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A key step in conserving biological diversity is to understand biodiversity patterns based on multiple independent lines of evidence. The aims of this dissertation were to investigate the morphological variability, and by applying DNA barcoding, provide first insights into genetic diversity of two invertebrate lineages: pseudoscorpions and stoneflies, both characterized by high degree of endemism and cryptic diversity. Generated DNA barcodes, 499 for pseudoscorpions and 355 for stoneflies, revealed DNA barcoding as an effective tool for specimen identification. Integrative approach identified 47 putative new species of pseudoscorpions and six species of stoneflies, including recently described stenoendemic Isoperla popijaci. Several species and generic hypotheses in both groups need to be re-tested when additional data become available. Phylogenetic reconstructions confirmed high genetic diversity in both groups, while this diversity in pseudoscorpions correlates with geomorphological features and consists of evolutionarily young lineages. Highlighting localities with emphasized biodiversity in fragmented karstic microhabitats, which promote speciation and endemism, creates preconditions for further conservation management Ključni korak u očuvanju biološke raznolikosti je razumijevanje njenih obrazaca uz korištenje višestrukih neovisnih pokazatelja. Ciljevi ove doktorske disertacije bili su utvrditi morfološku varijabilnost i, primjenom metode DNA barkodiranja, pružiti prvi uvid u genetsku raznolikost dviju skupina beskralješnjaka: lažištipavaca i obalčara, obje karakterizirane visokim stupnjem endemizma i kriptičnom raznolikošću. Generirani DNA barkodovi, 499 za skupinu lažištipavaca i 355 za skupinu obalčara, potvrdili su mogućnost primjene DNA barkodiranja u svrhu identifikacije uzoraka. Integrativnim pristupom otkriveno je postojanje 47 potencijalno novih vrsta lažištipavaca i šest vrsta obalčara, uključujući nedavno opisanu stenoendemsku vrstu Isoperla popijaci. Za razrješavanje taksonomskih statusa i odnosa među nekoliko vrsta i rodova u obje skupine, potrebno je prikupiti dodatan materijal. Filogenetska rekonstrukcija potvrdila je veliku genetsku raznolikost i kod lažištipavaca i kod obalčara, pri čemu je genetska raznolikost lažištipavaca u korelaciji s geomorfološkim značajkama Dinarskog krša i sastoji se uglavnom od evolucijski mladih genetskih linija. Isticanjem lokaliteta s izraženom bioraznolikošću u fragmentiranim krškim mikrostaništima, koja potiču specijaciju i endemizam, stvaraju se preduvjeti za daljnje upravljanje očuvanjem vrsta i staništa

Podrijetlo i odnosi među vrstama proučavaju se stoljećima. Kako je vrijeme prolazilo, pojavile su se mnoge nove tehnologije koje su povukle nova područja studija. Jedno od tih područja je bioinformatika, disciplina koja se bavi prikupljanjem i analizom bioloških podataka. Potpodručje bioinformatike koje se naziva filogenija usredotočuje se na odnose među organizmima. Koristi različite metode za izračunavanje razlika u genetskom materijalu vrsta, a zatim ih vizualizira u obliku filogenetskih stabala. U ovom radu implementirala sam dvije metode koje koriste udaljenosti sekvenci aminokiselina različitih podtipova HIV virusa za konstruiranje filogenetskog stabla: UPGMA i Metodu povezivanja susjeda. Udaljenost dviju sekvenci izračunata je primjenom Jukes-Cantorovog korekcijskog modela na broj vidljivih razlika. Stabla se vizualiziraju jednostavnom manipulacijom i ispisom znakova koji predstavljaju čvorove i grane. Na kraju uspoređujem svoju vizualizaciju s onom iz Biopython modula. The origin and relations between species have been studied for centuries. With the passage of time, many new technologies have emerged dragging along new fields of studies. One of those fields is bioinformatics, a discipline concerned with collecting and analyzing biological data. A subfield of bioinformatics called phylogeny focuses on relations between organisms. It uses different methods for calculating differences in the genetic material of species and then visualizes them in the form of phylogenetic trees. In this paper I have implemented two methods that use the distances of amino acid sequences of different HIV virus subtypes to construct a phylogenetic tree: UPGMA and Neighbor-Joining method. The distance of two sequences is calculated by applying Jukes-Cantor's correction model to the number of visible differences. Trees are visualized by simple manipulation and printing of characters that represent nodes and branches. In the end I compare my visualization to that of Biopython module.

Selecting appropriate candidates for genetic rescue mostly relies on previous genetic research and monitoring, while ecological and behavioural traits of the remnant and source populations are rarely considered for such conservation measures. Because of their slow recovery, Eurasian lynx Lynx lynx populations in Central and Western Europe have been a repeated target of genetic reinforcements and reintroductions in the past 50 years. Once inhabiting much of south-eastern Europe, the Balkan lynx L. l. balcanicus is now critically endangered and confined to a small population. Long-term isolation has caused loss of genetic diversity and has possibly led to inbreeding depression. Immediate actions need to consider genetic reinforcement to increase the genetic diversity and secure population viability. Here, we compared the Balkan lynx with two neighbouring populations: Dinaric population originating from the Carpathian subspecies (L.l. carpathicus) and Anatolian population of Caucasian subspecies (L.l. dinniki) to determine is better suited source from an ecological standpoint. Main findings suggest that the L.l. carpathicus is ecologically more similar to the L.l. balcanicus and therefore likely better suited for the environment of south-western Balkans on the basis of prey preference (roe deer being the main prey), local prey availability (lower lagomorph and higher ungulate availability) and habitat use (predominant use of the mixed and broadleaved forests). We discuss the contrasting results of genetic and ecological analyses from both the evolutionary and conservation perspective and provide potential solutions that would take into account both aspects to pave the road towards potential genetic rescue of the Balkan lynx.

Rijeke jadranskog slijeva kratke su i izolirane, što je potaklo alopatrijsku specijaciju i rezultiralo visokom razinom endemizma. Endemske ribe roda Chondrostoma koje nastanjuju jadranski slijev su vrste Chondrostoma knerii (Heckel, 1843), u hrvatskim krajevima poznata pod nazivom podustva, i Chondrostoma phoxinus (Heckel, 1843), poznata kao podbila. Nedostatak podataka o njihovom životnom ciklusu, ekološkim nišama i nedostatak samih kriterija za određivanje rijetkosti vrsta otežava njihovu konzervaciju. Cilj je ovog istraživanja odrediti filogenetske odnose i genetsku raznolikost podustve, podbile i samog roda Chondrostoma. Uzorci tkiva trbušne peraje su korišteni za umnažanje s tri genska markera (citokrom b, beta-aktin, BMP 4) lančanom reakcijom polimeraze (PCR). Provedena je filogenetska rekonstrukcija temeljena na sekvencama navedenih markera korištenjem sljedećih metoda: metoda najveće parsimonije (MP), metoda najveće vjerojatnosti (ML) i metoda susjednog povezivanja (MJ). Dobivena filogenetska stabla i mreže potvrdili su nekadašnji bliski kontakt hrvatskih vrsta s talijanskom vrstom Chondrostoma soetta. Rodovi Achondrostoma, Iberochondrostoma i Pseudochondrostoma relativno su nedavno odvojeni od roda Chondrostoma. Niska genska raznolikost vrste C. knerii ukazuje na ugroženost ove vrste pod djelovanjem antropogenih čimbenika. The rivers of the Adriatic Basin are short and isolated, which encouraged allopatric speciation and resulted in high degree of endemism. Endemic species of Chondrostoma genus which inhabit Adriatic Basin are species Chondrostoma knerii (Heckel, 1843), known under the name Dalmatian nase in Croatia, and Chondrostoma phoxinus (Heckel, 1843), known as Minnow nase. Lack of data about their life cycle, ecological niches and lack of criteria for determining rare species makes their conservation difficult. The aim of this study was to determine phylogenetic relationship and genetic diversity of Dalmatian nase, Minnow nase and Chondrostoma genus in general. Samples of pelvic fins tissue were used for multiplication of three genetic markers (cytochrome b, beta-actin and BMP4) using the polymerase chain reaction (PCR). Phylogenetic reconstruction was done based on sequences of aforementioned genetic markers using the methods of maximum parsimony, maximum likelihood and median joining. Resulting phylogenetic trees and networks confirmed close contact of Croatian and Italian species in the recent past. Achondrostoma, Iberochondrostoma and Pseudochondrostoma gemus were relatively recently separated from the genus Chondrostoma. Low genetic diversity of D

Taxonomy of Pseudohydnum gelatinosum and its sister taxa is revised via morphological data and a four-gene dataset (ITS, nc LSU rDNA, TEF1, RPB1). Identity of P. gelatinosum and Tremellodon pusillus is re-established based on newly collected and sequenced material from their type localities. Pseudohydnum alienum from Europe; P. umbrosum from temperate East Asia; P. cystidiatum, P. meridianum, and P. placibile from Vietnam; and P. omnipavum from North America are described as new to science; P. translucens and P. brunneiceps from East Asia are redescribed. Most Pseudohydnum collections from North America belong to P. gelatinosum ssp. pusillum. A significant divergence of TEF1 sequences in P. gelatinosum is discussed.

