Four populations of Heliotrope milkvetch have been identified on the Wasatch Plateau. Although these populations face few threats at present, they are restricted to a highly specific habitat type which may face increased threats in the future due to energy development. Heliotrope milkvetch is currently ranked as G3, indicating a globally vulnerable species. (NatureServe, 2024; https://explorer.natureserve.org/). This indicates it is a species of relatively minor conservation concern for the purposes of federal listing under the Endangered Species Act. However, resolution of the proper placement of Heliotrope milkvetch relative to both varieties of the Navajo Lake milkvetch would further clarify its conservation status. This project will provide additional information for conservation and management of Heliotrope milkvetch by producing a rigorous taxonomic treatment that would clarify the relationships of Heliotrope and Navajo milkvetch by combining genetic, morphological, and ecological data sources.

Heliotrope milkvetch (Astragalus montii) is a federally listed threatened species distributed on the central Wasatch Plateau in Sanpete and Sevier counties, where it is locally abundant in suitable habitat on the plateau and faces few threats at present. Taxonomic treatments of Heliotrope milkvetch have varied, with it being treated either as a distinct species or as a variety of another Utah-endemic species, the Navajo Lake milkvetch (Astragalus limnocharis). To date there has been no genetic study on either the Heliotrope or Navajo Lake milkvetches, meaning their relationship to each other is largely speculative. We propose collecting data on these two endemic species to guide management decisions regarding Heliotrope milkvetch via three objectives. Specifically, we plan to: (1) generate genetic data which can be used to assess phylogenetic relationships between these two species and produce population genetic data including genetic diversity within populations and effective population size; (2) collect morphological data for all taxa involved to develop updated descriptions and a dichotomous key reflecting the resulting taxonomy; (3) characterize ecological/environmental associations to assess similarities and differences between them.

Project Location: Field work will take place in the habitats of Starveling milkvetch (Astragalus jejunus), Navajo Lake Milkvetch (Astragalus limnocharis), and Heliotrope milkvetch (Astragalus montii) on the Wasatch Plateau, Table Cliff Plateau, Markagunt Plateau, and Monte Cristo Range in Utah. Project administration, DNA extraction, library preparation, data analysis, and preparation of deliverables will occur at Northern Arizona University (NAU) in Flagstaff, Arizona. Sequencing will be performed at the University of Oregon Genomics Core in Eugene, Oregon. Project Responsibilities: This project will be administered by the Hodel Lab at Northern Arizona University in Flagstaff, Arizona. Ph.D. Student Ava Brinkley will have primary responsibility for coordination and completion of the project and for generation of deliverables. This project will receive additional consultation on wet lab and data analysis protocols from Rob Massatti, Ph.D., and staff at the US Geological Survey's Southwest Biological Science Center in Flagstaff, Arizona. We propose to complete this project in the timeframe of two state fiscal years, from July 1, 2025 through June 30, 2027. An outline of project schedule by task is highlighted below. The tasks necessary to achieve these goals are broken out into five phases of the project, listed below. Phase 1: Finalizing Permitting, Morphological data collection, Scouting - Permitting will be requested from all relevant stakeholders, including USFWS, USFS, BLM, and NPS to enable minimally destructive tissue collections (~2 leaves) from live plants of all relevant taxa. During the summer of 2025, we will scout field sites to implement sample design and take morphological measurements for downstream applications in morphometric analyses and development of descriptions and dichotomous key. Phase 2: Tissue collections - We will collect tissue samples from pre-scouted field sites during the summer of 2026 to ensure positive identification of all specimens for the purposes of this study. Tissues will be promptly stored with silica desiccant to preserve DNA quality until lab processes begin in Phase 3. Phase 3: Wet lab and ecological variables - Once all tissues have been collected and sufficiently dried in silica desiccant, lab processes can begin. DNA Extractions and ddRADSeq library preparations will be performed in Flagstaff before sending samples off for sequencing. Ecological comparisons and morphometric analyses for the three entities will begin during this period to be completed by the beginning of Phase 4. Phase 4: Phylogenetic and population genetic analyses - When sequencing reads are returned, downstream genetic analyses can begin. Analyses will be performed using a variety of specialized software hosted on Northern Arizona University's high-performance computing cluster, MONSOON. Sequence assembly will be performed in ipyrad for downstream phylogenetic analyses using IQTree and SVDQuartets; population genetic work will be performed. Phase 5: Report drafting and data compilation - Once downstream analyses are completed, all collected data will be included in a report drafted for Utah DWR, USFWS, USFS, and other relevant stakeholders. All data and results will also be prepared for publication in a scientific journal.

Focal species listed in SGCN 2021-2022 Addendum to Wildlife Action plan.

