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Anguilla anguilla, the European eel, is an important species for aquaculture and fisheries. Its population has dropped dramatically in recent decades, reaching an all-time low. As a result, it has been listed as critically endangered on the IUCN Red List of Endangered Species since 2007. Therefore, constant population monitoring is essential to ensure the survival of this iconic species. Glass eel recruitment is declining worldwide, including the populations in the Mediterranean region. Despite the negative impact of man-made activities in Mediterranean coastal waters over the past few decades, data on spawning biomass escaping from the Mediterranean highlights the region’s importance for the global eel supply. Eel research and monitoring is done using conventional techniques, which have certain drawbacks. Therefore, the use of molecular-based detection as a credible choice for monitoring species in aquatic ecosystems was recently shown to be an effective management plan alternative. We present the first use of environmental DNA for monitoring eel populations in the Adriatic Sea and in the complex Dinaric karst freshwater ecosystem. The method has been demonstrated to be accurate and useful for detecting the presence of A. anguilla eDNA and identifying conservation areas. This is also the first study investigating the range and presence of the European eel in the Adriatic and in the Mediterranean Sea, as well as in underground karst systems, springs, and in the karst poljes of the Eastern Adriatic coast.

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.

Aim To use the method of meta-analysis to assess the influence of island population isolation on the sub-structuring of the Croatian population, as well as the influence of regional population groups on the sub-structuring of the Southeastern European population with regard to basic population genetic statistical parameters calculated by using STR locus analysis. Methods Bio-statistical analyses were performed for 2877 unrelated participants of both sexes from Southeastern Europe. Nine autosomal STR loci (D3S1358, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317, and D7S82) were analyzed by using standard F-statistics and population structure analysis (Structure software). Results Genetic differentiation of Croatian subpopulations assessed with the FST method was higher at the level of the Croatian population (0.005) than at the level of Southeastern Europe (0.002). The island of Vis showed the most pronounced separation in the Croatian population, and Albanians from Kosovo in the population of Southeast Europe, followed by Croatia, Bosnia and Herzegovina, and Hungary. Conclusion The higher structure of Croatian subpopulations in relation to Southeastern Europe suggest a certain degree of genetic isolation, most likely due to the influence of endogamy within rural island populations.

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.

Major histocompatibility complex contains some of the most variable genes in vertebrates whose diversity has been driven by evolutionary processes over long period of time. This large region of DNA through class I and II genes encodes cell-surface proteins that bind antigens derived from pathogens or parasites and present them to T-lymphocytes, thus playing a major role in immune response. Knowing wild boar's widespread distribution and potential to host many parasite species, makes it suitable for testing correlation between MHC haplotypes and susceptibility/resistance to diseases. The goal of this study was to investigate imunogenetic variability of major histocompatibility complex class II DRB1 locus in Croatian wild boars. We analysed 73 samples of muscle tissue of wild boars from whole Croatia, excluding region that belongs to Zagreb City. Samples were collected following regular hunting operations, cut to appropriate size, stored in 96% alcohol, signed and frozen. DNA was extracted using commercial kit. Polymerase chain reactions were used to amplify the exon 2 of DRB1 locus and successfully amplified sequences were sequenced. The exon 2 was used as an indicator of allelic diversity since it encodes the peptide-binding region of MHC receptor and is the site of most polymorphism. Suitable bioinformatics programs (BioEdit, SeqScape) were used for analyses. We identified 11 different DRB1 alleles in wild boars in Croatia, with allele SLA‐DRB1*02 being the most frequent. However, only four animals were heterozygous (5.5%), suggesting existence of null-alleles, which will be resolved in further study. The results of this preliminary study will be used for future research of correlation between the MHC haplotypes and specific parasitic diseases of wild boar.

The Major Histocompatibility Complex (MHC) contains genes that play a major role in immune system of vertebrates. This genomic region encodes cell-surface proteins that bind antigens derived from various pathogens and present them to T-cell lymphocytes which trigger the appropriate immune response. Some MHC genes are the most variable coding genes of the mammalian genomes and their variability reflects adaptive processes in a population, interactions with pathogens and the population ability to confront them. Wild boar is considered one of the most widespread species. As a host of many parasites, it is a suitable species for testing the correlation between MHC haplotypes and susceptibility to diseases. The goal of this study was to investigate immunogenic variability of MHC class II DRB1 gene in wild boar (n=49) from Medvednica NP. Extraction of DNA was performed from muscle samples using commercial kit. Polymerase chain reactions were used to amplify the exon 2 of DRB1 locus. This exon was used as an indicator of allelic diversity since it encodes the peptide-binding region of MHC receptor and is the major site of polymorphism. Successfully amplified DNA sequences were sent for sequencing service and analysed using bioinformatics programs (BioEdit, SeqScape). Seven different DRB1 alleles were identified, at least one of which was not previously identified. Results of this study will be used for further Research of the correlation between the MHC haplotypes and specific parasitic diseases of wild boars.

