conservation+genetics+of+the+central+newt+(notophthalmus...

The$final$publication$is$available$at$Conservation$Genetics,$http://link.springer.com$$$$$1$

Conservation+genetics+of+the+central+newt+(Notophthalmus+viridescens)+in+Iowa:+The+

importance+of+a+biogeographic+framework+

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Running$title:$Conservation$genetics$of$the$central$newt$in$Iowa$

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S.$Scott$Whitmore1,2,$Selena$Losee1,$Laurel$Meyer1,$and$Theresa$A.$Spradling1*$

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1Department$of$Biology,$University$of$Northern$Iowa,$Cedar$Falls,$IA$$50614R0421$

2Current$Address:$Center$for$Bioinformatics$and$Computational$Biology,$5315$SC,$The$

University$of$Iowa,$103$South$Capitol$St,$Iowa$City,$52242$$

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*Correspondence:$Theresa$A.$Spradling$Department$of$Biology$University$of$Northern$Iowa$Cedar$Falls,$IA$$50614R0421$Phone:$(319)$273R6214$Fax:$(319)$273R7125$Email:[email protected]$

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Keywords:$Notophthalmus+viridescens,$biogeography,$microsatellite,$mitochondrial$DNA$


$$Abstract++In$light$of$global$declines$in$amphibian$populations,$genetic$data$have$become$increasingly$important$for$understanding$population$structure$and$for$revealing$hidden$diversity.$At$the$species$level,$Notophthalmus+viridescens$is$an$IUCN$species$of$“least$concern”,$but$the$subspecies$N.+v.+louisianensis$(central$newt)$is$listed$as$“threatened”$in$Iowa,$a$state$on$the$western$periphery$of$the$species$range.$Genetic$data$were$collected$from$282$N.+v.+louisianensis$from$14$sites$in$Iowa.$Sequences$from$1,054$nucleotides$of$mitochondrial$DNA$from$Iowa$newts$revealed$unexpected$diversity$in$the$form$of$two$major$haplotype$groups$that$are$not$sister$clades,$with$southern$Iowa$N.+v.+louisianensis+being$more$closely$related$to+N.+v.+piaropicola$(peninsula$newt)$from$Florida$than$to$consubspecifics$in$Iowa.$Sequence$differentiation$indicates$that$the$two$lineages$of$newts$present$in$Iowa$diverged$near$the$beginning$of$the$Pleistocene.$Northern$and$southern$Iowa$haplotypes$were$found$together$at$one$site,$indicating$an$opportunity$for$hybridization$near$Remington’s$biogeographic$Suture$Zone$1,$a$hotspot$for$hybridization$in$other$species.$Three$microsatellite$loci$provided$additional$evidence$for$distinctness$of$northern$and$southern$Iowa$newt$populations.$This$study$highlights$the$relevance$of$historical$biogeography$to$conservation,$as$management$strategies$for$N.+v.+louisianensis$in$Iowa$must$reflect$previously$unrecognized$diversity$in$this$species.$Nuclear$and$mitochondrial$data$indicate$genetic$isolation$of$nearby$populations$on$the$same$drainage,$and$field$data$suggest$the$decline$of$one$study$population,$emphasizing$the$need$for$identification$and$protection$of$newt$breeding$sites$in$Iowa.$


Introduction++Global$declines$in$amphibian$numbers$are$so$profound$that$they$may$represent$a$sixth$mass$extinction$event$(Wake$&$Vredenburg$2008).$Numerous$factors$have$contributed$to$the$demise$of$amphibian$populations,$including$emerging$infectious$diseases$and$habitat$loss$and$fragmentation.$Cases$of$extinction$and$extirpation$are$well$documented$(Stuart$et$al.$2004;$Pounds$et$al.$2006).$However,$differentiating$catastrophic$declines$from$natural,$shortRterm$fluctuations$remains$challenging.$Not$all$amphibian$species$respond$to$environmental$pressures$or$disease$in$the$same$manner;$even$within$a$species,$responses$can$differ$between$disjunct$populations$(Blaustein$&$Kiesecker$2002).$In$order$to$evaluate$population$dynamics,$baseline$data$and$longRterm$monitoring$are$desirable$(Pechmann$et$al.$1991;$Pechmann$&$Wilbur$1994;$Travis$1994).$However,$prolonged$monitoring$often$is$not$an$option,$especially$in$crisis$scenarios$(Barinaga$1990).$Genetic$data$on$population$structure$are,$therefore,$of$increasing$importance$for$informing$conservation$planning$in$amphibians$(Emel$and$Storfer$2012).$$ Despite$global$declines$in$amphibians$as$a$whole,$the$eastern$newt,$Notophthalmus+viridescens,$is$one$species$that$appears$to$have$maintained$stable$numbers.$These$salamanders$require$breeding$ponds$in$close$proximity$to$woodlands$to$complete$their$lifeRcycle,$and$may$colonize$manRmade$farm$ponds$(Petranka$1998).$Throughout$their$distribution,$N.+viridescens$manifests$lifeRcycle$polyphenism$associated$with$pond$hydroperiod$(Takahashi$&$Parris$2008).$Historically,$the$species$has$been$divided$into$four$subspecies,$N.+v.+viridescens$(redRspotted$newt),$N.+v.+louisianensis$(central$newt),$N.+v.+dorsalis$(brokenRstripped$newt),$and$N.+v.+piaropicola$(peninsula$newt),$although$a$recent$allozymeRbased$study$has$raised$questions$about$the$validity$of$these$subdivisions$(Gabor$&$Nice$2004).$At$the$species$level,$N.+viridescens$is$listed$as$being$of$“least$concern”$by$the$IUCN$(Hammerson$2004).$However,$the$subspecies$N.+v.+louisianensis$(central$newt)$is$listed$as$“threatened”$in$Kansas$and$Iowa$(2011),$two$states$that$lie$on$the$western$periphery$of$the$species$range.$

Between$1982R1984,$field$surveys$for$newts$in$Iowa$were$conducted$at$several$sites$in$eight$counties$previously$known$to$support$newts$(Camper$1988).$At$that$time,$populations$were$confirmed$only$in$the$woodland$and$riparian$habitats$of$the$Cedar$and$Wapsipinicon$Rivers$in$Black$Hawk$and$Linn$Counties$(Camper$1988).$The$author$of$this$study$suggested$that$the$decrease$was$likely$the$result$of$habitat$loss$and$fragmentation$(Camper$1988).$Since$then,$no$peerRreviewed$publications$have$evaluated$the$status$of$central$newts$in$Iowa.$

