Water diviners described: six new species of the Niphargus aquilex complex (Crustacea, Amphipoda)

1 European Journal of Taxonomy 1011: 1–79 https://doi.org/10.5852/ejt.2025.1011.3023 europeanjournaloftaxonomy.eu ISSN 2118-9773 2025 · Weber D. et al. This work is licensed under a Creative Commons Attribution License (CC BY 4.0) Received: 8 January 2025 • Accepted: 11 June 2025 • Published: 25 August 2025 Topic editor: Magalie Castelin • Section editor: Fabio Stoch • Desk editor: Kristiaan Hoedemakers Monograph urn:lsid:zoobank.org:pub:09569CAD-967D-482F-A675-B4BCB7723F6F Water diviners described: six new species of the Niphargus aquilex complex (Crustacea, Amphipoda) Dieter WEBER 1,*,† , Traian BRAD 2,*,† & Alexander M. WEIGAND 3 1 Senckenberg Deutsches Entomologisches Institut, Eberswalder Straße 90, 15374 Müncheberg, Germany. 1,3 Fondation faune-flore, 25 rue Munster, 2160 Luxembourg, Luxembourg. 2 Institutul de Speologie “Emil Racoviță”, Department of Cluj-Napoca, Str. Clinicilor Nr. 5–7, 400006 Cluj-Napoca, Romania. 3 Musée national d’histoire naturelle du Luxembourg, 25 rue Munster, 2160 Luxembourg, Luxembourg. * Corresponding authors: dieter[email protected], [email protected] 3 Email: Alexander.W[email protected] † These authors contributed equally to this work. Abstract. With the onset of molecular taxonomy, it has been recognized that groundwater crustaceans in the family Niphargidae Bousfield, 1977 (Crustacea, Amphipoda) can demonstrate either highly variable or static phenotypes. Thus, it is recommended that new species descriptions follow an integrative taxonomic approach, considering morphotaxonomic and molecular characteristics to establish new taxa. The morphospecies Niphargus aquilex Schiødte, 1855 has its main distribution area in Central Europe, spanning southern England, the northern half of France, the Benelux countries, Germany and the Czech Republic. Genetic analyses have shown that it comprises a highly diverse species complex, including amongst others the well-recognized morphospecies Niphargus schellenbergi S. Karaman, 1932. In this study, we describe six new species based on available adult males and females – Niphargus luxemburgensis Weber, Weigand & Brad sp. nov., N. palatinensis Weber & Brad sp. nov., N. normandiensis Weber & Brad sp. nov., N. wasgauensis Weber & Brad sp. nov., N. saraviensis Weber & Brad sp. nov., N. lotharingiensis Weber & Brad sp. nov. – taking a re-evaluated published genetic backbone as a further reference into account. The six newly described species appeared pseudocryptic, as minor morphological differences were noticed. A short discussion of the morphological and molecular delimitations is provided. Keywords. Cryptic diversity, Niphargus, integrative taxonomy, groundwater fauna, species complex. Weber D., Brad T. & Weigand A.M. 2025. Water diviners described: six new species of the Niphargus aquilex complex (Crustacea, Amphipoda). European Journal of Taxonomy 1011: 1–79. https://doi.org/10.5852/ejt.2025.1011.3023 European Journal of Taxonomy 1011: 1–79 (2025) 2 Introduction The family Niphargidae Bousfield, 1977 (Crustacea, Amphipoda) comprises more than 440 formally described species (Horton et al. 2023) found nearly all over Europe and parts of Asia, from Spain (Karaman 2017) to Iran (Esmaeili-Rineh et al. 2017). They are usually stygobionts, that is, adapted and most often restricted to subterranean life in groundwater and groundwater-dependent habitats (e.g., springs, interstitials). Species show typical troglomorphic characteristics by lacking eyes and body pigmentation, but bearing elongated appendages in most cases. While the majority of the species were described based on their morphology alone, it was revealed that Niphargidae harbors a plethora of cryptic or pseudocryptic species (Fišer & Zagmajster 2009; Delić et al. 2017; Eme et al. 2018), which can be attributed to widely stable or largely overlapping morphological characteristics. Describing new species by integrating DNA sequences has become a standard for nearly ten years in Niphargus taxonomy (Brad et al. 2015; Delić et al. 2017; Fišer et al. 2017, 2018). The Niphargus aquilex species complex (“aquilex” “water diviner” in Latin) includes multiple cryptic species, and can serve as a prime example to witness past and current taxonomic challenges (McInerney et al. 2014; Weber et al. 2023). Westwood (1853) was the first to recognize N. aquilex Schiødte, 1855 as a species new to science, although he assigned his material to the first recording of Niphargus stygius (Schiødte, 1847) in England. Niphargus aquilex was redescribed based on specimens from the neotype locality Crowborough (United Kingdom), which were deposited in the British Museum (Karaman 1980). Another taxon of the N. aquilex species complex, Niphargus schellenbergi S. Karaman, 1932, was first described from Gännsbrünnle, in the south of Sendelbach (Bavaria, Germany). In the same year, N. schellenbergi was redescribed as a subspecies of N. aquilex (Schellenberg 1933), whereas from 1972 onwards, it was again considered a species distinct from N. aquilex sensu stricto (Straškraba 1972). Niphargus schellenbergi was always regarded as a single taxonomic entity (either at species or subspecies level), but even prior the age of DNA sequencing it was suspected that N. aquilex might actually represent a species complex (Gerecke et al. 2005). Several subspecies of N. aquilex were described: N. a. vejdowskyi (Wrześniowski 1890; Schellenberg 1932), N. a. tauri (Schellenberg 1933), N. a. moldavicus (Dobreanu et al. 1953), N. a. dobati (Sket 1999). While the systematic position of the first is still uncertain, the other three taxa have been elevated to the species level (Fišer et al. 2009a). Their taxonomic position was corroborated by molecular analyses performed by McInerney et al. (2014), who delineated several genetic lineages within the N. aquilex species complex. Recently, Weber et al. (2023) provided a comprehensive update on the taxonomic situation within the N. aquilex species complex. By investigating the mitochondrial cytochrome c oxidase subunit I (COI) and nuclear 28S ribosomal RNA (28S rDNA) genes and by applying a set of different molecular delineation methods, the authors were able to conservatively define eight molecular entities under the taxonomic umbrella of N. aquilex (annotated as lineages A, B, NDJ, F, G, H, KL, I). Two taxa were identified in the morphospecies N. schellenbergi (R, S) (Weber et al. 2023). Group B thereby referred to N. aquilex sensu stricto, and group S to N. schellenbergi sensu stricto. The nomenclature of the molecular lineages used herein follows that of McInerney et al. (2014) and extended by Weber et al. (2023). We recommend that all subsequent studies report the annotations of taxonomically undescribed molecular lineages. In this study, and by referring to the molecular delimitation results of Weber et al. (2023) as a taxonomic backbone, we provide here an integrative taxonomic framework and the formal description of six of the molecularly identified species within the N. aquilex species complex, that is, group A, NDJ, F, G, I, and R sensu Weber et al. (2023). At the same time, because a much larger set of DNA sequences was available (Weber et al. 2022; Weber & Weigand 2023), which was partly enriched by sequences obtained during fieldwork conducted within the scope of this study, we have reviewed all species delimitation results of Weber et al. (2023), but limited our work to a minimum interpretation necessary for the formal description of these six new species. WEBER D. et al., Six new species of the Niphargus aquilex complex (Amphipoda) 3 We provide a list of all available genetic sequences stored in GenBank (Sayers et al. 2021) for reference purposes, referring to the N. aquilex and N. schellenbergi morphospecies (Supp. file 1). Furthermore, genetic sequences for several clades of the N. aquilex species complex were named after and published using different annotation systems. We provide a compilation of this historic referencing (Supp. file 2), thus allowing readers to compare our data with those in older publications. Material and methods Sampling In addition to the published data in GenBank (Weber et al. 2023; Weber & Weigand 2023), extensive sampling of Niphargus specimens was performed in many regions of Germany, Belgium, Luxembourg, and France. Sampling of springs was performed by sieving the sediment and debris present at the point where water emerges from the subsurface through various meshes. Here, specimens of Niphargus, which are washed out with the groundwater, hide and live among sediment particles, dead leaves, or other types of debris. When the current of the spring water is not very strong, specimens of Niphargus can migrate towards the surface and back, most probably for feeding (Kureck 1967; Husmann 1976). Sampling of Niphargus in caves and artificial cavities was performed by simple manual picking of specimens using tweezers or by placing baits with chicken liver for one day. The specimens were individually sampled in 2 mL vials containing 96% ethanol. To simplify retrieving the type localities in the future, photographs of each of the six type localities of the newly described species, as well as a photograph of the site close to the type locality where we collected N. schellenbergi, were made (Supp. file 3). To estimate the preferred habitat of the individual species, we categorized the collection sites into springs, large underground cavities, and interstitial areas (Supp. file 4) for all species where we had more than ten occurrences. Among the springs, we distinguished four groups (cf. Thienemann 1922): 1. A rheocrene spring had a localized outlet with a directly visible discharge. 2. A helocrene spring has several small outlets of groundwater that are often not directly recognizable; they are collected in a sump to form an effluent. 