The bottlenose dolphin (Tursiops truncatus) is a cetacean distributed worldwide with an external morphology that varies between different populations. An endangered population of bottlenose dolphins inhabits the Adriatic Sea and is legally protected. The skulls of 95 adult bottlenose dolphins (47 females and 43 males, 5 of unknown sex) were morphometrically analyzed. They originated from bottlenose dolphins stranded dead from 1990 to 2011 in the Croatian part of the Adriatic Sea. For each animal a total of up to 53 skull measurements and meristic characters were taken using slide calipers to the nearest 0.01 cm. Sexual dimorphism within the Adriatic population was analyzed while the average morphometric values of Adriatic specimens were compared with published values for the genus Tursiops from different geographical areas. Male Adriatic bottlenose dolphins were signifi cantly larger in terms of 19 craniometric characteristics compared to females. The male skull is wider along the rostrum, at the level of the braincase and at the orbital region. Their braincase is higher and longer and their teeth are higher. Comparison of morphometrical values between Adriatic bottlenose dolphins and populations from other seas confi rms geographical polymorphism within the species T. truncatus. Our study showed that the skull size follows Bergmann’s rule, with larger skulls found in colder waters, while smaller skulls are found in populations from temperate and tropical seas. Our results represent referent craniometrical values for the Adriatic bottlenose dolphin and should be used when implementing morphometry in population conservation.

Numerous reports of hybridization between wild Canis species and domestic dog suggest that hybridization between golden jackal and dog is likely. Here we present an assay of a Y chromosome marker for discriminating between golden jackal (Canis aureus) and dog (Canis lupus familiaris) paternal ancestry. Taking advantage of an insertion found in a dog Zfy intron haplotype but not in a golden jackal haplotype, we developed a three-primer PCR system in which species are differentiated based on the number of amplicons. Two amplicons are produced from domestic dog DNA template, while one amplicon is produced from golden jackal DNA template. Both amplicons can be analyzed in a single agarose gel electrophoresis run, while the longer amplicon also serves as an internal control. The method was validated using 27 golden jackal and 25 dog samples ; all samples showed the expected amplicons. This simple and cost-effective method should prove useful for determining the direction of golden jackal - dog hybridization.

Divlja mačka (Felis silvestris) autohtona je i strogo zaštićena vrsta u Hrvatskoj, međutim objavljeno je vrlo malo znanstvenih podataka o hrvatskoj populaciji divlje mačke. Cilj ovog istraživanja bio je analizirati raznolikost kontrolne regije mitohondrijske DNA divlje mačke na području središnje Hrvatske, kao uvod u šire istraživanje na cjelokupnom području Hrvatske. Uspješno je sekvencirano ukupno 814 parova baza kontrolne regije, među kojima je utvrđena prisutnost tri do pet ponavljanja ponavljajućeg slijeda RS2 dugog 80 pb, koji su zbog učestale heteroplazmije isključeni iz daljnjih analiza. Populacijska analiza provedena je na 414 pb kontrolne regije iz 26 uzoraka mišića divljih mačaka te je utvrđena prisutnost 18 polimorfnih mjesta koji čine deset različitih haplotipova. Haplotipska raznolikost analiziranih slijedova iznosi 0, 895 +/- 0, 031, dok je nukleotidna raznolikost 0, 018 +/- 0, 010. Genetička raznolikost divlje mačke iz središnje Hrvatske na temelju kontrolne regije mitohondrijske DNA je relativno visoka te na razini raznolikosti dobivenih u istraživanjima divljih mačaka u Njemačkoj i Italiji, a usporedbom dobivenih haplotipova s 44 slijeda pohranjena u GenBank bazi podataka nije pronađeno niti jedno podudaranje. S obzirom da je istraživanje provedeno na manjem broju uzoraka i na ograničenom geografskom području, rezultati upućuju na potencijalno veću genetičku raznolikost divlje mačke na području cijele Hrvatske te odbacuju sumnje na ugroženost ove vrste niskom genetičkom raznolikošću.

