Filippo Barbanera

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.

A Quantitative Approach to Movement, Displacement, and Mobility of Protozoa

ABSTRACT Diffusion theory can completely describe the movement of a ciliate along a track of a certain length (L), travelled in a time (t), and with the extremes lying at a distance D. Three important descriptors of this behavior are: (1) the kinetic index (Ik= L/t), namely the average velocity in μm/s, which expresses the state of the “accelerator” of the ciliate; (2) the geometric index (Ig= D/L) measuring the straightness of the track by a dimensionless number. 0 ≤ Ig≤ 1, which expresses the state of the “steering wheel” and represents a sort of “directional efficiency”; and (3) the displacement rate (Rd= D/t), integrating the first two indices and expressing the combined effect of the “accelerator” and the “steering wheel” of the organism with a unique measure (in μm/s), which defines the average displacement rate or the effectiveness of the track in displacing the organism in space. A weighted estimate of general mobility is given by the mobility rate [Rmo= (R̄d.f)creeping‐ (R̄d.f)swimming], obtained by multiplying the average Rd of the creeping organisms and the average Rd of the swimming organisms by their relative frequencies of occurrence (f), and adding the two products. Values for experimental populations of Oxytricha bifaria (Ciliata, Hypotrichida) maintained at 24, 19, 14, and 9° C demonstrated both the appropriateness and the usefulness of these indices and rates to describe the tracks a posteriori, and to provide measures to reason about their possible adaptive significance.

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.

Breeding ecology of the Basra Reed Warbler, Acrocephalus griseldis, in Iraq (Aves: Passeriformes: Acrocephalidae)

The population of the Basra Reed Warbler, Acrocephalus griseldis, which has a limited range confined to the marshlands of central and southern Iraq, has declined substantially due to massive degradation of its main breeding habitat in the Iraqi marshes during the 1980s and 1990s. The breeding habitat and breeding biology is described here. We discovered a remarkable extension of the breeding range in the extreme west of Iraq.

Not-Adaptive Behavior of Isotropically Heated, Inert Populations of Oxytricha bifaria (Ciliophora, Stichotrichia)

Abstract The physiological effects on isotropically heated populations of Oxytricha bifaria cultured at 24 °C were investigated. At 34.6 °C ciliates became inert, and did not adaptively react to either cold or warm microgradients; they neither moved towards the favorable cold thermal source nor escaped from the unfavorable warm one. The inert oxytrichas were only able to perform the Side-Stepping Reaction (SSR) on the same spot. However, mobile ciliates at 31.6 °C reacted to the cold microgradient by immediately orienting themselves towards its source, without accelerating but reducing their SSR frequency. Moreover, in a warm microgradient such ciliates immediately increased their SSR frequency, then moved away from the thermal source. At 34.6 °C the behavior of ciliates was not-adaptive—not acting to guide the organisms to more favorable conditions—whereas at 31.6 °C it was still clearly adaptive. Therefore, the locomotory inertness of the oxytrichas at 34.6 °C was the result of thermal stress rather than their behavioral response to the environmental isotropy, in contrast to populations of the same species made inert at 9 °C.

Cold Microgradients Elicit Adaptive Behavior in Isotropically Cooled, Inert Populations of Oxytricha bifaria (Ciliophora, Hypotrichida)

Abstract To complete our investigations on the oriented behavioral response of isotropically cooled, inert populations of Oxytricha bifaria to a warm thermal gradient, their physiological potentialities under cold microgradient conditions arising at 8.5 °C were studied. We monitored the behavior of the experimental populations, both at the level of the passing cold wave front, and afterwards when the thermal gradient stabilized, evaluating (i) their distribution in general, (ii) their relative centroids, (iii) the percentage of both backward creeping and immobile ciliates, and (iv) the numerical indices and rates of their creeping tracks. At the arrival of the cold wave front, the oxytrichas react immediately to the thermal stimulus, creep backwards at very high velocity along uninterrupted linear tracks, and thus move away from the cooling source. No specific behavioral response was ever observed in the static microgradient conditions. At 8.5 °C, despite their inertness, the ciliates are still able to behave adaptively, reacting immediately and orientatedly, once a directional factor (the cold-repelling thermal gradient) arises in an isotropic environment. This is similar to their behavior in the symmetric warm attracting thermal gradient.