3. A limnocrene springs from the bottom of a pond, and the water flows over the water rim. 4. The term “basin” is not commonly used in the literature. Groundwater is often channeled artificially via a pipe into an artificial trough that has been investigated. Large underground cavities were divided into caves, which are of natural origin, mines, in which material was mined, wells, vertical pipes driven into the rock for water catchment, and souterrains, which refer to all other human-made objects not used for mining. Interstitial is a fine-grained system between gravels, where we only sampled interstitial habitats close to a river (Weber & Weigand 2023). DNA analysis DNA was extracted from one pereopod using the DNeasy Blood & Tissue Kit (Qiagen) or the NucleoSpin Tissue Kit (Macherey-Nagel, Düren, Germany), according to the manufacturer’s instructions. DNA isolates were stored at -20°C in the collections of the Evolutionary Biology and Ecology research unit of the Université libre de Bruxelles (Belgium), the Musée national d’histoire naturelle du Luxembourg (Luxembourg), and the Senckenberg German Entomological Institute (Germany). Polymerase chain reaction (PCR) was performed to amplify the standard barcoding fragment of the cytochrome c oxidase subunit 1 gene (COI) (Folmer et al. 1994) using the primer pair HCO2198 and LCO1490 (Folmer et al. 1994) or the primer pair HCO2198-JJ and LCO1490-JJ (Astrin & Stüben 2008) (10 pmol/μL), premixed at a ratio of 1 : 1. For COI, the PCR mix contained 1.5 μL DNA extract (with varying concentrations, not measured), 1 μL of primer mix, 5 µL of “DreamTaq DNA Polymerase” (Thermo Scientific, Macquarie Park, Australia) and 5 µL of ultrapure water. PCR cycling conditions were an initial 5 min denaturation step at 95°C, 38 cycles of 30 s of denaturation at 95°C, 90 s of annealing at 49°C, 60 s of extension at 72°C and a final elongation step of 30 min at 68°C. European Journal of Taxonomy 1011: 1–79 (2025) 4 A fragment of the nuclear 28S ribosomal RNA gene (28S rDNA) was analyzed using the PCR primers Niph15 and Niph16 (Verovnik et al. 2005). The PCR mixture for the 28S rDNA marker contained 2 μL of DNA extract (with varying concentration, not measured), 1 μL of each primer (10 pmol/μL), 0.2 μL of REDTaq polymerase (Sigma-Aldrich, St. Louis, MI, USA), 5 µL of REDTaq reaction buffer and 15.8 µL of ultrapure water. PCR cycling conditions for 28S rDNA were an initial 3-minute denaturation at 95°C, 56 cycles of 30 s of denaturation at 94°C, 60 s of annealing at 45°C, and 90 s of extension at 72°C. Bidirectional Sanger sequencing was performed at Genoscreen (Lille, France), Macrogen (Amsterdam and Maastricht, Amsterdam, The Netherlands), or at the Musée national d’histoire naturelle du Luxembourg. For COI, the same primers used for PCR amplification were used. Moreover, 28S rDNA was sequenced with three primers: Niph15 (Verovnik et al. 2005), alternatively Niph15i (Weber et al. 2023), Niph20, and Niph21 (Flot et al. 2010). The sequences were deposited in the NCBI GenBank database and the accession numbers are present successively in Supp. file 6 (COI) and Supp. file 9 (28S rRNA). A list of primers, together with their nucleotide sequence, for both PCR and sequencing is provided in Supp. file 5. Molecular species delimitation and diagnosis We used enriched molecular sequence data (COI and 28S rDNA) to re-evaluate the established molecular lineages within the N. aquilex species complex sensu Weber et al. (2023), which served as the taxonomic backbone for our integrative species descriptions using molecular and morphological data. Molecular sequence data were also analyzed to infer DNA characteristics (that is, individual pure diagnostic nucleotides or species-specific DNA sequence variants, that is, DNA barcodes or nuclear alleles), enabling a supporting differential molecular diagnosis of the delineated species in the N. aquilex species complex. On 26 August 2023, all available COI sequences for N. aquilex and N. schellenbergi were downloaded from Genbank, enriched by the dataset from Weber, Weigand & Weigand (unpublished) and our newly obtained sequences herein. Thus, the initial COI dataset thus contained 1284 entries. These entries corresponded to 1319 individual COI sequences collected from 894 sampling sites (Supp. file 6). This discrepancy in the number of COI sequences exists because McInerney et al. (2014) stored a single COI sequence in GenBank for specimens from different locations when their COI sequences were identical. All incomplete sequences (i.e., sequences missing nucleotides at one or both ends) were removed from dataset 1, as well as those that contained any ambiguous characters (e.g., a single ‘N’)), resulting in 1115 final COI sequences (dataset 2). Dataset 1 was used to generate distribution maps and establish a phylogenetic framework for the molecular identification of specimens missing in dataset 2. The phylogenetic tree hypothesis was constructed using MEGA-X (Kumar et al. 2018). Seven short sequences were removed from dataset 1 and a neighbor-joining tree with 1000 bootstrap replications was generated (Supp. file 7 with description of the method in Supp. file 8). Dataset 2 was used for the distance-based molecular species delimitation approach ASAP (assemble species by automatic partitioning) (Puillandre et al. 2021), referring to its online version (https://bioinfo.mnhn.fr/abi/public/asap/asapweb.html) and standard settings under the Jukes-Cantor (JC69) substitution model for computing genetic distances. We primarily used partition 1 for final species delimitation, but also compared these results with partitions 2 and 3. The data for the 28S rDNA dataset originated from multiple sources and comprised 404 sequences (referring to 415 specimens, since McInerney et al. (2014) deposited a single sequence for specimens with identical 28S rDNA sequences), all of which are provided in Supp. file 9, which comprises full 28S rDNA fragment available, 2 sequences (A in column N); only part of 28S rDNA fragment available, 78 sequences (B in column N); data from Weber et al. (2023), full 28S rDNA fragment and full COI available, 195 sequences (C in column N); new data within this study, full 28S rDNA fragment available, WEBER D. et al., Six new species of the Niphargus aquilex complex (Amphipoda) 5 138 sequences (D in column N); and new data within this study, only part of 28S rDNA fragment available, 2 sequences (E in column N). The COI dataset 2 was used to infer molecular diagnostic DNA characters and sequence variants for the delineated species. The’removedup’ function of the srnatoolbox webserver (https://arn.ugr.es/srnatoolbox/helper/removedup/; Aparicio-Puerta et al. 2022) was used to remove identical sequences (n = 674) in the COI alignment, resulting in 442 distinct COI sequence variants (= DNA barcodes). The alignment was manually inspected for pure diagnostic nucleotide positions (Jörger & Schrödl 2014), i.e., conserved nucleotides within a species, but with distinct character states in all sequence variants of all the other species. However, in addition to relying on (a few) individual diagnostic nucleotide positions, species-specific sets of diagnostic DNA barcodes are used in the differential diagnosis sections of the respective species. Additionally, the 28S rDNA dataset was investigated to identify diagnostic nuclear sequences (alleles) for the newly described species. Molecular diagnostic characteristics are provided in the diagnosis section of each species separately. Morphological analysis In the laboratory, one male and one female paratype were selected for further dissection, inspection, and description of the morphological characters. For inspection of the intraspecific morphological variability, we inspected 2–3 more paratypes for each species. The holotype and paratype specimens were not genetically analyzed but originated from a pool of specimens from a single locality, of which specimens have been genetically analyzed for their DNA sequence variants (COI and 28S). Special care was taken to ensure that the genetically analyzed specimens, the specimens selected for dissection, and the specimens deposited as intact holotypes and paratypes did not exhibit any morphological discrepancies. The specimens selected for morphological analysis were immersed in glycerol, dissected body appendages were mounted on slides in glycerol, and the slide cover glasses were sealed with varnish. The slides were inspected with an Olympus SZX16 stereo microscope equipped with an Olympus SC180 camera, and with an Olympus BX51 microscope equipped with an Olympus SC50 camera. The drawings were performed on printed photographs via manual inking and continuous inspection of the slides under the microscope. The terminology used for body parts and the choice of appendages considered for measurements are those previously described (Fišer et al. 2009b). The mandibular palp chaetotaxy nomenclature followed Karaman (1993). A table containing all measured appendages for all species described herein is presented in Supp. file 10. Repository material The specimens (types and paratypes), or microscope slides containing specimens appendages, used for species descriptions in this study were deposited in the Musée national d’histoire naturelle du Luxembourg (MNHNL) and in the Institutul de Speologie “Emil Racoviță” (ISER) in Cluj-Napoca, Romania. In the case of each described species, all specimens received an inventory number and are presented and explained in detail in the designated chapters in the results section below. Results Molecular species delimitation ASAP analysis of COI, partition 1 with recursion and an ASAP-Score of 4.0, identified 16 species; partition 2 with recursion and an ASAP-Score of 5.0, 21 species; and partition 3 with recursion and an ASAP-Score of 5.5, 23 species (Supp. file 6). ASAP analysis of 28S rDNA from a limited dataset resulted in the identification of 10 putative species (Weber et al. 2023). Species A, B, and I are undisputed in all methods. European Journal of Taxonomy 1011: 1–79 (2025) 6 The situation around species D + N is more complex. Eight specimens of the molecular entity D+N had identical 28S rDNA alleles, and therefore belonged to the same species. According to all models, it is undisputed that it is a separate species from all other species, but partitions 2 and 3 of the ASAP of COI define three species within the D+N complex, identical in partitions 2 and 3, and the 28S rDNA of all specimens under consideration are identical. Partition 1 of ASAP of COI of the species F + F’ assumes a single species. Only the less likely partitions 2 and 3 assume two separate species. 