The Roma are a minority group that do not share a common homeland, speak different languages and consist of individuals of various religions. Population-genetic studies of Roma, as a transnational isolate, have mostly sought to compare their genetic affinities with proposed parental populations. The aim of this study is to assess the genetic structure of the Bayash Roma population from northwestern Croatia, and in the general Croatian population, to investigate the extent of admixture between them. Population differentiation and structure was analyzed on a set of genetic microsatellite data from two original studies (100 Bayash Roma from northwestern Croatia and 195 individuals from the general Croatian population). Results demonstrated that two population clusters best explain the genetic structure. Most of the individuals of the Bayash Roma population were assigned to a single genetic cluster and most of the individuals of the general Croatian population were assigned to another. Admixture analysis revealed that the percentage of non- Croatian individuals in general Croatian population is approximately twofold higher than percentage of non-Romani individuals in Romani population. Higher percentages of admixed and non-Croatian individuals found in the general Croatian population and lower percentages of admixed and non-Roma individuals found in the Bayash Roma population is in line with the presence of ethnomimicry in Roma.

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.

The typical bottlenose dolphin skull is elongated antorbitally and compressed postorbitally. Many cranium bones show variation in size and shape between individuals, they are therefore used for differentiation of subspecies and populations. The aim of this study was to describe bottlenose dolphin population from Croatian part of the Adriatic Sea by craniometric data. In order to account for potential ontogenetic variation, juvenile and adult individuals were separated based on degree of fusion of maxillary and premaxillary bones. Fifty-nine cranial measurements were taken from 96 adult specimens of bottlenose dolphin collected in Croatia from October 1990 to May 2011. Measurements were conducted with 0.5 – 0.01 cm precision using a caliper. Only one morphotype was recognized ; however males and females significantly differed in 19 measurements, male skulls were more robust. Comparing the Croatian results with the same cranial measures of bottlenose dolphin from other seas showed that the skulls from the eastern Mediterranean Sea (Israeli coast) and from the Black Sea were significantly smaller. The western Mediterranean data did not differ from the Croatian. On a broader scale, populations from the eastern Atlantic Ocean, the North Sea, the north-western African coast, the South African coast and the Australian coast had significantly larger skulls compared to the Adriatic bottlenose dolphins. Populations from the Chinese waters and the eastern Floridian coast had significantly smaller skulls than the Adriatic dolphins.

The genes of the major histocompatibility complex (MHC) are a key component of the mammalian immune system and have become important molecular markers for measuring fitness-related genetic variation in wildlife populations. Because of human persecution and habitat fragmentation, the grey wolf has become extinct from a large part of Western and Central Europe, and remaining populations have become isolated. In Croatia, the grey wolf population, part of the Dinaric – Balkan population, shrank nearly to extinction during the 20th century, and is now legally protected. Using the cloning – sequencing method, we investigated the genetic diversity and evolutionary history of exon 2 of MHC class II DLA-DRB1, DQA1 and DQB1 genes in 77 individuals. We identified 13 DRB1, 7 DQA1 and 11 DQB1 highly divergent alleles, and 13 DLA-DRB1/DQA1/DQB1 haplotypes. Selection analysis comparing the relative rates of non-synonymous to synonymous mutations (dN/dS) showed evidence of positive selection pressure acting on all three loci. Trans-species polymorphism was found, suggesting the existence of balancing selection. Evolutionary codon models detected considerable difference between alpha and beta chain gene selection patterns: DRB1 and DQB1 appeared to be under stronger selection pressure, while DQA1 showed signs of moderate selection. Our results suggest that, despite the recent contraction of the Croatian wolf population, genetic variability in selectively maintained immune genes has been preserved.

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.

Roma population is the largest transnational minority in Europe, made of numerous endogamous groups which share common Indian origin. Based on the history of early migrations to Europe, they might be classified into the Balkan Roma, the Vlax Roma, and the Northern/Western European Roma. In order to capture genetic variation of Roma population in Croatia we analysed 15 autosomal STR loci (AmpFlSTR Identifiler) on 536 Roma belonging to two major migration categories: Balkan and Vlax. Vlax Roma samples comprise individuals from two different linguistic groups (Munteni and Ardeleni). Diversity indices pointed to the lowest diversity among Ardeleni Vlax Roma. Moreover, Fst and Rst genetic distances show closer relationship between Balkan and Munteni Vlax populations than between two Vlax Roma populations. STRUCTURE program analysis revealed the highest deltaK for K=2, assigning Ardeleni Vlax individuals separately. Although it would be expected that Vlax Roma groups cluster together according to migration data, the clustering of Munteni Vlax population closer to Balkan Roma suggests that despite of common migration history the gene pool of Vlax Roma population shows pronounced differentiation, probably due to strict socio- cultural rules of the endogamous groups within migration category. High level of endogamy in Ardeleni Vlax group is also confirmed with our socio-cultural data collected in regard to this population. Due to extremely polymorphic nature of analysed loci further analyses are needed to entirely resolve genetic relations among endogamous Roma groups.

