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.

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.

ABSTRACT Six blood groups (Rh, MN, Duffy, Kidd, Kell, and Lutheran) were investigated among three major ethnic groups (Bosniaks, Bosnian Croats, and Bosnian Serbs), as well as 10 regional subpopulations across Bosnia and Herzegovina (B&H): Krajina; Posavina; northeastern, eastern, middle, and central Bosnia; Sarajevo region; eastern, central, and western Herzegovina. This is the first study that introduces the molecular genetic typing of five blood groups within the B&H population, with the exception of the RhD blood group. The sample consisted of 450 buccal swabs from unrelated individuals. Five blood group systems (RhD, RhC, RhE, Kidd, MN) were genotyped by PCR with sequence specific primers, while three blood group systems (Kell, Duffy, Lutheran) were genotyped by the PCR-restriction-fragment-length polymorphism method. Minor variation of genetic diversity was observed within the three major B&H ethnic groups, as well as within the 10 subpopulations stratified according to geographical criteria. No genetic differentiation among ethnic groups was noticed. These results are in agreement with the results of previous studies based on different molecular genetics markers, which indicate that the three B&H ethnic groups belong to the same gene pool. A similar level of genetic variance was observed within regional subpopulations, with no significant genetic differentiation among them. Comparison of intrapopulation genetic diversity of the B&H population with other European and non-European populations, based on three loci (RHD, MN, and KEL), clearly show that the level of genetic diversity of the B&H population is within the European range.

Six blood groups (Rh, MN, Duffy, Kidd, Kell, and Lutheran) were investigated among three major ethnic groups (Bosniaks, Bosnian Croats, and Bosnian Serbs), as well as 10 regional subpopulations across Bosnia and Herzegovina (B&H): Krajina; Posavina; northeastern, eastern, middle, and central Bosnia; Sarajevo region; eastern, central, and western Herzegovina. This is the first study that introduces the molecular genetic typing of five blood groups within the B&H population, with the exception of the RhD blood group. The sample consisted of 450 buccal swabs from unrelated individuals. Five blood group systems (RhD, RhC, RhE, Kidd, MN) were genotyped by PCR with sequence specific primers, while three blood group systems (Kell, Duffy, Lutheran) were genotyped by the PCR-restriction-fragment-length polymorphism method. Minor variation of genetic diversity was observed within the three major B&H ethnic groups, as well as within the 10 subpopulations stratified according to geographical criteria. No genetic differentiation among ethnic groups was noticed. These results are in agreement with the results of previous studies based on different molecular genetics markers, which indicate that the three B&H ethnic groups belong to the same gene pool. A similar level of genetic variance was observed within regional subpopulations, with no significant genetic differentiation among them. Comparison of intrapopulation genetic diversity of the B&H population with other European and non-European populations, based on three loci (RHD, MN, and KEL), clearly show that the level of genetic diversity of the B&H population is within the European range.

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.

European plum cultivars (Prunus domestica L.) are hexaploid and partially self-fertile or self-sterile requiring compatible pollinizers with overlapping bloom times. Therefore, inter-planting of different pollinizer cultivars is recommended. In order to identify successful pollinizers of the plum cultivars ‘Edda’, ‘Opal’ (self-fertile), ‘Jubileum’, ‘Reeves’, ‘Mallard’, ‘Avalon’, ‘Cacanska Lepotica’ (self-fertile), and ‘Valor’, 60 fruits per cultivar were collected from nine orchards in 2017 and 2018, all of which were located in Ullensvang, western Norway. DNA extraction was subsequently conducted from the obtained embryos, followed by genetic characterization using seven microsatellite markers. Tissue samples from all possible pollinizers were collected during the summer of 2017 and the same DNA approach was conducted. Results showed that ‘Opal’ was the most successful pollinizer among the investigated plum cultivars. The main exception was ‘Cacanska Lepotica’, which consistently displayed very high level of self-pollination. The most successful foreign pollinizer of ‘Opal’ was ‘Mallard’. However, in more than two thirds of embryos extracted from ‘Opal’ fruits self-fertilization was determined. ‘Reeves’ was identified as the most successful pollinizer among embryos collected from ‘Valor’. Among the five cultivars (‘Edda’, ‘Jubileum’, ‘Reeves’, ‘Mallard’, and ‘Avalon’) that did not display self-pollination, the pollinizer success rate of ‘Opal’, ranged from 36.5% (‘Mallard’) to 93.5% (‘Edda’) in 2017, while in 2018 this rate ranged from 43.5% (‘Jubileum’ and ‘Reeves’) up to 96.5% (‘Edda’). Overall, genotyping embryos using SSRs (simple sequence repeats) proved an effective method in determining the success rate of individual pollinizers among European plum cultivars.

