Abstract
The genus Niphargus is the most diverse subterranean amphipod genus in the western Palearctic region but, owing to the presence of cryptic species and homoplasy, its taxonomy and biogeographic scenarios are complex, making molecular methods essential to understand its evolution. We conducted a study combining dna-based taxonomy with traditional morphotaxonomy to investigate Niphargus bihorensis Schellenberg, 1940, known from the Western Alps and Carpathians. We redescribed the type material of N. bihorensis from Bihor County, Romania, and revealed the presence of a cryptic species, N. absconditus n. sp., in the same karstic area (Pădurea Craiului Mountains). Additionally, the Alpine populations previously attributed to N. bihorensis turned out to belong to a new, not so closely related species, N. tizianoi n. sp. Phylogenetic analyses based on a concatenated dataset of one mitochondrial and two nuclear markers suggest that the N. bihorensis species complex belongs to a strongly supported clade, together with several species distributed from Switzerland to Iran.
Zoobank: urn:lsid:zoobank.org:pub:7E9D48F7-B055-45E3-81A5-73B3D6DAFD8B
Introduction
The genus Niphargus (Schiödte, 1849) is the most common and species-rich subterranean amphipod genus in the western Palearctic. Although this genus comprises more than 450 described species (Horton et al., 2023), many of its clades show low morphological disparity (Guillerme et al., 2020). Indeed, the subterranean environment presents several selective pressures caused by darkness and oligotrophy (Culver et al., 2023), which can lead to homoplasy (Fiser et al., 2008) and morphological stasis, resulting in frequent occurrence of cryptic species (Zagmajster et al., 2014). This hampers the possibility of drawing conclusions based solely on morphological characters (Trontelj et al., 2012), resulting in complex taxonomic and biogeographic situations.
This difficulty to identify niphargid species also makes it difficult to infer correctly their distribution ranges. Although, in groundwater-dwelling species, ranges > 200 km are rare (Trontelj et al., 2009), and although there are niphargids with larger distribution areas – Weber et al. (2020) reported that the range of Niphargus puteanus (Koch, 1836) reaches 756 km – species reported to have broad geographic ranges remain a puzzle questioning either the generality of the restricted range hypothesis of groundwater species, or suggesting taxonomic inaccuracy and the possible presence of cryptic diversity. For instance, Niphargus aquilex Schiödte, 1855 was first described in England but it is believed to be widely distributed in Central Europe, Italy, Slovenia, and other parts of the Balkan region, whereas N. tauri Schellenberg, 1933 was thought to have a distribution area extending from Italy and Slovenia as far east as Turkey (Taurus Mountains). Molecular species delimitation methods revealed that the species displaying a Niphargus aquilex morphotype in northwestern and central Europe may form a complex of undescribed cryptic species (Weber et al., 2023), but further research is needed. Similar cases have been found in several populations reported as N. puteanus in Italy, Germany, and Romania: the distribution of this species seems restricted to southern Germany and Austria (Weber et al., 2020). The difficulty in identifying distinctive morphological characters, especially when differences between species are small and intraspecific variability is high (Fišer et al., 2009b), has made the application of molecular methods essential for resolving such cases and reconstructing at least some aspects of the complex phylogeny of this species-rich genus.
In the present study, we investigated the intriguing case of a single species (Niphargus bihorensis Schellenberg, 1940) spread across vastly separated regions. Schellenberg (1940) described Niphargus foreli bihorensis from Meziad Cave (Peştera Meziad), Western Carpathians, Bihor County, Romania. Later, Karaman (1980), elevating this subspecies to species rank, documented its presence in an Italian locality in the Western Alps. The precise location in Italy was difficult to trace; Karaman (1980) reported “Val Sabbiola, Varallo, Como, May 2, 1953”. Fortunately, the specimens studied by Karaman were deposited at the Natural History Museum of Verona, where the complete label reports the name of the collector, Carlo Moscardini. By scrutinizing the grey biospeleological literature, and with the aid of a local biospeleologist (Tiziano Pascutto), we uncovered that Val Sabbiola (in the province of Vercelli, not Como) was studied by Moscardini (1955), who visited the sole cave present in the area (named “Böcc d’la Büsa Pitta”) and on 1 May 1953, collected numerous specimens of Niphargus sp. in a pool situated in the hall. The cave, which develops into schists, not limestones, was later visited by one of us (fs) together with T. Pascutto in 2002, and some specimens of Niphargus were found in the pool, corresponding exactly to Karaman’s description. The same species was later found in other localities in the Central and Western Alps in Italy, several sites in Switzerland, and, putatively, in an Austrian cave near Salzburg. Although the description of the Italian material by Karaman (1980) was quite accurate, allowing the identification of the newly found populations, the same cannot be said for Romanian N. bihorensis, whose original description (Schellenberg, 1940) and later illustrations (Cărăuşu et al., 1955) were rather imprecise. Additionally, a study based on dna sequences of populations from the type locality (Meziad Cave) and a nearby cave (Vadu Crişului cave), in the western Apuseni Mountains, suggested the presence of a cryptic species in the second cave (Meleg et al., 2013).
The availability of new material and dna sequences for several species of the genus Niphargus has made it possible to re-examine the above-mentioned taxonomic problems using a combined morphological and molecular approach (i.e., an integrative taxonomy approach sensu Dayrat, 2005). The aim of this study was to determine whether the Italian, Swiss, Austrian, and Romanian populations belong to the same species, and, if more than one species was recognized, whether they form a monophyletic group or whether their similarity is due to convergence. Additionally, this study aimed to clarify the position of the studied species in the phylogeny of the genus Niphargus to determine their relationships, and thus reconstruct the phylogeny and biogeography of the clade of membership.
Material and methods
Specimen collection and retrieval
The newly collected specimens are listed in supplementary table S1, including specimens collected during previous surveys and dna sequences that were retrieved from GenBank.
Italy
Samples were collected in seven sites during a survey to resolve the phylogeny and taxonomy of Italian amphipods (Stoch and Flot, 2017). Specimens were collected from caves (pools and subterranean brooks), mines, wells, and springs using a net with a handle for macrobenthic surveys (mesh size 0.25 mm, nhbs GmbH, Bonn, Germany) or commercial aquarium nets for small pools. Baited traps (plastic bottles using chicken liver as bait) were positioned for 24 h in wells and caves.
Switzerland
Swiss samples were collected in six sites by local drinking water providers as part of a countrywide campaign including citizen-science approaches (Altermatt et al., 2014; Alther et al., 2021; Couton et al., 2023). Drinking water providers accessed groundwater through perforated horizontal pipes (Alther et al., 2021; Studer et al., 2022). Specimens were collected using two different methods. First, by attaching a filter net (mesh size 0.8 mm, Sefiltec ag, Höri, Switzerland) to the inlet of the drainage pipe, to collect organisms from the passively flowing spring water. Second, by sampling the sedimentation/overflow basin of the spring box with a small hand net (mesh size 0.35 mm, jbl GmbH & Co. kg, Neuhofen, Germany).
