Monk seals (Monachus monachus) in the Mediterranean Sea: The threat of organochlorine contaminants and polycyclic aromatic hydrocarbons.

The Mediterranean monk seal (Monachus monachus) is an emblematic species of conservation concern. Anthropogenic pressures have led to severe population declines and significant fragmentation of the remaining populations. Because of their close relationship with coastal areas, Mediterranean monk seals may be potentially exposed to pollution from agricultural sources, as well as from oil tanker spills. Although monitoring of pollution has long been considered a priority for this species, data on monk seal contamination levels are scarce. In this study, 55 blubber samples of all genders and age classes collected during necropsies (1995-2013) from seals of the Eastern Mediterranean subpopulation were analyzed for organochlorine compounds (OCs), i.e., hexachlorobenzene (HCB), polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethane (DDTs), and polycyclic aromatic hydrocarbons (PAHs). Overall, PCBs > DDTs ≫ PAHs > HCB in all samples. Results showed a significant downward trend over the 19-year period for DDTs and HCB. No marked pattern was found for PAHs, even though relative abundance of cancerogenic PAH fraction rose in recent years. PCB levels in subadult specimens increased noticeably over time despite worldwide ban. Our findings did not suggest recent releases of DDT or new pesticides (e.g., Dicofol) in the environment, but may indicate an improper disposal of ancient storages of PCBs or a remobilization from reservoirs. OC levels exceeded thresholds that are commonly associated with immunosuppression and reproductive impairment in other pinnipeds. Hence, OCs could be responsible of potential toxicological effects in this subpopulation. This study represents the first report on PAH and one of the few reports on OC bioaccumulation in Mediterranean monk seals. Based on the levels found in the blubber of monk seals from the Eastern Mediterranean, and given the known harmful effects associated to the prolonged exposure to these substances and the reduced long-term expectations of species recovery, regular monitoring is advocated.

First Description of the Underwater Sounds in the Mediterranean Monk Seal Monachus monachus in Greece: Towards Establishing a Vocal Repertoire.

The Mediterranean monk seal Monachus monachus is one of the most endangered pinnipeds in the world, and is classified as "Endangered" by the International Union for the Conservation of Nature. Any additional knowledge about the species is invaluable to its effective conservation. In the present study, we deployed an autonomous underwater recorder in an important reproductive area of the Mediterranean monk seal in Greece to describe its underwater vocal repertoire. Over the 330 h of continuous recordings, 9231 vocalizations were labelled as potentially produced by Mediterranean monk seals, and 1694 good quality calls were analyzed. We defined 18 call types divided into three main call categories: harmonic, noisy, and pulsative calls. We also described the soundscape in which this endangered species lives and found that human activities around the two main pupping caves had a strong impact on the sonic environment of these seals: the noise level produced by boat traffic was high, and occurred on an hourly (25 to 50 min/hour) and daily basis (10.8 to 16.9 h/day). Such high levels of noise might not only impair the communication of the species, but also impact its survival, as chronic noise can induce physiological stress.

The antique genetic plight of the Mediterranean monk seal (Monachus monachus).

Disentangling the impact of Late Quaternary climate change from human activities can have crucial implications on the conservation of endangered species. We investigated the population genetics and demography of the Mediterranean monk seal (Monachus monachus), one of the world's most endangered marine mammals, through an unprecedented dataset encompassing historical (extinct) and extant populations from the eastern North Atlantic to the entire Mediterranean Basin. We show that Cabo Blanco (Western Sahara/Mauritania), Madeira, Western Mediterranean (historical range) and Eastern Mediterranean regions segregate into four populations. This structure is probably the consequence of recent drift, combined with long-term isolation by distance (R2 = 0.7), resulting from prevailing short-distance (less than 500 km) and infrequent long-distance dispersal (less than 1500 km). All populations (Madeira especially), show high levels of inbreeding and low levels of genetic diversity, seemingly declining since historical time, but surprisingly not being impacted by the 1997 massive die-off in Cabo Blanco. Approximate Bayesian Computation analyses support scenarios combining local extinctions and a major effective population size decline in all populations during Antiquity. Our results suggest that the early densification of human populations around the Mediterranean Basin coupled with the development of seafaring techniques were the main drivers of the decline of Mediterranean monk seals.

