In desert environments, unique communities depend on groundwater at springs. There is a diverse radiation of small (<5 mm) snails found across the desert southwest in North America. Nearly all springsnail species are considered critically imperiled with their existence depending on maintenance of spring-flows in regions of declining water availability. Extant, endemic, springsnails in the Trans-Pecos region of Texas include one species of Pseudotryonia Hershler, 2001, five nominal Tryonia W. Stimpson, 1865 (Cochliopidae) and seven Pyrgulopsis Call & Pilsbry, 1886 (Hydrobiidae). Four of these snails are classified as endangered under the US Endangered Species Act. Survey work was conducted at the type and previously reported localities of named springsnails and 116 previously unsampled spring sites to locate and identify additional populations. Sequences of the DNA barcoding region were used to establish a database of known sequences from the named species and confirm identifications of new populations encountered.

Our sampling plan included several potential methods, and they were deployed as appropriate depending on site conditions such as water depth and sediment type. The planned methods included drift net sampling over spring openings (1–2 days), dip net sampling, Surber sampling (3X per site), Bou-Rouch sampling (3X per site), and searching by eye and hand-collecting snails. We found snails primarily by hand-collections but at a few sites where the snails occurred in low abundances (Diamond Y spring, Karges Spring, Naegele Spring, Capote Creek Crossing) they were only found by dip net or Surber sampling. Our hand-collections included finding up to 60 individuals at a site for DNA barcoding and species characterization. Voucher specimens for new and resampled sites were placed into either the Philadelphia Academy of Natural Sciences at Drexel University (ANSP) or the Edwards Aquifer Research and Data Center (EARDC). Some occurrence records are also included from the United States National Museum (USNM) from taxonomic literature records. Due to concerns about privacy and conservation of fragile spring sites, as a condition of sampling, some landowners required omission of precise coordinates in published work. To allow scientific reproducibility, the coordinates can be obtained by researchers from the museum collection where the specimens are vouchered, from the TPWD Natural Diversity Database (https://tpwd.texas.gov/) or by contacting the study authors. For two occurrences, T. cheatumi from West Sandia Springs, and T. metcalfi from Capote Creek Crossing, the only available voucher materials were ground for DNA work and so museum vouchers are not available. Collections were conducted under TPWD permit #SPR-0116-011 and USFWS #TE802211-0.

Bulk samples were immediately preserved in 70% nondenatured ethanol in the field, then ethanol was replaced after 24 hours. Snails intended for DNA and taxonomic work were kept in cool spring water until processing the same day. Individuals for DNA work were killed by flash-boiling (Fukuda et al. 2008), followed by preservation in 95% ethanol. Individuals for morphological study were relaxed in mentholated water, then preserved in 70% ethanol. DNA was extracted using Qiagen DNAEasy Blood & Tissue Kit, followed by PCR using one of two primers for amplification of COI, the universal DNA barcoding primers (Folmer et al. 1994) or a derivative designed for spring snails (Liu et al. 2001). Samples were cleaned using the Qiagen QIAquick PCR Purification Kit, quantified using a Qubit (Invitrogen), and sequenced by Eton BioScience, Inc.

Contigs were made and sequences were aligned using Geneious 10.2.6 (Biomatters). 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. For Tryonia, the analysis was conducted with the Texas Pseudotryonia included as these genera are closely related. Previous analyses have placed Pyrgulopsis texana (Pilsbry, 1935) distant from the rest of the Texas Pyrgulopsis, therefore, that species was used to root the tree for analysis (Perez 2021). 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). Pairwise differences were calculated in MEGA 11 (Tamura et al. 2013) using the Kimura two-parameter model (Kimura 1980). The K2P model is the standard for DNA barcode studies, where distances are assumed to be relatively low (Hebert et al. 2003). To look for a barcoding gap, we compared the within- and among-species K2P distances within each genus.

The data file is in a commonly-used format, Phylip that can be translated to other formats for DNA sequence analysis. It can be opened in MEGA, BioEdit, Geneious.