Struttura genetica e rapporti filogenetici in popolazioni di pernici del genere Alectoris (Aves, Galliformes) del Mediterraneo: implicazioni per la conservazione

Il genere Alectoris (Aves, Galliformes)comprende sette specie distribuite nel Paleartico. In particolare, sono state oggetto di studio in questa tesi le quattro specie presenti nella regione del Mar Mediterraneo: la pernice rossa (A. rufa), la coturnice orientale (A. chukar), la pernice sarda (A. barbara) e la coturnice (A. graeca). Scopo di questa ricerca è l'analisi della struttura genetica e dei rapporti filogenetico-molecolari di popolazioni naturali e di allevamento di pernici del genere Alectoris del Mediterraneo e lo studio dell'affinità riproduttiva di esemplari ibridi ottenuti incrociando in cattività la pernice sarda e la pernice rossa, due specie per le quali non è riportato alcun caso di ibridazione in letteratura. Le analisi genetiche sono condotte impiegando tre diverse metodologie: (1) marcatori mitocondriali (mtDNA, Citocromo-b e Regione di controllo), (2) marcatori nucleari del DNA microsatellitare (STR, Short Tandem Repeats Sequences), (3) marcatori nucleari RAPD (Random Amplified polymorphic dNA)

non native and hybrid in a changing environment conservation perspectives for the last italian red legged partridge alectoris rufa population with long natural history

Abstract The ever-increasing biotic homogenization - especially when associated with introgressive hybridization - raises concern for the reduction of 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 to 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 assignation to the nominate subspecies found in 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

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

Preliminary data on the distribution, morphology and genetics of white-clawed crayfish and on their ectosymbionts in Lunigiana (Tuscany, Italy)

The white-clawed crayfish is a complex of species (Austropotamobius pallipes plus A. italicus, Crustacea) pivotal to riverine environments. Regrettably, it is endangered in many European countries due to a recent massive decline. We carried out a fine-scale survey on white-clawed crayfish distribution, morphology and genetics in an under-investigated region of central Italy (Lunigiana, Tuscany) to improve local knowledge and aid conservation management of the species. The torrential fauna of Lunigiana is scarcely known and habitat alterations due to the anthropic exploitation of the territory represent a potential threat to its persistence. We investigated crayfish occurrence in eight streams of the Magra River Basin. We performed nocturnal and diurnal transects, took morphometric measurements, collected samples for genetic analysis and checked for the occurrence of ectosymbionts. Crayfish were disclosed in two streams (Civasola, Verdesina). Morphometric and phylogenetic analysis (mtDNA COI gene Bayesian tree reconstruction) concurrently identified both populations as Austropotamobius italicus carinthiacus. In the Verdesina stream, crayfish were heavily infested by branchiobdellidans (Annelida). Verdesina crayfish were also significantly smaller than those found in the Civasola stream, where branchiobdellidans were absent. Hence, we hypothesized that such difference in size might be related to the high density of ectosymbionts. In the light of habitat features and of data herein provided, we propose that both Civasola and Verdesina streams should be considered as “natural ark sites” for A. italicus, with the Magra River Basin representing a regional stronghold for the conservation of the specie

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 resource

Investigation of ancient DNA to enhance natural history museum collections: misidentification of smooth-coated otter (Lutrogale perspicillata) specimens across multiple museums

Historical and modern natural museum collections are storehouses of extraordinary value for scientific research in a wide range of fields. Recent advances in molecular biotechnology (e.g., next generation genomics) have increased the range of collection material employable for DNA-based analyses to unprecedented levels. Nevertheless, the value of museum specimens strictly depends on reliability of data associated with them. We report on investigations of ancient DNA from specimens of smooth-coated otter (Lutrogale perspicillata, Mustelidae), the largest otter species living in Asia, in US and European mammal collections. Mitochondrial DNA Cytochrome-b gene sequencing proved that the studied specimens were not the expected taxon. Indeed, they actually belonged to three different species, namely the Asian small-clawed (Aonyx cinereus), Eurasian (Lutra lutra) and African clawless (Aonyx capensis) otters. This represents the first record of mustelid misidentification from museum collections. Detection of errors can be extremely difficult when based only on collectors’ notes and data. Hence, we warn scientists involved in otter research about potential challenges when dealing with museum specimens. We recommend curators pursue a multidisciplinary approach, including DNA analyses, to accurately catalogue the resources under their management and uphold the value of biodiversity information

Introductions over introductions: the genomic adulteration of an early genetically valuable alien species in the United Kingdom.