28S rDNA contains two heterozygous individuals that include both F and F’. According to all models, it is undisputed that species G is a separate species from all other species, but partitions 2 and 3 of the ASAP of COI define a single specimen as an additional separate species. Species R and S were identified as a single species by partition 1, as two separate species by partition 2, and as three different species by partition 3. The large number of 28S rDNA sequences, 29 for species R and 681 for species S, did not show any heterozygous individuals between the two putative species. In two cases, F+F’ for N. luxemburgensis sp. nov. and D+N for N. normandiensis sp. nov., molecular entities comprised two well-separated molecular clades each. For these two species, their respective molecular clades are annotated with different symbols but in the same species-specific color when presenting their distribution maps. Species descriptions In this chapter, six species of the N. aquilex species complex are formally described, and information on N. aquilex sensu stricto and N. schellenbergi sensu stricto is provided. Distribution maps were generated in QGIS ver. 3.34.8-Prizren (QGIS Development Team 2025) and Inkscape ver. 0.92 (2017), with colors corresponding to Weber et al. (2023) and follow https://www.w3schools.com/colors/colors_names.asp (Supp. file 2). We were able to define the type of habitat for 855 sampling sites (Supp. file 4), allowing for a rough inspection of the species’ preferred ecology. Class Malacostraca Latreille, 1802 Order Amphipoda Latreille, 1816 Family Niphargidae Bousfield, 1977 Genus Niphargus Schiødte, 1849 Niphargus aquilex Schiødte, 1855 Fig. 1 Remarks Weber et al. (2023) treated this species as N. aquilex B, which can be considered N. aquilex sensu stricto. The species was regularly found in nearly all types of biotopes, but surprisingly often in helocrene springs (Supp. file 4.1). It is widespread and common in Western Germany, with more scattered records in Luxembourg, Belgium, and along the Channel Coast in France and Great Britain (Fig. 1). A neotype locality was established by Karaman (1980) from Crowborough (United Kingdom) without mentioning precise coordinates. We sampled the region of Crowborough, and our specimens and sequences can therefore be considered to originate from the neotype locality. The drawing of Schiødte (1855) shows two of the characteristics of N. aquilex, that is, the single seta on the dactylus of the gnathopods and the rounded 3 rd epimeral plate. Karaman (1980) re-described N. aquilex in detail, but he did not take into consideration that N. aquilex might comprise a species WEBER D. et al., Six new species of the Niphargus aquilex complex (Amphipoda) 7 complex of morphological similar (pseudocryptic) or identical (cryptic) taxa. Karaman’s specimens are now > 40 years old, and we thus did not try to sequence them. Nevertheless, we obtained three freshly collected specimens from Crowborough and sequenced two COI and three 28S rDNA sequences. All the sequences corresponded to the former species B senus Weber et al. (2023), which was already delineated by McInerney et al. (2014). Niphargus schellenbergi Karaman, 1932 Fig. 2 Remarks Weber et al. (2023) treated this species as N. schellenbergi, which can be considered N. schellenbergi sensu stricto, given that we also included specimens very close to the type locality in our analyses. The species is widespread from Central to Western Germany, Luxembourg, Belgium (Wallonia), the Netherlands (Limburg), and Eastern France (Fig. 2). While in the first description it was assumed that N. schellenbergi mainly inhabits springs (Karaman 1932), we found it regularly in caves, artificial caverns, meadow drainages, springs, wells, and in the interstitial (Supp. file 4.2). Karaman (1932) set Fig. 1. Distribution of Niphargus aquilex Schiødte, 1855 sensu stricto, confirmed by molecular data. The neotype locality in Crowborough (United Kingdom) is marked by a yellow star, overlaying one site record. Very large freshwater bodies are indicated; country codes follow the ISO 3166 standard alpha-2 format. European Journal of Taxonomy 1011: 1–79 (2025) 8 the Gänsbrünnle in the South of Sendelbach (Bavaria, Germany) as type locality, referring to a well along the southern margin of its inferred distribution range in Central Germany, but he did not define any type material. We collected specimens in a spring very close to Gänsbrünnle (Supp. file 3.1). More southern distribution records, particularly in Central-East to South-Eastern France, most likely belong to the newly described sister species Niphargus lotharingiensis sp. nov. The drawing in Karaman (1932) shows well the characteristics of N. schellenbergi, i.e., the gnathopod II nearly in square-form with its 3 setae on its dactylus. The two drawings of the telson exhibit significant discrepancies and are of limited value for taxonomic determination. The same is true of the two drawings of uropod I, in which the outer ramus is longer in one instance and the inner ramus is longer in the other. A drawing of the entire specimen is missing. Fig. 2. Distribution of Niphargus schellenbergi S. Karaman, 1932 sensu stricto, confirmed by molecular data. The type locality at Gänsbrünnle (Germany) is marked by a yellow star, overlaying two site records. Very large freshwater bodies are indicated; country codes follow the ISO 3166 standard alpha-2 format. WEBER D. et al., Six new species of the Niphargus aquilex complex (Amphipoda) 9 Niphargus luxemburgensis Weber, Weigand & Brad sp. nov. urn:lsid:zoobank.org:act:1522BA40-BBC2-44B7-9E92-D0AE85085217 Figs 3–11 Diagnosis Medium-sized Niphargus species, poorly setose. Right posteroventral angle of epimeral plates. Outer 6 spines of maxilla I on outer lobe with 1 tooth each; 1 spine with several smaller teeth. Mandibular palp with small number (1–2) of B setae. Gnathopods with 1 seta along outer margin of dactylopodites. Pereiopod VII, the longest leg, almost half of total body length. Pleopods retinaculum with 2 hooks. Uropod I, longer exopodite. Uropod II, longer endopodite. Uropod III sexually dimorphic, exopodite elongated in males. Telson with 3 apical spines on each lobe. The COI marker shows two pure diagnostic sites at positions 55 (C) and 358 (C). Six 28S rDNA alleles are diagnostic (with 13 heterozygous specimens) as well as 79 COI barcodes. Etymology The species name derives from the Grand Duchy of Luxembourg, where it was first discovered. In Weber et al. (2023), this species was treated as N. aquilex F, F’. Material examined Holotype LUXEMBOURG • ♂; Girst, Gutland, basin of a spring in the center of the hamlet of Girsterklaus; 49.7847° N, 6.4989° E; 19 Mar. 2018; Dieter Weber leg.; kept intact in 96% ethanol; 180319-10; MNHNL130574. Paratypes LUXEMBOURG • 1 ♂; same collection data as for holotype; 30 Jan. 2018; dissected and appendages drawn; 180130-32; ISER microscope slide DW180130-32 • 1 ♀; same collection data as for preceding; dissected and appendages drawn; 180130-45; ISER microscope slide DW180130-45 • 1 ♂; same collection data as for holotype; 27 Mar. 2017; ISER DW170327-02 • 1 ♀; same collection data as for holotype; 19 Mar. 2018; ISER DW180319-09 • 1 ♀; same collection data as for holotype; 19 Mar. 2018; ISER DW180319-13 • 1 ♀; same collection data as for holotype; 27 Mar. 2017; ISER DW 170327-06; MNHNL130575. Molecular data COI and 28S rDNA sequences of specimens belonging to Niphargus luxemburgensis sp. nov. were deposited in GenBank. COI and 28S rRNA accession numbers are present in Supp. file 6 and Supp. file 9, respectively. Description (male paratype ISER DW180130-32) Measurements Total body length 7.7 mm (Fig. 3). Head Head (Fig. 3) 8.60% of total body length. Eyes and rostrum absent. Antennae Antenna I (Fig. 4A): with the main flagellum formed of 18 articles representing 41% of total body length. Peduncle length ⅓ of total length of antenna I. The accessory flagellum (Fig. 4B) biarticulated; proximal article slightly longer than first article of main flagellum; distal article ¼ of total length of accessory flagellum, with 2 apical setae of different lengths and 1 aesthetasc. Aesthetascs ⅓ of respective flagellum articles (Fig. 4C). European Journal of Taxonomy 1011: 1–79 (2025) 16 Uropods Uropod I (Fig. 10A): with two dorso-lateral rows of 4 spines on peduncle. Exopodite slightly longer than endopodite, and ratio of exopodite length : endopodite length 1.0 : 0.85. 1 strong spine at base of uropod I. Uropod II (Fig. 10B): with 1 dorsal spine and 3 apical spines. Endopodite longer than exopodite, endopodite length : exopodite length ratio 1.0 : 0.81, both rami with low number of spines. Uropod III (Fig. 10C): sexually differentiated, longer in males. Peduncle with 4 apical spiniform setae. Short endopodite about half the length of peduncle, with 2 apical setae. Proximal segment of exopodite longer than distal segment (ratio 1.0 : 0.77). Anterior margin of proximal segment of exopodite with 5 Fig. 8. Niphargus luxemburgensis Weber, Weigand & Brad sp. nov., paratype, ♂ (ISER DW180130-32). A. Pereopod V. B. Pereopod VI. C. Pereopod VII. WEBER D. et al., Six new species of the Niphargus aquilex complex (Amphipoda) 17 groups of 2–3 setae; posterior margin of exopodite with 3 groups of 1–2 setae; one group of 3 posteroapical setae. Distal segment of exopodite with 3 groups of 2 setae on anterior margin, 3 groups of 1–2 setae on posterior margin and 4 apical setae of different lengths. Telson Telson (Fig. 10E) nearly as wide as long (width : length ratio 0.95), with 1 subapical and 3 apical spines of different lengths on each lobe. Longest spine almost half of telson length. 2 thin setae of different lengths and plumose tip, 1 seta medially on 1 lobe. Sexual dimorphism The examined male and female are highly similar. Besides the usual sexual dimorphism (e.g., slightly smaller gnathopods, presence of oostegites, and slightly deeper coxal plates I–VI in females), in N. luxemburgensis sp. nov., female uropod III (Fig. 10D) is shorter compared to that of male. Female telson similar to that of the male, but smaller (Fig. 10F). Intraspecific and interspecific variability The intraspecific morphological variability of N. luxemburgensis sp. nov. appears to be very low. All morphological characters were nearly identical in all inspected specimens (data not shown). Type locality, ecology and distribution The type locality is the basin of a spring in the center of the hamlet of Girsterklaus (community of Girst, Gutland, Luxembourg) at 49.7847° N, 6.4989° E (Supp. file 3.2 & 3.3). The artificial basin is made of sandstone. The water contains algae; the bottom of the basin is covered with mud. Specimens of Niphargus luxemburgensis sp. nov. were found in the mud as well as among the algae. The water has a hardness of 15°GH (where one degree equals to 17.9 mg/l CaCO 3 ) and a slightly acidic pH of 6.7. There is no human access, neither to the actual spring, nor to the groundwater. We confirm the permanent Fig. 9. Niphargus luxemburgensis Weber, Weigand & Brad sp. nov., paratype, ♂ (ISER DW180130-32). A. Pleopod I, detail of the retinaculum. B. Detail of the epimeral plates. European Journal of Taxonomy 1011: 1–79 (2025) 18 presence of this species in the type locality as it was found on 10 July 2016, 4 September 2016, 30 January 2018, 17 February 2018 and 19 May 2018. In the same basin of the spring, N. schellenbergi sensu stricto was also found. We successfully collected 82 specimens of N. schellenbergi in 15 springs and 1 mine, at a distance of maximum 10 km around the type locality, in Luxembourg and in Germany, but could not find N. luxemburgensis there. The type locality was free of gammarids (Amphipoda, Gammaridae) during the periods of collection. Niphargus luxemburgensis