Research Article |
Corresponding author: Dieter Weber ( dieter.weber124@gmx.de ) Academic editor: Cene Fišer
© 2021 Dieter Weber, Traian Brad, Fabio Stoch, Jean-François Flot.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Weber D, Brad T, Stoch F, Flot J-F (2021) Rediscovery and redescription of Niphargus enslini Karaman, 1932 (Amphipoda, Niphargidae) in southern Germany. Subterranean Biology 40: 65-89. https://doi.org/10.3897/subtbiol.40.73017
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Niphargus enslini Karaman, 1932 was collected only once in 1905 from the Falkensteiner Höhle (Baden-Württemberg, Germany). Two years after its description, the species was synonymized with Niphargus virei and not studied any more. During recent surveys on German niphargids, further samples collected in this cave did not yield N. enslini specimens, but this species was collected in the Blätterteighöhle and in the Schwarzer Brunnen, two caves located in Baden-Württemberg and intercepting the same karstic aquifer feeding Falkensteiner Höhle. In an integrative taxonomic approach, we carefully studied the morphology of the newly collected specimens and sequenced two molecular markers (fragments of the cytochrome c oxidase subunit I (COI) and of the nuclear 28S rRNA gene) to test for possible conspecificity of N. enslini with N. virei. Morphological analysis confirmed that N. enslini is distinct from the N. virei species complex. We provide a redescription of newly collected material, together with new drawings of a more than 100 years old topotypic female. We briefly discuss the putative origin of N. enslini and the age of its split from the N. virei species complex.
28S rRNA, Baden-Württemberg, COI, integrative taxonomy, Niphargus orcinus, Niphargus virei, species delimitation
Species of the genus Niphargus Schiödte, 1849 have been intensively studied for more than 170 years and more than 400 species of this genus have been described so far (
Niphargus enslini Karaman, 1932 was described based on four specimens collected in 1905 in the Falkensteiner Höhle by Eduard Enslin.
One year later,
After a collecting gap of 112 years, we found again specimens that could be ascribed based on their morphology to N. enslini in two caves in the Swabian Alb, Southern Germany. This discovery gave us the opportunity to test
From 2016 to 2019, one of the authors (D.W.) collected 1,705 niphargid specimens from 85 springs, 13 natural caves, two artificial cavities, and two interstitial sites in Baden-Württemberg, Germany. Most specimens were collected by opportunistic sampling, putting mud and organic material onto sieves with a mesh size of 5 to 0.2 mm and washing with water to find and collect specimens. In few cases, niphargids were collected by eye using spring steel forceps or by means of baited traps.
Our sampling survey in Baden-Württemberg included Falkensteiner Höhle, the type locality of Niphargus enslini located between the city of Bad Urach and the village of Grabenstetten (both in the administrative district of Reutlingen, southern Germany). It is an active limestone cave more than 4,000 m long. Using three baited traps placed in the water basins in the first 400 m of the cave, we collected 77 specimens of Niphargus puteanus (
Four specimens morphologically attributable to Niphargus enslini were found on 26–27 August 2017 in the Blätterteighöhle (cave inventory number 8119/29; WGS 84 coordinates: 47.8463°N, 8.8589°E; altitude 478 m a.s.l., city of Aach, administrative district of Konstanz, Baden-Württemberg, southern Germany:
Another N. enslini specimen was found on 5 August 2017 in the Schwarzer Brunnen (literally ‘Black Well’; cave inventory number 7720/62, WGS 84 coordinates: 48.2746°N, 9.0606°E; altitude 799 m a.s.l.). The Schwarzer Brunnen (Fig.
In the Wimsener Höhle (Hayingen-Wimsen, Swabian Alb, Baden-Württemberg, Germany),
Finally, the Umweltgruppe Kirchheim (http://www.uwg-kirchheim.de/HTML/N2/tiereundpflanzen2_3.html, accessed 7th April 2020) mentioned N. enslini from the Buchbrunnenquelle close to Dischingen (WGS84 coordinates: 48.695°N, 10.363°E), based on a report from the Fachhochschule Magdeburg. The Hochschule Magdeburg-Stendal (Uta Langheinrich, email dated 17 April 2020) communicated that N. enslini was never found by the Fachhochschule Magdeburg or the Hochschule Magdeburg-Stendal. The report from the Buchbrunnenquelle is thus not confirmed.
The locations of the sampling sites of N. enslini are reported in Fig.
Distribution of Niphargus enslini. Black circles indicate the type locality and the new sampling sites; Wimsener Höhle where Niphargus virei is cited is reported in red. All the sites are clearly located in the same karstic aquifer. The record from Buchbrunnenquelle is not indicated here as it is probably erroneous.
