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High yields in crop production rely on a constant supply with high amounts of plant nutrients. Thus, inappropriate nutrient supply can cause significant economic losses. Nutrient deficiency alters plant morphological and physiological traits, i.e. cause specific symptoms which could be detected by non- destructive methods, using spectral reflectance analyses. This study aimed to describe and quantify the development of the nutrient deficit (nitrogen (N), phosphorus (P), potassium (K), magnesium (Mg) and iron (Fe)) in common bean (Phaseolus vulgaris L.) using multispectral and chlorophyll fluorescence imaging. Plants were grown in a control nutrient solution and in solutions lacking N, P, K, Mg or Fe and were analyzed every three days during 12 days of growth. Irrespective of measured traits, earliest symptoms and most pronounced changes were detected for K and N deficiency, whereas slowest and least pronounced symptoms development were found in Fe deficiency. From the fluorescence parameters, the earliest and most pronounced response was detected in electron transport rate, which decreased after six days for all treatments compared to control, except for the Fe deficiency. Of the morphological traits, the earliest response reduction in leaf area, found at sixth day in all treatments compared to the control. From multispectral traits, the earliest and most significant differences compared to control were reduction in green leaf index and increase in specific green reflection. These results show that nutrient deficiency could be detected and traced nondestructively using multispectral and chlorophyll fluorescence analysis.

Due to the availability and affordability of large scale genotyping, identification of genomic regions affected by the natural or artificial selection in the populations of wild and domesticated plants became standard procedure in modern population genetics. Identification of these regions and their association with traits of interest helps to understand evolutionary history of the species. The structure of cultivated common bean populations strongly resembles the structure of wild populations due to occurrence of at least two independent domestication events that occurred independently in Central and South America. Long- termed spatial isolation of two gene pools preserved their differential patterns of adaptation. Basic structure in collection of Croatian common bean landraces corresponds to the general pattern: first division is between two clusters of Central and South American origin, and later than divides into two clusters identified by different phaseolin types. In this study we used the differences in allele frequencies between populations (estimated by Fst values) to identify the SNP loci under adaptive selection.

The Balkan chamois (Rupicapra rupicapra balcanica) distribution goes from Croatia in the north to Greece in the south and Bulgaria in the east. The distribution of the subspecies and population density have been shaped by the natural and anthropological fragmentation of suitable habitats, the resulting restrictions on gene flow between different populations and human management. In Croatia, the Balkan chamois populations are both extant (Dinara Mountain), or (re)introduced (Velebit and Biokovo Mountains). Poaching, unsustainable hunting and the introduction of Alpine chamois are considered the main threats to the survival of Balkan chamois in the whole area of distribution, but conservation and management status varies from country to country and is subject to different national legislations. We combined 16 microsatellite loci and a partial mitochondrial control region to investigate the genetic structure and connectivity of Balkan chamois throughout its distribution range and to assess the accuracy of existing historical data on the origin of the Biokovo population 56 years after its reintroduction. We successfully genotyped 141 individuals from eight countries covering almost the entire distribution range (Croatia, Bosnia and Herzegovina, Serbia, Montenegro, Albania, North Macedonia, Greece and Bulgaria) and sequenced the partial control region in 44 individuals. Bayesian analysis revealed 3 genetic clusters throughout the distribution range and assigned individuals from Serbia and Bulgaria to two separate clusters, while all other individuals belonged to the same cluster. Thirty new haplotypes were obtained from partial mitochondrial DNA sequences, with private haplotypes in all analysed populations and two haplotypes shared among populations, indicating the possibility of past translocations. Additional analysis of past reintroduction in the genetic diversity of Balkan chamois on Biokovo using STRUCTURE and GENELAND algorithms showed clear separation of the reintroduced Biokovo population from the Prenj and considerable genetic similarity with the Čvrsnica-Čabulja populations in Bosnia and Herzegovina. This suggests that the current genetic composition of the Biokovo population is not exclusively derived from the Prenj population, as suggested by the available literature, but also from other source populations. The genetic composition of the Balkan chamois subspecies presented here provides the necessary starting point for the development of conservation strategies required for its sustainable management and conservation, and identifies the potential source populations for future (re)introduction plans.

