Project Details

Springsnails (herein, genus Pyrgulopsis) are tiny (1-5 mm) aquatic snails that are usually found in spring habitats. The Great Basin in Nevada and Utah is home to a surprising diversity of springsnails (approximately 103 species), and many are endemic to a single spring (Hershler 1998; SCT 2020). Springsnails are often highly abundant (e.g., >1,000 per square meter; Mladenka and Minshall 2001) in springs and can be found grazing on the periphyton that grows on rocks in the water (Lysne et al. 2007). The ecology of Great Basin springsnails is not well known, but their dominant presence in many springs suggests they likely have important roles in their ecosystem. Other freshwater prosobranch snails (those with gills and usually an operculum) are known to affect algal and periphyton biomass (Lamberti et al. 1987; Feminella and Hawkins 1995; Riley et al. 2008) and nutrient cycling (Hall et al. 2003). 
There are 18 species of springsnails in Utah, and 15 of them are listed as Species of Greatest Conservation Need (SGCN) in the Utah Wildlife Action Plan (UWAP; UWAP 2015 and 2020 minor amendment). Many of the historical localities for the 15 SGCN springsnails were visited by UDWR biologists in recent years (2018-2021; ESMF project numbers 23, 62, 126 and 187), but these surveys were not exhaustive. New localities still need to be explored to better understand the current distribution, status, and threats for these springsnails. However, a major hurdle toward a better understanding of these things is the inability to confidently identify individuals to species at new localities using shell and animal morphology. 
Springsnails from new localities cannot be reliably identified to species using morphology alone. Shell morphology can be highly variable within species (Hershler 1998). Male reproductive anatomy is useful for species level identification (Hershler 1998), but tools and techniques required for this level of examination is not practical for UDWR biologists. Genetic sequencing is currently the most reliable way to identify springsnails to species. However, the taxonomy of springsnails is still in flux, and additional phylogenetic research using mitochondrial and genomic data is still needed to verify which springsnail species are valid.
Taxonomic debate is as a crucial data gap in the Utah Wildlife Action Plan (UWAP; UWAP 2015); a clear understanding of taxonomy is fundamental to effective conservation of a species. Additional phylogenetic research may lead to the lumping of SGCN springsnails or it may identify additional sensitive springsnail species that need to be included on the Utah SGCN list. Either outcome will have major implications for effective conservation of Utah's springsnails. Clarifying the taxonomy for springsnails will also prevent inappropriate listing of species under the Endangered Species Act (see the recent example with the Kanab Ambersnail Oxyloma haydeni kanabensis, USFWS 2020).
A major uncertainty within springsnail taxonomy lies within the Toquerville Springsnail (Pyrgulopsis kolobensis) complex. Historically, Hershler considered this to be a widespread species in western Utah and eastern Nevada (Hershler 1998), but recent genetic analyses have found that some of the historical P. kolobensis localities actually represent distinct species. For example, Hershler et al. (2017) determined that P. kolobensis is only known at its type locality, and they resurrected one species and identified three new species from historical P. kolobensis localities. Many of the new (2018-present) springsnail populations in Utah have been tentatively identified as P. kolobensis, but that is not a valid name for such situations, and it is likely that many of these localities are inhabited by new species. 
Several other springsnail taxonomic questions still need to be resolved, and one of these is whether Southern Bonneville Pyrg (Pyrgulopsis transversa) and Ninemile Pyrg (Pyrgulopsis nonaria) are distinct species or whether they should be lumped with Bear Lake Springsnail (Pyrgulopsis pilsbryana; Liu et al. 2018). These springsnails were listed as unique species in the 2015 UWAP SGCN list, but they were lumped into P. pilsbryana in a minor amendment to the SGCN list in 2020. However, as UDWR has learned more about Utah's snails, it has become clear that this decision to lump the species was hasty, and additional research is needed to confirm the taxonomy of many of the springsnails and other mollusk groups in Utah. Conducting research to answer this question about the three springsnails above is the goal of this project because: 
*	the three springsnails are/were listed as SGCN in the UWAP,