Newt$populations$in$Iowa$provide$an$excellent$model$for$evaluating$the$conservation$status$of$a$common$species$at$the$periphery$of$its$range,$where$populations$may$be$especially$vulnerable$to$demographic$stochasticity$(Vucetich$&$Waite$2003).$Furthermore,$Iowa$newt$populations$transect$the$southern$edge$of$what$Remington$(1968)$identified$as$a$major$suture$zone$(“Suture$Zone$1”),$a$region$in$which$numerous$pairs$of$species$or$semispecies$come$together$and$hybridize$(Remington$1968).$$Swenson$and$Howard$(2004)$confirmed$that$this$geographic$region$bears$a$significant$association$with$hybridization$(Swenson$&$Howard$2004).$$Remington$(1968)$proposed$that,$for$many$species,$the$stage$was$set$for$hybridization$in$Suture$Zone$1$by$recent$anthropogenic$deforestation,$which$began$approximately$200$years$ago$(Remington$1968).$However,$for$newts$in$particular,$deforestation$is$more$likely$to$have$isolated$populations$rather$than$to$


have$driven$them$together.$Alternatively,$Hewitt$(2000)$proposed$that$Suture$Zone$1$resulted$from$patterns$of$postRPleistocene$recolonization$from$separate$glacial$refugia$common$to$multiple$species$(Hewitt$2000).$In$addition$to$historical$influences$on$Iowa$newt$populations,$present$day$differences$in$soil$type$and$habitat$type$in$this$portion$of$the$species$range$may$be$important.$Therefore,$genetic$analysis$of$Iowa$newt$populations$presents$an$opportunity$to$assess$population$structure$in$a$region$with$potentially$interesting$past$and$present$impacts$on$population$dynamics.$$$$Methods+$A$total$of$282$central$newts$(N.+v.+louisianensis)$were$sampled$from$14$sites$in$Iowa$over$the$course$of$this$study$(Fig.$1),$and$two$redRspotted$newts$(N.+v.+viridescens)$from$Tilhance$Creek,$Berkeley$County,$West$Virginia$were$also$used$for$comparison.$Iowa$fieldwork$was$conducted$under$Department$of$Natural$Resources$(IA$DNR)$permit$SC$553.$Field$and$laboratory$methods$were$approved$by$the$University$of$Northern$Iowa$Institutional$Animal$Care$and$Use$Committee.$In$the$field,$aquatic$newts$were$captured$by$dipRnet,$seine,$or$minnow$trap.$Terrestrial$newts$were$captured$by$hand.$$

Newts$in$Iowa$were$collected$during$their$active$period$from$April$until$late$October.$Sites$occupied$by$newts$were$classified$as$1)$riparian/floodplain$forest,$2)$isolated$wetland,$or$3)$wooded$upland.$This$classification$is$similar$to$that$used$by$Gates$and$Thompson$(1982)$for$their$study$of$newt$habitat$in$Maryland.$Sites$along$the$northern$Wapsipinicon$River$in$Chickasaw,$Bremer,$and$Black$Hawk$counties$as$well$as$Cutshall$Access$in$Buchanan$County$(localities$1R9;$Fig.$1)$are$best$described$as$riparian/floodplain$forests.$The$Natural$Resources$Conservation$Service$categorizes$soils$at$these$sites$as$loamy$glacial$till.$Most$of$these$locations$showed$evidence$of$recent$flooding.$Ham$Marsh$(locality$10;$Fig.$1)$is$a$10Racre$wetland$surrounded$by$cornfields,$cattle$pasture,$and$several$acres$of$prairie;$hence,$it$seems$isolated.$The$Natural$Resources$Conservation$Service$categorizes$soils$here$as$loamy$glacial$till.$Sites$in$Linn$and$Clinton$counties$(localities$11R14;$Fig.$1)$are$wooded$uplands.$None$of$these$sites$seem$likely$to$be$flooded$by$the$Cedar$or$Wapsipinicon$Rivers$due$to$the$difference$in$elevation.$However,$spring$flooding$may$occur$at$Behren’s$Ponds$or$at$the$newt$pools$in$Sherman$Park$due$to$runRoff$snow$melt.$Less$woody$debris$is$evident$at$these$sites$compared$to$the$riparian/floodplain$forests,$and$the$soil$type$at$these$localities$is$loess$or$shallow$loess$rather$than$loamy$glacial$till$as$seen$at$more$northern$localities.$

Tissue$was$collected$by$clipping$one$or$two$toes$from$each$newt.$Scissor$blades$were$disinfected$with$isopropanol$between$clippings.$Newt$feet$were$treated$with$antibiotic$prior$to$release$of$the$animal.$Clipped$toes$were$placed$in$absolute$ethanol$or$were$stored$on$ice$blocks$in$the$field$until$they$could$be$stored$at$R80˚$C.$

Whole$genomic$DNA$was$extracted$from$single$toe$clips$using$the$Qiagen$DNeasy$Blood$&$Tissue$Kit$(Quiagen,$Valencia,$California).$DNA$was$eluted$in$a$final$volume$of$200$µl.$$$


+Mitochondrial+DNA+Analysis+

$$ Two$regions$of$mitochondrial$DNA$(mtDNA)$were$amplified$and$sequenced.$Primers$SThr15300L$and$12S600H$(Zhang$et$al.$2008)$were$used$for$initial$amplification$and$sequencing$of$a$portion$of$mitochondrial$DNA$(mtDNA)$that$includes$the$control$region$(dRloop).$Based$on$these$sequences,$a$new$primer$was$created$(RVRml:$5'$GAGGGTGTGGCTAAACAAGR3')$for$use$with$SThr15300L$for$amplification$of$a$1233$base$pair$(bp)$region$of$mtDNA$(Table$1).$Sequencing$was$performed$with$the$SThr15300L$primer$on$all$individuals$to$cover$479$bp,$including$a$180$bp$region$of$noncoding$DNA,$a$72$bp$tRNARpro$gene,$and$a$227$bp$portion$of$the$control$region$for$all$newts.$Sequencing$of$an$additional$575$bp$of$the$mitochondrial$cytochromeRb$gene$was$accomplished$using$primers$Noto1$(5’RTGACCTACCAACACCATCAAATR3’)$and$Noto2$(5’RGTATTTGGTGTTAATAGGGAAATTR3’)$for$amplification$(Table$1;$Jonathan$Eastman,$pers.$comm.).$PCR$products$were$prepared$for$sequencing$using$ExosapRIT$(USB,$Cleveland,$OH),$and$sequencing$reactions$were$performed$at$the$Iowa$State$University$DNA$Facility$on$an$Applied$Biosystems$3730xl$DNA$Analyzer.$Chromatograms$were$verified$by$visual$screening;$unique$sequences$were$verified$by$doubleRstranded$sequencing$and$were$submitted$to$GenBank$(JX570765RJX570781).$

