According to historical records, the mouflon from Sardinia and Corsica were first introduced to Austria in 1731[14]. The next translocation to Northern Italy was in 1780[8]. The first introduction of significant numbers of mouflon into the wild in mainland Europe took place in 1868 in the area that is now part of Slovakia[14]. Our analysis revealed that mouflon from Croatia, Slovenia, Germany, and the Czech Republic share the most common haplotype, or have closely related country specific ones. Overall, mouflon from studied populations from these four countries are genetically closely related to haplotypes from Sardinia and Corsica.
A star-like pattern of haplotype network (Fig. 1), in which a very common haplotype, presumably ancestral, is located at the centre of a network and connected to rarer haplotypes by independent mutation steps, is usually regarded as an indication of a population that has recently expanded in size from one or a small number of founders[50]. Most of the haplotypes from Slovenia and the Czech Republic were closely related and separated by one mutation step from the central haplotype h2. The central position of h2 in the network indicates that this haplotype (originated from Germany) likely functions as one of the ancestors of the populations introduced in Slovenia and Croatia, as this haplotype is present in all studied populations in both countries, consistently with the historical record that the mouflon was introduced from Sardinia to Germany and from there to other European areas, including Slovenia and Croatia. Therefore, the results of the mtDNA CR are largely consistent with written historical data indicating Germany as the source population of mouflon in Slovenia and Croatia[29],[30]. However, our results also suggest the presence of additional sources, since both countries have haplotypes (M8, M14, M13) more closely connected to Sardinia than to Germany, which is probably a consequence of the additional translocation of the mouflon from Sardinia to the Brijuni Islands as a gift from Italian goverment to the former Yugoslav president Tito (Pers.comm. E. Kolić).
A high number of unique haplotypes (i.e., singletons) and low nucleotide diversity in populations from Slovenia and Croatia indicate that these populations likely underwent population expansion, probably as a consequence of the post-reintroduction management measures implemented in both countries[27],[28],[30],[31],[33],[34].
Overall, haplotype diversity for Slovenian and Croatian populations was high (0.776 and 0.333, respectively) while nucleotide diversity was low (0.007 and 0.002). Such combination of high haplotype and low nucleotide diversity can result from a rapid demographic expansion after a period of low effective population size[51], such in the case of rapid demographic expansion in novel distribution ranges after founding[52]–[54]. Additionally, higher nucleotide diversity of mouflon in Slovenia is probably the result of substantial introduction and translocation from different populations compared to Croatia, where most populations were established by individuals from the Brijuni Islands.
To position the mtDNA CR haplotypes within a broader evolutionary context, we performed a network analysis including additional sequences of Ovis sp. from Europe and Asia, which shows a separation between Asian and European haplotypes (Fig. S1). This haplotype network also revealed a 'star-like' genealogy for the most common haplotype h2.
Within the European cluster, populations showed mostly private haplotypes. Nevertheless, our results show the genetic differentiation between Corsico-Sardinian and introduced mouflon, as previously already described[7],[47]. Pairwise FST comparisons were significant and showed a strong degree of differentiation for all groups/countries except between Germany & Czech Republic and Croatia. This lack of differentiation is likely the consequence of the translocation history of mouflon in Croatia, where the population on the Brijuni Islands was founded by a German wildlife trader from his private zoo[29]. A positive relationship between genetic variation and number of sources is not only known from reintroduced endangered native species[55] but also from introduced populations of alien species[56]–[59].
Our study confirmed that mouflon populations (both introduced on the continent and historically introduced to Sardinia and Corsica) have low MHC diversity (only five alleles were found in our 95 samples), possibly due to a combination of historical founder effects and low effective population sizes[4],[7],[60],[61], which was further reduced by sequential translocations[8],[10],[27],[28],[30],[31],[33],[34]. In Slovenia and Croatia, the most common allele was Ovar-DRB1*07012, previously described as the second most common allele in the population from the Caroux-Espinouse Massif, where the allele Ovar-DRB1*0324 was the most common[45].
