Contributions to Zoology, 86 (3) 239-260 (2017) Molecular systematics of Haploginglymus, a genus of subterranean amphipods endemic to the Iberian Peninsula (Amphipoda: Niphargidae) José A. Jurado-Rivera1, 5, Genaro Álvarez2, José A. Caro2, 3, Carlos Juan1, 4, Joan Pons4, Damià Jaume4 1 Departament de Biologia, Universitat de les Illes Balears (UIB), Ctra. Valldemossa km 7’5, 07122-Palma de Mallorca, Spain 2 Sociedad Espeleológica Geos (Exploraciones e Investigaciones Subterráneas), C/ Dr. Miguel Ríos Sarmiento 74, 41020-Sevilla, Spain 3 Departamento de Geografía y Ciencias del Territorio, Universidad de Córdoba, Plaza del Cardenal Salazar 3, 14071-Córdoba, Spain 4 IMEDEA (CSIC-UIB), Instituto Mediterráneo de Estudios Avanzados, C/ Miquel Marquès 21, 07190-Esporles, Spain 5 E-mail: [email protected] Key words: Stygofauna, Crustacea, taxonomy, cave fauna, phylogenetic relationships Abstract References ...................................................................................... 248 Online Supplementary Information .......................................... 250 The molecular systematics of the subterranean amphipod genus Appendix ........................................................................................ 251 Haploginglymus is addressed through the phylogenetic analysis Systematics ............................................................................... 251 of three DNA gene fragments (nuclear ribosomal 28S and pro- Haploginglymus geos sp. nov. .............................................. 251 tein-coding Histone 3, plus mitochondrial Cytochrome c Oxi- dase subunit I). We take advantage of the description of a new species from southern Spain (Haploginglymus geos sp. nov.) to Introduction assess the singularity of this genus endemic to the Iberian Penin- sula and the inclusion of the morphologically aberrant H. more- noi within Haploginglymus. Our results corroborate the mono- Haploginglymus Mateus and Oliveira-Mateus, 1958, is phyly of the family Niphargidae but shows Niphargus to be para- a genus of niphargid amphipod endemic to the fresh phyletic as it currently stands, with Haploginglymus appearing inland subterranean waters of the Iberian Peninsula nested within it. A strongly supported sister-group relationship (Spain and Portugal). It is found in caves, wells and the between niphargids and the (thalassoid) pseudoniphargids is re- interstitial medium associated to riverbanks, but appar- covered as well, but we propose the Niphargidae should continue to be considered as a primary limnic group for biogeographic ently avoids the brackish waters of coastal aquifers. purposes despite its presumed marine derivation. Our findings First discovered in wells from the lower course of river are in agreement with previous studies that suggest the family Douro in NW Portugal, the genus displays a broad dis- Niphargidae originated in the late Cretaceous in the NE Atlantic, tribution across the entire Peninsula, where it is one of from where it eventually expanded across continental Europe. the most common subterranean crustaceans (Noten- boom, 1990). The genus was originally distinguished Contents from Niphargus Schiödte, 1849 – by far the largest ge- nus among freshwater amphipods (408 valid species; Introduction .................................................................................... 239 Lowry, 2010) – based on the display of an unsegmented Material and methods .................................................................. 240 Study site .................................................................................. 240 (vs. 2-segmented) exopod on uropod III. Nevertheless, Material examined ................................................................. 240 two additional niphargid genera described later on, DNA isolation, PCR amplification and sequencing ....... 240 both monotypic, viz. Carinurella Sket, 1971, and Niphar- Phylogenetic analyses ........................................................... 242 gobates Sket, 1981, share with Haploginglymus the Results ............................................................................................. 242 same condition of uropod III, although both can be Discussion ....................................................................................... 243 readily told apart from Haploginglymus based on other On the genus Haploginglymus ............................................. 243 Origin, intra-generic relationships and biogeography ... 243 features (Sket, 1971; 1981). Age and origin of the Niphargidae ..................................... 246 Despite its almost generalised distribution across Acknowledgements ....................................................................... 