Monica Guerrini

Molecular phylogeography of the asp viper Vipera aspis (Linnaeus, 1758) in Italy: evidence for introgressive hybridization and mitochondrial DNA capture

Owing to its temperature dependence and low vagility, the asp viper (Vipera aspis) is an interesting model species to study the effects of Pleistocene climatic fluctuations on vertebrate genomes. We genotyped 102 specimens from the whole Italian distribution range at three mitochondrial DNA regions (2278 characters, total) and six microsatellite DNA loci (Short Tandem Repeats, STR). The molecular phylogeny was constructed according to Bayesian, Neighbour Joining, Maximum Parsimony and Maximum Likelihood procedures. All methods grouped individuals of the three morphological subspecies (V. a. aspis, V. a. francisciredi, V. a. hugyi) into five different haploclades. Specimens assigned to hugyi clustered in two highly differentiated clades, one being sister group to the complex comprising the second clade of hugyi (i.e., a paraphyletic status), plus two clades of francisciredi. The Bayesian clustering of the STR variability disclosed only two groups, the first including aspis and francisciredi, the second all hugyi. Introgressive hybridization and capture of francisciredi-like lineages in the hugyi mitochondrial genome were suggested to explain the discordance between mitochondrial and nuclear data. The phylogeographic pattern was compatible with population contractions in three glacial refuges. Plausibility of derived hypothesis was checked using coalescence simulations as post hoc tests. Long-term drift and serial founder effects, rather than selection, appeared the main factors affecting the genetic make-up of the Italian asp viper.

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.

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.

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

Significance Human-mediated species dispersal stretching back at least 10,000 y has left an indelible stamp on present day biodiversity. A major contributing factor to this process was the trade in a wide range of exotic species that was fueled by elite demand. The black francolin—now extinct in the western Mediterranean but once a courtly gamebird prized for its flavor, curative, and aphrodisiac qualities by European aristocracy—was one of these species. Using historical sources and DNA analysis of modern and archival specimens, we show that this bird was not native to the western Mediterranean, and document its introduction to Cyprus and westward through the Mediterranean Basin via several trade routes that reached as far east as South Asia. 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 DNA identity of the mouflon of Cyprus (Ovis orientalis ophion, Bovidae): Near Eastern origin and divergence from Western Mediterranean conspecific populations

The mouflon population of Cyprus (Ovis orientalis ophion) comprises historically preserved feral descendants of sheep domesticated during the Neolithic. We determined genetic identity of this taxon in order to elucidate its systematic placement and enforce its protection. We used 12 loci of microsatellite DNA to infer genetic relationships between the Cypriot mouflon and either long-time isolated (Corsica, Sardinia) or recently introduced (central Italy) European mouflons (O. o. musimon). We also sequenced the mitochondrial DNA (mtDNA) Cytochrome-b gene to infer the origin of the Cypriot mouflon including many National Centre for Biotechnology Information (NCBI) entries of European and Near Eastern conspecifics. Microsatellites disclosed net divergence between Western Mediterranean and Cypriot mouflon. The latter was included in the highly heterogeneous Near Eastern O. orientalis mtDNA group, Iran representing the most credited region as the source for its ancient introduction to Cyprus. Both international and national legislation protect the mouflon of Cyprus as a wild taxon (O. o. ophion). However, the IUCN Red List of Threatened Species and NCBI include the Cypriot mouflon as subspecies of its respective domestic species, the sheep (O. aries). Unfortunately, people charged with crime against protected mouflon may benefit from such taxonomic inconsistency between legislation and databases, as the latter can frustrate molecular DNA forensic outcomes. Until a definitive light can be shed on Near Eastern O. orientalis systematics, we suggest that the Cypriot mouflon should be unvaryingly referred to as O. o. ophion in order not to impair conservation in the country where it resides.

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.

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.

DNA barcoding of bird species in Cyprus: a tool for conservation purposes

DNA barcoding has become a widely used and valuable tool in taxonomic research during the past three decades and its application is expanding to fields such as wildlife forensics (e.g. anti-poaching actions). One of the major advantages of DNA barcoding is that it allows for species identification from largely incomplete, deformed or cooked samples and even from traces of biological material. Cyprus is a well-known biodiversity hotspot, the island avifauna comprising around 400 species, 117 of which are listed in Annex I of the Birds Directive (2009/147/EC). Each year on average, 2.3 million individuals (mainly small migratory birds) are trapped illegally on the island and often served at restaurants. Consequently, reliable identification of specimens from processed tissues can be crucial for undertaking legal actions. Herein DNA barcoding of the large majority of bird species resident in Cyprus plus several migrants that were illegally captured was carried out to support local authorities in their anti-poaching actions. In this study, tissue from 103 specimens representing 81 bird taxa were sampled and a 648-bp long fragment of the Cytochrome Oxidase subunit Ι (COI) gene was targeted using a combination of standard, nested and semi-nested PCR depending on the condition of the tissue at hand. Sequences were obtained from all investigated samples and a total of 92 haplotypes was recovered. Phylogenetic reconstructions returned a pattern of relationships among taxa largely compatible with known avian phylogeny. Furthermore, diagnostic polymorphisms within the genus including one of the main targeted species, Sylvia atricapilla, were found. In conclusion, an avian genetic dataset is now available for the law enforcement authorities to identify specimens either confiscated (even when morphologically deformed or cooked) during raids in restaurants and in other premises suspected of serving illegally protected bird species or from trappers.

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.