Austria
A single site of a specimen morphologically similar to N. bihorensis was discovered during an extensive survey of the Austrian amphipod cave fauna conducted by Erhard Christian (University of Vienna) in collaboration with local speleological groups. Samples were collected using a hand net.
Romania
Specimen collection in the caves of the Pădurea Craiului Mountains, Western Carpathians, was detailed in the paper by Meleg et al. (2013). The type material of Niphargus bihorensis was requested for redescription to the Natural History Museum in Berlin (Germany).
dna isolation, amplification, and sequencing
The collected specimens were preserved in 75% or 96% ethanol and deposited in the Zoological Collection of the Department of Biology, Biotechnical Faculty, University of Ljubljana, Slovenia, and in the collection of the Université libre de Bruxelles (ulb), Belgium. All information on the specimens used in the analyses are available in supplementary table S1. Genomic dna was isolated from one of the pereopods using GenElute Mammalian Genomic dna (Sigma-Aldrich, USA) in Ljubliana and NucleoSpin@ Tissue kit (Macherey-Nagel) in Bruxelles, following the manufacturer’s protocol. Where possible, the rest of the specimen was used for further morphological studies. We amplified nuclear dna gene fragments (28S rRNA and histone H3) and a mitochondrial gene fragment (coi, i.e., Folmer’s fragment of cytochrome oxidase I; Folmer et al., 1994). A list of primers and pcr amplification protocols is available in supplementary table S2. pcr products were purified using Exonuclease I and FastAP (Thermo Fisher Scientific Inc., United States) according to the manufacturer’s instructions. Each fragment was sequenced in both directions using amplification primers from Macrogen Europe (Amsterdam, The Netherlands) and Genoscreen (Lille, France). The chromatograms were assembled and edited using Geneious 11.1. (Biomatters, New Zealand) and Sequencher 5.4.6 (Gene Codes). Some chromatograms of the nuclear marker H3 contained double peaks; these double peaks were coded in the resulting sequences using 1-letter iupac ambiguity codes (Cornish-Bowden, 1985).
Phylogenetic analysis
To assess the monophyly of the N. bihorensis species complex and establish interrelationships among its constituent members, we used a dataset consisting of 170 Niphargidae individuals (supplementary table S3) from a wide range of Niphargus species, including Haploginglymus Mateus & Mateus, 1958 nested within the genus Niphargus. The outgroup comprised two species of Pseudoniphargus Chevreux, 1901 and Microniphargus leruthi Schellenberg, 1934, both attributed to the family Pseudoniphargidae (Weber et al., 2021). The dataset comprised all available putative closely related species based on H3, 28S, and coi gene sequences (see supplementary table S1). 28S sequences were aligned prior to concatenation using the g-ins-i algorithm implemented in mafft v.7 (Katoh & Standley, 2013). The total length of the concatenated dataset was 2,070 bp. Considering the presence of heterozygous bases in the third codon position of H3 and the saturation (for transversions as well as for transitions) of the third codon positions of coi demonstrated for Niphargidae by Stoch et al. (2023), only the first and second H3 and coi codon positions were retained for phylogenetic analyses. The best partitioning scheme and optimal substitution models for the codon positions were searched using PartitionFinder 2.1.1 (Lanfear et al., 2017) and are listed in supplementary table S2. Phylogenetic relationships were reconstructed using the maximum likelihood and Bayesian inference. A maximum likelihood phylogeny using partition-specific settings and bootstrapping was constructed using iq-tree 2 (Nguyen et al., 2015). The node support for the maximum likelihood analysis was assessed using 1,000 ultrafast bootstrap replicates (Hoang et al., 2018). The Bayesian phylogeny with partition-specific settings was inferred in MrBayes 3.2.6 (Ronquist & Huelsenbeck, 2003) with two independent mcmc runs with four chains each for 20 million generations, and trees were sampled every 1,000 generations. After reaching the stationary phase, the first 25% of the trees were discarded as burnin, and a 50% majority rule consensus tree was calculated from the remaining trees. Phylogenetic analyses were run on the cipres Science Gateway V.3.3 (Miller et al., 2015; accessible at www.phylo.org).
Molecular species delimitation methods and haplotype networks
To delimit species in the focal clade identified by the phylogenetic trees, we applied asap and ptp to all available coi sequences (reported in tab. S1), checking the results by examining haplotype networks.
We ran Assemble Species by Automatic Partitioning (asap; Puillandre et al., 2021) with the Kimura two-parameter substitution model using the web server https://bioinfo.mnhn.fr/abi/public/asap/asapweb.html (last accessed 29 February 2024).
Poisson tree process (ptp) was performed by building a coi-based ml phylogenetic tree obtained using iq-tree 2. ptp analysis was run on the species delimitation server https://mptp.h-its.org/#/tree (last accessed 29 February 2024) assuming a probability of p < 0.001. We also ran multirate ptp (mPTP), but it lumped together several different species; for this reason, the results were not taken in consideration. More details on ptp are reported by Kapli et al. (2017).
Median-joining haplotype networks using all the available coi, 28S, its, and H3 sequences (supplementary table S1) were built using HaplowebMaker (Spöri & Flot, 2020; available online at https://eeg-ebe.github.io/HaplowebMaker/, last accessed 29 February 2024). The results of species delimitation methods were reported on these networks, as well as on the distribution map, attributing the same color to all the specimens belonging to the same species. To avoid clogging the network and allow displaying all the sequences (including older sequences that had likely not been scrutinized for the presence of double peaks in the chromatograms and for which chromatograms were not available anymore), the HaplowebMaker option “Mask” was used to deal with the ambiguity codes of H3 to build the network.
Morphological analysis
Identification and dissection were carried out using a Zeiss Stemi sv11 stereomicroscope. Specimens intended for deposition in collections were preserved in 75% ethanol with 10% glycerine. Selected specimens were placed on slides in glycerin and sealed with epoxy glue. Drawings and measurements were performed using a Zeiss Axioskop microscope equipped with a drawing tube (100–1000× magnification) and Nomarski’s differential interference contrast (dic). Drawings were scanned, converted to black and white bitmaps, and digitally inked following Coleman’s (2003, 2009) techniques and using Inkscape 1.2 (https://inkscape.org/, last accessed 29 February 2024).
