Two new phreatic snails (Mollusca: Caenogastropoda: Cochliopidae) from the Edwards and Edwards-Trinity aquifers, Texas

The Edwards and Edwards-Trinity Aquifers of Texas have diverse stygofauna, including fifteen species of snails found in phreatic and hyporheic habitats. These species have the hallmarks of adaptation to subterranean environments including extremely small body size and the loss of pigmentation and eyes. Here we use an integrative taxonomic approach, using shell, radula, and anatomical features as well as mitochondrial and nuclear DNA data, to circumscribe a new genus and two new cavesnail species from Central Texas. Vitropyrgus lillianae gen. et sp. nov. is described from Comal Springs (Comal County) and Fessenden Springs (Kerr County) and distinguished by a glassy, highly sculptured shell and distinctively simple, unornamented penial morphology. We also describe Phreatodrobia bulla sp. nov. from Hidden Springs (Bell County), and several other springs in Bell & Williamson Counties, Texas. This species has a smooth, unsculptured teleoconch, a reflected and flared lip, and deeply concave operculum. 

Methods
DNA was extracted using the Qiagen DNAEasy Blood and Tissue Kit, incubated at 65 °C for 24 hours. The same digestion was used to retain radula and opercula as well as DNA. PCR was conducted using the Platinum SuperFi DNA Polymerase Master Mix Kit (Thermo-Fisher). We followed the manufacturer protocol for PCR, conducting a temperature gradient between 48-54 °C for each species and proceeding with the temperature identified as the best, typically 51 °C. The primers used for COI were from Folmer COIH2198 and COIL1490 (Folmer et al. 1994) and the nuclear Large Ribosomal Subunit (LSU) LSU 1 & 3 (Wade et al. 2001). Amplicons were purified using the PCR DNA Fragment Extraction Kit (IBI Scientific, Peosta) and quantified with a Qubit 3.0 Fluorometer and Qubit dsDNA HS Assay Kit (Invitrogen). DNA sequencing was conducted at Eton Biosciences, inc. 

Following sequencing, Geneious 10.2.6 was used to assemble contigs, trim and visually inspect sequences, and align them using the MUSCLE algorithm. Model selection (Kalyaanamoorthy et al. 2017), Maximum likelihood analyses (Nguyen et al. 2015), and 1000 ultrafast bootstrap replicates (Hoang et al. 2018) were conducted in iqtree 1.6.12. For COI a 74 terminal alignment was assembled including all new sequences generated and members of all genera available in the Cochliopidae on Genbank. Pomatiopsis lapidaria was included as an outgroup. The COI alignment was partitioned to allow evaluation of 1st, 2nd, and 3rd codon positions separately. PartitionFinder and ModelFinder (via IQTREE) were used to assess whether partitions should be analyzed separately or merged and to determine the best fit model for each partition.

 For LSU an alignment with all available sequences (23) was generated using MUSCLE as implemented at https://www.ebi.ac.uk/Tools/msa/muscle/ and analyzed in IQ-TREE with 1000 ultra-fast bootstrap replicates. Other Cochliopidae were not available for this gene, so Pyrgulopsis species were used as the outgroup. Model selection was conducted in IQ-TREE using the Bayesian Information Criterion (BIC). For the 74-terminal alignment for COI, the following models were used for 1st positions: TNe+I+G4, 2nd positions: TPM3u+F+I+G4, and 3rd positions: HKY+F+G4. For the LSU alignment (23 terminals) HKY+F+I+G4 was identified.