Population genetics and forensic DNA for conservation management of the Cypriot mouflon (Ovis orientalis ophion)

The mouflon (Ovis orientalis ophion) is the largest wild terrestrial mammal of Cyprus. Considered as the flagship species of the island, its population (c. 3000 head) has a distribution range limited to the mountainous Paphos Forest and adjacent areas including parts of Troodos National Forest (> 700 Km2). The species is protected by a rigorous national legislation supported since 1996 by management plans carried out by the Game and Fauna Service, and, together with its habitat, by the National Law 152 (I)/2003 for the Protection & Management of Wild Birds and Game Species. The species is listed in the Annexes II and IV of 92/43 Habitats Directive, in the Appendix I of CITES, and classified as “vulnerable” by the IUCN. Poaching, habitat loss, road network building and livestock intrusion (i.e., increased risk of pathogen infection) represent the main threatening factors. We aimed at elucidating the systematic placement of the Cypriot mouflon to enforce its protection within an adaptive conservation framework. Therefore, we attempted to determine its genetic structure and relationships with either historically preserved (Corsica, Sardinia) or recently introduced (central Italy) populations including also GenBank entries from the historical range of the species (Near East). The Game and Fauna Service in collaboration with the Cyprus Veterinary Service collected 63 blood samples: 53 were from mouflons captured in the Paphos forest, eight from captive individuals and two of unknown origin. We also sampled 20 mouflons in Sardinia either in the wild (16) or in captivity (4), and collected scats of both Corsican (19) and central Italy (23: Tuscan Archipelago National Park, 13; Tuscan-Emilian Apennines National Park, 6; Apuan Alps Regional Park, 4) mouflon populations in order to increase geographical scope. We genotyped each sample at the entire mitochondrial DNA Cytochrome-b codifying gene (Cyt-b, 1140 bp) and up to 12 microsatellite DNA markers (Short Tandem Repeats, STR) isolated from goat, sheep and cattle genomes. We found that the Cypriot mouflon strongly diverged from western Mediterranean conspecifics, while North West Iran appeared as the most credited geographic region as the source for its ancient introduction to Cyprus. Although we disclosed much lower mitochondrial and nuclear DNA diversity in the Cypriot than in other island populations, neither evidence of genetic bottleneck nor significant low level of both average pairwise relatedness and inbreeding coefficient was detected. Overall, present mitochondrial and STR dataset worked reliably as crime-fighting tool to tackle illegal mouflon killing in Cyprus. Between 2008 and 2013, the Police and the Game and Fauna Service, in collaboration with the Cyprus Veterinary Services, confiscated 29 samples (meat, hairs, bloodstains) dealing with nine episodes of supposed poaching against the Cypriot mouflon. In all cases, we identified the species in point by sequencing the mtDNA Cyt-b gene. In one case, we were specifically requested to establish if there was a link between three dead mouflons recovered at a roadside and 12 bloodstains collected in the car of suspected poachers at the crime scene. With reference to this case, we were able to match nine bloodstains to two out of the three carcasses (seven with very strong support: Likelihood Ratio >3000 and Random Match Probability <10-3), overall assigning 22 out of 29 samples to the Cypriot mouflon and the remaining ones to wild boar, cow, domestic goat, horse and hare. These results included the first genetic reference for the Cypriot mouflon and the first published material of forensic wildlife investigations in Cyprus

Impacts of biological globalization in the Mediterranean: Unveiling the deep history of human-mediated gamebird dispersal

Humans have a long history of moving wildlife that over time has resulted in unprecedented biotic homogenization. It is, as a result, often unclear whether certain taxa are native to a region or naturalized, and how the history of human involvement in species dispersal has shaped present-day biodiversity. Although currently an eastern Palaearctic galliform, the black francolin (Francolinus francolinus) was known to occur in the western Mediterranean from at least the time of Pliny the Elder, if not earlier. During Medieval times and the Renaissance, the black francolin was a courtly gamebird prized not only for its flavor, but also its curative, and even aphrodisiac qualities. There is uncertainty, however, whether this important gamebird was native or introduced to the region and, if the latter, what the source of introduction into the western Mediterranean was. Here we combine historical documentation with a DNA investigation of modern birds and archival (13th–20th century) specimens from across the species’ current and historically documented range. Our study proves the black francolin was nonnative to the western Mediterranean, and we document its introduction from the east via several trade routes, some reaching as far as South Asia. This finding provides insight into the reach and scope of long-distance trade routes that serviced the demand of European aristocracy for exotic species as symbols of wealth and prestige, and helps to demonstrate the lasting impact of human-mediated long-distance species dispersal on current day biodiversity

Molecular taxonomy and intra-Palaearctic boundary: new insights from the biogeography of the black francolin (Francolinus francolinus) by means of microsatellite DNA

The transition zones between and within biogeographic realms are a topical issue. Long ascribed to eastern Palaearctic, Iran was recently proposed as entirely pertaining to its western counterpart (featuring theGreat Western Palaearctic). Molecular and phenotypic information from polytypic taxa may help untangle the bioclimatic and environmental dynamics that shaped faunal distributions. In this study, we collected 221 black francolin (Francolinus francolinus Linnaeus, 1766) samples from Cyprus to Bangladesh and compared the spatial genetic structure as inferred from nine microsatellite loci with publically available bioacoustic data. We found striking overlap between the two sources of information, which pointed to the occurrence of a western and an eastern subdivision (FST ¼ 0.435, P<0.001) with a break across central Iran. When framed within the palaeoclimatic and geomorphological history of this region, we found that high connectivity among local subspecies was facilitated by warmer and more humid conditions preceding the cold-arid climatic phase associated with the last glacial stage. The subsequent temperature increase likely led to the substantial isolation of eastern and western black francolin populations to moist refuges. Overall, these results point to an intra-Palaearctic boundary crossing Iran longitudinally, thus raising prospect of compromise between the two most debated views

