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DNA as evidence of distribution and vitality of endangered Balkan chamois

Goal: To analyze genetic structure of Balkan chamois in Dinaric Mountains, using neutral (SNP) and loci under selection (MHC complex) to estimate conservation status and to study mechanisms of molecular adaptation of local populations.

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Laura Iacolina
added a research item
Although the two species of chamois (Rupicapra rupicapra and R. pyrenaica) are currently classified as least-concern by the IUCN (International Union for Conservation of Nature), inconsistencies on the subspecies classification reported in literature make it challenging to assess the conservation status of the single subspecies. Previous studies relying on mitochondrial genes, sometimes in combination with nuclear or Y-chromosome markers, reported the presence of clusters corresponding to the geographic distribution but highlighting ambiguities in the genus phylogeny. Here we report novel de novo assembled sequences of the mitochondrial genome from nine individuals, including previously unpublished R. r. balcanica and R. r. tatrica subspecies, and use them to untangle the genus phylogeny. Our results based on the full mitogenome inferred phylogeny confirm the previously reported genus subdivision in three clades and its monophyletic positioning within the Caprinae. Phylogeny and taxonomy of Rupicapra species thus remain controversial prompting for the inclusion of archeological remains to solve the controversy.
Laura Iacolina
added a research item
The chamois, genus Rupicapra, is the most abundant mountain-dwelling ungulate in Europe and the Middle East and is currently recognised to be divided into two species: Rupicapra rupicapra (Northern chamois) and Rupicapra pyrenaica (Southern chamois), which are further subdivided into seven and three subspecies respectively. Although currently classified as least-concern in the IUCN Red List of Threatened Species, information on the status of the species need updating. To further complicate the assessment, the genus has been subject of reintroductions and translocation, sometimes without prior knowledge on the taxonomic status of individuals, and there is a molecular controversy over the subspecies subdivision based on morphological and behavioural characters. We combined previously published (N=5) with newly assembled (N=9) mitogenomes to further investigate the phylogeny of the genus. To avoid erroneous hypotheses on homology we excluded all indels, ambiguous positions and the portion of the control region presenting tandem repeats in the Caprini. The final alignment consisted of 15348 bp, 14 Rupicapra sequences and two outgroups (Ammotragus lervia and Arabitragus jayakari). Both Bayesian and maximum likelihood analyses showed the same subdivision in Eastern, Central and Western clades previously reported. Within the Eastern clade R. r. balcanica was the most differentiated and included both sequences from the known hybrid population of the Velebit Mountains, whereas R. r. tatrica and R. r. rupicapra had a sister group relationship. Although we observed differences in variability among genes, our results confirm the reliability of previous studies based on a reduced number of mitochondrial genes.
Laura Iacolina
added a research item
The chamois, genus Rupicapra, is the most abundant mountain-dwelling ungulate in Europe and the Near East and is currently recognized to be divided into two species: R. rupicapra (Northern chamois) and R. pyrenaica (Southern chamois) further divided into seven and three subspecies, respectively. While neither of the species is of conservation concern, some subspecies are protected at the national level in (part of) their distribution ranges. However, for most subspecies and populations the information necessary to assess the conservation status are lacking and several populations were subject to translocations and reintroductions, further complicating their general taxonomic picture. Here we combine nine newly sequenced mitochondrial genomes (mitogenomes) of four subspecies with previously published mitogenomes of Rupicapra (5) and related (sub)species in the Caprini, Ovibovini and Pantilopini families (39). The Bayesian and maximum likelihood phylogenetic analyses concordantly confirmed the monophyly of the genus Rupicapra and its positioning within the Caprini tribe. Both analyses identified the subdivision of the subspecies into three lineages in accordance with their geographical distributions (Eastern, Central and Western). The Central and Western lineages were more closely related, confirming previously reported results based on partial mitochondrial DNA regions. Within the Eastern lineage, R. r. balcanica was the most differentiated and included all individuals coming from the northern part of the Dinaric Karst mountain range, Mt. Velebit (Croatia), a known human-caused contact zone between R. r. rupicapra and R. r. balcanica, whereas R. r. tatrica and R. r. rupicapra showed a sister group relationship. Overall, our results, while providing an increased resolution, confirmed the phylogeny previously reconstructed using both whole and partial mitochondrial DNA sequences. The discrepancy among nuclear, mitochondrial and Y-chromosome markers in phylogenetic reconstruction of the genus thus remains. Further studies combining multiple markers and possibly including archeological remains are therefore warranted to solve the controversy.
