The brown bear was formerly widespread, but, during the last few centuries strongly declined and was eradicated almost everywhere in Europe, mainly in consequence of direct persecution and habitat loss. Bears survive in fragmented populations restricted to mountain ranges in the Cantabrian, Pyrenees and in Italy. In the Apennines there are about 100 bears, but the exact number is uncertain, and in the Alps there are 15 bears, which are sustained by an ongoing reintroduction project.

The phylogeography of the brown bear has been described in a number of papers [5,9,10]. In Europe there are two main mtDNA lineages: the eastern and the western mtDNA clades, which differ by ca. 7% mtDNA sequence divergence. The western lineage includes two clades, corresponding to the Iberian and Balkan refugia. The estimated mtDNA divergence time is about 800 000 years, which is older than postulated times of glacial population isolation. Therefore, it is likely that current brown bear phylogeographic structure is due to stochastic lineage sorting of pre-existing haplotypes. Leonard et al. [11] sequenced mtDNA extracted from permafrost-preserved brown bears, 14 000 to 42 000 yrBP. These authors found that three clades (II, III and IV), which now are separated into three different geographical areas, were present in the same populations in Alaska 35 000–45 000 yrBP. These data suggest that current phylogeographic structure of brown bear populations in North America is recent, and has been produced by lineage sorting after the LGM, and not by separate invasion across Beringia of pre-existing differentiated populations.

Small populations of brown bear in Europe are mostly monomorphic at their mtDNA. Kohn et al. [10] found that 23 over 28 distinct mtDNA haplotypes were fixed in only one locality. The current phylogeographic structure allows inferring two main post-glacial colonization routes in Europe: brown bears from the Iberian refuge dispersed to central Europe and reached southern Scandinavia; brown bears from south-eastern Europe dispersed northward and colonized northeastern Scandinavia. Therefore, the Alps and the Balkans were barriers to the dispersal of brown bears. The Scandinavian brown bear contact zone is maintained only by limited female dispersal. Male dispersal produced a moderate gene flow across the contact zone, which has been identified by microsatellite markers [12]. Isolation and longlasting demographic declines likely reduced the genetic diversity of Italian brown bears, which showed a mtDNA haplotype closely related to the Croatian brown bears mtDNAs, and reduced diversity at microsatellite markers. Genetic and demographic data suggest distinct conservation strategies for the Alpine and Apennine brown bear populations. The Apennine brown bears could be managed as a single unit, avoiding any restocking and supporting current trends of population expansion towards other neighbouring areas. The Alpine brown bear are geographically and genetically contiguous to the Croatian (plus other Balkan) populations. Alpine and contiguous brown bears should be managed as a conservation unit, by supportive reintroduction in the Alps, and by supporting the natural population expansion from Slovenia and Croatia. Reintroduced brown bears in the Alps and population fragments in the Apennines, are currently monitored by non-invasive genetics, using DNA obtained from scats or by hair-trapping.

Wolves were widespread and presumably distributed almost everywhere in Eurasia throughout the Holocene [13]. However, direct human persecution, deforestation and decrease of natural prey lead wolf populations in Europe to strongly decline during the last two centuries. After WWII wolves survived in scattered and isolated populations in Iberia, central Apennines, Greece and Finland, while populations in western Europe did not decline so strongly. Wolves were eradicated from the Alps in the 1920s and continued to decline until the 1970s, when about 100 individuals survived in the Apennines. From the 1980s wolves expanded northward along the Apennine ridge and recolonized parts of their historical range. Wolves crossed the northwestern Apennines and reached the southwestern Alps in 1992.

Wolves have no clear phylogeographic structure in America or in Eurasia. In fact, mtDNA CR sequences [7,14] showed that many populations have unique mtDNA haplotypes, but phylogenetic trees and parsimony networks have no structure, suggesting historically high rates of gene flow and worldwide population admixture. Some mtDNA haplotypes have restricted geographical distributions, likely due to the effects of recent, human-induced, population fragmentation and bottlenecks. Field observations, demographic and genetic studies concordant suggest that wolves may disperse rapidly over long distances. However, at smaller geographical scale, landscape features, prey availability and human-induced habitat disturbance may limit the rates of gene flow and induce population structuring. In northwestern Canada, wolves are strictly territorial or migratory, accordingly to the behaviour of their predominant prey, the moose or the caribou, and the Mackenzie River is a barrier to gene flow [15]. Italian wolves show a unique mtDNA CR haplotype, which is monomorphic in the population and different from any other mtDNA haplotype identified in any other wolf population and dog breed, so far. Analyses of microsatellite data [16] suggest that the Italian wolf population is sharply distinct from dogs and other wolf populations in Europe. A Bayesian analysis of past population dynamics [17] suggested that Italian wolves strongly declined and their effective population size (N_e) was reduced 1000-fold in the past 20 000–30 000 years. Demographic declines were comparatively much lower and recent in other central and northern European wolf populations (i.e., Bulgaria, northeastern Europe), which showed 10 to 100-fold reductions of N_e in the past 200–2000 years. The population decline of Italian wolves could have been caused by ancient events at LGM, while demographic fluctuations in other European populations appear to be much more recent. Wolves could have been isolated in Italy south of the River Po Valley during the LGM, when glaciated Alps, the Po and extensive wetlands in the Po Delta region, could have prevented any connection and gene flow with other continental wolf population.