Zoonotic Thelazia callipaeda eyeworm in brown bears (Ursus arctos): A new host record in Europe.

Thelazia callipaeda (Spirurida, Thelaziidae) eyeworm, a zoonotic parasite transmitted by lachryphagus drosophilids, causes subclinical to clinical ocular disease in a wide range of vertebrates, including humans. In the past 20 years, the infection spread primarily in areas where the vector thrives, and now is endemic in many European countries. Recently, this nematode has been reported also in the United States. Here, we describe the first record of the brown bear (Ursus arctos) as a host of T. callipaeda. Eight (26.7%) out of 30 bears were found to be infected with T. callipaeda adult worms. In addition, the parasite was detected in 13 wolves (Canis lupus) sharing the same environment with the brown bears. At the molecular characterization, all nematodes belonged to the haplotype 1, which is the only one recorded in Europe, as yet. This finding demonstrates that brown bears may act as hosts for this parasite, suggesting its potential role in the maintenance of the cycle not only in the wild but also in captivity. Under the above circumstances, the brown bear may represent a potential zoonotic risk for humans, both in rural and urban areas where other reservoirs may be infected.

First Report of Uncinaria hamiltoni in Orphan Eastern Mediterranean Monk Seal Pups in Greece and Its Clinical Significance.

The Mediterranean monk seal (Monachus monachus) is classified by the IUCN as "endangered," with a global population estimated to number fewer than 800 individuals. Our understanding of the biology and health status of the species is still limited, rendering every medical case a challenge for conservationists and veterinary clinicians. Although studying and managing disease in wild marine hosts is complex and challenging, studying and mitigating the effects of any disease to the Mediterranean monk seal is of utmost importance for conservation. The aim of this study was to document for the first time the presence of the hookworm Uncinaria hamiltoni in rehabilitated Mediterranean monk seal pups in Greece. A detailed examination protocol was followed for all pups that live-stranded over 30 years in 22 different locations, including physical, parasitological, and other examinations. Hookworms (adults and/or eggs) were detected in all the fecal samples, from all animals. Molecular identification using MtDNA (COI) and ribosomal DNA (D2/D3 28S and internal transcribed spacer [ITS] regions) identified the nematode species as Uncinaria hamiltoni. The clinical impacts and the benefits of anthelmintic treatment as a tool for the conservation management of the species are discussed.

Genetic and demographic history define a conservation strategy for earth's most endangered pinniped, the Mediterranean monk seal Monachus monachus.

The Mediterranean monk seal (Monachus monachus) is a flagship species for marine conservation, but important aspects of its life history remain unknown. Concerns over imminent extinction motivated a nuclear DNA study of the species in its largest continuous subpopulation in the eastern Mediterranean Sea. Despite recent evidence of partial subpopulation recovery, we demonstrate that there is no reason for complacency, as the species still shares several traits that are characteristic of a critically endangered species: Mediterranean monk seals in the eastern Mediterranean survive in three isolated and genetically depauperate population clusters, with small effective population sizes and high levels of inbreeding. Our results indicated male philopatry over short distances, which is unexpected for a polygynous mammal. Such a pattern may be explained by the species' unique breeding behavior, in which males defend aquatic territories near breeding sites, while females are often forced to search for new pupping areas. Immediate action is necessary to reverse the downward spiral of population decline, inbreeding accumulation and loss of genetic diversity. We propose concrete conservation measures for the Mediterranean monk seal focusing on reducing anthropogenic threats, increasing the population size and genetic diversity, and thus improving the long-term prospects of survival.

Ungulate management in European national parks: Why a more integrated European policy is needed.

