The Mojave desert tortoise (Gopherus agassizii) occupies the Mojave Desert regions of Arizona, Utah, Nevada, and California. This species is federally protected under the Endangered Species Act (USFWS, 1990). Desert tortoises are among the 4 reptile species listed in the 2015 Utah Wildlife Action Plan as species of greatest conservation need (SGCN). Desert tortoises have one of the most critical conservation rankings compared to other herpetological native species in the state of Utah, with a State Ranking of S2 (i.e., "imperiled"). While the global status of G. agassizii is G3 (vulnerable), the desert tortoise is listed as S2 in all states within its native range. This points to a significant disconnect between their global and provincial rankings. It should be noted that the last global status review of G. agassizii was conducted some time ago, on 9/6/2013 (Natureserve, 2022). To be returned to the Utah Landscape a desert tortoise must be: - From within the state of Utah - Taken from the wild - Free of communicable infections - In satisfactory overall physical health URTD is a serious infection caused by Mycoplasma agassizii that has been well recorded in desert tortoise populations for over 20 years (FWS 1990). This disease is easily spread within populations and great caution should be taken when relocating or returning desert tortoises that may have been exposed to URTD. Traditionally, antibody tests have been used to detect URTD. However, recent studies indicate that positive antibody tests are not a clear indicator of active disease, and positive antibody results are not invariably linked to mortality (Sandmeier, 2009). Additionally, individual animals tested concomitantly for Mycoplasma agassizii antibodies and the presence of Mycoplasma agassizii DNA may not test positive for both (Goessling et al., 2019). While TeHV-2 associated mortality is not well studied in wild populations, it has been documented to result in significant mortality rates in captive tortoises (Johnson et al, 2005). A primer set for TeHV-2 has been developed and validated in recent years (Braun et al., 2014). Primer sequence data has been made publically available for replication (Braun et al., 2014) (Attachment 1). The molecular diagnostics laboratory at The San Diego Zoo Institute for Conservation Research currently has a pipeline in place for receiving and processing samples from other institutions. The U.S. Fish and Wildlife Service (USFWS) also recently released an updated handbook to health assessment procedures for the Mojave desert tortoise (Averill-Murray et al., 2019). This handbook was an effort between the USFWS Desert Tortoise Recovery Office, the Arizona Exotic Animal Hospital, and the San Diego Zoo Institute for Conservation Research. This new guideline lists a health triage protocol for physical examination that is to be implemented alongside laboratory testing (attachment #2). The current translocation plan recommends rigorous visual screening prior to relocation. By comparing laboratory disease screening with the results of visual examinations we will be able to assess the accuracy of visual health screenings and improvements that could be made upon the current plan to detect active disease. By connecting individual health information with the population or origin we will be able to: ● Determine the origin of tortoises in the desert tortoise adoption program ● Identify populations that may be at risk or infected with communicable diseases ● Quantify the number of Utah born vs out of state tortoises found outside of the species range and turned in to UDWR ● Add actionable context to the disease screening data obtained in project #6647 This project will collaborate with the Kepas lab and the Klabacka lab at Utah Tech University.

Goal -- The goal of this project is to pair the disease screening data from project #6647 with genetic information that will link individual tortoises in the desert tortoise adoption program to their population of origin. The results of this testing will inform the state desert tortoise biologist of diseases present in individuals that are being considered for release, as well as their source population. Objective -- Assess the accuracy of the visual health screening process using molecular diagnostics and link that information to known populations in southern Utah.

This project does not propose any changes to the landscape or alter the ecology of any area.

Desert tortoises are currently assigned an S-rank of S2 (imperiled) in the state of Utah and all surrounding states that overlap with its range. There is no indication that this S-rank value will change in the upcoming Wildlife Action Plan 2025 revision. G. agassizii are found in the southwest corner of Utah, where rapid urban expansion is underway. This recent expansion may account for the increased number of desert tortoises found and surrendered to the state in recent years. The state and its partners are currently holding desert tortoises in multiple locations. While the desert tortoise program does function to remove some of these captive tortoises from holding facilities, there are currently many captive tortoises displaced throughout the state. The ultimate goal for animals that are turned in is release. By validating our visual health assessment with clear diagnoses through URTD and TeHV-2 testing, we will be able to accurately assess the efficacy of current protocols and determine the origin of infected and healthy tortoises. Potentially leading to informed release of healthy individuals currently in captivity and alerting management agencies of populations that may be experiencing declines due to infectious disease.

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This study proposes to work with previously collected desert tortoise samples under project #6647. No additional handling or sampling from desert tortoises is required.