Concatenated$mtDNA$sequences$(1,054$bp)$for$277$Iowa$newts,$two$West$Virginia$newts,$one$Florida$newt$(GenBank$accession$EU880323;$Zhang$et$al.$2008),$and$one$outgroup$specimen$(N.+meridionalis,$GenBank$accession$EU880322;$Zhang$et$al.$2008)$were$aligned$in$Geneious$Pro,$version$5.4.6$(Drummond$et$al.$2011),$using$the$MUSCLE$plugin$(Edgar$2004).$Alignments$were$verified$by$eye.$The$Florida$specimen’s$GenBank$ID$lists$a$University$of$California,$Berkeley,$Museum$of$Vertebrate$Zoology$specimen$voucher$(MVZ$205720),$which$corresponds$with$a$collecting$locality$11$mi.$south$of$Gainesville,$which$is$in$the$range$of$N.+v.+piaropicola$(Krysko$et$al.$2011).$Uncorrected$sequence$divergence$values$(p)$also$were$calculated$in$Geneious.$Twelve$individuals$that$represented$every$distinct$mtDNA$haplotype,$were$subjected$to$maximumRlikelihood$analysis$using$the$PAUP*$4.0$b10$(Swofford$2003)$and$ModelTest$plugins$(Posada$&$Crandall$1998)$of$Geneious$to$choose$the$best$model$of$molecular$evolution$for$subsequent$analyses$(GTR$+$I$+$G;$Nei$&$Kumar$2000)$according$to$AIC.$MaximumRlikelihood$analysis,$maximumRlikelihood$bootstrap$numbers$(1,000$replicates),$and$estimated$divergence$dates$(with$error$bars)$were$calculated$using$MEGA$5.0.5$(Tamura$et$al.$2011).$Monophyly$of$Iowa$newts$was$tested$by$constructing$a$parsimony$tree$structure$in$which$the$constraint$of$a$monophyletic$N.+v.+viridescens$was$enforced,$then$comparing$the$constrained$tree$structure$to$the$preferred$likelihood$tree$structure$using$a$ShimodairaRHasegawa$test$in$PAUP*$4.0$10b$(Swofford$2003)$with$likelihood$parameters$enforced$(GTR$+$I$$+$G),$full$optimization,$a$oneRtailed$test,$and$1,000$bootstrap$replicates.$Mega$5.0.5$was$used$to$perform$a$molecular$clock$test$by$comparing$likelihood$values$for$tree$topologies$with$and$without$molecular$clock$constraints$and$to$calculate$divergence$times$based$on$estimated$rates$of$sequence$divergence.$Arlequin$(version$3.5.1.2;$Excoffier$&$Lischer$2010)$was$used$to$calculate$pairwise$FST$and$ΦST$values$and$to$perform$AMOVA$analysis$on$ΦST$using$uncorrected$sequence$divergence;$10,000$random$permutations$were$performed$for$all$analyses.$Type$I$error$rate$was$controlled$by$correcting$a+priori+significance$values$


downward$from$p$≤$0.05$as$described$by$Benjamini$and$Yekutieli$(2001)$to$p$≤$0.009$for$91$comparisons.$Narum$(2006)$showed$this$approach$yields$a$biologically$meaningful$correction$to$experimentRwise$Type$I$error$rate$for$conservation$genetics$purposes.$$

$Microsatellite+Analysis+

$$ Microsatellite$loci$were$amplified$by$dyeRlabeled$PCR$(Schuelke$2000)$using$primers$for$N.+v.+viridescens$loci$Nvi2,$Nvi7,$Nvi11,$Nvi14,$and$Nvi19$(Croshaw$&$Glenn$2003)$and$primers$for$roughRskinned$newt$(Taricha+granulosa)$locus$Tgr14$(Jones$et$al.$2001;$Table$1).$Each$of$these$primer$sets$targets$tetranucleotide$repeats$except$for$Nvi2,$which$targets$a$trinucleotide$repeat,$and$Nvi7,$which$targets$a$dinucleotide$repeat.$For$each$25$µl$reaction,$1$µl$of$template$DNA$was$added$with$GoTaq$Green$Master$Mix$(Promega,$Madison,$WI)$or$GoTaq$Hot$Start$Master$Mix$(Promega,$Madison,$WI).$Thermocycling$conditions$and$fluorescent$dyes$are$listed$in$Table$1.$Amplified$fragments$were$analyzed$at$the$Iowa$State$University$DNA$Facility$on$an$Applied$Biosystems$3730xl$DNA$Analyzer.$$$ Output$from$capillary$electrophoresis$of$microsatellite$PCR$products$was$scored$using$GeneMarker$(version$1.90;$SoftGenetics,$State$College,$PA).$All$scoring$and$analysis$of$stutter$was$verified$by$visual$inspection.$The$software$Convert$(version$1.31;$Glaubitz$2004)$was$used$to$reformat$genotypic$data$for$several$common$software$packages.$In$order$to$screen$for$genotyping$errors$resulting$from$stutter$products,$null$alleles,$and$large$allele$dropout,$each$locus$was$analyzed$with$MicroRChecker$(version$2.3.3;$van$Oosterhout$et$al.$2004).$Linkage$disequilibrium$was$assessed$using$FSTAT$(version$2.9.3;$Goudet$1995,$2001),$with$the$a+priori+significance$value$adjusted$downward$from$p$≤$0.05$using$the$Bejamini$and$Yekutieli$(2001)$approach$suggested$by$Narum$(2006)$to$p$≤$0.012$for$42$comparisons.$Both$MicroRChecker$and$linkage$disequilibrium$tests$were$conducted$for$each$locus$and$for$each$sampling$site.$$$ Allelic$richness$(AR)$was$estimated$by$the$rarefaction$method$implemented$in$FSTAT.$This$procedure$scales$each$sample$to$the$smallest$sample$size.$AR$was$estimated$for$all$populations$and$for$only$those$populations$with$a$sample$size$greater$than$or$equal$to$15.$$ Slatkin’s$linearized$(1995)$FST$was$calculated$for$all$population$pairs$using$Arlequin$(version$3.5.1.2;$Excoffier$&$Lischer$2010).$Departures$from$Hardy$Weinberg$Equilibrium$(HWE)$also$were$assessed$using$Arlequin.$The$Markov$chain$method$was$used$to$calculate$probability+values$for$deviation$from$HWE$using$106$steps$in$the$forecast$chain$and$105$dememorization$steps.$FIS$for$microsatellite$data$was$estimated$using$FSTAT.$Probability$values$were$calculated$based$on$900$randomizations$to$determine$the$proportion$of$randomizations$larger$and$smaller$than$the$calculated$FIS.$$As$was$done$for$multiple$tests$of$significance$in$mtDNA$data,$Type$I$error$rate$was$controlled$using$the$correction$of$a+priori+significance$values$suggested$by$Narum$(2006)$downward$from$p$≤$0.05$$to$p$≤$0.009$for$91$comparisons.$

