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Abstract A genomic database of all Earth’s eukaryotic species could contribute to many scientific discoveries; however, only a tiny fraction of species have genomic information available. In 2018, scientists across the world united under the Earth BioGenome Project (EBP), aiming to produce a database of high-quality reference genomes containing all ~1.5 million recognized eukaryotic species. As the European node of the EBP, the European Reference Genome Atlas (ERGA) sought to implement a new decentralised, equitable and inclusive model for producing reference genomes. For this, ERGA launched a Pilot Project establishing the first distributed reference genome production infrastructure and testing it on 98 eukaryotic species from 33 European countries. Here we outline the infrastructure and explore its effectiveness for scaling high-quality reference genome production, whilst considering equity and inclusion. The outcomes and lessons learned provide a solid foundation for ERGA while offering key learnings to other transnational, national genomic resource projects and the EBP.

Echinococcus granulosus sensu lato is a group of tapeworm species known to cause cystic echinococcosis. Within this group, the Echinococcus canadensis cluster includes genotypes G8 and G10 that have a predominantly sylvatic life cycle – transmission occurs between wild cervids and wolves. Relatively few studies have explored the genetic variation of the elusive G8 and G10, and their extent of genetic variation is yet to be investigated at the complete mitochondrial (mt) genome level. The aim was to explore the genetic variation of these 2 genotypes in Europe using complete mtDNA sequences and provide a high-quality reference dataset for future studies. Sequences of complete mt genomes were produced for 29 samples of genotype G8 and G10 from wolves, moose, reindeer and roe deer, originating from Finland, Sweden, Russia, Poland, Latvia and Estonia. Genetic variation was explored based on phylogenetic network analysis, revealing marked differences between G8 and G10 (over 400 mutations), and more detailed patterns of variability within the 2 genotypes than previously observed. Understanding the mt genetic composition of a species provides a baseline for future studies aiming to understand whether this mt distinctiveness is mirrored in the nuclear genome and whether it has any impact on any phenotypic traits or parasite transmission.

Cystic echinococcosis (CE) is considered the most severe parasitic disease that ever affected the human population in Iceland. Before the start of eradication campaign in the 1860s, Iceland was a country with very high prevalence of human CE, with approximately every fifth person infected. Eradication of CE from Iceland by 1979 was a huge success story and served as a leading example for other countries on how to combat such a severe One Health problem. However, there is no genetic information on Echinococcus parasites before eradication. Here, we reveal the genetic identity for one of the last Echinococcus isolates in Iceland, obtained from a sheep 46 years ago (1977). We sequenced a large portion of the mitochondrial genome (8141 bp) and identified the isolate as Echinococcus granulosus sensu stricto genotype G1. As G1 is known to be highly infective genotype to humans, it may partly explain why such a large proportion of human population in Iceland was infected at a time . The study demonstrates that decades-old samples hold significant potential to uncover genetic identities of parasites in the past.

The genetic structure of modern-day brown bears mainly reflects contemporary population connectivity. Recent gene flow barriers have obscured the genomic signatures of postglacial recolonisation.

Summary: The Sicilian wolf remained isolated in Sicily from the end of the Pleistocene until its extermination in the 1930s–1960s. Given its long-term isolation on the island and distinctive morphology, the genetic origin of the Sicilian wolf remains debated. We sequenced four nuclear genomes and five mitogenomes from the seven existing museum specimens to investigate the Sicilian wolf ancestry, relationships with extant and extinct wolves and dogs, and diversity. Our results show that the Sicilian wolf is most closely related to the Italian wolf but carries ancestry from a lineage related to European Eneolithic and Bronze Age dogs. The average nucleotide diversity of the Sicilian wolf was half of the Italian wolf, with 37–50% of its genome contained in runs of homozygosity. Overall, we show that, by the time it went extinct, the Sicilian wolf had high inbreeding and low-genetic diversity, consistent with a population in an insular environment.

A high-quality genome for the parasitic tapeworm, Echinococcus granulosus, provides further insight into the EG95 vaccine target for cystic echinococcosis.

