Background: Patterns of biodiversity in the subterranean realm are typically different from those encountered on the Earth’s surface. The Dinaric karst of Croatia, Slovenia and Bosnia and Herzegovina is a global hotspot of subterranean biodiversity. How this was achieved and why this is so remain largely unresolved despite a long tradition of research. To obtain insights into the colonisation of the Dinaric Karst and the effects of the subterranean realm on its inhabitants, we studied the tertiary relictCongeria, a unique cave-dwelling bivalve (Dreissenidae), using a combination of biogeographical, molecular, morphological, and paleontological information.Results: Phylogenetic and molecular clock analyses using both nuclear and mitochondrial markers have shown that the survivingCongerialineage has actually split into three distinct species, i.e.,C. kusceri, C. jalzicisp. nov. andC. mulaomerovicisp. nov., by vicariant processes in the late Miocene and Pliocene. Despite millions of years of independent evolution, analyses have demonstrated a great deal of shell similarity between modernCongeriaspecies, although slight differences in hinge plate structure have enabled the description of the two new species. Ancestral plesiomorphic shell forms seem to have been conserved during the processes of cave colonisation and subsequent lineage isolation. In contrast, shell morphology is divergent within one of the lineages, probably due to microhabitat differences.Conclusions: Following the turbulent evolution of the Dreissenidae during the Tertiary and major radiations in Lake Pannon, species ofCongeriawent extinct. One lineage survived, however, by adopting a unique life history strategy that suited it to the underground environment. In light of our new data, an alternative scenario for its colonisation of the karst is proposed. The extantCongeriacomprises three sister species that, to date, have only been found to live in 15 caves in the Dinaric karst. Inter-specific morphological stasis and intra-specific ecophenotypic plasticity of the congerid shell demonstrate the contrasting ways in which evolution in the underground environments shapes its inhabitants.

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.

After being presumed extinct, the blind mole rat was rediscovered in Vučedol, Croatia. Phylogenetic analyses using mitochondrial markers (cytochrome b and 16S rRNA) were conducted to classify the population. The results confirmed a distinct separation between the Lesser mole rats (Nannospalax leucodon clade) and Western mole rats (N. monticola clade). Within the N. monticola complex, six lineages were identified, which likely diverged during the Middle to Late Pleistocene. The Vučedol sample belongs to the montanosyrmiensis cytotype subclade, which was also found in Fruška Gora, Serbia. This subclade may represent a new species, showing K2P genetic distances of 4.3% to 5.7% from the "Pannonian Plain" subclade located on both banks of the Danube River. In two Pannonian localities, secondary contacts between populations of both subclades have been documented. One of them is the Kelebia –Subotička peščara population, where a unique subclade "Kelebia" was identified. Populations with montanoserbicus cytotype cluster within two distinct subclades, montanoserbicus A and montanoserbicus B, with K2P distances ranging from 4.6% to 5.3%, indicating potential species status for montanoserbicus A. In the most parsimonious network, montanoserbicus B exhibits a close relationship with the Pannonian Plain sublineage and a single sample from Bosnia and Herzegovina, showing K2P distances of 1.9% to 3.2%, suggesting that they may belong to the same species, N. monticola. Significant divergence between populations in Bosnia and Herzegovina highlights the need for further exploration of local variability and taxonomic status. Detailed analyses with additional markers from more localities are necessary before final species delimitation and taxonomic revision can occur. For now, Nannospalax monticola (Nehring, 1898) remains the sole valid name in the N. monticola complex.

The systematics and taxonomy of the scorpion family Euscorpiidae are still unresolved, and, within it, the eastern Adriatic scorpiofauna is largely unknown and under-researched. Based on two mitochondrial sequences (COI and 16S rRNA) and one nuclear marker sequence (ITS1), we put 107 newly analyzed samples originating from the Alps, the Eastern Adriatic, and the adjacent Dinaric karst area into phylogenetic context. Several species delineation approaches were applied to reveal cryptic diversity. Divergence time dating was used to highlight the major events in the evolutionary history of the genera Euscorpius and Alpiscorpius. The deep intraspecific genetic divergences observed in some clades warrant taxonomic revision of several taxa (Euscorpius tergestinus, Euscorpius hadzii, Euscorpius biokovensis, and Euscorpius (Alpiscorpius) croaticus). In this study, the population of E. hadzii from Lastovo Island (formerly Euscorpius carpaticus lagostae) is elevated to species level as Euscorpius lagostae Di Caporiacco, 1950, stat. nov. Euscorpius croaticus is moved to the genus Alpiscorpius as Alpiscorpius croaticus (Di Caporiacco, 1950) comb. nov. The distribution ranges of several species are revised, and based on the new data, a more detailed revision of species distribution is necessary. We attribute the major divergence events to the impact of the Middle Miocene Climate Transition, the Messinian Salinity Crisis, and the Middle Pleistocene Climate Transition. The observed patterns are therefore a direct consequence of the geological history and complex topography of the region, which provided numerous microhabitats, as well as of the Pleistocene microrefugia that enabled their persistence.

Alpiscorpius liburnicus sp. n. is described from Krk Island, Croatia, as the first cave dwelling scorpion species in the genus. Clear differences from the genetically closest epigean species were found in metasomal morphometry. Its delineation is supported phylogenetically based on mitochondrial (COI and 16S rRNA) and nuclear (ITS1) markers. In the barcode fragment of the mitochondrial cytochrome oxidase subunit 1 gene (COI), the uncorrected pairwise genetic distances to other known taxa in “Alpiscorpius croaticus group” range between 4.2 and 4.8 %. The first molecular evidence of the species’ presence on the coastal part of Mt. Velebit implies its potential distribution in the whole of the eastern Adriatic coastal area, roughly from Rijeka City area to the source of the Zrmanja River. The new species is sympatric with common epigean taxa of the “Euscorpius tergestinus group” and with the synanthropic E. italicus in Mediterranean habitats. The phylogenetic relationships within the “A. croaticus group” are estimated. Localities of epigean A. croaticus are restricted to the continental side of southern and southeastern parts of Mt. Velebit. There it is sympatric and syntopic with the smaller A. omega in karst beech forests, with an extrazonal enclave in the Velika Paklenica canyon area on the southern slopes of the mountain. The third taxon in the group is known at the moment from a single locality in the middle part of Mt. Velebit, where it is also syntopic with A. omega in a beech forest near limestone cliffs.