The$software$Structure$(version$2.3;$Pritchard$et$al.$2000;$Falush$et$al.$2003)$was$used$to$cluster$individuals$into$putative$populations.$Population$admixture$and$correlated$allele$frequencies$were$assumed.$Sampling$sites$(POPDATA)$were$added$as$priors$for$the$LOCPRIORS$model.$This$model$enhances$clustering$when$genotypic$signal$is$weak$(Hubisz$


et$al.$2009).$BurnRin$period$was$set$to$106$steps$followed$by$105$repetitions.$Five$runs$were$evaluated$for$each$number$of$clusters$(K),$with$K$ranging$1R15.$The$appropriate$value$of$K$was$selected$by$considering$the$probability$of$each$run,$the$variance$of$the$probability$throughout$each$run,$and$the$change$in$K$(∆K)$between$each$set$of$five$runs$(Evanno$et$al.$2005).$CLUMPP$(version$1.1.2;$Jakobsson$&$Rosenberg$2007)$was$then$used$to$permute$clusters$across$the$five$runs$of$the$selected$K.$These$permuted$data$were$visualized$using$Distruct$(version$1.1;$Rosenberg$2004).$$$ Population$differentiation$was$assessed$following$the$approach$of$Kalinowski$(2010)$by$using$Slatkin’s$(1995)$linearized$FST$as$a$measure$of$differentiation.$For$this$analysis,$data$from$the$Behren’s$Pond$and$Ham$Marsh$populations$(n$≤$4),$were$omitted,$and$FST$was$calculated$using$a$stepwise$mutation$model$in$Arlequin$(version$3.5.1.2;$Excoffier$&$Lischer$2010).$These$FSTRbased$distances$were$then$clustered$using$neighbor$joining$in$MEGA$5$(Tamura$et$al.$2011).$AMOVA$analysis$was$performed$for$Iowa$newts$in$Arlequin$using$numbers$of$different$alleles$(FSTRlike)$and$1,000$permutations$of$the$data$for$significance$testing;$again,$the$two$small$populations$(n$≤$4)$were$omitted$from$this$analysis.$$$ Isolation$by$distance$was$assessed$using$Isolation$by$Distance,$Web$Service$(IBDWS,$version$3.15;$Jensen$et$al.$2005).$A$geographic$distance$matrix$was$calculated$using$GenAlEx$(version$6.3;$Peakall$&$Smouse$2006).$$++Results++Mitochondrial+data++$ Nine$unique$haplotypes$were$observed$for$newts$from$Iowa$(N.+v.+louisianensis;$Fig.$2;$Table$2).$Little$sequence$divergence$was$observed$among$the$10$northernmost$populations$(localities$1R10;$uncorrected$sequence$divergence$p$≤ 0.2%)$or$between$the$two$southernmost$populations$(localities$11R14;$p$≤$0.7%),$yet$divergence$between$northern$and$southern$haplotypes$was$relatively$high$(p$=$3.4R3.7%).$Only$the$Troy$Pond$population$(locality$12)$showed$both$northern$and$southern$haplotypes,$with$northern$haplotypes$being$more$common$(n$=$22$vs.$n$=$1).$The$Behren’s$Pond$population$(locality$11)$lies$farther$to$the$north$than$does$the$Troy$Pond$population$(Fig.$1),$yet$newts$at$Behren’s$Pond$showed$a$haplotype$characteristic$of$newts$at$more$southern$localities.$$$ Iowa$N.+v.+louisianensis$do$not$appear$to$be$monophyletic.$Southern$Iowa$haplotypes$formed$a$clade$with$N.+v.+piaropicola$from$Florida$with$strong$(99%)$bootstrap$support$and$a$low$level$of$sequence$divergence$(p$=$1.3R1.4%;$Fig.$2).$Additionally,$constraining$N.+v.+louisianensis$to$be$monophyletic$resulted$in$a$maximumRlikelihood$tree$with$a$significantly$poorer$likelihood$score$than$that$of$a$tree$in$which$paraphyly$was$allowed$(ShimodairaRHasegawa$test,$p$=$0.041).$Although$bootstrap$support$was$weaker$when$the$two$portions$of$mtDNA$were$analyzed$separately,$“DRloop”$and$cytochromeRb$data$each$provided$maximumRlikelihood$topologies$in$which$southern$Iowa$N.+v.+louisianensis$formed$a$clade$with$N.+v.+piaropicola$from$Florida$rather$than$with$other$N.+v.+louisianensis+(85%$and$93%$of$1,000$bootstrap$replicates,$respectively).$Uncorrected$mtDNA$sequence$divergence$between$northern$and$southern$Iowa$newts$(p$=$3.4R3.7%)$was$similar$to$the$level$of$divergence$observed$in$comparisons$of$Iowa$newts$(N.+v.+louisianensis)$to$other$subspecies$


of$newts:$N.+v.+viridescens$from$West$Virginia$differed$from$Iowa$N.+v.+louisianensis+by$3.1R3.8%$and$N.+v.+piaropicola+from$Florida$differed$from$northern$Iowa$populations$of$N.+v.+louisianensis$by$3.6R3.7%.$$$ We$used$two$approaches$to$molecularRclock$based$estimates$of$divergence$time$to$provide$an$approximate$framework$for$major$divergence$events$within$N.+viridescens.$Both$approaches$yielded$similar$divergence$time$estimates.$To$calibrate$a$clock,$we$first$determined$that$rate$of$cytochromeRb$evolution$did$not$differ$significantly$among$newts$included$in$this$study$(p$=$0.42),$so$a$strict$molecular$clock$could$be$applied$to$the$tree.$For$our$first$approach,$we$used$a$substitution$rate$for$cytochrome$b$that$was$estimated$for$European$newts$(Caccone$et$al.$1997):$0.77%$per$million$years$(my)$per$lineage.$The$majority$of$our$data$came$from$cytochromeRb$sequences$(575$bp),$but$we$also$included$480$bp$of$other$mitochondrial$sequences,$primarily$control$region.$$Comparisons$of$pairRwise$uncorrected$sequence$divergence$values$for$the$partitioned$data$indicate$a$slightly$higher$substitution$rate$for$the$combined$portions$of$the$dataset$that$do$not$include$cytochrome$b,$yielding$an$adjusted$rate$of$evolution$of$0.85%$per$lineage$per$my$for$these$portions$of$the$data.$Therefore,$the$adjusted,$overall$average$rate$of$evolution$for$our$complete$data$set$was$estimated$as$0.81%$per$lineage$per$million$years.$$This$rate$estimate$applied$to$the$maximum$likelihood$tree$placed$the$divergence$of$lineages$of$newts$that$currently$reside$in$northern$and$southern$Iowa$at$approximately$2.81$±$0.54$million$years$ago$(mya;$Fig.$2)$and$the$divergence$of$southern$Iowa$newt$populations$from$Florida$newts$(N.+v.+piaropicola)$at$0.84$±$0.35$mya$(Fig.$2).$Haplotypes$currently$in$southern$Iowa$likely$began$their$divergence$about$0.42$±$0.24$mya;$haplotypes$in$northern$Iowa$likely$began$their$divergence$more$recently,$but$the$error$surrounding$this$particular$estimate$was$exceptionally$large$(0.13$±$1.18$mya;$Fig.$2).$For$an$alternative$dating$approach,$we$used$an$estimated$divergence$date$for$N.+viridescens$and$N.+meridionalis$of$11.4$mya$(Zhang$et$al.$2008),$and$this$yielded$very$similar$date$estimates$with$the$northern$Iowa$and$southern$Iowa$haplotype$divergence$dated$at$3.2$±$0.75$mya.$$ Many$pairwise$comparisons$among$populations$yielded$FST$values$that$were$significantly$different$from$zero$(Table$3);$ΦST$values$indicated$similar$or$higher$levels$of$population$subdivision$with$the$same$pattern$of$statistically$significant$comparisons.$Population$subdivision$was$significant$even$between$some$neighboring$populations$such$as$Upper$Wapsi$(locality$1)$compared$to$Heffernan$WMA$(locality$2)$and$Syracuse$Wildlife$Area$(locality$13)$compared$to$Sherman$Park$(locality$14).$$ Standard$AMOVA$based$on$ΦST$values$indicated$that$within$population$genetic$variance$accounted$for$only$4%$of$total$variation,$while$amongRpopulation$variance$accounted$for$21%,$and$groupings$of$northern$vs.$southern$Iowa$populations$(populations$1R10$vs.$populations$11R14)$accounted$for$the$remaining$75%$of$genetic$variation$(p$<$0.01$for$all$comparisons).$$Microsatellite+data++$ Of$the$six$loci$initially$tested,$only$Nvi2,$Nvi7,$and+Nvi11$showed$reliable$scoring$patterns.$These$three$loci$were$used$for$all$downstream$analyses.$MicroRChecker$analysis$revealed$no$evidence$of$null$alleles$or$allelic$dropout$for$loci$Nvi2,$Nvi7,$and$Nvi11.$No$significant$linkage$disequilibrium$was$detected$between$pairs$of$loci$at$any$locality.$$