seems to be a typical species of the interstitial environment (Supp. file 4.3). It is widely distributed from the UK and North to Central France, Belgium, Germany to the Czech Republic in the East (Fig. 11). The frequent presence in Luxembourg and bordering Germany (Rhineland-Palatinate, Saarland) must be most likely attributed to a more intense sampling in those regions. Fig. 10. Niphargus luxemburgensis Weber, Weigand & Brad sp. nov., paratype, ♂ (ISER DW18013032) and paratype, ♀ (ISER DW180130-45). A. Male uropod I. B. Male uropod II. C. Male uropod III. D. Female uropod III. E. Male telson. F. Female telson. WEBER D. et al., Six new species of the Niphargus aquilex complex (Amphipoda) 19 Niphargus luxemburgensis sp. nov. is the only species described herein with two strongly divided subclades (F and F’). Although the genetic distance is relatively large, the connection in the haplotype network identifies both as belonging to the same species. The two clades cannot be distinguished morphologically. Nevertheless, they are geographically separated with only a small overlap: Clade F’, including the type locality, has its distribution in the North-Eastern half of France, Belgium, Luxembourg and the very West of Germany (Fig. 11). Clade F is distributed in southern England, the very North of France, Belgium, Luxemburg, all over Germany without the North and the South, up to the Czech Republic (Fig. 11). The geographic overlap of both clades includes Belgium, Luxembourg, and the very West of Germany. The higher number of sites for clade F might be a sampling artefact, as our dataset comprised many more sites in Germany than in France. It can be assumed that the two subclades of N. luxemburgensis are in the process of ecological speciation. Niphargus luxemburgensis sp. nov. has a moderate geographical overlap with Niphargus vejdovskyi Wrześniowski, 1890 in the very East of its distribution range, with the latter having been described as N. puteanus var. vejdovskyi from wells in Prague (Wrześniowski 1890), the village of Bechlín close to Roudnice and from the wells between Kralupy and Vodolka in Bohemia (Czech Republic). Wrześniowski’s drawings are difficult to understand but show some similarities to the N. aquilex species Fig. 11. Distribution of Niphargus luxemburgensis Weber, Weigand & Brad sp. nov. confirmed by molecular data. The type locality at Girsterklaus (Luxembourg) is marked by a yellow star, overlaying one site record. The two molecular clades F and F’ within N. luxemburgensis are referred to by circles and triangles, respectively. Very large freshwater bodies are indicated; country codes follow the ISO 3166 standard alpha-2 format. European Journal of Taxonomy 1011: 1–79 (2025) 20 group. This taxon was re-described as N. aquilex vejdovskyi and synonymised with N. aquilex var. bohemica Ortmann (Schellenberg 1932), based on material from Schneeberg in the Ore Mountains (Saxony, Germany), from Brunersdorf (Saxony, Germany). Schellenberg assumed that three females from Heligoland (Schleswig-Holstein, Germany) also belong to this species. The inner lobe of the maxilla I of the specimens from Heligoland has 3 to 6 apical setae (Schellenberg 1932), while N. luxemburgensis (as well as all other newly described species herein) has 1 apical seta only. Niphargus vejdovskyi is therefore not identical to N. luxemburgensis, but DNA sequences of the former are not yet available. Niphargus palatinensis Weber & Brad sp. nov. urn:lsid:zoobank.org:act:5DA10A74-7763-41C9-B0ED-76AD156C03D4 Figs 12–20 Diagnosis Medium-sized Niphargus species, poorly setose. Right postero-ventral angle of epimeral plates. Six spines of maxilla I outer lobe with 1–3 teeth each; 1 spine with several smaller teeth. Mandibular palp with small number (1–2) of B setae. Gnathopods with 1 seta along outer margin of dactylopodites. Pereiopod VII, the longest leg, almost half of total body length. Pleopods retinaculum with 3 hooks. Uropod I, longer exopodite. Uropod II, longer endopodite. Uropod III sexually dimorphic, exopodite elongated in males. Telson with 4 apical spines on each lobe. The COI marker shows six pure diagnostic sites at positions 31 (C), 184 (C), 223 (G), 353 (G), 358 (G) and 391 (A). A single 28S rDNA allele is diagnostic as well as 21 COI barcodes. Etymology The species name derives from the German Federal State of Rhineland-Palatinate, where the type locality is located. In Weber et al. (2023), this species was treated as N. aquilex A. Material examined Holotype GERMANY • ♂; Rhineland-Palatinate, Waldleiningen, Felsenbrunnen in the Palatinate Forest in the Middle Buntsandstein; rheocrene spring; 49.2881° N, 7.8467° E; 14 Dec. 2016; Dieter Weber leg.; kept intact in 96% ethanol; 161214-02; MNHNL130574. Paratypes GERMANY • 1 ♂; same collection data as for holotype; 14 Dec. 2016; dissected and appendages drawn; 161214-01; ISER microscope slide DW161214-01 • 1 ♀; same collection data as for holotype; 8 Aug. 2017; dissected and appendages drawn; 170808-13; ISER microscope slide DW170808-13. Molecular data COI and 28S rDNA sequences of specimens belonging to Niphargus palatinensis sp. nov. were deposited in GenBank. COI and 28S rRNA accession numbers are present in Supp. file 6 and Supp. file 9, respectively. Description (male paratype ISER DW161214-01) Measurements Total body length 6.92 mm (Fig. 12). Head Head (Fig. 12) 8% of total body length. Eyes and rostrum absent. WEBER D. et al., Six new species of the Niphargus aquilex complex (Amphipoda) 21 Antennae Antenna I (Fig. 13A): with main flagellum formed of 20 articles, representing 50% of total body length. Peduncle length 35% of total length of antenna I. The accessory flagellum (Fig. 13B). Biarticulated; proximal article shorter than first article of main flagellum; distal article 27% of total length of accessory flagellum, with 4 apical setae of different lengths and 1 aesthetasc. Aesthetascs ¾ of respective main flagellum articles (Fig. 13C). Antenna II (Fig. 13D): flagellum formed of 10 articles and representing 50% of total length of antenna II. Most flagellum articles bear 1 aesthetasc, longer than half of respective flagellum articles. Mouthparts Labrum (Fig. 14A): typical, subovoid shape. Labium (Fig. 14B). Large inner lobes with 1 row of fine setae on inner sides. Outer lobes with 2 rows of fine setae subapically on inner sides. Maxilla I (Fig. 14C): with 3 apical setae on distal article of palp. 6 spines of outer lobe with 1–3 teeth each and 1 spine with several small teeth. Inner lobe with 1 apical seta. Maxilla II (Fig. 14D): with inner lobe slightly shorter than outer lobe. 1 apical row of setae and 4 setae on inner margin of each lobe. Left mandible (Fig. 14E): 6 teeth on incisor process. 5 teeth on lacinia mobilis. 8 serrated setae intercalated with 4 trifid setae, between lacinia mobilis and molar process. Right mandible (Fig. 14F): 5 teeth on incisor process. Lacinia mobilis with several smaller teeth. 7 serrated and 2 trifid setae between lacinia mobilis and molar process. A long seta on molar process. Fig. 12. Niphargus palatinensis Weber & Brad sp. nov., paratype, ♂ (ISER DW161214-01); general appearance. European Journal of Taxonomy 1011: 1–79 (2025) 22 Fig. 13. Niphargus palatinensis Weber & Brad sp. nov., paratype, ♂ (ISER DW161214-01). A. Antenna I. B. Antenna I, details of accessory flagellum. C. Antenna I, details of aesthetascs. D. Antenna II. WEBER D. et al., Six new species of the Niphargus aquilex complex (Amphipoda) 23 Fig. 14. Niphargus palatinensis Weber & Brad sp. nov., paratype, ♂ (ISER DW161214-01). A. Labrum. B. Labium. C. Maxilla I. D. Maxilla II. E. Left mandible. F. Right mandible. European Journal of Taxonomy 1011: 1–79 (2025) 24 Mandibular palps (Fig. 14E–F): highly similar and of same length. 3 articles account for 17% (article 1), 39% (article 2) and 44% (article 3) of total length of palp. Proximal article without setae, median article with 8 ventral setae. Distal article of palp with one group of 3 A setae, three groups with 1 B setae, 17 D setae and 5–6 E setae. Maxilliped (Fig. 15A): with palp formed of 4 articles. Article 1 with 2 setae on inner margin. Article 2 with 23 setae aligned along inner margin. Article 3 with 4 apical setae, one group of 6 dorsal setae and one group of 7 setae on inner margin. Article 4 with 1 seta located on outer margin and 2 fine setae at nail insertion. Outer lobe with 5 apical setae and 9 flattened setae on inner margin. Inner lobe provided apically with 3 setae, 1 flattened seta and 2 dorsal setae. Gnathopods Gnathopod I (Fig. 15B): coxal plate in shape of rectangular trapezoid, with depth larger than width (ratio depth : width 1.0 : 0.64). Basis length : width ratio 1.0 : 0.44. Ischiopodite with one posteroventral group of 3 setae. Basis : carpus length ratio 1.0 : 0.58. Carpus with row of 7 setae along ventral margin, group of 5 setae located anterodorsally and one group of 8 setae on carpus surface close to ventral margin. Propodite as long as wide, three groups of 3–4 setae on ventral margin, one group of 4 setae on dorsal margin, one group of 10 anteroapical setae, 3 mesial setae on lateral surface, and one group of 3 setae close to palmar corner. 1 strong palmar spine, 1 supporting spine and 2 denticulate spines present in palmar corner. Dactylopodite with claw 26% of te total dactylopodite length and 1 seta along outer margin. Gnathopod II (Fig. 15C): slightly larger than gnathopod I, with coxal plate in shape of trapezoid; coxal plate width larger than depth (ratio width : depth 1.0 : 0.7). Basis length : width ratio 1.0 : 0.31. Ischiopodite with one posteroventral group of 4 setae. Basis : carpus length ratio 1.0 : 0.54. Carpus with row of 6 setae along ventral margin, group of 4 setae located anterodorsally and 1 row of 4 setae on carpus surface close to ventral margin. Propodite as long as wide, with 5 groups of 3 setae on ventral margin, 4 setae on dorsal margin, 1 mesial seta on lateral surface, 8 anteroapical setae, one group of 5 setae on lateral surface close to ventral margin, and 3 setae close to palmar corner. 