Effects of Movement and Mating Patterns on Gene Flow among

Overwintering Hibernacula of the Timber Rattlesnake (Crotalus horridus)

Corey Devin Anderson1

Radiotelemetry and genetic markers have enabled herpetologists to test the hypothesis that gene flow is restrictedamong snake dens (a.k.a. ‘‘hibernacula’’). In the present study, observations of movement and mating were comparedto indirect inferences of gene flow based on genetic data (microsatellite DNA loci) for six neighboring hibernacula ofthe Timber Rattlesnake (Crotalus horridus) in western St. Louis County, Missouri. Behavioral results were consistent withprevious findings for this species, with most individuals exhibiting high fidelity to hibernacula, and males exhibitinglarger activity areas than females. Behavioral results also supported several long-standing assumptions: males tended tomate at longer distances from hibernacula than females and random pairing between males and females from differenthibernacula could not be rejected. Some gravid females moved considerable distances between locations, but mostreturned to an area near their own hibernaculum to give birth, and birth distances tended to be shorter than thenearest-neighbor distance between hibernacula. Genetic results indicated that the observed number of heterozygotesdid not deviate from Hardy-Weinberg expectations at the level of total sample population, but that a significantheterozygote excess was found within hibernacula. A small but significant proportion of the genetic variance in thetotal population was due to differences among hibernacula; however, Bayesian clustering algorithms could not sortindividuals (or groups of hibernacula) into discrete demes. Results suggest that patterns of genetic differentiationobserved among hibernacula of the Timber Rattlesnake are better explained by behavioral and demographic factorsresulting in natal philopatry, sex-biased dispersal, and/or a limited number of breeding adults, than by the balance ofgene flow and drift, per se.

POPULATION genetic studies of snake dens (hibernac-ula) have become increasingly common in theherpetological literature (Prior et al., 1997; Bushar et

al., 1998; Lougheed et al., 1999; Gibbs and Weatherhead,2001; Clark et al., 2008; Shine, 2008). Contemporaryinterest traces back to a paper by Gannon (1978), whoexamined factors limiting the distribution of the PrairieRattlesnake (Crotalus viridus viridus) in Saskatchewan, Ca-nada, a region where hibernacula are often large and widelyspaced. At the time, the prevailing idea was that snakes weredispersal limited, and Gannon (1978) thought it wasunlikely that much interbreeding occurred between hiber-nacula. Unfortunately, suitable behavioral and molecular-genetic technologies did not exist at the time to testGannon’s hypothesis, and nearly 20 years passed beforepopulation-genetic studies emerged (Bushar et al., 1998).Inferences from early radiotelemetry studies suggested thatcontact between males and females from different hiber-nacula was probably frequent enough to prevent geneticdrift, even in regions where hibernacula are distantly spacedand most individuals mate upon emergence (Gregory,1984).

Recent studies have used microsatellite DNA loci to makeinferences about mating patterns among hibernacula(Bushar et al., 1998; Lougheed et al., 1999; Clark et al.,2008), but no study to date has found evidence that matingis restricted among hibernacula within local landscapes.While genetic studies have been insightful, interpretingpatterns of genetic variation has been difficult because fewstudies have employed direct observations of dispersal andmating behavior (Blouin-Demers and Weatherhead, 2002).Although behavioral studies may be logistically difficult,direct assessment of mating and dispersal among popula-tions remains the only valid approach for the interpretationof gene flow in an ecological context (Bossart and Prowell,

1998). For example, genetic homogeneity among hibernac-ula may be maintained by juvenile dispersal, adult dispersal,or gamete exchange between males and females fromdifferent hibernacula; discriminating among these variousfactors can only be accomplished via assessment of behaviorand genetics. The goal of the present study was to evaluatethe effects of movement and mating patterns on geneticvariation within and among hibernacula of the TimberRattlesnake (Crotalus horridus). Behavioral inferences aboutmating were compared with indirect inferences of gene flowbased on patterns of genetic variation (microsatellite DNAloci) and results interpreted with respect to the idea thatgene flow is restricted among hibernacula. Based on existinginformation, it was hypothesized that adult dispersal andjuvenile dispersal among hibernacula would be low, but thatmating between males and females from different hibernac-ula would be frequent enough to prevent differentiation viagenetic drift.