$ Nvi11$showed$the$most$polymorphism,$followed$by$Nvi7$then$Nvi2.$Unique$alleles$for$Nvi11$were$present$at$Cutshall$Access$and$Syracuse$Wildlife$Area$(Supplementary$Material).$Sweet$Marsh$and$Smoketown$Access$(N)$shared$an$allele$for$locus$Nvi7$that$was$not$found$in$the$other$populations.$Two$unique$alleles$were$present$in$the$Tilhance$Creek,$WV$population$(Nvi11,$Nvi7).$All$other$alleles$were$present$in$at$least$four$populations.$Mean$genetic$diversity$values$ranged$0.44R0.52$in$northern$populations$(localities$1R10)$and$0.45R0.67$in$southern$populations$(localities$11R14).$Mean$allelic$richness$(sample$size$scaled$to$15$individuals)$ranged$4.1R5.3$in$northern$populations$(localities$1R10)$and$4.0R4.7$in$southern$populations$(localities$11R14).$$Hence,$there$is$not$a$clear$pattern$of$higher$microsatellite$diversity$in$one$set$of$populations$or$the$other.$$ Many$pairwise$FST$values$were$significantly$greater$than$0$following$correction$for$multiple$tests$(Table$3).$The$majority$of$significant$FST$values$were$between$the$northern$and$southern$populations,$but$significant$FST$values$also$were$observed$between$some$neighboring$populations$such$as$Upper$Wapsi$(locality$1),$which$is$7.5$km$from$Heffernan$WMA$(locality$2),$and$Syracuse$Wildlife$Area$(locality$13),$which$is$6.2$km$from$Sherman$Park$(locality$14).$$$ HardyRWeinberg$equilibrium$tests$indicated$lower$than$expected$numbers$of$heterozygotes$for$Nvi11$at$Sweet$Marsh$(p$=$0.01),$Nvi7$at$Bruggeman$County$Park$(p$=$0.03),$and$Nvi2$at$C50$Public$Hunting$Area$(p$=$0.03)$and$Sherman$Park$(p$=$0.04).$Only$Troy$Pond$showed$greater$observed$heterozygosity$than$expected$for$Nvi7$(p$=$0.04).$With$correction$for$multiple$tests$of$significance$(Narum$2006),$the$Nvi11$locus$at$Sweet$Marsh$was$the$only$marker$indicative$of$departure$from$HardyRWeinberg$expectations.$Per$population$FIS$values$for$all$three$loci$were$near$zero$for$all$adequately$sampled$populations$(R0.15$to$0.14).$None$of$these$values$were$significant$at$the$5%$nominal$level.$$$ In$Structure$analysis$of$microsatellite$data,$both$log$transformed$probability$and$∆K$values$indicated$two$major$clusters$(K$=$2;$Fig.3).$Because$unequal$sample$sizes$can$affect$the$outcome$of$Structure$analyses$(Kalinowski$2010),$small$populations$(n$≤$4)$were$eliminated$from$the$analysis$for$comparison,$but$clustering$was$not$affected.$One$cluster$included$the$northern$ten$populations$along$tributaries$of$the$Wapsipinicon$River.$The$other$cluster$included$southern$populations,$although$the$genetic$affinity$of$newts$from$the$Syracuse$WMA$population$was$equivocal,$with$individuals$showing$nuclear$alleles$that$may$be$primarily$northern$in$origin.$NeighborRjoining$analysis$of$microsatelliteRbased$FST$values$(Fig.$4)$also$indicated$that$northern$populations$were$undifferentiated$from$one$another,$yet$were$genetically$distinct$from$the$southern$populations$(Troy$Pond,$Sherman$Park,$and$Syracuse).$Genetic$variance$between$northern$and$southern$populations$accounted$for$a$statistically$significant$4.4%$of$the$overall$genetic$variation$(AMOVA$analysis,$populations$1R9$vs.$populations$12R14,$p$=$0.004).$$$$ Isolation$by$distance$was$assessed$for$all$populations,$the$twelve$largest$populations$(n$≥$15),$and$the$nine$northern$populations$(n$≥$15).$Mantel$tests$and$reduced$major$axis$(RMA)$regression$analyses$were$conducted$for$raw$and$log$transformed$FST$and$geographic$distance.$Significant$trends$(p$<$0.05)$were$observed$for$all$combinations.$Excluding$the$WV$individuals$and$the$two$IA$populations$with$fewer$than$five$individuals$increased$data$fit.$The$best$fit$(r$=$0.6185,$p$<$1eR4)$with$the$most$even$distribution$of$populations$was$observed$for$the$twelve$populations$with$n$≥$15$(log[FST]$vs.$log[km];$Fig.$5A).$For$the$nine$northern$populations$with$n$≥$15,$the$best$fit$(r$=$0.4046,$p$<$0.05)$was$


