DOI: 10.5061/dryad.qbzkh18m4
title: Male dispersal drives gene flow in Timber Rattlesnakes (Crotalus horridus)

description: 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.
description: 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. <br>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.<br>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. <br>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).<br>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/).
description: The data are in an Excel file, and can be opened by a variety of
 Open Office softwares as well as the free Microsoft Excel
 Viewer.