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Using environmental DNA (eDNA) to assess the presence of cavefish and cave crayfish populations in caves of the Ozark Highlands

Many cavefishes and cave crayfishes are considered of conservation concern; however, sampling these species is inherently difficult given their occupied environments. The goal of our project was to verify the presence of select karst organisms while developing the foundation for sampling approaches that might be useful to conservation and management agencies. Our project objectives were to develop assays to amplify deoxyribonucleic acid (DNA) from several species of Ozark cavefishes and cave crayfishes and complete an initial surveillance of locations across the Ozark Highlands using environmental DNA (eDNA). Using DNA either provided by agency cooperators or that we extracted from tissue samples, we PCR amplified and then sequenced the Cytochrome Oxidase 1 (CO1) gene for cave crayfishes and the NADH Dehydrogenase Subunit 2 (ND2) gene for cavefishes. We developed species-specific primers and probes for five cave crayfishes and two cavefishes. From February 2017 to May 2017, we sampled 1–5 sampling units from 42 caves, wells, and springs (i.e., sites) using eDNA and traditional visual surveys. We measured physicochemical parameters at each sampling unit to estimate detection probability associated with both techniques. We also calculated two occupancy covariates for each site using geospatial data. We successfully amplified Troglichthys rosae DNA from the environment and detected DNA representing this species at 24 of 40 sites. At 16 of the sites where we detected T. rosae DNA, we did not visually observe the species. Although our assay for Typlichthys eigenmanni successfully amplified the target DNA from the environment, it also resulted in false absences where the species was visually confirmed. Using eDNA to detect cave crayfishes was much more difficult. The assay for Cambarus subterraneus did not work for eDNA samples and we were unable to pick up DNA from the environment, even at locations where it was visually confirmed. Alternatively, the eDNA surveys worked well for C. tartarus and we were able to amplify DNA at every site where it was visually observed. Our assay for C. aculabrum was based on a single sample obtained from GenBank, and did not amplify eDNA from field samples. Lastly, our eDNA results from samples in the known range of Orconectes stygocaneyi suggested the species may be found at an additional cave. Detection using eDNA based on our O. stygocaneyi assay was likely low because it was designed from a pseudogene; however, positive eDNA samples were sequenced to confirm species-specific DNA. Detection probability of both cavefishes and cave crayfishes varied by survey technique and was influenced by water volume, water clarity, water velocity, and substrate. Detection of cavefishes and cave crayfishes via visual surveys decreased when water volume increased, whereas detection using eDNA increased with greater water volume. Detection between taxa using either sample method was highest in habitats classified by fine substrates, except for eDNA detection of crayfishes which was greatest in coarse substrates. Detection of cavefishes increased with water clarity, but detection of cave crayfishes increased with turbidity. Detection probability of both cavefishes and crayfishes using eDNA increased slightly with water velocity, but decreased with visual surveys as water velocity increased. Occupancy by both taxa was positively related to particular geologic series. Crayfish occupancy was negatively related to fine-scale anthropogenic disturbance (i.e., 500-m buffer around the site), whereas crayfish showed no relationship with disturbance. Our results suggest possible range extensions, provide insights to factors driving detection using both sample techniques, and suggest areas where recharge zones may be shared among caves. Future efforts focused on a comprehensive evaluation of genetic diversity among cave crayfishes to improve assay design could improve detection and the applicability of eDNA as a supplemental and non-invasive sampling approach.

Cooperator Science Series

Understanding the impacts of surface-groundwater conditions on stream fishes under altered baseflow conditions