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Abstract This study offers the first report on variation sequence of the mitochondrial cytochrome b (MT-CYTB) gene in populations from Bosnia (northeastern Bosnia). This study was designed on the analysis of the genetic diversity of two populations of different cultural-anthropological and genetic origin, Roma population and native/non-Roma population. The main aim of our study was to estimate the usefulness of the CYTB sequence in the analysis of genetic categorization of different populations and intergroup diversity, as well as to provide some additional information on haplogroup-associated polymorphisms within the CYTB region in defining haplogroup status. Estimation of the genetic diversity was done using intra and intergroup genetic indices. The population-specific polymorphisms have been found in both categories of the populations. The results of the analysis of genetic differentiation show significant pairwise Fst differences between the Romani and native populations. Also, registered significant genetic differentiation is illustrated on the level of genetic variation between subpopulations of the Roma and non-Roma origin. The important result in our study is the confirmation of the significance of the triad of polymorphisms T14783C-G15043A-G15301A, indicating the influence of Asian component of the maternal gene pool on the genetic structure of the studied population of the Roma. Our data show that the haplogroup polymorphisms exist in the CYTB region and can provide useful information on the haplogroups that were defined only by the control region of the mtDNA. The results of this study indicate the region of CYTB gene can be a benefit in providing some additional information in the analysis of genetic structure of human populations and can be additionally applied in population studies.

Of the four species of the genus Satureja (Lamiaceae) that are recognized in Bosnia and Herzegovina, S. subspicata has the the widest distribution. It is taxonomically challenging species of geographically limited distribution and little data on its genetic diversity throughout its range is available. We sampled six geographically distinct populations from Bosnia and Herzegovina and applied nrDNA (ITS1, ITS2), chloroplast markers (matK and trnL) and AFLP to examine genetic diversity of S. subspicata in the center of its distribution range and to explore the possibility of establishing the species DNA barcode. AFLP analysis showed large genetic differentiation among populations as well as moderate correlation between genetic distance among populations and geographic distance among locations. MatK has not proven useful in distinguishing S. subspicata from sympatric species. However, nrDNA sequences provided necessary resolution power, with ITS2 being more informative. Estimates of evolutionary divergence between nrDNA sequences obtained in our research and homologous sequences of sympatric Satureja deposited in the GenBank reveal closer relationship between geographically proximate populations of different species and slight divergence within S. subspicata sequences pool. This outcome highlights the importance of considering overall genetic diversity across the distribution range of a species when assigning DNA barcode.

Apart from its physiological role in the cellular oxidation of ethanol interesting feature of the ADH1B gene locus is its characteristic geographical distribution in which certain variants of ADH1B peak in different parts of the world.  Therefore, ADH1B rs2066701 polymorphism is exploited as a genetic marker in tracing of the evolutionary processes and human migrations in the past. Taking into consideration the complexity of population genetic structure and several migrations in the history of the Balkan populations, including Bosnian and Herzegovinian, this study aimed to estimate the frequency of ADH1B rs2066701 polymorphism in the population of Bosnia and Herzegovina. The total of 101 randomly sampled individuals was genotyped for rs2066701 polymorphism in ADH1B gene using PCR-RFLP method. The obtained frequencies were used to calculate heterozygosity, fixation indices and Hardy-Weinberg equilibrium. Observed population-structure parameters were compared with other population values available in ALFRED database. Dimensional relations between the investigated populations were visualised with the NM-MDS (non metric multidimensional scaling) analysis using PAST. The minor allele frequency for rs2066701 was 0,257. Inter-population analysis including other European and non-European populations from the ALFRED database proved the above-mentioned European genetic background of the B&H population.

Rhyacophila Pictet 1834 is globally distributed and highly diverse genus of caddisflies (Trichoptera), characterized by numerous regionally endemic species. In the Balkan Peninsula, the highest number of Rhyacophila species (23) was recorded for Bosnia and Herzegovina. Rhyacophila bosnica Schmid, 1970 is found only in the Balkan Dinaric region, with a locus typicus in Vučja Luka, Bosnia and Herzegovina. Like with many species of Trichoptera, the morphology of its larva is still unknown. Therefore, DNA barcoding approach was used to link two developmental stages. In this paper, we report on the first DNA barcode record for this species.