MATERIALS AND METHODS

Study site and field sampling methods.—Behavioral data andtissue samples were collected between the years 2000 and2005 in and around the Tyson Research Center in westernSt. Louis County, Missouri. Because the Timber Rattlesnakeis difficult to observe, a prolonged sampling period andmultifaceted search strategy were needed to capture indi-viduals and to identify hibernacula. To find samplerattlesnakes: (1) known hibernacula were searched; (2)known parturition sites were searched during the peakparturition period; (3) roads and suitable habitat werescanned while driving or walking; (4) the vicinity ofradiotelemetry locations were searched; and (5) dead snakeswere salvaged.

1 Washington University in St. Louis, Department of Biology, Campus Box 1229, St. Louis, Missouri 63130. Present address: Arizona StateUniversity, The Biodesign Institute, Evolutionary Functional Genomics, P.O. Box 875301, Tempe, Arizona 85287-5301; E-mail:corey.d.anderson@asu.edu.

Submitted: 16 July 2008. Accepted: 19 October 2009. Associate Editor: D. Kizirian.F 2010 by the American Society of Ichthyologists and Herpetologists DOI: 10.1643/CH-08-121

Copeia 2010, No. 1, 54–61

Live individuals were captured using a snake stick or snaketongs and were transported in a covered, ventilated bucketto a secure location for processing. During processing allrattlesnakes were handled using the hook and tube method.Individuals were sexed via cloacal probing. Live animalswere given a unique scale clip on their tail and a passiveintegrated transponder (PIT) tag was implanted midbody,under the first row of dorsal scales (right side). To obtaingenomic material, a blood sample was taken with a syringevia the caudal vein and then transferred to a vacuum-sealedlithium-heparin tube for temporary storage at 220uC. Long-term storage was in cryogen tubes at 280uC. For deadsalvaged snakes, liver samples were dissected and stored incryogen tubes at 280uC.

All snakes were released at the point of capture, usuallywithin one day. Some snakes were temporarily housed toimplant radio transmitters (Holohil SI-2T and AI-3T) using avariant of the body-cavity implantation procedure ofReinert and Cundall (1982). All surgeries were conductedby the staff surgeon at the Washington University School ofMedicine, in a sterile surgical facility using an anesthesiamachine with isoflurane gas (Anderson and Talcott, 2006).One female rattlesnake did not recover from anesthesia andexpired (it should be noted that this individual wasemaciated and may not have been a good surgicalcandidate). Snakes with radio transmitters were released attheir capture location and then located via homingthroughout the active season. For each location a surveygrade GPS (Trimble GeoExplorer III) was used to logapproximately 25 points, which were later differentiallycorrected and averaged for maximum precision (usingUnited States Geological Survey [USGS] and ContinuouslyOperating Reference Stations [CORS] base files with thePathfinder software program). The accuracy of correctedlocations was inspected by overlaying the locations on anaerial photograph and cross-checking each location with thedescription (and/or distance measurements) from the corre-sponding field data. Inaccurate GPS locations were retaken,eliminated, or re-estimated using the data sheets. Newhibernacula were found by visually searching potentialhabitat during egress and ingress (‘‘egress’’ 5 departurefrom hibernacula, ‘‘ingress’’ 5 return to hibernacula), aswell as by following individuals implanted with radiotransmitters that were captured away from their hibernaculaduring the active season.

Behavioral analyses.—As a general representation of TimberRattlesnake activity areas, Hawth’s Analysis Tools, ver.3.27(http://www.spatialecology.com/htools) in ArcGIS 9.1(ESRI, Redlands, CA; http://www.esri.com) was used togenerate 100% minimum convex polygons (MCPs) for allrattlesnakes that were tracked at least one entire activeseason. To test the hypothesis that the size of male activityareas tended to exceed the size of female activity areas,MCP estimates for each sex were compared using a one-tailed Mann-Whitney U test. Because some individualswere tracked for multiple seasons, a resampling procedurewas used to determine the range of U-scores when onlyone sample season at a time was chosen for eachindividual. The resampling procedure was also used witha one-tailed Wilcoxon signed-ranks test to determine therange of W-scores associated with the hypothesis thatactivity areas of nongravid females were smaller in yearsthey were gravid.

To characterize the spatial distribution of sexual activity,snakes with radiotransmitters were followed during themating season (approximately late July through earlySeptember, peaking in early August). During the matingseason, if individuals with radio transmitters were foundnear (, 1 m) individuals of the opposite sex they wereconsidered to be engaged in a mating event. Behavioralindicators were also used to infer sexual activity (e.g., pre-copulatory shedding by females, mate guarding behavior bymales, courting by males, and coitus).

