Population genomic history of the endangered Anatolian and Cyprian mouflons in relation to worldwide wild, feral and domestic sheep lineages.

Once widespread in their homelands, the Anatolian mouflon (Ovis gmelini anatolica) and the Cyprian mouflon (Ovis gmelini ophion) were driven to near extinction during the 20th century and are currently listed as endangered populations by the IUCN. While the exact origins of these lineages remain unclear, they have been suggested to be close relatives of domestic sheep or remnants of proto-domestic sheep. Here, we study whole genome sequences of n = 5 Anatolian mouflons and n = 10 Cyprian mouflons in terms of population history and diversity, comparing them to eight other extant sheep lineages. We find reciprocal genetic affinity between Anatolian and Cyprian mouflons and domestic sheep, higher than all other studied wild sheep genomes, including the Iranian mouflon (Ovis gmelini). Studying diversity indices, we detect a considerable load of short runs of homozygosity (ROH) blocks (<2 Mb) in both Anatolian and Cyprian mouflons, reflecting small effective population size (Ne). Meanwhile, Ne as well as mutation load estimates are lower in Cyprian compared to Anatolian mouflons, suggesting the purging of recessive deleterious variants in Cyprian sheep under a small long-term Ne, possibly attributable to founder effects, island isolation, introgression from domestic lineages, or differences in their bottleneck dynamics. Expanding our analyses to worldwide wild and feral Ovis genomes, we observe varying viability metrics among different lineages, and a limited consistency between viability metrics and IUCN conservation status. Factors such as recent inbreeding, introgression, and unique population dynamics may have contributed to the observed disparities.

First Detection of Cryptosporidium parvum in the Endemic Cyprus Mouflon (Ovis gmelini ophion).

PURPOSE: Cryptosporidium is an intestinal zoonotic protozoan parasite that infects domesticated and wild animals. There are no reports on the prevalence and molecular characterisation of Cryptosporidium in the endemic Cyprus mouflon. The mouflon is strictly protected by national and international legislation. Its main distribution is Paphos State Forest and surrounding areas, where it may share the same water sources as free-ranging domestic goats. Therefore, the present study aimed to determine the prevalence of Cryptosporidium spp. and genotypes in mouflon and free-ranging goats within the mouflon range. METHODS: Faecal samples of 70 mouflons and 34 free-ranging goats were screened for Cryptosporidium by PCR amplification and sequencing. RESULTS: Only one sample (1/70) belonging to a mouflon was PCR positive for Cryptosporidium. Based on sequencing of the 18S rRNA locus, this species was identified as Cryptosporidium parvum (C. parvum). No positive sample was detected in the free-ranging goats (0/34). CONCLUSION: This is the first report on the molecular identification of this Cryptosporidium species in a Cyprus mouflon. The results indicate that the prevalence of Cryptosporidium in Cyprus mouflon is low.

Lead contamination of soils, sediments, and vegetation in a shooting range and adjacent terrestrial and aquatic ecosystems: A holistic approach for evaluating potential risks.

It is well accepted that shooting ranges constitute hotspots of Pb contamination. This study evaluated the degree of Pb contamination of soils, sediments and vegetation within the boundaries of a highly visited shooting range, as well as the fluvial transport and dispersal of Pb, and therefore the contamination of adjacent river and water reservoir. Soils in the shooting range were severely contaminated with Pb, as indicated by the values of enrichment and contamination factor. The concentration of Pb in these soils ranged from 791 mg kg-1 to 7265 mg kg-1, being several dozens or even hundreds of times higher compared with control background samples. A temporary stream being in close proximity was also polluted, though to a much lesser extent. The degree of Pb contamination was negatively correlated with the distance from the shooting range. To this effect, the degree of contamination of the river and the water reservoir being in the vicinity of the shooting range was negligible, as sediments and water samples preserved similar Pb concentrations with control samples. However, cultivated (olives) and wild native plant species grown in the area of the shooting range were found to uptake and accumulate high concentrations of Pb in their tissues (even 50 times higher compared with control samples). The severe contamination of soils, sediments and vegetation in the studied shooting range can provoke very high ecological risks. Overall, results suggest that management measures should be undertaken within the boundaries of the studied shooting range.

First mtDNA Sequences and Body Measurements for Rattus norvegicus from the Mediterranean Island of Cyprus.

Invasive species are the primary driver of island taxa extinctions and, among them, those belonging to the genus Rattus are considered as the most damaging. The presence of black rat (Rattus rattus) on Cyprus has long been established, while that of brown rat (Rattus norvegicus) is dubious. This study is the first to provide molecular and morphological data to document the occurrence of R. norvegicus in the island of Cyprus. A total of 223 black rats and 14 brown rats were collected. Each sample was first taxonomically attributed on the basis of body measurements and cranial observations. Four of the specimens identified as R. norvegicus and one identified as R. rattus were subjected to molecular characterization in order to corroborate species identification. The analyses of the mitochondrial control region were consistent with morphological data, supporting the taxonomic identification of the samples. At least two maternal molecular lineages for R. norvegicus were found in Cyprus. The small number of brown rats collected in the island, as well as the large number of samples of black rats retrieved in the past years might be an indication that the distribution of R. norvegicus is still limited into three out of the six districts of Cyprus.

