Shivwits milkvetch is a federally listed species at high risk of extinction. It is currently comprised of six populations, most of which are very small (<300 plants including seedlings) and at least two of which (Harrisburg and Pahcoon Wash) have been documented to be in decline over the last 20 years (FY2019 BLM long-term monitoring report). Studies of reproductive output (Houghton unpublished data) indicate an abnormally high percentage of aborted ovules, resulting in sometimes much-reduced seed set in comparison with other more successful Astragalus species, possibly indicating inbreeding depression. A genetic analysis carried out on samples collected in 2005/2006 suggested that these populations were formerly connected through gene flow but were showing reduced genetic diversity due of fragmentation, loss of connectivity, and small population size (Breinholt et al. 2009). It is likely that populations that formerly occurred in suitable habitat on the Chinle formation in the intervening area have been extirpated due to human activity. We propose to augment three small populations directly through transplanting to increase numbers, and also to explore the option of reciprocal transplanting among these populations in an attempt to restore genetic diversity that has been lost.

The goal of this project is to reduce extinction risk for Shivwits milkvetch. We hope to take first steps toward this goal through meeting the following short-term objectives: (1) Use next-generation sequencing methodology to determine the advisability of carrying out genetic rescue through reciprocal transplanting to increase genetic diversity at three small populations by evaluating their current population genetic status, (2) Carry out pilot augmentation experiments at each of the populations using greenhouse-propagated transplants produced from onsite-collected seeds of each population. If deemed advisable based on results of Objective 1, include reciprocal transplanting among the sites as part of the pilot augmentation experiment, (3) Evaluate success of the augmentation experiments the following spring in terms of survival, growth, and reproductive output.

This project directly addresses actions outlined in the US Fish and Wildlife Service recovery plan for this species (USFWS 2006), including maintaining or augmenting populations to achieve long-term trajectories that show stable or improving trends. As the long-term trends in at least two populations have been shown to be neither stable nor improving, active management is needed to achieve a reversal of these trends. Our objectives in the short term are to increase numbers and if possible increase genetic diversity in these small populations. In the longer term, we will evaluate the effects of these management actions in terms of genetic diversity and population health. All three populations are on Bureau of Land Management land; our project also addresses BLM management objectives and priorities for this species.

The scientists who will carry out the field component of this study are permitted by both US Fish and Wildlife Service and by the BLM to collect seeds and tissue of Shivwits milkvetch, and our plans for this study have been vetted by management within each agency.

The timeline for this study includes a jump-start this spring to enable us to meet our objectives within the ESMF one-year time frame. We have initiated propagation efforts to produce the plants for outplanting and plan to collect tissue for genetic analysis in April 2020. Genetic analysis and interpretation will be carried out during the summer of 2020, and outplanting of dormant transplants will take place in autumn. We will evaluate the short-term success of the planting in spring 2021 and prepare our report the following summer. To meet Objective 1, we will collect ca. 1 cm2 of fresh leaf tissue for genetic analysis from approximately 30 actively growing individuals in each population and preserve the labeled samples in silica gel for later extraction. We will also include tissue collection from one population of the close congener A. eremeticus for use as an out-group in the analysis. If there are insufficient actively growing individuals in Shivwits milkvetch wild populations, we will supplement with tissue from greenhouse-grown individuals. DNA extraction, quantification, and library preparation will be carried out by genetics summer interns at Southern Utah University under the direction of Dr. Jacqualine Grant. DNA extraction will utilize a Qiagen DNeasy Plant Pro kit. We plan to use a methodology called ddRADseq (double digest restriction-associated DNA sequencing) to obtain sequence information from each sample to use in the genetic analysis (Peterson et al. 2012). This method works well for non-model species because there is no need for a reference genome. Because plants have very large genomes, it is necessary to include a genome reduction step (in this case digestion with restriction enzymes) to obtain just a small subset of the genome prior to sequencing, most likely using an Illumina Hi-seq instrument. This methodology is complex and will be outsourced to a commercial genotyping company. Dr. Grant will carry out the analysis and interpretation once we have the sequence dataset in hand using available bioinformatics software. Population structure, inbreeding coefficients, and heterozygosity are calculated with GENALEX 6.5, and estimated pairwise genetic differentiation (FST) with SPAGeDi software. This will permit us to evaluate whether reciprocal transplanting would likely have the desired effect of increasing genetic diversity without high risk of negative effects (e.g., outbreeding depression). We will also be able to evaluate how much the situation has changed as a consequence of continued decline in these populations since the last genetic analysis. To meet Objectives 2 and 3, we modify a propagation protocol developed by Laura Schrage and Kathy Dilley of Zion National Park to obtain plants of Shivwits milkvetch for transplanting. We will use seeds from each population collected as part of our reproductive output study last spring (2019). We plan to try to obtain 100 individuals from each collection. Seeds will be scarified if necessary, moist-chilled in petri dishes for 2 weeks, then permitted to initiate germination at room temperature. Newly germinated seeds will be planted into Ray Leach conetainers containing a coarse-textured soilless mix amended with field soil from an occupied site. The young plants will be grown from emergence in March through early summer. Shivwits milkvetch has a spring ephemeral life history; the plants will go into dormancy as temperatures warm in mid-summer. The dormant plants will be held until outplanting in October. Thirty to 100 plants of each population will be planted back into their population of origin, depending on the results of Objective 1. If we go ahead with the reciprocal transplanting experiment, approximately 30 plants of each population will be outplanted at each of the three planting sites. Otherwise we will do simple augmentation, with all plants outplanted at their site of origin. The plants will be tagged at the time of outplanting in the field. To meet Objective 4, we will evaluate these outplantings the following spring. Surviving plants will emerge in March or April on the planting sites. They will then be evaluated nondestructively for survival, growth, and reproductive success.

