• Cyprus mouflon
• conservation management
• adaptive
• molecular genetics

The mouflon (Ovis gmelini ophion, Caprinae) is the largest terrestrial mammal and an endemic taxon of theisland of Cyprus, which has remained separated with respect to other similar Mediterranean and Middle East populations for about 10,500 years. Nowadays, the Cyprus mouflon is threatened by poaching, habitat loss and fragmentation, and infection from pathogens transmitted by livestock. As consequence, the species and its habitat are protected under the National Law 152 (I)/2003 for the Protection & Management of Wild Birds and Game Species and listed in the Annexes II and IV of 92/43 Habitats Directive. The Cyprus mouflon is also included in the Appendix I of CITES, and based on a recent survey (2016), about 3,000 heads inhabit the Paphos forest and adjacent forested areas of the island. In this thesis, we attempted at carrying out the first in-depth genetic study of the mouflon of Cyprus to gain useful information and offer cogent management recommendations for its long-term protection. The Cyprus Veterinary Services in collaboration with the Game and Fauna Service (Ministry of Interior) collected (2007 to 2018) 92 GPS-referenced samples of wild mouflons from 39 localities across the entire island distribution range and 14 samples of captive mouflons from seven ex situ facilities.When unknown, the gender of each investigated mouflon was inferred by molecular sexing. Then, the entire sample was genotyped at a panel of 13 loci of microsatellite DNA (biparentally inherited, nuclear DNA) to determine genetic diversity, inbreeding coefficient, spatial genetic structure and gene flow pattern in males and females separately as well as in the whole population. The entire mitochondrial DNA Control Region gene (mtDNA CR: uniparental, maternal DNA) and SRY/SRYM18 Y-chromosome genes (uniparental, paternal DNA) were sequenced in 40 and 10 individuals, respectively, to assess the phylogenetic placement of the Cyprus mouflon. This task was accomplished through the comparison with GenBank records [284, CR sequences; 27, (SRY+SRYM18) combined haplotypes] and a few samples from West Mediterranean (3) and Armenian (3) individuals available at the University of Pisa. Seven mouflons whose gender was unidentified turned out to be three males and four females; therefore, 69 males and 37 females were available to this study. Microsatellite DNA genotyping indicated that the Cyprus population held low genetic variability, which, however, was not associated with an alarming value of the inbreeding coefficient (FIS = 0.145, wild group). A weak spatial genetic structure was disclosed. The most frequent cluster (II, with 56% of total membership across all localities) occurred mainlyNorth-to-South across the eastern side as well as in some locality of the western part of the Paphos forest. The rarest genetic cluster (I, with 10.9% of total membership) was found across the entire investigated area, with a high frequency especially in the northwestern and southwestern regions. The geographic pattern of these genetic clusters was approximately the same when males and females were investigated on their own, this being an unexpected result as female mouflons are generally known to hold more structured social pattern and lower mobility than males.As far as the seven island ex situ facilities are concerned, remarkably, we found a few private alleles in two animals kept in captivity at Bird Park (Paphos District). Although the lack of information on the origin in the wild of these animals (or of their ancestors) prevent us from discussing further this result, it could be in the best interest of the Cyprus Game and Fauna Service moving these mouflons to the most important enclosure (Stravros tis Psokas) of the country and investigating their entire genome. The gene flow in the wild (males + females) population was maximum between the eastern edge and the core of the forest, whereas it was minimum across the edge in the northernmost part of the study area. Again, this result was similar to that found when males and females were taken into account separately, although some difference was discernible. Overall, the lower gene flow disclosed at the edge than in the core of the Paphos forest was likely due to the higher level of human disturbance (recent road network building) occurring especially in the north/north-eastern part of the investigated area. Finally, microsatellite DNA genotyping has not confirmed that the mouflon of Cyprus has suffered in the 1930s from a demographic collapse (<1% of the present-day population), as we did not find any reliable evidence for a genetic bottleneck. With reference to the mtDNA CR gene sequencing, we found eight heteroplasmic Cyprus mouflons with five (six individuals) and six (two individuals) repeated motifs. The remaining mouflons were homoplasmic and held four repeated motifs, which is the most common pattern in domestic sheep. When compared against all available GenBank records, the motif including six repetitions was private to Cyprus, a result stressing the genetic divergence of this island population. Concordantly, the four mtDNA CR haplotypes disclosed in the Cyprus mouflons were all private to this population. The Bayesian clustering analyses of (40 + 284 =) 324 investigated mtDNA CR sequences and the calculation of average pairwise TN93 genetic distances with respect to known haplogroups from the literature (HGA to HGE, plus HGX) pointed to HGX as the closest one to the Cyprus mouflon. However, the Median-Joining network indicated that the Cyprus population and HGX were separated by almost the same amount of mutational changes as those occurring between HGX and HGC (11 and 12, respectively). Hence, we suggested that the Cyprus mouflon could be considered as an independent and new haplogroup. Finally, we found a single (SRY + SRYM18) Y-chromosome haplotype in the Cyprus mouflon that was never reported previously in any wild or domestic sheep. Following the nomenclature available in the literature, this new haplotype was herein referred to as “h18”. The latter diverged from haplotypes held by West Mediterranean (Sardinia: O. aries musimon), Anatolian (O. gmelini anatolica) and Armenian (O. gmelini gmelini) mouflons, a very interesting finding that pointed further to the genetic uniqueness of the Cyprus population. In conclusion, based on our combined genetic results, we eagerly recommend that the conservation management of the Cyprus mouflon take place within a strict adaptive conservation framework, thusavoiding any contact with any allopatric Ovis resources. The Cyprus mouflon population would also certainly benefit from a constant genetic monitoring and the application of genetically controlled breeding techniques in order to sustain or even increase its genetic variability.