found$using$the$same$log$transformations$(Fig.$5B).$Southern$populations$were$not$separately$assessed$due$to$a$limited$number$of$comparisons$between$populations.$$$$Discussion+$$$ Our$study$of$N.+v.+louisianensis$in$Iowa$highlights$the$relevance$of$biogeographic$history$to$conservation$genetics$and$provides$an$example$of$the$possibility$for$genetic$data$to$reveal$cryptic$diversity$within$a$species.$Adequate$knowledge$of$genetic$diversity$and$of$population$structure$is$a$requirement$for$making$wellRinformed$management$recommendations$for$any$species.$$ In$view$of$past$reports$of$central$newt$population$declines$in$Iowa,$we$were$surprised$to$find$these$amphibians$in$significant$numbers$at$most$sites$(15$or$more$individuals).$We$discovered$several$unknown$or$previously$unpublished$newt$populations$(e.g.,$Upper$Wapsi$WMA,$Heffernan$WMA,$Sweet$Marsh,$Little$Buck$WMA,$and$the$Jurgenson$Access$of$the$Wapsi$River$Greenbelt).$Genetic$diversity$estimates$were$roughly$equivalent$among$sites$and$inbreeding$coeffients$were$near$zero$for$all$sites;$however,$these$tentative$data$are$based$on$a$relatively$small$number$of$nuclear$loci.$Our$surveys$indicate$that$the$Behren’s$Pond$population$of$newts,$which$exists$on$a$Nature$Conservancy$site,$may$be$particularly$vulnerable$to$extirpation.$$Our$sample$size$was$lowest$there$despite$extensive$sampling$during$a$period$in$which$newts$should$have$been$active.$LongRterm$sampling$at$Behren’s$Pond$for$the$purpose$of$monitoring$blueRspotted$salamander$(Ambystoma+laterale)$populations$has$yielded$large$numbers$of$central$newts$as$inadvertent$captures$in$the$past,$but$not$so$in$recent$years,$coinciding$with$deforestation,$plowing,$and$agriculture$in$the$immediate$vicinity$of$the$ponds$(H.$Hadow,$pers.$comm.).$The$longRterm$viability$of$this$newt$population,$therefore,$is$particularly$uncertain.$Moreover,$the$apparently$rapid$decline$in$population$numbers$at$Behren’s$Pond$may$signal$the$vulnerability$of$newt$populations$throughout$the$state$to$anthropogenic$disturbance.$

Management$strategies$for$N.+v.+louisianensis$in$Iowa,$where$the$species$is$listed$as$threatened,$must$reflect$the$fact$that$newt$populations$in$Iowa$consist$of$two$groups$with$unique$histories.$Our$mitochondrial$DNA$analyses$revealed$the$existence$of$two$reciprocally$monophyletic$haplotype$groups$in$Iowa,$with$southern$newts$showing$a$more$recent$common$ancestry$with$N.+v.+piaropicola$from$Florida$than$with$northern$Iowa$newts.$Both$of$our$approaches$at$dating$divergence$between$northern$and$southern$Iowa$N.+v.+louisianensis$indicated$an$early$Pleistocene$divergence,$approximately$2.8R3.2$mya$(Fig.$2).$Although$he$did$not$include$samples$of$the$Iowa$populations$studied$herein,$Reilly$(1990)$placed$the$earliest$divergence$among$extant$members$of$Notophthalmus$viridescens$at$2.8$mya$based$on$allozyme$genetic$distances.$$Therefore,$based$on$our$sampling,$the$two$forms$of$N.+viridescens$present$in$Iowa$likely$diverged$in$allopatry$early$in$the$history$of$the$species,$then$recolonized$Iowa$some$time$following$glacial$retreat,$as$did$many$other$species$and$semispecies$that$meet$at$Suture$Zone$1$(Hewitt$2000).$$Our$data$on$relationships$of$currently$recognized$subspecies$of$N.+viridescens$points$to$a$need$for$wider$geographic$sampling$of$the$species$and$study$of$what$appears$to$be$a$complicated$phylogeographic$history$and$its$relationship$to$life$history.$$ Our$analyses$of$population$structure$based$on$nuclear$data$lend$support$to$a$management$strategy$that$protects$both$northern$Iowa$newt$populations$and$southern$


populations,$which$are$genetically$distinct.$Where$these$populations$meet,$our$mitochondrial$and$nuclear$data$indicate$sympatry$and$possible$genetic$introgression$of$the$northern$and$southern$lineages$of$newts.$The$Troy$Pond$population$(locality$12;$Fig.$1)$showed$primarily$the$northern$mitochondrial$form$(Fig.$2)$with$nuclear$genotypes$that$appeared$more$southern$in$origin$(Fig.$3),$possibly$indicating$introgression.$Slightly$farther$north$at$Behren’s$Pond$(locality$11;$Fig.$1),$which$lies$in$the$same$drainage$system,$we$recovered$only$southern$mtDNA$haplotypes$(Fig.$2)$and$southern$nuclear$genotypes$(Fig.$3),$but$sample$size$was$exceptionally$low$(n$=$4)$despite$the$fact$that$sampling$occurred$in$late$summer$when$newts$are$typically$captured$at$this$location$(H.$Hadow,$pers.$comm.).$Northern$mtDNA$haplotypes$and$nuclear$alleles$may$have$been$present$at$Behren’s$Pond$when$population$numbers$were$higher,$and$it$remains$to$be$seen$whether$southern$alleles$will$remain$prominent$at$that$site$in$the$future.$Finally,$Syracuse$newts$(locality$13;$Fig.$1)$showed$only$southern$haplotypes$(Fig.$2),$but$showed$a$pattern$of$nuclear$genotypes$that$did$not$fit$neatly$in$one$group$or$the$other$and$may$have$more$affinity$to$northern$populations$(Fig.$3).$Study$of$potential$hybridization$between$genetically$divergent$lineages$of$newts$at$these$intermediate$sites$would$be$interesting$and$may$become$more$feasible$when$larger$portions$of$the$genome$can$be$analyzed.$$

LongRterm$monitoring$of$these$populations$could$allow$assessment$of$whether$the$zone$of$contact$between$northern$and$southern$lineages$of$central$newts$is$stationary$or$moving,$with$one$lineage$geographically$displacing$the$other.$The$current$zone$of$contact$between$the$two$forms$of$N.+v.+louisianensis$in$Iowa$corresponds$to$shifts$in$habitat$and$soil$type,$with$northern$populations$(localities$1R10,$Fig.$1)$occurring$in$riparian/floodplain$forest$or$isolated$wetland$on$loamy$glacial$till,$while$southern$populations$(localities$11R14,$Fig.$1)$occur$in$wooded$uplands$with$loess$or$shallow$loess$soils.$Therefore,$habitat$discontinuity$may$act$to$hold$the$northern$and$southern$lineages$apart$to$some$degree,$yielding$the$observed$narrow$zone$of$parapatry.$Field$observations$and$experimental$studies$should$be$conducted$to$investigate$life$cycle$patterns$for$newts$in$the$state.$Given$the$differences$we$observed$in$habitat$used$by$northern$and$southern$genetic$groups,$newts$in$Iowa$may$display$variation$in$their$response$to$environmental$cues,$such$as$pond$hydroperiod.$

Identification, evaluation, and preservation of breeding sites will be crucial for management of the species. Our$data$point$to$a$significant$amount$of$population$isolation$among$northern$populations$and$among$southern$ones,$even$between$neighboring$populations$6R7$km$apart$on$the$same$drainage,$as$exhibited$by$high$FST$in$nuclear$and$mtDNA$data,$significant$AMOVA$comparisons$among$populations$for$both$nuclear$and$mtDNA$data,$and$strong$patterns$of$isolation$by$distance$in$nuclear$data.$$Therefore,$our$genetic$data$indicate$that$N.+v.+viridescens$populations$are$somewhat$isolated$from$one$another,$even$at$neighboring$localities.$$Genetic$patterns$such$as$these$likely$arise$from$either$reduced$dispersal$abilities$over$inhospitable$terrain$or$from$breeding$site$fidelity$(Savage$et$al.$2010),$which$is$strong$in$N.+viridescens$(Gill$1978).$One$of$the$next$steps$for$conservation$purposes$should$be$to$assess$the$ecological$factors$associated$with$site$occupancy$(Gates$&$Thompson$1982;$Rinehart$et$al.$2009)$so$that$any$cryptic$populations$of$newts$can$be$recognized;$however,$it$already$is$apparent$from$our$genetic$data$and$the$philopatric$nature$of$the$species$(Gill$1978)$that$maintenance$and$protection$of$many$neighboring$breeding$sites$will$be$important$to$the$viability$of$this$species.$

