Introduced and extinct: neglected archival specimens shed new light on the historical biogeography of an iconic avian species in the Mediterranean.

Collection specimens provide valuable and often overlooked biological material that enables addressing relevant, long-unanswered questions in conservation biology, historical biogeography, and other research fields. Here, we use preserved specimens to analyze the historical distribution of the black francolin (Francolinus francolinus, Phasianidae), a case that has recently aroused the interest of archeozoologists and evolutionary biologists. The black francolin currently ranges from the Eastern Mediterranean and the Middle East to the Indian subcontinent, but, at least since the Middle Ages, it also had a circum-Mediterranean distribution. The species could have persisted in Greece and the Maghreb until the 19th century, even though this possibility had been questioned due to the absence of museum specimens and scant literary evidence. Nevertheless, we identified four 200-year-old stuffed black francolins-presumably the only ones still existing-from these areas and sequenced their mitochondrial DNA control region. Based on the comparison with conspecifics (n = 396) spanning the entirety of the historic and current species range, we found that the new samples pertain to previously identified genetic groups from either the Near East or the Indian subcontinent. While disproving the former occurrence of an allegedly native westernmost subspecies, these results point toward the role of the Crown of Aragon in the circum-Mediterranean expansion of the black francolin, including the Maghreb and Greece. Genetic evidence hints at the long-distance transport of these birds along the Silk Road, probably to be traded in the commerce centers of the Eastern Mediterranean.

Wild Avian Gut Microbiome at a Small Spatial Scale: A Study from a Mediterranean Island Population of Alectoris rufa.

This research is one of the few comparative descriptions at an intraspecific level of wild non-passerine microbiomes. We investigated for the first time the gut microbiome of red-legged partridges (Alectoris rufa) using fecal pellets in order to provide a more informed management. We focused on a small Italian population consisting of two demes (WEST, EAST) separated by about 20 km on the opposite sides of Elba Island. Given the small spatial scale, we set up a sampling protocol to minimize contamination from environmental bacteria, as well as differences due to variations in-among others-habitat, season, and age of feces, that could possibly affect the investigation of the three Elban sites. We found a significant divergence between the WEST and EAST Elban subpopulations in terms of microbial composition and alpha diversity. Although most represented bacterial phyla were the same in all the sites (Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes), microbiomes displayed a much higher diversity in western than in eastern partridges. This result might be related to locally diverging individual physiological needs and/or to different intensities in past releases of captive-bred birds between the two sides of Elba. We suggest that the two subpopulations should be treated as distinct management units.

Unexpected genetic integrity boosts hope for the conservation of the red-legged partridge (Alectoris rufa, Galliformes) in Italy.

The red-legged partridge (Alectoris rufa) is a medium-sized galliform endemic to southwestern Europe. In the easternmost part of the species' range, the population inhabiting Elba Island (Tuscan Archipelago National Park, Italy) is of undisputed conservation value. While we found nuclear and maternal DNA introgression with the exotic chukar partridge (A. chukar) in previous studies based on microsatellite DNA (n = 25) and two mitochondrial markers (n = 103), respectively, we disclosed a limited or null admixture in a few Elban partridges (n = 4) in a recent genomic investigation relying on 168,675 Single Nucleotide Polymorphisms (SNPs). We herein carried out an extended microsatellite DNA survey including additional 65 samples (total, 90) and six loci (total, 11) to determine both spatial structure and genetic integrity of local A. rufa. A sharp divergence between the subpopulations inhabiting the two sides of the island was disclosed, and the microsatellites indicated that all Elban partridges were not admixed with the chukar, thus fully reflecting the picture inferred using SNPs. We hypothesized that the spreading of chukar genes was constrained by negative selection, with the persistence of only the maternal lineage being indicative of thermal adaptation. The two subpopulations should be treated as distinct Management Units, and an envisaged plan to secure a stock onto nearby Pianosa Island could not only warrant endurance of the Elban population but also establish a source of valuable founders for the ex-situ management of the species in Italy. Our study exemplifies how a suitable samples/loci combination is the key to solve wildlife issues dealing with introgression.