Hybridization with free-ranging dogs is thought to threat the genetic integrity of wolves in Europe. However, genetic data (mainly mtDNA sequences) published in the recent past failed to detect any admixed individual in the extensively studied Italian and Spanish wolves [7,14]. A few cases of dog × wolf hybrids were observed in nature or detected by DNA analyses elsewhere in Europe [7,14]. Randi and Lucchini ([16], and additional unpublished data) reported results of population assignment and genetic admixture analyses in wild-living Italian (and other Eurasian) wolves, dogs and captive-reared wolves of hybrid origins, which were genotyped at 18 microsatellites, and were a-priori identified using non-genetic information. While most of the Italian wolves showed the usual coat colour pattern and “standard” phenotypic traits and body size, a few wild-living animals showed unusually dark coats (“black wolves”), or a vestigial first toe with spur in their hindlegs (“fifth finger wolves”), or whitish fingernails, suggesting possible hybridization. Admixture analyses were performed using Pritchard's software STRUCTURE [18] with two approaches. First, we assumed that all samples belong to an unknown number of “populations”. The individual classification based on phenotypic information was not used and all wolves and dogs were pooled into a hypothetical single “population”. The probability of the number of populations (K) for the pooled data was estimated by fixing prior values of K from 1 to n, and comparing the log-likelihood of the data. If the hypothetical single “population” is admixed and includes more than one sub-population, it will be not in Hardy–Weinberg (HWE) and linkage equilibrium (LE). The software will, therefore, “split” the population into a number of sub-populations (genetic clusters), assigning the individuals to the clusters until departure from HWE and LE are minimized. Results showed that dogs and wolves were split into different clusters based only on their genetic make up and independently on any prior population information. Known hybrids were associated partially to both dog and wolf clusters, in accordance with their admixed ancestry. In a second modelling approach we assumed that samples should belong to one of the following pre-defined “groups”: Italian wolves, wolves from other populations, dogs, captive-reared hybrids. Then we asked the software to assign the individuals and infer the ancestry of hybrids using prior population information. Results showed that all dogs and wolves were assigned to distinct clusters and had no significant ancestries in the other clusters, except for some Italian “wolves” showing anomalous phenotypes. Three “black wolves” (two from northern and one from central Apennines), and three “fifth finger wolves” (one of them showing also whitish fingernails, all collected in northern Apennines), showed significant ancestry in dogs' first and second past generations. It is remarkable that two additional Italian “black wolves” (both from northern Apennines) did not show any evidence of admixed ancestry. All the “wolves” with anomalous phenotypes showed the Italian wolf mtDNA haplotype. Therefore, these animals should be F1, or recent backcrossings of F1 between male dogs and female Italian wolves. These hybrids were collected in areas of recent recolonization, which are separated from the main distribution range of wolves in Italy. Dark coats are common in some North America wolf populations, but they were never observed in Europe, except for occasional observations in Italy. The spur on the hindlegs was never observed in North American or Eurasian wolves, while it is frequent in several breeds of domestic dogs. These anomalous phenotypic traits might be controlled by “domestic” alleles that were introduced into the wild-living wolf population by crossbreeding with dogs. However, despite occasional crossbreedings, dogs and wolves in Italy and elsewhere in Europe diverge significantly in their microsatellite allele frequencies, suggesting that there has been little gene flow between the two groups, at least during the most recent generations. Wolves and dogs are genetically differentiated, individuals cluster separately and can be reliably identified using Bayesian procedures without any prior population information. These data do not support the hypothesis that frequent crossbreeding with free-ranging dogs have increased the genetic diversity of the Italian wolf population after the bottleneck. High pup mortality, which was observed in feral dogs in Italy [19], may reduce survival and backcrossing rates of F1s. Phase shifting in the breeding season of F1s may restrict their chances to backcross into the wolf population. Therefore, the probability of introgression may be low also in presence of a substantial rate of hybridization. We can not exclude that typing additional microsatellites [20] would reveal admixed ancestry also in other wolves.

Despite international protection in most European countries, the wolf is still threatened throughout most of its range due to habitat destruction, direct persecution, accidental killing and hybridization with dogs. The availability of diagnostic morphological, behavioural and molecular traits would help to map the distribution of wolf × dog hybrids and locate eventual areas of introgression where populations of free-ranging dogs should be carefully controlled.

