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The Balkan terrapin (Mauremys rivulata) is an endangered species protected by the Habitats Directive. Its range in Croatia is restricted to the southernmost karstic aquatic habitats. Most of these habitats are antropogenic modifications of lentic and lotic water used historically for agriculture. The aim of this study was to investigate the available genetic tools for conservation management of the species and to conduct a preliminary analysis of the genetic diversity of M. rivulata in Croatia. The survey of literature and openly accessible genetic repositories revealed the knowledge on complete and incomplete genes, microsatellite markers, rRNA and complete mtDNA for the species. Ampliffication of all 18 microsatellite markers available for the species on 56 samples resulted in 15 successfully amplified markers. Nine of them were estimated to have high probability of null alleles and were excluded. Preliminary genetic diversity analysis of two populations from Croatia (Majkovi and Konavle) and one from Montenegro (Sutorina) based on six microsatellite markers revealed a high level of polymorphism (24 alleles per locus on average). The values of expected heterozygosity ranged from 0.85 (Sutorina) to 0.93 (both Majkovi and Konavle populations). Bayesian analysis of population structure revealed the existence of three gene pools, the first dominant in the Suturina population, and the second in both Croatian populations, while the third was present to a lesser extent in all populations. Although they provide insights into the population genetics of M. rivulata, the available microsatellite markers are limited due to the high proportion of null aleles, especially for parentage analysis. SNP markers developed using the ddRAD method would provide a more precise and cost-effective estimate of effective population size and efficient population management of this threatened species.

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.

Interakcije kitova i ljudi zabilježene su u svim svjetskim morima i mogu utjecati na stopu smrtnosti određene populacije kitova. Cilj ovog istraživanja bio je analizirati tipove i zastupljenost interakcija kitova i ljudi u hrvatskom dijelu Jadranskoga mora na temelju podataka prikupljenih u okviru dugogodišnjeg praćenja smrtnosti kitova koje od 1990. provodi Veterinarski fakultet Sveučilišta u Zagrebu. Pregledali smo i analizirali podatke o uginulim kitovima sakupljene od listopada 1990. do travnja 2022., uključujući i podatke dobivene razudbom. U tom razdoblju pronađene su ukupno 522 uginule jedinke iz reda kitova. Utvrđene interakcije kitova i ljudi klasificirane su kao: sumnja na prilov (N=47), ingestija ribarskog alata (N=22), potvrđen prilov (N=21), strangulacija grkljana (N=18), namjerno ubijanje (N=11), ingestija otpada (N=4) i kronično zaplitanje u ribolovni alat (N=3). Od ukupnog broja uginulih kitova u njih 111 utvrđena je najmanje jedna interakcija, a najčešće pogođena vrsta bio je dobri dupin (Tursiops truncatus) (N=105). Ovo istraživanje potvrđuje da u hrvatskom dijelu Jadrana postoji znatan negativni utjecaj ljudskih čimbenika na jedinke iz porodice kitova te u prezentaciji raspravljamo i predlažemo mjere za zaštitu.

The major histocompatibility complex (MHC) are highly polymorphic genes important for adaptive immunity. Due to their association with adaptation and fitness traits, MHC genes have been largely explored in evolutionary ecology and conservation genetics. The maintenance of high genetic diversity on MHC genes is attributed to the action of balancing selection driven by pathogens. Here, analysis of MHC class II DQA and DQB loci in striped dolphin from the Mediterranean Sea revealed extraordinary diversity, with 29 unique genotypes detected in 31 individuals. Allelic diversity represented by 17 DQA and 18 DQB alleles, which form 27 two-locus haplotypes, was further extended to substantial functional diversity. Namely, translated alleles formed ten DQA and seven DQB structural supertypes capable of binding and presenting a broad range of antigens. The acting of balancing selection on both genes was indicated by homogenised allele and supertype frequencies, presence of rare alleles, and several tests of long-term positive selection. We have found intriguing results on DQA diversity comparable to DQB locus, which is atypical for usually less polymorphic MHC alpha genes. Moreover, Ewens-Watterson test provided compelling evidence of recent selective pressure acting on the striped dolphin’s DQA gene. Indeed, the Mediterranean striped dolphin population faced severe recurrent morbillivirus epizootics over the last decades, of which the first one recorded during 1990-1992 was particularly severe. We hypothesize that morbillivirus exerted selective pressure that shaped extensive MHC class II DQ genes diversity in striped dolphin, which potentially might be an interesting model to observe contemporary adaptation to environmental challenges.

