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Species range expansions and (re)colonization of landscapes variously dominated by humans occur on a global scale. Understanding such range enlargements and subsequent changes in the composition of ecological communities is important for conservation management, and the golden jackal (Canis aureus) can be considered a model species for regional and continental range expansion. Although this mesopredator has been known from the Adriatic Coast of southeastern Europe for over 500 years, the species is a recent arrival further north, including in Slovenia where jackals were first confirmed in the 1950s. Research from eastern Italy found jackals with ancestry from the Dalmatian region on the Adriatic Coast and the Pannonian region further east. We predicted similar ancestry for Slovenian jackals, and examined samples from Croatia, including Dalmatia and interior regions, Serbia, Hungary, and Slovenia with 25 microsatellite markers to determine population genetic structure. We detected two distinct genetic clusters, representing the Dalmatian and Balkan-Pannonian (Pannonian) jackal populations (F$_{ST}$ = 0.157, 95% CI: 0.112–0.209). Contrary to expectations, only few individuals in Slovenia exhibited signs of Dalmatian ancestry, and none appeared to be direct immigrants. Some results suggested a third cluster centered in northern Hungary. These divergent profiles might indicate immigration from outside the study area, and samples from regions further east are required for additional resolution. Based on our results, we hypothesize that Dalmatia has not been a substantial source for recent range expansion of the species, which has likely occurred from the east. Further investigation can help resolve the ancestry and current distribution of the Dalmatian and Pannonian populations, and the ecological relationships resulting from progressively overlapping distributions of canid species. Finally, genomic research could illuminate whether genetic variants from eastern areas might have facilitated jackal expansion into regions characterized by a colder climate, the presence of snow, and extensive forest cover habitats seemingly avoided by the jackals occupying the Adriatic Coast and surrounding areas in southeastern Europe.

Poaching is one of the main threats to the strictly protected grey wolf (Canis lupus) population in Croatia. It is generally difficult to obtain hard evidence to convict individuals suspected of illegal hunting or trading threatened species. Veterinary forensics often includes species determination from trace evidence like blood, saliva, hair or bone fragments. We report a case of species determination from two canid skulls (marked as H1 and H2). Both animals were hunted near Zagreb, Croatia in 2010. Hunter reported a legal harvest of two golden jackals (Canis aureus), but hunting inspection doubted species identification. Our task was to determine which of the three canid species present in Croatia these skulls belong to – strictly protected grey wolf, golden jackal which is a game species or a dog. Because of similar morphology and evolutionary relatedness distinguishing the three canid species is particularly difficult, even on a DNA level. For species determination we used craniometrical analysis, combined with analysis of several DNA markers - mitochondrial DNA control region, Y chromosome and MHC genes. As skulls were exposed to high temperatures during trophy preparation, DNA (extracted from tooth rooth) was damaged and aggravated DNA analysis. Both craniometrical measurements and DNA analysis suggested that the skull marked as H1 belongs to the wolf. Based on craniometrical analysis we concluded that skull marked as H2 is not a purebred jackal or a purebred wolf, while DNA analysis of maternally inherited mitochondrial DNA control region confirmed dog maternal ancestry. Due to the damaged DNA amplification of other markers was not successful and we were not able to confirm paternal ancestry of this individual.

Detecting the hybridization between species is important for evolutionary studies of speciation processes, conservation biology and management, but we still lack a general empirical perspective of hybridization problems in canids. Hybridization may occur between many species of the canid family, sometimes threatening the survival of endangered species or populations. Mitochondrial DNA control region haplotypes can be used to discriminate between Croatian wolves, dogs and jackals, and the goal of our research was to identify species specific Y chromosome haplotypes that could be used in detection of paternal origin in possible hybrids. We analyzed three non- overlapping Y chromosome fragments of the grey wolf, golden jackal and dog in the total length of 1, 898 base pairs. Two haplotypes were identified, one shared among grey wolf and dog, and one specific golden jackal haplotype. We did not find polymorphic sites that could be used to distinguish the paternal line in wolf - dog hybrids, but six polymorphic sites were identified that can be used to discriminate golden jackal from grey wolf and domestic dog.