Persistence of aquatic fauna depends on the conditions and connectivity of surface water and groundwater. In light of altered baseflows and both current and future predicted increases in stream temperatures, it is important to assess current thermal conditions, examine thermal responses of aquatic fauna, and evaluate water-management practices. Our study objectives were to determine (1) how changes in baseflow levels in the Kiamichi River influence hyporheic exchange, which correspondingly influences temperature at the reach scale; (2) temperature tolerances of stream fishes as a means for predicting how habitat complexity influences stream-fish populations; and (3) assess how dam releases influence the downstream temperature and dissolved oxygen regime during the low-flow period. We quantified hyporheic exchange at four reaches and, as expected, found higher groundwater exchange via transient storage occurred at the upstream sites. The net groundwater flux estimation was negative for the majority of reaches indicating that surface water is lost to groundwater during summer (i.e., losing), baseflow conditions. We determined critical thermal maximum (CTMax) for 17 stream fishes and thermal tolerances ranged 32-38°C. We determined the average thermal tolerance for two habitat fish guilds to calculate changes in thermal stress due to hypothetical reservoir release scenarios. We developed a process-based Water Quality Analysis Simulation Program model to predict downstream temperature conditions over 74-km of river in response to reservoir releases that corresponded to discharges of 0.00 (control), 0.34, 0.59, 0.76, 1.13, and 1.50 m3/s. Based on the dissolved oxygen conditions observed in 2015 and 2017 and biological oxygen demand sampling results, reservoir releases did not directly reduce dissolved oxygen concentrations in the Kiamichi River (though dissolved oxygen concentrations are limited to current water-release strategies by the managing agency). We simulated three scenarios using three water-release temperatures: 27.64°C, 26.00°C and 24.07°C that corresponded to average reservoir temperatures at gate locations on the dam. We compared the predicted temperature time series with CTMax of two fish-habitat guilds to quantify the cumulative time when stream fishes experienced severe thermal stress downstream from Sardis Reservoir. According to our simulations, reservoir releases would be capable of regulating downstream water temperature during the summer baseflow period. The 0.00 m3/s scenario resulted in 130 h of thermal stress for benthic fishes, and 73 h for mid-column fishes. As expected, thermal relief increased with increasing release magnitude and decreasing release water temperature. The 0.34 m3/s release scenario reduced thermal stress (range is simulations from the top and bottom gate) by 11-18% for mid-column fishes and 8-12% for benthic fishes with an effective distance (where the cumulative time above CTMax was reduced by half) of 1-2 km for both guilds. The 0.59 m3/s release scenario reduced thermal stress by 18-25% for mid-column fishes and 12-20% for benthic fishes with effective distances of 4-8 km and 2-7 km, respectively. Three releases representing pre-dam flow magnitudes (0.76, 1.13 and 1.50 m3/s released from top gate) reduced thermal stress up to 46% for mid-column fishes and 41% for benthic fishes with an effective distance of 13-16 km, respectively. Lastly, we quantified temperature-induced stress via whole-body cortisol concentration of six stream fishes in response to prolonged thermal exposure at two temperatures (27°C and 32°C). We found no difference in cortisol levels between temperatures for any of the six species, indicating acclimation to elevated temperatures during the test period. However, Highland Stoneroller Campostoma spadiceum expressed cortisol concentrations greater than typical basal levels at both temperatures, suggesting stress from factors other than temperature (i.e., captivity). Our results suggest different reservoir-release options could improve downstream thermal-fish habitat during the summer baseflow period.

Cooperator Science Series

Adult survival, apparent lamb survival, and body condition of desert bighorn sheep in relation to habitat and precipitation on the Kofa National Wildlife Refuge, Arizona

The decline of desert bighorn sheep on the Kofa National Wildlife Refuge (KNWR) beginning in 2003 stimulated efforts to determine the factors limiting survival and recruitment. We 1) determined pregnancy rates, body fat, and estimated survival rates of adults and lambs; 2) investigated the relationship between precipitation, forage conditions, previous year’s reproductive success, and adult body condition; 3) assessed the relative influence of body condition of adult females, precipitation, and forage characteristics on apparent survival of lambs; and 4) determined the prevalence of disease. To assess the influence of potential limiting factors on female desert bighorn sheep on the KNWR, we modeled percent body fat of adult females as a function of previous year’s reproductive effort, age class, and forage conditions (i.e., seasonal NDVI and seasonal precipitation). In addition, we assessed the relative influence of the body condition of adult females, precipitation, and forage conditions (NDVI) on length of time a lamb was observed at heel. Adult female survival was high in both 2009 (0.90 [SE = 0.05]) and 2010 (0.96 [SE = 0.03]). Apparent lamb survival to 6 months of age was 0.23 (SE = 0.05) during 2009-2010 and 0.21 (SE = 0.05) during 2010-2011 lambing seasons. Mean body fat for adult females was 12.03% (SE = 0.479) in 2009-2010 and 11.11% (SE= 0.486) in 2010-2011 and was not significantly different between years. Pregnancy rate was 100% in 2009 and 97.5% in 2010. Models containing the previous year’s reproductive effort, spring NDVI and previous year’s reproductive effort and spring precipitation best approximated data on percent body fat in adult females in 2009-2010. In 2010-2011, the two highest-ranking models included the previous year’s reproductive effort and winter NDVI and previous year’s reproductive effort, and winter and spring NDVI. None of the models assessing the influence of maternal body fat, precipitation, or forage conditions were particularly useful for predicting apparent lamb survival. The high pregnancy rates and body fat levels in excess of 11% do not indicate that this population of desert bighorn was nutritionally stressed during our study and are thus likely not contributing to the low lamb survival estimates we observed. However, body condition data during the population decline is not available and whether this population was nutritionally limited during the initial population decline remains unknown. The prevalence of disease in the Kofa herd may be a limiting factor; however, due to a lack of disease monitoring during the population decline it is uncertain if disease contributed to the decline. Further research is needed to fully understand the complex interaction of disease in this population at the individual and population level and determine to what extent disease predisposes individuals to predation or other causes of mortality.