Geni glavnog sustava tkivne podudarnosti (engl. Major histocompatibility complex, MHC) prepoznati su kao važni lokusi za proučavanje adaptivne genetske raznolikosti u kralježnjaka. Ovi geni kodiraju za molekule koje limfocitima T predstavljaju antigene izvedene iz patogena, pokrećući tako odgovarajuću imunosnu reakciju. Pretpostavlja se da se visoki stupanj varijabilnosti gena MHC održava selekcijom posredovanom parazitima. Obični jelen (Cervus elaphus) pripada redu Cetartiodactyla, i jedan je od najvećih predstavnika porodice Cervidae. Nedavno širenje alohtonog parazita metilja Fascioloides magna predstavlja sve veću prijetnju europskim populacijama jelena. Cilj ovog istraživanja bio je genotipizirati lokus MHC-DRB 81 jedinke jelena iz Vojvodine, područja zahvaćenog invazijom F. magna, kako bi se istražio stupanj varijabilnosti gena MHC-DRB i time dobio uvid u imunogenetičku kompetentnost ove populacije. Za to smo koristili Illumina sekvenciranje nove generacije i web alat AmpliSAT za analizu dobivenih podataka. Otkrili smo visoku razinu varijabilnosti, s ukupno 32 alela, od kojih je 10 bilo novih. Broj alela po jedinki bio je 1-6 (prosjek = 4, 27), što ukazuje na postojanje najmanje tri lokusa DRB. Broj varijabilnih mjesta bio je 90 (36, 1%), a nukleotidna raznolikost po mjestu bila je 0, 11. Najučestaliji alel bio je Ceel-DRB*HR04, prisutan u 49, 6% jedinki, što bi moglo ukazivati na njegovu zaštitnu ulogu protiv F. magna.

Goal was to determine the influence of island population isolation on the sub structuring of the Croatian population, and the influence of regional population groups on the sub structuring of Southeast Europe with regards to basic population genetic statistical parameters calculated using STR locus analysis. Bio-statistical analyzes were performed for a total sample of 2877 unrelated participants of both sexes from the area of Southeastern Europe was analyzed. Nine autosomal STR loci (D3S1358, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317, D7S82) were analyzed using standard F-statistics and population structure analysis (program STRUCTURE). The total coefficient of genetic differentiation of Croatian subpopulations calculated by the FST method is higher at the level of the Croatian population (0.005) than at the level of Southeast Europe (0.002). In the population of Croatia, the subpopulation of the island of Vis shows the most pronounced separation, and in the population of Southeast Europe the population of Albanians from Kosovo, then the populations of Croatia, Bosnia and Herzegovina and Hungary. The established higher structure of Croatian subpopulations in relation to the populations of Southeast Europe suggests the existence of a certain degree of genetic isolation, most likely due to the influence of endogamy within rural island populations. The total genetic differentiation coefficient of Croatian subpopulations calculated by the FST method is higher at the level of the Croatian population (0.005) than at the level of Southeast Europe (0.002). The established higher structure of Croatian subpopulations in relation to Southeast Europe suggest the existence of a certain degree of genetic isolation, most likely due to the influence of endogamy within rural island populations.

