DNA isolation, PCR amplification and sequencing
Genomic DNA was purified from 13 specimens corresponding to nine different Haploginglymus taxa, viz. the new species described herein, the aberrant H. morenoi, and seven not yet formally described species coming from several Iberian locations (v. Table 1 & Fig. 1). In addition, sequences from 14 specimens of Niphargus from four different populations from the Iberian western edge of the Pyrenees (Basque Country), as well as eight specimens belonging to six different Pseudoniphargus species from the Iberian Peninsula, Portugal and the Canary Islands were also obtained and included in the dataset as potentially close outgroups. DNA extraction was performed using the DNeasy Tissue kit (Qiagen, West Sussex, UK) following the manufacturer’s protocol. Elutions were done in 100 μL volume and 1 μL was used in PCR reactions. Three different molecular markers were selected for the study, namely: a partial sequence of the mitochondrial Cytochrome c Oxidase subunit 1 gene (cox1; primers LCO1490 and HCO2189; Folmer et al., 1994), a partial sequence of the nuclear ribosomal 28S (LSU; primers 28S lev2; Verovnik et al., 2005 Zakšek et al., 2007), and a fragment of the nuclear Histone 3 gene (H3; primers H3aF and H3aR; Colgan et al., 1998). PCR conditions included 0.2 μM of each primer and 3.5 mM MgCl2 using a standard protocol of 35 cycles, with annealing temperature ranging from 50 to 45 °C (60s) depending on the sample. Denaturation (94 °C) and elongation (72 °C) lasted 30 and 60s, respectively. PCR products were inspected by electrophoresis in 1% agarose gel and purified using MSB Spin PCRapace (Invitek, Berlin, Germany). Sanger sequencing was performed with the same primers using the BigDye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA). Sequences were edited and contigs were assembled using CodonCode Aligner (CodonCode Corporation, Dedham, MA, USA), and deposited at GenBank under the accession numbers referred in Table S1.