Arizona

Assessing the feasibility of using acoustic monitoring for Burbot conservation, management, and production

Burbot Lota lota is the sole freshwater representative of the cod-like fishes and supports subsistence, commercial, and recreational fisheries worldwide above approximately 40° N. It is a difficult species to manage effectively due to its preference for deep-water habitats and spawning activity under the ice in winter. Like other gadiform fishes, Burbot use acoustic signaling as part of their mating system, and while the acoustic repertoire of the species has been characterized under artificial conditions (i.e., net pen suspended under ice in a natural lake), there has been no work to determine whether the species is as vocal in natural spawning aggregations. Our objective was to assess the feasibility of collecting and using acoustic data to characterize the spawning activity and locations of Burbot under field conditions. We recorded audio and video of Burbot spawning aggregations through holes drilled into the ice at known spawning grounds at Moyie Lake in British Columbia, Canada. Acoustic recordings (call counts and audiograms) were analyzed using Raven Pro v 1. 4 software. Acoustic behavior was also related to video data to determine how acoustic activity correlated to any observed spawning behavior. In general, wild Burbot spawning in Moyie Lake did not vocalize as frequently as counterparts spawning under artificial conditions. Further, Burbot vocalizations were not recorded in conjunction with spawning activity. While it may be feasible to use passive acoustic monitoring to locate Burbot spawning grounds and identify periods of activity, it does not seem to hold much promise for locating and quantifying spawning activity in real time.

Cooperator Science Series

Northern Mexican gartersnake demographics and movement ecology

The northern Mexican gartersnake ( Thamnophis eques ; hereafter NMGS) is a federally threatened species occurring in riparian areas in central and southeast Arizona and west-central New Mexico. While previous studies have examined the ecology of this species in central Arizona, less is known about NMGS ecology in grassland landscapes of southeast Arizona. This project continues a long-term mark-recapture study of NMGS in the upper Santa Cruz River in the San Rafael Valley of southeast Arizona. We analyzed mark-recapture data collected by Arizona Game and Fish Department from 2008–2019 and included data we collected during 2022 and 2023. We also studied NMGS movement ecology using a combination of externally attached GPS transmitters and surgically implanted VHF transmitters. We had 517 NMGS captures over 26 survey sessions across the entire study, 186 of which were captures of previously marked snakes. Most of our captures were females and evidence of a statistically significant female-biased sex ratio was present on three survey sessions. Trapping success was generally highest June-September and catch-per-unit-effort (CPUE) was strongly correlated with the number of individuals captured. We used mark-recapture data from 288 individuals to estimate annual apparent survival and session-specific recapture probabilities. Annual apparent survival was higher for females (0.72, 95% highest posterior density intervals [HPDI] = 0.630.81) than for males (0.58, 95% HPDI = 0.44–0.72). Session-specific recapture probabilities were similar for males and females and were highly correlated with CPUE. During 2023, we monitored the movements of 13 adult females using GPS transmitters, two adult males using VHF transmitters, and one adult female using a GPS transmitter followed by a VHF transmitter. GPS transmitters were deployed from 7-18 days at a time as limited by battery life. After filtering out low-quality GPS locations, we obtained a total of 1,688 hourly GPS locations. Mean and maximum distances moved per hour ranged from 5.54–10.92 m and 15.24–135.71 m, respectively, across GPS transmitter attachment periods. Space use estimates from 100% minimum convex polygons ranged from 0.04–1.16 ha across GPS transmitter attachment periods. All documented NMGS movements during 2023 were relatively close (< 100 m) to the Santa Cruz River. These results continue to build upon our knowledge of NMGS ecology and suggest the continued presence of a relatively robust population within the upper Santa Cruz River.