1 strong palmar spine, 1 supporting spine and 2 denticulate spines present in palmar corner. Dactylopodite with claw 28% of total dactylopodite length and 1 seta along outer margin. Pereopods Pereopod III (Fig. 16A): coxal plate in shape of rectangular, with width larger than depth, width : depth ratio 0.7. Propodite : dactylus length ratio 1.0 : 0.45. Dactylus, with nail measuring half of total length of dactylus, 1 dorsal seta with plumose tip, and 1 seta at nail base. Pereopod III nearly equal to pereopod IV (pereopod III : pereopod IV length ratio 1.0 : 0.98). Pereopod IV (Fig. 16B): relatively rectangular coxal plate, with concavity on posterior margin, width : depth ratio 1.0 : 0.7. Propodite : dactylus length ratio 1.0 : 0.54. Robust dactylus, with nail half of total length of dactylus; 1 dorsal seta with plumose tip and 1 seta at nail base. Pereopod V (Fig. 17A): coxal plate of irregular shape, with deep concavity on ventral side, 4 anterior setae and 1 posterior seta. Basis rectangular, length : width ratio 1.0 : 0.57, 4 setae on anterior margin, 9 setae on posterior margin, 3 anteroapical setae of different lengths. Dactylus with 1 dorsal seta with plumose tip, 1 seta at nail base, which represents 45% of total dactylus length. Propodite length : dactylus length ratio 1.0 : 0.36. Pereopod VI (Fig. 17B): coxal plate smaller than that of pereopod V, with concavity on ventral side less deep than that of pereopod V, and 1 posterior seta. Basis rectangular, length : width ratio 1.0 : 0.55, 4 setae on anterior margin, 8 setae on posterior margin, 2 anteroapical setae. Dactylus with 1 plumose seta on WEBER D. et al., Six new species of the Niphargus aquilex complex (Amphipoda) 25 outer margin and 2 setae of different lengths near nail base. Nail 36% of total dactylus length. Ratio propodite : dactylus length 1.0 : 0.33. Pereopod VII (Fig. 17C): longest leg (3.22 mm) of inspected paratype male. Coxal plate trapezoidal, with 1 seta on posterior margin. Basis rectangular, ratio length : width 1.0 : 0.5, 3 setae on anterior margin, 14 setae on posterior margin and 2 small anteroapical setae. Dactylus with 1 plumose seta on outer margin and 2 setae near nail base. Nail length 31% of total dactylus length. Ratio propodite : dactylus length 1.0 : 0.3. Pereopods V : VI : VII ratio 1.0 : 1.35 : 1.42. Fig. 15. Niphargus palatinensis Weber & Brad sp. nov., paratype, ♂ (ISER DW161214-01). A. Maxilliped. B. Gnathopod I. C. Gnathopod II. European Journal of Taxonomy 1011: 1–79 (2025) 32 Antennae Antenna I (Fig. 22A): with main flagellum formed of 17 articles, representing 55% of total body length. Peduncle length 40% of total length of antenna I. Accessory flagellum (Fig. 22B) biarticulated; proximal article slightly longer than first article of main flagellum; distal article 28% of total length of accessory flagellum, with 3 apical setae of different lengths and 1 aesthetasc. Aesthetascs ⅓ of respective main flagellum articles (Fig. 22C). Antenna II (Fig. 22D): flagellum formed of 8 articles and representing 42% of total length of antenna II. Most flagellum articles bear 1 aesthetasc, ¼ length of respective flagellum articles. Mouthparts Labrum (Fig. 23A): typical, subovoid shape. Labium (Fig. 23B). Large inner lobes with 1 row of fine setae on inner sides. Outer lobes with 1 row of fine setae subapically on inner sides. Maxilla I (Fig. 23C): with 3 apical setae on distal article of palp. Six spines of outer lobe with 1 tooth each and 1 spine with several small teeth. Inner lobe with 1 apical seta. Maxilla II (Fig. 23D): with inner lobe slightly shorter than outer lobe. 1 apical row of setae on each lobe. Left mandible (Fig. 23E): 5 teeth on incisor process. 3 teeth on lacinia mobilis. 10 serrated setae between lacinia mobilis and molar process. Right mandible (Fig. 23F): 4 teeth on incisor process. 2 teeth on lacinia mobilis. 5 serrated setae between lacinia mobilis and molar process. Long seta on molar process. Mandibular palps (Fig. 23E–F): highly similar and of same length. 3 articles account for 18% (article 1), 36% (article 2) and 46% (article 3) of total length of palp. Proximal article without setae, median article with 6–7 ventral setae. Distal article of palp with one group of 5 A setae, four groups with 1 B setae each, 22–23D setae and 6 E setae. Maxilliped (Fig. 24A): with palp formed of 4 articles. Article 1 asetose. Article 2 with 23 setae aligned along inner margin. Article 3 with 6 apical setae, one group of 6 dorsal setae and one group of 4 setae on inner margin. Article 4 with 1 seta located on outer margin and 2 setae at nail insertion. Outer lobe with 5 apical setae and 8 flattened setae on inner margin. Inner lobe provided apically with 5 setae and 1 flattened seta. Gnathopods Gnathopod I (Fig. 24B): coxal plate in shape of rectangular trapezoid, with depth larger than width (ratio depth : width 1.0 : 0.53). Basis length : width ratio 1.0 : 0.64. Ischiopodite with one posteroventral group of 8 setae. Basis : carpus length ratio 1.0 : 0.75. Carpus with row of 9 setae of various lengths along ventral margin, group of 3 setae located anterodorsally and one group of 2 setae on carpus surface close to ventral margin. Propodite nearly as long as wide, five groups of 3–4 setae on ventral margin, two groups of 2 setae each on dorsal margin, one group of 5 setae on propodite surface close to dorsal margin, one group of 10 anteroapical setae, 2 mesial setae on lateral surface, 2 setae on lateral surface close to ventral margin and one group of 3 setae close to palmar corner. 1 strong palmar spine, 1 supporting spine and 2 denticulate spines present in palmar corner. Dactylopodite with claw 30% of total dactylopodite length and 1 seta along outer margin. Gnathopod II (Fig. 24C): slightly larger than gnathopod I, with coxal plate in shape of trapezoid; coxal plate wider than deep (ratio width : depth 1.0 : 0.7). Ovoid gill and of same length as coxal plate width. Basis length : width ratio 1.0 : 0.38. Ischiopodite with one posteroventral group of 6 setae. Basis : carpus WEBER D. et al., Six new species of the Niphargus aquilex complex (Amphipoda) 33 Fig. 22. Niphargus normandiensis Weber & Brad sp. nov., paratype, ♂ (ISER DW180526-36). A. Antenna I. B. Antenna I, details of the accessory flagellum. C. Antenna I, details of the aesthetascs. D. Antenna II. European Journal of Taxonomy 1011: 1–79 (2025) 34 Fig. 23. Niphargus normandiensis Weber & Brad sp. nov., paratype, ♂ (ISER DW180526-36). A. Labrum. B. Labium. C. Maxilla I. D. Maxilla II. E. Left mandible. F. Right mandible. WEBER D. et al., Six new species of the Niphargus aquilex complex (Amphipoda) 35 Fig. 24. Niphargus normandiensis Weber & Brad sp. nov., paratype, ♂ (ISER DW180526-36). A. Maxilliped. B. Gnathopod I. C. Gnathopod II. European Journal of Taxonomy 1011: 1–79 (2025) 36 length ratio 1.0 : 0.57. Carpus with row of 9 setae of various lengths along ventral margin, group of 6 setae located anterodorsally and 1 row of 5 setae on carpus surface close to ventral margin. Propodite width larger than length (ratio width : length 1.0 : 0.94), with 7 groups of 3–5 setae on ventral margin, one group of 6 setae on dorsal margin, one group of 2 setae on lateral surface close to dorsal margin, 1 mesial seta on lateral surface, one anterodorsal group of 8 setae, and one group of 3 long setae in vicinity of palmar corner. 1 strong palmar spine, 1 supporting spine and 3 denticulate spines present in palmar corner. Dactylopodite with claw 30% of total dactylopodite length and 1 seta along outer margin. Pereopods Pereopod III (Fig. 25A): coxal plate in shape of rectangular trapezoid, wider than deep (ratio width : depth 1.0 : 0.75). Propodite : dactylus length ratio 1.0 : 0.4. Dactylus, with nail measuring half of total length of dactylus, 1 dorsal seta with plumose tip, and 2 setae at nail base. Pereopod III slightly longer than pereopod IV (pereopod III : pereopod IV length ratio 1.0 : 0.95). Pereopod IV (Fig. 25B): coxal plate in shape of rectangular trapezoid, width : depth ratio 1.0 : 0.68. Propodite : dactylus length ratio 1.0 : 0.4. Robust dactylus, with nail measuring almost half of total length of dactylus; 1 dorsal seta with plumose tip and 2 setae of different lengths at nail base. Pereopod V (Fig. 26A). Shortest leg (2.64 mm) of inspected male paratype. Coxal plate of irregular shape, with deep concavity on ventral side, and 2 anterior setae. Basis rectangular, length : width ratio 1.0 : 0.62, 3 setae on anterior margin, 6 setae on posterior margin, 3 anteroapical setae of different lengths. Dactylus with 1 dorsal seta with plumose tip, 2 setae of different lengths at nail base, which representing 42% of total dactylus length. Propodite length : dactylus length ratio 1.0 : 0.49. Pereopod VI (Fig. 26B): coxal plate relatively similar to that of pereopod V, with 1 posterior seta. Basis rectangular, length : width ratio 1.0 : 0.58, 4 setae on anterior margin, 10 setae on posterior margin, 3 anteroapical setae of different lengths. Dactylus with 1 plumose seta on outer margin and 2 setae of different lengths near nail base. Nail ⅓ of total dactylus length. Propodite : dactylus length ratio 1.0 : 0.42. Pereopod VII (Fig. 26C): longest leg (3.90 mm) of inspected paratype male. Coxal plate trapezoidal, with 1 seta on posterior margin. Basis rectangular, ratio length : width 1.0 : 0.52, 5 setae on anterior margin, 11 setae on posterior margin and 2 anteroapical setae of different lengths. Dactylus with 1 plumose seta on outer margin and 1 seta near nail base. Nail length 35% of total dactylus length. Ratio propodite : dactylus length 1.0 : 0.38. Pereopods V : VI : VII ratio 1.0 : 1.31 : 1.47. Pleopods Pleopods similar (pleopod III depicted in Fig. 27A), with unequal rami and 5 hooks on retinaculum. One of pleopods III laterally with 2 additional hooks on retinaculum. Epimeral plates (Fig. 27B) Epimeral plate I with right postero-ventral angle, straight ventral margin, relatively straight posterior margin with 4 setae. Epimeral plate II with right postero-ventral angle, convex ventral margin with 2 spines, convex posterior margin with 3 spines. Epimeral plate III with right posteroventral angle, straight ventral margin with 2 spines, convex posterior margin with 5 spines. Uropods Uropod I (Fig. 28A): with two dorso-lateral rows of 3–4 spines on peduncle. Exopodite longer than endopodite, exopodite : endopodite length ratio 1.0 : 0.66. 