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 Ćurčić, 1988 = Neobisium, syn. nov., and Archaeoroncus Ćurčić and Rađa, 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.

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.

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. Popijač’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. popijacisp. 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. popijacisp. 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.

Despite the recent advent of molecular data to assess the phylogeny of many invertebrate groups, the systematics of the pseudoscorpion family Chernetidae is unresolved, even though it comprises a quarter of the world’s generic pseudoscorpion diversity. We derive a preliminary molecular phylogeny of chernetids to assess subfamilial and generic monophyly using mitochondrial (COI) and nuclear (28S rRNA, 18S rRNA, and H3) markers. Three chernetid subfamilies have previously been recognized and Lamprochernetinae, originally defined based on T-shaped spermathecae, is recovered here but expanded to include the Old-World tropical genus Verrucachernes. In contrast, the genera Conicochernes and Calymmachernes of the subfamily Goniochernetinae are nested within the largest subfamily, Chernetinae. Three new subterranean species of the Palearctic genus Lasiochernes are also described from the Dinaric Karst: L. marinaeHlebec & Harvey, sp. nov., L. jalziciHlebec & Harvey, sp. nov., and L. pavlekaeHlebec & Harvey, sp. nov.. The former two species are single-cave endemics but L. pavlekae sp. nov. is more widespread. The relative lack of genetic structuring in this species, including haplotype sharing between sampling localities, together with a patchy distribution, suggests that its biogeography is probably shaped by multiple vector-mediated dispersal events, rather than geomorphological history. Due to their rarity, we discourage further collecting of Lasiochernes in this subterranean biodiversity hotspot.

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 forCapnopsis schilleri balcanicaZwick, 1984. A total of 84% morphological species could be unambiguously identified usingCOIsequences. 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 clusteredCOIsequences 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.

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.