Abstract Background This study aimed to fill a gap of knowledge by providing a quantitative measure of molecularly identified species and genotypes belonging to Echinococcus granulosus sensu lato (s.l.) causing human cystic echinococcosis (CE) in Europe during the period 2000–2021. As these species and genotypes are characterized by genetic, animal host and geographical differences, studying the E. granulosus s.l. complex is epidemiologically relevant. Methods A systematic review (SR) was conducted on the basis of both scientific and grey literature considering primary studies between 2000 and 2021 in four databases. From a total of 1643 scientific papers, 51 records were included in the SR. The main inclusion criterion for this study was the molecular confirmation of E. granulosus s.l. at the genotype/species level as a causative agent of human CE cases in selected European countries. Results Relevant data were obtained from 29 out of 39 eligible European countries. This SR identified 599 human molecularly confirmed echinococcal cysts: 460 (76.8%) identified as E. granulosus sensu stricto (s.s.), 130 (21.7%) as E. canadensis cluster (G6/7 and G10), 7 (1.2%) as E. ortleppi (G5), and 2 as E. vogeli (0.3%). Three geographical hotspots of human CE caused by different species of the E. granulosus s.l. complex were identified: (1) E. granulosus s.s. in Southern and South-eastern Europe (European-Mediterranean and Balkan countries); (2) E. canadensis (G6/7) in Central and Eastern Europe; (3) E. ortleppi in Central and Western Europe. This SR also identified data gaps that prevented a better definition of the geographical distribution of the Echinococcus granulosus s.l. species complex in Europe: western Balkan countries, part of Central Europe, and Baltic countries. Conclusions These results mandate longitudinal, multi-centre, intersectoral and transdisciplinary studies which consider both molecular and clinical epidemiology in animals and humans. Such studies would be valuable for a better understanding of the transmission of the E. granulosus s.l. species complex and their potential clinical impact on humans. Graphical Abstract

Abstract In human‐dominated landscapes, connectivity is crucial for maintaining demographically stable mammalian populations. Here, we provide a comprehensive noninvasive genetic study for the brown bear population in the Hellenic Peninsula. We analyze its population structuring and connectivity, estimate its population size throughout its distribution, and describe its phylogeography in detail for the first time. Our results, based on 150 multilocus genotypes and on 244‐bp sequences of the mtDNA control region, show the population is comprised by three highly differentiated genetic clusters, consistent with geographical populations of Pindos, Peristeri, and Rhodope. By detecting two male bears with Rhodopean ancestry in the western demes, we provide strong evidence for the ongoing genetic connectivity of the geographically fragmented eastern and western distributions, which suggests connectivity of the larger East Balkan and Pindos‐Dinara populations. Total effective population size (Ne) was estimated to be 199 individuals, and total combined population size (NC) was 499, with each cluster showing a relatively high level of genetic variability, suggesting that migration has been sufficient to counteract genetic erosion. The mtNDA results were congruent with the microsatellite data, and the three genetic clusters were matched predominantly with an equal number of mtDNA haplotypes that belong to the brown bear Western mitochondrial lineage (Clade 1), with two haplotypes being globally new and endemic. The detection of a fourth haplotype that belongs to the Eastern lineage (Clade 3a1) in three bears from the western distribution places the southernmost secondary contact zone between the Eastern and Western lineages in Greece and generates new hypotheses about postglacial maxima migration routes. This work indicates that the genetic composition and diversity of Europe's low‐latitude fringe population are the outcome of ancient and historical events and highlight its importance for the connectivity and long‐term persistence of the species in the Balkans.

Cystic Echinococcosis (CE) caused by Echinococcus granulosus sensu lato (s.l.) is one of the most important parasitic zoonotic diseases in the world and it represents an important public health and socio-economic concern. In the Mediterranean basin, CE is widespread and it is endemic in Italy, with major prevalence in southern areas. Several studies have investigated CE in domestic pigs, however, such data in wild boars are scant. In the last decades the wild boar population in Italy has increased and this ungulate could play an important role in the spreading of CE in the wild. Here we report on the prevalence and fertility rate of hydatid cysts in wild boars that were shot during two hunting seasons (2016–2017) in the Campania region of southern Italy.For each animal, a detailed inspection of the carcass and organs (lungs, liver and spleen) was performed and when cysts were found, their number, morphology and fertility were determined by visual and microscopic examination. Cysts were classified morphologically as fertile, sterile, caseous and calcified. Protoscoleces and germinal layers were collected from individual cysts and DNA was extracted to identify different strains/genotypes of E. granulosus s.l.Out of a total of 2108 wild boars 93 (4.4%) were found positive for CE. Infected animals were 45 males and 48 females, aged between 1 and 8 years. The average number of cysts per wild boar was 1.3 (min 1 - max 13). The total number of cysts collected was 123, of which 118 (95.9%) in the liver, 4 (3.3%) in the lungs and 1 (0.8%) in the spleen. Of all analyzed cysts, 70 (56.9%) were fertile and 53 (43.1%) sterile/acephalous. The presence of fertile cysts in 19.4% of CE-positive animals is noteworthy. Overall, molecular diagnosis showed 19 wild boars infected with the pig strain (G7). Keywords: Cystic echinococcosis, Wild boar, Genotyping, Drive hunting, Southern Italy

