Assessment of Eastern Hellbender Populations in Kentucky: Distribution, Recruitment, and Landscape Genetics

1. eDNA surveys to understand drivers of Eastern Hellbender (Cryptobranchus a. alleganiensis) occupancy and detection across Kentucky A. Sampling will be conducted at 48 study sites across Kentucky. These sites have been selected by KDFWR because they have not been surveyed sufficiently yet they have current and/or historical records for Eastern Hellbenders. B. Replicate water samples will be collected from these 48 sample sites; replicates are needed to assess probability of detection via eDNA methods (See below). We will collect 3 replicate water samples at each site to reflect pre-breeding (15 May – 15 July); breeding (15 August - 31 September) and eggs and hatching period (1 October-30 November). Established eDNA sample collection protocols will be followed. Laboratory methods, based on Spear et al. (2015), will be conducted in collaboration with Purdue University. We will also collect water samples to evaluate water quality (i.e., conductivity, turbidity, water temperature, etc.) during each eDNA sample at each site. C. We will use an occupancy modeling framework (McKenzie et al. 2017) to evaluate drivers of Eastern Hellbender occupancy and detection in Kentucky streams. We will examine the relationship between Eastern Hellbender occupancy (eDNA positive or negative) and 1) current and historic land-cover at the catchment and at riparian scale, 2) in-stream habitat conditions, 3) water chemistry parameters (i.e., conductivity, pH, dissolved oxygen). Understanding these relationships may provide specific management recommendations for hellbender conservation in Kentucky. In addition, we will examine factors that may influence detection of Eastern Hellbender eDNA including day-of-year and environmental conditions (i.e., water temperature, water speed, turbidity etc.) when each sample was taken. This examination will allow for precise recommendations on sampling protocol. Collectively, this analysis will provide a robust assessment of the factors related to Eastern Hellbender distribution in Kentucky. 2. Examining Eastern Hellbender nesting and recruitment A. We will use the results of our eDNA surveys during the first year of our project to develop a subset of sites to examine Eastern Hellbender nesting and recruitment. This subset will be chosen in such a way to allow for an assessment of recruitment across major drainages in Kentucky and to examine the relationship between recruitment and habitat factors (i.e., riparian forest cover). For example, Bodinof Jachowski et al. (2018) found that reduction in riparian forest cover is associated with reduced recruitment in Virginia streams. Knowing this information for Kentucky streams will provide concrete management recommendations, including habitat restoration. B. We will use two methods to assess nesting and recruitment in Kentucky streams. a. Artificial structures (i.e., nest boxes) arrays. We will place artificial structures in 10 in study sites (across major drainages ). Specifically, structures will be placed in arrays. Bodinof Jachowski and Hopkins (unpublished data) suggest 0.6 shelters per 100 m2 of wet in-stream habitat. Construction will follow the guidelines provided by Indiana Department of Natural Resources and Purdue Extension Specialists. Artificial structures will be placed in the field May-early July. Structures will be checked from mid-August through October for nests and adults. b. Study sites without artificial structures will be surveyed using trapping, snorkeling and judicious use of rock-turning methods. Specifically, we will be looking for nests and hellbenders of various size classes. C. All adults will be weighed, measured and tagged via passive integrated transponder. Tissue samples or eggs will also be procured during processing for Step 3 (below). Nests encountered in artificial structures will be periodically monitored until hatching. Ultimately, a long-term Eastern Hellbender monitoring program in Kentucky will be established via the continue surveillance of permanent artificial structures and marking salamanders with PIT tags. D. Analyses will examine relationships between recruitment measures (number of nests, nest presence/absence) to habitat factors. We may also be able to examine the relationship between size classes and habitat factors within sampled reaches, if sample size permits. Expected Results, Conservation Implications, and Broader Significance: Our integrative approach seeks to inform and focus conservation management of Eastern Hellbenders and will have direct and immediate applicability for population managers. First, our eDNA sampling design will provide guidance for survey methodology while examining factors driving patterns of distribution in Kentucky. Second, our focus on when and where recruitment occurs addresses an important but elusive issue in Eastern Hellbender conservation; most importantly, minimal recruitment may lead to population augmentation whereas evidence of recruitment suggests limited funds may not be necessary for Eastern Hellbenders population augmentation efforts in Kentucky.