Genome‐wide nuclear data confirm two species in the Alpine endemic land snail Noricella oreinos s.l. (Gastropoda, Hygromiidae)

Abstract The Austrian endemic land snail species Noricella oreinos (formerly Trochulus oreinos) occurs in the Northeastern Calcareous Alps at high elevations. Two morphologically highly similar subspecies N. o. oreinos and N. o. scheerpeltzi have been described. First analyses of mitochondrial and nuclear marker sequences indicated a high genetic divergence between them. In the present study, we aimed to assess gene flow between the two subspecies which should help to re‐evaluate their taxonomic status. Sequence data and amplified fragment length polymorphism (AFLP) markers of 255 Noricella specimens covering the whole distribution range were analyzed. A clear geographic separation was found within the potential contact zone, the Haller Mauern mountain range. Samples of all western sites were part of the clade representing N. o. scheerpeltzi and almost all samples from the eastern sites clustered with N. o. oreinos. However, within two sampling sites of the eastern Haller Mauern, a few individuals possessed a COI sequence matching the N. o. oreinos clade whereas at the ITS2 locus they were heterozygous possessing the alleles of both taxa. Contrary to the ITS2 results indicating historical and/or ongoing hybridization, AFLP analyses of 202 individuals confirmed a clear separation of the two taxa congruent with the mitochondrial data. Although they occur on the same mountain range without any physical barrier, no indication of ongoing gene flow between the two taxa was found. Thus, we conclude that the two taxa are separate species N. oreinos and N. scheerpeltzi.

the ΔK values for each K as computed in STRUCTURE for the datasets E1-E4.

Method S1
Sequence generation -Control reactions for DNA extraction and PCR amplification were included in mt COI amplification reactions to detect contaminations. The COI amplification reactions were performed following the QIAGEN TopTaq manufacturer's protocol with 1x TopTaq PCR Buffer, 1x Q-Solution™, 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.5 µM of each primer, 0.5 U TopTaq DNA polymerase, 1 µl template DNA and nuclease free water to a total volume of 25 µl. PCR conditions were: 94°C for 3 min, 35 cycles of 94°C for 30 sec, 54°C for 30 sec, 72°C for 30 sec, followed by 72°C for 7 min.
The ITS2 sequences were amplified using the TopTaq DNA polymerase (Qiagen). In case of length polymorphisms or more than one allele at the ITS2 locus, direct sequencing of the PCR was unsuccessful. PCR reactions were repeated from such specimens using the Q5® High-Fidelity DNA polymerase (NEB). The ITS2 master mix included 1x Q5 Reaction Buffer, 0.2 mM of each dNTP, 0.5 µM of each primer, 0.5 U Q5 High-Fidelity DNA Polymerase, 0.5 µl template DNA and nuclease free water to a total volume of 25 µl. PCR conditions were: 98°C for 30 sec, 30 cycles of 98°C for 10 sec, 63°C for 20 sec, 72°C for 30 sec, followed by 72°C for 7 min. Subsequent cloning and sequencing was performed according to Kruckenhauser et al. (2014). In general, up to four clones per individual were sequenced. From the nine individuals which possessed the ITS2 allele of both N. oreinos subspecies, DNA extraction and cloning of the ITS2 fragment was repeated. Cloned sequences that occurred more than once for an individual were removed in the final dataset.

Method S2
Increasing DNA concentration for AFLP generation -If the concentration of available or newlyextracted DNA samples was below 10 ng/µl, the samples were post-processed: The AE-based DNA samples were precipitated using a conventional protocol, whereas samples, which had been eluted in nuclease free water, were concentrated using a vacuum centrifuge (UniEquip UniVapo 150 ECH). For precipitation, 1/10 Vol. 3M sodium acetate (pH 5.2), 9/10 Vol. DNA and 3x Vol. 96% ethanol were used according to a standard precipitation protocol. The pellet was dissolved in 15-30 µl nuclease free water (Qiagen). For the vacuum centrifuge concentration, DNA samples were incubated at 50°C for 3.25 hours and 15 µl sterile ddH2O were added.

Method S3
Amplified Fragment Length Polymorphism -The various steps of restriction-ligation, PCR amplification and purification were carried out simultaneously for 48 samples in a GeneAmp® PCR System 9700 thermocycler (Thermo Fisher Scientific Inc.). Unless otherwise indicated, all chemicals were purchased from VWR. The initial selective PCR primer pairs were chosen after a primer test using samples of 16 specimens and nine selective primer pair combinations with three selective nucleotides each (data not shown).
For each reaction, 5 µl of template DNA (minimum of 50 ng DNA) was digested for three hours at 37 °C with 6 µl of the corresponding master mix. The master mix contained 1x T4 Ligase Following the protocol of (Bendiksby, Tribsch, Borgen, Trávníček, & Brysting, 2011), 6 µl of the NED-labelled PCR products were pooled with both 3 µl of FAM and VIC-labelled product and purified using Sephadex TM G-50 Superfine resins (GE Healthcare BioSciences). For MegaBACE preparation, 12.9 µl AD, 0.1 µl ET-ROX 400-R MegaBACE size standard and 2 µl (in case of the first run) or respective 1.6 µl of the purified PCR products were denatured at 95°C for 3 min and immediately cooled on ice until capillary electrophoresis.