Abstract Background Echinococcus tapeworms cause a severe helminthic zoonosis called echinococcosis. The genus comprises various species and genotypes, of which E. granulosus (sensu stricto) represents a significant global public health and socioeconomic burden. Mitochondrial (mt) genomes have provided useful genetic markers to explore the nature and extent of genetic diversity within Echinococcus and have underpinned phylogenetic and population structure analyses of this genus. Our recent work indicated a sequence gap (> 1 kb) in the mt genomes of E. granulosus genotype G1, which could not be determined by PCR-based Sanger sequencing. The aim of the present study was to define the complete mt genome, irrespective of structural complexities, using a long-read sequencing method. Methods We extracted high molecular weight genomic DNA from protoscoleces from a single cyst of E. granulosus genotype G1 from a sheep from Australia using a conventional method and sequenced it using PacBio Sequel (long-read) technology, complemented by BGISEQ-500 short-read sequencing. Sequence data obtained were assembled using a recently-developed workflow. Results We assembled a complete mt genome sequence of 17,675 bp, which is > 4 kb larger than the complete mt genomes known for E. granulosus genotype G1. This assembly includes a previously-elusive tandem repeat region, which is 4417 bp long and consists of ten near-identical 441–445 bp repeat units, each harbouring a 184 bp non-coding region and adjacent regions. We also identified a short non-coding region of 183 bp, which includes an inverted repeat. Conclusions We report what we consider to be the first complete mt genome of E. granulosus genotype G1 and characterise all repeat regions in this genome. The numbers, sizes, sequences and functions of tandem repeat regions remain to be studied in different isolates of genotype G1 and in other genotypes and species. The discovery of such ‘new’ repeat elements in the mt genome of genotype G1 by PacBio sequencing raises a question about the completeness of some published genomes of taeniid cestodes assembled from conventional or short-read sequence datasets. This study shows that long-read sequencing readily overcomes the challenges of assembling repeat elements to achieve improved genomes.

Anthropogenic hybridization is widely perceived as a threat to the conservation of biodiversity. Nevertheless, to date, relevant policy and management interventions are unresolved and highly convoluted. While this is due to the inherent complexity of the issue, we hereby hypothesize that a lack of agreement concerning management goals and approaches, within the scientific community, may explain the lack of social awareness on this phenomenon, and the absence of effective pressure on decision-makers. By focusing on wolf x dog hybridization in Europe, we hereby (a) assess the state of the art of issues on wolf x dog hybridization within the scientific community, (b) assess the conceptual bases for different viewpoints, and (c) provide a conceptual framework aiming at reducing the disagreements. We adopted the Delphi technique, involving a three-round iterative survey addressed to a selected sample of experts who published at Web of Science listed journals, in the last 10 years on wolf x dog hybridization and related topics. Consensus was reached that admixed individuals should always be defined according to their genetic profile, and that a reference threshold for admixture (i.e., q-value in assignment tests) should be formally adopted for their identification. To mitigate hybridization, experts agreed on adopting preventive, proactive and, when concerning small and recovering wolf populations, reactive interventions. Overall, experts' consensus waned as the issues addressed became increasingly practical, including the adoption of lethal removal. We suggest three non-mutually exclusive explanations for this trend: (i) value-laden viewpoints increasingly emerge when addressing practical issues, and are particularly diverging between experts with different disciplinary backgrounds (e.g., ecologists, geneticists); (ii) some experts prefer avoiding the risk of potentially giving carte blanche to wolf opponents to (illegally) remove wolves, based on the wolf x dog hybridization issue; (iii) room for subjective interpretation and opinions result from the paucity of data on the effectiveness of different management interventions. These results have management implications and reveal gaps in the knowledge on a wide spectrum of issues related not only to the management of anthropogenic hybridization, but also to the role of ethical values and real-world management concerns in the scientific debate.