1. Primary objectives of national parks usually include both, the protection of natural processes and species conservation. When these objectives conflict, as occurs because of the cascading effects of large mammals (i.e., ungulates and large carnivores) on lower trophic levels, park managers have to decide upon the appropriate management while considering various local circumstances. 2. To analyse if ungulate management strategies are in accordance with the objectives defined for protected areas, we assessed the current status of ungulate management across European national parks using the naturalness concept and identified the variables that influence the management. 3. We collected data on ungulate management from 209 European national parks in 29 countries by means of a large-scale questionnaire survey. Ungulate management in the parks was compared by creating two naturalness scores. The first score reflects ungulate and large carnivore species compositions, and the second evaluates human intervention on ungulate populations. We then tested whether the two naturalness score categories are influenced by the management objectives, park size, years since establishment, percentage of government-owned land, and human impact on the environment (human influence index) using two generalized additive mixed models. 4. In 67.9% of the national parks, wildlife is regulated by culling (40.2%) or hunting (10.5%) or both (17.2%). Artificial feeding occurred in 81.3% of the national parks and only 28.5% of the national parks had a non-intervention zone covering at least 75% of the area. Furthermore, ungulate management differed greatly among the different countries, likely because of differences in hunting traditions and cultural and political backgrounds. Ungulate management was also influenced by park size, human impact on the landscape, and national park objectives, but after removing these variables from the full model the reduced models only showed a small change in the deviance explained. In areas with higher anthropogenic pressure, wildlife diversity tended to be lower and a higher number of domesticated species tended to be present. Human intervention (culling and artificial feeding) was lower in smaller national parks and when park objectives followed those set by the International Union for the Conservation of Nature (IUCN). 5. Our study shows that many European national parks do not fulfil the aims of protected area management as set by IUCN guidelines. In contrast to the USA and Canada, Europe currently has no common ungulate management policy within national parks. This lack of a common policy together with differences in species composition, hunting traditions, and cultural or political context has led to differences in ungulate management among European countries. To fulfil the aims and objectives of national parks and to develop ungulate management strategies further, we highlight the importance of creating a more integrated European ungulate management policy to meet the aims of national parks.

Assessing SNP genotyping of noninvasively collected wildlife samples using microfluidic arrays.

Noninvasively collected samples are a common source of DNA in wildlife genetic studies. Currently, single nucleotide polymorphism (SNP) genotyping using microfluidic arrays is emerging as an easy-to-use and cost-effective methodology. Here we assessed the performance of microfluidic SNP arrays in genotyping noninvasive samples from grey wolves, European wildcats and brown bears, and we compared results with traditional microsatellite genotyping. We successfully SNP-genotyped 87%, 80% and 97% of the wolf, cat and bear samples, respectively. Genotype recovery was higher based on SNPs, while both marker types identified the same individuals and provided almost identical estimates of pairwise differentiation. We found that samples for which all SNP loci were scored had no disagreements across the three replicates (except one locus in a wolf sample). Thus, we argue that call rate (amplification success) can be used as a proxy for genotype quality, allowing the reduction of replication effort when call rate is high. Furthermore, we used cycle threshold values of real-time PCR to guide the choice of protocols for SNP amplification. Finally, we provide general guidelines for successful SNP genotyping of degraded DNA using microfluidic technology.

Trace element concentrations in the Mediterranean monk seal (Monachus monachus) in the eastern Mediterranean Sea.

The Mediterranean monk seal (Monachus monachus) is one of the most endangered marine mammals in the world. The biggest sub-population of the species survives in Greece, where understanding the effects of pollution on the survival of the species has been identified as a national research and conservation priority. From 1990 to 2013 we collected tissue samples from 59 deceased monk seals in order to: (i) Define the concentration of trace elements (As, Pb, Cd, Hg, Se, Cr, Ni) in three different matrices (i.e., blubber, liver and kidney), (ii) Determine whether differences in trace element concentrations are age- or gender-related, (iii) Evaluate the potential effects of these pollutants. The study recorded differences in trace element concentrations among matrices, but in general, trace element exposure in Mediterranean monk seals in Greece was low and within the non-acutely toxic levels for Pinnipeds. Only arsenic concentrations were at the upper limit of the normal range observed in other marine mammals (0.69±0.55mg/kg w.w. in blubber, 0.79±0.62mg/kg w.w. in liver and 0.79±0.59mg/kg w.w. in kidney). We recorded also exceptionally high Hg concentrations in a single adult female (24.88mg/kg w.w.). Age- and gender-related differences were also recorded and were due to various biological, ecological and chemical factors. Based on the results of the study, potentially adverse effects on the immune and endocrine system of the Mediterranean monk seal from some pollutants (e.g., As, Cd, Se, Ni, Cr) cannot be ruled out, which may expose the Mediterranean seal population in Greece to epizootics and stochastic phenomena of mass mortality. It is therefore of utmost importance that pollutant monitoring becomes an integral component of the standard monitoring protocol of the endangered Mediterranean monk seal in the eastern Mediterranean.