To determine the differences between the two populations from the Bihor Mountains, we selected 5 adult males and 5 adult females from each population, partially dissected them in glycerol, and mounted them on temporary slides in glycerol. In addition to 11 quantitative traits for which we found no differences in a previous study (Meleg et al., 2013), we checked or quantified other 27 traits (counts/presence absence), which proved useful for the discrimination of many closely related species in previous studies (Fišer et al., 2009b). Importantly, many of these traits were not used in taxonomy before 1990 and could have been overlooked by previous researchers.
Molecular diagnosis of species
Molecular diagnoses were based on coi Folmer’s fragment sequences and constructed using MolD (Fedosov et al., 2022). The software searches for a minimum number of nucleotides to generate a robust diagnosis for a species. The robustness of the diagnosis is assessed as a bootstrap value, calculated in simulation of mutations of the so called “redundant nucleotides”, which are unique and sufficient to distinguish a query taxon from all reference taxa in dataset. We employed the following settings: maximum of five undetermined nucleotides, maximum length of the minimum number of nucleotides set to 100 and 1000 randomizations. The dataset of the aligned coi sequence fragments used is listed in tab. S5 to ensure reproducibility of the diagnosis (Jörger & Schrödl 2013).
Results
Maximum likelihood (iq-tree; supplementary fig. S1) and Bayesian (MrBayes; supplementary fig. S2) phylogenetic analyses were congruent in suggesting that the western Alpine populations and Romanian N. bihorensis form a strongly supported clade which, considering its oldest described species, is herein referred to as the Niphargus tauri clade. Furthermore, asap and ptp results (supplementary table S4) as well as visual inspection of the coi haplotype networks (fig. 1) suggested that this clade comprised at least 22 species, among which eight potentially new to science. The species of the N. tauri clade showed a distribution area spanning from Switzerland to Iran (fig. 2a); a detailed maximum likelihood phylogeny of the clade is presented with the map (fig. 2b).
Median-joining network of different sequences of the members of the Niphargus tauri clade: (a) coi; (b) 28S, its in the box; and (c) h3. Every color indicates a different putative species; mutations are reported on branches as short segments; circle diameter is proportional to the number of individuals sharing the same haplotype. Colors are the same as in the map in fig. 2, except for the largest circle in (c) that includes more than one species sharing the same H3 haplotype.
Citation: Contributions to Zoology 93, 4 (2024) ; 10.1163/18759866-bja10064
(a) Map showing the distribution of the species of the Niphargus tauri clade. The glacier extension during the Last Glacial Maximum (lgm, 21 Kya: Clark et al., 2009) is displayed. Habitus of N. tizianoi n. sp. displayed in the left upper pane (topotype male, 8 mm). (b) Maximum-likelihood phylogenetic tree (iq-tree2) of the N. tauri clade using a concatenated set of markers, i.e., H3 (1st and 2nd codon positions), 28S, and coi (1st and 2nd codon positions). The tree is rooted using the N. bajuvaricus clade as outgroup. Node support is expressed as ultrafast bootstrap values/Bayesian posterior probabilities.
Citation: Contributions to Zoology 93, 4 (2024) ; 10.1163/18759866-bja10064
Morphological analysis revealed that five of these species (N. tizianoi n. sp., N. sp. Mulini, N. sp. Salzburg, N. sp. Huda luknja, N. absconditus n. sp.) are very similar to N. bihorensis, forming a species complex, whereas other species (N. sp. Podutik, N. dobati, N. sp. aff. dobati A and B, and N. tauri) resemble a member of another clade, Niphargus aquilex. Additionally, the its marker (fig. 1b) studied in the western Alpine populations supported species differentiation based on asap, clearly distinguishing N. tizianoi n. sp. from the closest N. sp. Mulini. The distribution map (fig. 2a) indicated that Slovenia and northeastern Italy, particularly along the Slovenian border, had the highest concentration of species (8 of 22), followed by northwestern Italy. The other representatives of this clade are localized in few sites distributed over a wider range.
Limiting our analysis to the N. bihorensis species complex identified on a morphological basis and given the extensive material available for morphological and molecular studies, we herein describe N. tizianoi n. sp. from the western Alps and N. absconditus n. sp. from Romania. Unfortunately, the material from the other sites hosting the other potential new species was insufficient for a thorough morphotaxonomic analysis, precluding their morphological description.
For the molecular diagnosis of the three species in the Niphargus bihorensis species complex defined above, we compared the coi sequences of each of the three species with all available sequences of the Niphargus tauri clade. The alignment comprised 57 sequences, of which 7, 13, and 15 belonged to N. bihorensis, N. absconditus sp. n., and N. tizianoi sp. n., respectively (supplementary table S5). Molecular diagnoses are reported in the species descriptions below.
Species description
Order Amphipoda Latreille, 1816
Family Niphargidae Bousfield, 1977
Genus Niphargus Schiödte, 1849
Niphargus bihorensis Schellenberg, 1940
Type series. Meziad Cave, Romania (original label “Rumänien, Pesterea Meziadului, ca. 480 m. in einem grossen Tümpel”), 21.08.1938, legit R. Leruth, 7 males, 4 females; deposited in the Museum für Naturkunde, Berlin, Germany, catalogue number 25164. Considering that Schellenberg did not select a holotype but wrote on the label included in the vial “typus”, we selected one male (6.5 mm length) as lectotype, and 10 specimens (6 males and 4 females) as paralectotypes.
Type locality. Meziad Cave (= Peştera Meziad, coordinates wgs84: longitude 22.478754 °E, latitude 46.763105°N), Apuseni Mountains (Western Carpathians), Bihor district, Romania.
Other material examined. Meziad Cave, 29.09.2011, legit I. Meleg, several specimens (5 specimens used for morphological analysis, 9 specimens used for molecular analysis).
Description of male lectotype. Body length of 6.5 mm. Habitus as in fig. 3a. Head length less than 10% of body length; rostrum absent. Pereonites i–vii with 2‒3 postero-ventral sensorial setae. Pleonites i–iii with 2‒3 small dorsal sensorial setae and 2 postero-distal setae (fig. 3a).
Niphargus bihorensis, male (lectotype). (a) Habitus; (b) antenna i; (c) antenna ii; (d) right mandible; (e) mandibulary palp; (f) maxilla i; (g) maxilla ii; (h) maxilliped, palp; (i) maxilliped, inner lobe; (j) maxilliped, outer lobe.
Citation: Contributions to Zoology 93, 4 (2024) ; 10.1163/18759866-bja10064
Antenna I (fig. 3b) about 30% of body length. Flagellum of 18 articles; each article with 1 aesthetasc, its length about one third of article length; aesthetasc accompanied by 3‒4 setae. Distal article shorter than the aesthetasc of penultimate article, bearing 6 distal setae and 1 posterodistal seta. Peduncle triarticulated (fig. 3b), proportions of articles length 1:2:3 as 1.0:0.7:0.2. Accessory flagellum biarticulated; distal article as long as one third of proximal article.