Major Histocompatibility Complex (MHC) genes play a pivotal role in host defence system. Interaction between parasite infection and MHC genes are explained by two hypotheses, first that propose greater resistance in heterozygotes, and other that promotes the role of specific alleles. In this study, we have analysed relation between MHC genes and non-native trematode, Fascioloides magna. In total, we have analysed 91 red deer livers. DNA was isolated from 40 selected samples, exon 2 of the DRB loci of MHC class II was amplified by PCR, and sequenced by Next Generation Sequencing (NGS). We have found 38 alleles, with only four of them been previously known (DRB_NS36, DRB_NS38, DRB_ref01, DRB_ref02). The presence of the DRB_ref01 allele was significantly higher in the area of Lipovljani compared to Baranja (χ2 = 4.928, p=0.026411). The DRB_ref02 allele was significantly more frequent in Lipovljani compared to Baranja (χ2 = 3.931, p = 0.473), and DRB_ref03 in Baranja than in Lipovljani (χ2 = 5.1481. p = 0.023272) and Spačva (χ2 = 4.5365, p = 0.033). The differences in infection between animals with DRB_ref01 and DRB_ref06 alleles were statistically significant (χ2 = 0.939, df = 1, χ2/df = 0.94, p = 0.332). The remaining alleles appeared rarely and could not be related to F. magna infection. The high allelic diversity observed in this research is in accordance with other studies conducted on deer species. The relationship between the DRB_ref01 and DRB_ref06 alleles suggests the potential evolution of host-parasite interaction in the case of red deer fascioloidosis.

Crvenokljuna čigra (Sterna hirundo) je kolonijalna ptica iz porodice galebova (Laridae) koja se uglavnom gnijezdi na niskim morskim obalama, ali nastanjuje i slatkovodna staništa, poput šljunčanih otoka na rijekama te umjetnih gnjezdilišta. U Hrvatskoj morske kolonije broje svega nekoliko desetaka parova, dok kontinentalne populacije uz rijeku Savu i Dravu mogu imati i do 130 parova. Procjena genetičke raznolikosti populacija važna je za uspostavu odgovarajućih koraka njihove zaštite. Cilj ovog istraživanja bio je odrediti haplotipove kontrolne regije mitohondrijske DNA kopnenih i morskih populacija crvenokljune čigre iz Hrvatske, te usporediti njihovu genetičku raznolikost. Analizirali smo 78 uzoraka s tri različite populacije (38 sa Save, 17 s Drave i 23 s jadranske obale) sekvenciranjem dijela kontrolne regije mtDNA duljine 709 pb. Pronašli smo 20 haplotipova s 13 polimorfnih mjesta, od kojih sedam haplotipova nije pronađeno u prethodnim istraživanjima. Najučestaliji haplotip je Stehi03 frekvencije iznad 43 %. Ukupna raznolikost haplotipova je relativno visoka (0, 795), dok je nukleotidna raznolikost niska (0, 00249). Populacija Save imala je najveći broj haplotipova (16). Indeksi raznolikosti najviši su za populaciju Drave te najniži za morsku populaciju. Ovi rezultati ukazuju na potencijalno veću genetičku raznolikost slatkovodnih populacija, ali potrebna su dodatna istraživanja za planiranje budućih smjernica očuvanja čigri u Hrvatskoj.

Context: Genes of the major histocompatibility complex (MHC) have been extensively used for estimation of genetic diversity in wild vertebrate populations on account of their extreme polymorphism and fundamental role in pathogen resistance. The roe deer (Capreolus capreolus) was the most abundant game species in Croatia, however, a decrease in population number was observed recently, which is probably multifactorial but coincides with the spread of Fascioloides magna in Croatia. Aims: In this study, we aimed to determine the degree of MHC allelic diversity in roe deer from Bjelovarsko-bilogorska county in order to evaluate the adaptation potential of this population. Methods: We amplified and sequenced 249 bp long fragment of MHC-DRB locus in 55 individuals by utilisation of Illumina MySeq technology. AmpliSAT integrated web tool was used for allele calling. Key results: Eight unique alleles were detected in 55 individuals. Previously acknowledged codon deletion was present in four alleles. The number of variable nucleotide positions was 22, nucleotide diversity was estimated at 0.04, and the average number of nucleotide differences at 10.6. The most common allele was Caca- DRB*0301 with a frequency of 42.7%. Detected alleles formed 18 different genotypes, the prevalent being Caca-DRB*0301/Caca-DRB*0303 which was found in 16.4% of individuals. A large number of individuals were heterozygous (83.6%). Conclusion: We detected moderate to high levels of genetic diversity in roe deer from Bjelovarsko-bilogorska county. A substantial proportion of heterozygotes detected in this study can potentially bring a selective advantage to the population. Implications: The assessment of adaptive genetic diversity can offer valuable clues regarding the population’s long-term survival prospects and will provide a basis for future association studies between MHC polymorphism and F. magna infection.