The Italian wolf population is expanding and wolves are recolonizing the Alps. Colonizing wolves presumably originate from a natural expansion of the Italian source population. However, local shepherds and hunters claim that illegal releases of captive non-indigenous wolves have contributed to support the fast growth rate of the Alpine population. The current Alpine wolf population size is small (and unknown), and population dynamics, inter-pack connectivity and dispersal rates of wolf packs can not be estimated using only field observations. Wolf colonization of the Alps represents an opportunity to apply non-invasive genetic methods [21]. DNA samples extracted from scats potentially permit to identify and map the presence of individual genotypes, and estimate their home ranges and relatedness. We are using excremental DNA as a source of information for molecular tracking of wolf packs in two study areas the Alps. DNA samples are genotyped by nucleotide sequencing of the diagnostic hypervariable part of the mtDNA control-region (for species identification), RFLP analyses in sex-linked canine ZFX/ZFY DNA sequences (for molecular sexing), and by multilocus microsatellites (for individual genotyping). All sequenced mtDNAs showed the exclusive presence of the Italian wolf haplotype in the Alps. The assignment test also showed that all individual microsatellite genotypes identified in the Alps belong to the Italian wolf population. Thus, DNA markers suggest that the ongoing recolonization of the Alps is due to the natural expansion of the Italian wolf population. We did not identify any putative case of admixed wolf-dog genotype in the study areas. The alleged presence of illegally released non-indigenous wolves was also not documented. The average relatedness in colonizing wolf packs was higher than among randomly selected Italian wolves, indicating that colonizing wolf packs likely include mainly related individuals. Genotypes in the two study areas were distinct, indicating the presence of two distinct wolf packs. Wolves sampled within each area were more related one each other than wolves between the two study areas and were subdivided into two distinct group of relatedness, mostly concordant with sampling locations and with the inferred patterns of kinship.

The European wildcat is widely distributed in Europe in a number of fragmented populations [22]. The extent of genetic diversification in wildcat populations and the eventual presence of phylogeographic structuring are currently unknown, and we have no information about the postglacial population dynamics and patterns of recolonization of the wildcat in Europe. In Italy there are three subspecies of cats [6]: the European wildcat (Felis silvestris silvestris), the Sardinian wildcat (F. s. libyca) and the domestic cat (F. s. catus). Cats were domesticated by the Egyptians about 6000–4000 yrBP, from north African or Near Eastern African wildcat populations [23]. The Sardinian wildcats originated from African wildcats that were introduced in the island about 6000 yrBP. Home and free-ranging domestic cats are distributed worldwide and virtually sympatric with European and African wildcats almost everywhere. They can interbreed with wildcats and produce fertile offspring in captivity and in nature. The fear of widespread hybridization lead to suppose that “pure” wildcat populations would eventually no longer exist in parts of Europe, Near East and South Africa, and made uncertain any identification of “pure” wildcats to be used as references for taxonomy. Morphologic and morphometric studies did not evidence diagnostic traits suitable to identify hybrids and/or introgressed cat populations [24]. Bayesian clustering and admixture analyses [6,25] showed that hybridization and introgression of domestic cat genes into the gene pool of wild living cat populations is widespread in Scotland, and rare, but not absent, in Italy. We analysed allele frequency variation at 12 feline microsatellites in samples of wild and domestic cats collected from various localities in Europe, and which were pre-classified using morphological traits. Genetic variability was significantly partitioned among taxonomic groups, suggesting that morphological diversity reflects the existence of distinct gene pools. Admixture analyses simultaneously assigned the European, Sardinian wildcats and domestic cats to different clusters, independent of any prior information, and pointed out the admixed gene composition of some known captive-reared hybrids, which were assigned to more than one cluster. Among the putative wildcats, only one Sardinian wildcat was assigned to the domestic cat cluster and one presumed European wildcat showed mixed ancestry in the domestic cat gene pool.

Admixture analyses suggest that wild and domestic cats in Italy are distinct, reproductively isolated gene pools, and that introgression of domestic alleles into the wild-living population is very limited and geographically localized. However, other wildcat populations in Europe show instances of widespread introgression. It is unclear if ecological conditions or historical factors might differentially affect the reproductive interactions among wild and domestic cat populations in different areas of Europe.

We are using microsatellites to estimate within and among population genetic diversity in otters collected from some European localities. Genetic diversity is moderately high within populations and significantly partitioned among sampling locations. Bayesian cluster analyses of multilocus genotypes split the otters into several genetically distinct populations, which are partially concordant with the geographical origin of the samples. Bottleneck tests and coalescent inference of past demographic fluctuations, suggest that otter populations likely declined several thousand years ago, but do not show evidence of recent bottlenecks. Some otter populations started to decline from 8000 to 10 000 yrBP, when permafrost and dry steppe habitat conditions could have been limited the presence of this species in northern and central Europe. Post-glacial founder events and re-colonization of northern Europe after the last glacial maximum, or more recent population declines during the early Holocene in central Europe, might have produced the population and genetic bottlenecks. Until a comprehensive phylogeographic framework for otters in Europe is available, we suggest that recovery plans should favour the expansion of existing natural populations through processes of re-naturalization of rivers and wetlands. Despite past population decline, otters in Europe are still widespread and locally viable. Therefore, we suggest avoiding the reintroduction of otters from mixed captive-reared stocks of unknown geographical and genetic origin, at least until the relevant otter populations from putative south European glacial refuges are genotyped, and eventual phylogeographic units are identified.