Antenna ii (fig. 3c) as long as one half of antenna I. Flagellum of 9 articles; each article bearing an aesthetasc; distal article bearing 4 distal setae accompanied by 1 shorter aesthetasc. Peduncle with proportions of articles length 1:2:3 as 1.0:3.0:2.9; flagellum 70% of peduncle length.
Mandibles (fig. 3d). Right mandible: incisor process with 4 teeth, lacinia mobilis with 2 teeth; between lacinia mobilis and pars molaris a row of thick, serrated setae is present; 3 small spines accompanying pars molaris. Left mandible (fig. 3d): incisor process with 4 teeth, lacinia mobilis with 2 teeth; between lacinia mobilis and pars molaris a row of 7 thick, serrated setae is present; 3 small spines accompanying pars molaris; long seta accompanying pars molaris present. Proportions of mandibular palp articles (fig. 3e) 1:2:3 as 1.0:2.5:2.4. Proximal palp article without setae; second article with 7 setae; distal article with 4 A setae on the outer side and 2 B setae on the inner side; C setae absent; 9 D setae and 4 E setae present, the distal one longer than the article.
Maxilla I (fig. 3f). Palp articles ratio 1:3, with distal article bearing 4 apical setae. Outer lobe with 6 spines with 1 small tooth and the 7th, inner spine with several small teeth. Inner lobe elongated (3 times longer than wide), with 1 distal seta.
Maxilla ii (fig. 3g). Inner lobe 0.8 times as long as outer lobe; both of them with the apical and subapical setae as usual in the genus.
Maxilliped. Palp (fig. 3h) article 1 bearing 1 inner seta; article 2 with 26 inner setae; article 3 with 4 outer setae and 5 inner setae in the distal part, and a row of 6 distal medial setae; article 4 with 1 outer seta and with only 1 short seta close to the insertion of nail. Outer lobe (fig. 3i) with 5 flattened, thick inner spines and 6 distal setae, proximal part of inner margin bearing 3 setae. Inner lobe (fig. 3j) bearing 6 stout apical setae accompanied by 3 thick spines.
Gnathopod i (fig. 4a). Coxa subquadrangular, with 7 short setae on anterior margin. Basipodite short and stout, 1.75 times longer than wide; ischiopodite, meropodite and carpopodite shape and setation as illustrated in fig. 4a. Propodite with convex, slightly inclined palm, bearing along posterior margin 4 groups of 3 setae each; anterior margin with sparse setae, and a row of 5 facial setae close to the insertion of palmar spine; antero-distal group of 5 setae close to the insertion of dactylopodite. Palmar corner with a strong palmar spine, accompanied by 2 short spines. Dactylopodite bearing 1 seta along anterior margin; distal nail as long as 2/3 of dactylopodite.
Niphargus bihorensis, male (lectotype) and female (paralectotype). (a) Male gnathopod i; (b) female gnathopod i; (c) male gnathopod ii; (d) male pereopod iii; (e) male pereopod iv.
Citation: Contributions to Zoology 93, 4 (2024) ; 10.1163/18759866-bja10064
Gnathopod ii (fig. 4c). Coxa subrounded; anterior margin with 6 short setae. Gill (epipodite) narrow, as long as basipodite. Basipodite elongated, 2.7 times longer than wide; ischiopodite, meropodite and carpopodite shape and setation as illustrated in fig. 4c. Propodite 1.3 times longer than propodite of gnathopod I; palm convex and less inclined than palm of gnathopod I, bearing along posterior margin 5 dense rows of setae; palmar anterior margin with few setae, and an irregular row of 4 facial setae close to the insertion of palmar spine; antero-distal group of 6 setae close to the insertion of dactylopodite. Palmar corner with a strong palmar spine accompanied by 1 short and stout spine. Dactylopodite bearing 1 seta along anterior margin; distal nail longer than one half of dactylopodite.
Pereopods iii–iv (fig. 4d, e). Coxal iii‒iv plates subquadrate with 5 marginal setae each; gill iii and iv as long as basis, and longer than basis, respectively; gills narrow. Pereopods iii and iv approximately subequal (ratio iii:iv as 1.1:1), shape and setation as in fig. 4d, e. Dactylopodites iii–iv with a single dorsal plumose seta, and one short spine on ventral side, close to the insertion of nail.
Pereopods v–vii (fig. 5). Proportions of pereopods v: vi: vii as 1.00:1.7:1.8, shape and setation as in fig. 5. Pereopod vii (fig. 5c) length about 40% of body length. Coxa v–vi narrow and elongated, ratio width:maximum length about 2.2, bearing few short setae; coxa vii small, subrounded, 2.5x wider than long with 2 posterior setae. Gills (epipodites) on pereopod v and vi as long as coxal width. Basipodites v‒vii, with straight or slightly concave posterior margins, without distal lobes; posterior margins with a regular row of short setae; anterior margins with setae only. Ischiopodites v‒vii with a marked notch. Dactylopodites v-vii with nails length about one third of total dactylus length; dorsal margins with a single plumose seta; ventral margin bearing only one short spine, accompanied by a tiny seta, near nail insertion (fig. 5d).
Niphargus bihorensis, male (lectotype). (a) Pereopod v; (b) pereopod vi; (c) pereopod vii; (d) dactylopodite of pereopod vii.
Citation: Contributions to Zoology 93, 4 (2024) ; 10.1163/18759866-bja10064
Pleopods (fig. 6c). Pleopods i–iii protopodites (peduncles) with 3 hooked retinacles. Rami (exopods and endopods) of 7 articles with 2 long plumose setae each.
Niphargus bihorensis, male (lectotype: a-g) and female (paralectotype: h). (a) Pleonite 3, dorsal side in lateral view; (b) epimeral plates i-iii; (c) pleopod iii; (d) uropod iii (male); (e) endopodite of uropod iii (male); (f) urosome and uropods i and ii (male); (g) telson, dorsal view; (h) uropod iii (female).
Citation: Contributions to Zoology 93, 4 (2024) ; 10.1163/18759866-bja10064
Uropods. Uropod i (fig. 6f) protopodite with 2 longitudinal rows of 3‒4 dorsal spines and 2 distal spines close to the insertion of exopodite; length ratio endopodite:exopodite as 1.0:0.9, rami quite straight; endopodite with 1 dorsal spine and 3 terminal spines; exopodite with 1 dorsal spine and 4 terminal spines. Uropod ii (fig. 6f) protopodite short, one-half length of uropod I protopodite, with 2 dorsal spines, and 2 distal spines close to the insertion of exopodite; endopodite and exopodite subequal in length, bearing 1 dorsal spine and 3‒4 distal spines. Uropod iii (fig. 6d) as long as 45% of body length; protopodite 1 distal spine close to the insertion of exopodite; endopodite (fig. 6e) short (2 times longer than wide), 30% of protopodite length, apically with 2 setae and no spines; exopodite 2-articulated, distal article elongated, approximately 70% of proximal article; proximal article with 4 groups of 2‒3 spines; distal article with setae only, as in fig. 6d.
