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NEST SITE SELECTION AND EMBRYONIC SURVIVALIN FOUR-TOED SALAMANDERS, HEMIDACTYLIUMSCUTATUM (CAUDATA: PLETHODONTIDAE)Author(s): George W. Wahl III, Reid N. Harris, Tracie NelmsSource: Herpetologica, 64(1):12-19. 2008.Published By: The Herpetologists' LeagueDOI: http://dx.doi.org/10.1655/06-082.1URL: http://www.bioone.org/doi/full/10.1655/06-082.1

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NEST SITE SELECTION AND EMBRYONIC SURVIVAL INFOUR-TOED SALAMANDERS, HEMIDACTYLIUM SCUTATUM

(CAUDATA: PLETHODONTIDAE)

GEORGE W. WAHL, III, REID N. HARRIS1, AND TRACIE NELMS

Department of Biology, MSC 7801, James Madison University, Harrisonburg, VA 22807, USA

ABSTRACT: Nest site selection occurs when females place eggs at sites that differ from random sites within adelimited area. Selection of components of the microhabitat that increase embryonic survival is expected.Female four-toed salamanders, Hemidactylium scutatum, at two montane study ponds in Virginia selectednesting locations that had more moss, grass, and other living vegetation than found in other locations around thepond. In addition, selected nest sites had steeper bank angles and average lower pH’s than other sites and weremore often found facing north than expected by chance. Of the variables selected by H. scutatum whenchoosing a nest site, bank angle, nest aspect, and pH were correlated with embryonic survival. We found thatnorth-facing nests were cooler than south-facing nests. Embryonic survival was highest in nests on steep slopesthat faced north with lower average temperatures and that had lower pH’s. A third pond that historically hadfewer nesting females and was characterized by low embryonic survival did not have nesting habitats thatcombined steep bank angles with a northerly aspect. When constructing ponds for wildlife in areas where H.scutatum is of special concern, we suggest that steep bank angles be situated so that they face north.

Key words: Embryonic survival; Hemidactylium scutatum; Nest site selection; Plethodontidae

NEST SITE selection occurs when femalesplace eggs at sites that differ from randomsites within a delimited area (Wilson, 1998). Insome species, nest selection can lead toenhanced offspring survival. For example, anumber of field surveys of reptile nests havedetected nest site selection where one orseveral characteristics were highly prevalentin nests but not in random sites, andembryonic survival was greater in selectedmicrohabitats (Madsen and Shine, 1999;Spencer and Thompson, 2003; Wilson,1998). Studies of nest site selection arerelatively rare for amphibians; however, nestconstruction and nest site selection does occurin some species of salamanders (Guy et al.,2004; Jackson et al., 1989; Petranka andPetranka, 1981). We studied nest site selec-tion and embryonic survival in four-toedsalamanders, Hemidactylium scutatum, atthree montane ponds in Virginia. Nest siteselection and its relationship to embryonicsurvival have not been previously studied inthis species.

Different nesting habitats of H. scutatumhave been observed in the field (Bishop, 1941;Breitenbach, 1982; Gilbert, 1941; Wood,1955). In general, females lay eggs in terres-

trial nests that are on the edge of ponds andbogs. Hatchlings must travel from their nest tothe aquatic environment in order to completea relatively short larval period. Breitenbach(1982) described the nesting habitats in eachof his study ponds in Michigan with respect totypes of vegetation. At one pond where moss,grasses, and sedges were observed, he found89% of the nests in clumps of Sphagnummoss. Observations from Gilbert (1941) in thearea of Ithaca, New York, found five nests ofH. scutatum in Sphagnum moss and 27 inmosses of the genera Climacium, Thuidium,and Mnium. Wood (1955) listed 13 genera ofmosses in which H. scutatum are known tonest in eastern Virginia as well as a list of othernesting substrates including the following:sedges, liverworts, loose bark, mounds of pineneedles, and rotten wood. Recently, Chalmersand Loftin (2006) found that H. scutatum nestsites were distinguished from available, unoc-cupied microhabitats by steeper shore, greaternear-shore water depth, and presence ofmoss. The ponds used in this study had avariety of nesting substrates, and we surveyedthree ponds to determine (1) whether femaleH. scutatum nest in a nonrandom subset ofmicrohabitats, and (2) whether embryonicsurvival was correlated with attributes of nestmicrohabitat.1 CORRESPONDENCE: e-mail, harrisrn@jmu.edu

Herpetologica, 64(1), 2008, 12–19

E 2008 by The Herpetologists’ League, Inc.