Glavni sustav tkivne podudarnosti (MHC) sudjeluje u adaptivnom imunosnom odgovoru sisavaca. Veća raznolikost gena MHC omogućuje limfocitima T da prepoznaju širi spektar antigena, što u konačnici daje jedinci, ali i populaciji veću šansu za preživljavanje napada patogena. Dobar su molekularni marker za proučavanje genetičke raznolikosti vrsta i populacija zbog svoje poligenosti i polimorfnosti. Čagalj (Canis aureus) je zvijer (red Carnivora) koja pripada porodici pasa (Canidae). Istraživanja raznolikosti gena MHC kod čaglja tek su nedavno započela unatoč njegovoj širokoj rasprostranjenosti na euroazijskom i afričkom kontinentu. Cilj ovog istraživanja je određivanje raznolikosti tro-lokusnih haplotipova DQA/DQB/DRB te pojedinih lokusa skupine II sustava MHC u populaciji čagljeva iz Srbije. U ovom istraživanju na 29 uzoraka mišića čagljeva pronađeno je tri alela lokusa DQA i DQB, četiri alela lokusa DRB te šest haplotipova. Sedam je alela dosad pronađeno samo kod čaglja, a tri su trans-specijski polimorfizmi. Tri su haplotipa pronađena samo u srpskoj populaciji, a tri haplotipa su zajednička s istočnoeuropskom populacijom. Potvrđena je postavljena hipoteza djelovanja pozitivne selekcije na svim lokusima koristeći dN/dS test, odnosno omjer nesinonimnih i sinonsimnih supstitucija na nukleotidnom slijedu alela. The genes of the major histocompatibility (MHC) system takes part in the adaptive immune response in mammals. Greater diversity of MHC genes enables T lymphocytes to recognise a wider spectrum of antigens, which in turn gives the individual a greater chance of surviving a pathogen attack. They are a good molecular marker for studying genetic diversity of both species and populations because they are polygenic and polymorphic. The golden jackal (Canis aureus) is a carnivore from the dog family (Canidae). Studies of MHC gene diversity of golden jackals have only recently emerged despite its wide distribution on the Eurasian and African continent. The aim of this graduate thesis is to determine variability of three-locus DQA/DQB/DRB haplotypes and individual Class II MHC loci in golden jackals from Serbia. In this study of 29 jackal muscle samples we have found three DQA and DQB alleles, four DRB alleles and six haplotypes. Seven alleles have only been found in jackals so far, while three alleles are trans-species polymorphisms. Three haplotypes are private to the serbian populations, while three haplotypes are shared with the Eastern European population. We have confirmed the proposed hypothesis of existing positive selection on all loci using the dN/dS test, i.e. the ratio of non-synonymous and synonymous substitutions on the allelic nucleotide sequences.

Geni unutar glavnog sustava tkivne podudarnosti (MHC) kodiraju receptore koji su odgovorni za preradu i prikazivanje antigena limfocitima T, čime se pokreće adaptivni imunosni odgovor organizma. Veća varijabilnost genskih lokusa MHC omogućuje prepoznavanje šireg spektra antigena, pa posljedično i bolju obranu od patogena. Varijabilnost lokusa MHC održava ravnotežna selekcija, koju karakterizira veći broj raznolikih alela u populaciji. Geni MHC pokazali su se kao dobar marker za proučavanje adaptivne evolucije vrsta i populacija. Čagalj (Canis aureus) široko je rasprostranjen ali jedan od dosad najmanje istraženih pripadnika porodice pasa (Canidae). U ovom istraživanju utvrdila sam i opisala alele na lokusima DLA-DQA i DQB gena MHC skupine II u uzorku od 27 čagljeva iz istočne Europe. Pronašla sam 2 alela po lokusu, a svi su poznati iz prethodnih istraživanja. Unatoč malom broju pronađenih alela, evolucijske udaljenosti ukazuju na veliku raznolikost među njima. Potvrdila sam djelovanje ravnotežne selekcije na održavanje varijabilnosti oba istražena lokusa koristeći omjer stopa nesinonimnih i sinonimnih mutacija na čitavom nukleotidnom slijedu, kao i na pojedinim kodonima. Genes within the major histocompatibility complex (MHC) encode receptors that are responsible for processing and presentation of antigens to T lymphocytes, which initiates the adaptive immune response of the organism. Increased variability of MHC gene loci enables identification of a broader range of antigens, and consequently a better defense against pathogens. The variability of the MHC loci is maintained by the balancing selection, which is characterized by a larger number of divergent alleles in the population. MHC genes proved to be a good marker for the study of adaptive evolution of the species and populations. Golden jackal (Canis aureus) is widespread, but so far one of the least studied members of the dog family (Canidae). In this research, I found and described the alleles of the DLA-DQA and DQB MHC class II loci in a sample of 27 jackals from Eastern Europe. I found two alleles per locus, all of which are known from previous research. Despite the small number of alleles found, evolutionary distances indicate a considerable diversity among them. I confirmed the role of balancing selection on maintaining variability of the investigated loci, using the ratio of synonymous and nonsynonymous mutations both on the entire nucleotide sequence, and the particular codons.