Abstract Background Echinococcus granulosus and E. multilocularis are tapeworm parasites of major medical and veterinary importance, causing cystic and alveolar echinococcosis, respectively. Both diseases are listed among the most severe parasitic diseases in humans, representing 2 of the 17 neglected diseases prioritised by the World Health Organisation. However, little is known about the role of urban animals in transmission of both parasite species. Findings A sensitive non-invasive genetic method was used to monitor E. granulosus and E. multilocularis infection among dog faecal samples collected from an urban area in Estonia in 2012–13. Out of 181 dog faecal samples analysed, 2.2% tested positive for E. granulosus, determined by sequencing as genotype G1. None of the samples tested positive for E. multilocularis. Conclusions We report contamination of an urban environment with highly pathogenic E. granulosus G1 disseminated by dogs, and a potential risk to human health.

The survival of isolated small populations is threatened by both demographic and genetic factors. Large carnivores declined for centuries in most of Europe due to habitat changes, overhunting of their natural prey and direct persecution. However, the current rewilding trends are driving many carnivore populations to expand again, possibly reverting the erosion of their genetic diversity. In this study we reassessed the extent and origin of the genetic variation of the Italian wolf population, which is expanding after centuries of decline and isolation. We genotyped wolves from Italy and other nine populations at four mtDNA regions (control-region, ATP6, COIII and ND4) and 39 autosomal microsatellites. Results of phylogenetic analyses and assignment procedures confirmed in the Italian wolves a second private mtDNA haplotype, which belongs to a haplogroup distributed mostly in southern Europe. Coalescent analyses showed that the unique mtDNA haplotypes in the Italian wolves likely originated during the late Pleistocene. ABC simulations concordantly showed that the extant wolf populations in Italy and in south-western Europe started to be isolated and declined right after the last glacial maximum. Thus, the standing genetic variation in the Italian wolves principally results from the historical isolation south of the Alps.

Capercaillie (Tetrao urogallus) and other grouse species represent conservation concerns across Europe due to their negative abundance trends. In addition to habitat deterioration, predation is considered a major factor contributing to population declines. While the role of generalist predators on grouse predation is relatively well known, the impact of the omnivorous wild boar has remained elusive. We hypothesize that wild boar is an important predator of ground-nesting birds, but has been neglected as a bird predator because traditional morphological methods underestimate the proportion of birds in wild boar diet. To distinguish between different mammalian predator species, as well as different grouse prey species, we developed a molecular method based on the analysis of mitochondrial DNA that allows accurate species identification. We collected 109 wild boar faeces at protected capercaillie leks and surrounding areas and analysed bird consumption using genetic methods and classical morphological examination. Genetic analysis revealed that the proportion of birds in wild boar faeces was significantly higher (17.3%; 4.5×) than indicated by morphological examination (3.8%). Moreover, the genetic method allowed considerably more precise taxonomic identification of consumed birds compared to morphological analysis. Our results demonstrate: (i) the value of using genetic approaches in faecal dietary analysis due to their higher sensitivity, and (ii) that wild boar is an important predator of ground-nesting birds, deserving serious consideration in conservation planning for capercaillie and other grouse.

Knowledge of space use is central to understand animals' role in ecosystems. The raccoon dog Nyctereutes procyonoides is considered as one of the most influential alien mesopredator species in Europe, having the potential to cause loss of local biodiversity and act as a vector for zoonotic diseases. We collared 12 animals to study their home range and habitat use in two areas with different management regimes in Estonia: in a protected natural area and in an intensively managed area. From May to October raccoon dogs inhabiting the natural area had considerably smaller home ranges compared to the managed area, 193.3ha±37.3SD and 391.9ha±292.9SD, respectively. This result contradicts somewhat earlier findings in other European raccoon dog populations, where the home range sizes in natural areas in summer and autumn period have usually been larger compared to managed areas. In both study areas raccoon dogs preferred watersides, where amphibians and other semi-aquatic prey are abundant, to other habitats available in their home ranges. We also studied movements of a raccoon dog pair in the managed study area in winter period. Due to mild weather conditions during the study period, raccoon dogs changed their resting sites quite often, covering a relatively large 599 ha area from November 2012 to January 2013, indicating the absence of usual winter lethargy during the mild winters.