V magistrskem delu smo karakterizirali 11 striktno anaerobnih sevov, izoliranih iz ovčjega vampa v Sloveniji v dvomesečnem obdobju (Stare, 2021). Preučevani sevi se uvrščajo v rod Prevotella in oblikujejo šest domnevnih vrst. Eno izmed vrst, v katero se poleg šestih preučevanih sevov uvrščata tudi dva na Japonskem izolirana seva, smo izbrali za formalni opis. Z uporabo sekvenc celotnih genomov smo ugotovili, da sta dva izmed sevov, izolirana v različnih časovnih točkah, identična. Ostali sevi oblikujejo jasno ločene razvojne linije znotraj vrste. Drugi preučevani sevi pripadajo petim novim vrstam Prevotella, kar smo potrdili s primerjavo z vsemi znanimi črevesnimi in vampnimi sevi rodu. Preučili smo zmožnosti razgradnje rastlinskih polisaharidov vseh šestih domnevnih vrst in ugotovili, da vsaka od njih razgrajuje drugačen nabor polisaharidov. Zmožnost uporabe polisaharidov je predvideno ena od bistvenih prilagoditev vampnih Prevotella. Raznolikost uporabe hranil po vsej verjetnosti prispeva k manjšanju kompeticije med vrstami. Sevi nove vrste razgrajujejo ksilane in pektine in ne razgrajujejo škroba, slednje je pri rodu Prevotella nepričakovano. Eden izmed sevov je izgubil zmožnost razgradnje inulina, kar smo potrdili tudi z genomsko analizo. Kot je značilno za rod Prevotella, sevi nove vrste fermentativno proizvajajo sukcinat in ocetno kislino ter ob prisotnosti žolčnih kislin v gojišču ne rastejo. Z uporabo testov API in analizo membranskih dolgoverižnih maščobnih kislin smo opisali diferencialne označevalce, ki novo vrsto Prevotella jasno ločijo od že opisanih. Nova vrsta je v vampu pogosta, saj je bila najdena v vseh preučenih metagenomskih vzorcih goveda in ovc. Z delom smo želeli razširiti nabor gojenih in karakteriziranih vampnih vrst rodu Prevotella, saj je kljub njihovi abundanci trenutno priznanih le pet vrst, izoliranih iz vampa. In the master thesis, we characterized 11 strictly anaerobic strains isolated from the sheep rumen in Slovenia during a two-month period. The studied strains belonged to the genus Prevotella and formed six putative species. One of the species, comprising six studied strains and two other strains isolated in Japan, was selected for a formal species description. Using whole genome sequences, we found that two strains, isolated at two time points, were identical. The other strains formed clearly diverged intraspecies lineages. Other studied strains belonged to five new Prevotella species, which we confirmed by comparison with all known gut and ruminal strains of the genus. We examined the ability of all six species to degrade plant polysaccharides and discovered that each putative species utilizes a different set of polysaccharides. The capability of polysaccharide utilization is predicted to be one of the essential Prevotella adaptations. The diversity of nutrient usage likely contributes to decreasing the competition between species. The strains from the new species degraded xylans and pectins but not starch, which is unexpected for Prevotella. One of the strains lost its ability to degrade inulin, which was explained also by genomic analysis. The strains of the new species produced succinate and acetic acid fermentatively and did not grow in presence of bile acids, as is typical for Prevotella. We used API tests and the membrane long chain fatty acids profiles to describe differential markers that clearly distinguish the new Prevotella species from the previously described species. The new species was common in the rumen, as it was found in all studied metagenome samples of cattle and sheep. With the thesis, we aimed to expand the collection of cultured and characterized ruminal Prevotella species, because despite their abundance, there are currently only 5 published isolated rumen species.

Tanacetum cinerariifolium is an endemic species of the eastern Adriatic coast that synthesizes the natural insecticide pyrethrin. We have characterized the complete chloroplast genome of the species and analyzed its phylogeny within the Asteraceae family. The complete chloroplast genome of T. cinerariifolium has a size of 150, 136 bp, including a large single-copy (LSC) region of 82, 717 bp, a small single-copy (SSC) region of 18, 411bp, and a pair of inverted repeats (IRs) of 24, 504 bp. The chloroplast genome of T. cinerariifolium encodes 108 genes, including 77 protein-coding genes (PCGs), 27 tRNA genes, and 4 rRNA genes. Phylogenetic analyses based on the complete chloroplast genomes placed T. cinerariifolium in a sister position to species of the genera Artemisia and Chrysanthemum.

Genomi su sve češće korišteni podaci u proučavanju biologije i evolucije organizama, a broj dostupnih genoma u Banci gena se u posljednih nekoliko godina udvostručio kao posljedica razvoja tehnologije sekvenciranja. Razvoj ovih tehnologija utjecao je na populariziranje područja genomike i to prvenstveno zbog značajnog pada cijene sekvenciranja. Rekonstrukcija genoma provodi se u tri koraka: sekvenciranje, sastavljanje i anotacija, a za svaki korak postoji više različitih pristupa. Sastavljanje genoma je računalno i vremenski nazahtjevniji korak te je jedan od glavnih fokusa istraživanja u području genomike. Tri su trenutno dostupne metode za sastavljanje genoma (mapiranje, de novo i hibridna metoda), a odabir metode ovisi o nekoliko glavnih parametara koji uključuju: vrstu organizma koji se proučava, pokrivenost genomskih podataka, dostupnost referentne sekvence, broj uzoraka, dostupnost računalnog servera za provođenje analiza i sl. Prema tome, svaki genomski projekt je jedinstven i teško je odabrati samo jednu metodu koja će dati najbolje rezultate, pogotovo kada se proučavaju nemodelne vrste. Divokoza (Rupicapra spp.) je zbog svoje rasprostranjenosti i predložene sistematike dobar model za proučavanje utjecaja povijesnih i evolucijskih događaja. U ovoj se disertaciji koristilo nekoliko metoda za sastavljanje i anotaciju mitohondrijskih i jezgrinih genoma divokoze, a dobiveni su se rezultati usporedili. Na temelju usporedbi rezultata metoda za sastavljanje mitohondrijske i jezgrine DNA, procijenile su se pogodnosti različitih metoda za sastavljanje i anotaciju genoma, uspoređen je utjecaj korištenja osam genoma divokozi srodnih vrsta kao referenci u metodi mapiranja te su se rekonstruirali filogenetski odnosi s ciljem boljeg razumijevanja povezanosti taksonomskih jedinica roda Caprini i vrste Rupicapra. Uz navedeno, testirana je točnost novosastavljenih genoma divokoze usporedbom izoliranih fragmenata introna s intronskim sekvencama divokoza dostupnih u Banci gena. Rezultati ovog istraživanja pridonijet će boljem poznavanju raznolikosti i evolucije genoma divokoze, razjašnjavanju taksonomskih odnosa podvrsta, a sastavljeni genomi pružit će dobru referentnu osnovu za buduće populacijske i genomske analize divokoze i njenih srodnika. The genome is a collection of all biological information necessary for the functioning of an organism, and in humans and animals consists of the mitochondrial genome (mtDNA) and the nuclear genome (nDNA). With the development of genomic technologies, the genome data are increasingly being used to study the biology and evolution of organisms, which is confirmed by the fact that the number of available genomes in the Gene bank has doubled in recent years. In addition, the development of these technologies has influenced the popularization of the field of genomics, mainly due to the significant decrease in the cost of sequencing. Genomic analysis can be used: to identify genes responsible for inherited diseases or adaptations to the environment, to study structural changes, to identify common conserved sites, to find genes specific to a group of organisms, etc. To perform any of these analyzes, the genome of the species under study must be reconstructed. Reconstruction of a genome involves three steps: sequencing, assembly, and annotation. There are different approaches for each step, and the choice of method depends primarily on whether the reconstruction is of the mitochondrial or nuclear genome. Genome assembly is a computationally intensive and time-consuming step, and there are currently three available methods for genome assembly (mapping, de novo, and hybrid methods). The choice of method depends on several key parameters that include: the type of organism studied, the coverage of genomic data, the availability of reference sequences, the number of samples, the availability of a computer server for analysis, etc. Therefore, each genome project is unique and it is difficult to determine the method that will be most successful, especially when studying non-model species. The chamois (Rupicapra spp.) is a good model for studying the effects of historical and evolutionary events because of its wide distribution and proposed systematics. In this dissertation, 12 completely sequenced chamois samples were used and their genome reconstruction was performed using different methods for assembling and annotating mtDNA and nDNA. Mapping and de novo methods were used for mtDNA assembly, while GeSeq and MITOS annotators were used for annotation. All sequences obtained with both assembly methods were compared and validated with the web application BLAST. In this process, each sequence was compared to the chamois mtDNA reference sequences. For nDNA assembly, eight available genomes of closely related species were used as references for the mapping method, followed by SNP calling procedure. Based on the filtered SNPs and references, 56 combinations were identified (newly assembled chamois genomes) that were validated and annotated using BUSCO tools. Then, three smaller sets of genes were defined from the common set of annotated genes, based on which distance matrices were calculated and the relationships were visualized using the multidimensional scaling (MDS) method. Newly assembled genomes generated by mapping chamois samples against the domestic goat genome were used to verify structure by comparing them with the chamois genome sequences (23 sets of introns) available in Gene bank. Phylogenetic analyses of mtDNA (maximum likelihood and Bayesian methods) were performed using a dataset containing 10 mtDNA sequences from this dissertation, 5 chamois mtDNA sequences from Gene bank, and two related sequences as outgroup. Phylogenetic analyses (maximum likelihood and Bayesian methods) of the genus Caprinae were performed on a data set containing 40 sequences of the genus Caprinae and 5 sequences of Bovidae as outgroup. The final phylogenetic analysis of chamois was performed using the program BEAST on a common alignment of an intron data set consisting of 21 chamois sequences and three sequences representing an outgroup. After performing two methods for reconstructing complete mtDNA sequences (mapping and de novo) and comparing the obtained mtDNA sequences, it was clearly determined that both methods were suitable for reconstruction. The de novo method proved to be the better choice because of its speed and simpler procedure. In addition, the de novo methods successfully isolated complete mtDNA sequences from samples that had failed quality control (Gams53, Gams85, OSIL-06). For this reason, all mtDNA analyses were performed on a larger number of samples. In other words, if only the mapping method had been used, these three samples could not have been included in the further analysis. The mtDNA annotation tools MITOS and GeSEQ gave very similar results for all 10 samples, and variations in START and STOP codons were detected in four genes (ND1, ND2, ND3, ND5). The variations found refer to two or three bases found in the START and STOP codons. Their occurrence can be interpreted as a consequence of the different algorithms used by the annotators in the analyzes and as a consequence of the larger variation in these genes. Phylogenetic analyses (maximum likelihood and Bayesian methods) performed on ten mtDNA sequences from chamois reconstructed in this dissertation, in combination with five mtDNA sequences from Gene bank, yielded identical phylogenetic trees for the genus Rupicapra. These results confirmed previous research on chamois in which they were divided into three mtDNA clusters (W, C, and E). Performed phylogenetic analyses (maximum likelihood and Bayesian method) on 40 mtDNA sequences of the genus Caprinae (including the four sequences obtained in this dissertation) and five sequences of the genus Bovidae also revealed the same topology as presented in previous studies. From the comparison of the newly assembled chamois genomes with the genomes of related species, it was concluded that almost all of the related genomes used can serve as good references. Although all the species used were non-model species, the best results were obtained with the genomes of domestic goats and domestic sheep, which was to be expected since these species are extremely important species in agriculture and are often the focus of research. The number of genes found for most combinations of chamois and related species was very high, confirming that these genomes can be used for mapping processes. However, during the mapping processes it was found that some of the genomes used were of low quality, while some genomes were found to have irregularities in the information available in the Gene bank. This confirms once again that not all available genomes are of good quality. In other words, any sequence available in the Gene bank should be verified before use. From the similarity analyses, it can be concluded that the relationships between all combinations depend primarily on the gene or genome fragments used for these analyses. Although the number of polymorphisms found had a greater impact on the results when single gene fragments were used, this number was negligible when longer portions of the genome (100 and 500 genes) were used, with differences between samples of approximately 1 %. In other words, larger distances were calculated between combinations from shorter alignments. The results of the MDS for a set of 100 and 500 genes clearly showed that samples from chamois samples mapped to different references were more similar to each other, while still exhibiting some differences in amino acid composition. Smaller differences between samples were found for combinations with domestic sheep and American mountain goat (about 1 % and 000,5 %, respectively). The comparisons of the intron regions of the newly assembled chamois genomes with the introns available in the Gene bank suggest that the intron sequences obtained from the newly assembled genomes are of satisfactory quality and have been grouped with other chamois samples at the species and subspecies level. The results of this research will contribute to a better knowledge of the diversity and evolution of the chamois genome, elucidate the taxonomic relationships among subspecies, and assembled genomes will provide a good reference base for future population and genome analyses of chamois and its relatives.

