Utilising public sequence databases to investigate genetic diversity of stoneflies in Medvednica Nature Park.

In Medvednica Nature Park, near Croatia's capital Zagreb, urbanisation significantly impacts the fauna. Comprehensive field research has never been conducted in this area, despite the presence of diverse microhabitats and the discovery of several rare species previously unknown in the Croatian fauna. This study provides the Park with first insight into the genetic and morphological diversity of stoneflies, one of the most endangered groups of organisms. Phylogenetic reconstructions and species delineation methods revealed intraspecific haplotype variation in most species (e.g. Brachypteraseticornis, Isoperlagrammatica and Leuctrabraueri), except for Leuctraprima. Additionally, our study has identified isolated populations that merit further in-depth investigation concerning morphology, genetics and ecology.

Molecular analyses of pseudoscorpions in a subterranean biodiversity hotspot reveal cryptic diversity and microendemism.

Nested within the Mediterranean biodiversity hotspot, the Dinaric Karst of the western Balkans is one of the world's most heterogeneous subterranean ecosystems and renowned for its highly diverse and mostly endemic fauna. The evolutionary processes leading to both endemism and diversity remain insufficiently understood, and large-scale analyses on taxa that are abundant in both subterranean and surface habitats remain infrequent. Here, we provide the first comprehensive molecular study on Croatian pseudoscorpions, a lineage of arachnids that is common and diverse in both habitats. Phylogenetic reconstructions using 499 COI sequences derived from 128 morphospecies collected across the Dinaric Karst show that: (i) occurrence in karstic microhabitats boosters speciation and endemism in the most diverse genera Chthonius C.L. Koch, 1843 (37 morphospecies) and Neobisium Chamberlin, 1930 (34 morphospecies), (ii) evidence for ongoing diversification is found in many species and species complexes through low optimal thresholds (OTs) and species delineation analyses, and (iii) landscape features, such as mountain ranges, correlate with patterns of genetic diversity in the diverse genus Neobisium. We present two synonymies: Protoneobisium Curcic, 1988 = Neobisium, syn. nov., and Archaeoroncus Curcic and Rada, 2012 = Roncus L. Koch, 1873, syn. nov. Overall, our study suggests that karstic microhabitats promote diversification in soil- and cave-dwelling arthropods at all taxonomic levels, but also provide important refugia for invertebrates in past and present periods of environmental change.

DNA barcoding for biodiversity assessment: Croatian stoneflies (Insecta: Plecoptera).

Background: The hemi-metabolous aquatic order Plecoptera (stoneflies) constitutes an indispensable part of terrestrial and aquatic food webs due to their specific life cycle and habitat requirements. Stoneflies are considered one of the most sensitive groups to environmental changes in freshwater ecosystems and anthropogenic changes have caused range contraction of many species. Given the critical threat to stoneflies, the study of their distribution, morphological variability and genetic diversity should be one of the priorities in conservation biology. However, some aspects about stoneflies, especially a fully resolved phylogeny and their patterns of distribution are not well known. A study that includes comprehensive field research and combines morphological and molecular identification of stoneflies has not been conducted in Croatia so far. Thus, the major aim of this study was to regenerate a comprehensive and taxonomically well-curated DNA barcode database for Croatian stoneflies, to highlight the morphological variability obtained for several species and to elucidate results in light of recent taxonomy. Methods: A morphological examination of adult specimens was made using basic characteristics for distinguishing species: terminalia in males and females, head and pronotum patterns, penial morphology, and egg structures. DNA barcoding was applied to many specimens to help circumscribe known species, identify cryptic or yet undescribed species, and to construct a preliminary phylogeny for Croatian stoneflies. Results: Sequences (658 bp in length) of 74 morphospecies from all families present in Croatia were recovered from 87% of the analysed specimens (355 of 410), with one partial sequence of 605 bp in length for Capnopsis schilleri balcanica Zwick, 1984. A total of 84% morphological species could be unambiguously identified using COI sequences. Species delineation methods confirmed the existence of five deeply divergent genetic lineages, with monophyletic origin, which also differ morphologically from their congeners and represent distinct entities. BIN (Barcode Index Number) assignment and species delineation methods clustered COI sequences into different numbers of operational taxonomic units (OTUs). ASAP delimited 76 putative species and achieved a maximum match score with morphology (97%). ABGD resulted in 62 and mPTP in 61 OTUs, indicating a more conservative approach. Most BINs were congruent with traditionally recognized species. Deep intraspecific genetic divergences in some clades highlighted the need for taxonomic revision in several species-complexes and species-groups. Research has yielded the first molecular characterization of nine species, with most having restricted distributions and confirmed the existence of several species which had been declared extinct regionally.