One female specimen from the Falkensteiner Höhle stored in the crustacean collection of the Natural History Museum of Berlin, collection GBIF Crustacea – ZMB Berlin, No. 24795, leg. Enslin, determined by Schellenberg as Niphargus orcinus virei. This specimen, a badly preserved female, is probably one of those described as Niphargus enslini by
One pereopod, removed from each of four specimens of Niphargus enslini, was used for DNA extraction, and the remaining body parts and appendages of each specimen were stored in 96% ethanol at -20 °C at the Université libre de Bruxelles (ULB), Belgium. Genomic DNA was extracted using the NucleoSpin@ Tissue kit by Macherey-Nagel, following the manufacturer’s protocol. The eluted DNA was stored at 4 °C until amplification then long-term stored at -20 °C.
A fragment of the nuclear 28S rRNA gene and a 658 bp fragment of the mitochondrial cytochrome c oxidase subunit I (COI) were amplified using the primers described by
Direct sequencing was performed using the same primers as for amplification and/or using internal primers (see
Chromatogram inspection and sequence editing were performed in Sequencher version 4.1.4 (Gene Codes). No double peaks suggesting 28S heterozygotes were observed in this set of chromatograms.
To assess the position of the N. enslini species complex within the phylogenetic tree of Niphargidae, 232 Niphargidae species were used together with four species of the family Pseudoniphargidae (genera Pseudoniphargus and Microniphargus) as outgroups, since the latter was suggested to be the sister group of Niphargidae in recent studies (
28S sequences were aligned using the E-INS-i algorithm implemented in MAFFT 7 (
COI sequences (Suppl. material
All chromatograms and FASTA alignments were uploaded to Zenodo (
One sexually mature male and one female with well developed oostegites from the Blätterteighöhle as well as the adult female from the Falkensteiner Höhle preserved in the Berlin Museum were dissected in glycerol; their appendages were mounted in Kaiser’s glycerol gelatine (Merck, Germany) and inspected with an Olympus SZX16 stereomicroscope and an Olympus BX51 microscope. Drawings were performed either manually by reproducing on transparent paper the photographs taken with the microscope camera or using a camera lucida and digitally inking the pencil drawings using Adobe Illustrator drawing software. Systematic microscope inspection of the slides with specimens’ appendages ensured the addition of finer details that could not be seen on photographs or preliminary drawings due to the thickness of mouthparts and appendages.
The phylogenetic analyses based on 28S rDNA using 232 Niphargidae species (and representatives of the family Pseudoniphargidae as an outgroup), reported in Suppl. material
A subset of the phylogenetic tree with Niphargus enslini and its closest neighbours of the Niphargus virei sister group, reconstructed using maximum likelihood (IQTree2), based on a fragment of the 28S rRNA gene. Ultrafast bootstrap supports are shown on nodes. The N. virei clade, which includes N. enslini (100% support), is reported in red. A full tree is shown in the Suppl. material
The haplotype network based on COI (Fig.
Family Niphargidae Bousfield, 1977
syn: Niphargus orcinus enslini
Male. Germany, 1 ♂; Blätterteighöhle; 26–27 August 2017; Bernd Hugger, Rafael Grimm, Dieter Weber leg. The specimen was dissected and used for species redescription. The dissected body appendages were transferred to permanent slides and stored in the collection of the Université libre de Bruxelles (ULB) under voucher number: 170827-004.
Females. Germany, 3 ♀♀; Blätterteighöhle; 26–27 August 2017; Bernd Hugger, Rafael Grimm, Dieter Weber leg. One female (Voucher number: 170827-001) was dissected and used to study the sexual dimorphism. Two females were not dissected and are preserved in 96% ethanol at -20 °C in the collection of the Université libre de Bruxelles (ULB), Belgium. Voucher numbers: 170827-002 and 170827-003. Germany, 1 ♀, Falkensteiner Höhle, leg. Enslin, partially dissected and mounted on a slide stored in the crustacean collection of the Natural History Museum of Berlin (GBIF Crustacea – ZMB Berlin, No. 24795).
Juveniles. Germany, 1 juv. (voucher number 170805-029); Schwarzer Brunnen; 5 August 2017; Harald Knupfer leg. The specimen was far too small for morphological determination and was completely used for DNA extraction.
All DNA isolates are stored at –20 °C at the Université libre de Bruxelles (ULB) in the research unit Evolutionary Biology & Ecology. Voucher numbers: 170827-001, 170827-002, 170827-003, 170805-029.
Robust, mid-sized Niphargus. Right to acute postero-ventral angle of epimeral plates. Pleon with 3–6 spines on the postero-dorsal side of each segment. Gnathopods coxae trapezoidal with only one thin seta along the outer margin of dactylus. Coxal plate IV sub-rectangular, with no posterior protrusion. Pereopod VI reaching more than half of the total body length. Uropod I rami subequal. Uropod III sexually dimorphic; exopod elongated in males. Telson relatively elongated in males, bearing 4 apical, 4–5 lateral and 3–5 smaller dorsal spines on each lobe.