For mating pairs, ArcGIS was used to measure theEuclidean distance from the location of the mating eventto the resident hibernaculum of the male and female. Forcases where the resident hibernaculum of the male andfemale differed, the hypothesis that male mating distancesexceeded female mating distances was tested using a one-tailed Wilcoxon signed-ranks test. For mating events thatincluded cases where the residency of only one member ofthe pair was identified, a one-tailed Mann-Whitney U testwas used to test the hypothesis that male mating distancesexceeded female mating distances (before testing, to yieldone mating distance for each individual, mating distanceswere averaged within years, then over years).

To test whether individuals mated at random with respectto hibernaculum, a randomization procedure was appliedfor pairs where the hibernaculum of both individuals wasdetermined. The number of pairs was held constant andthen males and females were pulled randomly from the dataset. P-values were generated based on the proportion ofrandomizations (9,999), where the number of mating pairsfrom the same hibernaculum was equal to or larger than inthe observed data set.

To examine the spatial distribution of birth locationsrelative to communal hibernacula, a one-sample sign testwas used to determine whether birth distances tended toexceed the average nearest-neighbor distance betweenhibernacula. To determine dispersal patterns of neonatesfrom parturition sites to hibernacula, neonates were cap-tured and marked at birth locations.

Genomic extraction and microsatellite amplification.—Com-mercial kits from Qiagen and Viogene were used to extracttotal genomic DNA from blood samples, diluting aliquotsfor PCR 103 with nanopure water. Primer sets for 12microsatellite loci were ordered from Qiagen: six specificallydeveloped for the Timber Rattlesnake by Villarreal et al.(1996); six developed for the Massasauga Rattlesnake byGibbs et al. (1998), but optimized for the Timber Rattlesnakeby Anderson (2006).

Twenty-five microliter PCR amplifications were conduct-ed with a Biometra T1 thermocycler containing 2.5 ml dNTPs(2 mM/nucleotide), 2.5 ml 103 PCR buffer, and 5 ml primers(forward and reverse at 2 pmol/L, in a 3:1 ratio of unlabeledto fluorescent labeled primer), the remaining volumeconsisting of Taq polymerase (homemade lab stock volumeoptimized by batch) and nanopure water, with 1 ml dilutedgenomic DNA added to the 25 ml reaction volume in thefinal step. For the six primer sets developed by Villarreal etal. (1996), a cycling profile similar to that of Bushar et al.(1998) was used but with a final extension for all loci at 72uCfor 10 min. For the six primer sets developed by Gibbs et al.(1998), a similar cycling profile was used, but with a colderannealing temperature (57uC) and locus specific bufferconditions as determined by Anderson (2006). Following

Anderson—Gene flow in Crotalus horridus 55

amplification, the 25 ml reactions were diluted 63 withnanopure water and visualized using a BaseStation DNAFragment Analyzer and Cartographer software (MJ Re-search). Alleles were scored at each locus for each individualmanually by fragment size.

Population-genetic analyses.—Allele sizes for all microsatelliteloci were entered into Microsoft Excel for preprocessingusing the Excel Microsatellite Toolkit (Park, 2001). To avoidbias in population-genetic analyses associated with sam-pling a high proportion of newborn (‘‘neonate’’) siblings,genotypes from newborns collected from parturition sites orin the vicinity of den sites prior to hibernation were notincluded in population-genetic analyses. To evaluate theutility of the microsatellite loci for population-geneticstudies, GENEPOP was used (Raymond and Rousset, 1995)to generate summary statistics, to perform Hardy-Weinbergexact tests, and to test for linkage disequilibrium amongpairs of loci.

FSTAT (Goudet, 1995) was also used to calculate Weir andCockerham (1984) F-statistics, bootstrapping over loci todetermine 95% confidence intervals. Weir and CockerhamF-statistics were chosen because the method does not makeassumptions about sample size and is well suited to smalldata sets. To test for a pattern of isolation by distance amonghibernacula, pairwise FST and pairwise distance semimatriceswere entered in GENEPOP to conduct Mantel’s nonpara-metric test of matrix correlation (Mantel, 1967). To examinerelatedness within hibernacula relative to the total sample,FSTAT was used to calculate Hamilton’s relatedness coeffi-cient (Queller and Goodnight, 1989).