Srna obična (Capreolus capreolus) rasprostranjena je na većem dijelu Europe i prilagođena raznolikim staništima. Zbog socioekonomskih promjena u ruralnim područjima i šumskim nasadima, te reintrodukcija, brojnost populacije povećava se u zadnjih 30 godina, s iznimkom područja odnedavno zaraženih fasciolidozom (Fascioloides magna) gdje je zabilježen pad brojnosti. Rast populacije u većini staništa i pojava alohtonog parazita povećali su izloženosti parazitima. Raznolikost gena glavnog sustava tkivne podudarnosti (engl. Major histocompatibility complex – MHC) pogodan je pokazatelj kapaciteta populacije za suprotstavljanje novim patogenima i spososobnosti za prilagodbu. Geni MHC kodiraju membranske molekule čija je glavna funkcija prezentacija antigena T limfocitima te stoga imaju središnju imunoregulacijsku ulogu. Cilj istraživanja bio je utvrditi varijabilnost lokusa DRB1 klase II gena MHC u srne obične iz Hrvatske. DNA je izolirana iz 50 uzoraka mišićnog tkiva. Rezultati sekvenciranja nagovjestili su značajno veći udio heterozigota u odnosu na naše preliminarno istraživanje (70 % naspram 25 %). Međutim, pojava nukleotidne delecije kod nekih heterozigota onemogućila je potpunu identifikaciju alela isključivo putem Sangerovog sekvenciranja, te smo upotrijebili molekularno kloniranje kako bi potvrdili novopronađene sekvence. Ova metoda je dugotrajna i skupa pa bi ciljano sekvenciranje sljedeće generacije u budućnosti moglo reducirati vrijeme i trošak razdvajanja alelnih faza.

Common tern is a colonial seabird species whose breeding grounds include both freshwater and marine sites. Freshwater colonies are under a significant negative anthropogenic influence, mainly due to river regulations. Consequently, freshwater colonies throughout Europe are currently located mostly on artificial breeding sites, including rafts and gravel islands. Common terns in inland Croatia and Slovenia breed in the areas of the rivers Sava and Drava, whereas numerous marine colonies are scattered along the Adriatic coast. In order to explore their population potential for adaptation and environmental change, it is important to assess genetic diversity indices. Furthermore, the spatial pattern of genetic structure could contribute to future management decisions. Therefore, we aimed to inspect genetic diversity and degree of differentiation among common tern colonies. We analysed 52 samples from freshwater colony sites in Croatia and Slovenia and a coastal colony site in Slovenia. We amplified 7 previously published microsatellite loci. The mean allelic diversity per microsatellite locus was 6.095, ranging from 5 to 8 alleles and the mean expected heterozygosity was 0.72, ranging from 0.638 to 0.782. The results indicate relatively high genetic variability of the study populations. Structure analysis revealed two distinctive clusters - corresponding to the areas of the rivers Sava and Drava, and the coastal colony site Sečovlje Salina. Our findings illustrate the importance of those habitats as the reservoirs of genetic diversity and calls for their further protection and management.