1 strong spine at base of uropod I. WEBER D. et al., Six new species of the Niphargus aquilex complex (Amphipoda) 37 Uropod II (Fig. 28B): with 4 dorsal and 2 apical spines on peduncle. Endopodite longer than exopodite, endopodite : exopodite length ratio 1.0 : 0.84, both rami provided with 1–2 dorsal and 5 apical spines. Uropod III (Fig. 28C) sexually differentiated, longer in males. Peduncle with 4 posteroapical setae. Endopodite 67% of length of peduncle, with 4 small apical setae. Distal segment of exopodite slightly longer than proximal segment (ratio 1.0 : 0.95). Anterior margin of proximal segment of exopodite with Fig. 25. Niphargus normandiensis Weber & Brad sp. nov., paratype, ♂ (ISER DW 180526-36). A. Pereopod III. B. Pereopod IV. European Journal of Taxonomy 1011: 1–79 (2025) 38 Fig. 26. Niphargus normandiensis Weber & Brad sp. nov., paratype, ♂ (ISER DW180526-36). A. Pereopod V. B. Pereopod VI. C. Pereopod VII. 3 groups of 3–4 setae; posterior margin of exopodite with 4 groups of 2–4 setae; 1 anteroand 1 posteroapical seta. Distal segment of exopodite with 3 groups of 2 setae on anterior margin, 4 groups of 2 setae on posterior margin, and 8 apical setae of different lengths. Telson Telson (Fig. 28E) wide (width : length ratio 1.0 : 0.76), with 3 long apical spines, 2 spines and 2 thin setae with plumose tip located medially on each lobe. Longest spine 78% of telson length. WEBER D. et al., Six new species of the Niphargus aquilex complex (Amphipoda) 39 Sexual dimorphism The examined male and female are highly similar. Besides the usual sexual dimorphism (e.g., slightly smaller gnathopods, presence of oostegites, and slightly deeper coxal plates I–VI in females), in N. normandiensis sp. nov., the female uropod III is shorter comparted to that of the male (Fig. 28D). The female telson is highly similar to that of the male, but smaller (Fig. 28F). Type locality, ecology and distribution The species comprises three distinct genetic clades (clade N, D and non-annotated) which are distributed in the Normandy (France), with further scattered records on the Channel Islands (Guernsey, Jersey), on two islands off the Brittany Coast of France and in more Central France (Fig. 29). Thereby, clade D is restricted to Normandy (France), whereas both other clades occur on islands in the Atlantic Ocean and in more Central France. The type locality is the Carrière souterrain de Saint-Vaast-en-Auge, Calvados, France at 49.2910° N, 0.00090° W, a subterranean quarry in sandstone outside the village (Supp. file 3.6). The 50 m long cavity is completely dry with a small lake with stagnant water at 40–45 m distance from the entrance, behind a collapsed mine gallery. The water level varies seasonally from 20–70 cm. Specimens of N. normandiensis sp. nov. were found swimming on the clayey bottom of this lake. We confirm the permanent presence of this species in the type locality as it was found on 26 May 2018 as well as on 20 May 2019. We found this species in several springs and in one subterranean quarry. Ecological details are not inferred due to the small number of sampling sites. Fig. 27. Niphargus normandiensis Weber & Brad sp. nov., paratype, ♂ (ISER DW180526-36). A. Pleopod III, detail of the retinaculum. B. Detail of the epimeral plates. European Journal of Taxonomy 1011: 1–79 (2025) 40 Fig. 28. Niphargus normandiensis Weber & Brad sp. nov., paratype, ♂ (ISER DW180526-36) and paratype, ♀ (ISER DW180526-31). A. Male uropod I. B. Male uropod II. C. Male uropod III. D. Female uropod III. E. Male telson. F. Female telson. WEBER D. et al., Six new species of the Niphargus aquilex complex (Amphipoda) 41 Niphargus wasgauensis Weber & Brad sp. nov. urn:lsid:zoobank.org:act:E36BC687-B276-47E9-B685-24FC0718F3DA Figs 30–38 Diagnosis Medium-sized Niphargus species, poorly setose. Right postero-ventral angle of epimeral plates. 6 spines of maxilla I outer lobe with 1–3 teeth each; 1 spine with several smaller teeth. Mandibular palp with small number (1–2) of B setae. Gnathopods with 2 setae along outer margin of dactylopodite. Pereiopod VII, the longest leg, almost half of total body length. Pleopods retinaculum with 2–3 hooks. Uropod I, longer exopodite. Uropod II, longer endopodite. Uropod III sexually dimorphic, exopodite elongated in males. Telson with 5 apical spines on each lobe. The COI marker shows a single pure diagnostic site at position 586 (C). Two 28S rDNA alleles are diagnostic (with 1 heterozygous specimen) as well as 12 COI barcodes. Fig. 29. Distribution of Niphargus normandiensis Weber & Brad sp. nov. confirmed by molecular data. Clade D is marked by green circles; clade N is marked by light-green triangles; a so far non-annotated clade is marked by black circles. The type locality at the Carrière souterrain de Saint-Vaast-en-Auge (France) is marked by a yellow star, overlaying one site record (clade D). Very large freshwater bodies are indicated; country codes follow the ISO 3166 standard alpha-2 format. European Journal of Taxonomy 1011: 1–79 (2025) 48 Pleopods Pleopods I, II and III similar in shape (pleopod III, with 3 hooks on retinaculum, depicted in Fig. 36A), with unequal rami. Pleopods II and III with 2 hooks on retinaculum. Epimeral plates (Fig. 36B) Epimeral plate I with right postero-ventral angle, straight ventral margin, convex posterior margin with 4 setae. Epimeral plate II with right postero-ventral angle, convex ventral margin with 1 spine, convex posterior margin with 5 spines. Epimeral plate III with right posteroventral angle, convex ventral margin with 2 spines, convex posterior margin with 4 spines. Fig. 35. Niphargus wasgauensis Weber & Brad sp. nov., paratype, ♂ (ISER DW170101-22). A. Pereopod V. B. Pereopod VI. C. Pereopod VII. WEBER D. et al., Six new species of the Niphargus aquilex complex (Amphipoda) 49 Uropods Uropod I (Fig. 37A): with two dorsolateral rows of 2–4 spines on peduncle. Exopodite slightly longer than endopodite, exopodite : endopodite length ratio 1.0 : 0.91. 1 strong spine at base of uropod I. Uropod II (Fig. 37B): with 3 dorsal and 3 apical spines on peduncle. Endopodite longer than exopodite, endopodite : exopodite length ratio 1.0 : 0.89, both rami provided with 2 dorsal and 5 apical spines of different lengths. Uropod III (Fig. 37C): sexually differentiated, longer in males. Peduncle with 3 small apical setae. Short endopodite, 47% of length of peduncle, with 2 apical setae. Proximal segment of exopodite longer than distal segment (ratio 1.0 : 0.85). Anterior margin of proximal segment of exopodite with 3 groups of 2–3 setae; posterior margin of exopodite with 4 groups of 1–2 setae; 2 anteroand 2 postero-apical setae. Distal segment of exopodite with 2 setae on anterior margin, 1 seta on posterior margin, 2 subapical and 5 apical setae of different lengths. Telson Telson (Fig. 37E): wide (width : length ratio 1.0 : 0.9), with 5 long apical spines alongside small seta with plumose tip. Two thin setae with plumose tip on outer margin of 1 lobe. Longest spine 43% of telson length. Sexual dimorphism The examined male and female are highly similar. Besides the usual sexual dimorphism (e.g., slightly smaller gnathopods, presence of oostegites, and slightly deeper coxal plates I–VI in females), in N. wasgauensis sp. nov., the female uropod III is shorter than that of male (Fig. 37D). The telson of the Fig. 36. Niphargus wasgauensis Weber & Brad sp. nov., paratype, ♂ (ISER DW170101-22). A. Pleopod III, detail of the retinaculum. B. Detail of the epimeral plates. European Journal of Taxonomy 1011: 1–79 (2025) 50 inspected female paratype (Fig. 37F) is smaller, with 6 spines on one lobe, and 2 fine setae with plumose tip on the outer margin of both lobes. Type locality, ecology and distribution The species shows a very narrow distribution limited to the Wasgau region, i.e., Franco-German uplands covering parts of the Federal State of Rhineland-Palatinate (Germany) and the Departments of BasRhin and Moselle (both France) (Fig. 38). The type locality is the Sülzlochquelle 2 in the community of Godramstein (Rhineland-Palatinate, Germany) at 49.2152° N, 8.0842° E (Supp. file 3.7). It is a natural rheocrene spring with a permanent moderate flow, 21°GH hardness and 380 ppm TDS. No other species Fig. 37. Niphargus wasgauensis Weber & Brad sp. nov., paratype, ♂ (ISER DW170101-22) and paratype, ♀ (ISER DW160716-25). A. Male uropod I. B. Male uropod II. C. Male uropod III. D. Female uropod III. E. Male telson. F. Female telson. WEBER D. et al., Six new species of the Niphargus aquilex complex (Amphipoda) 51 of either Niphargus or gammarids (Amphipoda; Gammaridae) were found in this spring. Across its known distribution range, it was found in five springs, two abandoned mines and in one gallery of the Siegfried Line. The species seems to be absent in the interstitial environment. Previous published records from the Stollen am Sachsenstein (Germany), which referred to N. aquilex aquilex (Weber 1988, 1989), also belong to this species. Fig. 38. Distribution of Niphargus wasgauensis Weber & Brad sp. nov. confirmed by molecular data. The type locality at Sülzlochquelle 2 (Germany) is marked by a yellow star, overlaying one site record. Very large freshwater bodies are indicated; country codes follow the ISO 3166 standard alpha-2 format. European Journal of Taxonomy 1011: 1–79 (2025) 52 Niphargus saraviensis Weber & Brad sp. nov. urn:lsid:zoobank.org:act:C7E58FC7-564C-410B-ADBE-EE2AC9BA06AD Figs 39–47 Diagnosis Medium-sized Niphargus species, poorly setose. Right postero-ventral angle of epimeral plates. Six spines of maxilla I outer lobe with 1–2 teeth each; one spine with several smaller teeth. Mandibular palp with small number (1–2) of B setae. Gnathopods with 1 seta along outer margin of dactylopodite. Pereiopod VII, the longest leg, almost half of total body length. Pleopods retinaculum with 2 hooks. Uropod I, longer exopodite. Uropod II, longer endopodite. Uropod III sexually dimorphic, exopodite elongated in males. Telson with 4 apical spines on each lobe. The COI marker shows two single pure diagnostic sites at positions 94 (A) and 357 (A). Four 28S rDNA alleles are diagnostic (with 7 heterozygous specimens) as well as 23 COI barcodes. Etymology The species name derives from the German Federal State of Saarland, in which the type locality is located. In Weber et al. (2023), this species was treated as N. aquilex G. Material examined Holotype GERMANY • ♂; Saarland, Zilla’s Keller in Nunkirchen; 49.4826° N, 6.8317° E; 30 Dec. 2017; Dieter Weber leg.; kept intact in 96% ethanol; 171230-02; MNHNL130580. Paratypes GERMANY • 1 ♂; same collection data as for holotype; 30 Dec. 2017; dissected and appendages drawn; 171230-01; ISER microscope slide DW171230-01 • 1 ♀; same collection data as for preceding; dissected and appendages drawn; 171230-05; ISER microscope slide DW171230-05 • 1 ♂; same collection data as for holotype; 12 Mar. 2018; ISER DW180312-01 • 1 ♂; same collection data as for holotype; 12 Mar. 2018; ISER DW180312-06 • 1 ♀; same collection data as for holotype; 12 Mar. 2018; ISER DW18031203 • 1 ♀; same collection data as for holotype; 30 Dec. 2017; ISER DW171230-07 • 1 ♂; same collection data as for holotype; 12 Mar. 2018; 180312-02; MNHNL130581 • 1 ♂; same collection data as for holotype; 12 Mar. 2018; 180312-04; MNHNL130582 • 1 ♀; same collection data as for holotype; 30 Dec. 2017; 171230-04; MNHNL130583 • 1 ♀; same collection data as for holotype; 30 Dec. 2017; 171230-06; MNHNL130584. Molecular data COI and 28S rDNA sequences of specimens belonging to Niphargus saraviensis sp. nov. were deposited in GenBank. COI and 28S rRNA accession numbers are present in Supp. file 6 and Supp. file 9, respectively. Description (male paratype ISER DW171230-01) The total body length is 7.91 mm (Fig. 39). Head Head (Fig. 39) 6.95% of total body length. Eyes and rostrum absent. Antennae Antenna I (Fig. 40A): with main flagellum formed of 18 articles, representing 50% of total body length. Peduncle length ⅓ of total length of antenna I. The accessory flagellum (Fig. 40B) biarticulated; proximal article almost twice as long as first article of main flagellum; distal article ¼ of total length of accessory WEBER D. et al., Six new species of the Niphargus aquilex complex (Amphipoda) 53 flagellum, with 2 apical setae of different lengths and one aesthetasc. Aesthetascs slightly more than half of respective flagellum articles (Fig. 40C). Antenna II (Fig. 40D): flagellum formed of 6 articles and representing 40% of total length of antenna II. Most flagellum articles bear one short aesthetasc. Mouthparts Labrum (Fig. 41A): typical, subovoid shape. Labium (Fig. 41B). Relatively small inner lobes with 1 row of fine setae located subapically on inner sides. Outer lobes each with 1 row of fine setae on outer sides, 1 row of fine setae subapically on inner sides and 1 row of fine apical setae. Maxilla I (Fig. 41C): with 4 apical setae on the distal article of palp. Six spines of outer lobe with 1–2 teeth each and one spine with several smaller teeth. Inner lobe with 1 apical seta. Maxilla II (Fig. 41D): with inner lobe slightly shorter than outer lobe. 1 apical row of setae each lobe. Inner lobe with 2 subapical setae on inner margin. Outer lobe with 3 smaller setae on outer margin. Left mandible (Fig. 41E): 5 teeth on incisor process. 