Bayesian K-means clustering algorithms in STRUCTURE2.1 (Pritchard et al., 2000; Falush et al., 2003) and BAPS 5(Corander and Marttinen, 2006) were also used to testwhether hibernacula represent discrete genetic subpopula-tions. For STRUCTURE, ten independent runs (burn in 5

105, repetitions 5 106) were conducted for each K under amodel of admixture with correlated allele frequencies andno prior information about hibernaculum residency. BAPS5.1 was run in the ‘‘clustering of groups of individuals’’mode using genotype data from individuals of knownhibernaculum residency. A vector of replicate K values wasentered (ten runs per K, from K 5 2 to K 5 7); the softwarereports the set of estimates with the ‘‘best’’ partition and theprobability associated with different priors.

RESULTS

At the beginning of the study, two communal hibernaculawere known at the study site; however, by the end of the2001 active season, a total of six communal hibernacula hadbeen confirmed (Fig. 1A). In 2002 a male Timber Rattlesnakewas radiotracked to an additional overwintering location inWest Tyson County Park, but no other individuals werefound in this location despite repeated searches throughoutthe remainder of the project. Residency at communalhibernacula was determined for 82 of 158 individuals: 5(den #1), 4 (den #2), 16 (den #3), 19 (den #4), 19 (den #5),and 19 (den #6).

Three of 82 identified residents were known to haveswitched hibernacula over the study period. Two radio-tracked adult males moved to den #2 after previouslyoverwintering at different hibernacula (one at den #4, oneat den #5); a third male, originally captured as a neonate atden #3 in 2002, was recaptured at den #5 in 2004. Of the 28

individuals that received radio transmitters, 23 (12 males, 11females) were tracked for at least one entire active seasonyielding complete activity areas for 54 sample years(Table 1). In 52 of the 54 sample years rattlesnakes returnedto the same hibernaculum at the end of the active season.

For both males and females, the maximum number ofactive seasons collected for an individual equaled four.Males were tracked for an average of 2.1 active seasons(median 5 2) and females were tracked for an average of 2.6active seasons (median 5 3). For all combinations ofindividuals and years (Umax 5 25, P 5 0.02), the size ofmale activity areas (Fig. 1B; nmales 5 12; 100% MCP: max 5

465 ha, min 5 6 ha) tended to exceed the size of femaleactivity (Fig. 1C; nfemales 5 9; 100% MCP: max 5 17 ha, min5 2 ha). Likewise for all combinations of individuals andyears (Umax 5 20, P 5 0.002), the size of male activity areastended to exceed the size of gravid female activity areas(Fig. 1D; ngravid females 5 11; 100% MCP: max 5 19 ha, min 5

1 ha). When female activity areas were compared betweenyears they were gravid and years they were non-gravid, thesize of non-gravid female activity areas tended to exceed thesize of gravid female activity areas for some combinations ofthe data (Wmax 5 35, P 5 0.025), but not for others (Wmin 5

11, P . 0.05).

Between 2000 and 2005, 21 radiotracked individuals wereobserved in a total of 40 mating events (Fig. 1C). For the 12pairs where the resident hibernaculum of the male andfemale was identified, in 4 cases individuals were from thesame hibernaculum. Random mating could not be rejected(P 5 0.19) when the number of mating pairs was heldconstant and males and females were pulled randomly fromthe data 9,999 times. Data on the spatial distribution ofmating pairs relative to hibernacula indicated sex-biasedmovement patterns (Table 2). Euclidean distances betweenhibernacula ranged from 306 m to 1,298 m (mean 5

520 m). Euclidean distances from the mating male’s hiber-naculum to the mating female’s hibernaculum ranged from0 m to 2,349 m. For the eight pairs in which hibernaculadiffered for males and females, distances were longer formales in all cases (P 5 0.01). For pairs where the hibernac-ulum of at least one individual was known, Euclideandistances from the mating location to the mating male’shibernaculum ranged from 172 m to 2,240 m (n 5 14, mean5 1,001 m); Euclidean distances from the mating location tothe mating female’s hibernaculum ranged from 195 m to1,148 m (n 5 10, mean 5 511 m); male mating distancestended to exceed female mating distances (P 5 0.05).