Plavobijeli dupin (Stenella coeruleoalba) iz reda kitova (Cetacea) je u Sredozemnom moru široko rasprostranjena vrsta. U Jadranskom moru ova vrsta nije rezidentna, iako su opažanja plavobijelih dupina tijekom posljednjih godina sve učestalija. U sklopu sustavnog praćenja uginuća kitova u hrvatskom dijelu Jadranskoga mora, koje se provodi od 1990. godine, ukupno je pronañeno 255 mrtvih kitova, od toga 30 (11, 8 %) plavobijelih dupina. U proteklih godina prosječno su pronañena dva mrtva plavobijela dupina godišnje (1 do 3 životinja), izuzev 2002. godine, kada je zabilježeno 7 uginulih plavobijelih dupina. Početkom 2012. godine u kratkom vremenskom razdoblju, a na većem zemljopisnom području, pronañeno je 8 uginulih plavobijelih dupina u hrvatskom dijelu Jadrana. Po tri dupina pronañena su u siječnju (Susak, Korčula i Dubrovnik) i u veljači (Mljet, Čiovo i Privlaka). U ožujku i u travnju je pronañen po jedan dupin (Šćedro i Pašman). Od ukupno 8 pronañenih plavobijelih dupina postmortalno je pregledano 6 jedinki. Sve pregledane jedinke bile su fizički zrele životinje, jedna ženka i pet mužjaka. Potvrñen je nalaz povećanog broja uginulih plavobijelih dupina u istom vremenskom razdoblju na talijanskoj obali Jadranskoga mora. Ovim izlaganjem biti će prikazani nalazi postmortalne pretrage, te istaknuta složenost obrade lešina i dijagnostike. Raspravit će se uzroci uginuća plavobijelih dupina i njihov utjecaj na rezidentnu populaciju dobrih dupina (Tursiops truncatus).

In the bottlenose dolphin pelvic rudiment is all that remains of the hind limb skeleton. It is positioned dorsal to the anus and serves as attachment point for muscles acting on the genitalia and the abdominal body wall. The aim of our study was to determine morphological and morphometrical characteristics of pelvic rudiments and correlate them to the sex of an individual. Pelvic rudiments of 132 bottlenose dolphins were studied. They originated from the collection of the Faculty of Veterinary Medicine University of Zagreb, Croatia which hosts skeletons of whales found dead from 1990 to 2011 in the Croatian part of the Adriatic Sea. Ten measurements were used in the morphometrical analysis (vestigial hip bone length, length from cranial tip to highest point, length from cranial tip to middle of the crest, crest width, crest height, height at the cranial 1/8 of the length, width at the cranial 1/8 of the length, height at the caudal 1/8 of the length, width at the caudal 1/8 of the length, length from highest point to caudal tip). There were taken from each pelvic rudiment with 0.01 cm precision using a caliper. Sex dependent shape of the pelvic rudiment is observed in animals older than 12 years and with a body length > 266 cm. Female pelvic rudiment of adult bottlenose dolphins is significantly shorter and gentler in shape. The most accentuated morphological difference lies in the central part of the pelvic rudiment. Here, in adult males a lateral, prominent crest is arising from the dorsal, convex edge. This crest is significantly wider and higher in males than in females. Our study shows that pelvic rudiment of adult bottlenose dolphins is sexual dimorphic and its use in sex assignment is reliable.

Dobri dupin (Tursiops truncatus) je posljednja vrsta morskih sisavaca koja obitava u hrvatskom dijelu Jadrana, a populacija mu je procijenjena na 220-250 jedinki. Smatra se ugroženom vrstom i u Hrvatskoj je zakonom zaštićen. Genetička raznolikost unutar populacije je meñu najvažnijim ciljevima zaštite ugroženih vrsta. Geni glavnog sustava tkivne podudatnosti (MHC) su ključni za funkcioniranje imunološkog sustava kod sisavaca i danas se smatraju najznačajnijm molekularnim biljezima za utvrñivanje genetičkog statusa ugroženih populacija. Cilj našeg istraživanja bio je utvrñivanje raznolikosti gena DRA MHC klase II u dobrog dupina iz Jadranskog mora. DNA je izolirana iz 63 uginule jedinke koje su pronañene na hrvatskoj obali Jadrana. Koristeći se metodama sekvenciranja i kloniranja istražili smo raznolikost 189 pb dugog drugog egzona DRA gena MHC klase II. Pronašli smo tri alela, meñu kojima je jedan opisan po prvi put. Novi alel nosi sinonimnu mutaciju u odnosu na divlji tip. Niska raznolikost DRA gena utvrñena u ovom istraživanju usporediva je sa rezultatima ranijih istraživanja na srodnim vrstama. Nastavak istraživanja na ostalim lokusima klase II pružit će bolji uvid u imunološki status dubrog dupina u Jadranu, što je važno zbog utvrñene povećane učestalosti zaraznih bolesti u populaciji.