Potamophylax idliri sp. nov. (Trichoptera: Limnephilidae), a new species from the Jastrebac Mountains in Serbia, with molecular and ecological notes.

In this paper, we describe a new species, Potamophylax idliri sp. nov., from the Jastrebac Mountains in Serbia, which is morphologically most similar to Potamophylax coronavirus Ibrahimi, Bilalli Vitecek 2021 (in Ibrahimi et al. 2021) and Potamophylax juliani Kumanski 1999 (in Kumanski Malicky 1999). The males of the new species differ from all known species of the Potamophylax winneguthi Species Group by morphological characters of the male genitalia. The genetic distinctiveness calculated based on sequencing of the barcode region of the cytochrome c oxidase subunit I gene (COI) between Potamophylax idliri sp. nov. and other species of the Potamophylax winneguthi Species Group is on par with those of other morphologically recognized species in the group and especially the other four species of the P. winneguthi Species Cluster. Potamophylax winneguthi Species Cluster is erected for the following species: P. idliri sp. nov., P. winneguthi, P. haidukorum Malicky 1999 (in Kumanski Malicky 1999), P. juliani, and P. coronavirus, characterized primarily by the bilobed apical margins of inferior appendages with pointed corners, in lateral and caudal views. The new species was found at two localities in the Jastrebac Mountains, at 988 and 891 m above sea level. Considering the knowledge about distribution and ecology of the other species of this group, we posit that P. idliri sp. nov. is a microendemic of the Jastrebac Mountains in Serbia.

Morphological and molecular characterisation of the Popijac's Yellow Sally, Isoperlapopijaci sp. nov., a new stenoendemic stonefly species from Croatia (Plecoptera, Perlodidae).

A new species of the Yellow Sally genus (Isoperla Banks, 1906) is described, based on morphological (males and females adults, larval and egg) and molecular (the barcode region of the cytochrome c oxidase subunit I gene (COI)) features. Popijac's Yellow Sally, I.popijaci Hlebec & Sivec, sp. nov. inhabits two karstic sources of the Krasulja rivulet in Croatia. Male and female of the new species are characterised by colouration patterns of the head and pronotum; the dimensions of the female subgenital plate; the medial penial armature and oval-shaped egg without collar and anchor. The larvae differ from their congeners by the uniquely coloured head and pronotum. Based on morphological characteristics I.popijaci sp. nov. belongs to the I.tripartita species group. Phylogenetic and taxonomic relationships were reconstructed using three methods of phylogenetic inference and three species delimitation methods. As I.popijaci sp. nov. occurs at a narrow area of the Krasulja rivulet in Krbava field, the study puts emphasis on the conservation and hotspot importance of the temporary rivers in the Dinaric karst. Furthermore, the study accentuates the necessity for further research on the genetic diversity of Plecoptera in Croatia.

Mineral Composition of Elements in Walnuts and Walnut Oils.

Walnuts (Juglans nigra) are considered to be a functional food. In the present study, twenty one macro-, micro-, and trace elements (Al, As, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Mo, Na, Ni, Pb, Se, Sr, and Zn) were selected to be determined in walnuts and walnut oils. The beneficial content of essential elements could be shown by the obtained results, the macro-elements Ca, K, Mg, and Na being present in nuts with 1062 mg/kg, 2771 mg/kg, 1426 mg/kg, and 42.3 mg/kg, respectively. Regarding micro- and trace elements, the following order (decreasing content) was found: Mn > Fe > Zn > B, Mo > Cu > Ni > Co > Al > Sr > Ba > Li > Pb > Se > Cr > As > Cd. Furthermore, the potentially toxic trace elements (As, Cd, Pb) determined were below the maximum allowed values in most of the investigated walnut samples. A comparison of oils and nuts revealed that the former contain lower concentrations of all elements analyzed except for Fe and Zn. This suggested the origin from contamination during oil processing. Fe influences the oil oxidation rate, thus its entry during production should be avoided.