The extent of gene flow during the range expansion of non-native species influences the amount of genetic diversity retained in expanding populations. Here, we analyse the population genetic structure of the raccoon dog (Nyctereutes procyonoides) in north-eastern and central Europe. This invasive species is of management concern because it is highly susceptible to fox rabies and an important secondary host of the virus. We hypothesized that the large number of introduced animals and the species' dispersal capabilities led to high population connectivity and maintenance of genetic diversity throughout the invaded range. We genotyped 332 tissue samples from seven European countries using 16 microsatellite loci. Different algorithms identified three genetic clusters corresponding to Finland, Denmark and a large 'central' population that reached from introduction areas in western Russia to northern Germany. Cluster assignments provided evidence of long-distance dispersal. The results of an Approximate Bayesian Computation analysis supported a scenario of equal effective population sizes among different pre-defined populations in the large central cluster. Our results are in line with strong gene flow and secondary admixture between neighbouring demes leading to reduced genetic structuring, probably a result of its fairly rapid population expansion after introduction. The results presented here are remarkable in the sense that we identified a homogenous genetic cluster inhabiting an area stretching over more than 1500km. They are also relevant for disease management, as in the event of a significant rabies outbreak, there is a great risk of a rapid virus spread among raccoon dog populations.

Although sheep (Ovis aries) have been one of the most exploited domestic animals in Estonia since the Late Bronze Age, relatively little is known about their genetic history. Here, we explore temporal changes in Estonian sheep populations and their mitochondrial genetic diversity over the last 3000 years. We target a 558 base pair fragment of the mitochondrial hypervariable region in 115 ancient sheep from 71 sites in Estonia (c. 1200 BC-AD 1900s), 19 ancient samples from Latvia, Russia, Poland and Greece (6800 BC-AD 1700), as well as 44 samples of modern Kihnu native sheep breed. Our analyses revealed: (1) 49 mitochondrial haplotypes, associated with sheep haplogroups A and B; (2) high haplotype diversity in Estonian ancient sheep; (3) continuity in mtDNA haplotypes through time; (4) possible population expansion during the first centuries of the Middle Ages (associated with the establishment of the new power regime related to 13th century crusades); (5) significant difference in genetic diversity between ancient populations and modern native sheep, in agreement with the beginning of large-scale breeding in the 19th century and population decline in local sheep. Overall, our results suggest that in spite of the observed fluctuations in ancient sheep populations, and changes in the natural and historical conditions, the utilisation of local sheep has been constant in the territory of Estonia, displaying matrilineal continuity from the Middle Bronze Age through the Modern Period, and into modern native sheep.

After a long and deliberate persecution, the grey wolf (Canis lupus) is slowly recolonizing its former areas in Europe, and the genetic consequences of this process are of particular interest. Wolves, though present in mainland Estonia for a long time, have only recently started to recolonize the country's two largest islands, Saaremaa and Hiiumaa. The main objective of this study was to analyse wolf population structure and processes in Estonia, with particular attention to the recolonization of islands. Fifteen microsatellite loci were genotyped for 185 individuals across Estonia. As a methodological novelty, all putative wolf-dog hybrids were identified and removed (n = 17) from the dataset beforehand to avoid interference of dog alleles in wolf population analysis. After the preliminary filtering, our final dataset comprised of 168 "pure" wolves. We recommend using hybrid-removal step as a standard precautionary procedure not only for wolf population studies, but also for other taxa prone to hybridization. STRUCTURE indicated four genetic groups in Estonia. Spatially explicit DResD analysis identified two areas, one of them on Saaremaa island and the other in southwestern Estonia, where neighbouring individuals were genetically more similar than expected from an isolation-by-distance null model. Three blending areas and two contrasting transition zones were identified in central Estonia, where the sampled individuals exhibited strong local differentiation over relatively short distance. Wolves on the largest Estonian islands are part of human-wildlife conflict due to livestock depredation. Negative public attitude, especially on Saaremaa where sheep herding is widespread, poses a significant threat for island wolves. To maintain the long-term viability of the wolf population on Estonian islands, not only wolf hunting quota should be targeted with extreme care, but effective measures should be applied to avoid inbreeding and minimize conflicts with local communities and stakeholders.