Red deer (Cervus elaphus) are large indigenous mammals in Croatia. Even though Major Histocompatibility Complex (MHC) genes play a pivotal role in initiating immune response, there are no reports on their variability in red deer from Croatia. In this study, the variability of the DRB locus of MHC genes class II was analysed in 23 red deer from a mountain region of Croatia (Gorski Kotar). NGS sequencing revealed 23 unique DRB exon 2 sequences in 23 individuals, of which only six had been previously identified. The number of allelic variants per individual ranged from 1 to 6 (mean 3.09), with one individual having only one allele and one individual having all six allelic variants recognised. The most frequent allele, Ceel-DRB*HR01, was identified in 12 individuals (16.9%). The number of variable nucleotide sites was 84 (33.7%), and the number of variable amino acid positions in translated sequences was 44 (53%). The mean nucleotide evolutionary distance, based on the Jukes-Cantor model, with the gamma distribution shape parameter, was 16.5%, while the pairwise nucleotide distance ranged from 0.04% to 14.24%. The amino acid evolutionary distance, based on the Jones-Taylor-Thornton model, was 34.7%. Compared to the literature, it is clear that the mountain population of red deer from Croatia possesses considerable variability on the DRB locus, which is further confirmed by the notable nucleotide evolutionary distance., Jelen obični (Cervus elaphus) jest zavičajna vrsta divljači u Republici Hrvatskoj. Unatoč činjenici da glavni sustav tkivne podudarnosti ima vodeću ulogu u pokretanju imunosnog odgovora, nema dostupnih podataka o njegovoj varijabilnosti u jelena običnog na području Hrvatske. U ovom je radu istražena raznolikost lokusa DRB gena MHC klase II na uzorku od 23 jelena iz gorskoga staništa (Gorski kotar). Sekvenciranje nove generacije (NGS) potvrdilo je postojanje 23 jedinstvene sekvencije DRB egzona II, od kojih je prethodno identificirano svega 6. Broj alela kolebao je od 1 do 6 (prosjek 3,09) po životinji, dok je jedna jedinka imala svega 1 alel, a jedna svih šest alela. Najčešći alel, Ceel-DRB*HR01, utvrđen je u 21 jedinci (16,9 %). Broj varijabilnih nukleotidnih mjesta bio je 84 (33,7 %), a broj varijabilnih pozicija aminokiselina 44 (53 %). Prosječna evolucijska udaljenost nukleotida, temeljena na Jukes- Cantorovu modelu, s gama-distibucijskim parametrom, bila je 16,5 %, dok je nukleotidna udaljenost sekvencija (temeljena na baznim parovima) iznosila od 0,04 % do 14,24 %. Aminokiselinska evolucijska udaljenost, temeljena na Jones-Taylor-Thorntonovu modelu, iznosila je 34,7 %. Usporedbom s literaturom vidljivo je da gorska populacija jelena običnoga u Hrvatskoj posjeduje zadovoljavajuću raznolikost na lokusu DRB, što je nadalje potvrđeno primjetnom nukleotidnom evolucijskom udaljenošću.

Golden jackal is a canid species currently distributed throughout the Southeastern, Central and Northern Europe. Serbian population has been growing rapidly since the early 1980s. Previous research was done solely on neutral markers and the results indicated lower genetic variability of the Serbian population. Given the jackals recent expansion, it is important to investigate variability of the adaptive loci in order to explore population’s potential for adaptation. Genes of the major histocompatibility complex (MHC) are one of the most polymorphic coding regions of the vertebrate genome. The role of MHC genes is to encode receptors responsible for presentation of antigens to T lymphocytes, which is crucial for specific immune response. DNA was extracted from 36 individuals that were legally shot in northwestern, eastern and southeastern Serbia. MHC class II loci DLA- DRB1, DQA1 and DQB1 were amplified and sequenced. Newly found allele was confirmed by molecular cloning and three-locus haplotypes were assigned to each individual. Analysis revealed four DQA, three DQB and four DRB alleles. Allele DQA1*00101 was found for the first time in golden jackal species, exclusively in one individual and surprisingly, in homozygotic form. Found alleles formed six different three-locus haplotypes. Our results suggest that Serbian jackal population might have a higher MHC diversity than the Croatian population as two additional alleles were detected on DQA locus despite the significantly smaller sample size.

The genes of the major histocompatibility complex (MHC) code for receptors that recognize and bind antigens in order to present them to T cells, thus they have a major role in determining the immune response. MHC region includes some of the most variable loci in the vertebrate genome, reflecting the strong association between MHC diversity and resistance/susceptibility to disease. With their well-characterized function and exceptional diversity, they are a key genetic marker for studying the processes of species adaptive evolution. The goal of our study was to examine spatial distribution of allelic diversity at exon 2 of MHC class II DRB locus across the majority of distribution range of the genus Rupicapra using next generation approach (Ion Torrent S5, Thermo Fisher). We identified 20 alleles in 96 individuals. Twelve alleles had been previously identified in chamois while the remaining 8 are novel. The number of variable nucleotide sites in detected alleles was 23 (10%) and the number of variable amino acid positions in translated sequences was 11 (14%). Out of 20 identified alleles, 18 translated into different amino acid sequences. The number of alleles per individual ranged from 1 to 6 (mean = 3.1), indicating the presence of at least 3 loci resulting from gene duplication events. The most frequent allele, Ruru-DRB*01, was identified in 68 individuals (71%) while 5 alleles were present only in a single individual each. Our results provide evidence of multiple co-amplifying copies, showing that MHC class II DRB is a complex multilocus system with a high level of polymorphism in chamois. Finally, our study represents the first assessment of immunogenetic map of chamois populations that can be used in future conservation management of this species.

