Bosnia and Herzegovina has valuable natural resources with a high percentage of endemic and autochthonous species (Kučinić et al. 2008, Đug and Drešković 2012). The freshwater fauna of Trichoptera in this area is under-investigated, with a lack of morphological description of different life stages and DNA barcode data. Public data show 58,993 barcode entries for Trichoptera in the Barcode of Life Data Systems (BOLD) submitted from 92 countries, and none from Bosnia and Herzegovina (B&H) (BOLD 2021). Previous research in Bosnia and Herzegovina has provided the first DNA barcode for the endemic species Rhyacophila bosnica, stored in GeneBank, under accession number MK211322 by a domestic institution (Kalamujić Stroil et al. 2018). A few DNA barcodes of adult individuals of Trichoptera from Bosnia and Herzegovina were found in BOLD. However, these specimens were collected on B&H territory, but analyzed, processed, and stored by foreign institutions. To change the current state of DNA barcoding of Trichoptera in Bosnia and Herzegovina, we aimed to employ this approach in investigating caddisflies in selected habitats in the Sarajevo Canton. Our fieldwork was done in all five protected areas (spring of the Bosna River, Bijambare, Trebević, Skakavac, and Bentbaša) in which larvae samples were collected according to ‡,§ ‡,§ ‡,§ ‡,§ ‡ © Destanović D et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. the AQEM sampling methodology. The standard animal DNA barcode was successfully obtained using degenerated primers LCO1490JJ and HCO2198-JJ (Astrin and Stüben 2008). Out of 684 collected individuals (313 Trebević, 130 spring of the Bosna River, 117 Bijambare, 71 Bentbaša, 53 Skakavac), a subset of specimens were sequenced. We uncovered 14 different taxa, 11 genera and six families (Limnephilidae, Glossosomatidae, Rhyacophilidae, Goeridae, Hydropsychidae, Polycentropodidae). The preliminary data of Trichoptera composition in the Sarajevo Canton indicated species richness. Based on our sequential data, a new subspecies was discovered in two investigated areas (Valladolid et al. 2020), proving that Trichoptera species diversity in our country is far from entirely uncovered. The benefit and power of the DNA barcoding approach are that it can pinpoint the areas of vast and unknown species diversity more economically, both financially and temporarily, than the morphological approach. Therefore, we believe that it is critical to support the development of DNA barcoding for the bioassessment of freshwater ecosystems in Bosnia and Herzegovina. Several problems prevented us from exploiting sequential data to the fullest. Despite a general notion among scientists that European Trichoptera species are well covered in the BOLD database, most of the sequences we obtained were absent from the database. Secondly, we recognized that morphological data about the larval developmental stage of B&H Trichoptera species are largely missing. The unified, updated, and complete data on this order of insects is urgently needed. However, insufficient financial support by governmental institutions and lack of systematic approach to barcoding the wildlife of Bosnia and Herzegovina hampers this process. Further attempts to collaborate with the stakeholders can be crucial with profound and substantial implications for biomonitoring of aquatic macroinvertebrates in general. New approaches, such as novel DNA barcoding-based methodology can fill an important gap in our knowledge of Balkan caddisflies haplotypes, lineages, and their diversification and distribution patterns.

Chaetopteryx villosa (Fabricius, 1798) is a caddisfly species distributed throughout Europe, except in the Balkan and Apennine Peninsula. However, phylogenetically close species belonging to the C. villosa group are widespread throughout entire Europe. Species of this group (C. villosa, C. gessneri, C. fusca, C. sahlbergi, C. atlantica, C. bosniaca, C. vulture, and C. trinacriae) have distinct distributions with some overlaps. Adult forms of these species are morphologically similar, whereas larval morphology is only known for some species. There are also indications of species hybridization (e.g., C. villosa x fusca). Presumably, the molecular approach for the species determination of this group would be highly beneficial. In the BOLD database, there are 154 specimens with COI-5P barcodes of C. villosa species. Out of the remaining species, C. sahlbergi has 27 specimens with a barcode, C. fusca 20, C. gessneri 5, C. bosniaca 5, and C. atlantica 1, whereas sequences from the species C. vulture and C. trinacriae are missing. Therefore, we tested the power of discrimination of the COI-5P marker in the C. villosa group, as the most common barcoding markers for species identification in animals. Only sequences from public records originating from experienced research groups or taxonomists and containing a specimen photograph were taken as input. A total of 75 ‡,§ ‡,§ ‡,§ ‡,§ ‡,§ © Destanović D et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. sequences from the BOLD database were obtained. Out of these sequences, 11 belonged to C. fusca, 5 to C. gessneri, 52 to C. villosa, 5 to C. bosniaca, and 2 to C. sahlbergi. For the generation of overview trees, COI-5P barcodes of Rhyacophila fasciata and Rh. nubila were used as outgroups. All sequences were trimmed at 5’ and 3’ ends, resulting in a final alignment length of 516 base pairs. Multiple sequence alignments and editing were done in the MEGA-X software. Analysis of nucleotide polymorphism was done in DNASP6 software. MEGA-X was used to calculate the pairwise distance and overall mean pdistance, and to construct the overview trees. Analysis of DNA polymorphism revealed 14 haplotypes of C. villosa, 3 haplotypes of C. fusca, 2 haplotypes of C. gessneri, and one for species C. bosniaca and C. sahlbergi. There were no significant interspecific and intraspecific differences among haplotypes based on pairwise distances. The p-distance between one of the haplotypes of C. fusca and C. villosa was 0.000, whereas the p-distance among haplotypes of C. villosa varied from 0.001 to about 0.055. The mean overall p-distance among haplotypes of all species equaled 0.03. No species-specific clusters were observed when phylogenetic trees were constructed except for C. gessneri, regardless of the method used (i.e., NJ, UPGMA, ML, ME, or MP). To minimize the possibility of species misidentification, we used only records submitted by NTNU-Norwegian University of Science and Technology (Norway), SNSB-Zoologische Staatssammlung Muenchen (Germany), Zoologisches Forschungsmuseum Alexander Koenig (Germany), University of Oulu, Zoological Museum (Finland), prof Hans Malicky and prof Mladen Kučinić. No records identified as hybrids were included in the analyses. With the exception of C. gessneri, COI-5P marker failed to separate the species of the C. villosa group. However, it is highly unlikely that poor species determination was the basis for such a result. To enable the comprehensive and unbiased evaluation of the relationships within this group, data coverage in BOLD database for most of the studied species should be enhanced, encompassing different geographical distribution of samples. Further studies are needed to detect the array of molecular markers suitable for the species delineation in a complex group such as C. villosa.