In this study, 96 samples from 48 soil profiles (Gleysols, Chernozems, Anthrosols) on arable land in eastern Croatia were analyzed. The results were used to build local (specific) pedotransfer functions (PTFs) for predicting soil water retention at field capacity (FC) and wilting point (WP). The built multiple linear regression models are user-friendly and perform very well (RMSEs 2.7% and 1.8%, MAEs 2.1% and 1.5% ; for FC and WP, respectively) in spite having only the contents of organic C, clay and silt as predictors (selected as three basic properties for which the data is widely-accessible). Since in this study the soils on the most common parent materials in continental Croatia (fluvial deposits and loess derivates with mostly silt loam, silt clay loam, and silt clay textures) were considered, we expect our PTFs to be widely applicable. Prediction performance of the universal regression-based PTFs (EU-PTFs), developed by Toth et al. (2015) at the European scale, was also tested. Although Croatian soils are not part of the EU-HYDI database that was used for creating EU-PTFs, the universal model predicts the FC of the here-analyzed soils almost equally well (RMSE=3.3%, MAE=2.7%) as our local model. However, while predicting the WP of the local soils, the universal model was not as accurate – it overestimated WP (RMSE=4.9%, MAE=4.4%) more than the local model did. Because in many soils WP is usually low, such prediction errors may be unacceptable. Nevertheless, in some cases, the EU- PTFs can be used instead of local PTFs, at least in the European regions that have soilscapes similar to these considered herein. However, to increase EU-PTFs reliability, the soil database for their development should incorporate more European countries and regions.

Aoudad, Ammotragus lervia, is an ungulate species native to the mountain ranges of North Africa. Six subspecies have been described in its native range, differing in distribution and morphological characteristics. The aoudad was successfully introduced into Europe in the second half of the twentieth century, mainly for hunting purposes, with established populations in Croatia, Italy, and Spain. Previous analysis of mitochondrial DNA showed that the aoudad was introduced to Europe from at least four maternal lineages, but it is not known from which evolutionary units/subspecies the introduced aoudads originated. The aim of our study was to gain insight into adaptive genetic diversity in European populations of aoudad by analyzing exon 2 of the major histocompatibility complex (MHC) class II DRB gene. Between 2016 and 2018, aoudad samples were collected from free- ranging populations from Croatia (Mosor Mountain) and Spain (Sierra Espuña and La Palma Island), and from captive populations from the Czech Republic (near the city of Plzeň) and Spain (Almeria). Using next-generation Ion Torrent sequencing, we detected eight highly divergent alleles in 68 individuals, with three to four alleles present per population. The number of variable nucleotide sites in the detected alleles was 42 out of 236 (18%) and the number of variable amino acid positions in the translated sequences was 23 out of 78 (29%). The high average proportion of shared alleles within the population (0.51) is consistent with the expectations for recently introduced populations funded by a small number of individuals. The two most common alleles had frequencies of 37% and 29%, with the first present in all populations and the second present in all populations except Almeria. We found private alleles in populations from Croatia and the Czech Republic, for which previous study at neutral loci showed a common origin. These private alleles are probably a consequence of the founder effect. In Almeria, a population thought to represent a subspecies of Saharan aoudad, a private allele accounted for 60% of the allele frequency, and this population was genetically the most distinct from the others. The observed differences in DRB alleles between populations are inevitably the result of DRB alleles carried by the founder individuals. Because previous study showed that mitochondrial haplotypes are highly variable in European aoudad populations, more so than is common for the same subspecies of ungulates, observed MHC variability in these populations may also reflect different evolutionary lineages from which the founder individuals originated.