Genome-wide analyses suggest parallel selection for universal traits may eclipse local environmental selection in a highly mobile carnivore.

Ecological and environmental heterogeneity can produce genetic differentiation in highly mobile species. Accordingly, local adaptation may be expected across comparatively short distances in the presence of marked environmental gradients. Within the European continent, wolves (Canis lupus) exhibit distinct north-south population differentiation. We investigated more than 67-K single nucleotide polymorphism (SNP) loci for signatures of local adaptation in 59 unrelated wolves from four previously identified population clusters (northcentral Europe n = 32, Carpathian Mountains n = 7, Dinaric-Balkan n = 9, Ukrainian Steppe n = 11). Our analyses combined identification of outlier loci with findings from genome-wide association study of individual genomic profiles and 12 environmental variables. We identified 353 candidate SNP loci. We examined the SNP position and neighboring megabase (1 Mb, one million bases) regions in the dog (C. lupus familiaris) genome for genes potentially under selection, including homologue genes in other vertebrates. These regions included functional genes for, for example, temperature regulation that may indicate local adaptation and genes controlling for functions universally important for wolves, including olfaction, hearing, vision, and cognitive functions. We also observed strong outliers not associated with any of the investigated variables, which could suggest selective pressures associated with other unmeasured environmental variables and/or demographic factors. These patterns are further supported by the examination of spatial distributions of the SNPs associated with universally important traits, which typically show marked differences in allele frequencies among population clusters. Accordingly, parallel selection for features important to all wolves may eclipse local environmental selection and implies long-term separation among population clusters.

Stable isotopes confirm a coastal diet for critically endangered Mediterranean monk seals.

Understanding the ecology and behaviour of endangered species is essential for developing effective management and conservation strategies. We used stable isotope analysis to investigate the foraging behaviour of critically endangered Mediterranean monk seals (Monachus monachus) in Greece. We measured carbon and nitrogen isotope ratios (expressed as δ(13)C and δ(15)N values, respectively) derived from the hair of deceased adult and juvenile seals and the muscle of their known prey to quantify their diets. We tested the hypothesis that monk seals primarily foraged for prey that occupy coastal habitats in Greece. We compared isotope values from seal hair to their coastal and pelagic prey (after correcting all prey for isotopic discrimination) and used these isotopic data and a stable isotope mixing model to estimate the proportion of coastal and pelagic resources consumed by seals. As predicted, we found that seals had similar δ(13)C values as many coastal prey species and higher δ(13)C values than pelagic species; these results, in conjunction with mean dietary estimates (coastal=61 % vs. pelagic=39 %), suggest that seals have a diverse diet comprising prey from multiple trophic levels that primarily occupy the coast. Marine resource managers should consider using the results from this study to inform the future management of coastal habitats in Greece to protect Mediterranean monk seals.

North-South differentiation and a region of high diversity in European wolves (Canis lupus).