Eleven confirmed parturition events were observed byfollowing females with radio transmitters that were gravid.Euclidean distances from the mother’s parturition site to themother’s hibernaculum ranged from 17 m to 661 m (mean5 246 m [Table 2]). In 10 of the 11 events, females gavebirth at sites that were closest to their own hibernaculum,and birth distances tended to be less than the nearest-neighbor distance between hibernacula (P 5 0.01). Fifty-twoneonates captured at parturition locations used by gravidfemales were marked. Six neonates were recaptured at den#3, all originally captured at a road escarpment 199 m fromthe hibernaculum.

Population-genetic analyses.—Full or partial genotype datawere collected for a total of 146 individuals, but data for 57neonates captured in the late summer or early fall were notincluded in population-genetic analyses. In total, genotype

56 Copeia 2010, No. 1

data were obtained for 58 (30 males, 28 females) of 82known residents of communal hibernacula: 5 (den #1), 3(den #2), 7 (den #3), 17 (den #4), 12 (den #5), and 14 (den#6). Genotype data were not obtained for all knownresidents because: 1) some identified residents were consid-ered neonates and were therefore not included in popula-tion-genetic analyses; 2) tissues were not taken for someindividuals captured early in the study; or 3) full or partialgenotype data could not be obtained from blood or tissue.Nevertheless, it should be noted that not all analysesrequired a priori categorization of individuals by hibernac-ula.

Results of PCR amplifications revealed that five of 12 loci(7-87, 7-144, Scu01, Scu05, and Scu07) exhibited significantheterozygote deficits (Table 3). For locus 7-87, the nullhypothesis of no difference in the number of observed andexpected heterozygotes could not be rejected (P 5 0.601)when male 5946 (who was homozygous for rare allele 142)was removed from the analysis. When female 076A (whowas homozygous for rare allele 140 at Scu01 and homozy-gous for rare allele 134 at Scu07) was removed from the

analysis, Scu01 became monomorphic, and the null hypoth-esis of no difference between the number of observed andexpected heterozygotes could not be rejected for Scu07 (P 5

1). For subsequent analyses, loci 7-144 and Scu05 wereremoved due to extreme heterozygote deficits that might beindicative of null alleles, and loci 7-150 and Scu01 wereremoved due to low polymorphism, yielding eight poly-morphic loci (3-155, 5-183, 7-87, 5A, Scu26, Scu16, Scu11,and Scu07). For the eight loci, Hardy-Weinberg equilibriumover all hibernacula could not be rejected, indicatingrandom mating at the level of the total sample.

The deviation from Hardy-Weinberg expectations in thetotal sample population (F < FIT) measured 20.011 (95% CI:20.067 to 0.046). The proportion of genetic variance in thetotal population due to variation among hibernacula (h <FST) measured 0.029 (95% CI: 0.024 to 0.082). The deviationfrom Hardy-Weinberg expectations within hibernacula (f <FIS) measured 20.048 (95% CI: 20.109 to 20.014). Pairwisegenetic subdivision between hibernacula (measured via h)ranged between 20.030 and 0.109, but there was nocorrelation between genetic subdivision and geographic

Fig. 1. (A) Location of known hibernacula and representative 100% minimum convex polygons (MCPs) for (B) males, (C) females, and (D) gravidfemales that were radiotracked for at least one complete active season between 2000 and 2004 at the Tyson Research Center. For individuals thatwere radio-tracked for more than one complete active season, the largest MCP is presented.

Anderson—Gene flow in Crotalus horridus 57

distance. Relatedness (R) within hibernacula relative to thetotal sample measured 0.106 (95% CI: 0.050 to 0.162).

The Bayesian clustering algorithms implemented inSTRUCTURE 2.1 and BAPS 5 revealed no substructuring.For STRUCTURE 2.1 runs, the value of alpha (the Markovchain parameter) varied between 0 and 10 and did notconverge. The mean proportion of the genome estimated tohave originated from each population was always symmetric(,1/K). For BAPS 5 runs in the ‘‘clustering of groups ofindividuals’’ mode, the program always returned an errormessage with the graphical output yielding the same colorfor all specified groups. This is an indication that nostructure exists in the data set (J. Corander, pers. comm.).