Invasive alien species are a major cause of biodiversity loss. Nevertheless, non-native species can also contribute to conservation objectives. In 1673, the red-legged partridge (Alectoris rufa), a galliform native to southwest Europe, was introduced from France (A. r. rufa) into the UK for hunting purposes. Nowadays, hunters constantly supplement natural populations of A. rufa in its native range with stocks of captive-bred individuals. Such birds are usually genetically unscreened, and human-mediated hybridization with the exotic chukar (Alectoris chukar) has undermined genomic integrity of the species. Alectoris rufa in the UK has never been genetically investigated, and birds from East Anglian estates with no modern history of supplementation offer a potential genomic backup for the highly polluted native-range A. r. rufa. We genotyped modern and ancient (1824–1934) birds at the mitochondrial DNA (mtDNA) level to determine present and past kinship between East Anglian and native-range A. rufa. We used Short Tandem Repeats (STR) and Random Amplified Polymorphic DNA (RAPD) markers to identify A. rufa × A. chukar hybrids. The kinship of East Anglian birds with A. r. rufa was confirmed. No A. chukar introgression was found in ancient East Anglian A. rufa. Among modern partridges, we found birds with A. chukar mtDNA, and both STRs and RAPDs disclosed many A. rufa × A. chukar hybrids. While the genetic analysis pointed to the increase of diversity and decline of disparity over time within and among A. rufa populations, respectively, the conservation value of the resource historically introduced to the UK proved to have been quashed by three decades of recent releases of A. chukar and its A. rufa hybrids. © 2014, Springer International Publishing Switzerland

Bergamot (Citrus bergamia Risso) fruit extracts and identified components alter expression of interleukin 8 gene in cystic fibrosis bronchial epithelial cell lines

<p>Abstract</p> <p>Background</p> <p>Cystic fibrosis (CF) airway pathology is a fatal, autosomal, recessive genetic disease characterized by extensive lung inflammation. After induction by TNF-α, elevated concentrations of several pro-inflammatory cytokines (i.e. IL-6, IL-1β) and chemokines (i.e. IL-8) are released from airway epithelial cells. In order to reduce the excessive inflammatory response in the airways of CF patients, new therapies have been developed and in this respect, medicinal plant extracts have been studied. In this article we have investigated the possible use of bergamot extracts (<it>Citrus bergamia </it>Risso) and their identified components to alter the expression of IL-8 associated with the cystic fibrosis airway pathology.</p> <p>Methods</p> <p>The extracts were chemically characterized by ¹H-NMR (nuclear magnetic resonance), GC-FID (gas chromatography-flame ionization detector), GC-MS (gas chromatography-mass spectrometry) and HPLC (high pressure liquid chromatography). Both bergamot extracts and main detected chemical constituents were assayed for their biological activity measuring (a) cytokines and chemokines in culture supernatants released from cystic fibrosis IB3-1 cells treated with TNF-α by Bio-Plex cytokine assay; (b) accumulation of IL-8 mRNA by real-time PCR.</p> <p>Results</p> <p>The extracts obtained from bergamot (<it>Citrus bergamia </it>Risso) epicarps contain components displaying an inhibitory activity on IL-8. Particularly, the most active molecules were bergapten and citropten. These effects have been confirmed by analyzing mRNA levels and protein release in the CF cellular models IB3-1 and CuFi-1 induced with TNF-α or exposed to heat-inactivated <it>Pseudomonas aeruginosa</it>.</p> <p>Conclusions</p> <p>These obtained results clearly indicate that bergapten and citropten are strong inhibitors of IL-8 expression and could be proposed for further studies to verify possible anti-inflammatory properties to reduce lung inflammation in CF patients.</p

