DOI: 10.5061/dryad.vhhmgqp0r
title: Two new phreatic snails (Mollusca: Caenogastropoda: Cochliopidae) from the Edwards and Edwards-Trinity aquifers, Texas

description: 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. 
description: 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
 1<sup>st</sup>, 2<sup>nd</sup>, and
 3<sup>rd</sup> 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.
description: Files are submitted in .fasta or .phylip format which are
 universally translatable for phylogenetics work.
description: # Title of Dataset: *** New phreatic snails (Mollusca: Caenogastropoda:
 Cochliopidae) from the Edwards-Trinity aquifer system, Texas. We developed
 a dataset of COI and nuclear LSU to distinguish species of phreatic snails
 of Texas and understand their relationship. ## Description of the Data and
 file structure These files include 2 DNA sequence alignments of the COI
 DNA barcoding region and one of the nuclear large subunit. They are in
 Phylip relaxed format. The first two numbers are the number of sequences,
 then the number of characters, followed by all the sequences in a row,
 headed by an identifer. The identifier is either the GenBank number or a
 laboratory identifier that can be matched with the GenBank data. The data
 is positioned so that the reading frame starts with codon #1. All
 available sequences from GenBank appearing in previous literature were
 included with new sequences generated during this study. GenBank sequences
 must be used with caution as misidentifications and outdated metadata are
 typical. For that reason, the GenBank sequences that were included are
 only those from the taxonomic literature that described these species, or
 from topotypic individuals. For type localities where we were not able to
 sample and sequence from fresh materials, these were used to confirm
 species identity. Sequences were aligned in Geneious R10 using the MUSCLE
 alignment algorithm (Edgar 2004). Phylogenetic analyses were conducted in
 IQTree 1.6.12 (Minh et al. 2013, Nguyen et al. 2015, Hoang et al. 2018).
 Missing data is identified by a hyphen. ## Sharing/access Information
 Links to other publicly accessible locations of the data: Was data derived
 from another source? If yes, list source(s):GenBank