Arizona

Maintaining frog monitoring and population on the Las Cienegas National Conservation Area

This report summarizes and analyzes the data collected on the Chiricahua Leopard Frog (CLF) populations on the Las Ciénegas National Conservation Area (LCNCA) in southeast Arizona, U.S.A., during the reporting period covered under this funding agreement from September 2020 through October 2024 and place this analysis in historical context of the previous monitoring efforts. The major findings of this report are: 1. We conducted a total of 465 surveys across 46 sites on the LCNCA and the associated Elgin Buffer Zone during September 2020 through October 2024. We conducted 323 surveys across the 19 core CLF monitoring sites on the LCNCA and 112 surveys across 13 core buffer zone sites. 2. We detected CLF at each of our 19 core LCNCA monitoring sites during at least one year in which we conducted surveys across the year’s entire field season (i.e., 2021-2024). We detected CLF reproduction in each of these four years at four sites and suspected reproduction to have occurred during all four years at a fifth site. We did not detect CLF reproduction at seven of these 19 core monitoring sites during the report period. We assisted with six CLF translocations to sites on the LCNA during the report period. 3. Site occupancy rates across the LCNCA during the report period remain consistent with those reported during previous years and continue to indicate the presence of a stable CLF metapopulation within the LCNCA. 4. Monitoring efforts in the Elgin Buffer Zone continue to be successful at preventing reinvasion by invasive American bullfrogs to the LCNCA. We detected and removed bullfrogs from three sites in the Elgin Buffer Zone and no bullfrogs were detected during 2024. The LCNCA continues to remain bullfrog free since 2014.

Arizona

Biological feasibility of introducing bighorn sheep to the Jicarilla Apache Nation

The biological feasibility of introducing Rocky Mountain bighorn sheep ( Ovis canadensis canadensis ) to the Dulce area of the Jicarilla Apache Nation (JAN) depends on availability and condition of potential habitat and the potential for disease risk, as pneumonia is the largest current threat to wild sheep populations. We modeled quality and quantity of potential bighorn sheep habitat incorporating the three most recent fire scars around Dulce, determined potential winter range within preferred habitat, assessed on the ground vegetation characteristics, and examined potential for disease transmission via risk of contact with domestic sheep and goats. Most of the area of interest for this study has a suitability value ≥ 50%, with approximately 23-29% of the study area considered preferred, or high-quality habitat for bighorn sheep. High-quality habitat for Rocky Mountain bighorn sheep is defined as being within 300 m of escape terrain, within 1.6 km of water, and containing ≤ 30% shrub and tree cover. Of this, approximately 43-56% of potential preferred habitat qualifies as winter range, which is mostly concentrated in the narrow valley bottoms where roads are commonly located and the south-facing slopes surrounding valleys. Analysis of field-collected vegetation data indicate most of the existing forage within the surveyed area to be of moderate or high forage value to bighorn sheep, but horizontal visibility, predominantly in the form of shrubs, is more obscured than what bighorn sheep prefer for most of the area of interest. Maximum shrub and tree cover is also the most limiting factor in the suitability model, primarily due to the prevalence of dense shrub regeneration, particularly Gambel oak, which occurs after high severity burns. The largest quantities of high-quality potential bighorn sheep habitat within the study area occur in unburned areas or those burnt by the predominantly low-moderate severity Amargo fire in 2021. Relative to risk of contact with domestic sheep and goats that can transmit lethal pneumonia-causing pathogens, potentially causing an introduction effort to fail, there is high risk because of the proximity to two hobby-subsistence herds (< 3 km away).