*	this question can be addressed in one year, whereas multiple years may be needed to resolve P. kolobensis taxonomy. The relatively small size and low cost of this project will make it a good starting project that leads to future springsnail taxonomic projects in Utah, and
*	results are expected to provide multiple benefits to springsnail conservation in Utah.
Expected benefits of this project include:
*	Genetically verified species-level identification for springsnails from new localities.
*	Validity of the three SGCN springsnail species.
*	Clarification of species distribution, status, and threats for at least three SGCN springsnails. 
*	Address some of the taxonomic uncertainty around historic P. kolobensis localities and newly discovered springsnail populations.
*	Project results will be used to generate more accurate s-ranks for SGCN springsnails, which will lead to better prioritization of mollusk conservation actions across Utah. 
*	A crucial data gap in the UWAP (taxonomic debate) will be addressed. 
*	Objectives in the Springsnail Conservation Agreement and Strategy for Springsnails in Nevada and Utah and the new UDWR Statewide Mollusk Strategy will be addressed (see relation to management plans section). 
*	Published project results in a peer-reviewed journal will provide a significant scientific contribution toward clarifying taxonomy for springsnails in the Great Basin.
It will be important to implement this project by FY23 so that the findings can be used to update s-ranks for the revised UWAP in 2025. Results of this project may suggest that the three SGCN springsnails are more stable than previously thought, leading to a higher (better) s-rank and removal from the SGCN list. Conversely, this study may find that they are more imperiled than previously thought. Either outcome will allow UDWR to better understand the current status of the three SGCN springsnails and move forward with appropriate conservation actions. 
Taking steps to prevent springsnails from being listed under the Endangered Species Act will be important to reduce economic impacts to Utahns. Since springsnails are usually associated with springs, federally listing them could affect acquisition of water rights, groundwater pumping rates, cattle ranching practices, and housing development, though the extent of the economic impact would depend on which springsnail species are listed.
References
Feminella, J. W. and C. P. Hawkins. 1995. Interactions between stream herbivores and periphyton: a quantitative analysis of past experiments. Journal of the North American Benthological Society 14(4):465-509.
Hall Jr., R. O., J. L. Tank, and M. F. Dybdahl. 2003. Exotic snails dominate nitrogen and carbon cycling in a highly productive stream. Frontiers in Ecology and the Environment 1(8): 407-411.
Hershler, R. 1998. A systematic review of the Hydrobiid snails (Gastropoda: Rissooidea) of the Great Basin, Western United States. Part I. Genus Pyrgulopsis. The Veliger 41(1): 1-132.
Hershler, R., L. Hsiu-Ping, C. Forsythe, P. Hovingh, and K. Wheeler. 2017. Partial revision of the Pyrgulopsis kolobensis complex (Caenogastropoda: Hydrobiidae), with resurrection of P. pinetorum and description of three new species from the Virgin River drainage, Utah. Journal of Molluscan Studies 83:161-171.
Lamberti, G. A., L. R. Ashkenas, S. V. Gregory, and A. D. Steinman. Effects of three herbivores on periphyton communities in laboratory streams. Journal of the North American Benthological Society 6(2): 92-104.
Liu, H-P., R. Hershler, and P. Hovingh. 2018. Molecular evidence enables further resolution of the western North American Pyrgulopsis kolobensis complex (Caenogastropoda: Hydrobiidae). Journal of Molluscan Studies 84: 103-107.
Lysne, S. J., L. A. Riley, and W. H. Clark. 2007. The life history, ecology, and distribution of the Jackson Lake Springsnail (Pyrgulopsis robusta Walker 1908). Journal of Freshwater Ecology 22(4): 647-653.
Mladenka, G. C. and G. W. Minshall. 2001. Variation in the life history and abundance of three populations of Bruneau Hot Springsnails (Pyrgulopsis bruneauensis). Western North American Naturalist 61(2): 204-212.
Riley, L. A., M. F. Dybdahl, and R. O. Hall, Jr. 2008. Invasive species impact: asymmetric interactions between invasive and endemic freshwater snails. Journal of the North American Benthological Society 27(3): 509-520.
Utah Wildlife Action Plan [UWAP]. 2015. Utah Wildlife Action Plan: a plan for managing native wildlife species and their habitats to help prevent listing under the Endangered Species Act. Publication number 15-14. Utah Division of Wildlife Resources, Salt Lake City, Utah, USA.