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 flagship 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. anatolica, 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.

Novel genome reveals susceptibility of popular gamebird, the red-legged partridge (Alectoris rufa, Phasianidae), to climate change.

We produced a high-quality de novo genome assembly of the red-legged partridge A. rufa, the first reference genome of its genus, by utilising novel 10× Chromium technology. The estimated genome size was 1.19 Gb with an overall genome heterozygosity of 0.0022; no runs of homozygosity were observed. In total, 21,589 protein coding genes were identified and assigned to 16,772 orthologs. Of these, 201 emerged as unique to Alectoris and were enriched for positive regulation of epithelial cell migration, viral genome integration and maturation. Using PSMC analysis, we inferred a major demographic decline commencing ~140,000 years ago, consistent with forest expansion and reduction of open habitats during the Eemian interglacial. Present-day populations exhibit the historically lowest genetic diversity. Besides implications for management and conservation, this genome also promises key insights into the physiology of these birds with a view to improving poultry husbandry practices.

Genome-wide markers redeem the lost identity of a heavily managed gamebird.

Heavily managed wildlife may suffer from genetic homogenization and reshuffling of locally adapted genotypes with non-native ones. This phenomenon often affects natural populations by reducing their evolutionary potential and speeding up the ongoing biodiversity crisis. For decades, the red-legged partridge (Alectoris rufa), an intensively managed gamebird of conservation concern and considerable socio-economic importance, has been subjected to extensive releases of farm-reared hybrids with the chukar partridge (Alectoris chukar) and translocations irrespective of subspecific affinity. These practices have led to serious concerns that the genetic integrity and biogeographic structure of most red-legged partridge populations are irreversibly affected, as suggested by previous studies based on few genetic markers. Using over 168 000 genome-wide loci and a sampling across the entire A. rufa range, we detected unexpectedly limited and spatially uneven chukar introgression as well as significant intraspecific structure. We demonstrate that species widely feared to have irretrievably lost their genetic identity are likely to be much less affected by unsuitable management practices than previously assumed. Our results spell the need for a radical re-think on animal conservation, possibly restoring native status to populations long treated as compromised. Our study exemplifies how the application of innovative conservation-genomic methods is key to solving wildlife management problems dealing with introgressive hybridization worldwide.

Non-native and hybrid in a changing environment: conservation perspectives for the last Italian red-legged partridge (Alectoris rufa) population with long natural history.

The ever-increasing biotic homogenization - especially when associated with introgressive hybridization - raises concern for the reduction of the spatial component of diversity in wildlife worldwide. Nonetheless, there is a growing attention to the potential conservation value of hybridization in fastening the adaptive evolutionary responses to rapidly changing selective pressures. Under these premises, we investigated the genetic affinity of the red-legged partridge (Alectoris rufa) population inhabiting Elba Island (Italy) in the context of the overall species phylogeography and particularly of the nominate subspecies it is traditionally ascribed to. Although notoriously hybrid with the congeneric A. chukar, this island population is of undisputedly value because of its long natural history and self-sustainability. As such, its adaptive conservation management calls for a comprehensive knowledge including the assessment of its geographic origin. For this purpose, 110 fecal samples were collected across Elba, genotyped at their joint Cytochrome-b and Control Region genes (2,249 characters), and compared with 149 conspecifics from all over the species distribution range. We confirmed a widespread A. chukar mitochondrial DNA introgression in Elba partridges, whereas their expected formal assignment to the nominate subspecies from Italy and France was rejected, since these turned out to be closely related to conspecifics from the Iberian Peninsula. This counterintuitive result found support in a large variety of literary sources and compelling evidences from personal testimonies revealing recent intense management with farm-reared birds of Spanish origin. Although the nativeness of Elba partridges was disproved, we advise local authorities to keep warranting the ongoing conservation efforts - and especially restore the connectivity between the western and eastern island sub-populations - as this resource may still be conceived as the ultimate repository for part of the otherwise extinct Italian A. r. rufa genome. Indeed, the admixture with conspecifics from the Iberian Peninsula does not necessarily mean that the entirety of the native nuclear genome of Elba partridges has been wiped out. Furthermore, these latter represent an interesting case study in conservation biology to investigate the possible role played by introgressive hybridization in the adaptation to recent land use and vegetation cover changes associated with rural abandonment in an insular yet heavily anthropized context.