European wolves (Canis lupus) show population genetic structure in the absence of geographic barriers, and across relatively short distances for this highly mobile species. Additional information on the location of and divergence between population clusters is required, particularly because wolves are currently recolonizing parts of Europe. We evaluated genetic structure in 177 wolves from 11 countries using over 67K single nucleotide polymorphism (SNP) loci. The results supported previous findings of an isolated Italian population with lower genetic diversity than that observed across other areas of Europe. Wolves from the remaining countries were primarily structured in a north-south axis, with Croatia, Bulgaria, and Greece (Dinaric-Balkan) differentiated from northcentral wolves that included individuals from Finland, Latvia, Belarus, Poland and Russia. Carpathian Mountain wolves in central Europe had genotypes intermediate between those identified in northcentral Europe and the Dinaric-Balkan cluster. Overall, individual genotypes from northcentral Europe suggested high levels of admixture. We observed high diversity within Belarus, with wolves from western and northern Belarus representing the two most differentiated groups within northcentral Europe. Our results support the presence of at least three major clusters (Italy, Carpathians, Dinaric-Balkan) in southern and central Europe. Individuals from Croatia also appeared differentiated from wolves in Greece and Bulgaria. Expansion from glacial refugia, adaptation to local environments, and human-related factors such as landscape fragmentation and frequent killing of wolves in some areas may have contributed to the observed patterns. Our findings can help inform conservation management of these apex predators and the ecosystems of which they are part.

Ecoregion-based conservation planning in the Mediterranean: dealing with large-scale heterogeneity.

Spatial priorities for the conservation of three key Mediterranean habitats, i.e. seagrass Posidonia oceanica meadows, coralligenous formations, and marine caves, were determined through a systematic planning approach. Available information on the distribution of these habitats across the entire Mediterranean Sea was compiled to produce basin-scale distribution maps. Conservation targets for each habitat type were set according to European Union guidelines. Surrogates were used to estimate the spatial variation of opportunity cost for commercial, non-commercial fishing, and aquaculture. Marxan conservation planning software was used to evaluate the comparative utility of two planning scenarios: (a) a whole-basin scenario, referring to selection of priority areas across the whole Mediterranean Sea, and (b) an ecoregional scenario, in which priority areas were selected within eight predefined ecoregions. Although both scenarios required approximately the same total area to be protected in order to achieve conservation targets, the opportunity cost differed between them. The whole-basin scenario yielded a lower opportunity cost, but the Alboran Sea ecoregion was not represented and priority areas were predominantly located in the Ionian, Aegean, and Adriatic Seas. In comparison, the ecoregional scenario resulted in a higher representation of ecoregions and a more even distribution of priority areas, albeit with a higher opportunity cost. We suggest that planning at the ecoregional level ensures better representativeness of the selected conservation features and adequate protection of species, functional, and genetic diversity across the basin. While there are several initiatives that identify priority areas in the Mediterranean Sea, our approach is novel as it combines three issues: (a) it is based on the distribution of habitats and not species, which was rarely the case in previous efforts, (b) it considers spatial variability of cost throughout this socioeconomically heterogeneous basin, and (c) it adopts ecoregions as the most appropriate level for large-scale planning.

The structure of Mediterranean rocky reef ecosystems across environmental and human gradients, and conservation implications.

Historical exploitation of the Mediterranean Sea and the absence of rigorous baselines makes it difficult to evaluate the current health of the marine ecosystems and the efficacy of conservation actions at the ecosystem level. Here we establish the first current baseline and gradient of ecosystem structure of nearshore rocky reefs at the Mediterranean scale. We conducted underwater surveys in 14 marine protected areas and 18 open access sites across the Mediterranean, and across a 31-fold range of fish biomass (from 3.8 to 118 g m(-2)). Our data showed remarkable variation in the structure of rocky reef ecosystems. Multivariate analysis showed three alternative community states: (1) large fish biomass and reefs dominated by non-canopy algae, (2) lower fish biomass but abundant native algal canopies and suspension feeders, and (3) low fish biomass and extensive barrens, with areas covered by turf algae. Our results suggest that the healthiest shallow rocky reef ecosystems in the Mediterranean have both large fish and algal biomass. Protection level and primary production were the only variables significantly correlated to community biomass structure. Fish biomass was significantly larger in well-enforced no-take marine reserves, but there were no significant differences between multi-use marine protected areas (which allow some fishing) and open access areas at the regional scale. The gradients reported here represent a trajectory of degradation that can be used to assess the health of any similar habitat in the Mediterranean, and to evaluate the efficacy of marine protected areas.