Telson (fig. 6g). Telson 2.5 times longer than wide, cleft 80% of length; lobes apically bearing 4 spines each, as long as 1/3 of lobe; lateral margin with 2 plumose setae; dorsal surface without setae and spines.
Epimeral plates (fig. 6b). Epimeral plate I with 2 posterior setae; plate ii and iii with 2 submarginal ventral spines and 2 posterior spines; plate iii with posterior margin slightly convex, posterior ventral corner slightly rounded.
Urosomites (fig. 6f) with 0‒2 dorso-lateral setae. One short spine present near uropod I insertion.
Description of female paralectotype. Habitus as in male; adult females with large oostegites, illustrated for gnathopod ii in fig. 4b. Gnathopod ii slightly smaller than in male (fig. 4b). Uropod iii exopodite shorter than in male (fig. 6h), distal article flattened, about 40% of proximal article in length. Setation as in fig. 6h.
Remarks. In the original description by Schellenberg (1940) the four drawings were quite poor and with few details, taken from a 6.5 mm long male, i.e., the same described above. For this reason, and given the presence of a cryptic species, the first detailed description of the species is reported above. As regards variability, the description of 27 morphological characters is reported in table 1 for 3 males and 2 females (topotypes). The holotype agrees in all characters with the other topotypes apart the number of articles of antenna I flagellum (18 instead of 21‒22) and the number of ventro-posterior setae on pereonite vii (2‒3 instead of 1‒2), that are within the variation reported for N. absconditus n. sp. Up to now, the species is known only from Meziad cave; details of its ecology are reported in Meleg et al. (2013).
Diagnostic molecular characters. The species can be distinguished from N. absconditus n. sp. and from all other niphargid species on the basis of coi and 28S dna sequences (supplementary table S1). coi: kf218661-666 and ok156517; 28S: kf218727 (GenBank Accession Numbers).
Molecular diagnosis (coi). ‘G’ at site 424, ‘C’ at site 514; support: 97%.
Niphargus absconditus n. sp. Fišer, Stoch & Salussolia
https://zoobank.org/Nomenclatural Acts/3167505F-7C27-441F-8709-5A23A56EE867
Type series. Vadu Crişului Cave, Apuseni Mountains (Western Carpathians), Bihor County, Romania, 29/09/2011, leg. I. Meleg, 2 males, 3 females. Holotype (male) and paratypes (1 male, 3 females) deposited in the amphipod collections of the University of Ljubliana, code n. na797 (holotype), na798 (male paratype) and na794-na796 (female paratypes).
Type locality. Vadu Crişului cave (wgs84 coordinates: longitude: 22.511003° E, latitude 46.962406° N), Romania.
Derivatio nominis. The adjective ‘absconditus’ in Latin means ‘hidden’, ‘cryptic’.
Description and remarks. Being the morphological characters very similar to those of N. bihorensis, a complete description is not given, focusing on few relevant aspects.
Holotype male, body length 6.0 mm. Antenna i (fig. 7a), flagellum of 20 articles; distal article shorter than in the type of N. bihorensis. Antenna ii (fig. 7b), flagellum of 7 articles. Mouthparts as in N. bihorensis. Maxilla 1 (fig. 7c) with palp articles ratio 1:2, distal article bearing 5 apical setae. Gnathopod i (fig. 7d) as in N. bihorensis.
Niphargus absconditus n. sp. male (holotype). (a) Antenna i; (b) antenna ii; (c) maxilla i; (d) gnathopod i; (e) gnathopod ii; (f) urosome with uropods i and ii, basipodite and endopodite of uropod iii, telson (lateral view); (f) urosomites ii and iii (right to left) with epimeral plates.
Citation: Contributions to Zoology 93, 4 (2024) ; 10.1163/18759866-bja10064
Gnathopod ii with elongated basipodite. 3 times longer than wide. Pereopods iii (fig. 8a), iv (fig. 8b) and v (fig. 8c) as in N. bihorensis. Pereopod vi (fig. 8d) and vii (fig. 8e) slender than in N. bihorensis; basipodites with straight posterior margins, without distal lobes, approximately 2 times longer than wide.
Niphargus absconditus n. sp. male (holotype). (a) Pereopod iii; (b) pereopod iv; (c) pereopod v; (d) pereopod vi; (e) pereopod vii.
Citation: Contributions to Zoology 93, 4 (2024) ; 10.1163/18759866-bja10064
Pleopods as in N. bihorensis. Uropods 1 and 2 (fig. 7f) with rami more elongated than in the type of N. bihorensis; distal spines longer and more gracile, but in equal numbers than in the previous species. Endopod of uropod 3 more elongated than in the type of N. bihorensis, about 2 times longer than wide. Epimeral plates (fig. 7g) and telson as in N. bihorensis.
Apart from the slightly more elongated pereopods and uropodal rami, no sound morphological differences could be found between the population from Vadu Crişului Cave and the population from Meziad Cave (typical N. bihorensis); thus, the new species can be considered cryptic with regards to N. bihorensis. All the morphological traits studied showed a considerable degree of overlap between the two populations (table 1). The most promising characters appeared to be the dorsal spine on the telson lobes, found only in males. Most males from the Meziad Cave had this spine, but only one male from the Vadu Crişului Cave had it; however, the N. bihorensis holotype in Schellenber’s collection lacked this spine. Even this trait, polymorphic in both populations, cannot be used for a diagnosis. It is possible that other diagnostic traits or diagnostic combinations of traits were overlooked; however, with our current knowledge of morphology, we were unable to discriminate between these two populations.
Up to now, the new species is known only from Vadu Crişului Cave; details of its ecology are reported in Meleg et al. (2013).
Diagnostic molecular characters. The species can be distinguished from N. bihorensis and from all other niphargid species on the basis of coi and 28S dna sequences (supplementary table S1). coi: kf218667-679; 28S: kf218731-732 (GenBank Accession Numbers).
Molecular diagnosis. ‘G’ at site 292, ‘G’ at site 325, ‘G’ at site 502; support 100%.