Mitochondrial D-loop Sequence Variability in Three Native Insular Griffon Vulture (Gyps fulvus) Populations from the Mediterranean Basin.

The islands of Sardinia, Crete, and Cyprus are hosting the last native insular griffon populations in the Mediterranean basin. Their states have been evaluated from "vulnerable" to "critically endangered". The sequence analysis of molecular markers, particularly the mtDNA D-loop region, provides useful information in studying the evolution of closely related taxa and the conservation of endangered species. Therefore, a study of D-loop region sequence was carried out to estimate the genetic diversity and phylogenetic relationship within and among these three populations. Among 84 griffon specimens (44 Sardinian, 33 Cretan, and 7 Cypriot), we detected four haplotypes including a novel haplotype (HPT-D) that was exclusively found in the Cretan population with a frequency of 6.1%. When considered as a unique population, haplotype diversity (Hd) and nucleotide diversity (π) were high at 0.474 and 0.00176, respectively. A similar level of Hd and π was found in Sardinian and Cretan populations, both showing three haplotypes. The different haplotype frequencies and exclusivity detected were in accordance with the limited matrilineal gene flow (FST = 0.07097), probably related to the species reluctance to fly over sea masses. The genetic variability we observe today would therefore be the result of an evolutionary process strongly influenced by isolation leading to the appearance of island variants which deserve to be protected. Furthermore, since nesting sites and food availability are essential elements for colony settlement, we may infer that the island's colonization began when the first domestic animals were transferred by humans during the Neolithic. In conclusion, our research presents a first contribution to the genetic characterization of the griffon vulture populations in the Mediterranean islands of Sardinia, Crete and Cyprus and lays the foundation for conservation and restocking programs.

Identification of an ancestral haplotype in the mitochondrial phylogeny of the ovine haplogroup B.

BACKGROUND: European mouflon (Ovis orientalis musimon) has been reintroduced in mainland Europe since the 18th-century sourcing from the Sardinian and Corsican autochthonous mouflon populations. The European mouflon is currently considered the feral descendent of the Asian mouflon (O. orientalis), and the result of first wave of sheep domestication occurred 11,000 years ago in the Fertile Crescent, and brought to Corsica and Sardinia ca. 6,000 years ago, where they still live as autochthonous populations. However, this phylogeny is based on mitogenome sequences of European mouflon individuals exclusively. METHODS: We sequenced the first complete mtDNA of the long-time isolated Sardinian mouflon and compared it with several ovine homologous sequences, including mouflon from mainland Europe and samples representative of the five known mitochondrial domestic sheep haplogroups. We applied Bayesian inference, Maximum Likelihood and Integer Neighbour-Joining network methods and provided a robust, fully-resolved phylogeny with strong statistical support for all nodes. RESULTS: We identified an early split (110,000 years ago) of the Sardinian mouflon haplotype from both sheep and mainland European mouflon belonging to haplogroup B, the latter two sharing a more recent common ancestor (80,000 years ago). Further, the Sardinian mouflon sequence we generated had the largest genetic distance from domestic sheep haplogroups (0.0136 ± 0.004) among mouflon species. Our results suggest the Sardinian mouflon haplotype as the most ancestral in the HPG-B lineage, hence partially redrawing the known phylogeny of the genus Ovis.

The First Mitogenome of the Cyprus Mouflon (Ovis gmelini ophion): New Insights into the Phylogeny of the Genus Ovis.

Sheep are thought to have been one of the first livestock to be domesticated in the Near East, thus playing an important role in human history. The current whole mitochondrial genome phylogeny for the genus Ovis is based on: the five main domestic haplogroups occurring among sheep (O. aries), along with molecular data from two wild European mouflons, three urials, and one argali. With the aim to shed some further light on the phylogenetic relationship within this genus, the first complete mitochondrial genome sequence of a Cypriot mouflon (O. gmelini ophion) is here reported. Phylogenetic analyses were performed using a dataset of whole Ovis mitogenomes as well as D-loop sequences. The concatenated sequence of 28 mitochondrial genes of one Cypriot mouflon, and the D-loop sequence of three Cypriot mouflons were compared to sequences obtained from samples representatives of the five domestic sheep haplogroups along with samples of the extant wild and feral sheep. The sample included also individuals from the Mediterranean islands of Sardinia and Corsica hosting remnants of the first wave of domestication that likely went then back to feral life. The divergence time between branches in the phylogenetic tree has been calculated using seven different calibration points by means of Bayesian and Maximum Likelihood inferences. Results suggest that urial (O. vignei) and argali (O. ammon) diverged from domestic sheep about 0.89 and 1.11 million years ago (MYA), respectively; and dates the earliest radiation of domestic sheep common ancestor at around 0.3 MYA. Additionally, our data suggest that the rise of the modern sheep haplogroups happened in the span of time between six and 32 thousand years ago (KYA). A close phylogenetic relationship between the Cypriot and the Anatolian mouflon carrying the X haplotype was detected. The genetic distance between this group and the other ovine haplogroups supports the hypothesis that it may be a new haplogroup never described before. Furthermore, the updated phylogenetic tree presented in this study determines a finer classification of ovine species and may help to classify more accurately new mitogenomes within the established haplogroups so far identified.