Provide evidence about the nature of the problem and the need to address it. Identify the significance of the problem using a variety of data sources. For example, if a habitat restoration project is being proposed to benefit greater sage-grouse, describe the existing plant community characteristics that limit habitat value for greater sage-grouse and identify the changes needed for habitat improvement.

This project will be completed by the University of Texas Rio Grande Valley (UTRGV) with assistance from UDWR biologists. UDWR biologists will address objective 1 (funded by proposed ESMF project #6142); UTRGV will address objectives 2-5.
1)	Conduct field surveys and compile existing preserved specimens for the three SGCN springsnail species and other unidentified nearby springsnail populations. Prepare these specimens for genetic and morphological analyses (to be conducted by UDWR biologists; see ESMF project # 6142)
2)	Verify the species identity of the springsnail specimens using mitochondrial and genomic sequencing techniques. 
3)	Conduct a phylogenetic analysis to clarify taxonomy for the springsnails.
4)	Examine and describe the anatomy of the springsnails.
5)	Use the results of the genetic and morphologic work above to determine whether a) the three SGCN springsnail species should be lumped or remain unique, b) new localities were identified for the three SGCN species, and c) new species were identified.

Provide an overall goal for the project and then provide clear, specific and measurable objectives (outcomes) to be accomplished by the proposed actions. If possible, tie to one or more of the public benefits UWRI is providing.

LOCATION: Justify the proposed location of this project over other areas, include publicly scrutinized planning/recovery documents that list this area as a priority, remote sensing modeling that show this area is a good candidate for restoration, wildlife migration information and other data that help justify this project's location.

TIMING: Justify why this project should be implemented at this time. For example, Is the project area at risk of crossing an ecological or other threshold wherein future restoration would become more difficult, cost prohibitive, or even impossible.

1)	Utah Wildlife Action plan (UWAP 2015) -- Taxonomic debate is one of the critical data gaps identified in this plan. Implementing this project will help clarify the taxonomy of springsnails in the Great Basin, which will have a multitude of benefits to the conservation of springsnails (see list of expected benefits in the Needs section above).
2)	UDWR Statewide Mollusk Conservation Strategy (Holcomb in prep.) -- This proposed project will benefit all objectives of this Strategy since taxonomy is fundamental to conservation, but it will specifically address objective 1 (clarify the species number and boundaries for springsnails). 
3)	Conservation Agreement and Strategy (CAS) for Springsnails in Nevada and Utah (SCT 2020) -- The purpose of this CAS is to ensure long-term persistence of springsnails and their habitats to help prevent species listings under the Endangered Species Act. This project will help address objective 1 (Compile known springsnail distribution, status, and habitat), but since taxonomy is the foundation of all conservation efforts, it will also support all of the other CAS objectives.

Holcomb, K. in prep. Utah Division of Wildlife Resources Statewide Mollusk Conservation Strategy. Utah Division of Wildlife Resources, Salt Lake City, Utah.

Springsnail Conservation Team [SCT]. 2020. Conservation Strategy for Springsnails in Nevada and Utah, Version 1.0. Nevada Department of Wildlife, Reno, and Utah Division of Wildlife Resources, Salt Lake City.

Utah Wildlife Action Plan [UWAP]. 2015. Utah Wildlife Action Plan: a plan for managing native wildlife species and their habitats to help prevent listing under the Endangered Species Act. Publication number 15-14. Utah Division of Wildlife Resources, Salt Lake City, Utah, USA.

List management plans where this project will address an objective or strategy in the plan. Describe how the project area overlaps the objective or strategy in the plan and the relevance of the project to the successful implementation of those plans. It is best to provide this information in a list format with the description immediately following the plan objective or strategy.

If applicable, detail how the proposed project will significantly reduce the risk of fuel loading and/or continuity of hazardous fuels including the use of fire-wise species in re-seeding operations. Describe the value of any features being protected by reducing the risk of fire. Values may include; communities at risk, permanent infrastructure, municipal watersheds, campgrounds, critical wildlife habitat, etc. Include the size of the area where fuels are being reduced and the distance from the feature(s) at risk.

Describe how the project has the potential to improve water quality and/or increase water quantity, both over the short and long term. Address run-off, erosion, soil infiltration, and flooding, if applicable.