New Mexico

Science summary in support of Manatee Protection Area (MPA) design in Puerto Rico

The Antillean manatee (Trichechus manatus manatus), a subspecies of the West Indian manatee, is listed as endangered by the US Department of Interior. In accordance with its listing, the U.S. Fish and Wildlife Service’s Caribbean Field Office (USFWS) is mandated to create one or more Manatee Protection Areas (MPAs) for Puerto Rico. Designation of these areas must comply with the legal definition of an MPA’s purpose: to prevent or reduce take of manatees (CFR 50: 44 FR 60964, Oct. 22, 1979). To meet this goal, we pursued two objectives: 1) identify areas which include the specific ecological attributes necessary to support manatee populations, and 2) identify areas where take can be reduced through approved MPA regulatory frameworks. We achieved these objectives through literature review, expert elicitation, and geospatial modeling. This report delivers to USFWS a set of nine potential MPA regions. These regions represent the spatial realization of experts’ hypotheses regarding manatee requirements and threats, and the potential to implement MPA strategies (e.g. watercraft access, speed regulations, signage and boater education). The nine regions are compared based on a number of factors, including their potential to reduce take, quality of the habitat encompassed, and total area. These maps and statistics serve as suitable starting points to select one or more MPA sites, but we recommend that the mapped attributes and threats (i.e., boating activity) of MPAs be ground-truthed to visually confirm the local presence of resources, threats, and manatees before any area is selected. Once established, the effectiveness of MPAs can be monitored and updated through processes of adaptive monitoring and management. Aerial surveys, radio tracking studies, and public surveys are all valuable tools to assess the success of an MPA. Establishing MPAs is a management action that, integrated within the species Recovery Plan, should enhance the conservation of manatees.

Cooperator Science Series

Environmental DNA reveals invasion of Puerto Rican waterways by non-native Clarias catfish

The Sharptooth walking catfish ( Clarias gariepinus ) is native to parts of the Middle East, Asia, and Africa but is one of the world's most invasive freshwater fish species. The species’ ability to invade is partly due to its ability to breathe atmospheric oxygen, thereby avoiding hypoxia or poor water quality in receiving waterbodies; further, it can crawl over moist land to disperse to new systems. In 2018, two individuals purported to be Sharptooth walking catfish were collected and identified within the Canal de Patillas, a canal in the Guayama municipality in southeastern Puerto Rico. Several other specimens were also collected the following year. Given that this invasion was in its infancy, environmental DNA (eDNA) surveillance was employed as a rapid and efficient surveillance tool to help delineate the extent of this invasion. Water samples were collected and analyzed for Clarias eDNA from 66 sites encompassing a wide range of habitats in canals, lakes, and a river surrounding the area where they were first reported. Using eDNA-metabarcoding, we detected Clarias eDNA at 40 of the 63 sites throughout the sampled waterbodies. Furthermore, our molecular analysis indicated that the Whitespotted Clarias ( C. fuscus ) is the likely invader, and the original specimens, classified as C. gariepinus , were misidentified based on the use of morphological rather than genetic features for species classification. Environmental DNA surveillance indicated that the distribution of Clarias catfish in this system is substantially more extensive than previously documented. These findings suggest that additional survey efforts could help determine the invasion's full spatial extent and leading edge to inform the scope and scale of any potential management responses.

Puerto Rico

A comprehensive freshwater mussel database for the Duck River Drainage, Tennessee

We have developed a comprehensive database for freshwater mussels for the Duck River drainage in Tennessee, including its largest tributary, the Buffalo River. This database is intended to serve as an expandable template that could be applied statewide. The Duck River is one of the most biologically diverse rivers in the world, with historically over 70 mussel species, and it has been selected as a priority watershed by multiple management and conservation entities. The database for this system compiles over 7,000 mussel records, spanning 200 years, from multiple Federal, State, academic, and private entities, representing 77 native species. The database is spatially explicit and includes temporal and methodological data for each record, and notes of negative survey data were made when possible. The database can facilitate the creation of distribution maps for each species and temporal maps of species richness to show watershed-wide trends. This project addresses the present lack of a centralized mussel database in Tennessee for a critical system. It will be available to facilitate species status assessments, inform conservation planning, and serve as a model for similar databases for other Tennessee watersheds.