Prsne peraje različitih vrsta dupina razlikuju se po svojim svojstvima i pokazatelj su sposobnosti manevriranja u prostoru. Cilj istraživanja je usporedba morfometrijskih i denzitometrijskih karakteristika prsnih peraja dvije vrsta dupina, koje se razlikuju po svojim biološkim osobinama i okolišu koji nastanjuju. Istraživanje obuhvaća ukupno 24 prsne peraje odraslih životinja od čega je 12 prsnih peraja dobrih dupina i 12 prsnih peraja plavobijelih dupina oba spola. Izmjerene su vanjske mjere prsnih peraja i ukupna dužina tijela, koje su izražene indeksom mjera prsne peraje (IFM) i indeksom mjera prsne peraje prema dužini tijela (IFBL). Denzitometrijska obrada prsnih peraja učinjena je DEXA metodom i izmjerena je mineralna gustoća nadlaktične kosti kao glavnog mjesta prihvata mišića koji pokreću prsnu peraju. Statističkom analizom je utvrñena visoka povezanost indeksa prsne peraje (IFM) s mineralnom gustoćom nadlaktične kosti. Taj odnos je veći u dobrog dupina nego u plavobijelog dupina, čime se potvrñuje intenzivnija mišićna aktivnost i njen utjecaj na morfološka svojstva prsne peraje. Rezultati istraživanja potvrñuju veći utjecaj prsne peraje na pokretanje tijela životinje, a time i veću manevarsku sposobnost u dobrog dupina nego u plavobijelog dupina.

The bottlenose dolphin (Tursiops truncatus), is the only resident marine mammal species of the Adriatic Sea. This species is considered endangered due to intensive eradication operations in the middle 20th century and is legally protected. Genetic variability is considered prerequisite for the long - term persistence and adaptability of populations. The aim of this study was to investigate the level of mitochondrial DNA control region diversity in the bottlenose dolphin population from the Adriatic Sea. Total genomic DNA from muscle samples of 108 bottlenose dolphins found dead during the period from October 1997 to September 2011 both on Croatian (106 individuals) and Italian (2 individuals) coast of the Adriatic Sea was extracted. 739 bp –long fragments of the mtDNA control region were amplified and sequenced with universal MTCRf primer and a newly-designed primer DUPr. Totally, 11 haplotypes with 26 polymorphic sites were identified, with a nucleotide diversity of 0.010452 ± 0.005435 and gene diversity of 0.6108 ± 0.0494. Haplotype DD01 was the most frequent (65 individuals, 60.2%) and was shared between Croatian and Italian samples. The second Italian sample was separated into its unique haplotype. Both estimates of haplotype diversity and nucleotide diversity for Adriatic bottlenose dolphin population were lower than those found in the western Mediterranean Sea.

Vodoravno položena repna peraja i mišići repa, vezani za repnu kralježnicu, čine glavnu pokretačku snagu u gibanju kitova (Cetacea). U zbirkama diljem svijeta uočene su upalne i degenerativne promjene repne kralježnice kitova, no o njihovim uzrocima i utjecaju na opće stanje životinje malo se zna. Ovim istraživanjem pregledano je 116 kostura dobrih dupina (Tursiops truncatus) koji su od 1990. do 2009. godine pronañeni u hrvatskom dijelu Jadranskoga mora, a čuvaju se u zbirci Veterinarskog fakulteta Sveučilišta u Zagrebu. Makroskopskim pregledom uočene su promjene na repnim kralješcima u 49 (41, 2%) dobra dupina. Neke životinje imale su više tipova promjena, a one su se očitovale u vidu spondilitis/spondiloze u 20 (17, 2%) dupina, ankilozirajuće spondiloartroze u 6 (5, 2%) životinja, zadebljanja i povijanja vrhova trnastih izdanaka u 36 (31, 0%) životinja, prijeloma vrhova trnastih izdanaka i kralježničkih lukova u 11 (9, 5%) životinja i prijeloma poprečnih izdanaka u 4 (3, 4%) životinje. Dorzoventralno gibanje repnog područja vjerojatno je jedan od razloga čestih povreda i nastajanja ovih promjena. Uočene promjene utjecale su na pomičnost kralježnice, a time i na sposobnost životinje da lovi. Bol koja se pri kretanju javljala vjerojatno je ograničavala kretanje cijele kralježnice. Radi boljeg razumijevanja zdravstvenog stanja jadranske populacije dobrih dupina nužna su daljnja istraživanja o uzrocima ovih promjena s obzirom da se radi o zaštićenoj i ugroženoj životinjskoj vrsti.