Ciljevi ove disertacije bili su utvrditi morfološku i genetsku raznolikost populacija potočnog i plemenitog raka u Hrvatskoj te razviti modele povoljnosti staništa koji će predvidjeti kako invazivne strane vrste rakova te klimatske promjene utječu na njihovu raznolikost i dugoročni opstanak. Geometrijska morfometrija ustanovila je postojanje morfološke raznolikosti i razlikovanje populacija ovisno o pripadnosti različitim linijama, genetskim grupama, riječnim slivovima i tipu staništa. Filogenetska rekonstrukcija potvrdila je veliku genetsku raznolikost, s filogeografskom strukturom i rasprostranjenošću evolucijskih linija oblikovanih geoklimatskim procesima. Populacijsko-genetičke analize ustanovile su visoku razinu genetske raznolikosti i diferencijacije populacija, s ograničenim protokom gena. Modeli povoljnosti staništa pokazali su da će populacije plemenitog i potočnog raka biti ugroženije budućim klimatskim promjenama, nego širenjem invazivnih stranih vrsta rakova. Preklapanjem podataka o genetskoj raznolikosti s budućim povoljnim staništima identificirane su populacije i područja najveće vrijednosti kojima treba dati prioritet u budućim konzervacijskim planovima. Aims of this dissertation were to determine the morphological and genetic diversity of the stone crayfish and noble crayfish in Croatia, and to develop habitat suitability models that will predict how the invasive crayfish species and climate change affect their biodiversity and long-term survival. Geometric morphometrics revealed the existence of morphological diversity and differentiation of populations based on the crayfish affiliation to different genetic lineages, genetic clusters, river basins and habitat types. Phylogenetic reconstruction confirmed the existence of high genetic diversity, with geo-climatic processes shaping the contemporary geographic distribution of evolutionary lineages. Population genetics revealed high genetic diversity and differentiation of populations, with limited gene flow. Habitat suitability models suggested that climate change-driven habitat loss represents a greater threat than the potential future distribution of the invasive crayfish species. The obtained results enabled identification of populations and areas with the highest conservation value and which should be given the highest priority in conservation planning.

The complete mitochondrial genome of the Carniolan honeybee (Apis mellifera carnica) from Slovenia, a homeland of this subspecies, was acquired in two contigs from WGS data and annotated. The newly obtained mitochondrial genome is a circular closed loop of 16,447 bp. It comprises 37 genes (13 protein coding genes, 22 tRNA genes, and 2 rRNA genes) and an AT-rich control region. The order of the tRNA genes resembles the order characteristic of A. mellifera. The mitogenomic sequence of A. m. carnica from Slovenia contains 44 uniquely coded sites in comparison to the closely related subspecies A. m. ligustica and to A. m. carnica from Austria. Furthermore, 24 differences were recognised in comparison between A. m. carnica and A. m. ligustica subspecies. Among them, there are three SNPs that affect translation in the nd2, nd4, and cox2 genes, respectively. The phylogenetic placement of A. m. carnica from Slovenia within C lineage deviates from the expected position and changes the perspective on relationship between C and O lineages. The results of this study represent a valuable addition to the information available in the phylogenomic studies of A. mellifera—a pollinator species of worldwide importance. Such genomic information is essential for this local subspecies’ conservation and preservation as well as its breeding and selection.

Naša raziskava sovpada s porastom zanimanja za »novo porajajočo se« bolezen virusa hepatitisa E (HEV) v svetu in prinaša nove izsledke o razširjenosti okužbe v slovenskih rejah prašičev. Ker gre za zoonozo, smo z raziskavo želeli ugotoviti, kakšno je stanje v slovenskih klavnicah in ali hepatitis E virus ogroža zdravje ljudi. Vzorčenje prašičev je potekalo v štirih klavnicah po Sloveniji med junijem in septembrom 2014. Vzorce blata, jeter in žolča smo odvzeli 811 klinično zdravim prašičem različnih starostnih kategorij. Ob vzorčenju v klavnici smo jemali tudi brise površin na klavni liniji, vzorčili mleto meso in pečenice. Z metodo RT-PCR v realnem času smo dokazovali prisotnost nukleinske kisline HEV v odvzetih vzorcih. Istim prašičem smo odvzeli tudi vzorec krvi in določali prisotnosti protiteles IgG proti HEV. Največ HEV pozitivnih živali smo ugotovili med 322 odojki, in sicer 17 %. Med 400 pitanci smo ugotovili en pozitiven vzorec jeter in en pozitiven vzorec žolča, kar je bistveno manj, kot smo pričakovali. Med 89 plemenskimi svinjami nismo ugotovili pozitivne živali. Med 63 brisi površin smo odkrili dva pozitivna vzorca. V vzorcih pečenic in mletega mesa prisotnosti HEV nismo ugotovili. Z genetsko tipizacijo 45 HEV pozitivnih vzorcev in filogenetsko primerjavo nukleotidnih zaporedij slovenskih sevov skupaj z zaporedji sevov HEV, ki so dostopni v podatkovni zbirki GenBank, smo ugotovili pojav velike genetske raznolikosti med vzorci ter prisotnost štirih različnih podtipov genotipa 3 (3a, 3b, 3e in 3f) HEV v Sloveniji. V izvedeni študiji genotipa 4 nismo ugotovili. V določanje nukleotidnega zaporedja smo vključili tudi pet humanih vzorcev, pozitivnih na prisotnost HEV. Zaradi premajhne količine virusne RNA v HEV pozitivnih humanih vzorcih nam pri nobenem ni uspelo določiti nukleotidnega zaporedja. Z dvema komercialnima encimskoimunskima testoma (ID Screen® Hepatitis E Indirect Multi-species proizvajalca IDvet in HEV-IgG ELISA porcine proizvajalca Axiom) smo pregledali vzorce krvi domačih prašičev na prisotnost protiteles IgG proti HEV. Pregledali smo 351 vzorcev, naključno izbranih iz vzorcev odvzetih na celotnem območju Slovenije v okviru rednega monitoringa kužnih bolezni pri prašičih v letih 2013 in 2014, ter 359 vzorcev, odvzetih v klavnici ob zakolu leta 2014. Primerjava rezultatov obeh testov je pokazala, da je encimskoimunski test proizvajalca Axiom ustreznejši za presejalna testiranja. Za razjasnitev 38 rezultatov, ki se pri vzorcih iz klavnice pri obeh encimskoimunskih testih niso ujemali, smo uporabili modificiran humani imunoblot test recomLine HEV IgG/IgM (Mikrogen diagnostik). Izvedena raziskava je prva tovrstna študija, ki dokazuje prisotnost HEV pri prašičih na klavni liniji v Sloveniji, prav tako je prva izvedena sistematična raziskava v tako širokem obsegu v Sloveniji. Rezultati raziskave prispevajo k boljšemu razumevanju stanja okužb prašičev s HEV ob vstopu na klavno linijo, in podajajo natančnejšo sliko o stanju prekuženosti prašičev v rejah. Glede na številčnost pregledanih vzorcev in razpršenost rej po državi, iz katerih so izhajali vzorčeni prašiči, lahko z gotovostjo trdimo, da rezultati podajajo natančen podatek o razširjenosti okužb s HEV med domačimi prašiči v Sloveniji. Our research coincides with increasing interest in the newly emerging zoonosis, the hepatitis E virus, and provides new information regarding infection distribution in Slovenian pig herds. Since HEV is a zoonosis, our goal was to investigate the existing condition in Slovenian slaughterhouses and to assess associated health risks posed to consumers and personnel. Sampling of pigs was conducted in four Slovenian slaughterhouses, between June and September 2014. Feces, liver and bile samples were collected from 811 healthy pigs (different age categories). At the time of slaughter, swab samples of different surfaces and samples of minced meat and sausages were also taken. All samples were analyzed with real time PCR (RT-PCR) for the presence of HEV. Blood samples taken from the same pigs were analyzed for the presence of IgG HEV antibodies. The highest number of HEV positive animals was found in the 3 month old age group of pigs (322 animals), where 17% of animals were HEV positive. In the age group of 6 month old pigs (400 animals), only one liver sample and one bile sample was positive, which was significantly less than we would have expected. Out of 89 pigs that were older than 1 year, none was HEV positive. Out of 63 surface swabs, two were positive for the presence of HEV. None of the minced meat and sausage samples was positive. A phylogenetic tree, generated combining sequences obtained from Slovenian strains (45 positive samples) and nucleotide sequences of HEV strains derived from GeneBank, revealed high genetic variability between samples, allowing classification of the identified Slovenian strains into four different subtypes of genotype 3 (3a, 3b, 3e and 3f). Genotype 4 was not found. Additionally, five HEV positive human samples were sequenced, although sequencing was unsuccessful due to an insufficient amount of viral RNA. With two commercial ELISA (ID Screen® Hepatitis E Indirect Multi-species (IDvet) and HEV-IgG ELISA porcine (Axiom)) we analyzed blood samples from domestic pigs for the presence of IgG HEV antibodies. We analyzed 351 randomly selected serum samples collected from pigs located in different parts of Slovenia (these samples were collected within the scope of annual monitoring of infectious diseases in 2013 and 2014) and 359 samples collected from pigs at slaughter in 2014. Comparison of results from both ELISA showed that Axiom ELISA is more suitable as a screening method. A human immunoblot test was optimized and standardized to test 38 serum samples that showed discrepancies in results obtained with Axion and IDvet ELISA kits. The work presented herein represents the first systematic, wide scope analysis on the presence of HEV in pigs at slaughter in Slovenia. Results of the research have contributed to a better understanding of pig HEV infection status at slaughter and have shown a more detailed picture of seroprevalence status in pig farming. Since our research included a great number of samples and dispersion of different farms around the country, we can safely say that the obtained results show accurate information about pig HEV infections in Slovenia.

