (last glacial maximum), and parameters of demographic expansion for northern lineages are consistent with postglacial warming (5-11 000 years before present).

Phylogeographic studies of highly mobile large carnivores suggest that intra-specific genetic differentiation of modern species might be the consequence of the most recent Pleistocene glaciation. However, the relative influence of biogeographical processes and subsequent humaninduced population fragmentation requires a better understanding. Poland represents the western edge of relatively continuous distributions of many wide-ranging species, e.g. lynx (Lynx lynx), wolves (Canis lupus), moose (Alces alces) and, therefore, a key area for understanding historic and contemporary patterns of gene flow in central Europe. We examined wolf genetic structure in Poland and in a recently recolonized area in eastern Germany using microsatellite profiles (n = 457) and mitochondrial DNA sequencing (mtDNA, n = 333) from faecal samples. We found significant genetic structure and high levels of differentiation between wolves in the Carpathian Mountains and the Polish lowlands. Our findings are consistent with previously reported mtDNA subdivision between northern lowlands and southern mountains, and add new and concordant findings based on autosomal marker variation. Wolves in western Poland and eastern Germany showed limited differentiation from northeastern Poland. Although the presence of private alleles suggests immigration also from areas not sampled in this study, most individuals seem to be immigrants from northeastern Poland or their descendants. We observed moderate genetic differentiation between certain northeastern lowland regions separated by less than 50 km. Moreover, mtDNA results indicated a southeastern subpopulation near the border with Ukraine. The observed structure might reflect landscape fragmentation and/orecological differences resulting in natal habitat-biased dispersal.

Hybridisation between a domesticated species and its wild ancestor is an important conservation problem, especially if it results in the introgression of domestic gene variants into wild species. Nevertheless, the legal status of hybrids remains unregulated, partially because of the limited understanding of the hybridisation process and its consequences. The occurrence of hybridisation between grey wolves and domestic dogs is well documented from different parts of the wolf geographic range, but little is known about the frequency of hybridisation events, their causes and the genetic impact on wolf populations. We analysed 61K SNPs spanning the canid genome in wolves from across Eurasia and North America and compared that data to similar data from dogs to identify signatures of admixture. The haplotype block analysis, which included 38 autosomes and the X chromosome, indicated the presence of individuals of mixed wolf–dog ancestry in most Eurasian wolf populations, but less admixture was present in North American populations. We found evidence for male‐biased introgression of dog alleles into wolf populations, but also identified a first‐generation hybrid resulting from mating between a female dog and a male wolf. We found small blocks of dog ancestry in the genomes of 62% Eurasian wolves studied and melanistic individuals with no signs of recent admixed ancestry, but with a dog‐derived allele at a locus linked to melanism. Consequently, these results suggest that hybridisation has been occurring in different parts of Eurasia on multiple timescales and is not solely a recent phenomenon. Nevertheless, wolf populations have maintained genetic differentiation from dogs, suggesting that hybridisation at a low frequency does not diminish distinctiveness of the wolf gene pool. However, increased hybridisation frequency may be detrimental for wolf populations, stressing the need for genetic monitoring to assess the frequency and distribution of individuals resulting from recent admixture.

1. The Eurasian lynx Lynx lynx occupies a variety of environmental and climatic conditions, and the majority of present-day European populations have either recovered from severe demographic bottlenecks, or are living in fragmented habitat. These factors may have affected the genetic variability of lynx populations. We summarize available data on genetics, population status and ecology of these felids to shed light on the pattern and mechanisms behind their genetic variability and population differentiation in Europe. 2.Genetic studies conducted so far, based on mtDNA and microsatellites, have shown that the Eurasian lynx has low to moderate genetic variability. Variability is lowest in the north (Scandinavian bottlenecked population), but is also low in the Carpathian region. A trend towards loss of genetic variation has been noted in fragmented and reintroduced populations. Genetically, the populations are highly differentiated from each other. 3. There are clear relationships between the pattern of lynx genetic variability, differentiation between the populations, and such factors as population history (demographic bottlenecks), social interactions and habitat fragmentation. The genetic divergence between lynx populations is also strongly correlated with the depth and duration of snow cover. 4. Our review provides evidence that the lynx is undergoing significant genetic differentiation, due to several factors. To enable better planning of conservation programmes for the Eurasian lynx, researchers should identify the Evolutionarily Significant Units among its populations, using different classes of molecular markers.

Endangered species worldwide exist in remnant populations, often within fragmented landscapes. Although assessment of genetic diversity in fragmented habitats is very important for conservation purposes, it is usually impossible to evaluate the amount of diversity that has actually been lost. Here, we compared population structure and levels of genetic diversity within populations of spotted suslik Spermophilus suslicus, inhabiting two different parts of the species range characterized by different levels of habitat connectivity. We used microsatellites to analyze 10 critically endangered populations located at the western part of the range, where suslik habitat have been severely devastated due to agriculture industrialization. Their genetic composition was compared with four populations from the eastern part of the range where the species still occupies habitat with reasonable levels of connectivity. In the western region, we detected extreme population structure (F ST = 0.20) and levels of genetic diversity (Allelic richness ranged from 1.45 to 3.07) characteristic for highly endangered populations. Alternatively, in the eastern region we found significantly higher allelic richness (from 5.09 to 5.81) and insignificant population structure (F ST = 0.03). As we identified a strong correlation between genetic and geographic distance and a lack of private alleles in the western region, we conclude that extreme population structure and lower genetic diversity is due to recent habitat loss. Results from this study provide guidelines for conservation and management of this highly endangered species.

