Threatened across much of their range, timber rattlesnakes (Crotalus horridus) exhibit patterns in movement and genetic diversity that are shaped by many aspects of their environments in ways that may foster or constrain conservation. We combine movement data with nuclear and mitochondrial population genetic data to understand the population structure of snakes in four overwintering sites (hibernacula) in central Pennsylvania. Our analyses support the conclusion that hibernacula separated by only a few kilometers can represent genetically distinct populations. In addition, as the first dataset to compare nuclear and mitochondrial patterns in C. horridus, we provide novel evidence for how likely asymmetry in gene flow (with males responsible for inter-hibernaculum matings) shapes timber rattlesnake population genetics.

Between 2015-2017, we collected data on the demographics and ecology of Crotalus horridus at a location in central Pennsylvania using mark-recapture and radio-telemetry data in a cluster of four hibernacula. We captured 310 C. horridus individuals. We brought each captured snake back to the laboratory where we identified sex, and marked individuals. We used passive integrated transponder (PIT) tags injected subcutaneously to mark each snake. As a secondary marking technique, we clipped ventral scales in a unique series to confirm the identity of recaptured snakes in the case of lost PIT tags. Scale clips were preserved in 70% ethanol and stored in a -20ºC freezer. 
Over the course of this study, we tracked 19 individuals (11 females and 8 males) using radio transmitters (model SB-2 and model SI-2, Holohil Systems, Carp, Ontario, Canada) surgically implanted into the body cavities based on the methods of Reinert and Cundall (1982). Transmitters weighed between 5.2-9.0 g and had a battery life between 13 and 20 months. Snakes were radio-tracked on average every 3 days between egress in May and ingress in October. Nine snakes were tracked for two years, four snakes were tracked for three years, and six snakes were tracked for the entire four years of the study.
We sampled DNA from 148 rattlesnakes, extracting DNA from scale clips using Qiagen DNEasy Blood & Tissue kits (Venlo, Netherlands). Individual snakes were assigned to one of four hibernacula based on where the specimen was collected. We assigned a snake to a particular hibernaculum if it was (1) captured early in the year (April or May) near a particular hibernaculum, (2) recaptured during egress or ingress at a particular hibernaculum, or (3) was radio-tracked back to a particular hibernaculum (Fig. 1). Based on radio-telemetry data collected during this project, we know that snakes remain near hibernacula prior to their first shed early in the active season. Once snakes shed (early June), they increase movement rates. Based on these radio-telemetry data, we felt confident in assigning snakes to a particular hibernaculum early in the active season (before June). Any snake that we could not confidently assign to a hibernaculum was not included in the genetic analyses. 
We genotyped 90 snakes at the ND2 mitochondrial locus. We genotyped snakes sampled as evenly as possible across hibernacula. The loci were amplified using previously described primers (Kumazawa et al. 1996; Ashton and de Queiroz 2001), with thermocycler settings as described in Kumazawa et al. (1996). Samples were sent to Eurofins Genomics (Louisville, KY) for sequencing. Sequences were aligned and edited using Geneious Prime 2020.1 (https://www.geneious.com).
We genotyped 113 snakes at 6 microsatellite loci using the primers CwA29, Scu07, CwB23, Scu01, 7-144, and CwC24 (Villarreal et al. 1996; Gibbs et al. 1998; Holycross et al. 2002). We amplified the markers in a single multiplexed PCR reaction as described in Januszkiewicz et al. (2018) using the Qiagen Multiplex PCR kit (Venlo, Netherlands). We sent the amplified DNA to the Cornell Institute of Biotechnology (Cornell University, Ithaca NY) for fragment analysis and called the microsatellites using the Geneious Microsatellite Plugin 1.4.6 (https://www.geneious.com/plugins/microsatellite-plugin/).

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