Niphargus tizianoi n. sp. Stoch, Knüsel & Flot
https://zoobank.org/NomenclaturalActs/1B8A28B7-AE46-4C48-98C6-277D70AD4F1F
Type series. Böcc d’la Büsa Pitta, Italy (original label “Val Sabbiola, Varallo”), 02.05.1953 legit C. Moscardini; holotype male specimen (mounted by Karaman on slide n. 3592- 93); paratypes one female (mounted on slide 3594) and 8 specimens preserved in alcohol 75° (Ruffo collection, Museo Civico di Storia Naturale, Verona, Italy). Type locality. Böcc d’la Büsa Pitta (cadastre number 2517 Pi/vc, coordinates wgs84: 8.255532°E, 45.873458°N), Val Sabbiola, municipality of Varallo, province of Vercelli, Piedmont, Italy (in the paper by Moscardini, 1955, the municipality was Sabbia, suppressed in 2018 and aggregated to Varallo). Furthermore, Moscardini (1955) named the cave as “Boeuce dla Büsa pitta”, unknown to speleology up to that time, and the date of collection as 01.05.1953.
The specimens were collected in a pool of water in the entrance hall. The cave developed in schists.
Other material examined. Böcc d’la Büsa Pitta (2517 Pi/vc), 05.07.1997, legit M. Bodon, T. Pascutto, A. Balestrieri, 5 specimens; 22.04.2000, legit. S. Bugalla, 6 specimens; 06.10.2002, legit T. Pascutto, F. Stoch, G. Tomasin, 10 specimens; 28.03.2012, legit E. Lana (10 specimens, 4 of them preserved for dna sequencing); all the specimens collected in the pool of percolating water located in the entrance hall. Mine Alpe Cima l’Ert, Valle Cervo, Quittengo, Biella (coordinates wgs84: 8.015303° E, 45.668795° N), 22.02.1997, legit T. Pascutto (1 male, collected in a pool). Specimens used for dna sequencing: Spring in Val di Rabbi, Trento (Italy), 19/08/18, legit F. & S. Stoch; Costa Borgnone Spring, Switzerland, 29/05/2019, legit N. Bongni, R. Alther, F. Altermatt; Spring at Baerenburg, Andeer, Switzerland, 24/08/2021, 02/12/2021, legit M. Knüsel, R. Alther, F. Altermatt; Spring 1, Gordevio, Switzerland, 26/07/2021, 02/08/2021, legit M. Knüsel, R. Alther, F. Altermatt; Spring Anzonico, Faido, Switzerland, 02/12/2021, legit M. Knüsel, R. Alther, F. Altermatt; Spring at Chantarella, St. Moritz, Switzerland, 26/07/2021, legit M. Knüsel, R. Alther, F. Altermatt; Spring above Quellenberg, St. Moritz, Switzerland, 02/08/2021, legit M. Knüsel, R. Alther, F. Altermatt.
Derivatio nominis. The new species is dedicated to the speleologist Tiziano Pascutto, who helped trace the correct type locality and recollect topotypic material.
Description and remarks. The description reported by Karaman (1980) is complete and well illustrated; considering that the slides used for the drawings by Karaman (1980) are considered as the type material of the new species, there is no reason to repeat the description herein.
Remarks. The new species differs from N. bihorensis and N. absconditus n. sp. in several morphological characters. Antenna I slightly longer, exceeding half of body length; inner lobe of maxilla I with 2, occasionally with 3 setae (versus 1‒2 setae in the other two species); distal article of maxilla I palp bearing 8‒9 setae (versus 6‒7). Gnathopods with broader propodite, especially in gnathopod 2 broader than long, easily distinguishable from those of the previous species. Pereopods iii‒vii slender than in N. bihorensis and N. absconditus n. sp.; pereopods vi and vii with a narrow basipodite (up to 3 times longer than wide), margins subparallel and bearing longer anterior setae. Uropod i with outer ramus slightly longer than inner ramus in males (versus subequal rami in the other two species), with longer distal spines (similar to those of N. absconditus n. sp.). Telson with each lobe bearing 5‒7 long distal spines, as long as one half of lobe length, longer in female (versus 4‒5 shorter spines in the other two species, as long as one third of lobe length); a slender spine is sometimes present on the inner margin of each lobe, in the proximal part of the notch (feature never observed in the other two species). The above-mentioned characters are diagnostic and allow an easy distinction of N. tizianoi n. sp. from N. bihorensis and N. absconditus n. sp.
N. tizianoi n. sp. is distributed in the Western and Central Alps where it inhabits non-calcareous terrains (mainly subsurface groundwaters flowing in metamorphites); the type locality is a tectonic cave, where it inhabits small pools of percolating waters; all the other sites are Alpine springs. Some on the sites are located in areas covered by glaciers during the Last Glacial Maximum (lgm, 21 Kya: Clark et al., 2009).
Diagnostic molecular characters. The species can be distinguished from N. bihorensis and from all other niphargid species on the basis of coi and 28S dna sequences. All available sequences are reported in supplementary table S1.
Molecular diagnosis. ‘T’ at site 282, ‘C’ at site 313, ‘G’ at site 454; support: 99%
Discussion
Our research clearly indicated that the distribution of N. bihorensis and its cryptic sister species N. absconditus n. sp. is restricted to Romania, with its closest known relative being N. gebhardti from Hungary. The Italian population previously attributed to N. bihorensis by Karaman (1980) belonged to a distinct species, N. tizianoi n. sp., present exclusively in the Western Alps in Italy and Switzerland. The redescription of the type material of N. bihorensis from Schellenberg’s collection allowed for an easy distinction between the two species, and phylogenetic analysis showed that they belonged to distinct, albeit closely related, subclades within the N. tauri clade.
The subclade including N. tizianoi n. sp. comprised three undescribed species from Piedmont (N. sp. Mulini), Austria (N. sp. Salzburg), and Slovenia (N. sp. Huda Luknja), and N. ambulator, which is endemic to a karstic massif in the Western Prealps (Karaman, 1975).
However, the taxonomic structure of the N. tauri clade remains unclear and prevents biogeographical speculation on the origin of N. bihorensis and N. tizianoi n. sp. Indeed, our analyses revealed that this clade includes species showing strong morphological similarities with Niphargus aquilex, a species complex distributed in central and northern Europe (Weber et al., 2023), which does not belong to the N. tauri clade. Therefore, this similarity can be attributed to convergence. The distribution of N. aquilex tauri (raised to species rank by Karaman S., 1950) is restricted to Turkey (Trontelj et al.; 2009; Ipek & Şirin, 2009); the specimens reported from Italy (Pesce & Vigna Taglianti, 1973, later attributed to N. aquilex by Karaman, 1993) and the Balkans (Karaman, 1973) have an uncertain taxonomic status; a N. tauri population from Slovenia was described as Niphargus fongi Fišer & Zagmaister, 2009, and Niphargus aquilex dobati Sket, 1999, also from Slovenia, was raised to species rank (Fišer et al., 2009a). Moreover, our analysis revealed that N. dobati is a species complex.