Large terrestrial carnivores are particularly prone to factors constraining levels of population genetic diversity because of their low densities and high spatial requirements. We studied the pattern of Eurasian lynx Lynx lynx population genetic variability in the westernmost part of its natural range from Scandinavia to the Carpathian Mountains (north-central Europe) based on 190 samples using 613 base pair-long sequences from the mitochondrial DNA control region (mtDNAcr). We examined whether the population history or contemporary habitat constraints of this large and mobile carnivore could have significantly affected its genetic structure. We recorded nine mtDNA haplotypes, including five not previously reported. Lynx from Latvia and Estonia had the highest variability with haplotype and nucleotide diversities of 0.81-0.88% and 0.44-0.47%, respectively. In contrast, there was no polymorphism present in peripheral populations from Norway and the Carpathian Mountains. Lynx populations were strongly differentiated [analysis of molecular variance (AMOVA): FST = 0.570, P < 0.001, FST = 0.464, P < 0.001)]. Spatial Analysis of Molecular Variance identified four separate groups of populations: (1) Norway, Finland and Estonia; (2) Latvia and North Eastern Poland; (3) the Białowieża Primeval Forest; (4) the Carpathians. The patterns of genetic diversity and differentiation suggested a number of discrete populations that are poorly connected by contemporary gene flow and could therefore be considered demographically independent. The peripheral location of these populations, habitat fragmentation and the strict territorial structure of lynx populations are factors likely contributing to the observed patterns. The study provides suggestions for active conservation/management decisions including translocations or reintroductions of lynx. bs_bs_banner Animal Conservation. Print ISSN 1367-9430 Animal Conservation 15 (2012) 603-612

Background The Shannon diversity index has been widely used in population genetics studies. Recently, it was proposed as a unifying measure of diversity at different levels—from genes and populations to whole species and ecosystems. The index, however, was proven to be negatively biased at small sample sizes. Modifications to the original Shannon’s formula have been proposed to obtain an unbiased estimator. Methods In this study, the performance of four different estimators of Shannon index—the original Shannon’s formula and those of Zahl, Chao and Shen and Chao et al.—was tested on simulated microsatellite data. Both the simulation and analysis of the results were performed in the R language environment. A new R function was created for the calculation of all four indices from the genind data format. Results Sample size dependence was detected in all the estimators analysed; however, the deviation from parametric values was substantially smaller in the derived measures than in the original Shannon’s formula. Error rate was negatively associated with population heterozygosity. Comparisons among loci showed that fast-mutating loci were less affected by the error, except for the original Shannon’s estimator which, in the smallest sample, was more strongly affected by loci with a higher number of alleles. The Zahl and Chao et al. estimators performed notably better than the original Shannon’s formula. Conclusion The results of this study show that the original Shannon index should no longer be used as a measure of genetic diversity and should be replaced by Zahl’s unbiased estimator.

The initiation of adaptive immune responses to protein antigens has to be preceded by their uptake by antigen presenting cells and intracellular proteolytic processing. Paradoxically, endocytic receptors involved in antigen uptake do not bind the majority of proteins, which may be the main reason why purified proteins stimulate at most weak immune responses. A shared feature of different types of adjuvants, capable of boosting immunogenicity of protein vaccines, is their ability to induce acute inflammation, characterized by early influx of activated neutrophils. Neutrophils are also rapidly recruited to sites of tissue injury or infection. These cells are the source of potent oxidants, including hypochlorous acid (HOCl), causing oxidation of proteins present in inflammatory foci. We demonstrate that oxidation of proteins by endogenous, neutrophils-derived HOCl increases their immunogenicity. Upon oxidation, different, randomly chosen simple proteins (yeast alcohol dehydrogenase, human and bovine serum albumin) and glycoproteins (human apo-transferrin, ovalbumin) gain the ability to bind with high affinity to several endocytic receptors on antigen presenting cells, which seems to be the major mechanism of their increased immunogenicity. The mannose receptor (CD206), scavenger receptors A (CD204) and CD36 were responsible for the uptake and presentation of HOCl-modified proteins by murine dendritic cells and macrophages. Other scavenger receptors, SREC-I and LOX-1, as well as RAGE were also able to bind HOCl-modified proteins, but they did not contribute significantly to these ligands uptake by dendritic cells because they were either not expressed or exhibited preference for more heavily oxidised proteins. Our results indicate that oxidation by neutrophils-derived HOCl may be a physiological mechanism of conferring immunogenicity on proteins which in their native forms do not bind to endocytic receptors. This mechanism might enable the immune system to detect infections caused by pathogens not recognized by pattern recognition receptors.