Tennessee

Quality assessment of past spawning mark estimations from a long-term survey in the Connecticut River watershed

The calcified structures of fishes provide insight into their periodic growth rates and can be combined with other biological variables to identify metrics such as size or age at maturity and mortality rates. Collecting this information on growth and life history can help evaluate the success of conservation efforts and inform future management decisions for a species in need. However, before these life history data can be applied to larger stock assessments that direct management decisions, confidence in the validity of the data needs to be reported through metrics of accuracy and precision. For this report, we assessed the bias and precision of paired reader estimations of spawning marks on scales of Blueback herring (Alosa aestivalis) collected in the U.S. Fish & Wildlife (USFWS) Annual Adult River Herring Stock Assessment for the lower Connecticut River basin. The paired reads on scales from a total of 8,698 fish over the ten years of the long term monitoring program were evaluated for the annual presence of systematic bias and precision using a combination of qualitative (i.e., frequency tables) and quantitative (i.e., Evan’s and Hoenig’s Test of Symmetry, and Coefficient of Variation (CV) calculations) analyses. While seven out of the ten survey years had systematic bias detected by the tests of symmetry, only three years (2013, 2016, 2018) had imprecision values >10% CV threshold. Data were further categorized into specific age classes within survey years to increase our resolution on where bias and imprecision was most prevalent. While the ability for accurate bias detection was limited by sufficient sample sizes (>25 fish), average imprecision values increased with age, and median age classes (4 through 6) commonly had bias detected. However, the removal of insufficient age classes prior to calculating average annual CV did not significantly change the initial average. Lastly, 2023, which was the first year to implement a standardized training procedure prior to production estimating, had the highest precision for both the annual average and specific age classes compared to all prior survey years. This standardized training procedure will continue to be used by USFWS for the lower Connecticut River tributaries, and can be modified for other river systems. Overall, this report’s results highlight the importance of assessing precision and encourage the standardization of spawning mark identification quality control and assurance for future studies. With more quality assessments and baseline information on precision and bias, there can be more beneficial discussion on defining thresholds and how to implement spawning history variability into catch curve analyses.

Connecticut, Massachusetts

Gambel’s quail (Callipepla gambelii) nest site selection and habitat use in Oracle Junction, Arizona

Gambel’s quail ( Callipepla gambelii ) is the most widespread, abundant, and heavily harvested species of game quail in Arizona. Gambel’s quail population dynamics in Arizona are strongly influenced by seasonal weather conditions. This is particularly relevant for Gambel’s quail management as drought conditions are expected to increase in Arizona, and this may exacerbate inter-annual variation in Gambel’s quail abundance and recruitment. The goal of this study is to provide information on Gambel’s quail population demographics, habitat use, and movement to inform land management decisions for Gambel’s quail in southeast Arizona in a way that mitigates the effect of drought. We used multiple trapping methods to capture Gambel’s quail from February–July 2024 and February–15 May 2025. We found noose mats (n = 57) to be more effective than drop traps (n=14) in capturing Gambel’s quail, with multiple types of walkin traps proving ineffective. We deployed 33 GPS backpack transmitters on female Gambel’s quail to monitor their movements and locate nests. We documented eight mortalities of tracked quail from apparent predation. Quail home range sizes ranged from 3 ha-100 ha (mean = 25 ha) in 2024 and from 7–57 ha (mean = 29 ha) in 2025. We identified four nest sites with 6-15 eggs and all nests were predated. We collected vegetation measurements at nest and brood sites and corresponding random points to examine nest and brood site selection although our small sample sizes limited our ability to make inferences about Gambel’s quail habitat selection.

Arizona

Brook Floater restoration: Identifying locations to reintroduce or augment populations with propagated mussels

In February 2020, we held a workshop where we sought to identify where states should reintroduce or augment brook floater to minimize the probability of extinction within a state. We focused on Massachusetts and Connecticut, two states with only a few, small populations still extant, that likely need population restoration to prevent statewide extirpation. We identified that restoration actions aimed at redundancy (number of populations), representation (number of occupied basins), and resiliency (population size) were constrained by resource availability such as limited broodstock , staff time, and budgets. Optimal restoration locations depended on habitat conditions, the status (viability) of nearby mussel populations, population size (number of individuals), and the location within watersheds; all important considerations in addressing population persistence. Restoration actions also accounted for the risk of disease transmission among mussels and fish, and the genetic health and diversity of mussel populations. The workshop identified the multiple, compounding uncertainties related to population restoration, identified information gaps critical to decision making, and charted a path forward to make decisions given uncertainties. The optimization approach developed can be used to select specific watersheds for restoration in any state, province, or region and can easily be adapted as new information becomes available.

