Hyporheic interstitials are ecologically dynamic and often neglected aquatic environments. In this study, groundwater amphipods (Niphargidae, Pseudoniphargidae and Crangonyctidae) were sampled in hyporheic interstitials throughout Luxembourg and The Greater Region and specimens were analyzed by DNA barcoding. Sites characterized by gravel or coarse sand and high flow velocities of incoming water were the most species- and specimen-rich. A total of 11 species were detected, of which the Niphargus aquilex lineage EF of the N. aquilex cryptic species complex and juveniles of N. schellenbergi dominated the data set, but an unknown lineage of the N. aquilex-complex was also found. Some regions appeared to be devoid of groundwater amphipods. We hypothesize that underlying sandstone formations resulting in low sediment porosity may prevent physical colonization, but also that historical water pollution may have a long-lasting effect, either through the persistence of contaminants in the sediment or low recolonization rates of affected populations. In summary, our approach expanded regional species inventories, confirmed known occurrences, and validated previously questionable or historical morphology-based detections. In addition, the collection of absence data provided valuable insights into local extinctions. Finally, DNA-based distribution data are needed to gather information on the ecological affinities of groundwater amphipods to understudied hyporheic interstitial environments.

The genus Microniphargus (Crustacea, Amphipoda): evidence for three lineages distributed across northwestern Europe and transfer from Niphargidae to Pseudoniphargidae

Niphargus amphipods were collected from 2007 to 2018 at 98 sites comprising artificial caverns, springs and interstitial waters in the Grand Duchy of Luxembourg. Opportunistic sampling was combined with passive trapping. Specimen identification was achieved using morphological keys and molecular data. Initial morphological determination and literature data suggested five species, whereas sequencing of fragments of the mitochondrial cytochrome c oxidase subunit 1 gene and nuclear 28S rDNA marker supported the presence of seven species: Niphargus schellenbergi, Niphargus puteanus, Niphargus fontanus, one species of the Niphargus kochianus complex, and three species of the Niphargus aquilex complex. Niphargus schellenbergi was by far the most abundant and widespread species. Limited overlap was observed between literature-based records, our initial morphological determinations based on classical taxonomic characters, and genetic sequence data. In general, the combination of phenotypically variable taxa, such as N. schellenbergi, and cryptic or near-cryptic species, as in the N. aquilex complex, renders morphological identification of niphargids from Luxembourg a challenging or even impossible task. DNA taxonomy will therefore have to be used in future studies of the fauna of this region.

Niphargus enslini Karaman, 1932 was collected only once in 1905 from the Falkensteiner Höhle (Baden-Württemberg, Germany). Two years after its description, the species was synonymized with Niphargus virei and not studied any more. During recent surveys on German niphargids, further samples collected in this cave did not yield N. enslini specimens, but this species was collected in the Blätterteighöhle and in the Schwarzer Brunnen, two caves located in Baden-Württemberg and intercepting the same karstic aquifer feeding Falkensteiner Höhle. In an integrative taxonomic approach, we carefully studied the morphology of the newly collected specimens and sequenced two molecular markers (fragments of the cytochrome c oxidase subunit I (COI) and of the nuclear 28S rRNA gene) to test for possible conspecificity of N. enslini with N. virei. Morphological analysis confirmed that N. enslini is distinct from the N. virei species complex. We provide a redescription of newly collected material, together with new drawings of a more than 100 years old topotypic female. We briefly discuss the putative origin of N. enslini and the age of its split from the N. virei species complex.