Collembola is one of the major functional groups in soil as well as a model taxon in numerous disciplines. Therefore the accurate identification of specimens is critical, but could be jeopardised by cases of cryptic diversity. Several populations of six well characterised species of springtails were sequenced using the COI barcode fragment as a contribution to the global Collembola barcoding campaign. Each species showed high intraspecific divergence, comparable to interspecific sequence divergence values observed in previous studies and in 10 congeneric species barcoded here as a reference. The nuclear marker, 28S, confirmed all the intraspecific lineages found with COI, supporting the potential specific status of these entities. The implications of this finding for taxonomy and for disciplines relying on species names, such as evolution and ecology, are discussed.

Parisotoma notabilis is the most common species of Collembola in Europe and is currently designated as ubiquist. This species has been extensively used in numerous studies and is considered as well characterized on a morphological ground. Despite the homogeneity of its morphology, the sequencing of the barcoding fragment (5′ end of COI) for several populations throughout Europe and North America revealed four distinct genetic lineages. The divergence found between these lineages was similar to the genetic distance among other species of the genus Parisotoma included in the analysis. All four lineages have been confirmed by the nuclear gene 28S. This congruence between mitochondrial and nuclear signals, as well as the geographical distribution pattern of lineages observed in Europe, supports the potential specific status of these lineages. Based on specimens from the type locality (Hamburg), the species name was successfully assigned to one of these lineages. This finding raises several problems as Parisotoma notabilis has been widely used in many ecological studies. Accumulation of new data for the different lineages detected, especially ecological information and life history traits, is needed to help resolve this situation.

The species Heteromurus (Verhoeffiella) absoloni Kseneman, 1938 is redescribed in detail and characterized by its barcode, based on specimens from its type locality in Montenegro. A neotype is designated. Dorsal S-chaetotaxy is given for the first time in the subgenus Verhoeffiella. Chaeta morphology and distribution are thoroughly analyzed, in particular on antennae where 12 chaetal types are recognized. Several morphological features are newly described for the genus and for Heteromurinae. The widely disjunct distribution of the species is approached through morphological and molecular comparison of specimens from the type locality in Montenegro and from the Catalan population. We established that this last record is a new species described here as  Heteromurus (Verhoeffiella) gamae sp. nov. New combination is proposed Heteromurus (Verhoeffiella) constantinellus (Ćurčić & Lučić in Lučić, Ćurčić & Mitić 2007) comb. nov. A table of all species of the subgenus is provided. The taxonomic status of Verhoeffiella and the problems of species discriminations in the subgenus are discussed.

DdPCR is becoming one of the most widely used tool in the field of eDNA-based aquatic monitoring. Although emulsion PCR used in ddPCR confers a partial mitigation to inhibition due to the high number of reactions for a single sample (between 10K and 20K), it is not impervious to it. Our results showed that inhibition impacts the amplitude of fluorescence in positive droplets with a different intensity among rivers. This signal fluctuation could jeopardize the use of a shared threshold among samples from different origin, and thus the accurate assignment of the positive droplets which is particularly important for low concentration samples such as eDNA ones: amplification events are scarce, thus their objective discrimination as positive is crucial. Another issue, related to target low concentration, is the artifactual generation of high fluorescence droplets (‘stars’). Indeed, these could be counted as positive with a single threshold solution, which in turn could produce false positive and incorrect target concentration assessments. Approximating the positive and negative droplets distribution as normal, we proposed here a double threshold method accounting for both high fluorescence droplets (‘stars’) and PCR inhibition impact in delineating positive droplets clouds. In the context of low concentration template recovered from environmental samples, the application of this method of double threshold establishment could allow for a consistent sorting of the positive and negative droplets throughout ddPCR data generated from samples with varying levels of inhibitor contents. Due to low concentrations template and inhibition effects, Quantasoft software produced an important number of false negatives and positive comparatively to the double threshold method developed here. This case study allowed the detection of the invasive crayfish P. leniusculus in 32 out of 34 sampled sites from two main rivers (Alzette and Sûre) and five of their tributaries (Eisch, Attert, Mamer, Wiltz and Clerve).