Connecticut, Massachusetts

Direct effects of pesticides and other grassland management practices on the North American Monarch Butterfly (Danaus plexippus plexippus): A systematic review

This review synthesizes recent scientific literature on the effects of grassland management practices and pesticide applications on monarch butterflies ( Danaus plexippus ), focusing on studies published since the 2020 U.S. Fish and Wildlife Service Species Status Assessment (SSA). While the SSA highlighted habitat conservation efforts to enhance monarch populations, there has been limited empirical research on the direct effects of these management strategies on monarch survival and fitness. Following PRISMA guidelines, we conducted a systematic review to evaluate research published during and after the SSA (2019-2024) on the direct effects of common grassland management practices (mowing, grazing, burning, haying) and pesticides (herbicides, insecticides, fungicides) across monarch life stages. This search identified 29 studies that met our criteria. Of these, only two studies addressed the impacts of grassland management on monarchs, indicating a substantial and important information gap. The other 27 studies reviewed indicate significant risks of pesticide exposure to monarchs, particularly at immature life stages from lethal and sublethal effects. The effects of chronic insecticide exposure, particularly neonicotinoids, are especially critical to consider, as research shows insecticides can result in impairment of growth, development, and reproductive behaviors. Additionally, fungicides pose overlooked risks to monarchs due to their toxicity to non-target organisms. Notably, most studies evaluating pesticide effects were conducted in a laboratory setting and thus it is unclear how these results relate to fieldrealistic exposure, as well as exposure over multiple life history stages and potential synergistic effects of pesticides. Additional relevant information gaps remain, especially on studies of southern migratory paths critical for monarch population persistence. Data gaps at relevant scales for grassland management combined with growing evidence of pesticide persistence and subsequent threats to insects, highlight how more ecologically applicable metrics for monitoring environmental chemicals could help improve understanding of the effects of pesticides on monarchs and characterize contamination of milkweed and nectar resources.

Cooperator Science Series

Assessing the spawning movement and habitat needs of riverine Neosho Smallmouth Bass

Stream fishes are vulnerable to a variety of natural and anthropogenic stressors. Information on fish movements and habitat use is essential to conserve and manage populations, particularly at the edges of distributions and novel habitats. The Neosho Smallmouth Bass Micropterus dolomieu velox is endemic to the southwestern Ozark Highlands ecoregion, where the riverscape is highly dissected by impoundments. Our study objectives were to determine the pre-spawn, spawn, and post-spawn movements of adult, radio-tagged Neosho Smallmouth Bass, and identify the habitat factors at multiple spatial scales related to suitable spawning habitat. Movements by tagged fish in the Elk River of Oklahoma and Missouri and two Oklahoma tributaries draining to lower Elk River and Grand Lake O’ the Cherokee were greatest during the spring spawning period and were positively related to discharge and fish size; however, we observed considerable individual and stream-specific variability. Temperature and fish movement rate in the Elk River were positively related in all seasons except for winter, although temperature was less important for Smallmouth Bass movement in the smaller streams. Tagged fish were never detected using active or passive telemetry in the reservoir or reservoir-river interface except during periods of lotic character (i.e., the reservoir was not pooled above the Buffalo Creek-Elk River confluence). Nests were typically located at intermediate depths (mean = 0.8 m; SD = 0.3) and in low velocity habitats (0.0–0.2 m/s). Most of the nests examined comprised gravel substrates; however, 2.5% of the nests observed were located on full or partial bedrock substrate. We also documented nest clustering behavior by Neosho Smallmouth Bass (i.e., adjacent nests within 2 m of each other); 66 nest clusters were identified across 22 stream reaches. Cluster presence was more prevalent in warmer stream reaches with wide, shallow channels, and less likely in groundwater-gaining reaches, whereas overall nest abundance was greater in warmer streams and reaches with deeper pools. We showed the importance of both warmer streams and deep pools of small streams for Smallmouth Bass rearing (i.e., young-of-year abundance). We found negative relationships between floods and first-year juvenile survival and show the importance of stream network position (i.e., adjacency to larger streams) for mitigating the negative effects of July floods. Our analyses of both nesting and young-of-year habitat use suggest small streams, typically not considered important to many fisheries, are responsible for a proportion of Neosho Smallmouth Bass production. Further, consideration of management actions restricting take during the early spawning season may be warranted due to the unique nesting behavior (i.e., clustering) exhibited by this subspecies.