Hrvatska je raznolika vrstama gujavica, a teritorij Hrvatske kao dio Balkanskog poluotoka smatra se jednim od centara divergencije vrsta rodova Octodrilus i Octodriloides. Dosad je u Hrvatskoj zabilježeno 15 vrsta roda Octodrilus i 5 vrsta roda Octodriloides. Istraživanje je provedeno u svrhu utvrđivanja genske raznolikosti i raznolikosti vrsta spomenutih rodova DNA barkodiranjem 177 uzoraka s 77 lokaliteta širom Hrvatske. Iz tkiva uzoraka izolirao sam, umnožio i poslao na sekvenciranje sekvence COI regije mtDNA, a upotrebom računalnih programa SEQUENCHER (4.1.4.) i MEGA-X sekvence sam sravnio i obradio. Uz malobrojne, javno dostupne sekvence konstruirao sam filogenetsko stablo prema metodi najveće vjerojatnosti, pomoću kojeg sam izdvojio ukupno 28 linija. Linije sam svrstao u 9 vrsta roda Octodrilus i 3 vrste roda Octodriloides. 3 linije roda Octodrilus zbog specifičnosti u građi ili juvenilnog stadija primjeraka nisu svrstane u opisane vrste. 5 vrsta čini kompleks od najmanje dvije linije sa značajnim genskim udaljenostima. Istraživanje je pokazalo i prednost određivanja vrsta gujavica molekularnim metodama u odnosu na određivanje vrsta prema građi tijela koje je kod gujavica vrlo složeno. Potrebno je provesti istraživanja na daleko većem broju uzoraka kako bi se molekularno-filogenetskim metodama detektirale preostale opisane vrste na području Hrvatske i kako bi se usporedbom s rezultatima ovog istraživanja odredio njihov filogenetski položaj. Croatia is diverse with earthworm species and the Croatian territory, as part of Balkan Peninsula, is considered to be one of the divergency centers of genera Octodrilus and Octodriloides. So far, 15 species of the genera Octodrilus and 5 species of the genera Octodriloides have been recorded in Croatia. The research was conducted in order to determine the genetic and species diversity of the mentioned generas by DNA barcoding 177 samples from 77 localities throughout Croatia. The COI region from mtDNA was isolated, amplified and sequenced from the sample’s tissue. Using the SEQUENCHER (4.1.4) and MEGA-X computer programs, the sequences were aligned and processed and a phylogenetic tree was constructed according to the maximum-likelihood method. A total number of 28 lineages were singled out from a tree and classified to 9 species of the genera Octodrilus and 3 species of the genera Octodriloides. 3 lineages of the genera Octodrilus were not classified to described species due to specificity in structure or juvenile stage of samples. 5 species form a complex of at least two lineages with significant genetic distances. The research also showed the advantage of determining earthworm species by molecular methods instead of determination according to morphology and anatomy, which is very complex. It is necessary to conduct researches on a far larger number of samples in order to detect the remaining described species in Croatia by molecular-phylogenetic methods and to determine their phylogenetic position by comparing them with the results of this research.

Acipenseridae su uz porodicu Polyodontidae jedini živi predstavnici reda Acipenseriformes. One su među najprimitivnijim ribama koštunjačama te ujedno i zadnji preživjeli predstavnici gotovo potpuno izumrlog nadreda zrakoperki - Chondrostei, tzv. štitonoša. Razni antropogeni pritisci, ali i njihova relativna ekološka osjetljivost, doveli su Acipenseridae u prirodi u vrlo nepovoljan položaj te ih brojne zemlje počinju štititi. Na području Hrvatske, kroz prošlost je zabilježeno 8 vrsta iz porodice Acipenseridae te 1 vrsta iz porodice Polyodontidae, od kojih samo 2 vrste trenutno opstaju u prirodi te su obje klasificirane u ugrožene kategorije prema IUCN-u. Biologija porodice Acipenseridae na našim prostorima nije dovoljno istražena, što predstavlja značajan problem u zaštiti ovih vrsta. Uvid u taksonomsku i gensku raznolikost ovih vrsta kao i njihovu rasprostranjenost prvi je korak prema razvoju daljnjih istraživanja koja će omogućiti njihovu zadovoljavajuću zaštitu. U ovom istraživanju korištene su metode molekularne analize genskog markera citokrom b na uzorcima jedinki jesetre i kečige iz divljine i uzgajališta. Napravljene su rekonstrukcije filogenetskog stabla metodama najveće parsimonije i najveće vjerojatnosti, rekonstrukcija filogenetske mreže metodom susjednog povezivanja, kao i rekonstrukcija stabla evolucijske prošlosti bayesovom metodom. Također su izračunate mjere genske raznolikosti. Rezultati potvrđuju pripadnost uzorkovanih jedinki vrstama Acipenser ruthenus i A. baerii te ukazuju na zamjetnu razliku u stupnju genetske raznolikosti između populacija iz prirode i onih iz uzgajališta. With the family Polyodontidae, Acipenseridae are the only living members of the order Acipenseriformes. They are amongst the most primitive bony fish and are also the last surviving members of the almost extinct superorder of Actinopterygii - Chondrostei. Different anthropogenic pressures, in combination with their relatively high ecological vulnerability, have led Acipenseridae to a quite unfavorable position, so most countries have started implementing measures for their protection. Historically, there have been 8 recorded species from the family Acipenseridae and 1 from the family Polyodontidae in Croatia, of which only 2 still exist in the wild, both being classified in endangered IUCN categories. The biology of Acipenseridae in Croatia isn't researched enough, which poses a problem in conservation efforts. Gaining insights into their taxonomic and genetic diversity and geographic distribution is the first step for conducting research which will lead to adequate protection of these endangered species. This research uses methods of molecular analysis of the gene marker cytochrome b on species of sterlet and sturgeon captured in the wild and from a fishery. Reconstructions of phylogenetic trees using maximum parsimony and maximum likelihood methods, as well as a reconstruction of their phylogenetic network using mutual joining method and a reconstruction of their evolutionary history using Bayesian methods were made. Measures of their genetic diversity were also calculated. The results confirm that the sampled individuals belong to Acipenser ruthenus and A. baerii species and indicate a much higher degree of genetic diversity of wild populations in comparison to populations bred in fisheries.

Some wild, morphologically diverse taxa of the genus Iris in the broad Alpine-Dinaric area have never been explored molecularly, and/or have ambiguous systematic status. The main aims of our research were to perform a molecular study of critical Iris taxa from that area (especially a narrow endemic accepted species I. adriatica, for which we also analysed genome size) and to explore the contribution of eight microsatellites and highly variable chloroplast DNA (ndhJ, rpoC1) markers to the understanding of the Iris taxa taxonomy and phylogeny. Both the microsatellite-based UPGMA and plastid markers-based maximum likelihood analysis discriminated three main clusters in the set of 32 analysed samples, which correspond well to the lower taxonomic categories of the genus, and support separate status of ambiguous regional taxa (e.g., I. sibirica subsp. erirrhiza, I. x croatica and I. x rotschildii). The first molecular data on I. adriatica revealed its genome size (2C = 12.639 &plusmn, 0.202 pg) and indicated the existence of ecotypes. For future molecular characterisation of the genus we recommend the utilisation of microsatellite markers supplemented with a combination of plastid markers.

Vodene muhe plesačice (Empididae ; Clinocerinae i Hemerodromiinae) su važna sastavnica slatkovodnih zajednica, naročito u tekućicama. Ličinke i odrasli su predatori te su stoga od iznimne važnosti za razumijevanje slatkovodnih hranidbenih mreža. One nisu taksonomska, već ekološka grupa te se dijele na dvije zasebne potporodice, Clinocerinae i Hemerodromiinae. Potporodica Clinocerinae sadrži više od 350 vrsta uključenih u 16 rodova. Prijašnja klasifikacija bazirana je isključivo na morfologiji vrsta, s iznimkom roda Wiedemannia za kojeg je napravljena filogenija na temelju molekularnih podataka. U ovom radu napravljena je prva molekularna filogenija potporodice Clinocerinae koja uključuje predstavnike svih rodova. DNA je izolirana iz svježih i muzejskih jedinki iz područja svih svjetskih biogeografskih regija. Sekvencirana su dva mitohondrijalna gena, gen za podjedinicu citokrom c oksidaze I (COI) i citokrom β (Cytβ) te tri nuklearna gena, domena karbamoil fosfat sintetaze (rudimentarna CAD), gen za elongacijski factor (EF-1α) te izocitrat dehidrogenaza (IDH). Filogenetska analiza napravljena je koristeći metode najveće vjerojatnosti i Bayesian analizom na matrici spojenih sekvenci. Naši preliminarni rezultati ne pokazuju dovoljno filogenetskog signala kako bi potvrdili pripadnost rodova Proagomya i Asymphiloptera potporodici Clinocerinae, za razliku od ostalih rodova koji tvore monofiletsku grupu.

In total 27 lectures and 51 poster presented by 209 authors from 13 European countries (Croatia, Serbia, Finland, Slovenia, Italy, Estonia, Austria, Bosnia and Herzegovina, Montenegro, Portugal, Slovakia, Spain and Sweden) during a two-day programme.