Human-modified biogeographic patterns and conservation in game birds: The dilemma of the black francolin (Francolinus francolinus, Phasianidae) in Pakistan.

The ever-increasing human-mediated wildlife reshuffling is raising concern for the conservation of biodiversity. The loss of biological distinctiveness among regions lessens the genetic diversity and consequently the evolutionary potential of local biotas to tackle present-day global change and human disturbance. This process may be sometimes cryptic unless investigated by means of a molecular approach. In this respect, game birds are a paradigmatic case. The black francolin (Francolinus francolinus, Phasianidae) is a medium-sized galliform whose distribution range stretches from Cyprus to the Gulf of Bengal. Six morphologic subspecies are known, with three of which occurring in Pakistan, where the species is heavily hunted and used as pet for chirping competitions. We genotyped 98 samples (feathers) at both the entire mitochondrial DNA Control Region gene and nine microsatellite loci to get a deeper insight into the genetic diversity of the black francolin in Pakistan in order to offer cogent recommendations for its conservation management. We identified several mtDNA lineages that were consistent with the currently described subspecies/taxonomy whose pattern of co-occurrence is compatible with the geological history and the faunal movement routes of the region under study. However, the biparentally inherited microsatellites returned a quite discordant picture of an extensive, sex-biased genetic mixing due to the intensive relocations of already overharvested male individuals for chirping competitions. Our results indicated that the genetic integrity of the black francolin in Pakistan could be seriously at risk and call for monitoring and limiting its trade other than enhancing the public awareness of the importance of local biodiversity resources.

Phylogeography of the smooth-coated otter (Lutrogale perspicillata): distinct evolutionary lineages and hybridization with the Asian small-clawed otter (Aonyx cinereus).

We investigated the phylogeography of the smooth-coated otter (Lutrogale perspicillata) to determine its spatial genetic structure for aiding an adaptive conservation management of the species. Fifty-eight modern and 11 archival (dated 1882-1970) otters sampled from Iraq to Malaysian Borneo were genotyped (mtDNA Cytochrome-b, 10 microsatellite DNA loci). Moreover, 16 Aonyx cinereus (Asian small-clawed otter) and seven Lutra lutra (Eurasian otter) were sequenced to increase information available for phylogenetic reconstructions. As reported in previous studies, we found that L. perspicillata, A. cinereus and A. capensis (African clawless otter) grouped in a clade sister to the genus Lutra, with L. perspicillata and A. cinereus being reciprocally monophyletic. Within L. perspicillata, we uncovered three Evolutionarily Significant Units and proved that L. p. maxwelli is not only endemic to Iraq but also the most recent subspecies. We suggest a revision of the distribution range limits of easternmost L. perspicillata subspecies. We show that smooth-coated otters in Singapore are L. perspicillata x A. cinereus hybrids with A. cinereus mtDNA, the first reported case of hybridization in the wild among otters. This result also provides evidence supporting the inclusion of L. perspicillata and A. cinereus in the genus Amblonyx, thus avoiding the paraphyly of the genus Aonyx.

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.

Large-scale patterns of genetic variation in a female-biased dispersing passerine: the importance of sex-based analyses.