Arkansas, Missouri, Kansas, Oklahoma

Environmental DNA (eDNA) surveillance of the federally threatened Slender Chub (Erimystax cahni) in the Clinch River and Powell River

The slender chub ( Erimystax cahni ) is a federally threatened fish native to and geographically restricted to eastern North America. More specifically, the Powell, Clinch, and lower Holston Rivers in Tennessee were historical collection areas. Habitat degradation from multiple sources, including surface mining, agriculture, dams, and urbanization, is associated with the decline of this species - an obligate inhabitant of gravel shoals in large rivers. As of 1964, only 15 voucher specimens were known and no living specimens were documented for decades. A federal recovery plan and Species Status Assessment were developed to determine if slender chub still exists and could be restored. Given the lack of recent observations using conventional sampling to search for its presence, we used environmental DNA sampling to determine their potential presence. Our specific objectives were to (1) develop a quantitative polymerase chain reaction (qPCR) assay aimed at species-specific detection and (2) sample historically known areas of collection. We sampled 43 sites in the Clinch and Powell Rivers. For the first time in almost two decades, we provide evidence for the continued existence of a putatively extinct species. We detected evidence of E. cahni in both the Clinch and Powell Rivers, but only at a few sites. We cannot confirm the presence of E. cahni , and positive eDNA matches could be attributed to amplification from a hybrid. Our results do indicate slender chub DNA perseveres in the Clinch and Powell Rivers and can inform resource agencies of localities to pursue on-the-ground searches for the slender chub with conventional methods (e.g., snorkeling) and potential restoration and recovery sites.

Tennessee, Virginia

Assessing the distribution and habitat needs of the Least Darter and sympatric species of the Ozark and Arbuckle Mountain ecoregions

Limited information is known about factors driving the distribution of Least Darter in Oklahaoma. The Least Darter occurs in the Ozark Highland and Arbuckle Uplift ecoregions of Oklahoma which represent the southern extent of its range. Least Darter was historically recorded in Oklahoma from groundwater-fed streams. Our study objectives were to determine the distribution of Least Darter and a subset of congeners across the two ecoregions of Oklahoma and assess factors driving patch occupancy of Least Darter at a fine spatial scale. We used temporally replicated snorkel surveys conducted in July through October 2018-2019 to determine occupany by Least Darter. We snorkeled and seined for four species in each reach including two life stages of Smallmouth Bass (subadult and adult). We sampled 153 sites (i.e., riffle-pool complexes) nested within 61 stream reaches (i.e., 200-500-m long) in the Arbuckle Uplift and Ozark Highland ecoregions. Detection probability was similar between ecoregions. Least Darter was detected at more sites when snorkeling compared to seining (24 versus 18). Smallmouth Bass, Redspot Chub and Southern Redbelly Dace were typically 2-3 times more likely to be detected by snorkeling than by seining. We found relationships between occupancy and habitat parameters that were both shared among species but also species-specific. Least Darter occurrence probability in the Ozark Highlands was lower than in the Arbuckle Uplift. Occurrence probability was higher for subadult Smallmouth Bass and Southern Redbelly Dace in 2018 compared to 2019. Occurrence probabilities of both Least Darter and Southern Redbelly Dace were higher in cooler habitat patches. Southern Redbelly Dace was negatively associated with a higher proportion of pool habitat across a reach. Lastly, subadult Smallmouth Bass and Redspot Chub were more likely to occur in deeper pools and in larger streams (i.e., drainage area). We sampled one study reach (~150-m long with shallow riffles or a waterfall on each end) in the Arbuckle Uplift (winter and summer sampling) and Ozark Highland (winter sampling) ecoregions to determine fine-scale habitat selection during the thermally harsh seasons. We developed transects across the reaches to quantify depth, velocity, substrate, cover and water temperature. We found Least Darter used higher water column velocities and shallower water depths with little vegetation during the winter. The average water depth used was similar during summer and winter (~ 20 cm deep). Least Darter used denser vegetation during the summer and tended to avoid coarse substrates in both seasons. If the conservation of Least Darter is a management goal, actions to mitigate increasing stream water temperatures (e.g., protection of springs and riparian corridors) and protecting stream morphologies that facilitate species separations (i.e., allow for a wide range of water depth and velocities) may be beneficial (e.g., fencing cattle from streams, promoting natural bankful flows during spring)

Oklahoma