Description of efforts, both completed and planned, to bring the proposed action into compliance with any and all cultural resource, NEPA, ESA, etc. requirements. If compliance is not required enter "not applicable" and explain why not it is not required.

This project will be completed by the University of Texas Rio Grande Valley (UTRGV) with assistance from UDWR biologists. UDWR biologists will conduct Part I of the methods (funded by proposed ESMF project #6142); UTRGV will conduct Part II of the methods.
Part I 
UDWR biologists will compile springsnail specimens for genetic and phylogenetic analysis. The first priority is to use existing specimens in UDWR collections or museum collections (e.g., Smithsonian National Museum of Natural History). If specimens do not already exist, or if their quality is not sufficient for this research, UDWR biologists will conduct field surveys to collect specimens. Specimens are needed from 30-40 locations, and these locations will be spread across the UDWR's Northern, Central, and Southern regions (see map in the Images/Documents tab). 
Preservation of springsnails for genetic and morphologic analyses will follow standard procedures. Thirty springsnails per locality will be flash boiled and preserved in 70-95% non-denatured ethanol for genetic analysis (Perez et al. 2021). Approximately 25 springsnails from each locality will be relaxed in a saturated solution of menthol and then preserved in 70% denatured ethanol for morphological analysis (Perez et al. 2021). 
Part II

For snails targeted for DNA sequencing, shells will be photographed, then total genomic DNA will be extracted from the entire animal by digestion with Qiagen DNeasy Blood and Tissue Kit following the
manufacturer's protocol. We will amplify the mitochondrial locus cytochrome oxidase subunit 1 (COI or cox1) and the nuclear Large Ribosomal Subunit (LSU). COIH2198 and COIL1490 (Folmer et al. 1994). Details of primers and PCR reaction conditions will follow Perez et al. 2021, recent work on Pyrgulopsis. These genes are being targeted as there is existing data on COI for Pyrgulopsis and recent work has shown the nuclear LSU has value for distinguishing Pyrgulopsis species. PCR products will be sequenced at the UT Texas Genomic Sequencing and Analysis Facility (https://research.utexas.edu/cbrs/cores/genomics/services/).

For molecular data analysis, DNA sequences will be assembled in Geneious version 10.2.3, (Kearse et al. 2012) then aligned using MUSCLE. MEGA will be used to calculate genetic distances using the Kimura two-parameter model (K2P, Kimura 1980). To look for a barcoding gap, we will compare the within- and between-species K2P distances. In addition to a barcoding gap, we will also use standard maximum likelihood phylogenetic analyses in IQTree followed by species delimitation analyses to delineate species-level groups and look for concordance with the barcoding analysis. This additional step is necessary to ensure the barcode-assigned groups meet our expected criteria for species delineation (monophyly and diagnosability). All sequences and the complete alignment will be deposited with sequence and analysis repositories GenBank (https://www.ncbi.nlm.nih.gov/genbank/) and Dryad (https://datadryad.org/) so they are available for future use.

For morphometric comparison of shell shapes, twenty mature individuals of each species to be examined will be photographed and landmarked with landmarks around the perimeter of their shells using tpsDig 2.32 (Rohlf 2017). Landmarks will be converted to Procrustes coordinates using least squares superimposition in CoordGen 8 (Sheets 2014). The Procrustes coordinates will be imported to PAST 3.16 (Hammer et al. 2001) where shell shapes are compared with Principal Components Analysis (PCA), Canonical Variate Analysis (CVA), MANOVA, and post-hoc tests using Bonferroni-corrected pvalues. The results of PCA and CVA will be visualized in JMP Pro 15.1.0 (1989-2019) and tpsRelw (Rohlf 2003).

For anatomical dissections, shells will be dissolved by immersion in 50% HCL for 1 minute for investigation with the tissues stained and dissected in Bouin's solution. Digital photographs using light microscopy will be taken with an Olympus SZX9 or Olympus CX41 using AmScope v. 3.7 and images will be stacked in Helicon Focus 6.7.1.