Dispersal affects the distribution, dynamics and genetic structure of natural populations, and can be significantly different between sexes. However, literature records dealing with the dispersal of migratory birds are scarce, as migratory behaviour can notably complicate the study of dispersal. We used the barn swallow Hirundo rustica as model taxon to investigate patterns of genetic variability in males and in females of a migratory species showing sex-biased dispersal. We collected blood samples (n = 186) over the period 2006 to 2011 from adults (H. r. rustica subspecies) nesting in the same breeding site at either high (Ireland, Germany and Russia) or low (Spain, Italy and Cyprus) latitude across Europe. We amplified the Chromo Helicase DNA gene in all birds in order to warrant a sex-balanced sample size (92 males, 94 females). We investigated both uniparental (mitochondrial ND2 gene) and biparental (microsatellite DNA: 10 loci) genetic systems. The mtDNA provided evidence for demographic expansion yet no significant partition of the genetic variability was disclosed. Nevertheless, a comparatively distant Russian population investigated in another study, whose sequences were included in the present dataset, significantly diverged from all other ones. Different to previous studies, microsatellites highlighted remarkable genetic structure among the studied populations, and pointed to the occurrence of differences between male and female barn swallows. We produced evidence for non-random patterns of gene flow among barn swallow populations probably mediated by female natal dispersal, and we found significant variability in the philopatry of males of different populations. Our data emphasize the importance of taking into account the sex of sampled individuals in order to obtain reliable inferences on species characterized by different patterns of dispersal between males and females.

Conservation of endemic and threatened wildlife: molecular forensic DNA against poaching of the Cypriot mouflon (Ovis orientalis ophion, Bovidae).

Molecular DNA techniques in combination with appropriate reference population database and statistical methods are fundamental tools to forensic wildlife investigations. This is even more relevant when taxa with uncertain systematics are involved, as is the case of the genus Ovis (Bovidae), whose evolution has been influenced by multiple events of domestication. The Cypriot mouflon, Ovis orientalis ophion, a protected subspecies endemic to Cyprus, is threatened by poaching. This study deals with a case of alleged poaching that occurred in Cyprus (September, 2010). A car did not stop at a checkpoint and when finally blocked by the police, several bloodstained exhibits (n=12) were recovered. Three recently deceased mouflons were found by game wardens at the roadside. The Cyprus Veterinary Services established that these animals had been killed by gunshot. As part of the investigation, DNA testing was performed to establish if there was a link between the dead mouflons and the bloodstained exhibits. The mitochondrial Cytochrome-b gene (Cyt-b) and 12 loci of microsatellite DNA were used as markers. The Cyt-b sequences were obtained from 11 exhibits. They were the same as each other and the same as the single haplotype obtained from the three dead mouflons and all the investigated wild Cypriot mouflons (20 individuals). A database of wild mouflons (47 individuals) from which the unknown samples may have originated was generated. The probability of identity (P(ID)) of the microsatellite panel, computed by genotyping all 47 wild mouflons (10 selected loci, P(ID)=10(-5)), allowed us to assign nine exhibits to two out of the three carcasses (seven with very strong support: Likelihood Ratio, LR>3000 and Random Match Probability, RMP, <10(-3)). This study represents the first genetic reference for the Cypriot mouflon and the first published material of forensic wildlife investigations in Cyprus.

Sequenced RAPD markers to detect hybridization in the barbary partridge (Alectoris barbara, Phasianidae).

In the Alectoris partridges (Phasianidae), hybridization occurs occasionally as a result of the natural breakdown of isolating mechanisms but more frequently as a result of human activity. No genetic record of hybridization is known for the barbary partridge (A. barbara). This species is distributed mostly in North Africa and, in Europe, on the island of Sardinia (Italy) and on Gibraltar. The risk of hybridization between barbary and red-legged partridge (A. rufa: Iberian Peninsula, France, Italy) is high in Sardinia and in Spain. We developed two random amplified polymorphic DNA (RAPD) markers to detect A. barbara × A. rufa hybrid partridges. We tested them on 125 experimental hybrids, sequenced the relative species-specific bands and found that the bands and their corresponding sequences were reliably transmitted through a number of generations (F1, F2, F3, BC1, BC2). Our markers represent a highly valuable tool for the preservation of the A. barbara genome from the pressing threat of A. rufa pollution.