12

MATERIALS AND METHODS

The three ponds used in this study werePond Ridge Pond (PR), Clines Hacking Pond(CH), and White Oak Flat Pond (WOF) onthe Shenandoah Mountain range in theGeorge Washington National Forest, Rock-ingham County, Virginia. An overview of thegeneral characteristics of these ponds is foundin Gill (1978). The elevations of the threeponds are 1024 m at PR, 1033 m at CH, and1030 m at WOF. Based on long-term esti-mates of nesting females between 1991 and2005, two of our study ponds—PR and CH—consistently had large numbers of nestingfemales and were considered to be preferredponds for nesting (Harris, 2005). These werethe only two ponds within a broader studyarea that had a large number of nests, andthey were used to test whether females chosemicrohabitats for nesting and to test for theeffects of microhabitat variables on embryonicsurvival. An additional pond (WOF) thatconsistently had many fewer nests between1991 and 2005 and was considered a lesspreferred pond was used as a comparison inorder to test for pond-specific differences inthe quality of nesting habitat.

We searched three study ponds by standingin the pond, moving around the perimeter, andexamining vegetation for H. scutatum nests.Ponds were visited three times a week frombefore any nest was initiated until all embryoshad hatched, and nests were continuallydiscovered and marked. All living vegetationand nonliving substrates were searched inorder to find the embryos and/or females.When a female was found either on a nest ornot associated with a nest, she was capturedand her ventral spot pattern was photographedfor later identification (Harris and Ludwig,2004; Harris et al., 1995). When a new nest wasfound, the eggs were counted, nest initiationdate was recorded, and distance from nest towater was measured. The location of nests andthe location of females not associated with anest were marked with a flag.

Random points were established at thebeginning of the H. scutatum nesting period.For each pond, a random distance around thecircumference of the pond was generated aswell as a random distance away from thewater’s edge within the nesting area of H.

scutatum for each of 20 random sites. Thedistances from the water’s edge of the randompoints did not differ from the distance from thewater’s edge of nests at the three study ponds(Kruskal-Wallis test; P’s for each pond . 0.05).Measurements of microhabitat temperature,distance to water, and depth of water weretaken at all nest sites and random sites once aweek for four weeks. All temperature mea-surements at each pond were taken within anhour during constant weather conditions. Thevariable ‘‘variance in distance to water’’ wasdefined as the variance in the distancemeasurements for a nest over time.

Several measurements were recorded justonce over the nesting period at nest sites andrandom sites. Digital photographs were takenof the microhabitat in order to describevegetation coverage for each site. Photographsof nest sites and random sites were taken onthe same day after the majority of nests wereinitiated. A wooden frame that measured15 cm 3 15 cm, which is an estimate of nestsize that included the size of joint nests(Blanchard, 1934; Harris and Gill, 1980), wasplaced over each nest before it was photo-graphed. The digital photograph was viewedon a computer monitor, with the image sizeset in AdobeH Photoshop to 2100 3 2800 pix-els. A transparent grid containing 400 ran-domly spaced dots was placed over thecomputer screen so that the dots spread overthe entire photograph. Counters were used totally the number of dots that fell on each ofnine categories of vegetation and nonlivingmaterial. The nine categories were moss,grass, fallen leaf, dirt, reed, dead grass, deadreed, other vegetation, and other nonlivingmaterials. Moss in our study sites was almostuniformly in the genus Thuidium. The ‘‘othervegetation’’ category included live vegetationthat was not moss or grass, such as small dicotplants, and reeds. The fallen leaf, dirt, deadgrass, dead read, and other nonliving materialsoccurred infrequently and were pooled into a‘‘nonliving’’ category. Therefore the ninecategories were reduced to four for analyses:moss (Thuidium), grass, other vegetation, andnonliving materials.