DISCUSSION

In the present study, fidelity to hibernacula was high, but itis not clear whether hibernacula were ecologically anddemographically cohesive units. While continued refine-ment of technologies for tracking small snakes (Cobb et al.,2005) is starting to make long-term observations possible,following a sufficiently large sample of individuals for alifetime is beyond the resources of most researchers.Estimates of short-term fidelity from the present studysupport the idea that most individuals return to the samehibernaculum at the end of the active season; however,evidence of size constraints at some hibernacula and

Table 1. Activity Area Estimates (100% MCP) in Hectares (1 hectare 5 10,000 m2) for the Timber Rattlesnake at the Tyson Research Center from2000 to 2004. Snake ID: alphabetical identification of individual with transmitter (uppercase 5 male, lowercase 5 female; * 5 indicates that a femalewas gravid; i: incomplete data (‘ 5 received implant during active season, ! 5 transmitter failure during active season, !! 5 transmitter removedduring active season, { 5 natural mortality during active season, { 5 intentionally killed by human during active season; D 5 death due tosurgical complication).

Snake ID 2000 2001 2002 2003 2004

A i‘ 27.78 — — —B i‘ i{ — — —C i‘ i{ — — —D i‘ 76.82 iD — —E i‘ 465.02 i! — —f i‘ i! — — —G — 145.25 i{ — —h — 1.68* 3.81 6.91 9.18i — 19.00 8.86 3.45* 7.37J — 35.75 7.72 7.19 28.51k — 11.22 9.69* 10.03 4.97l — 6.20 2.53* i!! —m — i‘ i! — —n — i‘ 18.57* i! —O — i‘ 150.88 12.11 19.24P — i‘ 36.12 20.79 61.81q — i‘ 5.74* 11.56 14.64r — i‘ 6.85* 12.84 11.19s — i‘ 0.67* 2.03 13.78T — i‘ 132.07 87.19 127.45U — — 30.43 14.14 i‘

v — — 9.63* 17.46 i‘

w — — 11.65* 14.93 —x — — 9.65* i{ —Y — — i‘ 238.20 282.12Z — — i‘ 36.16 61.45AA — — i‘ i{ —BB — — i‘ 5.51 16.46

Table 2. Distances Relevant to Mating and Dispersal Exhibited by the Timber Rattlesnake at the Tyson Research Center. n: number of samples (atotal of 40 consort bouts were observed; if individuals engaged in more than one consort in a season [or over multiple seasons], distancemeasurements were averaged per year, then over years to yield n); mean: average distance; stdv: standard deviation of the sample; max: maximumdistance; min: minimum distance.

Distance (meters) n mean stdv max min

Den to closest den 6 520 399 1298 306Mating location to mating male’s den 14 1001 669 2240 172Mating location to mating female’s den 10 511 285 1148 195Mating male’s den to mating female’s den 10 1058 1030 2349 0Female’s parturition site to female’s den 11 246 221 661 17

58 Copeia 2010, No. 1

observed bouts of hibernaculum-switching suggest thatlifetime fidelity to hibernacula may be lower.

Even if lifetime fidelity to hibernacula were high enoughto result in ecologically and demographically cohesivepopulation units, the frequency of mating among malesand females from different hibernacula was presumablyhigh enough to prevent differentiation due to genetic drift.This is because the Timber Rattlesnake breeds after dispersalfrom hibernacula in the late summer and early fall (Martin,1993; Aldridge and Brown, 1995), and activity areas of malesand females from different hibernacula regularly overlap(Reinert and Zappalorti, 1988a). Overlap among activityareas may be increased by the behavior of males during themating season; some make long-distance movements fromsummer foraging ranges to find receptive females (Coupe,2002). While long-distance movements by males have beennoted for this species, the present study is the first todemonstrate a statistical effect of sex on mating distancefrom hibernacula; namely, that males tend to mate at longerdistances from hibernacula than females.

Interestingly, movement patterns of gravid females dif-fered slightly from previously described accounts for theTimber Rattlesnake (Reinert and Zappalorti, 1988a). As inprevious studies, most gravid females gave birth in alocation that was closest to their own hibernaculum;however, as compared to previous studies, gravid femaleactivity areas were not necessarily smaller than those ofnongravid females; in fact, the three largest activity areasexhibited by females occurred during years they were gravid.This finding could be explained by the observation thatsome gravid females moved considerable distances amongbasking sites before returning to a location near their ownhibernaculum to give birth. This finding may also beexplained by the fact that activity areas of nongravidfemales appear to be smaller than reported in earlier studies(Reinert and Zappalorti, 1988a; Brown, 1993).