3 teeth on lacinia mobilis. 6 serrated setae between lacinia mobilis and molar process. Right mandible (Fig. 41F): 5 teeth on incisor process. 5 small teeth on lacinia mobilis. 4 serrated setae alternated with 4 trifid setae between lacinia mobilis and molar process. Long seta on molar process. Mandibular palps (Fig. 41E–F): highly similar and of same length. 3 articles account for 21% (article 1), 37% (article 2) and 42% (article 3) of total length of palp. Proximal article with 1 apical seta. Median Fig. 39. Niphargus saraviensis Weber & Brad sp. nov., paratype, ♂ (ISER DW171230-01); general appearance. European Journal of Taxonomy 1011: 1–79 (2025) 54 article with 7 ventral setae. Distal article of palp with one group of 3 A setae, two groups with 1 B seta each, 23–24 D setae and 6–7 E setae. Maxilliped (Fig. 42A): with palp formed of 4 articles. Article 1 asetose. Article 2 with 14 setae aligned along inner margin. Article 3 with 5 apical setae, one group of 4 dorsal setae and one group of 5 setae on inner margin. Article 4 with 1 seta located on outer margin and 2 setae at nail insertion. Outer lobe with 3 apical setae and 9 setae on inner margin, of which 7 flattened. Inner lobe provided apically with 1 flattened seta and 5 setae. Fig. 40. Niphargus saraviensis Weber & Brad sp. nov., paratype, ♂ (ISER DW171230-01). A. Antenna I. B. Antenna I, details of accessory flagellum. C. Antenna I, details of aesthetascs. D. Antenna II. WEBER D. et al., Six new species of the Niphargus aquilex complex (Amphipoda) 55 Gnathopods Gnathopod I (Fig. 42B): coxal plate in shape of rectangular trapezoid, with depth larger than width (ratio depth : width 1.0 : 0.62). Basis length : width ratio 1.0 : 0.40. Ischiopodite with one posteroventral group of 3 setae. Basis : carpus length ratio 1.0 : 0.56. Carpus with row of 6 setae along ventral margin, group of 5 setae located anterodorsally and two groups of 2–6 setae on carpus surface close to ventral margin. Propodite nearly as long as wide, 4 groups of 1–4 setae on ventral margin, one group of 3 setae on dorsal margin, one group of 5 anterodorsal setae, one group of 6 anteroapical setae, 1 mesial seta on lateral surface, 1 seta on lateral surface close to ventral margin and one group of 2 setae close to palmar corner. Fig. 41. Niphargus saraviensis Weber & Brad sp. nov., paratype, ♂ (ISER DW171230-01). A. Labrum. B. Labium. C. Maxilla I. D. Maxilla II. E. Left mandible. F. Right mandible. European Journal of Taxonomy 1011: 1–79 (2025) 56 One strong palmar spine, one supporting spine and two denticulate spines present in palmar corner. Dactylopodite with claw 33% of total dactylopodite length and 1 seta along outer margin. Gnathopod II (Fig. 42C): slightly larger than gnathopod I, with coxal plate in shape of trapezoid; coxal plate slightly wider than long (ratio width : depth 1.0 : 0.80). Basis length : width ratio is 1.0 : 0.29. Ischiopodite with one posteroventral group of 4 setae. Basis : carpus length ratio 1.0 : 0.56. Carpus with Fig. 42. Niphargus saraviensis Weber & Brad sp. nov., paratype, ♂ (ISER DW171230-01). A. Maxilliped. B. Gnathopod I. C. Gnathopod II. WEBER D. et al., Six new species of the Niphargus aquilex complex (Amphipoda) 57 row of 8 setae of various lengths along ventral margin, group of 5 setae located anterodorsally and one group of 5 setae on carpus surface close to ventral margin. Propodite almost as long as wide, 5 groups of 2–3 setae on ventral margin, 1 seta on dorsal margin, 1 seta on lateral surface close to ventral margin and 5 anteroapical setae of different lengths. 1 mesial seta on the lateral surface, 3 setae close to palmar corner. 1 strong palmar spine, 1 supporting spine and 2 denticulate spines present in palmar corner. Dactylopodite with claw 40% of total dactylopodite length and 1 seta along outer margin. Pereopods Pereopod III (Fig. 43A): coxal plate in shape of trapezoid, ratio width-depth 1.0 : 0.73. Propodite : dactylus length ratio 1.0 : 0.4. Dactylus with nail measuring half of total length of dactylus, 1 dorsal seta with plumose tip. Pereopod III equal slightly longer than pereopod IV (pereopod III : pereopod IV length ratio 1.0 : 0.95). Pereopod IV (Fig. 43B): relatively rectangular coxal plate, width : depth ratio 1.0 : 0.75. Propodite : dactylus length ratio 1.0 : 0.41. Robust dactylus, with nail measuring half of total length of dactylus; 1 dorsal seta with plumose tip. Pereopod V (Fig. 44A): coxal plate of irregular shape, with deep concavity on ventral side, 2 anterior setae and 1 posterior seta. Basis rectangular, length : width ratio 1.0 : 0.64, 6 setae on posterior margin, 3 setae on anterior margin, 2 anteroapical setae of different lengths. Dactylus with dorsal seta with plumose tip, 2 setae at nail base, which represents 44% of total dactylus length. Propodite length : dactylus length ratio 1.0 : 0.31. Pereopod VI (Fig. 44B): coxal plate smaller than that of pereopod V, but similar in shape and 1 posterior seta. Basis rectangular, length : width ratio 1.0 : 0.55, 6 setae on posterior margin, 3 setae on anterior Fig. 43. Niphargus saraviensis Weber & Brad sp. nov., paratype, ♂ (ISER DW171230-01). A. Pereopod III. B. Pereopod IV. European Journal of Taxonomy 1011: 1–79 (2025) 64 Fig. 49. Niphargus lotharingiensis Weber & Brad sp. nov., paratype, ♂ (ISER DW160703-02). A. Antenna I. B. Antenna I, details of accessory flagellum. C. Antenna I, details of aesthetascs. D. Antenna II. WEBER D. et al., Six new species of the Niphargus aquilex complex (Amphipoda) 65 Fig. 50. Niphargus lotharingiensis Weber & Brad sp. nov., paratype, ♂ (ISER DW160703-02). A. Labrum. B. Labium. C. Maxilla I. D. Maxilla II. E. Left mandible. F. Right mandible. European Journal of Taxonomy 1011: 1–79 (2025) 66 with 9–11 ventral setae. Distal article of palp with one group of 4A setae, 4 groups with 1–2 B setae, 17–22 D setae and 7 E setae. Maxilliped (Fig. 51A): with palp formed of 4 articles. Article 1 asetose. Article 2 with 31 setae aligned along inner margin. Article 3 with 6 apical setae, one group of 6 dorsal setae and one group of 9 setae on inner margin. Article 4 with 1 seta located on outer margin and 2 setae at nail insertion. Outer lobe with 4 apical setae and 15 flattened setae on inner margin. Inner lobe provided apically with 3 setae, 1 flattened seta and 2 plumose setae. Gnathopods Gnathopod I (Fig. 51B): coxal plate in shape of rectangular, with depth larger than width (ratio depth : width 1.0 : 0.8). Basis length : width ratio 1.0 : 0.41. Ischiopodite with one posteroventral group of 5 setae. Basis : carpus length ratio 1.0 : 0.56. Carpus with row of 14 setae of various lengths along ventral margin, group of 7 setae located anterodorsally and one group of 5 setae on carpus surface close to ventral margin. Propodite nearly as long as wide, seven groups of 2–5 setae on ventral margin, one group of 6 setae on dorsal margin, one group of 3 setae on propodite surface close to dorsal margin, one group of 5 anteroapical setae, 3 mesial setae on lateral surface, 2 setae on lateral surface close to ventral margin and one group of 4 setae close to palmar corner. One strong palmar spine, one supporting spine and two denticulate spines present in palmar corner. Dactylopodite with claw 26% of total dactylopodite length and 3 setae along outer margin. Gnathopod II (Fig. 51C): slightly larger than gnathopod I, with coxal plate in th shape of rectangular trapezoid; coxal plate width equal to its depth (ratio width : depth 1.0 : 1.0). Ovoid gill and of same length as basis. Basis length : width ratio 1.0 : 0.27. Ischiopodite with one posteroventral group of 3 setae. Basis : carpus length ratio 1.0 : 0.54. Carpus with row of 12 setae of various lengths along ventral margin, group of 4 setae located anterodorsally and 1 row of 10 setae on carpus surface close to ventral margin. Propodite as long as wide, with 8 groups of 1–5 setae on ventral margin, 5 setae on dorsal margin, 3 mesial setae on lateral surface, 1 seta on lateral surface close to dorsal margin, 6 anteroapical setae and 4 setae close to palmar corner. 