Our results add to the mounting evidence that although large ranges sometimes exist in groundwater amphipods, they are most of the time artefacts caused by poor taxonomy and/or cryptic species. We emphasizes once more that morphological characters alone are not sufficient in depicting the phylogenetic affinities of niphargid species, hence the need for an integrative approach including dna taxonomy to put the pieces of such ‘taxonomic puzzle’ in place.
editor: r. vonk
Acknowledgments
We would like to thank all water providers who contributed samples for this study in Switzerland. Special thanks to the collectors (reported in supplementary table S1) of specimens from Italy, Austria, Slovenia, and Romania, who put the material at our disposal for study. Florence Rodriguez Gaudray and Laurent Grumiau provided critical assistance with labwork at ulb. We thank three anonymous reviewers for their constructive review that greatly improved the manuscript.
Funding
AS was supported by a DarCo (biodiv21_0006) PhD grant. FS and JFF were supported by the Belgian Fond de la Recherche Scientifique (F.R.S. ‒ fnrs) via “Chargé de recherches” fellowship n° fc43267 to FS and “Projet de Recherches” grant n° T.00078.23 to JFF. MK, RA, and FA were supported by the Swiss Federal Office for the Environment foen/bafu (project “AmphiWell”). VZ and CF were supported by the Slovenian Agency for Research and Innovation, through the core programme P1-0184 and project J1-2464. This study is part of the DarCo project funded by Biodiversa+, the European Biodiversity Partnership under the 2021‒2022 BiodivProtect joint call for research proposals, co-funded by the European Commission (ga N°101052342) and with the funding organisations Ministry of Universities and Research (Italy), Agencia Estatal de Investigación – Fundación Biodiversidad (Spain), Fundo Regional para a Ciência e Tecnologia (Portugal), Suomen Akatemia – Ministry of the Environment (Finland), Belgian Science Policy Office (Belgium), Agence Nationale de la Recherche (France), Deutsche Forschungsgemeinschaft e.V. (Germany), Schweizerischer Nationalfonds (Grant N° 31bd30_209583, Switzerland), Fonds zur Förderung der Wissenschaftlichen Forschung (Austria), Ministry of Higher Education, Science and Innovation (Slovenia), and the Executive Agency for Higher Education, Research, Development and Innovation Funding (Romania).
Supplementary material
Supplementary material is available online at: https://doi.org/10.6084/m9.figshare.25674435
References
Altermatt, F., Alther, R., Fišer, C., Jokela, J., Konec, M., Küry, D., Mächler, E., Stucki, P. & Westram, A. M. (2014). Diversity and distribution of freshwater amphipod species in Switzerland (Crustacea: Amphipoda). PLoS ONE 9: e110328.
Alther, R., Bongni, N., Borko, Š., Fišer, C. & Altermatt, F. (2021). Citizen science approach reveals groundwater fauna in Switzerland and a new species of Niphargus (Amphipoda, Niphargidae). Subterr. Biol., 39: 1–31.
Cărăuşu, S., Dobreanu, E. & Manolache, C. (1955). Amphipoda. Forme salmastre şi de apă dulce. Fauna Republicii Populare Romîne, 4. Academiei Republicii Populare Romîne, Bucarest.
Clark, P. U., Dyke, A. S., Shakun, J. D., Carlson, A. E., Clark, J., Wohlfarth, B., Mitrovica, J. X., Hostetler, S. W., McCabe, A. M. (2009). The last glacial maximum. Science 325: 710–714.
Coleman, C. (2003). “Digital inking”: how to make perfect line drawings on computers. Org. Divers. Evol. 3: 1–14.
Coleman, C. O. (2009). Drawing setae the digital way. Zoosystematics Evol. 85: 305–310.
Cornish-Bowden, A. (1985). Nomenclature for incompletely specified bases in nucleic acid sequences: recommendations 1984. Nucleic Acids Res. 13: 3021–3030.
Couton, M., Studer, A., Hürlemann, S., Locher, N., Knüsel, M., Alther, R. & Altermatt, F. (2023). Integrating citizen science and environmental dna metabarcoding to study biodiversity of groundwater amphipods in Switzerland. Sci. Rep. 13: 18097.
Culver, D. C., Kowalko, J. E. & Pipan, T. (2023). Natural selection versus neutral mutation in the evolution of subterranean life: A false dichotomy? Front. Ecol. Evol. 11: 1080503.
Dayrat, B. (2005). Towards integrative taxonomy. Biol. J. Linn. Soc. 85: 407–415.
Fedosov, A., Achaz, G., Gontchar, A. & Puillandre, N. (2022). mold, a novel software to compile accurate and reliable dna diagnoses for taxonomic descriptions. Mol. Ecol. Res. 22: 2038–2053.
Fišer, C., Binindaemonds, O., Blejec, A. & Sket, B. (2008). Can heterochrony help explain the high morphological diversity within the genus Niphargus (Crustacea: Amphipoda)? Org. Divers. Evol. 8: 146–162.
Fišer, C., Çamur-Elipek, B. & Özbek, M. (2009a). The subterranean genus Niphargus (Crustacea, Amphipoda) in the Middle East: A faunistic overview with descriptions of two new species. Zool. Anz. 248: 137–150.
Fišer, C., Trontelj, P., Luštrik, R. & Sket, B. (2009b). Toward a unified taxonomy of Niphargus (Crustacea: Amphipoda): a review of morphological variability. Zootaxa 2061: 1–22.
Folmer, O., Black, M., Hoeh, W., Lutz, R. & Vrijenhoek, R. (1994). dna primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3: 294–299.
Guillerme, T., Cooper, N., Brusatte, S. L., Davis, K. E., Jackson, A. L., Gerber, S., Goswami, A., Healy, K., Hopkins, M. J., Jones, M. E. H., Lloyd, G. T., O’Reilly, J. E., Pate, A., Puttick, M. N., Rayfield, E. J., Saupe, E. E., Sherratt, E., Slater, G. J., Weisbecker, V., Thomas, G. H. & Donoghue, P. C. J. (2020). Disparities in the analysis of morphological disparity. Biol. Lett. 16: 20200199.
Hoang, D. T., Chernomor, O., Von Haeseler, A., Minh, B. Q. & Vinh, L. S. (2018). UFBoot2: Improving the ultrafast bootstrap approximation. Mol. Biol. Evol. 35: 518–522.