Primo dato genetico per il fratino (Charadrius alexandrinus) in Italia e confronto su scala continentale

The Kentish Plover (Charadrius alexandrinus, Charadriformes) is a shorebird with a huge range including beaches and salt pans across northern temperate to subtropical latitudes of Europe, Asia, and Africa. In the last decades, the availability of breeding sites for this species has been greatly reduced through direct destruction or functional loss of dune areas due to a remarkable expansion of coastal tourism. Altogether this has often prevented the Kentish Plover from settling along the shore and, wherever possible, its reproductive success has in any case been negatively impacted by human disturbance. In Italy, the species is in sharp decline and reported as Endangered in the national Red List of vertebrates. Three well-established nuclei are known in Tuscany, with one occurring in the municipality of Castagneto Carducci (Province of Livorno). In this area, after a strong storm a cracked egg was recovered in 2020 on the beach of the Special Area of Conservation/Special Protection Area Padule di Bolgheri. A 523 bp-long fragment of the mitochondrial DNA Control Region was amplified by PCR. The sequence was aligned with those available (n = 198) in the GenBank (i) to determine relationships between the Italian representative and conspecifics from the entire range based on both their geographical origin and affiliation to mainland/island populations, (ii) to identify genetically homogeneous clusters, and (iii) to test a hypothesis of sudden demographic expansion across the entire range of the species. Overall, the haplotypic diversity (h) was 0.86 ± 0.02 while the nucleotide diversity (π, %) was 0.46 ± 0.28, with the island populations holding higher values than the mainland ones (except Azores: h = 0.12 ± 0.11, π = 0.24 ± 0.43). Thirty-five (e.g., Taiwan, 8; Ukraine, 7; Russia, 5) over a total of 47 haplotypes (H) turned out to be private, while all the remaining ones were shared by several populations. Of these, two (H3, H14) showed up with a remarkably high frequency (18.5% and 31.6%, respectively): H3 occurred almost exclusively in Europe while H14 across the whole Eurasia (more to the East than to the West). The Median Joining network reflected a double-star structure with most of the haplotypes being a single-mutational step away from H3 and H14, a pattern that did not change when the birds from the mainland and island populations were investigated separately. The single Italian representative was assigned to H3 as well as most of the individuals from the Iberian Peninsula and Macaronesia. The Analysis of the Molecular Variance indicated that the 25.53% of the genetic diversity was partitioned among populations and the 74.47% among individuals within populations (φST = 0.25; P &lt; 0.001). The values of Fs and D statistics of Fu’s and Tajima tests were - 26.782 (P &lt; 0.001) and 1.680 (P = 0.016), respectively, and the curve of Mismatch Distribution showed a markedly unimodal trend (Harpending index: r = 0.027, P = 0.45). Therefore, the hypothesis of a sudden demographic expansion across the whole species’ range could not be rejected. A Bayesian clustering analysis of the 199 investigated sequences pointed to the occurrence of three genetic groups. Clusters 1 (red, n = 86; mean membership value, 0.43) and 3 (green, n = 83, with the Italian representative: mean membership value, 0.46) occurred mostly across Europe and in the eastern part of the species’ range, respectively, whereas cluster 2 (yellow, n = 30: mean membership value, 0.11) included birds from Cape Verde, Russia, and the United Arab Emirates. However, these groups did not reflect a significant spatial genetic structure across the Kentish Plover’s range, thus confirming the well-known high levels of intraspecific gene flow. In conclusion, while this study provided the first contribution to the knowledge of genetic relationships of the Italian Kentish Plover population, a national scale investigation of this species to draw up more adequately informed conservation strategies cannot be delayed any longer

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 (&gt; 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 &amp; 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 &gt;3000 and Random Match Probability &lt;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