As in previous population-genetic studies of this species,behavioral and genetic results were consistent with a patternof increased pedigree relatedness within hibernacula due tothe behavior of gravid females and neonates (Bushar et al.,1998; Clark et al., 2008). For the Timber Rattlesnake, gravidfemales typically give birth near their own hibernaculum(Reinert and Zappalorti, 1988a; Brown, 1993), and manyoffspring presumably recruit to the same hibernaculum as

their mother (Brown and Maclean, 1983; Reinert andZappalorti, 1988b; Cobb et al., 2005). In contrast, neonataldispersal among hibernacula of the Black Rat Snake isenhanced by the behavior of gravid females, which typicallymove extended distances away from their own hibernacu-lum to oviposit (Blouin-Demers and Weatherhead, 2002).Such differences in the behavior of gravid females arethought to explain why modest levels of genetic differen-tiation have been detected among hibernacula of the TimberRattlesnake (Bushar et al., 1998; Clark et al., 2008), whereasno significant genetic differentiation has been detectedamong hibernacula of the Black Rat Snake (Prior et al., 1997;Lougheed et al., 1999), despite broad similarities in adultfidelity and mating patterns (Weatherhead and Hoysak,1989; Gibbs and Weatherhead, 2001).

Interestingly, patterns of genetic differentiation observedamong communal hibernacula of the Timber Rattlesnakeare analogous to those described among social groupswithin local populations of mammals (Storz, 1999). Popu-lation genetic data from a taxonomically diverse array ofsocial mammals supports low to moderately high levels ofgenetic differentiation among social groups (FST 5 0.006–0.227), coupled with consistently high levels of within-group heterozygosity indicated by negative FIS-values. Thisrelationship between intergroup divergence and heterozy-gote excess runs counter to predictions designed forpopulation-genetic ‘‘demes’’ (Wright, 1969), where restrict-ed gene flow leads to the gradual loss of genetic variationand increased correlations between uniting gametes as aresult of inbreeding. Hence for the Timber Rattlesnake,negative FIS may reflect the consequences of male dispersaland female philopatry (rather than avoidance of inbreed-ing). Female philopatry should result in positive alleliccorrelations between female gametes within a hibernacu-lum, but negative allelic correlations between femalegametes and those of the immigrant male, resulting inexcess heterozygosity proportional to the genetic varianceamong female family groups from different hibernacula(Chesser, 1991). Excess heterozygosity within hibernaculacould also be explained simply by binomial sampling erroryielding allele frequency differences among male and femaleparents (Balloux, 2004). In general, behavioral and demo-graphic factors that result in natal philopatry, sex-biaseddispersal, or a limited number of breeding adults can

Table 3. Results of PCR Amplifications for Microsatellite DNA Primer Sets Developed by Villarreal et al. (1996) and Gibbs et al. (1998) on GenomicSamples from the Timber Rattlesnake Captured at the Tyson Research Center. Nn: number of individuals sampled; Hexp: heterozygosity expected;Hobs: heterozygosity observed. P-values were estimated via GENEPOP (Raymond and Rousset, 1995) using the Markov chain method in the defaultsetting (100 batches with 1000 iterations per batch); ***P , 0.001, **P , 0.01, *P , 0.05.

Locus Nn Hexp Hobs Allele sizes (bp)

3-155 80 0.58 0.60 123, 131, 133, 1475-183 81 0.40 0.41 131, 139, 1467-144*** 56 0.74 0.30 106, 110, 114, 116, 1207-150 54 0.02 0.02 146, 1487-87** 82 0.22 0.20 142, 152, 1565A 79 0.53 0.54 145, 147, 151, 153, 155Scu26 84 0.82 0.82 150, 152, 160, 164, 168, 170, 172, 174, 176, 178, 180Scu16 79 0.58 0.59 151, 153, 157, 159Scu11 83 0.83 0.84 160, 162, 164, 172, 178, 180, 188, 194, 202, 204Scu07** 83 0.14 0.13 134, 149, 153Scu05*** 69 0.76 0.42 199, 201, 205, 211, 213, 215, 217Scu01** 85 0.02 0.00 104, 140

Anderson—Gene flow in Crotalus horridus 59

produce the same genetic patterns as those associated withgroup living sociality, even in taxa characterized by morecontinuously distributed populations and lesser degrees ofsocial cohesion (Storz, 1999).