Angle of the bank on which a nest orrandom site occurred was measured once.Two wooden rods connected at one end by a

March 2008] HERPETOLOGICA 13

hinge were used to measure the angle. Therods were held perpendicular to the water,directly in front of a nest or random site, andthen one rod swiveled down toward the bank.Once the rod fell flush against the bank, theangle between the rods was measured with aprotractor. Zero degrees was the maximumsteepness, and 90u was flat.

Aspect was measured for each nest byholding a compass directly over the eggs andreading the direction the bank was facing atthat point. Aspect measurements ranged from0–359 degrees and were recorded to theprecision of one degree. Measurements above180 degrees were subtracted from 360 to testhypotheses that changes along a north-southaxis in either direction (east or west) werecorrelated with other measured variables,such as embryonic survival.

We measured pH at random sites and nestsites. ColorpHastH pH indicator strips wereplaced onto the vegetation in the site. At nestsites, indicator strips were placed as close toeggs as possible. Strips were placed invegetation located at the flag for random sites.Once the strip became moist (,1–3 min), thepH was recorded. The pH measurements hada precision of 0.5.

A detailed initial egg count was made ateach nest site. Additional egg counts at eachnest were made to check for the deposition ofnew eggs or the formation of a joint nest sincejoint nesting is common (Harris et al., 1995).A detailed final egg count was determined foreach nest when the embryos were in their lastdevelopmental stage before hatching. Theinitial egg count was divided by the final eggcount to calculate egg survival per nest. Therewas one nest where the final count was higherthan the initial count. This nest was notdeleted from the data set because although amistake was made in the initial egg count dueto the difficulty in counting cryptic eggs in adeep nest, there was still an obvious highsurvival for the nest and the data point wouldbe ranked appropriately for a nonparametricanalysis. Therefore, the axes for survival goabove 1.0 because of this one nest.

Statistical Analyses

To determine if nest characteristics differedbetween nest sites and random sites, we used

a logistic regression with the response variablebeing one for nest site used and zero forrandom site not used. Habitat variables thathad P-values . 0.25 in univariable analyseswere not included in the multivariable analysis(Hosmer and Lemeshow, 2000, p. 95). Theproportion of a nest site that included moss,grass, and other living vegetation determinedthe proportion of the nest site with nonlivingvegetation, so the latter variable was notincluded in the statistical model. We pooleddata from the two ponds with large populationsizes, PR and CH, because the interactions ofpond with habitat variables did not approachsignificance. For the nest aspect variable, weused a Rayleigh test to test the null hypothesisthat nests were uniformly distributed aroundthe ponds, which are approximately circular inshape (Zar, 1996, p. 615).

All nest characteristics were plotted againstthe proportion of embryos that hatched and aSpearman rank correlation was used todetermine statistical significance. The Spear-man rank correlation was used becausesurvival was not normally distributed, and thistest does not depend on any statisticaldistribution. In order to test for predictors ofsurvival in a multivariable analysis, we used amaximum likelihood approach (PROC GEN-MOD in the statistical software programSAS). The gamma distribution was chosenbecause it is flexible enough to fit thefrequency distributions of the habitat vari-ables. We used the habitat variables with P ,0.25 in the Spearman correlation analysis intoa multiple regression model in PROC GEN-MOD. Only solitary nests could be used forlength of the embryonic period analysesbecause joint nests had more than onecomplement of eggs laid at different timesand embryonic periods were not the same. Itwas not possible to distinguish each female’sembryos in a joint nest throughout the nestingperiod. All nests with zero survival had to betaken out of the length of embryonic periodanalyses because embryonic period is unde-fined if all eggs die before hatching. Meansare given with standard errors.

We compared a third pond that had anhistorically low density of nests (WOF) to PRand CH to test the hypothesis that a pond’snest density was associated with the quality of

14 HERPETOLOGICA [Vol. 64, No. 1

nesting habitat. To compare ponds withregard to two important variables in nestselection, we measured aspect and bank angleevery meter at the three study ponds. To testfor homogeneity of slopes among ponds whenbank angle was regressed on nest aspect, ananalysis of covariance was used. All statisticaltests were conducted using Statistical AnalysisSystem (SAS) software.