Noninvasive genotyping of the red-legged partridge (Alectoris rufa, Phasianidae): semi-nested PCR of mitochondrial DNA from feces.

DNA-based studies using avian feces are scarce and deal only with large-sized species. The red-legged partridge, Alectoris rufa, is a medium-sized member of the order Galliformes. Our goal was to set up a fast, noninvasive procedure for mitochondrial DNA (mtDNA) genotyping of A. rufa fecal samples. We focused on the protected population from Elba Island (Tuscan Archipelago National Park, Italy). Dry A. rufa fecal samples (n = 30) were collected in winter. Both the cytochrome b gene (1,092 bp) and the control region (ca. 1,155 bp) were amplified by means of semi-nested PCRs. Twenty-five samples were successfully sequenced for both genes: 8 showed A. rufa mtDNA lineage and 17 chukar partridge (A. chukar), an exotic species. Mixed maternal ancestry suggests A. rufa x A. chukar hybridization. Our protocol allows noninvasive mtDNA genotyping of any Alectoris species and appears suitable to investigate protected populations as well as those existing either at very low density or inhabiting poorly accessible regions.

Genetic structure of Mediterranean chukar (Alectoris chukar, Galliformes) populations: conservation and management implications.

The chukar (Alectoris chukar, Galliformes) is a species hunted throughout its native range from the East Mediterranean to Manchuria and in the USA, which hosts the world's largest introduced population. This study aims to investigate the genetic structure of Mediterranean chukar populations to aid management decisions. We genotyped 143 specimens at two regions of the mitochondrial DNA (mtDNA: cytochrome b, control region) and eight loci of the microsatellite DNA. Samples were collected in northern (Limnos, Lesvos, Chios) and southern (Crete) Aegean islands (Greece) and Cyprus. We also carried out mtDNA-based comparison with chukars (n = 124) from Asia (16 countries) and the USA (five states). We propose six management units for Mediterranean populations. Given their genetic integrity, Limnos and Cyprus, which host different subspecies, proved to be of primary conservation interest. We found exotic A. chukar mtDNA lineages in Lesvos, Chios and Crete and produced definitive genetic evidence for the Asian origin of the US chukars.

Genetic insight into Mediterranean chukar (Alectoris chukar, Galliformes) populations inferred from mitochondrial DNA and RAPD markers.

The chukar (Alectoris chukar, Galliformes) is one of the most important game birds as it is widely distributed and hunted over the whole of its range. The aim of this work was to assess the genetic differentiation as well as the possible presence of hybrid specimens in A. chukar populations from Italy, Greece and Cyprus. To provide phylogenetic context, conspecific, allopatric specimens from Israel, Georgia, Armenia, Kazakhstan, Afghanistan, Pakistan, Mongolia, China and USA were compared. Sequencing of the mitochondrial DNA (mtDNA) Control Region supplied information on the ancestry of A. chukar populations, whereas Random Amplified Polymorphic DNA (RAPD) fingerprinting was used to assess whether hybridization had occurred. The Italian population was found to be an inter-specific mixture of A. chukar and A. rufa (i.e., the red-legged partridge) mtDNA lineages, whereas the representatives from Greece and Cyprus showed only the A. chukar maternal line. RAPD markers revealed introgression with A. rufa genes in the Italian population, whereas no A. chukar x A. rufa hybrid specimens were detected in the eastern Mediterranean populations. The genetic data obtained from the Italian A. chukar population as well as from a few Greek specimens pointed against their Mediterranean kinship, suggesting relationships with A. chukar subspecies from the easternmost part of the Asian continent.