Kužni ektim povzroča virus Orf, ki spada v družino Poxviridae, rod Parapoxvirus. V doktorski nalogi opisujemo analizo dveh genomov izolatov virusov Orf pri gamsih (sev ORFV_82/13 in sev ORFV_12/17). Z metodo sekvenciranja naslednje generacije (angl. next-generation sequencing, NGS) smo prvi določili skoraj celotno nukleotidno zaporedje genomov (angl. nearly complete genome) virusov Orf pri gamsih. Analiza genomov virusov Orf pri gamsih je pokazala visoko stopnjo sorodnosti z virusi Orf pri ovcah in kozah. Genoma sta na nivoju nukleotidnega zaporedja med seboj 99,93-odstotno podobna in se od genomov drugih virusov Orf, dostopnih v podatkovni zbirki NCBI GenBank, razlikujeta od 7,61 % (ovčji sev NZ2) do 11,62 % (kozji sev NP). Filogenetska analiza genov osrednje regije je pokazala, da sta virusa Orf pri gamsih tvorila svojo filogenetsko podskupino. V doktorski nalogi smo z ugotavljanjem prisotnosti protiteles proti virusom Orf s serum nevtralizacijskim testom ugotavljali prekuženost populacije gamsov z virusom Orf. Testirali smo 40 vzorcev serumov gamsov. Rezultati testa so bili negativni pri 38 vzorcih, dveh rezultatov ni bilo mogoče odčitati zaradi citotoksičnega učinka vzorca seruma na celično kulturo. S pregledom arhivskih vzorcev zadnjih dveh desetletij, upoštevajoč posredovane podatke lovskih družin ter izkušnje Inštituta za patologijo, divjad, ribe in čebele, smo ugotovili ciklično pojavljanje kužnega ektima pri gamsih. Fibropapilomi so benigni tumorji kože, ki jih povzročajo virusi iz družine Papillomaviridae. Bolezen z izraženimi kliničnimi znaki kaže v Evropi tendenco širjenja med populacijo srn, medtem ko je pri navadnem jelenu opažena redko. V nalogi opisujemo primere fibropapilomatoze pri srnah in navadnem jelenu z značilno klinično in histopatološko sliko. Z metodo NGS smo določili nukleotidno zaporedje celotnih genomov sedmih papilomavirusov (PV), in sicer šestim sevom PV pri srnah (CcaPV1 sev 31-16, CcaPV1 sev 32-16, CcaPV1 sev 37-14, CcaPV1 sev 63-15, CcaPV1 sev 103-16 in CcaPV1 sev 84-14) in enemu sevu PV pri navadnem jelenu (CePV1v sev 64-14). Pri srnah smo potrdili okužbo s tipom CcaPV1 in pri navadnem jelenu s podtipom CePV1v. Primerjava virusnih genomov z že znanimi genomi iz NCBI GenBank je pokazala visoko genetsko stabilnost genoma v času in prostoru. S pregledom arhivskih vzorcev zadnjih dveh desetletij smo ugotovili pojavljanje fibropapilomatoze pri srnah na letni ravni, pri navadnem jelenu pa izredno redko. Contagious ectyma is a disease caused by Orf virus, a member of the family Poxviridae, genus Parapoxvirus. In the doctoral dissertation, we describe the analysis of two genomes of Orf virus isolates in chamois (strain ORFV_82/13 and strain ORFV_12/17). Using next-generation sequencing (NGS), we were the first ones to identify nearly complete genome of Orf viruses in chamois. The analyses revealed a high degree of similarity to Orf virus found in sheep and goats. At the nucleotide sequence level, the genomes are 99.93 % identical to each other and differ from other Orf viruses from 7.61 % (sheep strain NZ2) to 11.62 % (goat strain NP). Phylogenetic analysis of genes in the central region has shown that chamois Orf viruses form a distinct phylogenetic group. In the doctoral dissertation, the presence of antibodies against Orf viruses by serum neutralization assay was used to determine the seroprevalence of chamois population with Orf virus. Forty serum samples from chamois were tested. Test results were negative for 38 samples, and two results could not be read due to the cytotoxic effect of the serum sample on the cell culture. By reviewing the archival samples of the last two decades, considering the data provided by hunting organisations and the experience of the Institute of Pathology, wild animals, fish and bees, we found cyclic occurrence of contagious ecthyma in chamois. Fibropapillomas are benign skin tumours caused by viruses of the Papillomaviridae family. The disease with pronounced clinical signs shows a tendency to spread among the roe deer population in Europe, whereas it is rarely observed in red deer. The dissertation describes cases of fibropapillomatosis in roe deer and red deer with a characteristic clinical and histopathological picture. NGS method was used to determine the nucleotide sequence of the whole genome of seven papillomaviruses (PV), namely the sixth PV strain in roe deer (CcaPV1 strain 31-16, CcaPV1 strain 32-16, CcaPV1 strain 37-14, CcaPV1 strain 63-15, CcaPV1 strain 103-16 and CcaPV1 strain 84-14) and one PV strain in red deer (CePV1v strain 64-14). Infections with the CcaPV1 type were confirmed in roe deer and with the CePV1v subtype in red deer. Comparison of the viral genomes with previously known genomes from NCBI GenBank showed high genetic stability of the genome in time and space. Reviewing archival samples from the last two decades, it was found that fibropapillomatosis occurs annually in roe deer but extremely rarely in red deer.

Rod Vibrio sadrži važne patogene školjkaša i drugih beskralješnjaka, riba i ljudi. Kako bi se očuvalo njihovo zdravlje bitna je njihova identifikacija do razine vrste. Cilj ovog istraživanja je bio identificirati odabrane Vibrio uzorke do razine vrste, odrediti filogenetske odnose i utvrditi sličnosti, tj. razlike u strukturi zajednica Vibrio bakterija između akvakulture školjkaša Limskog kanala i Malostonskog zaljeva. Analizirani su uzorci morske vode, sedimenta kao i tkiva školjkaša mediteranske dagnje (Mytilus galloprovincialis) i europske plosnate kamenice (Ostrea edulis). Metodom lančane reakcije polimerazom (PCR) ciljane su različite kodirajuće regije šest konstitutivno eksprimiranih gena (engl. housekeeping genes) i 16S rRNA. Uzorci su poslani na sekvenciranje nakon čega je provedena metoda multilokusnog sravnjivanja sljedova (MLSA). Za filogenetsku analizu korištene su metode najveće vjerojatnosti i susjednog sparivanja. Eksperimentalni uzorci unutar Harveyi podskupine su se razlučili do razine vrste, a unutar Splendidus podskupine do razine grupacije. Na području uzgajališta školjkaša Limski kanal identificirano je šest vrsta te pet Splendidus grupacija, dok je na području uzgajališta školjkaša Malostonski zaljev identificirano pet vrsta i šest Splendidus grupacija. Dokazana je prisutnost više patogenih i uvjetno patogenih Vibrio vrsta za na području istočnog Jadrana. The genus Vibrio consists of important shellfish and other invertebrate, fish and human pathogens. In order to preserve their health, identification to species level is essential. The aim of this study was to identify selected Vibrio samples to species level, to determine phylogenetic relationships and to determine similarities and differences in the structure of the Vibrio community between the shellfish aquaculture of the Lim Bay and Mali Ston Bay. Samples of seawater, sediments, tissues of Mediterranean mussel (Mytilus galloprovincialis) and European flat oyster (Ostrea edulis) were analyzed. Different coding regions of six housekeeping genes and 16S rRNA were targeted by the polymerase chain reaction (PCR). Samples were sent for sequencing after which a multilocus sequence alignment (MLSA) was performed. For phylogenetic analysis two methods were used: Maximum likelihood and Neighbour-joining. Experimental samples within the Harvey clade were identified to the species level while experimental samples within the Splendidus clade to the grouping level. In Lim Bay, six species and five Splendidus groups have been identified, while five species and six Splendidus groups in Mali Ston Bay. The presence of several pathogenic and conditionally pathogenic Vibrio species in the eastern Adriatic has been proven.

Ignacio Bolívar Urrutia je Cladonotinae zamislio kao potporodicu koju čine vrste koje posjeduju široki scutellum koje se od drugih potporodica mogu razlikovati i po brojim drugim manje uočljivim morfološkim karakteristikama. Iako se danas koriste brojna svojstva kako bi se pokazalo da nova vrsta ne pripada drugim potporodicama, svojstvo širokog scutelluma je gotovo jedino svojstvo koje se koristi ne bi li se opravdalo smještanje novoopisane vrste baš u potporodicu Cladonotinae. Zbog toga danas potporodica Cladonotinae sadrži vrste izuzetno različitih morfologija kojima je jedina sličnost relativno širok nosni štitić, iako čak ni to nije uvijek tako. Ovaj diplomski rad je kladistički analizirao gotovo sve ikada korištene morfološke karakteristike jedinki potporodice Cladonotinae ne bi li pokazao koja svojstva su potencijalno dobri pokazatelji holofiletskih taksona unutar potporodice. Rezultati pokazuju da su svojstva pronotuma, ponajviše projekcije, slabi pokazatelji holofiletskih taksona. Svojstva koja su se pokazala kao najbolji kandidati za daljnja istraživanja povezanosti morfologije i stvarnog stanja evolucijskih odnosa su svojstva nogu. Pokazalo se da korištenje isključivo morfologije za opisivanje i determinaciju vrsta potporodice Cladonotinae daje rezultate upitne ispravnosti. Mnoge vrste danas smještene u Cladonotinae morfologijom ne pokazuju pripadnost potporodici te su nužno potrebna daljnja molekularna istraživanja kako bi se moglo odrediti koja su svojstva praktični pokazatelji pripadnosti vrsta potporodici Cladonotinae. Ignacio Bolívar Urrutia envisioned Cladonotinae as a subfamily composed of species bearing broad scutellum, distinguished from other subfamilies by other less noticeable morphological characteristics. Although many traits are used today to show that new species do not belong to other subfamilies, the broad scutellum is almost the only trait used to justify the placement of the newly described species in the Cladonotinae subfamily. Therefore, subfamily Cladonotinae contains species of extremely different morphologies, the only similarity being relatively wide scutellum, although even that is not always the case. This thesis cladistically analyzed almost every historically used morphological characteristic of individuals of the subfamily Cladonotinae in order to show which characters are potentially good indicators of holophyletic taxa within the subfamily. Results show that characters of pronotum, mostly projections, are weak indicators of holophyletic taxa. The properties that have proven to be the best candidates for further research into the relationship between morphology and the actual state of evolutionary relationships are the properties of legs. The exclusive use of morphology to describe and determine the species of the subfamily Cladonotinae has been shown to give results of questionable correctness. Many species placed in Cladonotinae do not show morphological similarities to other Cladonotinae species, hence further molecular research is necessary to determine which characters are good practical indicators of species belonging to the Cladonotinae subfamily.