The sterrhine loopers Timandra griseata and T. comae have been treated as distinct species since 1994. However, morphological differences between the taxa are minor and therefore their status has often been disputed. Here, we present a molecular phylogenetic study, which separates T. griseata and T. comae into different clades. Altogether, 43 Timandra specimens from eight European countries were studied. The phylogeny is based on a comparative sequence analysis of mitochondrial genes coding for the cytochrome C oxidase subunit I (COI) and NADH dehydrogenase subunit 1 (ND1). Nevertheless, a single individual of both species was assigned to the "wrong" clade. The symplesiomorphy of T. griseata and T. comae is considered to be a result of introgressive hybridization. Conditions that could lead to the hybridization of T. griseata and T. comae are discussed, as well as the likely distribution history of these taxa in Northern Europe. Results of the current analysis are in favour of retaining the species status of T. griseata and T. comae.

Concordant differences in morphology, phenology and RAMS markers, as well as in sequenced mtDNA (COI, COII, cytb) and nuclear DNA (ITS2) fragments, indicate that Dolerus asper Zaddach, 1859 and Dolerus brevicornis Zaddach, 1859 are valid species. On the basis of morphology, molecular markers, and distributional records, both species are distinct from Dolerus gibbosus Hartig, 1837 (= Dolerus planatus Hartig, 1837). Taxonomy of the species is clarified and the neotypes of Dolerus asper Zaddach, 1859 and Dolerus brevicornis Zaddach, 1859 are designated. The synonymies of Dolerus asper Zaddach, 1859, to Dolerus planatus Hartig, 1837 and Dolerus derzavini Malaise, 1931, spec. rev. to D. asper Zaddach, 1859 are abandoned. Dolerus carbonarius Zaddach, 1859 and Dolerus fumosus Zaddach, 1859 are considered to be species inquirendae. Phylogenetic analyses of the ITS2 fragment and fragments of ITS2 + COI and ITS2 + COII yielded the topology [D. asper, (D. brevicornis, D. gibbosus)], while those of all other markers and their combinations resulted in the topology [D. brevicornis, (D. asper, D. gibbosus)]. In the latter hypothesis the clade asper + gibbosus is also supported by structural synapomorphies.

Urban areas are becoming increasingly important for wildlife as diminishing natural habitats no longer represent a suitable environment for many species. Red foxes (Vulpes vulpes) are nowadays common in many cities worldwide, and in recent years they have colonized urban areas in Estonia. We used a public web-based questionnaire approach to evaluate the distribution and behaviour of Estonian urban foxes, to detect related problems and to assess health risks to humans and domestic animals. In total, 1205 responses were collected throughout the country. Foxes have colonized the majority of Estonian towns (33 out of 47) in a relatively short period of time, and have already established breeding dens in several towns. Despite their recent arrival, the behaviour of Estonian urban foxes is similar to that reported in longer-established urban fox populations: they are mostly active during night-time, often visit city centres and some also have dens in such locations. Certain characteristics of urban foxes serve as a basis for conflict with humans: foxes have entered houses and attacked domestic animals, killing cats and poultry. About 8% of reported foxes exhibited symptoms of sarcoptic mange, a disease that also infects domestic animals, especially dogs. The proportion of mange-infected foxes was higher in large urban areas. In addition to mange, a substantial fraction of red foxes in Estonia are known to be infected with the life-threatening tapeworm Echinococcus multilocularis, the causative agent of alveolar echinococcosis. Therefore, urban foxes may represent a source of serious infectious disease for pets and humans.