$


Acknowledgements+

We$thank$Jeffrey$W.$Tamplin$for$providing$newt$samples$from$West$Virginia$and$Harlo$Hadow$for$providing$newt$samples$from$Behrens$Pond.$Neil$Bernstein$offered$advice,$equipment,$and$samples$for$the$project.$$Terry$Van$De$Walle$and$Jeff$LeClere$provided$records$of$known$newt$populations.$James$W.$Demastes$advised$specimen$collection$and$provided$critical$review$of$this$manuscript.$Jonathan$Eastman$made$many$helpful$suggestions$for$data$analysis$and$presentation.$We$thank$Laura$Whitmore,$Andrew$Heise,$and$Esther$Heise$for$help$in$the$field.$Lauren$Dougall,$Sheree$Harper,$Shantra$Neuhring,$and$Kayla$Peiffer$assisted$in$the$lab.$We$thank$Matt$Fisher$(Nature$Conservancy)$and$other$landowners$for$access$to$their$respective$properties.$We$thank$Andrew$Storfer,$Jason$Baumsteiger,$Mizuki$Takahashi,$and$Jonathan$Eastman$for$Noto1$and$Noto2$primer$sequences.$Jack$Kosmicki$drew$Fig.$1.$Comments$from$two$anonymous$reviewers$and$the$editor$of$this$article$improved$the$quality$of$this$manuscript.$This$work$was$funded$by$a$Wildlife$Diversity$Small$Grant$from$the$Iowa$Department$of$Natural$Resources$and$by$grants$from$the$College$of$Natural$Sciences$and$the$Intercollegiate$Academic$Fund$of$University$of$Northern$Iowa.$


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Figure+1.$Map$of$collecting$localities$in$Iowa.$(1)$Upper$Wapsi$Wildlife$Management$Area$(WMA),$(2)$Heffernan$WMA$and$Powder$Horn$Farms,$(3)$Sweet$Marsh,$(4)$Smoketown$Access$(N$site),$(5)$Smoketown$Access$(S$sites),$(6)$Little$Buck$WMA,$(7)$C50$Public$Hunting$Area,$(8)$Bruggeman$County$Park,$(9)$Cutshall$Access,$(10)$Ham$Marsh,$(11)$Behren’s$Ponds$and$Woodland,$(12)$Troy$Pond,$(13)$Syracuse$Wildlife$Area,$and$(14)$Sherman$Park.$

$Figure+2.+Maximum$likelihood$tree$(GTR+I+G$model)$based$on$mtDNA$sequence$data$(575$

bp$of$cytochrome$b$plus$479$bp$of$dRloop).$$Newts$from$Iowa$(Notophthalmus+viridescens,$n=277)$exhibited$9$haplotypes$(H1RH9;$Table$2),$which$are$shown$here$with$haplotypes$from$N.+v.+viridescens$(n=2),$and$N.+v.+piaropicola$(Zhang$et$al.$2008;$GenBank$EU880323).$Population$(pop)$numbers$correspond$with$Fig.$1.$Population$12$is$the$only$population$with$both$“northern”$and$“southern”$alleles.$The$tree$is$rooted$with$N.+meridionalis$(Zhang$et$al.$2008;$GenBank$EU880322).$$Bootstrap$values$(1,000$replicates)$are$shown$at$nodes.$The$scale$bar$shows$corrected$sequence$divergence$values$(number$of$substitutions$per$site)$on$bottom$and$estimated$divergence$dates$(millions$of$years)$on$top$(bars$at$nodes$indicate$95%$confidence$intervals).$

$Figure+3.+Population$clustering$results$from$Structure$analysis.$Sampling$sites$and$mtDNA$

haplotype$groups$are$listed$along$the$bottom$and$top$of$each$graph,$respectively.$Vertical$bars$indicate$individual$genotypes,$and$proportion$of$shading$indicates$cluster$membership$coefficient$when$k$=$2.$

$Figure+4.$NeighborRjoining$tree$based$on$nuclear$microsatellite$data.$“Northern”$

populations$show$low$levels$of$population$subdivision,$but$are$quite$differentiated$from$the$three$southernmost$populations$based$on$Slatkin’s$(1995)$linearized$FST$(stepwise$mutation$model).$Iowa$populations$10$and$11$and$the$West$Virginia$specimens$were$eliminated$from$this$analysis$due$to$small$sample$sizes$(n$≤$4).$

$Figure+5.+Isolation$by$distance$plots.$Distance$along$the$xRaxis$is$in$log$(km).$Isolation$by$

distance$was$significant$for$(A)$the$twelve$largest$populations$(y$=$1.268x$–$3.482,$r$=$0.6185,$p$<$0.0000)$and$(B)$nine$northern$populations$(y$=$1.945x$–$4.133,$r$=$0.4046,$p$<$0.05).$


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A B


Table&1.&PCR$thermocycling$conditions.$Locus DNA polymerase Fluorescent Denaturation Annealing Annealing Number of Extension Name (Promegaa) label temperature (°C) time temperature(s) cycles at each temperature and time (s) (°C) temperature (°C) (°C) and time (s) Nvi2 GoTaq Hex 94, 45 45 47, 48 10, 30 65, 45 Nvi7 GoTaq Hot Start Hex 94, 45 45 47, 48 10, 30 65, 45 Nvi11 GoTaq Hot Start 6-FAM 94, 45 45 43, 48 10, 30 72, 45 Nvi14 GoTaq Hot Start Hex 94, 45 45 41, 48 10, 30 65, 45 Nvi19 GoTaq 6-FAM 94, 45 45 50, 48 10, 30 72, 45 Tgr14 GoTaq 6-FAM 94, 45 45 50 38 72, 45 d-loop GoTaq 95, 45 45 65 36 72, 45 cyt-b GoTaq Hot Start 94, 30 30 50 30 72, 60 a$Madison,$WI$$


$Table&2.&Mitochondrial$haplotype$frequencies$by$population.$Populations$are$listed$in$geographic$order$(north$to$south).$Haplotype$(H)$numbers$correspond$to$Fig.$2;$haplotypes$H1PH4$are$“northern$Iowa”$and$haplotypes$H5PH9$are$more$closely$related$to$a$Florida$newt$haplotype.$$Locality na H1 H2 H3 H4 H5 H6 H7 H8 H9 1 Upper Wapsi Wildlife Management Area (WMA)23 0.65 0.35 2 Heffernan WMA and Powder Horn Farms 26 1.00 3 Sweet Marsh 19 0.37 0.63 4 Wapsi River Greenbelt at C33 35 1.00 5 Smoketown Access 21 1.00 6 Little Buck WMA 15 0.07 0.93 7 C50 Public Hunting Area 17 0.94 0.06 8 Bruggeman County Park 18 0.89 0.11 9 Cutshall Access 25 1.00 10 Ham Marsh 1 1.00 11 Behren’s Ponds and Woodland 4 1.00 12 Troy Pond 23 0.96 0.04 13 Syracuse Wildlife Area 25 0.28 0.16 0.56 14 Sherman Park 25 0.04 0.88 0.08$aSample$size$