Testicular germ cell tumours (TGCT) are the most common malignancy in men aged 15–35 years and its incidence is increasing worldwide. Tumour markers commonly used in the diagnosis and prognosis of TGCT have limited sensitivity and specificity and additional, minimally invasive biomarkers for disease management are needed. Metabolomic-based profiling of amino acids in urine may identify changed metabolites as potential biomarkers of testicular cancer. Impaired metal(loid) homeostasis may increase susceptibility to cancer development. We compared a quantitative profile of 30 amino acids and 18 metal(loid)s in urine samples between 86 men with newly diagnosed TGCT and 68 control subjects, aged 18 to 55 years. The concentration of free amino acids was determined by GC-MS using Phenomenex EZ:faast kit for sample preparation, multi-element analysis was performed by ICP-MS, and all results were corrected for creatinine levels. Testicular cancer patients had significantly higher levels of aspartic acid (Asp), manganese (Mn) and zinc (Zn), and significantly lower levels of threonine (Thr), serine (Ser), histidine (His) and cobalt (Co) when compared to control subjects. Linear discriminant analysis using all 30 amino acids successfully separated subjects with TGCT from control subjects and the discrimination rate was 86%. Metabolic pathway analysis (MetaboAnalyst 4.0) showed that Asp, Thr, Ser, and His were related to alterations in 12 metabolic pathways and those with the highest impact were metabolism of alanine, aspartate and glutamate (0.26) and glycine, Thr and Ser (0.23). Our results indicated that disorders in the citric acid cycle, the urea cycle and amino acid metabolism play a role in the pathogenesis of testicular cancer. The impaired urinary profile of Mn, Co and Zn may be highly relevant with regard to their role in amino acids synthesis, antioxidant defense and genetic repair processes. More research is needed to verify our findings

In this study, we analysed telemetry data from GPS monitoring of the European mouflon Ovis aries musimon in two areas of the Mediterranean region in Croatia. Four males were collared on the island of Dugi Otok (43°56'44.5"N 15°07'30.0"E), while two females and one male were collared on the mainland coast near the town of Senj (44°59'31.2"N 14°54'54.7"E). GPS collars programmed to take 24 locational fixes per day were deployed on seven European mouflons to provide insights into their space use. The study period was divided into two seasons - winter (January - March) and spring (April - June). The results showed that home ranges of the island population (IP) were considerably smaller than those of the mainland coast population (MP) in both seasons: 294.78 ha ± 153.34 ha for IP and 440.28 ± 31.45 ha for MP in winter, and 168.66 ha ± 60.33 ha for IP and 326.76 ± 62.69 ha for MP in spring (based on Minimum Convex Polygon method). The daily range value was estimated as 1.59 ± 0.31 km/day for IP and 2.49 ± 0.72 km/day for MP in winter, and 1.41 ± 0.22 km/day for IP and 2.81 ± 0.94 km/day for MP in spring. A possible reason for the larger seasonal home ranges of MP individuals may be explained by the presence of large predators and higher anthropogenic pressures on the mainland coast, such as hunting pressure and proximity to urban areas. In addition, variation in daily ranges among populations may be explained by food-dependent movement behaviour driven by differences in resource availability between two habitats. However, to obtain more precise daily range values, a higher sampling frequency of GPS collars is needed to increase the precision of population-specific daily range estimates. Further analyses should be conducted with a larger sample and include natural and anthropogenic covariates, such as resource availability and predation, that may potentially influence the movement ecology of European mouflon.

Literature, historical and archaeological data confirm that chamois once lived in the Biokovo Mountain (first records ca. 12 thousand years ago). The population became extirpated in the early 20th century due to many reasons such as overhunting, poaching, competition with and disturbance by livestock. The population was later reintroduced (1964-1969), 48 individuals were released from the neighbouring mountains in Bosnia Herzegovina (BIH). The main objective of this study was to determine the accuracy of historical data on the origin of the Balkan chamois population on Biokovo Mountain and to assess the population genetic status of both the initial and translocated populations 56 years after the reintroduction. We used 16 microsatellite loci to analyse the genetic structure of Balkan chamois populations. We screened three populations from BIH: Prenj (12), Čvrsnica (12) and Čabulja Mountains (5), and one from Biokovo Mountain (20). Independent analysis of the BIH populations in STRUCTURE revealed a clear separation of the population of Prenj from the populations of Čvrsnica and Čabulja, with the latter two forming a single population. This suggests the Neretva River and the highway M-17 may be effective barriers for the species as the constitute a genetic boundary. By including the Biokovo population in STRUCTURE analysis, it built a separated cluster. This divergence may be due to the historical founder effect and more recent genetic drift due to isolation, and local adaptation. Interestingly, we report a considerable genetic similarity between the Biokovo and Čvrsnica-Čabulja populations. This suggests that the Biokovo population may have multiple origins, not only from Prenj Mountain, as primarily assumed based on both available literature and personal communications.