Results from population-genetic studies of mammaliansocial groups and snake hibernacula indicate that classicmeasures such as Wright’s F-statistics may be sensitive todifferences in allele frequencies among hibernacula irre-spective of the origin of those differences, i.e., whether theyderive from the balance of gene flow and drift or whetherthey derive from other behavioral and demographic factors.Bayesian clustering algorithms have become a popularalternative for identifying demes, but may be less sensitiveto differentiation when molecular information is limitedand genetic differences between sample populations areweak. In this situation, Bayesian algorithms that enableclustering of hibernacula (rather than strictly individuals)and that do not simultaneously estimate admixture propor-tions can be used (e.g., BAPS), but in the present studyyielded the same result: no structure. Some have suggestedthat contingency tests be used as an upper bound for thenumber of demes, and that F-statistics and Bayesianclustering algorithms be used as a lower bound (Funk,2005). However, problems arise when genetic structuring isweak and not detectable across methods; in such cases, thebiological basis of genetic differentiation may be lessapparent.

Population genetic studies of hibernacula would benefitfrom a closer look at other models of population structurethat are appropriate for hibernacula. For example, Bushar etal. (1998) suggested that hibernacula exhibit several attri-butes characteristic of metapopulations (Levins, 1969), suchas a small number of migrants between certain geographi-cally close hibernacula, small population sizes (which couldincrease the likelihood of extinction), and the potential forrecolonization from neighboring hibernacula. While in-triguing, the complicated annual life cycle of the TimberRattlesnake makes the application of the metapopulationmodel tenuous. In particular, the fact that most normalactivities occur away from hibernacula in relatively contin-uous habitats taxes most traditional metapopulation mod-els. In addition, metapopulation models in populationgenetics are based on the assumption that gene flow isrestricted among fragments to the point that they representdiscrete demes (Olivieri et al., 1990), an assumption thatdoes not appear to be realistic for most snake hibernacula.

A lesser known model of population structure wasproposed by D. S. Wilson (1975, 1977, 1980), who notedthat many organisms have complex life histories involvingidentifiable dispersal and non-dispersal stages. During non-dispersal stages, movements are often spatially restricted,but during dispersal stages, individuals move on to mateover a much larger area. Wilson (1975) coined the term‘‘trait group’’ to contrast the ecologically homogeneouspopulation associated with non-dispersal, from the popula-tion-genetic deme associated with mating during dispersalstages. Hibernacula might be considered trait groupsbecause they represent ecologically homogeneous popula-tions associated with seasonal non-dispersal, located withina deme.

While information about patterns of mating and geneticvariation among hibernacula is starting to accumulate forsome species such as the Timber Rattlesnake, informationfor most species and populations remains unknown. In

particular, we have little information about patterns ofgenetic variation in habitat that is discontinuous, inlandscapes where individuals do not hibernate communally,or in landscapes where individuals hibernate communallybut mate immediately after emergence. Although manyquestions remain, information from existing studies castdoubt on the old idea that gene flow is restricted amongneighboring hibernacula. This hypothesis was couched inthe context of a population near the northern limit of itsgeographic range, before the advent of radio telemetry, andis unlikely to hold true for most populations in continuouslandscapes.

ACKNOWLEDGMENTS

Many people contributed to the labor required for thismanuscript. Six undergraduates (J. Ballantyne, H. Blum, A.Delevan, P. Garner, E. Koop, A. Sajadi), one high schoolstudent (R. Turnquist), and one full-time volunteer (W.Drda) assisted with fieldwork; W. Drda in particular made alarge contribution in terms of captures and telemetrylocations. I would also like to thank: my graduate advisor,A. Templeton, for the independence, patience, and resourc-es he granted that made this study possible; M. Talcott forlending his surgical expertise to implant radiotransmitters;and O. Sexton for encouraging me to conduct this study atthe Tyson Research Center. J. Cheverud deserves thanks fortelling me about trait groups, as does W. Brown, A. Larson,M. Rosenberg, and T. Dowling for comments on earlierdrafts of this manuscript. The American Museum of NaturalHistory (Theodore Roosevelt Grant), the Webster GrovesNature Study Society (Menke Scholarship), and the Friendsof Tyson contributed funds for this project. Animalhandling required for this project was done in strictaccordance with procedures approved by the WashingtonUniversity Animal Care and Use Committee, under collect-ing permits issued by the Missouri Department of Conser-vation.

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