Sunčica Stipoljev
added a research item
Genes of the major histocompatibility complex (MHC) code for cell surface proteins essential for adaptive immunity. They show the most outstanding genetic diversity in vertebrates, which has been connected with various fitness traits and thus with the long-term persistence of populations. In this study, polymorphism of the MHC class II DRB locus was investigated in chamois with Single-Strand Conformation Polymorphism (SSCP)/Sanger genotyping and Ion Torrent S5 next-generation sequencing (NGS). From eight identified DRB variants in 28 individuals, five had already been described, and three were new, undescribed alleles. With conventional SSCP/Sanger sequencing, we were able to detect seven alleles, all of which were also detected with NGS. We found inconsistencies in the individual genotypes between the two methods, which were mainly caused by allelic dropout in the SSCP/Sanger method. Six out of 28 individuals were falsely classified as homozygous with SSCP/Sanger analysis. Overall, 25% of the individuals were identified as genotyping discrepancies between the two methods. Our results show that NGS technologies are better performing in sequencing highly variable regions such as the MHC, and they also have a higher detection capacity, thus allowing a more accurate description of the genetic composition, which is crucial for evolutionary and population genetic studies.
Nikica Sprem
added a research item
Genetic characterisation of wild ungulates can be a useful tool in wildlife management and in obtaining a greater understanding of their biological and ecological roles in a wider spatiotemporal context. Different ways of optimising methodologies and reducing the costs of genetic analyses using widely available bone tissues collected within regular hunting allocations were examined. Successful isolation and analysis of DNA from widely available bones can be cheap, fast and easy. In particular, this study explored the possibility of using bones for extracting high quality nuclear DNA for microsatellite analysis. The utility of applying a modified demineralisation process using two commercially available DNA isolation kits, which differ significantly in price, was evaluated. The sample sets included bones and, for comparison, muscle tissues from four wild ungulate species: chamois (Rupicapra rupicapra), roe deer (Capreolus capreolus), wild boar (Sus scrofa), and Alpine ibex (Capra ibex). For the recent bones, these results confirmed that the DNA concentrations and microsatellite amplification were sufficiently high, even when using low-cost kits, after prior demineralisation. For old bones, prior demineralisation and use of a specially designed isolation kit led to a more successful extraction of DNA. Besides reducing kit-related costs, low-cost kits are much faster and therefore make genetic analysis more efficient.
Nikica Sprem
added 2 research items
During the early 1900s, Northern chamois (Rupicapra rupicapra) populations in the northern Dinaric Mountains were extirpated. During the 1960s and 1970s there were several reintroductions of individuals from two Northern chamois subspecies (Alpine chamois, R. r. rupicapra and Balkan chamois, R. r. balcanica) from neighbouring areas in the attempt to re-establish the population. Accurate taxonomic classification, at subspecies level, of the autochthonous extirpated population was not known. To clarify which subspecies was present before reintroduction, we genotyped four male chamois skulls originating from Velebit Mountain, collected around 25 years before the population local extinction. DNA was successfully extracted from middle layer and outer sheath of horns. Assignment based on microsatellite loci, using both Bayesian clustering in STRUCTURE (with q values between 0.55 and 0.73) and DAPC (with individual membership probabilities of 0.99 and 1.00) indicated higher assessed likelihood for the Alpine subspecies.