RESULTS

Nest Site Selection

Female H. scutatum exhibited nest siteselection using the data set that combined thetwo preferred ponds (PR and CH). Logisticregression indicated that females selectednests with a steeper bank angle, a higherproportion of moss, grass, and living vegeta-tion, and a lower pH than random sites(Table 1, Fig. 1). Moss, grass, and othervegetation were significantly more commonat nest sites than at random sites and nonlivingsubstrates were less common at nest siteswhen compared to random sites (Fig. 1). Nestbank angles were significantly steeper thanbank angles at random sites (nest sites: 43.33degrees 6 2.05; n 5 66; random sites: 50.75degrees 6 3.74; n 5 40). The pH of thevegetation moisture was significantly lower atnest sites than random sites (nest sites: 5.26 60.07; n 5 65; random sites: 5.58 6 0.08; n 539). Water depth did not differ between nestsites and random points. Nests were notuniformly distributed around the ponds (Ray-leigh’s z 5 6.475, P , 0.0002). The meanaspect was 31.9 degrees; thus nests were morelikely than expected to face toward the north-northeast.

Nest Site Selection and Embryonic Survival

Of the variables selected by H. scutatumwhen choosing a nest, bank angle, nest aspect,and pH were negatively correlated withembryonic survival (Table 2). Survival de-creased as the bank became less steep. Asthe aspect moved from north to south ineither direction (North–East–South or North–West–South), survival also decreased (Fig. 2).As pH decreased at a nest site, embryonicsurvival increased. The proportion of moss at anest site tended to be positively correlatedwith embryonic survival, but was not statisti-cally significant (P 5 0.068). Some variableswere less likely to be directly selected bynesting females. Among these variables, thevariance of nest distance to water and averagenest temperature were negatively correlatedwith embryonic survival (Table 2). A multipleregression analysis using a maximum likeli-hood approach revealed that the following

TABLE 1.—Variables that predicted the presence ofHemidactylium scutatum nest sites along the banks oftwo small ponds in montane Virginia. Parameters from alogistic regression are given for the model that includes allpredictor variables with a P , 0.25. Nest aspect wasanalyzed separately. Sample sizes 5 65 nest sites and 39

random sites.

Nest Variable b SE Wald x2 P

Moss 6.723 2.56 6.91 0.009Grass 5.700 1.75 10.59 0.001Live 9.336 3.85 5.88 0.015Angle 0.457 0.25 3.39 0.066pH 27.695 3.59 4.59 0.032

FIG. 1.—Proportion of each category of vegetation typeand nonliving material at nest sites and at random sites.Nest sites and random sites differed significantly for eachcategory.

TABLE 2.—Spearman correlations (rs) of survival andnest variables.

Nest Variable rs df P

Moss 0.232 61 0.068Grass 0.048 61 0.711Live 0.043 61 0.735Dead 20.189 61 0.137Angle 20.255 62 0.042Aspect 20.377 62 0.002ph 20.387 62 0.002Variance in Distance to the water 20.387 62 0.002Initiation date 0.245 62 0.051Depth 0.163 62 0.205Temp 20.314 61 0.012

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variables predicted survival well after all othervariables were accounted for (moss, x2 5 3.14,df 5 1, P 5 0.0764; aspect, x2 5 3.43, df 5 1,P 5 0.0642; pH, x2 5 5.02, df 5 1, P 50.0250; variance in nest distance to water, x2

5 3.29, df 5 1, P 5 0.0698; depth, x2 5 3.70,df 5 1, P 5 0.0544.

Many habitat variables were correlated witheach other. Variance in distance to water andbank angle variables were highly correlated (rs

5 0.601; df 5 65; P , 0.0001), meaning thatrelatively steep nest sites had a more constantdistance to water over time. Aspect waspositively correlated with temperature at nestsites. Average temperature increased as theaspects approached south (rs 5 0.432; df 5 64;P 5 0.0003). This correlation appeared to bedriven by the north facing nests being thecoolest. The proportion of grass at nest sitesincreased as aspects approached south (rs 50.293; df 5 64; P 5 0.017). Moss tended tobecome more frequent at north-facing nests,but this correlation was not significant (rs 520.184; df 5 64; P 5 0.139). The nests initiatedlater in the spring tended to have higherembryonic survival, although this was a ratherweak correlation (Table 1). In addition, initia-tion date was highly negatively correlated withlength of embryonic period for solitary nests (rs

5 20.872; df 5 56; P , 0.0001) and initiationdate was positively correlated with average nesttemperature (rs 5 0.275; df 5 64; P 5 0.026).