As with many research projects with rare perennial plants, the work described here is only the first step in evaluating whether population augmentation and/or genetic rescue will really improve the conservation status of Shivwits milkvetch. Yearly population monitoring will be needed to determine whether transplants survive and contribute seeds to future generations as well as whether seed set of each population overall is improved.

Dr. Michael Stevens of Utah Valley University will provide overall guidance and also the staff to carry out all the necessary fieldwork, including tissue collection, outplanting, and evaluation. Dr. Susan Meyer of the US Forest Service Shrub Sciences Laboratory will take responsibility for producing the transplants and will also participate actively in interpretation of the genetics data, decision-making, and report and possibly manuscript preparation. Dr. Jacqualine Grant of Southern Utah University will provide the expertise and student help to complete the genetics component of the study in an expeditious manner. We will also work closely with the Stephanie Root of the BLM St. George Field Office and Jena Lewinsohn of USFWS to make sure that we carry out all phases of the study with their continued cooperation and blessing.

At some point in the future, genetic reevaluation will be needed to determine whether the influx of new genes due to reciprocal transplanting had any effect on overall genetic diversity. It may also be possible to use the knowledge gained in this study to design and execute additional reciprocal transplant activities and also introductions into unoccupied suitable habitat in an effort to improve connectivity and gene flow among existing populations.

07/01/2020

06/30/2021

2021

Shivwits milkvetch (Astragalus ampullarioides) is a federally listed endangered species endemic to the Chinle Formation and restricted to Washington County, Utah. It is currently considered to be at very high risk of extinction. It is comprised of six populations, most of which are very small (<300 plants including seedlings) and at least two of which (Harrisburg and Pahcoon Wash) have been documented to be in decline over the last 20 years. Studies of reproductive output at three small populations on BLM land indicate an abnormally high percentage of aborted ovules, resulting in sometimes much-reduced seed set, possibly indicating inbreeding depression. An earlier genetic analysis suggested that these populations were formerly connected through gene flow but were showing reduced genetic diversity due to loss of connectivity and small population size. The goal of this project is to reduce extinction risk for Shivwits milkvetch. It includes the following short-term tasks: (1) Use next-generation sequencing methodology to determine the advisability of carrying out genetic rescue through reciprocal transplanting to increase genetic diversity at three small populations on BLM land by evaluating their current population genetic status, (2) Carry out pilot augmentation experiments at each of these populations, including reciprocal transplanting if advisable, using greenhouse-propagated transplants produced from onsite-collected seeds of each population. (3) Evaluate success of the augmentation experiments the following spring in terms of survival, growth, and reproductive output.