The sequence and phylogenesis of the ?-globin genes of Barbary sheep (Ammotragus lervia), goat (Capra hircus), European mouflon (Ovis aries musimon) and Cyprus mouflon (Ovis aries ophion).

In order to investigate the polymorphism of ?-globin chain of hemoglobin amongst caprines, the linked (I)? and (II)? globin genes of Barbary sheep (Ammotragus lervia), goat (Capra hircus), European mouflon (Ovis aries musimon), and Cyprus mouflon (Ovis aries ophion) were completely sequenced, including the 5? and 3? untranslated regions. European and Cyprus mouflons, which do not show polymorphic ? globin chains, had almost identical ? globin genes, whereas Barbary sheep exhibit two different chains encoded by two nonallelic genes. Four different ? genes were observed and sequenced in goat, validating previous observations of the existence of allelic and nonallelic polymorphism. As in other vertebrates, interchromosomal gene conversion appears to be responsible for such polymorphism. Evaluation of nucleotide sequences at the level of molecular evolution of the (I)?-globin gene family in the caprine taxa suggests a closer relationship between the genus Ammotragus and Capra. Molecular clock estimates suggest sheep-mouflon, goat-aoudad, and ancestor-caprine divergences of 2.8, 5.7, and 7.1 MYBP, respectively.

Introgression of Alectoris chukar genes into a Spanish wild Alectoris rufa population.

In order to detect introgression of other Alectoris genus species into wild populations of Spanish Alectoris rufa, we studied a sample of 93 red-legged partridges (supposed to be A. rufa) captured in the island of Majorca. A set of 31 chukar partridges (Alectoris chukar) from Cyprus and 33 red-legged partridges (A. rufa) from one Spanish farm were also studied to provide suitable populations for comparison. Factorial correspondence analysis on microsatellite genotypes supported a clear distinction of birds from Cyprus, whereas partridges from Majorca and the Spanish farm overlapped in a wide area. The existence of A. chukar mitochondrial DNA in 16 individuals from Majorca indicated introgression into their maternal lineage even if their phenotypes were not different from A. rufa. Bayesian inference based on microsatellite analysis indicated a noticeable degree of genetic proximity to A. chukar only for one of these hybrids.

Structure and function of sheep hemoglobin Chios: A novel allele at the HBBB locus with two Lys-->Arg substitutions at positions beta66(E10) and beta144(HC1).

A novel hemoglobin variant was observed in pure sheep (Ovis aries) breeds of the island of Chios (Greece), Egypt and Hungary. This silent variant was identified by gel electrophoresis and RP-HPLC of dissociated globin chains. Two Arg for Lys substitutions were detected, by means of MALDI TOF electrospray mass spectrometric analysis for the intact globins, at positions beta66(E10) and beta144(HC1) of a globin chain having the sequence of the beta(B) chain. Sequencing of the beta-globin gene confirmed the variant gene as being an allele of the HBBB locus having the AAG-->AGG and the AAA-->AGA mutations at codons 66 and 144, respectively, both corresponding to the Lys-->Arg substitution. The intrinsic oxygen affinity of the variant Hb (logP(50)=0.79 at pH 7.0) was found to be intermediate between that of the sheep Hb B (logP(50)=0.92) and that of Cypriot mouflon (O. a. ophion) Hb (logP(50)=0.53), the latter having only the Lys-->Arg change at beta144, whereas nearly no differences were observed in the presence of the Cl(-) physiological effector. Result supports the indication that Arg at beta144 enhances the role of the ligand in decreasing oxygen affinity, this effect being partially counteracted when Arg is at beta66. Data also shows that the Lys-->Arg change at beta66 is responsible for 1.49 fold reduction in the intrinsic oxygen affinity. This hitherto undescribed variant increases to seven the number of alleles at the sheep HBBB locus. Following the nomenclature used for human Hb variants, the new allele was termed as the Hb Chios or [beta(B)66(E10) Lys-->Arg, 144(HC1)Lys-->Arg], whereas the proposed genetic nomenclature of the locus is HBBK.

[A new species of wild mice on the Island of Cyprus]. / Découverte d'une nouvelle espèce de souris sur l'île de Chypre.

A mitochondrial and nuclear gene analysis allowed us to precise the taxonomical position of the two sympatric species of mice known to be present on Cyprus. One of them is the commensal house mouse M. m. domesticus, and the other revealed to be a new taxon that is a sister species of M. spicilegus and M. macedonicus. The new species is equidistant from each of these, the divergence dating around 0.5-1 Myr. Its origin either results from an ancient accidental colonisation of the island or from a recent transportation by the first epipalaeolithic settlers. In this last eventuality, the new species would also exist somewhere else in Asia Minor.