Habitus as in Fig.
Head (Fig.
Antenna I (Fig.
Antenna II (Fig.
Labrum of typical, subovoid shape.
Lower lip (Fig.
Left mandible (Fig.
Right mandible (Fig.
Mandibulary palps (Figs
Maxilla I (Fig.
Maxilla II (Fig.
Maxilliped (Fig.
Gnathopod I (Fig.
Gnathopod II (Fig.
Pereopod III (Fig.
Pereopod IV (Fig.
Pereopod V (Fig.
Pereopod VI (Fig.
Pereopod VII (Fig.
Pereopods V:VI:VII ratio 1.0:1.32:1.27.
Pleopods similar each other (pleopod I as in Fig.
Uropod I (Fig.
Uropod II (Fig.
Uropod III (Fig.
Epimeral plates (Fig.
Urosomite I (Fig.
Telson (Fig.
Male and female highly similar except for the presence of oostegites (Fig.
Comparison between the female from the Falkensteiner Höhle (type locality) and the female from the Blätterteighöhle could not detect marked differences. The small differences that could be noticed observing
Niphargus enslini is a distinct species from the Niphargus virei species complex; the distinction is supported both by molecular and morphotaxonomical data. The 28S phylogenetic tree clearly shows that N. enslini is the sister taxon of the N. virei species complex. COI haplotype network analyses and ABGD species delimitation method confirmed that the known populations of this clade can be ascribed to four putative species, i.e., N. enslini and N. virei A, B, and C (
Around the Blätterteighöhle,
Interestingly, all three caves where N. enslini was reported discharge in different river watersheds (the Neckar River and the Rhine River – flowing to the North Sea – and the Danube River – flowing into the Black Sea: Fig.
Moreover, N. enslini was only found in sites that were not covered by glaciers during the Last Glacial Maximum (
Two biogeographic scenarios can be proposed to explain the isolation of the whole species group into two clades (i.e., N. enslini and the N. virei species complex).
In the first scenario, the orogenesis of the Jura massif in late Tortonian and early Messinian (around 6.6–8.6 Ma:
In the second scenario, the most recent common ancestor of the N. virei species complex and of N. enslini could have transgressed the Jura watershed, and the split should have occurred later during Quaternary glaciations.
Previous attempts to date the split of the N. virei species complex clade from the other niphargids, based on different molecular clocks, support the hypothesis of a rather old event: McInerney et al. (2013) suggested around 23 Ma, Delić et al. (2019) found a value of 15 (6.5–19) Ma, whereas the most recent multimarker chronogram built by
Bernd Hugger and Rafael Grimm assisted in collecting in the Blätterteighöhle. Harald Knupfer collected in the Schwarzer Brunnen. Bernd Hugger took a photo of one specimen alive. The Freunde der Aachhöhle gave the permission of the use of the cave map. Uta Langheinrich gave information on niphargids collected by the Hochschule Magdeburg-Stendal.
Traian Brad was supported by a grant of the Ministry of Research and Innovation, project number PN-III-P4-ID-PCCF-2016-0016 (DARKFOOD), and by EEA Grants 2014–2021, under Project contract no. 4/2019 (GROUNDWATERISK). Molecular analyses were supported by the Belgian Fonds de la Recherche Scientifique (FNRS) via research credit J.0272.17 and by the Fédération Wallonie-Bruxelles via an ‘Action de Recherche Concertée’ (ARC) grant, both to Jean-François Flot.
Tables S1–S4, Figure S1
Data type: pdf. file
Explanation note: Table S1. List of primers used for amplification and sequencing and PCR amplification conditions. Table S2. Species (in alphabetical order) and GenBank accession numbers of the 28S sequences (GB 28S) used in the phylogenetic analysis. Outgroup genera and species used in the analysis are reported in bold. Table S3. Species, voucher codes, sites, geographical coordinates (WGS84), collectors and date of collection and GenBank accession numbers of the COI sequences used in the haplotype network. Table S4. Morphometric data (length of body, head, telson and length and width of articles of appendages) of the male of Niphargus enslini collected in the Blätterteighöhle (measures in mm). Figure S1. Phylogenetic tree positioning Niphargus enslini within the wider phylogeny of the genus Niphargus, constructed using maximum likelihood (IQTree2), based on a fragment of the 28S rRNA gene. Ultrafast bootstrap supports are shown on nodes. The N. virei clade, which includes N. enslini (100% support), is reported in red. Four Pseudoniphargidae were used as outgroups.