Table&3.&FST$based$on$uncorrected$distances$in$mtDNA$sequence$data$(below$diagonal)$and$numbers$of$different$alleles$in$microsatellite$data$(above$diagonal).$$Numbers$significantly$different$from$zero$after$correction$for$multiple$tests$(p$≤$0.0094;$Narum$2006)$are$shown$in$bold$font;$underlined$values$emphasize$population$pairs$with$significant$FST$for$both$mtDNA$and$microsatellites.$Grey$boxes$highlight$comparisons$between$northern$and$southern$populations.$$$$ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 Upper Wapsi WMA ———— 0.01 0.06 0.00 0.01 -0.01 0.06 0.10 0.06 0.04 0.16 0.10 0.03 0.09 2 Heffernan WMA 0.34 ———— 0.06 0.01 0.02 0.03 0.04 0.08 0.04 -0.06 0.12 0.07 0.07 0.05 3 Sweet Marsh 0.11 0.65 ———— 0.03 0.01 0.03 0.01 0.03 0.03 -0.01 0.13 0.08 0.15 0.16 4 Smoketown Access N 0.38 0.00 0.70 ———— -0.01 0.00 0.03 0.06 0.03 -0.05 0.15 0.09 0.06 0.10 5 Smoketown Access S 0.31 0.00 0.62 0.00 ———— 0.00 0.01 0.04 0.02 -0.06 0.14 0.08 0.08 0.11 6 Little Buck 0.20 0.03 0.52 0.05 0.02 ———— 0.06 0.10 0.06 0.01 0.16 0.08 0.05 0.11 7 C50 Public Hunting 0.21 0.03 0.52 0.05 0.01 -0.06 ———— 0.01 0.00 -0.02 0.11 0.08 0.17 0.14 8 Bruggeman County Park 0.18 0.09 0.48 0.12 0.07 -0.00 -0.00 ———— 0.00 -0.02 0.15 0.10 0.17 0.15 9 Cutshall Access 0.33 0.00 0.65 0.00 0.00 0.03 0.02 0.09 ———— 0.02 0.16 0.09 0.13 0.13 10 Ham Marsh 0.27 1.00 -0.33 1.00 1.00 0.88 0.88 0.79 1.00 ———— -0.02 -0.14 0.14 -0.15 11 Behren’s Pond 0.63 1.00 0.63 1.00 1.00 0.90 0.90 0.83 1.00 1.00 ———— -0.01 0.24 0.06 12 Troy Pond 0.25 0.00 0.57 0.02 -0.00 -0.02 -0.02 0.02 0.00 0.91 0.93 ———— 0.15 0.05 13 Syracuse Wildlife Area 0.46 0.70 0.45 0.74 0.68 0.60 0.61 0.57 0.70 0.39 0.54 0.64 ———— 0.10 14 Sherman Park 0.65 0.89 0.65 0.90 0.88 0.81 0.82 0.78 0.89 0.77 0.81 0.83 0.41 ————

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$Supplement&1.&Microsatellite$allele$frequencies$per$locus$per$sampling$locality.$$$

Allele frequencies per locus

Nvi11 Nvi7 Nvi2

size (bp) size (bp) size (bp)

N 174 182 186 190 194 198 202 206 210 214 218 222 226 230 234 238 242 n 133 135 137 139 141 n 177 183

Upper Wapsi WMA

23 0.130 0.261 0.065 0.065 0.109 0.109 0.043 0.109 0.043 0.022 0.043 23 0.370 0.630 24 0.021 0.979

Heffernan WMA 26 0.077 0.077 0.231 0.115 0.058 0.038 0.288 0.077 0.038 25 0.440 0.560 24 0.104 0.896

Sweet Marsh 19 0.053 0.289 0.237 0.079 0.026 0.026 0.079 0.158 0.053 19 0.632 0.342 0.026 19 1.000

Smoketown Access (N) 35 0.029 0.157 0.200 0.086 0.029 0.100 0.057 0.043 0.129 0.114 0.043 0.014 35 0.429 0.529 0.043 35 0.057 0.943

Smoketown Access (S) 21 0.024 0.048 0.167 0.214 0.095 0.024 0.048 0.071 0.048 0.048 0.071 0.071 0.071 21 0.500 0.500 21 0.048 0.952

Little Buck (E)

15 0.033 0.300 0.100 0.033 0.067 0.033 0.100 0.167 0.067 0.067 0.033 15 0.400 0.600 15 1.000

C50 Public Hunting Area

17 0.059 0.029 0.235 0.147 0.088 0.029 0.118 0.088 0.118 0.029 0.059 17 0.706 0.294 17 0.059 0.941

Bruggeman County Park 17 0.029 0.118 0.265 0.059 0.265 0.088 0.088 0.029 0.029 0.029 18 0.028 0.722 0.250 18 0.111 0.889


Cutshall Access 24 0.021 0.083 0.021 0.063 0.104 0.146 0.146 0.146 0.042 0.021 0.042 0.104 0.063 25 0.660 0.340 25 0.080 0.920

Ham Marsh 1 0.500 0.500 1 0.500 0.500 1 0.500 0.500

Behren's Ponds and Woodland

3 0.333 0.667 3 0.333 0.500 0.167 3 0.333 0.667

Troy Pond 25 0.080 0.180 0.320 0.040 0.020 0.280 0.040 0.040 25 0.140 0.500 0.360 25 0.320 0.680

Syracuse Wildlife Area

25 0.200 0.080 0.040 0.040 0.260 0.120 0.120 0.020 0.040 0.020 0.060 24 0.083 0.188 0.729 25 0.020 0.980

Sherman Park 25 0.120 0.260 0.020 0.100 0.140 0.240 0.080 0.040 25 0.160 0.260 0.580 25 0.420 0.580

Tilhance Creek, WV 2 0.250 0.250 0.500 2 0.750 0.250 2 0.500 0.500

All (weighted) 0.002 0.002 0.029 0.085 0.210 0.121 0.074 0.090 0.113 0.049 0.065 0.058 0.049 0.027 0.011 0.013 0.005 0.045 0.468 0.002 0.478 0.007 0.116 0.884

All (unweighted) 0.017 0.001 0.038 0.086 0.276 0.134 0.059 0.075 0.088 0.041 0.054 0.045 0.039 0.021 0.010 0.011 0.004 0.100 0.454 0.017 0.425 0.005 0.172 0.828

$$$

conservation+genetics+of+the+central+newt+(notophthalmus...

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