The diploid Celina/QTee® (‘Colorée de Juillet’ × ‘Williams’), one of the most promising pear cultivars developed by the Norwegian breeding program Graminor, was launched in 2010. In Norway, the flowering is medium to late, while the fruits ripen in the beginning of September. The fruits are attractive with an intense red blush (50%) on a green background. Although, ‘Celina’ is cultivated in the most climatically suitable regions for fruit cultivation, present in Norway, unfavorable environmental conditions for pear pollination can have a very negative effect on fruit set and consequent yield. The aim of this study was to determine the S-alleles of ‘Celina’, as well as its frequently used pollinizers, and, through paternity testing of ‘Celina’ seeds, give a recommendation regarding the most important pollinizers of this pear cultivar. In order to accomplish this, ‘Celina’ and its potential pollinizers were all S-genotyped. After harvest, seeds collected from ‘Celina’ fruit in 2017 and 2018 were genotyped using eleven microsatellite markers. Genomic DNA was also extracted from leaf material collected from ‘Celina’, as well as from five pear cultivars used as pollinizers in the three examined orchards, and analyzed using the same marker set. Subsequently a simple sequence repeat (SSR) database was constructed and used for gene assignment analyses with the aim of quantifying pollen donor contribution from individual pollinizers. The obtained results indicate that ‘Anna’, the only examined pollinizer that was fully cross-compatible with ‘Celina’, together with ‘Fritjof’, the genotype which had the highest flowering overlap with ‘Celina’, proved to be the most successful pollinizers across all seasons and orchards. Although both cultivars were ubiquitous in the examined orchards, either as planted trees or as branches introduced during the flowering period, they were the most abundant pollinizers in only one orchard each. It is therefore possible to conclude that pollinizer abundance has a secondary significance in pollinizer success within investigated ‘Celina’ orchards.

The main goal of any DNA extraction procedure is to ensure reliable and reproducible results in a simple, fast and inexpensive manner. When it comes to plant tissues, this goal is challenging to achieve due to the presence of a variety of metabolites that interfere with DNA during isolation and downstream analysis. In this study, we compared the efficiency of three methods for DNA extraction from plum kernels: 1) the standard CTAB Soltis method which is the most common protocol for DNA extraction from various plant tissues (seeds, young leafs, mature leafs, root); 2) CTAB-based method originally described for DNA isolation from medicinal plants with high levels of secondary metabolites; 3) and one of various commercially available kits. The usefulness of the obtained DNA was evaluated by SSR analysis with seven microsatellite markers. Although the latter two extraction protocols retrieved genomic DNA that gave positive PCR results, only DNA isolated by kit produced full SSR profile

UDK: 582.675.1:575(497.6) Helleborus multifidus Vis. is endemic Illyric-Adriatic species with distribution range in Italy, Slovenia, Croatia, Bosnia and Herzegovina, Montenegro and Albania. Although few studies reported different taxonomic categories for H. multifidus, this one is the first using molecular-genetic markers (trnL region and matK of chloroplast DNA and nuclear ITS1 and ITS2 region) for genetic characterization of H. multifidus presented at three localites in Bosnia and Herzegovina. The results revealed that PCR-RFLP on trnL intron was not informative for testing inter- or intrapopulation diversity. Contrary, analysis of matK, ITS1 and ITS2 sequences showed differences between populations from Trebinje region and Kupreško polje, pointing to the need to include additional analyses in order to confirm these findings.