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We will obtain permits via all relevant stakeholders, including USFWS, USFS, BLM, and NPS to enable minimally destructive tissue collections (~2 leaves) from live plants of all relevant taxa. Heliotrope milkvetch is ESA listed.

The overarching goal of this project is to provide novel data to support decision-making processes regarding the conservation status of Heliotrope milkvetch by clarifying its proper taxonomic treatment and quantifying measures significant to management. (1) Genetic characterization: Double-digest restriction site-associated DNA sequencing (ddRADSeq) provides a cost-effective and scientifically sound method for phylogenetic resolution of shallow-level (i.e., intrageneric) evolutionary relationships, and has been used to successfully inform taxonomic decisions for other morphologically challenging groups in previous studies (Anderson et al., 2023; Chapin et al., 2021; Spriggs et al., 2019). Population-level sampling will be conducted for Heliotrope milkvetch and both varieties of Navajo Lake milkvetch, with a handful of Starveling milkvetch samples to be used as an outgroup for analyses. We plan to collect 60 individuals of Heliotrope milkvetch and 60 individuals of each variety of Navajo Lake milkvetch and an additional 12 samples of Starveling milkvetch, for a total of 192 samples. Field-collected leaf tissue will be stored with silica desiccant and transported to the Science Lab Facility at Northern Arizona University (NAU) in Flagstaff, AZ for DNA extraction and library preparation before being sent for sequencing at the University of Oregon Genomics Core. Downstream analyses of genetic data will be completed using the MONSOON high-powered computing cluster at NAU. For this project, we will follow a practical phylogenetic species concept similar to that of Mishler and Theriot (2000). We will define species as the least inclusive monophyletic group (that does not render a broader group non-monophyletic) that has distinctive biological attributes making it worthy of formal recognition (e.g., apomorphies, unique combinations of phenotypic features, or ecological distinctness). While the latter criterion is somewhat arbitrary, it will allow us to use species recognition as a tool for conservation, while abiding by the constraint of monophyly. In addition to phylogenetic work, resultant genetic data will be useful for quantifying critical information for effective management including genetic variation, effective population size, and gene flow and connectivity patterns. (2) Morphometric Study: To ensure an accurate understanding of the morphological variation present within and between populations and taxa, we will collect in situ morphological data for proposed diagnostic characters. The description of Heliotrope milkvetch included in A Utah Flora was based on six herbarium specimens, making it challenging for the full range of morphological variation to be captured in the existing description (Welsh, 2015). Morphological data will be used to inform the species delimitation process as well as the writing of updated descriptions and a dichotomous key corresponding to the taxonomic treatment indicated by this study. In addition, we will use field-collected morphological data in statistical analyses to assess morphological differentiation within and between populations and putative taxa. (3) Ecological Comparison: The three milkvetch taxa investigated in this study are endemic to limestone plateaus and accordingly have distinct ecological characteristics (NatureServe, 2024; https://explorer.natureserve.org/). If the three entities exist in distinct habitats, it is possible that different environmental conditions can impact species boundaries (Givnish, 2010; Jones et al., 2021). To quantify the environmental distinctness (or lack thereof) among sampling locations, we will use precipitation and temperature data associated with previously documented and geolocated specimens, as well as the geographic data from those sampled during the course of this project. Specifically, we will use the 19 bioclimatic layers from WorldClim (https://www.worldclim.org/data/bioclim.html), which include measurements of temperature and precipitation ranges and seasonality. We will use a Principal Components Analysis to distill the precipitation and temperature variation into three axes of environmental variation following Hodel et al. (2022).

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While this project will primarily be conducted out of the Biological Sciences department at NAU, additional support from USGS Southwest Biological Science Center will be available for wet lab and computational tasks in Phases 3 and 4. Additionally, we will coordinate field work with the Utah State University/Utah DWR Rare Plants Team.

Anticipated Results Anticipated outcomes of this study include a rigorously evaluated taxonomic placement for Heliotrope milkvetch along with all populations of Navajo Lake Milkvetch based on genetic, morphological, and ecological characteristics of these putative taxa. Additionally, detailed accounts of genetic diversity, effective population size, ecological characteristics, and morphological variation in this group will be generated, providing critical information for effective management and field identification of resultant taxa.

Deliverables for this project will include: A final report compiling all methods and findings for the three main objectives, which will be submitted for publication to a peer-reviewed journal pursuant to the requirements of Ava Brinkley's PhD dissertation. All DNA assemblies, sequencing reads, and other genetic data collected will be furnished to the state and published on NCBI GenBank, a public-access genetic data repository. A report describing field work undertaken and notable findings made in the process, along with high-resolution DSLR photographs of Heliotrope and Navajo Lake milkvetches for use in the state field guide.