Mladica je autohtona vrsta i endem dunavskog slijeva te se nalazi na Crvenom popisu IUCN-a kao ugrožena vrsta. Najveći razlog njezine ugroženosti je fragmentacija staništa uzrokovana izgradnjom brana i pregrada na vodotocima što je uzrokovalo smanjenje i nestajanje populacija. Iako se brojnost mladice od kraja 19. stoljeća smanjila za dvije trećine, stabilne populacije mladica u Hrvatskoj zabilježene su na granici sa Slovenijom u Kupi i na granici s Bosnom i Hercegovinom u Uni. Cilj ovog istraživanja bio je opisati gensku strukturu mladice u Hrvatskoj, utvrditi stupanj intraspecijske i intrapopulacijske genske raznolikosti, procijeniti zavičajnost pojedinih populacija mladica i njihovu različitost u odnosu na ostale europske populacije te istražiti evolucijsku povijest mladice u Hrvatskoj. Provedena je filogenetska rekonstrukcija temeljena na sekvencama za gen citokrom b (cyt b) i rodopsin (rhod) metodama najveće parsimonije (MP) i najveće vjerojatnosti (ML). Dobivena filogenetska stabla i mreža utvrdili su postojanje evolucijski odvojene linije mladice. Utvrđeno je jasno odvajanje roda Hucho unutar Salmonidae. Utvrđeno je da je genska raznolikost mladice na području dunavskog slijeva vrlo niska te da su migracije između populacija minimalne ili ne postoje. Niska genska raznolikost rezultat je prelova te izgradnje pregrada u vodotocima što uzrokuje fragmentaciju staništa. Danube salmon is an indigenous species, endemic to the Danube basin and it is on the IUCN Red List as an endangered species. The biggest reason for its endangerment is habitat fragmentation caused by the construction of dams and barriers on watercourses, which has caused the reduction and disappearance of populations. Although population size has decreased by two thirds since the end of the 19th century, stable populations of Danube salmon in Croatia have been recorded on the border with Slovenia in Kupa and on the border with Bosnia and Herzegovina in Una. The aim of this study was to describe the genetic structure of Danube salmon in Croatia, to determine the degree of intraspecific and intrapopulation genetic diversity, to assess the origin of individual populations of Danube salmon and their diversity in relation to other European populations and to investigate the evolutionary history of Danube salmon in Croatia. Phylogenetic reconstruction based on sequences for the cytochrome b (cyt b) and rhodopsin (rhod) gene was performed using the maximum parsimony (MP) method and maximum probability (ML) method. The resulting phylogenetic trees and network determined the existence of an evolutionarily separate lineage for Danube salmon. A clear separation of the genus Hucho within Salmonidae was determined. It was found that the genetic diversity of Danube salmon in the Danube basin is very low and that migrations between populations are minimal or non-existent. Low genetic diversity is the result of overfishing and the construction of dams in watercourses, which causes habitat fragmentation.

Glavni cilj ovog diplomskog rada bio je utvrditi i analizirati raznolikost i gensku strukturu populacija vrste Alburnus sarmaticus (velika pliska) u Hrvatskoj. Ova vrsta u Europi obitava samo u crnomorskom slijevu, i to u nizvodnim dijelovima rijeke Dunav, te u Dnjeparu i Južnom Bugu na području Rumunjske, Ukrajine i Bugarske, dok se u Hrvatskoj može naći potpuno izolirana populacija ove vrste u rijekama Savi, Kupi i Mrežnici. Uzorci su prikupljeni na rijekama Mrežnica i Kupa. Analiza podataka je uključivala izolaciju ukupne genomske DNA, umnažanje odabranih genskih markera lančanom reakcijom polimerazom, sravnjivanje sekvenci, što sam provela pomoću programa BioEdit, prilikom čega su uočene i pojedine mutacije među sekvencama. Također sam izradila izradila filogenetska stabla pomoću programa PAUP, prilikom čega sam koristila metode najveće parsimonije i najveće vjerojatnosti. Utvrdila sam da sekvence velike pliske pripadaju monofiletskoj liniji. Zatim sam izradila filogenetsku mrežu dobivenih haplotipova korištenjem programa Network. Za utvrđivanje stupnja intrapopulacijske genske raznolikosti koristila sam program DnaSP. Intraspecijsku i interpopulacijsku strukturu velike pliske u Hrvatskoj detaljno sam analizirala korištenjem programa MIGRATE. Filogenetska stabla pokazuju kako vrsta A. sarmaticus pripada staroj monofiletskoj liniji. Rezultati su također pokazali visoku gensku raznolikost, što je vrlo pozitivan pokazatelj za budući povoljan razvitak ove vrste. The main goal of this thesis was to determine and analyze the diversity and genetic structure of populations of Alburnus sarmaticus (Pontian shemaya) in Croatia. In Europe, this species lives only in the Black Sea basin, in the downstream parts of the Danube, and in the Dnieper and Southern Bug in Romania, Ukraine and Bulgaria, while in Croatia a completely isolated population of this species can be found in the Sava, Kupa and Mrežnica Rivers. Samples were collected from the Mrežnica and Kupa Rivers. Data analysis includes isolation of total genomic DNA, amplification of selected gene markers by polymerase chain reaction, sequence alignment, which I performed using the BioEdit program, during which individual mutations between sequences were observed. I also made phylogenetic trees using the PAUP program, using the methods of the highest parsimony and the highest probability. I determined that the sequences of the Pontian shemaya belong to the monophyletic line. I then constructed a phylogenetic network of the obtained haplotypes using the Network program. I used the DnaSP program to determine the degree of intrapopulation gene diversity. I analyzed the intraspecific and interpopulation structure of the Pontian shemaya in Croatia in detail using the MIGRATE program. Phylogenetic trees show that the species A. sarmaticus belongs to the old monophyletic line. The results also showed high genetic diversity, which is a very positive indicator for the future favorable development of these species.

Rod Delminichthys sadrži četiri vrste koje žive na području Hrvatske i Bosne i Hercegovine. To su: imotska gaovica (Delminichthys adspersus Steindachner, 1882), popovska gaovica (Delminichthys ghetaldii Heckel, 1843), jadovska gaovica (Delminichthys jadovensis Zupančič i Bogutskaya, 2002) te krbavska gaovica (Delminichthys krbavensis Zupančič i Bogutskaya, 2002). Ciljevi ovog istraživanja su bili potvrđivanje filogenetskog položaja svih vrsta unutar roda Delminichthys te utvrđivanje njihovih međusobnih srodstvenih odnosa, opisivanje intraspecijske i intrapopulacijske genske raznolikosti, procjena efektivne veličine pojedinih populacija, razine protoka gena između pojedinih populacija te konačno, utvrđivanje mogućih podzemnih migracijskih puteva između populacija istih vrsta. Kako bih analizirao filogenetsku strukturu roda Delminichthys, koristio sam programe MEGA-X, PAUP i Network. Metodama najveće parsimonije (MP), najveće vjerojatnosti (ML) i susjednog povezivanja (MJ) su se analizirale sekvence kontrolne regije (CR) i mitohondrijskog gena citokroma b (cyt b) kako bi se mogla izraditi filogenetska stabla i mreže kojima bi se utvrdila filogenetska struktura ovog roda. Potvrđena je struktura roda na četiri vrste, a pomoću programa DnaSP i MIGRATE su otkriveni visoki genski poliformizam na intraspecijskoj i intrapopulacijskoj razini, efektivne veličine populacija te protok gena između njih. Genus Delminichthys consists of four species that live on the territory of Croatia and Bosnia and Herzegovina. Those are: spotted minnow (Delminichthys adspersus Steindachner, 1882), southern dalmatian minnow (Delminichthys ghetaldii Heckel, 1843), jadova minnow (Delminichthys jadovensis Zupančič i Bogutskaya, 2002) and krbava minnow (Delminichthys krbavensis Zupančič i Bogutskaya, 2002). The aims of this study were confirming the phylogenetic position of all species within the Delminichthys genus and the phylogenetic relationships between them, the determination of the intraspecific and intrapopulation diversity, the estimation of the effective population sizes, rates of gene flow between the populations and finally, the determination of the possible underground migratory routes between the populations of the same species. In order to analyze the phylogenetic structure of the Delminichthys genus, I used programs MEGA-X, PAUP and Network. The sequences of the control region (CR) and the mitochondrial gene for the cytochrome b (cyt b) were analyzed with the maximum parsimony (MP), maximum likelihood (ML) and median-joining (MJ) methods in order to build the phylogenetic trees and the networks that would confirm the phylogenetic structure of this genus. I confirmed the structure of four species within the genus, and with the use of the programs DnaSP and MIGRATE, I discovered that the genetic polymorphism on both intraspecific and intrapopulation levels were high, while also determining their effective population sizes as well the gene flow between their populations.

Paklare (porodica Petromyzontidae) koje se i dalje prema mnogim publikacijama svrstavaju među ribe, zapravo su skupina kralješnjaka koja se prije 500 milijuna godina na temelju fundamentalne razlike, nepostojanja čeljusti, odvojila od riba i ostalih razreda kralješnjaka koji imaju razvijenu čeljust. Njihova taksonomija nije do kraja razriješena na globalnoj razini pa tako ni na području Hrvatske uz nedovoljno proučenu taksonomiju, rasprostranjenost i ugroženost. Cilj ovog istraživanja bio je: utvrditi srodstvene odnose proučavane porodice, odrediti točan taksonomski položaj vrsta unutar porodice, napraviti filogenetsku rekonstrukciju skupine na proučavanom području te procijeniti gensku raznolikost i strukturu pojedinih utvrđenih vrsta i linija s krajnjim ciljem zaštite ako je ona potrebna. Kako bih utvrdila taksonomski položaj prisutnih vrsta na području dunavskog slijeva Hrvatske, provela sam filogenetsku rekonstrukciju koja se temeljila na sekvencama gena za citokrom b pomoću metode najveće parsimonije, metode najveće vjerojatnosti i metode susjednog povezivanja. Dobivena filogenetska stabla i filogenetska mreža potvrdili su postojanje 4 odvojene linije unutar vrste Eudontomyzon vladykovi Oliva i Zanandrea, 1959 te prisutnost vrste Eudontomyzon danfordi Regan, 1911 na području Hrvatske. Pomoću testova genske raznolikosti i genske različitosti uz analize molekularne dijagnostike utvrdila sam umjerenu do visoku razinu genske raznolikosti unutar i između utvrđenih vrsta i linija te duboku strukturiranost unutar vrste Eudontomyzon vladykovi. Lampreys (family Petromyzontidae), which according to many publications are still classified as fish, are actually a group of Vertebrates. 500 million years ago they separated from fish and other classes of Vertebrates that have developed jaws based on the absence of a jaw. Their taxonomy has not been fully resolved on the global level, nor in Croatia, with an insufficiently studied taxonomy, distribution and endangerment. The aim of this study was to: determine the relationships of the studied family, determine the exact taxonomic positions of species within the family, make a phylogenetic reconstruction of the group in the study area and astimate the genetic diversity and structure of individual identified species and lineages. In order to determine the taxonomic position of the species present in the Danube basin of Croatia, phylogenetic reconstruction was performed based on cytochrome b gene sequences using the maximum parsimony method, maximum likelihood method and the median joining method. The obtained phylogenetic trees and phylogenetic network confirmed the existence of 4 separate lineages within the species Eudontomyzon vladykovi Oliva i Zanandrea, 1959 and the presence of the species Eudontomyzon danfordi Regan, 1911 in Croatia. Using tests of genetic diversity and genetic differentiation in addition to molecular diagnostic analyzes, moderate to high levels of genetic diversity within and between identified species and lineages and deep structuring within the species Eudontomyzon vladykovi were determined.