During dissection, opercula and radula will be set aside for scanning electron microscopy (SEM). These will be obtained using a JEOL JSM-6010 PLUS/LA. Shells, opercula, and radula will be air-dried. Radula will be sputter-coated for imaging using the SEM as detailed in Perez et al. (2021). 
The outcome of this project will be a submitted scientific journal article that addresses whether: a) the three SGCN springsnail species should be lumped or remain unique, b) new localities were identified for the three SGCN species, and c) new species were identified. This publication will be beneficial to the mollusk community and can be used by the Freshwater Mollusk Conservation Society to support any needed updates to their list of accepted aquatic snail species (https://molluskconservation.org/
MServices_Names.html). 
References

Folmer, O.M., Black, W., Hoeh, R., Lutz, R. & Vrijenhoek, R. (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology, 3, 294--299.
 
Hammer, ÃÂ., Harper, D.A.T. & Ryan, P.D. (2001) PAST: Paleontological statistics software package for education and data analysis. Palaeontologia Electronica, 4, 1--9.
 
Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M. & Sturrock, S. (2012) Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics, 28, 1647--1649.
https://doi.org/10.1093/bioinformatics/bts199
 
Kimura, M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111--120 (1980). https://doi.org/10.1007/BF01731581
 
Perez, K. E., M. S. Leal, H. Glover, H. Glover, R. T. Chastain, B. T. Hutchins, and B. Schwartz. 2021. Two new species of Pyrgulopsis Call & Pilsbry, 1886 (Mollusca: Caenogastropoda: Hydrobiidae) from springs in the Rio Grande watershed in Texas. Zootaxa 5071(3):384-402.
 
Rohlf, F.J. (2003) TpsRelw, relative warps analysis. http://life.bio.sunysb.edu/morph/
 
Rohlf, F.J. (2017) TpsDig2. http://life.bio.sunysb.edu/morph/.
 
Sheets, H.D. (2014) Integrated Morphometrics package (IMP) 8. https://www.animal-behaviour.de/imp/.

Describe the actions, activities, tasks to be implemented as part of the proposed project; how these activities will be carried out, equipment to be used, when, and by whom.

Describe plans to monitor for project success and achievement of stated objectives. Include details on type of monitoring (vegetation, wildlife, etc.), schedule, assignments and how the results of these monitoring efforts will be reported and/or uploaded to this project page. If needed, upload detailed plans in the "attachments" section.

List any and all partners (agencies, organizations, NGO's, private landowners) that support the proposal and/or have been contacted and included in the planning and design of the proposed project. Describe efforts to gather input and include these agencies, landowners, permitees, sportsman groups, researchers, etc. that may be interested/affected by the proposed project. Partners do not have to provide funding or in-kind services to a project to be listed.

This project is expected to be completed within one year. Once completed, it will provide a multitude of benefits to the future conservation of SGCN springsnails throughout Utah: 
*	Genetically verified species-level identification for springsnails from new localities.
*	Validity of the three SGCN springsnail species.
*	Clarification of species distribution, status, and threats for at least three SGCN springsnails. 
*	Address some of the taxonomic uncertainty around historic P. kolobensis localities and newly discovered springsnail populations.
*	Project results will be used to generate more accurate s-ranks for SGCN springsnails, which will lead to better prioritization of mollusk conservation actions across Utah. 
*	A crucial data gap in the UWAP (taxonomic debate) will be addressed. 
*	Objectives in the Springsnail Conservation Agreement and Strategy for Springsnails in Nevada and Utah and the new UDWR Statewide Mollusk Strategy will be addressed (see relation to management plans section). 
*	Published project results in a peer-reviewed journal will provide a significant scientific contribution toward clarifying taxonomy for springsnails in the Great Basin.
We expect this project to be completed within one year. However, this project will not answer all of the existing springsnail taxonomic questions, so continued research will be needed in the future. The following funding sources will be considered for future research: Competitive State Wildlife Grant with Nevada and Idaho, Recovering America's Wildlife Act (if available), BLM, USFS, and ESMF.

Detail future methods or techniques (including administrative actions) that will be implemented to help in accomplishing the stated objectives and to insure the long term success/stability of the proposed project. This may include: post-treatment grazing rest and/or management plans/changes, wildlife herd/species management plan changes, ranch plans, conservation easements or other permanent protection plans, resource management plans, forest plans, etc.

Potential for the proposed action to improve quality or quantity of sustainable uses such as grazing, timber harvest, biomass utilization, recreation, etc. Grazing improvements may include actions to improve forage availability and/or distribution of livestock.