Asclepias welshii was listed as threatened because of its restriction to CPSD and perceived threats from off-road vehicle use in its active sand dune habitat, but the discovery of smaller outlier populations and the apparent long-term persistence of the species in the face of recreational disturbance at CPSD has raised the question of its possible delisting. A recent drone census of the population indicated high stem numbers in 2021 but ambiguous evidence for shifts in distribution and local decline within the dunes. A concomitant population genetic analysis indicated reasonably high levels of genetic diversity and high rates of outcrossing, but the molecular markers used did not have sufficient resolution to definitively address clone structure or to distinguish clearly between clone members and their closely related seed progeny (Meyer et al. 2024). Distinguishing between juveniles established from seed and new offshoots from rhizomes in the field has been difficult, making it hard to evaluate the importance of seedling recruitment in extending existing patches or establishing new ones. This study will contribute substantially to our understanding of the establishment ecology of the species and provide a solid basis for interpretation of ongoing and future monitoring data, both on the ground and from drone imagery. This will better inform management decisions for the species, including any decision regarding delisting by the US Fish and Wildlife Service.

1) Develop a set of microsatellite (SSR or simple sequence repeat) markers for A. welshii that are suitable for fine-scale population genetic analysis and that can be used to genotype large numbers of stems/clumps at low cost, and SSR-genotype stem samples using these markers with an amplicon sequencing approach. 2) Determine the clonal structure of discrete patches of stems by genotyping all stems/clumps within a patch (rather than a restricted subsample as carried out earlier) using more precise microsatellite markers. 3) Determine the relative importance of clonal (asexual) reproduction and reproduction from seed (sexual reproduction) at the patch level for A. welshii at CPSD. *Carry out morphological studies in the field to reliably distinguish between clonal offshoots and juvenile plants produced from seed using nondestructive methods. *Carry out field evaluations to determine the relative abundance of juvenile plants from seed and new clonal offshoots within patches in habitats with different levels of disturbance (ORV-accessible vs. protected, active dunes vs. dunes vegetated to different degrees and with different plant communities).

The only known large population of Asclepias welshii is at CPSD, so this is the only location where this study could be performed. There are good reasons for carrying out the proposed study on a short time frame. First, with the initiation of drone monitoring of this population, there will be a great deal of spatial information on its distribution and how it changes over time relative to both recreational vehicle disturbance and dune movement and stabilization, which are thought to be the main drivers. However, the emphasis in the past has been on clonal migration. No one has considered the role of seedling establishment and its potential for establishing new, genetically unique clones far from both parents and from other clones. By focusing the lens on the seedling stage, we will be in a better position to understand patterns of colonization and decline observed in the imagery. Second, we now have a team of interested researchers at Southern Utah University who can combine their talents in the short run to make a quantum leap in our understanding of this plant. This team in this configuration likely has a limited amount of time to accomplish this work, increasing their motivation to tackle a challenging research agenda now.

Managing use, especially recreational use, to minimize negative impacts on A. welshii and other rare organisms at CPSD is a high priority for both the BLM Kanab Field Office and Utah State Parks. Substantial new information on the population ecology of A. welshii will be useful to them in designing and implementing management strategies that include this species. For US Fish and Wildlife Service, this information will also be very useful as they consider the conservation status of the species and the case for delisting. In addition, the microsatellite markers that are developed can subsequently be used to perform low-cost characterization of genetic diversity and population genetic structure for outlying populations of the species.

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This work will be carried out with the full support of Utah State Parks and the Bureau of Land Management Kanab Field Office as part of continuing research by the Southern Utah University group. A permit for Dr. Meyer to collect Utah listed species is in place with US Fish and Wildlife Service through the BLM Utah State Office. This includes collecting seeds, obtaining tissue samples, and otherwise carrying out field studies that may directly impact individual plants.