Samo posljednja 4 desetljeća, arheje su prepoznate kao zasebna domena života, prije čega su bile svrstavane pod domenu bakterija. Inicijalna saznanja su se oslanjala isključivo na uzgojive ekstremofilne predstavnike arheja, sve do razvoja sofisticiranijih molekularnih metoda pretraživanja genoma u okolišu. Njima se ispostavilo da arheje obitavaju u gotovo svim okolišima i to u znatnoj zastupljenosti. Recentnijih desetljeća se ponajviše proširilo znanje o njihovoj genetskoj raznolikosti i sličnostima s eukariotskom domenom. Monofilija svih grupa još uvijek je predmet istraživanja te veoma često se pronalaze dublja grananja grupa ili novi razredi. Fiziološki, arheje sadrže karakteristike nalik bakterijama, karakteristike nalik eukariotima te potpuno individualne značajke. Štoviše, sličnije su eukariotima nego bakterijama na molekularnoj razini. Otkriće Asgard supergrupe arheja približava nas spoznaji događaja tijekom postanka i ranog razvoja eukariota. Sukladno tome, ustanovljeno je da su eukarioti ogranak arhealne domene, kao potomci Asgard arheja i bakterijskih endosimbiota. Potpuni opseg eukariotskih značajci koje možemo pripisati arhealnom pretku predmet su mnogih teorija. Njih će jedino razlučiti daljnje proširenje filogenetske raznolikosti te buduće kultivacije arheja. Only for the past 4 decades, archaea have been recognised as a separate domain of life, being classified as bacteria beforehand. Our initial findings have exclusively relied upon cultivatable extremophilic representatives of archaea, up until the development of more sophisticated molecular methods for surveying environmental genomes. Their use made apparent that archaea appear in all environments and in substantial quantities. In the recent decades we have most expanded our knowledge about their genetic diversity and their similarities with the domain of eukaryotes. The monophyly of all groups if still a subject of research and, very often, deeper branchings or new phyla are being found. Physiologically, archaea possess bacterial-like characteristics, eukaryote-like characteristics and distinctly archaeal properties. Furthermore, they are more alike eukaryotes than bacteria on the molecular scale. The discovery of the Asgard superphylum has brought us closer to understanding events that happened during eukaryogenesis. Following that notion, it has been established that eukaryotes are a branch of the archaeal domain, as descendants of Asgard archaea and bacterial endosymbionts. The full extent of eukaryotic properties which we ascribe to an archaeal ancestor is a subject of many theories. They will only be resolved by further expansion of phylogenetic diversity and future archaeal culturing.

Namen magistrskega dela je bil potrditi obstoj vrst iz rodu Prevotella, ki jih je napovedala študija metagenomske analize vampnih ekosistemov (Stewart in sod., 2019), kar smo morda dosegli z izolacijo enega seva. Hkrati smo izolirali vsaj pet novih vrst rodu Prevotella. Primerjali smo genome pridobljene iz metagenomov s tistimi iz izoliranih Prevotella in ugotovili da imajo nekateri izmed prvih pomanjkanje številnih jedrnih genov za uspešno analizo MLSA, kot tudi nenavadno visoko število encimov CAZY in velikost genoma. Večina MAG se tako uvršča med genome srednje kvalitete, ki niso najprimernejši izbor za študije filogenije. Na podlagi TETRA in ANIb naj bi združeni metagenomski odčitki Stewart in sod. predstavljali 161 ločenih vrst znotraj Prevotellaceae, v vampu ovna s področja Slovenije pa smo izolirali zgolj eno potencialno vrsto. Kongruenca ribosomskih genov, ohranjena pri gojenih sevih, se ne odraža pri MAG, kjer so ti geni včasih najdeni celo v več kopijah. Mnenja smo, da je analizo surovih odčitkov Stewart in sod. (2019) nujno potrebno ponoviti z različnimi individualnimi pristopi združevanja in razvrščanja odčitkov za kontrolo kvalitete. The main purpose of Master's thesis was to confirm the existence of metagenome-derived Prevotella species of rumen ecosystems from Scotland (Stewart et al., 2019), which is speculated to be fulfilled with the isolation of one such specimen. At the same time, we managed to cultivate strains of at least five new Prevotella species. Comparison of Stewart metagenomes and cultivated isolates of Prevotella revealed a distinctive deviation with metagenome-derived species showing absence of multiple core genome genes needed for MLSA analysis. Moreover, many had high numbers of CAZY enzymes and genome sizes. The majority of MAG fall into a category of medium quality drafts, which are discouraged from using in phylogenetic studies. According to TETRA and ANIb, Stewart metagenomes represent 161 species within Prevotellaceae family, however we have presumably isolated only one of them. Congruency of genes coding for ribosomal proteins, conserved in cultivated isolates of Prevotella is missing in most metagenome-derived species. In our judgement, complete analysis from raw reads obtained in Stewart et al. (2019) should be repeated, this time with multiple algorithm approach to assembly and binning of reads in order to account for quality control.

The genetic diversity was studied of Cryptosporidium scrofarum (syn Cryptosporidium pig genotype II) of domestic pigs (Sus scrofa domesticus) from Punjab, India. Nested PCR amplification targeting the 18S rRNA and actin gene loci from Cryptosporidium positive samples was carried out, and the amplicons were sequenced. Phylogenetic comparison of a partial 18S rRNA gene revealed that they were genetically most similar to C. scrofarum isolated from other parts of the world. However, comparison of sequences representing a fragment of the genomic actin locus identified a new genotype conserved within the isolates sampled from India but distinct from other published sequences, suggesting the presence of a different Indian genotype., U ovom je radu istraživana genetička raznolikost Cryptosporidium scrofarum (syn Cryptosporidium pig genotype II) domaćih svinja (Sus scrofa domesticus) iz regije Punjab, Indija. Provedeno je umnožavanje 18S rRNA i lokusa aktin-gena pomoću ugniježđene PCR metode iz uzoraka pozitivnih na Cryptosporidium te su sekvencirani amplikoni. Filogenetska usporedba parcijalnog 18S rRNA gena pokazala je da su uzorci genetski najsličniji C. scrofarum izoliranom u drugim dijelovima svijeta. Također, usporedba sekvencija dijela lokusa genomskog aktina otkrila je novi genotip očuvan unutar izolata uzorkovanih u Indiji, ali različitih od drugih objavljenih sekvencija, upućujući na postojanje zasebnog indijskog genotipa.

Glavoči su najbrojnijih skupina riba zrakoperki. Također, oni su jedna od akustički najistraživanijih skupina zrakoperki današnjice, sa 23 poznate vrste koje se glasaju. U sklopu ove disertacije, u četiri znanstvene publikacije prvi je put istražena, upotrebom bioakustičkih metoda i analiza, sposobnost stvaranja zvukova i vokalni repertoar glavoča linije Gobius Neogobius melanostomus, Neogobius fluviatilis i Ponticola kessleri, kao i Perccottus glenii (Odontobutidae). Nadalje, ovom su disertacijom istražene anatomske strukture uključene u proces produkcije zvukova kod rotana, a rezultati su pokazali kako se mehanizam najvjerojatnije temelji na kontrakcijama kranijalno-pektoralnih mišića. Upotrebom molekularnih analiza koje uključuju metode filogenetske rekonstrukcije s mitohondrijskim i jezgrenim genetičkim biljezima, rekonstruirani su odnosi vokalnih glavoča linije Gobius te istražena korelacija između genetičke i akustičke divergencije. Rezultati ove disertacije naglašavaju kako su zvukovi glavoča specifični za pojedinu vrstu, dok njihova interspecijska akustička divergencija prati genetičku, što pokazuje kako evolucija zvukova i genotipova prati isti obrazac. Gobies are the most numerous fish group. They are also one of the most investigated acoustic cluster of actinopterygian fish nowadays, with 23 species vocally tested. In this dissertation, represented through four scientific publications, the acoustic communication and vocal repertoire was investigated in Gobius-lineage gobies Neogobius melanostomus, Neogobius fluviatilis and Ponticola kessleri, as well as in Perccottus glenii (Odontobutidae), using standardized bioacoustic methods and analyses. The presented dissertation investigated the anatomical structures involved in the sound production, and the conducted analyses indicate that the sonic mechanism could be based on the contractions of cranio-pectoral muscles. By using phylogenetic analyses with a multilocus approach, we reconstructed the interspecific relationships between vocal gobies from Gobius-lineage and correlated the acoustic and genetic divergences. Results of this dissertation emphasize that goby sounds are truly species-specific, while their interspecific acoustic divergence follows the genotypic divergence, which could indicate that acoustic signals in gobies from Gobius-lineage carry phylogenetic signal.