Glavni sustav tkivne podudarnosti (MHC) ima ključnu imunoregulacijsku ulogu kod sisavaca. Raznolikost MHC gena omogućuje prepoznavanje širokog spektra patogena, a time i povećava izglede za preživljavanje vrste. Važan su molekularni marker za proučavanje adaptivne evolucije populacija i vrsta. Čagalj (Canis aureus) pripada porodici Canidae (psi), podred Caniformia, redu Carnivora (mesojedi). Unatoč svojoj širokoj rasprostranjenosti, čagalj je slabo istraživana vrsta i samo je nekoliko genetičkih istraživanja ove vrste objavljeno do danas. Cilj ovog rada je odrediti raznolikost gena DRB lokusa i DQA1/DQB1/DRB1 haplotipova skupine II glavnog sustava tkivne podudatnosti na uzorcima čagljeva istočne Europe. Iz 21 uzorka čaglja, postupcima izolacije DNA, PCR metodom, elektroforezom, sekvenciranjem i molekularnim kloniranjem pronašla sam pet alela na DRB lokusu. Svi aleli imaju jedinstveni aminokiselinski slijed, a dva alela su novoutvrđena te sam im dala radne nazive DRB*14aEst i DRB*15aEst. Alel DRB1*00901 je poznat iz prethodnih istraživanja na ostalim vrstama porodice pasa što potvrđuje postojanje trans-specijskog polimorfizma. dN/dS testom je potvrđena pretpostavka djelovanja pozitivne selekcije na DRB lokusu. Pronašla sam ukupno sedam jedinstvenih haplotipova u istraženom uzorku, a koristila sam alele dobivene za DQA i DQB lokuse na istom uzorku u ranijem istraživanju. The genes of major histocompatibility (MHC) system are a key component of the immune system in mammals. The diversity of MHC genes enables the recognition of a wider range of pathogens, and thus increases the chances of survival. They are important molecular markers for studying adaptive genetic diversity in populations and species. Golden jackal (Canis aureus) belongs to the family Canidae (dogs), suborder Caniformia, order Carnivora (carnivores). So far there are only a few published genetic studies concerning golden jackal although it is a widespread species. The aim of this graduate thesis is to determine the diversity of the MHC DRB class II gene in golden jackals from Eastern Europe. Using the methods of DNA isolation, PCR method, electrophoresis, sequencing and molecular cloning, I have identified five alleles at DRB locus from 21 samples. All five alleles has unique amino acid sequence, and two alleles have been newly found and named DRB*14aEst and DRB*15aEst. The allele DRB*00901 is known from previous research of other species in the dog family, which confirms the existence of a trans-species polymorphism. The dN/dS (ratio of non-synonymous versus synonymous substitutions) test has confirmed the assumption that DRB loci are influenced by positive selection. In the examined sample, total of seven unique haplotypes have been identified. For inference of haplotypes I used alleles found at DQA and DQB loci of the same samples identified in previous research.

Geni glavnog sustava tkivne podudarnosti (MHC) ključna su komponenta imunološkog sustava kod sisavaca te su i važan molekularni marker adaptivne genetičke raznolikosti u divljim populacijama životinja. Veća raznolikost MHC gena unutar populacije povećava izglede populacije za obranu od patogena i parazita, a time i izglede za dugoročno preživljavanje. Čagalj (Canis aureus) pripada porodici Canidae (psi), podred Caniformia, redu Carnivora (mesojedi). Do sada nema objavljenih istraživanja raznolikosti MHC gena ove vrste. Cilj ovog diplomskog rada je odrediti razinu raznolikosti DQA, DQB i DRB gena skupine II MHC sustava u čagljeva iz Izraela. Iz 30 uzoraka uške čaglja, postupcima izolacije DNA, PCR metodom, elektroforezom, sekvenciranjem i molekularnim kloniranjem, pronađena su tri alela na DQA lokusu, pet alela na DQB lokusu i četiri alela na DRB lokusu. Našli smo jedan novi jedinstveni alel na DRB lokusu (DRB1*04802var). Ostali pronađeni aleli poznati su iz prethodnih istraživanja na ostalim vrstama porodice pasa, što potvrđuje postojanje trans-specijskog polimorfizma. DQB i DRB lokusi pokazuju veliku raznolikost alela i velike evolucijske udaljenosti te visok omjer nesinonimnih supstitucija naspram sinonimnih, što potvrđuje pretpostavku djelovanja pozitivne selekcije na te lokuse. The genes of major histocompatibility (MHC) system are a key component of the immune system in mammals and are important molecular markers of adaptive genetic diversity in wild animal populations. Greater diversity of MHC genes within the population increases the chances of defence against pathogens and parasites, and thus the prospects for long-term survival. Golden jackal (Canis aureus) belongs to the family Canidae (dogs), suborder Caniformia, order Carnivora (carnivores). So far there are no published studies of MHC gene diversity on this species. The aim of this graduate thesis is to determine the level of diversity of the MHC system class II DQA, DQB and DRB genes in golden jackals from Israel. Three alleles at DQA locus, five alleles at DQB locus and four alleles at the DRB locus were found in 30 samples of jackal's ear. Methods used were DNA isolation, PCR method, electrophoresis, sequencing and molecular cloning. We found one new unique allele on DRB locus (DRB1*04802var). The rest of the alleles found are known from previous research on the other species of the dog family, which confirms the existance of a trans-species polymorphism. DQB and DRB loci show great diversity of alleles, large evolutionary distances, high ratio of non-synonymous versus synonymous substitutions, confirming the assumption that loci are influenced by positive selection.