The chamois is widely perceived as a strictly high-elevation ungulate, though its spatial behavior suggests fairly high phenotypic plasticity and long-range dispersal abilities. Here, we describe and discuss five events of ‘unconventional’ habitat use by chamois, i.e., individuals found swimming in the sea of Croatia and Spain, between 2004 and 2019. In four cases, sex, age, swimming distance and direction were recorded. The chamois were young males up to 5 years of age, with swimming distances of ca 300 to 3300 m. In Croatia, two individuals were found swimming across sea stretches and one towards the coast; in Spain the chamois was seemingly swimming towards the open sea. The reasons for such unusual behavior remain unknown. Although it might represent the outcome of a maladaptive combination of physiological needs (e.g., individual attempts to disperse) and constraining casualties (e.g., environmental or anthropogenic barriers), the limited information suggests caution in interpretation. Swimming behavior in chamois is not new. To our knowledge, however, this is the first reported information on the use of sea stretches, which suggests that even medium sized water courses and lakes are unlikely to fully block chamois movements.
Laura Iacolina
added 3 research items
Balkan chamois (Rupicapra rupicapra balcanica) inhabits the south part of the species distribution in Europe. It prefers rocky habitat with steep slopes, thus low valleys tend to constitute a barrier to gene-flow. The subspecies is currently protected in some countries and hunted in others. Knowledge on the genetic composition of this subspecies is limited and confined to regional studies. We present the first investigation including samples from most of the subspecies range to evaluate the possible presence of metapopulations and barriers to gene-flow. Additionally, we included other subspecies from neighbouring countries to assess the geographic boundaries to its distribution. By combining nuclear (20 microsatellites) and mitochondrial (Dloop and cytochrome b) data we provide information on the connectivity levels of the different (meta)populations. Such knowledge will constitute the necessary starting point for a sustainable management of the Balkan chamois, being it the identification of potential source populations for re-introduction programmes or the evaluation of a viable harvest rate.
Nikica Sprem
added a research item
Chamois (Rupicapra rupicapra) is a mountain-dwelling ungulate inhabiting predominantly rocky habitat with steep slopes. Since it mostly inhabits boreal habitats at high altitudes, low valleys tend to separate populations, thereby limiting gene flow. In the present study, we genotyped 54 georeferenced chamois using 20 SSR loci to test the influence of the Kupa River on the spatial genetic structure of the population in the bordering area between Croatia and Slovenia in the northern Dinaric Mountains. Both GENELAND and STRUCTURE assigned all individuals to one spatial cluster, indicating that the Kupa River does not represent a barrier to gene flow for chamois. Unfortunately, in 2015, a razor wire fence was constructed along the Croatian-Slovenian border at the Kupa River. This fence represents a major threat to the chamois population as it may cause mortality, obstruct seasonal dispersal and reduce the effective population size. If the fence remains as it is, changes in the genetic structure and genetic diversity of the population due to the effect of drift and reduced effective population size can be predicted over the next generations.
Nikica Sprem
added a research item
In this research, we aimed to assess the influence of forest succession on the density of Balkan chamois population on Mt Biokovo. To calculate the percentage of forest cover increase, we analysed forest vegetation coverage from two periods (1968 and 2016). In 1968, 18% of the analyzed area was covered with forest/shrub, while in 2016 50% of the area was covered. We propose an average forest/shrub progressiveness of 0.66% per year on Mt Biokovo. Since there are various anthropogenic and non-anthropogenic factors that may affect population, it is hard to draw firm conclusions about the importance of forest succession on the changing chamois density trends. Further research based on feeding ecology in relation to existing habitat conditions should be conducted.
Nikica Sprem
added a research item
Hybridisation and gene introgression are important sources of diversification, the relevance of which in the evolutionary processes is well recognised. Their fitness consequences in animal populations, however, are not sufficiently well understood, despite hybridisation rates becoming increasingly important worldwide following human‐related activities such as domestication, game management and habitat alteration. In Europe, the density and distribution of native ungulates have largely been influenced by humans since pre‐historic times. This, alongside the introduction of non‐native and domesticated species, may bear major consequences at the genetic and population levels. We provide an updated overview of recent hybridisation events in wild European ungulates; we describe their ecological drivers, extent, current distribution, potential consequences and proposed management strategies. We reviewed the scientific literature published between 2000 and 2018 and found that confirmed hybridisation was described in 75 of the 89 references we included, involving nearly all the species that we investigated. Most researchers relied on genetic information for hybrid identification, which often involved a domestic counterpart. However, introductions and translocations also led to crossbreeding between wild ungulate (sub)species. Only 43 papers provided management recommendations, mostly focused on preventing hybridisation and removing hybrids. Hybridisation proved to be relatively common in several ungulate taxa in Europe. Despite reported changes in phenotype and fitness‐related traits in some species, the consequences of hybridisation for adaptation, life history, and evolutionary potential remain largely unknown. The current conservation paradigm aims to prevent the spread of domestic or non‐native genes in native populations; accordingly, conservation plans should: 1) determine the genetic origin of possible source populations; 2) protect native populations from the risk of crossbreeding with non‐native ones, and 3) establish permanent monitoring.