Dogs worldwide share a single genetic origin from Asian wolves. While dog and wolf lineages are difficult to separate for nuclear genes, mitochondrial lineages are clearly distinguishable for the two species, offering a good opportunity to evaluate the differences between them. Species identification from DNA is an important tool for wolf conservation in Croatia, and wolf – dog differentiation is necessary for forensic cases, wildlife management and scientific research. The goal of this paper was to provide a data set on Croatian dog and wolf mitochondrial DNA control region sequences and to research if these sequences can be used as a reference for species identification. We have analyzed 281 base pair sequence of the mitochondrial DNA (mtDNA) control region of 20 mixed breed dog blood samples, 91 grey wolf muscle samples and two muscle samples of wolf-like animals. We have identified 12 dog and 4 wolf mtDNA control region haplotypes. None of the haplotypes have been shared, confirming that mtDNA control region haplotypes can be used to discriminate between Croatian wolves and dogs, and to confirm maternal ancestry of putative hybrids. Sequences of two wolf-like animals clearly grouped into a dog cluster.

The structure of the pituitary gland was studied in 3 bottlenose dolphins (Tursiops truncatus) from the Adriatic Sea. This species is legally protected in Croatia. All examined animals died of natural causes and were found stranded along the eastern Adriatic coast. The pituitary gland is a rectangular body suspended at the base of the brain and situated in a depression of the sphenoid bone, clearly identififi ed as sella turcica. The gland was divided into the adenohypophysis and the eurohipophisis, interconnected by thick layer of connective tissue. Pars distalis and pars tuberalis were observed in the adenohypophysis while the pars intermedia was lacking. The pars distalis was built of three main cell types: acidophils, basophils and chromophobes. Most of the acidophils were situated in the centre of the gland while chromophobes occurred dorsal and basophils ventral. Pars tuberalis was composed of cords of polygonal and vacuolated cells, which were in close association with numerous blood vessels. Large numbers of colloid-containing follicles were observed throughout the pars tuberalis. The neurohypophysis of the bottlenose dolphin consisted of unmyelinated nerve fifi bres among which numerous pituicytes were scattered.

The measurement of bone mineral density (BMD) in dolphins was described in earlier investigations, but only by application of osteodensitometric methods used for man. The aim of this study was to test the possibilities of applying the method for small animals in the measurement of the bone mineral density in dolphins. The humeri of the right flfl ippers of 24 bottlenose dolphins (11 males and 13 females) were analyzed using the dual energy x-ray absorptiometry (DEXA) method for measurement and analysis of the bone mineral density in small animals. The tested method can be successfully applied for measuring bone mineral density in dolphins, but the range of measured values was lower than values that resulted from using the method for man. Statistical analysis indicated positive linear correlations between bone mineral density of dolphin humerus and the total body length and age of the dolphins.

The bottlenose dolphin (Tursiops truncatus) is an endangered and legally protected species in Croatia, whose demographic history is poorly known. It is the only resident marine mammal species in Croatian part of the Adriatic Sea, with number estimated at around 250 individuals. There are indications that there has been intensive eradication operations in the mid 20th century that might have caused reductions in the effective population size and might have resulted in a loss of genetic variation. Therefore, we aimed to investigate a possibility of recent population size contraction and to compare the level of genetic diversity in the bottlenose dolphin population from the Croatian part of the Adriatic Sea with other bottlenose dolphin populations. Thirty samples were genotyped at 12 dinucleotide microsatellite loci. Bottleneck analysis gave ambiguous evidence for a recent population decline in the investigated bottlenose dolphin population. M ratio test, with two sets of parameter values, suggested a recent bottleneck ; whereas analysis by Bottleneck program under two mutation models (TPM and SMM) showed no evidence for a genetic bottleneck. In addition, there was no evidence for a significant deviation from the normal L-shaped distribution of allele frequencies as expected for a stable population under mutation-drift equilibrium. Furthermore, both allelic richness (6.956) and expected heterozygosty (0.676) of the Adriatic population are above mean of all compared populations (5.993 and 0.658, respectively), confirming relatively high level of genetic diversity in the investigated Adriatic population. Due to ambiguous results, further research is needed to elucidate demographic history of this bottlenose dolphin population.