247 Iberia, only five species of Haploginglymus have been Downloaded from Brill.com10/06/2021 03:08:27PM via free access 240 Jurado-Rivera et al. – The Iberian subterranean amphipod genus Haploginglymus formally described thus far (Mateus and Oliveira- uncispina Notenboom, 1988; Salentinella seviliensis Mateus, 1958; Stock, 1980; Karaman, 1986; Pretus and Platvoet, 1987; Stenasellus escolai Magniez, 1977; Mi- Sabater, 1990; Iannilli et al., 2009). Aside the morpho- crocharon marinus Chappuis and Delamare, 1954; logically aberrant H. morenoi Iannilli, Minelli and Megacyclops brachypus Kiefer, 1954; Diacyclops bi- Ruffo, 2009, which displays a modified spatulate max- cuspidatus odessanus (Shmankevich, 1875); Cypria illipedal palp, slender gnathopods and strongly sexual- ophtalmica (Jurine, 1820) and a not yet determined har- ly-dimorphic gnathopod II, all show a rather uniform, pacticoid copepod. In addition, the bathynellid syncarid ordinary Niphargus-like aspect. Anyway, molecular Hexabathynella sevillaensis Camacho, 2005, was re- analyses under way suggest the genus mirrors the con- cently described from the same caves (Camacho, 2005). dition described elsewhere for Niphargus, where each species displays a very reduced distribution and the few Material examined presumed widespread species correspond in fact to complexes of stenochorous cryptic species (Trontelj Specimens were collected directly with a hand-held et al., 2009; Meleg et al., 2013). plankton net in the cave lakes and fixed in situ in 95% As a first step to explore the diversification ofHaplo - ethanol. Once in the laboratory, they were dissected in ginglymus in Iberia, we combine herein morphology lactic acid under the stereomicroscope, and appendages and molecular traits to describe a new species from figured with a Leica DM2500 microscope equipped southern Spain. We use DNA sequences of three gene with Nomarski differential interference contrast and a fragments (nuclear ribosomal 28S and protein-coding drawing tube. Body measurements were derived from Histone 3, plus mitochondrial Cytochrome c Oxidase the sum of the maximum dorsal dimensions of body subunit I) to assess the distinctiveness of this Iberian somites and exclude telson length. Type material is de- genus in front of the rest of niphargids, and the place- posited in the invertebrate collection of Naturalis Bio- ment of the morphologically aberrant H. morenoi with- diversity Center, Leiden (RMNH). Abbreviations and in Haploginglymus. In addition, we have found the nomenclature used in morphological descriptions are Pseudo niphargidae Karaman, 1993 to be the closest as follows: A1 (antennule); A2 (antenna); G1-G2 (gna- relative to niphargids, which gives a new perspective to thopods I and II, respectively); P3-P7 (pereiopods III to the debate on the origin, diversification and biogeogra- VII, respectively); and U1-U3 (uropods I to III, respec- phy of this extremely diversified family of continental tively). water amphipods. DNA isolation, PCR amplification and sequencing Material and methods Genomic DNA was purified from 13 specimens corre- sponding to nine different Haploginglymus taxa, viz. the Study site new species described herein, the aberrant H. morenoi, and seven not yet formally described species coming The new species is known only from phreatic waters from several Iberian locations (v. Table 1 & Fig. 1). In found in two caves located at Cerro de Santiago, a addition, sequences from 14 specimens of Niphargus Cambrian limestone outcrop within the municipality of from four different populations from the Iberian west- Cazalla de la Sierra (Seville, southern Spain). The area ern edge of the Pyrenees (Basque Country), as well as is next to Riera de Benalija, a tributary brook to the eight specimens belonging to six different Pseudo- Guadalquivir River that flows into El Pintado reservoir. niphargus species from the Iberian Peninsula, Portugal The cave lakes are hydrographically connected to the and the Canary Islands were also obtained and includ- reservoir so that their level follows the annual oscilla- ed in the dataset as potentially close outgroups. DNA tion of the reservoir’s water table. Specimens were extraction was performed using the DNeasy Tissue kit found crawling in high numbers on the bottom of the (Qiagen, West Sussex, UK) following the manufactur- cave lakes and were readily attracted by bait. Some er’s protocol. Elutions were done in 100 μL volume and specimens were observed venturing in open water, but 1 μL was used in PCR reactions. Three different mo- most remained associated to the substratum. Direct ob- lecular markers were selected for the study, namely: a servations did not suggest the species carried an inter- partial sequence of the mitochondrial Cytochrome c stitial life. Crustaceans found