Molecular biogeography and transitions within zoogeographic realms: the black francolin (Francolinus francolinus) as tool to elucidate the intra-palaearctic boundary

The transition zones between western and eastern Palaearctic is a topical and long debated issue among zoologists, with Iran recently proposed as entirely pertaining to the former (and featuring the “Great Western Palaearctic”) rather than to the latter. Further molecular and phenotypic information from sedentary and low-vagile taxa may help untangle the complex bioclimatic and environmental dynamics that shaped the distribution of faunal assemblages in this region and either validate the currently proposed boundaries or suggest their reappraisal. In this study, we collected and analyzed 221 samples of black francolin (Francolinus francolinus Linneus, 1766), a galliform whose distribution range in Asia entirely encompasses Iran and that, by virtue of limited dispersal capabilities and high habitat fidelity, represents an ideal model to address this issue. This secretive and still scarcely known species has been the object of recent investigations at the University of Pisa addressing phylogeographic and conservation as well as historic-anthropological issues. Here, we compared the genetic affinity of birds sampled across the entire range (from Cyprus eastward to Bangladesh) as inferred at nine microsatellite loci with publically available vocalizations (namely male advertising calls) analyzed in a recent study. We found a striking overlap between the two sources of information pointing to the occurrence of a western and an eastern cluster including two and four morphological subspecies, respectively. The divide between the two runs north to south across central Iran, and was found to match with that separating genetically dissimilar conspecific populations of other vertebrates. When framed within the paleoclimatic and geomorphological information of this region, this scenario suggested a general and extended higher connectivity favoured by a warmer and more humid conditions preceding the cold-arid climatic phase associated with the last glacial stage that restrained most taxa to moist refuges. The subsequent increase of temperatures led to the substantial isolation of eastern and western black francolin populations with a drastic decrease of the gene flow between them. While the taxonomic significance of this split will be hopefully investigated further in a more in-depth study relying on whole genome data, in light of these results we propose to consider a western-eastern Palaearctic boundary crossing Iran longitudinally, thus reaching a compromise between the two traditionally most debated views

Molecular taxonomy and acoustic data of the black francolin (Francolinus francolinus) support a reappraisal of the intra-Palaearctic boundary

The debate about the transition zone between eastern and western Palaearctic is still ongoing, with some zoologist recently ascribing the whole Iran – a key crossroad of faunal biodiversity – to the latter as part of the “Great Western Palaearctic”. Additional distributional and systematic information from low-vagile and sedentary taxa may help settle the dispute. For this purpose, we genotyped samples of black francolin 221 (Francolinus francolinus), a landbird distributed in the Middle East and southern Asia, comparing the genetic (affinity of individuals from the entire range at nine microsatellite loci with vocalisations characterised in a recent study.A stunning overlap emerged between the two sources of information, both indicating the occurrence of an eastern and a western cluster consisting of four and two morphological subspecies, respectively, with a divide running north to south across central Iran. Interestingly, the same scenario was found between genetically dissimilar populations of other vertebrates. Palaeoclimatic and geological data from Iran indicated an overall higher connectivity of the region thanks to milder and moister climatic conditions prior to the cold -arid climatic phase initiated by the last glacial stage and isolating most taxa to disjunct refuges. Subsequently, the temperature raise likely compounded this scenario by hampering gene flow between populations of species - black francolin included - located east and west of the Kavir and Lut deserts. Although the taxonomic significance of this split in the focal species awaits for further studies, these results produced weight for the occurrence of an eastern-western Palaearctic boundary crossing Iran longitudinall

Spatial genetic structure and Ovis haplogroup as a tool for an adaptive conservation management of the endangered Cyprus mouflon

The mouflon (Ovis gmelini ophion) is the largest wild terrestrial mammal of Cyprus. Regarded as the local flagshi species, its population range is limited to the mountainous Paphos Forest and adjacent areas including part of Troodos National Forest Park (> 700 Km2). This species, protected by both national and international legislation, is classified as Endangered by the IUCN, with livestock intrusion, poaching and habitat loss as the main threatening factors. We determined the spatial genetic structure by investigating sexes separately and shed further light on the Cyprus mouflon placement among Ovis haplogroups (HGs) to enforce its protection within an adaptive conservation framework. We genotyped 108 samples collected across the entire species’ range at both14 loci of the microsatellite DNA and the mitochondrial DNA Control Region (CR). Microsatellite genotyping indicated that the wild population retained low genetic variability, which, however, was not associated with a level of inbreeding raising particular concern (FIS = 0.12). An overall weak spatial genetic structure was disclosed, consistently with the limited extension of the mouflon range, the lack of significant physical barriers to dispersal and the intense gene flow mostly occurring along a northeast-southwest axis across the forest. Nevertheless, evidences of structure were found for females in compliance with their philopatric behaviour. The disclosure of unique features compared to Mediterranean and Near East conspecifics, such as the occurrence of a six-fold 76 bp-long repeated motif in the Cyprus mouflon CR, along with the outcome of a phylogenetic reconstruction (based on a far higher number of Ovis GenBank records and Cyprus haplotypes than in previous studies) inspired the proposal of a new haplogroup (HGC2) which included the Anatolian mouflon (O. g. ana tolica, former HGX) as sister taxon. While both high habitat heterogeneity and low human disturbance were the main drivers in determining the overall spatial structure, future conservation efforts to preserve this valuable genetic resource should focus on avoiding possible introgressive hybridisation with co-grazing livestock to the edge of its range