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The systematic status of the controversial crustacean family Leptobathynellidae is investigated using molecular and morphological methods in this study. Partial sequences of the nuclear 18S gene are studied from 28 genera of Bathynellacea from several continents. The analysis includes some of the most plesiomorphic genera of the family Parabathynellidae, such as Billibathynella and Brevisomabathynella from Australia; Habrobathynella and Parvulobathynella from India; the diverse Iberobathynella; the cosmopolitan genus Hexabathynella; and representative genera of two subfamilies of Bathynellidae (Gallobathynellinae and Bathynellinae). We used a molecular approach to analyse the systematic relationships amongst 64 species from Europe, North America, Australia and Asia, and review the morphological characters relevant at the family level. The molecular phylogeny clearly shows the presence of three highly divergent clades that could represent the three families. This is the first molecular phylogenetic reconstruction of Bathynellacea that can be used to: (1) verify the validity of Leptobathynellidae, (2) explore the diversity of the families and (3) explore the phylogenetic relationships among families. We propose a plausible evolutionary scenario for the order Bathynellacea.

The presence and distribution of Rhyacophila fasciata Hagen 1859 in Europe were revised, based on bibliographic study, collection specimens, and new material collected in different countries. The status of formerly synonymized species, Rhyacophila ferruginea (Scopoli 1763) and Rhyacophila septentrionis McLachlan 1865 was also assessed. The type of R. ferruginea is missing, the taxon is still unidentified, and thus we propose Rhyacophila ferruginea as a nomen dubium. Morphological features and genetic evidence revealed that R. septentrionis differs from R. fasciata, so we propose to change its status to status resurrectus. We therefore include new descriptions of the different stages (larva, pupa, male, and female) of R. fasciata and of R. septentrionis, together with a molecular analysis based on mitochondrial cytochrome oxidase I (mtCOI) and ecological notes. The species R. coppai Oláh 2020 NEW SYNONYM and R. soreda Coppa & Oláh 2020 NEW SYNONYM are synonyms of R. sociata Navás 1916; the species R. kopasa Oláh & Coppa 2020 NEW SYNONYM and R. rova Oláh & Coppa 2020 NEW SYNONYM are synonyms of R. denticulata McLachlan 1879; the species R. matrensis Oláh & Szczęsny 2020 is probably a synonym of R. fasciata, so more study of this species is needed in order to confirm or deny that it is a valid species.

The morphology of all postembryonic stages (larva, pupa, male, and female) of Rhyacophila macedonica Karaouzas, Valladolid, & Ibrahimi n. sp. from Greece, Republic of North Macedonia, Kosovo and Serbia was examined. Morphological data were supplemented by a molecular analysis of the mitochondrial cytochrome C oxidase subunit I (COI) and compared with samples of the nominate species Rhyacophila fasciata Hagen 1859, as well as with other species and subspecies in this group: Rhyacophila septentrionis McLachlan 1865, Rhyacophila denticulata McLachlan 1879, Rhyacophila sociata Navás 1916, Rhyacophila kykladica Malicky & Sipahiler 1993, Rhyacophila fasciata delici Kucinic & Valladolid 2020, and Rhyacophila fasciata viteceki Valladolid & Kucinic 2020. Our results revealed morphological differences between the nominate species and R. macedonica, as well as genetic differences among the taxa in the Rhyacophila fasciata Group, so we propose R. macedonica as a new species of the Group. Based on the new molecular data, we also elevate R. f. delici to the status of a distinct species, R. delici (status promotus).

The “Iberobathynella group”, or Iberobathynellini tribe, is a complex of six genera consisting of 33 nominal species and several cryptic species with an amphiatlantic distribution (in Europe, North Africa and North America). A modern systematic revision of this group of subterranean crustaceans is presented here. A phylogenetic and biogeographic study using morphological and molecular data (mitocondrial coi and nuclear 18S) was carried out and allowed to a) re-evaluate the taxonomic status and validity of previously erected subtribes, genera and subgenera that show congruence in the data; b) assess whether the identified mitochondrial lineages represent cryptic species; c) provide a plausible phylogenetic hypothesis for the relationships within Iberobathynellini and with the other two genera of the family Parabathynellidae that inhabit North America and Europe (Montanabathynella and Parabathynella, respectively); d) propose a plausible temporal and historical framework (paleobiogeographic scenario) for the diversification and evolution of the Iberobathynellini tribe based on the current distribution of morphotypes and their estimated times of divergence. Our results show that in parabathynellids, molecular and morphological divergences are not always congruent. Subtribe and subgenus are invalid categories so they must be eliminated. Paraiberobathynella genus needs to be revisited. The molecular dating results support the early divergence of the Iberobathynellini Tribe (Upper Cretaceous, around 78 Mya) and the vicariance by plate tectonics as main factor to explain the amphi-Atlantic distribution shown by this ancient subterranean crustacean group. Since there are species morphologically very similar to I. magna and I. imuniensis, but genetically different, we can ensure the existence of at least three cryptic species. Texanobathynella is undoubtedly a valid genus distinct from Iberobathynella. Montanabathynella and Parabathynella are two well-differentiated genera closely related to the Iberobathynellini tribe.