Comparison of CH and PR with WOF

A third pond in the study area is locatedbetween CH and PR, however it consistently

has a lower density of nests and nestingfemales. In 2005, PR and CH had a highnumber of females (48 and 36, respectively),whereas WOF had a low number of females(17), which is representative of the relativepopulation sizes between 1991 and 2004 aswell. In addition, embryonic survival differedamong ponds with WOF having much lowersurvival than CH and PR (MedianCH 5 0.227;MedianPR 5 0.286; MedianWOF 5 0.000;Kruskal-Wallis x2 5 6.147; df 5 2; P 50.046). As a way to test whether our studyponds had a favorable combination of steepbank angle and north-facing aspect, weplotted angle against aspect for each pond.For example, a positive relationship wouldmean that north-facing nests have steep bankangles. In particular, we tested whether WOFlacks a favorable combination of bank angleand aspect for successful nesting. By measur-ing angle and aspect every meter at the ponds,we determined that the relationship betweenbank angle and aspect differed among thethree ponds (Aspect * Pond: F(2,123) 5 5.29; P5 0.0062). The relationship between bankangle and aspect was positive at CH (rs 50.539; df 5 34; P 5 0.0007; Fig. 3). Aninspection of the data (and the pond) revealedthat there were parts of the pond that facednorth and had the favored steep bank anglesas well as parts of the pond that faced south

FIG. 2.—The statistically significant negative correlationbetween embryonic survival and aspect (direction that thenest faced). The regression line is shown to highlight thetrend in the data.

FIG. 3.—Correlation between bank angle and aspect(direction that potential nest sites faced) for PR and CH(closed circles and solid line) and for the less-preferredWOF (open circles and dotted line). Angle and aspectwere positively correlated for the pooled PR and CH data,but negatively correlated at WOF, indicating that femalesat WOF did not have nesting sites that combinedfavorable steep bank angles with a north-facing aspect.

16 HERPETOLOGICA [Vol. 64, No. 1

and had flatter bank angles. There was nosignificant relationship between bank angleand aspect at PR. At WOF, the relationshipbetween bank angle and aspect was actuallynegative (rs 5 20.365, df 5 49; P 5 0.008).An inspection of the data (and the pond)revealed that few parts of the pond existedthat both faced north and had the favoredsteep bank angle.

DISCUSSION

Evidence of Nest Site Selection

The hypothesis that microhabitats at nestsites would differ from microhabitats atrandom sites was supported. H. scutatumused several components when choosing anest site: moss, grass, and other vegetationcategories were more common at nest sites(Fig. 1). When compared to the other cate-gories of vegetation, presence of moss differedthe most between nest sites and random sites(Fig. 1). This quantitative measure of selec-tion of moss supports many of the previousqualitative observations in which nests of H.scutatum were found primarily in moss(Bishop, 1941; Breitenbach, 1982; Gilbert,1941; Wood, 1955).

Females also selected steeper banks aroundthe study ponds. Steep bank angles wouldlogically help H. scutatum hatchlings enter thepond, which is necessary as they begin anaquatic larval period. As well as aidinghatchlings, steep banks may also be anindicator of higher moisture. Bank angle washighly correlated with variance in distance towater. Nests on flatter areas had a highvariance in distance to water and were oftenfar (up to 2 m) from the pond edge andtherefore tended to dry out more than nestson steep banks, which had a more constantdistance to water and were always much lessthan 2 m away.

Selection of north-facing aspect may alsoindicate that females prefer moist environ-ments in which to establish their nests. In thenorthern hemisphere, north-facing slopes areassociated with moister environments whereassouthern slopes receive more direct sunlightand tend to be dryer. We found a significantpositive correlation between nest temperatureand aspect, which indicated that temperature

increased as nests faced south. Grass occurredon south-facing slopes and moss tended tooccur on north-facing slopes. Moss may alsocause nest sites to have a lower pH thanrandom sites. Whether it is moss, aspect, orbank angle that is the primary object ofselection by females or whether some vari-ables are selected together cannot be deter-mined from our survey. The multivariablelogistic regression model did not reveal one ortwo dominant factors that can explain whatfemales select. Females seem to choose aconstellation of habitat variables: locationswith steep bank angles that face north andhave higher proportions of moss, grass, andother vegetation, and that have a lower pH.These results are similar to those found in astudy of H. scutatum in Maine, where femalesnested in microhabitats with steeper banksand with the presence of moss (Chalmers andLoftin, 2006). Our study and the study ofChalmers and Loftin suggest that commonnest site characteristics will be found through-out the wide geographical range of H.scutatum.