Objective 1) Microsatellite marker development and genotyping. Approach: To examine fine-scale population structure in A. welshii we will use a genetics-based two-tier approach in which we first assemble the A. welshii genome and subsequently search for and sequence DNA microsatellites. The following workflow has been adapted from Feng et al. (2024) and Costa and Almeida (2022), who were successful in performing de-novo genome assembly and identifying DNA microsatellites in two Asclepias species using an affordable Oxford Nanopore Sequencing technology and open-sourced data analysis toolkits. Sample Collection: The sampling plan for each of the five patches will be developed based on current-year high-resolution drone imagery of each patch and each sampled stem will be accurately geolocated. Plant tissue samples will be collected nondestructively from all individual stems/clumps in each patch by cutting a segment ca. 1 x 4 cm from the tip of a healthy leaf. Samples will be placed in labeled coin envelopes and stored in silica gel until DNA extraction. DNA Extraction: Plant samples will be homogenized by crushing leaf tissue with a pestle and mortar and DNA will be extracted using a Qiagen DNeasy Plant Pro Kit. De-Novo Genome Assembly Library Prep and Sequencing: Extracted plant DNA will be fragmented into smaller sizes that are suitable for sequencing using a Fisherbrand Model 505 Sonic Dismembrator. To select the correct DNA fragment size for Oxford Nanopore Sequencing, we will use a Circulomics SRE kit v2.0 from Pacific Biosciences. Subsequently, DNA concentration and purity will be measured using a Qubit 4 Fluorometer. Size-selected DNA samples will be prepared for sequencing using an Oxford Nanopore Genomic DNA by Ligation kit. Finally, samples will be sequenced using a Nanopore PromethION flow cell on an Oxford Nanopore PromethION P2 Solo sequencer. De-Novo Genome Assembly and Microsatellite Identification: To estimate the genome size of A. welshii we will perform a k-mer analysis using the software provided by the Python Package Jellyfish v2.2.7 (Marais and Kingsford, 2011) and the Genoscope Online webtool (Vurture et al., 2017). Genome size estimation is an essential parameter for genome assembly that ensures accuracy and enables genetic diversity analyses. Subsequently, we will use the C++ toolkit MaSurCA v3.4.1 (Zimin et al., 2013) to perform de-novo genome assembly of A. welshii. Once we complete the first draft of the genome, we will correct the assembly, eliminate redundancy, and fill gaps by using the Pilon v1.23 (Walker et al., 2014), Purge Hapolotigs (Roach et al., 2018), and LR gapcloser v3 (Xu et al., 2019) toolkits, respectively. Upon completion of the first genome assembly, we will assemble four additional genomes using DNA collected from individual leaf segments from stems in discrete patches from different areas within the dune system. This will allow us to identify regions of genomic variation among A. welshii individuals. Finally, putative polymorphic microsatellite DNA sequences will be identified using the free web-based applications Tandem Repeat Analyzer and Repeat Explorer. Microsatellite PCR and Sequencing: Once a set of DNA microsatellites has been identified, we will design PCR primers to amplify each sequence. PCR products will be assessed for size by gel electrophoresis and subsequently purified by using a Qiagen QIAquick PCR Purification kit. Subsequently, DNA concentration and purity will be measured using a Qubit 4 Fluorometer. Purified PCR products will be prepared for amplicon sequencing using an Oxford Nanopore Native Barcoding 96 V14 Kit. Samples will be sequenced using a Nanopore PromethION flow cell on an Oxford Nanopore PromethION P2 Solo sequencer. Objective 2) Characterization of clone structure by examining genotypes of all individual stems/clumps in discrete patches. We will select five discrete patches from different parts of the dune system for genetic characterization and clone structure analysis. This will include the three patches partially characterized using SNP markers in our earlier study (Meyer et al. 2024) as well as two additional patches not yet characterized. Approximately 100 stems will be sampled from each patch, depending on patch area and density. The stems sampled in each patch will be geolocated on drone imagery to permit spatial analysis of observed patterns of differentiation. A genetic distance matrix will be calculated from the microsatellite data (Phylip software: Felsenstein 1989) and statistically compared with a geographic distance matrix based on geolocations in the drone imagery. We will determine from this analysis which stems are truly clonal (genetically identical), and also how closely related remaining stems are to each of the detected clones and to each other. This will enable us to determine which of the stems in the patch are genetically unique and thus likely the product of recent recruitment from seed rather than clonal spread. This will be confirmed by morphological examination in representative cases (Objective 3). Using microsatellite data from all individuals in a discrete patch, we will also attempt to determine the putative parentage of juveniles produced from seed. Microsatellite genotype data for the stems sampled from five patches at CPSD will also be analysed using standard population genetics tools (Arlequin software: Excoffier and Lischer 2010) to make the analysis comparable to the analysis based on SNPs (single nucleotide repeats) in Meyer et al. (2024). This will include calculation of expected and observed heterozygosity and spatial structure both within patches and across patches. Objective 3) Quantifying asexual versus sexual reproduction in A. welshii as a function of dune movement, disturbance regime, and degree of stabilization. The first step in determining the relative importance of clonal propagation versus establishment from seed in different environments is to be able to distinguish juvenile plants from clonal offshoots. Because the juvenile plants are known to have a very distinctive leaf morphology in comparison to adult plants and their typical clonal offshoots, this would appear to be straightforward. What is not known is how long plants retain their juvenile leaf form, and whether they can produce their first rhizome while still in this juvenile condition. This has not been examined directly, but some earlier monitoring reports state that these 'primary stems' can be either seedling stems or clonal offshoots with juvenile morphology (Palmer 1999). It seems more likely that juvenile stems associated with a rhizome are in the process of producing the rhizome rather than being the product of the rhizome. We will determine whether this is the case by nondestructively excavating both stems with juvenile leaf morphology and stems with typical rhizome offshoot morphology to determine whether a rhizome connection is present and how the morphology of the rhizome and its connection varies between the two stem types. Juvenile stems vary from tiny new seedlings with cotyledons still present to much more robust stems that potentially could be a year or more old. We predict that systematic excavation of stems across this range of size variation will show that stems with juvenile leaf morphology do not arise from preexisting rhizomes. If this is the case, it will be possible to classify young stems as seed-produced juveniles or clonal offshoots. To examine the relative importance of seedling recruitment versus clonal spread, we will select sampling areas that represent different disturbance regimes: active unprotected dunes, active dunes in protected areas (tiger beetle exclosures), stabilized dunes with different vegetative cover and composition (at least three types), and areas with high ORV impact. We will carry out walking transects through at least three representative areas of each type and score the numbers of juveniles and new offshoots encountered within a two-meter band along each transect. Mature stems/clumps of A. welshii will also be counted within each belt transect. Transects will vary in length depending on the configuration of the sample area but the total transect length in each sampled area will be at least 100 m. At least eighteen areas will be sampled (six area types x three representative areas per type). These data will be statistically analyzed to determine the relative abundance of juveniles vs. clonal offshoots in each area type. This will give us a general idea of the frequency of recruitment from seed and will also tell us whether the relative frequency of sexual reproduction (establishment from seed) varies by habitat type within the dunes.