Task 1) We collected leaf tissue samples for next-generation sequencing ahead of schedule as planned in April 2020. Samples were collected from 20 individuals at each of four Shivwits milkvetch populations (Pahcoon Wash, Silver Reef, Harrisburg, and Zion National Park). We also collected tissue from a nearby population of a close congener, Astragalus eremeticus, to use as an out-group in the analysis. These samples were submitted to a commercial laboratory (Floragenex) for genetic analysis in early summer 2020. Unfortunately, possibly due to COVID19 complications, we did not receive the sequence data back from Floragenex until the end of May 2021, nearly a year after submission. This made it impossible to evaluate the advisability of the reciprocal transplant design in time for any transplanting within the one-year time frame of the grant. We now are in the process of analyzing the sequence data and will be able to make a decision on the reciprocal transplant design prior to our fall transplanting, which is now scheduled for October 2021. Task 2) We encountered some problems with seed quality and unknown seed dormancy levels in our first efforts to propagate this species in spring 2020, but produced ca. 10-20 individuals of each of the three populations in container culture. The provisional plan was to hold these as dormant stock for a fall 2020 outplanting, but we decided instead to produce a much larger number of plants of each population during the fall and winter for a spring 2021 outplanting of actively growing stock. This was based on the idea that we would have the genetics data in time to evaluate the reciprocal transplant idea by early spring. Using an improved propagation protocol with longer moist chilling of hand-scarified seeds, we produced ca. 160 plants each of the Harrisburg and Silver Reef populations and ca. 40 plants of the Pahcoon Wash population. These were ready to plant out in late March 2021. However, we decided to postpone this planting because of extreme lack of soil moisture due to a very dry winter, which also created greatly increased risk of herbivory. In addition, we still lacked the genetics information to make a decision on how to implement the planting design. By holding these plants over as dormant stock, we will likely be able to implement the reciprocal transplant design as originally intended after a one-year delay, in fall 2021. We achieved an unplanned accomplishment when some of the Shivwits milkvetch container plants initiated flowering in the greenhouse in spring 2021. Thirty of these plants were potted up, allowed to establish in the pots, and transferred to an orchard in the foothills near Provo, where they were pollinated by a diversity of native bees. We were able to harvest 3500 seeds from these plants after they were moved to the greenhouse for the summer. The ability to produce seeds of this species in cultivation creates the possibility for much more extensive augmentation, genetic rescue, and introduction studies in the future. It also eliminates the problematic removal of seeds from wild populations where seed production may already be limiting. Task 3) We plan to evaluate the success of the fall 2021 outplanting in spring/early summer 2022 and to provide a supplemental report to ESMF that includes the genetic analysis and the immediate outcome of the transplant experiment at that time.

The three populations of Shivwits milkvetch in our study are at imminent risk from multiple stressors, so that effective action to prevent local extinction is a very high priority. If we can successfully reciprocally transplant and establish plants at these three populations, detailed studies of their subsequent reproductive success will be necessary to detect any positive effect on seed production. Measures will also need to be taken to protect these experimental plants, and possibly other plants in these small populations, from flower stalk herbivory due to rabbits, root herbivory due to ground squirrels and pocket gophers, and pre-dispersal seed loss due to insect damage to developing seeds. It may also be necessary to quantify these effects in order to detect the relative importance of inbreeding depression in limiting seed production. In the longer run, it could be possible to restore some genetic connectivity by establishing new populations via transplants or direct seeding into suitable habitat in the intervening area. As these plants are relatively long-lived perennials, it will be necessary to follow these and future outplantings/seedings for multiple years to obtain reliable results. In addition to monitoring survival, reproductive output, and population trends, at some point in the future following reciprocal transplanting it will be very interesting to engage in another genetic analysis to see whether genetic rescue, measurable as an increase in population-level genetic diversity, has resulted from our efforts. We have also noted serious encroachment of annual bromes into the edges of Shivwits milkvetch habitat, especially at Harrisburg, where a grass fire recently came dangerously close to occupied habitat. The Chinle clays generally support low vegetative cover, but the occupied patches are so small that wildfire remains a threat. The bromes are also potential competitors if they can successfully invade into the clay soils, and this becomes more likely if the occupied habitat is surrounded by brome-infested areas. Some form of chemical control of nearby source populations may be worth investigating. As explained above, several unforeseeable obstacles have prevented us from completing this project in a timely manner. The timeline was already very tight, requiring considerable up-front work to be ready to initiate the project in a way that could meet the completion deadline. The delay in receiving the sequence data coupled with the extremely dry winter of 2020-2021 precluded the installation of any transplants until after this completion deadline. However, we will soon have the genetic analysis and we already have the transplants ready to go in early this fall. We already know that the sequence data set is of high quality, so that the genetic analysis promises to be relatively simple and straightforward. We have funding and logistical support from the St. George Field Office of the Bureau of Land Management that will help us complete the outplanting and also the spring/early summer evaluation as originally planned. We will likely also have BLM funding for at least one follow-up year beyond next spring. It may be necessary to request further ESMF funding for longer-term studies in future years.