Objective -- Determine population origin of captive desert tortoises Task 1 -- Isolate DNA from blood already extracted from 30 captive tortoises. We will perform DNA isolations using a Qiagen DNEasy spin kit. Task 2 -- Amplify variable genetic regions (microsatellites) using PCR. Microsatellites are highly variable regions within the genomes of most organisms that can be used for examination of variation within a species. We will perform amplification of 15 microsatellites using DNA primers that were previously created specifically for tortoise genomes (Edwards et al., 2003; Schwartz et al., Edwards et al., 2011; Schwartz et al., 2003). Task 3 -- Identify genetic variants using microsatellite quantification. Amplified microsatellites (from Task 2) will be sent to the Arizona Molecular Clinical Core, where the sequence length of the microsatellites will be quantified via capillary electrophoresis. Task 4 -- Determine population assignments for each tortoise. In collaboration with Dr. Taylor Edwards, the Clinical Laboratory manager at the Arizona Molecular Clinical Core, we will run the results from Task 3 through an extensive genetic database for desert tortoises that has been created and curated from tortoise populations in Arizona, California, Nevada, and Utah for over 20 years. Microsatellites will be used to make genetic population assignments for each individual tortoise.

No field monitoring will be required. All samples are currently in storage at Utah Tech University.

Organizations: This collaborative effort will involve UDWR, Utah Tech University, Brigham Young University, the Great Basin Serpentarium, Best Friends Animal Sanctuary, and the Temporary Care Facility in Hurricane, Utah. It should be noted that all desert tortoises in captivity are in possession of UDWR. Individuals: This project will be managed by assistant professors Megen Kepas (Utah Tech Biol Sci Dept), Randy Klabacka (Utah Tech Biol Sci Dept, BYU Dept of Biol) and UDWR desert tortoise biologist Ann McLuckie (UDWR Washington County Field Office), and an undergraduate laboratory technician.

The findings and recommendations of this project will remain applicable in the coming years. We anticipate that with the increased urbanization in Washington County, human tortoise interactions and the likelihood of a tortoise being turned in to the state may subsequently increase. Additionally, URTD and TeHV-2 infections have also been widely detected in wild populations of G. agassizii throughout their range. Recommendations based on this project will be taken into account with respect to current health screening practices, and provide foundational information should major threats arise in the future.