Ki to vi (Ordo: Ce ta cea) od 1995. u Hr vat skoj su za ko nom za{;ti};ene `ivo tinj ske vr ste, a nji ho va smr tno st sus tav no se pra ti od 1990. Od lis to pa da 1990. do trav nja 2010. ut vr|eno 205 na la za uginulih mor skih si sa va ca u hr vat skom di je lu Jad ranskog mo ra. Pos tmor tal no su preg le dane 176 je di nke. U ra du su opi sana ~eti ri mor ska si sav ca ~ije su le{;ine pro na|ene na Mlje tu i u okol nom mo ru Mlje ta. U sklo pu pos tmor tal noga preg le da odre|eni su vr sta, spol, dob, vanj ske mor fo lo{;ke mjere `ivotinja te su obavljeni razudba i pregled organa. Analiziran je sadr`aj `eluca, in va di ra no st pa ra zi ti ma, opi sa ne su pa to lo{;ke prom je ne i od re|en je uz rok smr ti. Dvi je le{;ine pri pa da le su dob rom du pinu (Tur sio ps trun ca tus), jed na gla vatom dupinu (Grampus griseus) i jed na krup no zubom du pinu (Ziphius carvirostris).

Decline of grey wolf (Canis lupus L.) populations in Europe started some 250 years ago, causing almost complete eradication of wolves in Western Europe. As a consequence majority of remaining populations today exhibit unique haplotypes. Dinaric population has survived the eradication, representing the closest stable wolf gene pool to the West Europe. The grey wolf in Croatia came to the edge of extinction at the beginning of the ´90s, while during the last 10 years increase of population size has been observed and in 2008 it was estimated at around 200 individuals. We have analyzed 281 bp part of the mtDNA control region of 91 grey wolf samples from Croatia. Totally four haplotypes with 11 polymorphic sites have been identified, with haplotype diversity of 0.711 ± 0.018. One haplotype is new and so far has not been registered in any grey wolf population worldwide. Croatian wolf population has one of the highest mtDNA variability recorded until now, probably retaining a significant portion of genetic diversity of the formerly widespread and continuous European wolf population. Therefore, Croatian wolf population is a valuable source of gene diversity, which through immigration could help restore populations with reduced variability.

Difficult labour (dystocia) has an important role in human and veterinary obstetrics and often requires an assisted delivery to prevent complications, both in the mother and the newborn. Its main causes are abnormal fetal positions, fetal abnormalities, and disorders in the form and function of the reproductive tract of the mother. In animals in the wild difficult labours proceed unassisted and they can end fatal for the fetus, mother or both. As part of a long-term project to investigate marine mammal strandings, 139 bottlenose dolphin (Tursiops truncatus) carcasses found in the Croatian part of the Adriatic Sea were examined from October 1990 to November 2009. In five cases we observed dystocia with fatal consequences. The age of the females ranged from 6 to 22 years. The causes of dystocia were abnormal fetal positions (dolphin No. 8 and 159) and a fetal congenital abnormality (dolphin No. 183). In two dolphins the cause of dystocia could not be observed but there were evidences of a difficult labour in form of a prolapsed uterus (dolphin No. 17) and prolapsed vagina and urinary bladder (dolphin No. 35). The bottlenose dolphin is the only resident marine mammal species in the Adriatic Sea, with an estimated number of around 200 adult individuals and around 20 cubs living in the Croatian area. It is estimated that between 15 and 20 bottlenose dolphin births occur there annually, so the estimated total number of births since 1990 is between 270 and 360. This means that dystocia with fatal consequences appears in between 1.4% to 1.9% of all births in the bottlenose dolphin from the Croatian part of the Adriatic.

An unusual lymph vessel was found in a male dog of estimated age 5-6 years, probably a German shepherd and Rottweiler cross bred. At the level of the 11th right dorsal intercostal artery a common trunk arising from the cranial cisterna chyli divided into a very thin thoracic duct (ductus thoracicus) with a diameter of 1-2 mm and a much bigger lymphatic vessel with a diameter of 6 mm. The thoracic duct, very thin in its postcardiac segment, was situated in its normal anatomical position while the big lymphatic vessel extended cranially through the right mediastinum covered with the right mediastinal pleura (pleura mediastinalis dextra). It ran laterally to the origins of the right dorsal intercostal arteries, situated at the ventral border of the right azygos vein, the right surface of the oesophagus and the dorsal wall of the Sussdorff’ s cavity (cavum mediastini serosum). At the root of the right lung (radix pulmonis dextra) it crossed the ventral surface of the oesophagus, inclined to the left cranial medistinum and drained into the thoracic duct. We recognized this large lymphatic vessel and its unusual course as the accessory thoracic duct - an anatomical variation with its origin in the embryonic development of the thoracic duct.