Barbary sheep (Ammotragus lervia) is a non- native ungulate from North Africa which has been established in the wild beyond its natural range due to intentional introductions and illegal releases. In Europe, four countries are inhabited with free-ranging Barbary sheep population: Croatia, Czech Republic, Italy and Spain. While being threatened in their native area, the majority of its exotic populations have showed high adaptability and formidable capacity to settle and expand their ranges. Non-native species cause changes to the ecosystems in which they are introduced. To understand their potential to establish and spread in the novel range, it is crucial to study their genetic diversity and population genetic structure. Population genetic parameters affect population viability and adaptive potential under environmental change. Here, we report the first genetic study of European Barbary sheep populations. We used ten microsatellite loci previously published to characterize and compare the genetic diversity and spatial pattern of genetic structure among study populations. We aimed to understand how current levels of genetic diversity and structuring vary among Barbary sheep populations that differ with regard to the time and source of introduction. Our results present important knowledge about origins of these populations and are the basis for further management.

Fascioloidosis is a parasitic disease caused by non-native parasite Fascioloides magna. F. magna originally parasitize in the North American deer species. It was introduced to Europe at least twice, forming the three foci of infection: Italian, Czech and Danubian. The latter two gave origin to other, newly formed areas, including the Croatian one. F. magna was detected in Croatia in 2001 and since then have spread to the majority of its lowland areas. The fact that F. magna is a non-native parasite enables us to observe development of host- parasite interactions, but also to create conditions of case-control study in the wild, large mammal population. In this research we aimed to compare variability and presence of specific alleles of MHC class II – DRB exon 2 in relation to F. magna infection. A total of 117 red deer livers and faecal samples originating from lowland (Baranja, Lipovljani and Spačva) and mountain area (Gorski Kotar) were collected. Of them, based on location and parasitological analysis, 46 individuals were selected and tissue samples were sent for analysis using Next Generation Sequencing (NGS). We have detected 44 DRB alleles (2-4 alleles per individual). Four alleles were already known (our references - DRB_NS36, DRB_NS38, DRBref01 i DRBref02). Statistically significant difference was detected in the level of infection in the case of animals with DRB_ref01 and DRB_ref06 alleles (χ2 = 0, 939, p = 0, 332). This implicates on a potential development of host-parasite interaction in the case of red deer and Fascioloides magna.

The brown bear (Ursus arctos) is the most widely distributed ursid species, which historically inhabited the majority of Eurasian continent. However, due to extensive hunting and habitat destruction, brown bears in Europe have lost most of their range. Several remnant west-European populations are small and endangered, while large populations reside in Scandinavia, Baltic-Karelian region, Carpathian and Dinaric-Pindos Mountains. The Croatian population, which is estimated at approximately 1000 individuals and legally protected, belongs to the Dinaric-Pindos population. Genetic studies in endangered populations can significantly contribute to establish efficient conservation guidelines. The Major histocompatibility complex (MHC) region includes positively selected immune genes involved in pathogen recognition, mating preferences and susceptibility to infectious and autoimmune diseases. Due to their role in important biological traits, MHC genes represent valuable genetic markers for adaptive genetic variability that determines population capacity for environmental challenges. Using sequencing-cloning and single-strand conformation polymorphism methods in this preliminary research, we analysed 32 brown bear samples from Croatia at MHC class II DQB locus. We identified six unique DQB alleles, three of which were new variants. In six individuals, three alleles were detected and locus duplication was deduced. Alleles Urar-DQB*03 and Urar-DQB*04 were regularly found within the same genotype, and were also present in all three-allelic individuals. That finding indicates that DQB gene duplicates are linked and alleles are inherited as two-locus haplotype, which is presumably favoured by natural selection. We will continue our research with a larger sample set of individuals collected during several years, analysis of additional MHC class II loci and investigating association between MHC polymorphism and resistance / susceptibility to common pathogens that are present in bears.