Nikica Sprem
added a research item
The Dinaric region is the natural habitat of the Alpine chamois (Rupicapra rupicapra rupicapra) and Balkan chamois (Rupicapra rupicapra balcanica). Recently confirmed, these two subspecies hybridize in the contact zone on Mts. Velebit, coastal Croatia. Patterns of horn development in chamois can differ within populations and subspecies, and are mostly influenced by genetic structure, sex and resource availability. These factors control the direct ability of energy allocation to secondary sexual traits with diverse outcomes. This is the first study to investigate the horn growth patterns of Alpine and Balkan chamois populations and their admixed progeny. We explored the differences in horn growth and compensatory patterns among populations as a function of genetic background, separately for females and males. A significantly different pattern of horn development was detected in the hybrid population showing higher rates of initial horn growth – until 2.5 years, and much lower compensation rates in the first 4.5 years of life in both sexes in comparison to other chamois populations. Interestingly, differences in growth patterns were more expressed among males. Higher initial horn growth and low compensation rates in the hybrid population can be explained by both non-genetic and genetic factors, but may suggest an effect of heterosis, i.e. the occurrence of hybrid vigour. Therefore, this situation could reveal the extent to which life history patterns and evolutionary consequences can shape important traits of population outlook/vigour, such as horn size, in different chamois populations.
Nikica Sprem
added a research item
The Dinaric Mountains in Slovenia, Croatia and Bosnia and Herzegovina provide a unique system to address the effects of past hunting on the genetic structure of Northern chamois (Rupicapra rupicapra) and possible hybridization in the contact zone in the Velebit Mountains. The northern Dinaric Mountains should be occupied by Alpine chamois (Rupicapra rupicapra rupicapra) while the central and southern areas are inhabited by the Balkan chamois (R. rupicapra balcanica). This is the first study to characterize the genetic variation in chamois populations in the area. We used microsatellite and mitochondrial markers to analyze the genetic variation and structure of chamois populations from different geographical areas with different histories. Specifically, the influence of recent human translocations and geographical isolation on the genetic architecture of chamois populations was explored in the assumed contact zone. We successfully genotyped 74 individual samples and the number of alleles/locus ranged from 6 to 20 with a mean of 9.20. Allelic richness across populations ranged from 2.94 in the Prenj Mountains (Bosnia and Herzegovina) to 3.56 in the Biokovo Mountains (Croatia). A similar pattern was also observed for heterozygosity, ranging between 0.729 and 0.572, and expected heterozygosity, between 0.762 and 0.644 in the Prenj and Biokovo Mountains, respectively. The global FST for 7 population samples was 0.103 ± 0.047 (range = 0.0156 – 0.185). The Structure tree clusters separated samples from the northern Dinaric Mountains from those of the southern Dinaric Mountains into 2 clusters according to geographic location. The results obtained using a Bayesian clustering methodology was similar. By using mtDNA variation in chamois from Slovenia, Croatia and Bosnia and Herzegovina, the existence of Alpine chamois haplotypes in northern areas and Balkan chamois haplotypes in southern areas was confirmed. These results confirm the impact of recent human management (i.e., translocation) into the Velebit Mountains, which established a new contact (hybridization) zone between the subspecies. Therefore, future translocations must be planned carefully to avoid compromising genetic integrity and posing a serious risk to native species, as in this case.
Nikica Sprem
added a project goal
To analyze genetic structure of Balkan chamois in Dinaric Mountains, using neutral (SNP) and loci under selection (MHC complex) to estimate conservation status and to study mechanisms of molecular adaptation of local populations.