Sva dosadašnja istraživanja mineralne gustoće kostiju dupina su opisana samo na temelju rezultata dobivenih primjenom DEXA [dual energy x-ray absorptiometry) metode za mjerenje slabinske kralježnice čovjeka, a pokušaja mjerenja drugim metodama do sada nije bilo. Mineralna gustoća kosti kao svojstvo koštanog sustava kod dupina se može povezivati s različitim dijelovima morfologije, fiziologije, patologije, dijagnostike i liječenja, te s nizom bioloških svojstava. Istraživanje je provedeno na nadlaktičnim kostima desne prsne peraje ukupno 24 dobra dupina (11 mužjaka i 13 ženki). Koštani preparati su analizirani upotrebom osteodenzitometra s metodom mjerenja mineralne gustoće kosti za male životinje i s metodom za čovjeka. Cilj istraživanja je opisati razlike u izvedbi i tumačenju rezultata dobivenih primjenom metode za male životinje i primjenom metode za čovjeka te utvrditi mogućnost primjene metode za male životinje u mjerenju mineralne gustoće kosti dupina. Utvrđeno je da se metoda za mjerenje mineralne gustoće malih životinja može uspješno primjenjivati na dupinima, ali je raspon izmjerenih vrijednosti sadržaja minerala i mineralne gustoće kosti nešto niži nego kod primjene metode za čovjeka. Statistička analiza je pokazala pozitivnu linearnu korelaciju između mineralne gustoće nadlaktične kosti dupina i ukupne dužine tijela i dobi dupina.

As part of a long-term project to investigate marine mammal strandings, 131 bottlenose dolphin {;Tursiops truncatus) carcasses found in the Croatian part of the Adriatic Sea were examined from 1990 to April 2009. In 13 cases (five of them were either fetuses found in uteruses of dead mothers or aborted fetuses ; while eight were born alive and lived for various periods of time) we observed congenital umbilical hernia, a malformation of the ventral abdominal wall. This malformation is well known in animals and humans and causes death in-utero or soon after birth in terrestrial animals. In humans, the congenital umbilical hernia appears in 3.5 out of 10, 000 births. The bottlenose dolphin is the only resident marine mammal species in the Adriatic Sea, with an estimated number of around 200 adult individuals and around 20 cubs living in the Croatian area. It is estimated that between 15 and 20 bottlenose dolphin births occur there annually, so the estimated total number of births since 1990 is between 270 and 360. This means that congenital umbilical hernia appears in between 361 and 482 out of 10, 000 births of bottlenose dolphins in the Croatian Adriatic Sea. Our findings indicate that the incidence of congenital umbilical hernia in the Adriatic bottlenose dolphins is more than 100 times higher than that recorded in humans. The cause of such high incidence in the bottlenose dolphin remains unknown. Our future research will focus on determining a degree of relatedness among affected animals using molecular markers, in which we would test the hypothesis of the genetic basis of this malformation in the bottlenose dolphin. Furthermore, we do not know whether such high incidence of congenital umbilical hernia is found only in the Adriatic bottlenose dolphin population, or is it inherent to the bottlenose dolphin species.

Dobri dupin (Tursiops truncatus) je jedini rezidentni sisavac Jadranskoga mora i od 1995. godine je u Hrvatskoj zakonom zaštićen, čime je zabranjeno njegovo uznemiravanje i ubijanje. Smrtnost morskih sisavaca u Hrvatskoj se sustavno prati od 1990. godine. Do travnja 2009. godine postmortalno su pregledane 131 jedinke dobrog dupina. U četiri jednike, utvrđene su ozljede uzrokovane vatrenim oružjem. Jedna je životinja bila ženka, dok su ostale tri bili mužjaci, a prema starosnim skupinama jedna je životinja bila mladunče dok su ostale tri bile odrasle. Nastrijeljeni dupini nađeni su 2002. u Novigradskom moru, 2003. na Hvaru i 2008. godine kraj Makarske i u Istri. Dva su dupina ozljeđena puščanim metkom, a druga dva olovnom sačmom. U tri dupina smrt je nastupila neposredno nakon ranjavanja zbog ozljeda vitalnih dijelova tijela, dok u jednog dupina ozlijede nisu bile fatalne. Dupin oznake 76 napucan je sa 160 olovnih kugli u područje leđa i leđne peraje. U dupina oznake 109 puščani metak je ozlijedio slabinsku kralježnicu i rebra s okolnim mišićjem i krvnim žilama, te ošit i predželudac. Životinja je iskrvarila u lijevu pleuralnu šupljinu. U dupina 186 olovna kugla probila je svod lubanjske šupljine i oštetila desnu polutku velikog mozga. Dupin 195 uginuo je zbog paralize repa uzrokovane oštećenjem slabinske kralježnice i okolnog mišićja puščanim metkom. Svi slučajevi prijavljeni su nadležnim tijelima.