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09/19/2024

06/30/2025

2025

Study Tortoises Captive desert tortoises (Gopherus agassizii) were examined at three different holding and/or long-term care locations 1) The Great Basin Serpentarium, 2) The Red Cliffs Desert Reserve Temporary Care Facility (managed by the St George Habitat Conservation Plan), and 3) Best Friends Animal Society of Kanab. Blood Samples for Genetic Testing Blood samples were obtained by a licensed DVM for all three captive groups during the FY24 funding period. The blood samples from Best Friends Animal Society were drawn onsite in the clinic on the Kanab campus by Dr. Colleen Guilfoyle on 6/28/2024. Blood samples from the Temporary Care Facility tortoises were drawn at the St George Veterinary Hospital by Dr. Kelly Houston on 5/22/2024. Blood samples from tortoises housed in the Great Basin Serpentarium were drawn by Dr. Laurel Harris on 5/16/2024 at Wasatch Exotic Pet Care. Maximum blood collection volumes were set by the USFWS guidelines (USFWS, 2019). However, the volume of blood drawn, provided it remained below the maximum limit, was at the discretion of the veterinarians, who took into consideration the animal's health status, stress, and the ease of blood collection. Following retrieval from the veterinary facilities, the plasma phase was removed from each sample by centrifugation and sent to the ecophysiology lab at Utah State University (Dr. Susannah French). The solid (red blood cell) phase of each blood sample was frozen and transported on ice to Utah Tech University where the DNA was extracted in the lab of Dr. Randy Klabacka for genotyping. The extracted DNA samples are currently in storage in the -80-degree freezer of the Utah Tech molecular lab. DNA Extraction from Desert Tortoise Whole Blood Samples DNA was extracted from red blood cells using the Qiagen DNeasy spin kit for blood samples (Product ID 69504). DNA was extracted using the spin column extraction method outlined in the kit directions. We attempted to extract DNA from all blood samples provided. The minimum concentration needed to proceed to PCR amplification is 20ng/uL. Extraction was attempted up to three times for samples that did not yield over 20ng/uL. The A260/280 ratio was recorded for each sample following extraction to assess DNA purity. All DNA samples that passed the minimum yield had A260/280 ratios within 0.08 of the ideal value of 1.8, indicating high-purity and passing the quality check. The DNA concentration in the samples from tortoises 894, C2, and C6 were too low to proceed to the amplification step (Table 1) after three failed extractions. Low DNA yields may result from limited sample volume, premature cell lysis caused by small-gauge needles during blood collection, and/or DNA degradation occurring during sample transport. Table 1. Desert Tortoise ID and Maximum DNA Concentration Extracted in ug/uL ID Final Concentration (ng/uL) 857 34.2 869 71 872 78.3 873 82.9 887 110.7 890 113.6 894 Below Threshold 910 109.1 912 42.8 914 96.8 Thanos 49 Meenie 25 Moe 22.3 Minnie 65.4 Silver_Reef 201.2 A6 120.4 C2 Below Threshold C4 60.3 C6 Below Threshold PCR Amplification for Sequencing We amplified variable genetic regions (microsatellites) using PCR and verified the amplification of the target regions by their molecular weight using gel electrophoresis (Fig. 1). We amplified up to 15 total microsatellite regions per sample using the DNA primers that were created specifically for G. agassizii (Edwards et al., 2004; Edwards et al., 2003; Edwards et al., 2013). In brief, these amplified microsatellite regions are used to examine variation within a species because they are highly polymorphic, i.e., useful for telling closely-related populations apart. The loci amplified were: Goag4, Goag3, Goag5, Goag6, Goag7, Goag32, GP55, GP102, Gp19, Gp30, and Gp61. When additional DNA was available, the mitochondrial gene ND4 was also amplified using the appropriate primers (NAP2 and New Gly). Further details on primer sequences, PCR protocols, and previous assay performance are provided in the spreadsheet submitted with this report. Figure 1. Example of sequencing preparation output. The sample is from tortoise #857. Sequencing and Analysis Prepared samples awaiting sequencing are currently in storage at Utah Tech University. The samples will be shipped to Arizona Molecular Clinical Core (AMCC) upon the start of fall semester 2025, between August and September for fragment analysis and sequencing by Taylor Edwards. Post electrophoresis, Edwards and Klabacka will compare the microsatellite data from our study group to the existing desert tortoise genetic database. This database contains information from >997 wild desert tortoises from populations in Arizona, California, Nevada, and Utah, and spans over 20 years. We anticipate that we will be able to make genetic population assignments for each individual tortoise. We expect the analysis portion of the work to be completed by the spring of 2026. Manuscript preparation will begin in the spring of 2026 and will include Ann McLuckie and Alyssa Hoekstra as co-authors. References Edwards, T., & Berry, K. H. (2013). Are captive tortoises a reservoir for conservation? An assessment of genealogical affiliation of captive Gopherus agassizii to local, wild populations. Conservation Genetics. https://doi.org/10.1007/s10592-013-0458-y Edwards, T., Goldberg, C. S., Kaplan, M. E., Schwalbe, C. R., & Swann, D. E. (2003). PCR primers for microsatellite loci in the desert tortoise (Gopherus agassizii, Testudinidae). Molecular Ecology Notes, 3(4), 589--591. https://doi.org/10.1046/j.1471-8286.2003.00521.x Edwards, T., Schwalbe, C. R., Swann, D. E., & Goldberg, C. S. (2004). Implications of anthropogenic landscape change on inter-population movements of the desert tortoise (Gopherus agassizii). Conservation Genetics, 5(4), 485--499. https://doi.org/10.1023/B:COGE.0000041031.58192.7c U.S. Fish & Wildlife Service. (2019, March 28). Health assessment procedures for the Mojave desert tortoise (Gopherus agassizii): A handbook pertinent to translocation. Desert Tortoise Recovery Office, U.S. Fish & Wildlife Service.

Desert tortoises are listed as federally threatened under the Endangered Species Act. Due to habitat loss and degradation, displacement of tortoises occurs and the need to translocate individuals to areas within their range has become increasingly difficult. The UDWR has a desert tortoise adoption program for any tortoises that come from unknown locations or have been in captivity for a long period of time that do not often qualify for translocation. It is important to know where the tortoises from the adoption program have originated from in order to understand if release into suitable habitat will negatively impact wild populations by introducing genetics from distant or out-of-state populations. The results of this project will provide data to incorporate into desert tortoise management in the state and address any issues involving tortoises entering the adoption program. These data will also be compared with the previously analyzed disease data sampled from the same tortoises to understand which populations they came from that may have the presence of URTD. Some challenges that were encountered was having blood samples from three tortoises that did not meet the threshold to be able to amplify them for DNA analyses.

To correlate disease screening and health exam data from Utah desert tortoises with population of origin. Previous data collected by McLuckie and Edwards identified distinct desert tortoise populations within the state of Utah. Using blood samples collected from desert tortoises during the disease screening project (WRI project 6647), we aim to connect disease data with population of origin. Combining this genetic data with molecular diagnostic and physical exam data will allow us to 1) determine the resolution with which we can trace a displaced tortoise to its population of origin and 2) infer the population of origin for diseased tortoises. The findings and recommendations of this project will remain applicable in the coming years. We anticipate that with the increased urbanization in Washington County, human tortoise interactions and the likelihood of a tortoise being turned in to the state may subsequently increase. Additionally, URTD and TeHV-2 infections have also been widely detected in wild populations of G. agassizii throughout their range. Recommendations based on this project will be taken into account with respect to current health screening practices, and provide foundational information should major threats arise in the future.