The Barbary sheep is a caprine (sheep-antelope) ungulate whose native range covers mountains of North Africa where it is considered vulnerable by the IUCN. Its population has been decreasing due to habitat destruction and exposure to poaching. The species was introduced to Europe in the 20th century mainly as a game species since its morphological features make it attractive for trophy hunters. Since then a large free-ranging population was established in Spain and smaller ones were established in Croatia, the Czech Republic and Italy. Possible future negative impacts on the ecosystem were suggested through parasite transmission, competition with native fauna and alteration of the herbaceous plant community. To address populations dispersal potential in non-native habitats it is important to gain information regarding the genetic diversity of European populations. In order to investigate mitochondrial DNA diversity, 66 samples were analyzed, 32 of which were from Mosor Mountain in Croatia, 19 from the Iberian Peninsula (Almeria and Sierra Espuña) and 15 from the Canary islands. Four haplotypes were found in total. Haplotype Amle01 was the only one found in Croatian and the Canarian samples, indicating their possible common origin. Samples from Almeria were monomorphic for the Amle02 haplotype, while samples from Sierra Espuña exhibited three haplotypes (Amle01, 03 and 04) with a gene diversity of 0.71. Future implementation of microsatellite data analysis could reveal more information on illegally introduced Barbary sheep population origin and European population structure.

Major histocompatibility complex (MHC) class II genes code for protein receptors expressed on the surface of professional antigen-presenting cells. These receptors bind foreign antigens derived from extracellular pathogens and present them to T cells, which initiate an adaptive immune response. MHC genetic region contains some of the most polymorphic coding genes in the vertebrate genome and MHC genes represent some of the best candidates for studying the processes of adaptive evolution. The wild boar is an important game species and an ancestor of the domestic pig. It is considered one of the most widespread species. However, while many data are available on MHC variability in domestic pigs, only few studies have been performed in wild boars. The MHC region in swine, called the swine leukocyte antigen (SLA), is characterised based on research on domestic pig, and includes one functional gene (DRB1) and four pseudogenes. However, duplication of DRB1 gene was recently suggested. The goal of this study was to investigate variability and evolution of the fulllength MHC class II SLA-DRB1 locus exon 2. We analysed 133 wild boar samples from Croatia using direct sequencing and cloning methods. A considerable amount of nucleotide polymorphism of SLADRB1 in the Croatian wild boar population was detected, since 20 notably divergent alleles were found, eight of which were new. A recent discovery of duplication of the DRB1 gene in swine was confirmed by our results, as we identified eight individuals with three alleles. Furthermore, our phylogenetic reconstruction suggested the duplication event was recent, as paralogous sequences had not accumulated sufficient divergence to be apparent as distinct clusters. Since locus duplication has not been found in other investigations on the SLA region, the molecular organization of the MHC class II region in swine should be further investigated and probably reconsidered.

Major histocompatibility complex genes encode cell-surface glycoproteins that present antigens to T cells, a process essential for triggering an appropriate immune response. MHC contains some of the most variable genes in vertebrates whose diversity has been driven by evolutionary processes over long period of time. Their variability reflects the ability of a population to confront various pathogens and strong association between MHC variability and resistance to infectious organisms has been established. Wild boar's widespread distribution and potential to host many parasite species makes it suitable for testing that correlation. The goal of this study was to improve our method for assessing variability of the MHC class II DRB1 locus, since our preliminary analysis revealed only 5.5% of heterozygous individuals, which strongly indicated existence of null-alleles. In this study we compare results obtained by the two PCR methods, differing in primer sequences. We analysed 155 samples of wild boars from Croatia. Muscle samples were collected following regular hunting operations and DNA was extracted using commercial kit. Polymerase chain reaction was used to amplify the exon 2 of DRB1 locus with different primers than in our previous preliminary research. Furthermore, the primers used in this research enabled amplification of the whole exon 2 (267 bp), while previous method amplified 198 bp of the exon 2. We successfully amplified and sequenced 124 individuals of which 73.4% were heterozygotes. We identified 19 different DRB1 alleles, seven of which were new, while only 11 alleles were identified in our previous research. In conclusion, the PCR method with different primer set proved to be more efficient for identification of all/majority of alleles present in a population of wild boar from Croatia.