The complete Cytchrome b gene and partial mtDNA control region were sequenced for the Pakistani domestic yak (Bos grunniens) within the Bovidae family. A total of 300 samples were genotyped using 27 bovine microsatellite markers from the Gilgit-Baltistan and Skardu regions of Pakistan. We identified a total of 35 mutations and 9 haplotypes based on D-loop sequences, with a haplotype and nucleotide diversity of 0.9640±0.051 and 0.02172±0.00224, respectively. For the Cyt b gene, a total of 23 variable sites and six different haplotypes were observed with 0.885±0.067 haplotype and 0.00989±0.003 nucleotide diversity. Phylogenetic analysis of D-loop and Cyt b gene suggested that domestic yak sequences cluster into two highly divergent maternal lineages (lineages I and II), while three haplogroups A, C, and D were identified of the six previously known haplogroups. Haplogroups A and C were dominant and widely distributed among all investigated yak samples. All microsatellites were polymorphic and a total of 138 alleles were observed, with average polymorphic information content (PIC) of 0.56 indicating their effectiveness. The average heterozygosity was observed at 0.6071 with allele diversity of 5.1111 and gene diversity of 0.4830. The implications of these findings can be applied for yak conservation., Za pakistanskog domaćeg jaka (Bos grunniens) iz obitelji šupljorožaca (Bovidae), sekvenciran je cjelokupni citokromni b gen i djelomice kontrolna regija mtDNK. Genoti¬pizirano je tri stotine uzoraka uporabom 27 goveđih mikrosatelitskih markera pasmine Gilgit-Baltistan i Skardu iz Pakistana. Identifi¬cirali smo ukupno 35 mutacija i 9 haplotipova na temelju sekvencija D-petlje uz haplotipsku i nukleotidnu raznolikost od 0,9640±0,051, od¬nosno 0,02172±0,00224. Za citokromni b gen, ukupno 23 varijabilne lokacije i šest različi¬tih haplotipova zamijećeno je s 0,885±0,067 haplotipskom i 0,00989±0,003 nukleotidnom raznolikošću. Filogenetska analiza D-petlje i citokromnog b gena ukazala je da se sekven¬ce domaćih jakova grupiraju u dvije vrlo di¬vergentne loze po majci (loza I i II), dok su identificirane tri haplogrupe A, C i D od ranije poznatih šest haplogrupa. Haplogrupe A i C bile su dominantne i široko rasprostranjene među svim istraženim uzorcima jakova. Svi mikrosateliti bili su polimorfni te je zamijeće¬no ukupno 138 alela, s prosječnim sadržajem polimorfne informacije (PIC) od 0,56 ukazu¬jući na njihovu učinkovitost. Zamijećena je prosječna heterozigotnost od 0,6071 s alelnom raznolikošću 5,1111 i genetskom raznolikošću 0,4830. Implikacije ovih nalaza mogu se rabiti za očuvanje populacije jakova.

Dothistroma needle blight (DNB), caused by Dothistroma septosporum and D. pini, and Brown spot needle blight, (BSNB), caused by Lecanosticta acicola, belong to the most harmful and important pine diseases today. DNB has been present in Southeastern Europe since the 1950s and BSNB since the 1970s. Recent outbreaks of BSNB and the fact that DNB is widely spread, made it necessary to study the population structure of these disease agents by measuring their genetic variety in phylogenetic, mating type gene and microsatellite marker analyses. In addition, the diversity of certain morphological traits was determined and migration patterns evaluated through use of coalescent-based method. Populations from Slovenia, Serbia and Croatia, obtained from Pinus nigra, P. mugo and P. halepensis, were selected for the study and compared with populations from Europe (EU), North America (NA), Central America (CA), South America (SA), Africa (AF) and Australia (AU). Serbian and most of Slovenian D. septosporum isolates grouped in phylogenetic analyses based on elongation factor (EF1-α) and β-tubulin-2 (βT2) together with the isolates from EU and NA, while Celje (Slovenia) formed a so far unrecognized new EF1-α haplotype. Croatian D. septosporum isolates also formed a new EF1-α haplotype and a separate clade, while they grouped with isolates from NA, CA, SA, AU and AF in the βT2 gene region analysis. ITS analysis was uninformative. In population structure analyses, most of Slovenian and Serbian populations composed a single genetic cluster, while populations from Trenta (Slovenia) and Kožino (Croatia) formed separate clusters. Migration analyses showed that gene flow occurs between populations from Slovenia and Serbia. Slovenian D. pini isolates and isolates from SA have identical ITS, EF1-α in βT2 sequences. In population structure analyses, D. pini isolates displayed clonality and low genetic diversity, likely as a consequence of a single event introduction. L. acicola phylogenetic analysis revealed a novel EF1-α haplotype in Croatian population and showed that Slovenian and Croatian isolates share a common ancestry with individuals from Central and Northern Europe. Population structure analysis revealed four distinct genetic clusters, generally corresponding to their respective geographic location and hosts. Observed unequal ratio of mating types and low overall genetic diversity in the population indicates that L. acicola predominantly reproduces asexually. Low genetic variation within each of the four clusters and absence of or low gene flow suggests that the different L. acicola populations were independently introduced. Rdeča pegavost borovih iglic, ki jo povzročata glivi Dothistroma septosporum in D. pini ter rjava pegavost borovih iglic, ki jo povzroča gliva Lecanosticta acicola, sta dve izmed pomembnejših bolezni borov. Rdeča pegavost borovih iglic je v jugovzhodni Evropi prisotna od leta 1950, rjava pegavost od leta 1970, obe bolezni se v zadnjih letih razširjata po Evropi in širše. Na osnovi filogenetskih analiz treh genskih regij, analize mikrosatelitnih markerjev, ugotavljanja načina razmnoževanja ter na osnovi izbranih morfoloških lastnosti smo ugotavljali strukturo populacij, genetsko raznolikost ter vzorce migracij populacij D. septosporum, D. pini in L. acicola. Izbrane populacije iz Slovenije, Srbije in Hrvaške, pridobljene iz Pinus nigra, P. mugo in P. halepensis, so bile primerjane s izolati iz Evrope (EU), Severne Amerike (SA), Centralne Amerike (CA), Južne Amerike (JA), Afrike (AF) in Avstralije (AU). V filogenetskih analizah elongacijskega faktorja 1-α (EF1-α) in β-tubulina-2 (βT2) se srbski in večina slovenskih izolatov D. septosporum združujejo z izolatiiz EU in SA, medtem ko izolati D. septosporum iz Celja (Slovenija) predstavljajo doslej neznani EF1-α haplotip. Hrvaški izolati D. septosporum so ravno tako predstavljali novi haplotip EF1-α in ločeno skupino, so se pa v analizi genske regij βT2 združili z izolati iz SA, CA, JA, AU in AF. ITS analiza je bila neinformativna. Populacijske in migracijske analize so pokazale, da se večina preučevanih slovenskih in srbskih D. septosporum populacij uvršča v isto genetsko gručo, medtem ko se populacije iz Trente (Slovenija) in Kožina (Hrvaška) grupirajo posebej. Slovenski D. pini izolati in izolati iz SA so imeli enake ITS, EF1-α in βT2 sekvence. V populacijskih analizah so vsi izolati D. pini pokazali klonalnost, nizko genetsko pestrost, kar nakazuje na vnos iz enega vira. Hrvaška populacija glive L. acicola vsebuje EF1-α haplotip, ki ni opisan v drugih populacijah L. acicola. Filogenetska analiza na osnovi regije EF1-α pokaže, da imajo izolati L. acicola iz Slovenije in Hrvaške skupne prednike z izolati iz srednje in severne Evrope. Analiza populacijske strukture slovenskih in hrvaških populacij L. acicola je razkrila štiri različne genetske gruče, ki praviloma odražajo geografsko lokacijo in gostitelje. Opaženo neenakomerno razmerje tipov parjenja, nizko število haplotipov in nizka splošna genetska raznolikost v populaciji nakazujejo na močan vpliv nespolnega razmnoževanja. Nizka genetska variabilnost znotraj vsakega od štirih grozdov in odsotnost oziroma nizek genski pretok nakazujeta na to, da so različne L. acicola populacije bile vnesene neodvisno.

International audience; Avian influenza viruses of the H9N2 subtype continue to spread in wild birds and poultry worldwide. Infection with H9N2 avian influenza virus was detected for the first time in Morocco in January 2016. In this study, a total of 105 organ and tracheal swab samples from 21 broiler farms in Morocco were collected from July 2016 to October 2018 for H9N2 screening. The suspicion of disease was based on severe respiratory signs such as sneezing, coughing, rales and gasping, while H9N2 virus infection was confirmed by real-time RT-PCR. Hemagglutinin (HA) genes of four isolates were amplified by conventional RT-PCR, sequenced, and aligned for phylogenetic analyses. Among the 21 flocks, 48% (10/21) were qRT-PCR positive for H9, with the cycle threshold values ranging from18.6 to 34.8. The maximum similarity in nucleotide and protein sequences (96-98%) was observed between the Moroccan viruses and an H9 virus isolated from broiler chickens in 2017 in Burkina Faso (A/chicken/BurkinaFaso/17RS93-19/2017) and from a layer chicken in the United Arab Emirates in 2015 (A/chicken/Dubai/D2506/2015). The HA genes revealed the close relationship between the four Moroccan viruses, with 97.9%-99.9% nucleotide identity. Phylogenetic analysis showed that the Moroccan viruses belonged to the G1 lineage, and likely originated from the Middle East, as previously reported in 2016.; Virusi ptičje gripe H9N2 nastavljaju se širiti u peradi i divljih ptica širom svijeta. Infekcija niskopatogenim virusom influence H9N2 prvi je put otkrivena u Maroku u siječnju 2016. godine. U ovom je istraživanju za probir na H9N2 prikupljeno ukupno 105 organa i obrisaka iz dušnika s 21 farme brojlera od srpnja 2016. do listopada 2018. iz različitih regija Maroka. Sumnja na bolest temeljila se na teškim respiracijskim znakovima kao što su kihanje, kašljanje, hropanje i hripanje, a infekcija virusom H9N2 potvrđena je PCR-om obrnute transkripcije u stvarnom vremenu. Sekvencije gena za hemaglutinin (HA) od četiri izolata amplificirane su pomoću RT-PCR qRT-PCR poravnane za filogenetsku i analizu sličnosti aminokiselina. Od 21 uzorka jata 48 % (10/21) bilo je pozitivno na H9 s pragom broja ciklusa u rasponu od 18,6 do 34,8. Maksimalna sličnost u nukleotidnim i proteinskim sekvencijama (96 -98 %) uočena je između marokanskih virusa i virusa H9 izoliranih iz brojlerskih pilića u 2017. u Burkini Faso (A/piletina/BurkinaFaso/17RS93-19) i od kokošjeg pileta u Ujedinjenim Arapskim Emiratima u 2015. (A/piletina/Dubai/D2506/2015). HA geni otkrili su blisku vezu između četiriju virusa, s 97,9 % -99,9 % nukleotidnog identiteta. Filogenetska analiza pokazala je da marokanski virusi pripadaju lozi G1 i vjerojatno potječu s Bliskog istoka, kao što je objavljeno 2016. godine.