Mutations in the ABS domain are usually associated with differential disease resistance[45],[62]. In our dataset, we observed much higher evolutionary distances between nucleotides and amino acids in ABS than in non-ABS (19% vs. 6%; 49% vs. 10%), indicating a direct effect on binding properties for antigenic peptides[63] as it has been previously already described[64],[65].
Portanier et al.[45] found that specific alleles and heterozygosity of MHC exon 2 alleles have effects on faecal egg counts and faecal oocyte counts of gastrointestinal nematodes in female mouflon. This suggests the occurrence of heterozygote advantage, effects of rare alleles, and/or fluctuating selection in the Caroux-Espinouse mouflon population. On the contrary, no association was detected between genetic diversity and resistance to coccidia parasites, indicating that different parasite classes are impacted by different genetic drivers[66]. Portanier et al.[45] also found a marked gradient between the most parasitized MHC DRB genotype (Ovar-DRB1*07012/*07012) and the least parasitized genotype (Ovar-DRB1*0324/*0114), with a statistically significant difference also between Ovar-DRB10324/*0324 and Ovar-DRB1*0324/*0114 genotypes, leading to a 57.2% decrease in faecal egg abundance. The presence of the allele Ovar-DRB*0114 led to a 56% decrease in faecal oocyte of nematodes between individuals carrying or not carrying this allele.
In our sample set, we found Ovar-DRB*0114 allele in only 4 individuals. Overall, we also found only 4 individuals with advantageous genotype (Ovar-DRB1*0324/*0114). This genotype was present in three mouflons from Slovenia and in just one from Croatia. On the contrary, genotype Ovar-DRB1*07012/*07012 was present in 56 individuals: 36 in Slovenia, 12 in Croatia, 5 in Czech Republic, two in Sardinia, and one in Corsica. The genotype Ovar-DRB10324/*0324 was present only in one individual from Slovenia. In the present study, DRB1*0114 allele was the rarest allele, which indicates that mouflon populations in Slovenia and Croatia are not specifically adapted to the resistance against gastrointestinal nematodes.
Herrmann-Hoesing et al.[67] studied the impacts of genetic diversity of MHC DRB alleles on ovine progressive pneumonia virus resistance in domestic sheep. They evidenced that alleles DRB1*0324 and DRB1*0114 were associated with a higher provirus level, while DRB1*07012 allele was associated with a lower susceptibility for this virus. The authors explained that these differences were linked with specific amino acids encoded by the diverse alleles and determining the immune response. Provirus and macro-parasitic strongyles are very different pathogen types, and probably the functional links between genetics and resistance could indeed be expected when considering different parasite classes[68]–[70].
Considering that a positive relationship between genetic diversity and fitness-related traits such as parasite resistance can be explained by inbreeding depression with more inbred individuals having lower levels of heterozygosity and fitness[39], we can hypothesise that mouflon populations in both countries may have high susceptibility to disease.
The DRB locus showed signs of positive selection as relative rates of non-synonymous were higher comparing to synonymous mutations (the overall ratio ϖ = 3.21), which is similar to that found in other wild ungulates in Europe, for example northern chamois (Rupicapra rupicapra) and southern chamois (Rupicapra pyrenaica)[71]–[73]. Positive selection most likely affects only a few codons at a few time points but, due to the small number of alleles, we lack statistical power to test this hypothesis.
Genetic differentiation of the studied mouflon populations at MHC DRB gene revealed past demographic history of populations established by founder effect and expanded in isolation. The pairwise FST between populations from Slovenia and Croatia was high and significant. Additionally, the hierarchical AMOVA strongly supports differentiation within populations in the same geographic group/country, and also differentiation between Slovenian and Croatian mouflon.
The observed heterozygosity of mouflon in both countries (Slovenia: 0.102; Croatia: 0.135) was much lower compared to expected heterozygosity (Slovenia: 0.413; Croatia: 0.631). Additionally, nucleotide diversity was low (Slovenia: 0.035; Croatia: 0.034) compared to mouflon population in France (0.91) [45]. Allelic richness is slightly lower in Slovenia than in Croatia, which may also be related to the past translocation and management, due to probably smaller effective size of mouflon populations in Slovenia.