Mitochondrial DNA (mitogenome) is a good genetic marker for genetic variability analysis and for inferring evolutionary patterns. Mitogenome comparisons are used as tools in modern taxonomic studies since the taxonomic relationships between animal taxa have been traditionally defined based on geography and morphology. Northern chamois (Rupicapra rupicapra) is a mountain ungulate that is currently categorized into seven subspecies, but some genetic studies contradict this taxonomy. Two subspecies of Northern chamois inhabit Croatia: Balkan (Rupicapra rupicapra balcanica) and Alpine chamois (Rupicapra rupicapra rupicapra). In this study, we report the assembly of a complete mitogenome sequence of the Balkan chamois. The new sequence was directly assembled from Illumina next- generation sequencing short reads that were aligned to a reference genome of Northern chamois since they share approximately 99.8% mitogenome similarity. The newly assembled mitogenome is 16 443 bp in length and contains 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and D-loop region. The mitogenome is AT- biased with a nucleotide composition of 33.5% A, 30.0% T, 23.2% C, and 13.4% G. The assembly of the complete mitogenome sequence provided here, along with mitogenomes of other Northern chamois subspecies, is the initial step in resolving the taxonomy of the Genus Rupicapra.

The chamois, genus Rupicapra, is the most abundant mountain-dwelling ungulate in Europe and the Middle East and is currently recognised to be divided into two species: Rupicapra rupicapra (Northern chamois) and Rupicapra pyrenaica (Southern chamois), which are further subdivided into seven and three subspecies respectively. Although currently classified as least-concern in the IUCN Red List of Threatened Species, information on the status of the species need updating. To further complicate the assessment, the genus has been subject of reintroductions and translocation, sometimes without prior knowledge on the taxonomic status of individuals, and there is a molecular controversy over the subspecies subdivision based on morphological and behavioural characters. We combined previously published (N=5) with newly assembled (N=9) mitogenomes to further investigate the phylogeny of the genus. To avoid erroneous hypotheses on homology we excluded all indels, ambiguous positions and the portion of the control region presenting tandem repeats in the Caprini. The final alignment consisted of 15348 bp, 14 Rupicapra sequences and two outgroups (Ammotragus lervia and Arabitragus jayakari). Both Bayesian and maximum likelihood analyses showed the same subdivision in Eastern, Central and Western clades previously reported. Within the Eastern clade R. r. balcanica was the most differentiated and included both sequences from the known hybrid population of the Velebit Mountains, whereas R. r. tatrica and R. r. rupicapra had a sister group relationship. Although we observed differences in variability among genes, our results confirm the reliability of previous studies based on a reduced number of mitochondrial genes.

The expression of sexually selected traits in highly dimorphic ungulates may be influenced by environmental quality. Variations in habitat conditions can impose different constraints on the allocation of energy resources to male life‐history traits, and possibly alter the female preferences for specific features. Here, we compared the horn growth patterns in male European mouflon Ovis aries musimon living in different habitats (Mediterranean vs. continental) but sharing a common genetic origin. We hypothesized that the expression of sexually selected traits such as horn development should be promoted in more favorable habitat conditions (i.e., Mediterranean). Using linear mixed models on data retrieved from individuals harvested under the same hunting regime, we found longer horns and greater individual variance in horn segment length in the Mediterranean population than in the continental one. Furthermore, Mediterranean rams showed no evidence of compensatory horn growth, as opposed to the continental rams. Unexpectedly, horn base circumference was greater in the continental habitat than in the Mediterranean one. The overall results suggest different patterns of investment in horns in the two populations, with seemingly stronger pressure and consequences of sexual selection on mouflon rams living in more favorable environments. Although the role of hunters' selectivity cannot be excluded a priori, our data suggest that the differences in the expression of sexually selected traits in our study populations may be influenced by environmental conditions. Because sexual selection can impose substantial fitness costs on individuals, further investigations on the trade‐offs between reproduction and survival would improve our understanding of the dynamics of mouflon populations living in different environmental conditions., The expression of sexually selected traits in highly dimorphic ungulates may be influenced by environmental quality. We compared the horn growth patterns in male European mouflon living in different habitats but sharing a common genetic origin. Our results suggest different patterns of investment in horns in two populations, suggesting that the differences in the expression of sexually selected traits may be influenced by environmental conditions.