Effect of Female Selection onEmbryonic Survival

The hypothesis that embryonic survival isassociated with preferred components ofnesting microhabitat was supported. Thepreferred aspects and bank angles werepositively associated with survival. However,selected vegetation types were not correlatedwith embryonic survival, although the propor-tion of moss tended to be positively correlatedwith embryonic survival (P 5 0.068). Thesouth facing nests with higher average tem-peratures may have caused dryer conditionsand lower embryonic survival. The correlationbetween average temperature and initiationdate was weaker than the correlation betweenaverage temperature and aspect, althoughboth were significant. This comparison sug-gests that the microclimate was more impor-tant for maintaining nest temperature than theseasonal change in temperature. Wilson(1998) showed similar results of nest temper-ature in a nest site selection study on stripedmud turtles. The selected habitat variable, inthis case vegetation cover, kept the turtle nestsites at a lower average temperature. These

March 2008] HERPETOLOGICA 17

covered sites had higher embryonic survivalthan open sites.

Nests found with the preferred steep bankangle had higher survival than nests that werefound in flatter areas (Table 2). Steep bankangles may help with hatchling survival as theyenter the aquatic environment but steepbanks have not been hypothesized to increaseembryonic survival. Bank angle was correlatedwith variance in distance to water, which washighly correlated with embryonic survival.Therefore, nests with steep bank angles mayhave had high embryonic survival because ofthe low variance in distance to water duringthe embryonic period. These nests remainedcloser to the water more of the time, and thiseffect kept nest sites wetter. Nests with ahigher proportion of moss are likely to remainmoister and have a lower pH than nests withless moss. A higher moisture level can keepembryos from desiccating, and more acidicconditions might limit pathogenic fungi,thereby enhancing embryonic success.

Nests established later in the laying periodhad better survival (Table 2). Initiation datewas highly negatively correlated with length ofthe embryonic period. Shorter embryonicperiods might allow less time for eggs tosuccumb to factors that cause mortality, suchas fungus or desiccation. Analyzing thecorrelation of embryonic period and embry-onic survival would provide stronger evidencefor this hypothesis. Unfortunately, embryonicperiod cannot be determined for nests thathave no survival because embryonic period isundefined.

Management Implications for H. scutatum

A small number of nesting females and lowembryonic survival characterized WOF, whichdid not have areas that combined a favorableaspect and bank angle for nesting sites. Thespecies H. scutatum has special concern statusthroughout a portion of its native range, suchas in Maine and Minnesota (Harris, 2005).Understanding the species’ nesting habitatrequirements could help agencies bettermanage areas for the preservation of H.scutatum. The USDA Forest Service createdthe ponds used in this study in the mid 1950’sfor wildlife use (Gill, 1978). Future mainte-nance and creation of such ponds could be

designed so that the steepest part of the bankwould face north. Having steep banks face thenorth may also promote high moisture, loweraverage temperature, and the growth of moss.Results from this study strongly suggest thathaving steep pond edges, facing north, withmoss, high moisture, and lower averagetemperatures would support reproducingpopulations of H. scutatum. Constructingponds in such a way would help populationsof H. scutatum while not interfering withhabitat requirements of other amphibians orother wildlife. Modifying WOF such thatnorth-facing locations have steep banks andthen following the number of nesting femalesover time would provide an interesting test ofthis hypothesis.

Acknowledgments.—We thank E. Andre, J. Banning,and K. Duncan for help with various aspects of thefieldwork and J. Kastendiek for providing useful com-ments on earlier manuscript drafts. A permit from theVirginia Division of Game and Inland Fisheries allowed usto handle salamanders for this study. This work wassupported by National Science Foundation grant 04–13981.

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.Accepted: 31 August 2007

.Associate Editor: Nathan Mills

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