The raccoon dog (Nyctereutes procyonoides) is an introduced species in Europe with a continually expanding range. Since the species is capable of affecting local ecosystems and is a vector for a number of severe zoonotic diseases, it is important to understand its food habits. Raccoon dog diet was studied in Estonia by examining the contents of 223 stomach samples collected during the coldest period of the year, August to March, in 2010-2012. The most frequently consumed food categories were anthropogenic plants (e.g. cereals, fruits; FO = 56.1%) and carrion (e.g. carcasses of artiodactyls and carnivores; FO = 48.4%). Carrion was also the only food category that was consumed significantly more frequently by raccoon dogs exhibiting symptoms of sarcoptic mange than by uninfected animals. Small mammals, which represent intermediate hosts for the zoonotic tapeworm Echinococcus multilocularis, were more commonly recorded in samples also containing anthropogenic plants than expected by chance. Comparison of raccoon dog and red fox (Vulpes vulpes) diet in Estonia revealed higher overlap than found elsewhere in Europe, with 'carrion' and 'anthropogenic plants' making up the bulk of both species' diet; however, raccoon dogs were more omnivorous than red foxes. Our results suggest that while the use of most food categories reflects the phenology of natural food sources, 'anthropogenic plants' and 'carrion' provide an essential resource for raccoon dogs during the coldest period of the year, with the latter resource especially important for individuals infected with sarcoptic mange. Since both of these food categories and small mammals are often found at supplementary feeding sites for wild boar (Sus scrofa), this game management practice may facilitate high densities of mesocarnivores and promote the spread of some severe zoonotic diseases, including alveolar echinococcosis, trichinellosis, rabies and sarcoptic mange.

Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1-G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1-G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6-G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (

Spatial genetics is a relatively new field in wildlife and conservation biology that is becoming an essential tool for unravelling the complexities of animal population processes, and for designing effective strategies for conservation and management. Conceptual and methodological developments in this field are therefore critical. Here we present two novel methodological approaches that further the analytical possibilities of STRUCTURE and DResD. Using these approaches we analyse structure and migrations in a grey wolf (Canislupus) population in north-eastern Europe. We genotyped 16 microsatellite loci in 166 individuals sampled from the wolf population in Estonia and Latvia that has been under strong and continuous hunting pressure for decades. Our analysis demonstrated that this relatively small wolf population is represented by four genetic groups. We also used a novel methodological approach that uses linear interpolation to statistically test the spatial separation of genetic groups. The new method, which is capable of using program STRUCTURE output, can be applied widely in population genetics to reveal both core areas and areas of low significance for genetic groups. We also used a recently developed spatially explicit individual-based method DResD, and applied it for the first time to microsatellite data, revealing a migration corridor and barriers, and several contact zones.

Studies on hybridization have proved critical for understanding key evolutionary processes such as speciation and adaptation. However, from the perspective of conservation, hybridization poses a concern, as it can threaten the integrity and fitness of many wild species, including canids. As a result of habitat fragmentation and extensive hunting pressure, gray wolf (Canis lupus) populations have declined dramatically in Europe and elsewhere during recent centuries. Small and fragmented populations have persisted, but often only in the presence of large numbers of dogs, which increase the potential for hybridization and introgression to deleteriously affect wolf populations. Here, we demonstrate hybridization between wolf and dog populations in Estonia and Latvia, and the role of both genders in the hybridization process, using combined analysis of maternal, paternal and biparental genetic markers. Eight animals exhibiting unusual external characteristics for wolves - six from Estonia and two from Latvia - proved to be wolf-dog hybrids. However, one of the hybridization events was extraordinary. Previous field observations and genetic studies have indicated that mating between wolves and dogs is sexually asymmetrical, occurring predominantly between female wolves and male dogs. While this was also the case among the Estonian hybrids, our data revealed the existence of dog mitochondrial genomes in the Latvian hybrids and, together with Y chromosome and autosomal microsatellite data, thus provided the first evidence from Europe of mating between male wolves and female dogs. We discuss patterns of sexual asymmetry in wolf-dog hybridization.

Time-scales estimated from sequence data play an important role in molecular ecology. They can be used to draw correlations between evolutionary and palaeoclimatic events, to measure the tempo of speciation, and to study the demographic history of an endangered species. In all of these studies, it is paramount to have accurate estimates of time-scales and substitution rates. Molecular ecological studies typically focus on intraspecific data that have evolved on genealogical scales, but often these studies inappropriately employ deep fossil calibrations or canonical substitution rates (e.g., 1% per million years for birds and mammals) for calibrating estimates of divergence times. These approaches can yield misleading estimates of molecular time-scales, with significant impacts on subsequent evolutionary and ecological inferences. We illustrate this calibration problem using three case studies: avian speciation in the late Pleistocene, the demographic history of bowhead whales, and the Pleistocene biogeography of brown bears. For each data set, we compare the date estimates that are obtained using internal and external calibration points. In all three cases, the conclusions are significantly altered by the application of revised, internally-calibrated substitution rates. Collectively, the results emphasise the importance of judicious selection of calibrations for analyses of recent evolutionary events.