Horton, T., Lowry, J., De Broyer, C., Bellan-Santini, D., Copilaş-Ciocianu, D., Corbari, L., Costello, M. J., Daneliya, M., Dauvin, J.-C., Fišer, C., Gasca, R., Grabowski, M., Guerra-García, J. M., Hendrycks, E., Hughes, L., Jaume, D., Jazdzewski, K., Kim, Y.-H., King, R., Krapp-Schickel, T., LeCroy, S., Lörz, A.-N., Mamos, T., Senna, A. R., Serejo, C., Souza-Filho, J. F., Tandberg, A. H., Thomas, J. D., Thurston, M., Vader, W., Väinölä, R., Valls Domedel, G., Vonk, R., White, K., & Zeidler, W. (2023). World Amphipoda Database. Niphargus Schiödte, 1849. Retrieved from https://www.marinespecies.org (last accessed 29 February 2024).
Jörger, K. M. & Schrödl, M. (2013). How to describe a cryptic species? Practical challenges of molecular taxonomy. Front. Zool. 10: 59.
Kapli, P., Lutteropp, S., Zhang, J., Kobert, K., Pavlidis, P., Stamatakis, A. & Flouri, T. (2017). Multi-rate Poisson Tree Processes for single-locus species delimitation under Maximum Likelihood and Markov Chain Monte Carlo. Bioinformatics 33: btx025.
Karaman, S. (1950). Über die kleinen Niphargus-Arten Jugoslaviens. Srp. Akad. Nauka, Pos. Izd. Beograd 163: 87–99.
Karaman, G. S. (1973). Neubeschreibung der Art Niphargus tauri Schellenberg, 1933 (Gammaridae) aus dem Taurus Gebirge, Klein Asien. Crustaceana 24: 275–282.
Karaman, G. S. (1975). Three Niphargus species from Yugoslavia and Italy, N. ambulator n. sp., N. pupetta (Sket) and N. transitivus Sket (fam. Gammaridde). Poljopr. Šumar. 21: 13–34.
Karaman, G. S. (1980). First discovery of Niphargus bihorensis Schell. 1940 (fam. Gammaridae) in Italy with remarks to N. elegans Garb. 1894. Glasn. Rep. zav. zašt. Prir. Muz. Titograd 13: 71–80.
Karaman, G. S. (1993). Crustacea. Amphipoda di acqua dolce. Fauna d’Italia, 26. Calderini, Bologna.
Katoh, K. & Standley, D. M. (2013). mafft multiple sequence alignment software version 7: Improvements in performance and usability. Mol. Biol. Evol. 30: 772–780.
Knüsel, M., Borko, Š., Alther, R., Salussolia, A., Flot, J.-F., Altermatt, F., Fišer, C. & Stoch, F. (2023). Phylogenetic structure and molecular species delimitation hint a complex evolutionary history in an Alpine endemic Niphargus clade (Crustacea, Amphipoda). Zool. Anz. 306: 27–36.
Lanfear, R., Frandsen, P. B., Wright, A. M., Senfeld, T. & Calcott, B. (2017). PartitionFinder 2: new methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses. Mol. Biol. Evol. 34: 772–773.
Meleg, I. N., Zakšek, V., Fišer, C., Kelemen, B. S. & Moldovan, O. T. (2013). Can environment predict cryptic diversity? The case of Niphargus inhabiting Western Carpathian groundwater. PLOS ONE 8: e76760.
Miller, M. A., Schwartz, T., Pickett, B. E., He, S., Klem, E. B., Scheuermann, R. H., Passarotti, M., Kaufman, S. & O’Leary, M. A. (2015). A RESTful api for access to phylogenetic tools via the cipres Science Gateway. Evol. Bioinform. 11: ebo.S21501.
Moscardini, C. (1955). Primo contributo alla conoscenza della fauna della Val Sabbiola (Vercelli). Atti Soc. Nat. Mat. Modena 8:5–86: 38–47.
Nguyen, L.-T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. (2015). iq-tree: a Ffast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32: 268–274.
Pesce, G. L. & Vigna Taglianti, A. (1973). I Niphargus dell’Appennino Centrale (Amphipoda, Gammaridae). Quad. Mus. Speleol. “V. Rivera” 2: 109–120.
Puillandre, N., Brouillet, S. & Achaz, G. (2021). asap: assemble species by automatic partitioning. Mol. Ecol. Resour. 21: 609–620.
Ronquist, F. & Huelsenbeck, J. P. (2003). MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574.
Schellenberg, A. (1940). Subterrane Amphipoden Osteuropas, ihre Variabilität und ihre verwandtschaftlichen Beziehungen. Zool. Jahrb. Abt. Syst. Oekol. Geogr. Tiere 74: 243–268.
Spöri, Y. & Flot, J. (2020). HaplowebMaker and CoMa: Two web tools to delimit species using haplowebs and conspecificity matrices. Methods Ecol. Evol. 11: 1434–1438.
Stoch, F. & Flot, J. (2017). Molecular phylogeny and biogeography of freshwater amphipods in Italy: state of the art. Ecography 8: 551–552.
Stoch, F., Salussolia, A. & Flot, J.-F. (2023). Polyphyly of the Niphargus stygius species group (Crustacea, Amphipoda, Niphargidae) in the Southern Limestone Alps. Retrieved from http://biorxiv.org/lookup/doi/10.1101/2022.04.28.489871 (last accessed 29 February 2024).
Trontelj, P., Blejec, A. & Fišer, C. (2012). Ecomorphological convergence of cave communities. Evolution 66: 3852–3865.
Trontelj, P., Douady, C. J., Fišer, C., Gibert, J., Gorički, G., Léfebure, T., B., S. & V, Z. (2009). A molecular test for cryptic diversity in ground water: how large are the ranges of macro-stygobionts? Freshw. Biol. 54: 727–744.
Weber, D., Brad, T., Weigand, A. & Flot, J.-F. (2023). Water diviners multiplied: cryptic diversity in the Niphargus aquilex species complex in Northern Europe. Retrieved from https://www.biorxiv.org/content/10.1101/2023.08.13.553147v1 (last accessed 29 February 2024).
Weber, D., Flot, J.-F., Weigand, H. & Weigand, A. M. (2020). Demographic history, range size and habitat preferences of the groundwater amphipod Niphargus puteanus (C.L. Koch in Panzer, 1836). Limnologica 82: 125765.
Weber, D., Stoch, F., Knight, L.R.F.D., Chauveau, C. & Flot, J.-F. (2021). The genus Microniphargus (Crustacea, Amphipoda): evidence for three lineages distributed across northwestern Europe and transfer from Niphargidae to Pseudoniphargidae. Belg. J. Zool. 151: 169–191.
Zagmajster, M., Eme, D., Fišer, C., Galassi, D., Marmonier, P., Stoch, F., Cornu, J.-F. & Malard, F. (2014). Geographic variation in range size and beta diversity of groundwater crustaceans: insights from habitats with low thermal seasonality. Glob. Ecol. Biogeogr. 23: 1135–1145.