This project does not have a post-completion monitoring component, but our findings will definitely be applicable as the new monitoring protocol for A. welshii is further refined. It is doubtful that juvenile plants will be readily detectable or identifiable in the drone imagery, as they closely resemble several other narrow-leaved herbaceous species that co-occur with A. welshii in the dune habitat. Along with reproductive output studies on the ground, some on-the-ground data collection on juvenile plants will likely need to be included in the monitoring protocol. Our proposed work will greatly facilitate that effort.

A principal partner and colleague in this research is Kody Rominger, Research Fellow at Southern Utah University, who is engaged in developing and applying a drone monitoring protocol for A. welshii at CPSD and also carrying out the census of outlying populations. He receives funding and field support from the BLM Utah State Office and Kanab Field Office (Aaron Roe, BLM Utah State Botanist and Carson Gubler, Rangeland Management Specialist). Other partners include Lark Willey (Botanist, US Fish and Wildlife Service) and Mindy Wheeler (Coordinator, Utah Rare Plant Conservation Program). We also have an informal partnership with Park Manager Patrick Buhr at Coral Pink Sand Dunes State Park.

This project will provide valuable information on the population biology of A. welshii that will be useful in the future management of this species regardless of whether or not it is eventually delisted. In addition, the SSR markers we develop will be essential for examining among and within-population genetic diversity and population genetic structure of outlying populations of the species at nominal cost compared to other genotyping methods.

CPSD is a very popular area for legal recreational off-highway-vehicle use, and this is likely to continue. There have been considerable efforts to monitor the effects of OHV use on the dunes and to protect sensitive species and their habitat, particularly for the CPSD tiger beetle. There are currently conflicting perspectives on the impact of OHVs on A. welshii. Some are of the opinion that vehicle disturbance benefits the species by slowing dune stabilization, while others emphasize the direct negative impacts on actively growing plants. Better information on the role of clonal spread vs. seedling recruitment in colonizing new and formerly occupied areas within the dunes will be a powerful adjunct to long-term drone monitoring. This study will provide the tools to add this component to ongoing management efforts to maintain OHV recreation as a sustainable use at CPSD.