1 strong palmar spine, 1 supporting spine and 2 denticulate spines present in palmar corner. Dactylopodite with claw 33% of total dactylopodite length and 3 setae along outer margin. Pereopods Pereopod III (Fig. 52A): coxal plate in shape of rectangular trapezoid, as deep as wide. Propodite : dactylus length ratio 1.0 : 0.44. Dactylus, with nail measuring half of total length of dactylus, 1 dorsal seta with plumose tip, and 1 seta at nail base. Pereopod III slightly longer than pereopod IV (pereopod III : pereopod IV length ratio 1.0 : 0.92). Pereopod IV (Fig. 52B): relatively rectangular coxal plate, with concavity on posterior margin, width : depth ratio 1.0 : 0.94. Propodite length : dactylus length ratio 1.0 : 0.4. Robust dactylus, with nail measuring half of total length of dactylus; 1 dorsal seta with plumose tip and 2 setae of different lengths at nail base. Pereopod V (Fig. 53A): coxal plate of irregular shape, with deep concavity on ventral side, 6 anterior setae and 2 posterior setae. Basis ovoid-rectangular shaped, length : width ratio 1.0 : 0.62, 5 groups of 1–2 setae on anterior margin, 12 setae on posterior margin, 4 anteroapical setae of different lengths. Dactylus with 1 dorsal seta with plumose tip, 2 setae of different lengths at nail base, which represents 40% of total dactylus length. Propodite length : dactylus length ratio 1.0 : 0.28. Pereopod VI (Fig. 53B): coxal plate smaller than that of pereopod V, but highly similar in shape and 2 posterior setae. Basis ovoid-rectangular shaped, length : width ratio 1.0 : 0.51, 6 groups of 1–2 setae on anterior margin, 12 setae on posterior margin, 5 anteroapical setae of different lengths. Dactylus with 1 WEBER D. et al., Six new species of the Niphargus aquilex complex (Amphipoda) 67 plumose seta on outer margin and 2 setae of different lengths near nail base. Nail 35% of total dactylus length. Ratio propodite : dactylus length 1.0 : 0.34. Pereopod VII (Fig. 53C): longest leg (5.65 mm) of inspected paratype male. Coxal plate trapezoidal, with 1 seta on posterior margin. Basis ovoid-rectangular, ratio length : width 1.0 : 0.5, 5 groups of 1–2 setae Fig. 51. Niphargus lotharingiensis Weber & Brad sp. nov., paratype, ♂ (ISER DW160703-02). A. Maxilliped. B. Gnathopod I. C. Gnathopod II. European Journal of Taxonomy 1011: 1–79 (2025) 68 on anterior margin, 14 setae on posterior margin and 4 anteroapical setae of different lengths. Dactylus with 1 plumose seta on outer margin and 2 setae of different lengths near nail base. Nail length 40% of total dactylus length. Ratio propodite : dactylus length 1.0 : 0.3. Pereopods V : VI : VII ratio 1.0 : 1.22 : 1.52. Pleopods Pleopods: similar (pleopod I depicted in Fig. 54A), with unequal rami, 3 (pleopods I and II) and 4 (pleopods III) hooks on retinaculum. Fig. 52. Niphargus lotharingiensis Weber & Brad sp. nov., paratype, ♂ (ISER DW160703-02). A. Pereopod III. B. Pereopod IV. WEBER D. et al., Six new species of the Niphargus aquilex complex (Amphipoda) 69 Fig. 53. Niphargus lotharingiensis Weber & Brad sp. nov., paratype, ♂ (ISER DW160703-02). A. Pereopod V. B. Pereopod VI. C. Pereopod VII. European Journal of Taxonomy 1011: 1–79 (2025) 70 Epimeral plates (Fig. 54B) Epimeral plate I with right postero-ventral angle, relatively straight ventral margin, convex posterior margin with 2 setae. Epimeral plate II with right postero-ventral angle, convex ventral margin with 1 antero-ventral spine, convex posterior margin with 8 setae. Epimeral plate III with right posteroventral angle, relative straight ventral margin with 2 antero-ventral spines, straight posterior margin with 9 setae. Uropods Uropod I (Fig. 55A): with two dorso-lateral rows of 4–5 spines on peduncle. Exopodite slightly longer than endopodite, ratio exopodite : endopodite lengths 1.0 : 0.95. 1 strong spine at base of uropod I. Uropod II (Fig. 55B): with 2 dorsal spine and 3 apical spines on peduncle. Endopodite slightly longer than exopodite, endopodite : exopodite length ratio 1.0 : 0.96, both rami provided with 3–4 dorsal and 5–6 apical spines. Uropod III (Fig. 55C): sexually differentiated, longer in males. Peduncle with 3 apical and 3 posterior setae. Short endopodite, 40% of length of peduncle, with 1 apical seta. Proximal segment of exopodite longer than distal segment (ratio 1.0 : 0.7). Anterior margin of proximal segment of exopodite with 6 groups of 2–4 setae (including plumose setae); posterior margin of exopodite with 3 groups of 3–4 setae; 4 anteroand 4 postero-apical setae. Distal segment of exopodite with 3 groups of 1–2 setae on anterior margin, 3 groups of 1–2 setae on posterior margin, 4 apical and 4 subapical setae of different lengths. Fig. 54. Niphargus lotharingiensis Weber & Brad sp. nov., paratype, ♂ (ISER DW160703-02). A. Pleopod I, detail of the retinaculum. B. Detail of the epimeral plates. WEBER D. et al., Six new species of the Niphargus aquilex complex (Amphipoda) 71 Telson Telson (Fig. 55E): nearly as wide as long (width : length ratio 0.95), with 2 subapical and 5 apical spines of different lengths on each lobe. Longest spine slightly longer than half of telson length. 2 thin setae of different lengths and plumose tip, 1 seta medially on 1 lobe. Sexual dimorphism The examined male and female are highly similar. Besides the usual sexual dimorphism (e.g., slightly smaller gnathopods, presence of oostegites, and slightly deeper coxal plates I–VI in females), in Fig. 55. Niphargus lotharingiensis Weber & Brad sp. nov., paratype, ♂ (ISER DW160703-02) and paratype, ♀ (ISER DW160703-01). A. Male uropod I. B. Male uropod II. C. Male uropod III. D. Female uropod III. E. Male telson. F. Female telson. European Journal of Taxonomy 1011: 1–79 (2025) 72 N. lotharingiensis sp. nov., the female uropod III (Fig. 55D) is shorter than that of the male, and the telson of the inspected female paratype is provided apically with 4 setae only on each lobe (Fig. 55F). Type locality, ecology and distribution The species is widely distributed in eastern France (Fig. 56), with a slight geographical overlap with its sister species N. schellenbergi sensu stricto in the northeastern margin of its distribution range (Fig. 2). It comprises two distinct genetic clades, of which one is very rare and currently only known from two sites in the eastern part of the species’ range. The type locality is the Tunnel du Col des Croix (connecting Vosges and Haute-Saône, France) at 47.8632° N, 6.7464° E, in the very east of the species known distribution (Supp. file 3.10). It is a 1087 m long abandoned railway tunnel with a 1 m rail gage once connecting Lure (Haute-Saône, France) with Le Thillot (Vosges, France). After its closure in 1962, it was used for the supply of potable water and was therefore locked. Niphargus lotharingiensis sp. nov. can be found in a variety of subterranean biotopes (Supp. file 4.6). However, the inferred absence in the interstitial environment is most likely due to the fact that the species does not occur in the area where the interstitial environment was intensively sampled. Fig. 56. Distribution of Niphargus lotharingiensis Weber & Brad sp. nov. confirmed by molecular data. The type locality at the Tunnel du Col des Croix (France) is marked by a yellow star, overlaying one site record. Circles and diamonds refer to two distinct genetic clades. Very large freshwater bodies are indicated; country codes follow the ISO 3166 standard alpha-2 format. WEBER D. et al., Six new species of the Niphargus aquilex complex (Amphipoda) 73 Discussion Notes on crypsis and pseudocrypsis in the N. aquilex species complex With the recent integration of DNA characters, Weber et al. (2023) provided the first solid molecular framework to shed further light on the complex taxonomic situation within the N. aquilex species complex. In the present work, by adding new molecular data as well as an in-depth morphological (re)investigation, we newly described six of the molecular entities delineated by Weber et al. (2023). Morphologically, the six species described herein can be considered pseudocryptic because several minor morphological differences were observed. In general, species are counted as cryptic if they cannot be distinguished by their morphology, that is, if the morphological variation within a species is that high (i.e., variable) or low (i.e., static) that it widely overlaps with its congeners. Cryptic species of Niphargus can only be distinguished by means of methods other than morphology, which are currently mainly molecular characters in their DNA. By incorporating other lines of evidence (e.g., DNA sequences) and morphologically re-examining the specimens at hand according to the proposed genetic clustering, it may become apparent that other morphological characters can serve as diagnostic traits to allow robust species delineations (Knowlton 1993; Luttikhuizen & Dekker 2010). The N. aquilex species complex is an excellent example of a combination of cryptic and pseudocryptic species pairs. The six species described herein can be best distinguished by the nucleotide sequences of their COI and 28S rDNA gene fragments, along with a few morphological differences. Pure diagnostic nucleotide positions in their COI barcodes – allowing the unambiguous identification of a single species from one or a few single nucleotide polymorphisms (SNPs) – were scarce or even non-existing for some species (N. schellenbergi sensu stricto, N. normandiensis sp. nov., N. lotharingiensis sp. nov.). A notable exception was N. palatinensis sp. nov., which showed six pure diagnostic nucleotide positions. The reduced number of informative (diagnostic) SNPs to identify single species can be best explained by the combination of the following aspects: a) COI comprises a rather fast-evolving protein-coding gene, with the degeneracy of the genetic code allowing codons to mutate more easily while still coding for an identical amino acid, b) nucleotide data has only four character states (A, T, G, C), and homoplastic characters can arise over time due to parallel mutations at homologous sites, c) the number of unique DNA sequence variants was high in several species (213 for N. schellenbergi sensu stricto, 79 for N. luxemburgensis sp. nov., and 51 for N. aquilex sensu stricto), and d) the analysis of sister species pairs within a hyperdiverse species complex with more similar genetic sequence variants when compared to more distantly related congeners. All these aspects reduce the likelihood of observing pure diagnostic SNPs for a single species. Notes on molecular species delimitations In a more comprehensive ASAP species delimitation approach, the new genetic data yielded between 16 and 21 potential species, while initially only 10 to 15 potential species were delineated (Weber et al. 2023). However, not all potential species groups were affected by ASAP analysis of our comprehensive molecular dataset. As such, the newly described species N. palatinensis sp. nov. (former species A sensu Weber et al. 2023) and N. wasgauensis sp. nov (former species I sensu Weber et al. 2023) always formed a single distinct molecular cluster (Table 1). The same situation holds true for the former species B sensu Weber et al. (2023), which, based on three specimens found in the stated area of the type locality (Crowborough, UK) (Karaman 1980), can be assigned to N. aquilex sensu stricto. This taxon has not been re-described here because a comprehensive re-description is available (Karaman 1980). For the other taxa morphologically examined in the present study, the genetically enriched ASAP analysis provided a more complex situation. The two molecular lineages, S and R+R* sensu Weber et al. (2023), of the morphospecies N. schellenbergi were clearly separated by their 28S rDNA markers, but ASAP analysis failed to clearly separate them. This result, however, was reasonable given that ASAP is a genetic distance-based approach, and only two mutational steps are present between their nuclear 28S rDNA alleles. However, COI partitions also differed in their results. Partition 1 clustered both potential species