The Challenges of Detecting Subtle Population Structure and Its Importance for the Conservation of Emperor Penguins', Molecular Ecology, Vol

Citation for published version: Younger, JL, Clucas, GV, Kao, D, Rogers, AD, Gharbi, K, Hart, T & Miller, KJ 2017, 'The challenges of detecting subtle population structure and its importance for the conservation of emperor penguins', Molecular Ecology, vol. 26, no. 15, pp. 3883-3897. https://doi.org/10.1111/mec.14172

DOI: 10.1111/mec.14172

Publication date: 2017

Document Version Peer reviewed version

Link to publication

This is the peer reviewed version of the following article: Younger, JL, Clucas, GV, Kao, D, et al. The challenges of detecting subtle population structure and its importance for the conservation of emperor penguins. Mol Ecol. 2017; 26: 3883– 3897 which has been published in final form at https://doi.org/10.1111/mec.14172. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.

University of Bath

Alternative formats If you require this document in an alternative format, please contact: [email protected]

General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.

Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

Download date: 28. Sep. 2021 This article isprotected rights by All copyright. reserved. doi: lead todifferences version between this andthe ofPleasecite article Version Record. this as been and throughtypesetting, proofreadingwhich may thecopyediting, process, pagination This article has acc been Keywords: Antarctica, population genomics, RAD gemma.clucas@ *Corresponding authors: Jane Younger 6 5 4 3 2 1 Author affiliations: These† authors co * These authors contributed equally and share first Hart† Jane L. Younger* penguins emperor of conservation the for structure population subtle detecting of challenges The typeArticle : Original Article DR. JANE YOUNGER (Orcid :ID 0000 ...... Accepted Article of Western Australia (MO96) Australian Institute UK. Edinburgh Genomics, Ashworth Laboratories, University ofEdinburgh, Edinburgh EH9 3JT, Hall, Department of Natural Resources and the Environment, University of New Hampshire, James Southampton, SO14 Ocean & Earth Sciences, University ofSouthampton Waterfront Campus, European Way, 60660, USA. Department ofBiology, Loyola University Chicago, 1032 W. Sheridan Road, Chicago, IL, Department ofZoology, Uni 10.1111/mec.14172 1

and Karen J. Miller† 56 College Road, Durham, NH

unh.edu 1,2 , Gemma Clucas* V. ntributed equally and share final

of Marine Science, 3ZH, UK.

epted undergone for and buthas not review publication fullpeer 6 .

versity ofOxford, South Parks Road, Oxford, OX1 UK.3PS,

, 35 Stirling Highway, Crawley, WA 6009, Australia. - , 1,3 0001

03824, USA. - ,4

[email protected] and Gemma Clucas , Damian Kao Indian Ocean MarineResearch Centre - 5974

and its importance importance its and - - 7350) seq, Southern Ocean, Ross dispersalSea,

- authorship. authorship. 1 , Alex D. Rogers

1 , Karim Gharbi -

, The The University

5 , Tom

This article isprotected rights by All copyright. reserved. Accepted demographicof connectivity among populations can bemade. account when determining management strategiesfor threatened species population structure difficultiesthat molecular ecol recognitionthat its is vital for the effective conservation of the species. Wediscuss the many methods, havewe shown thatpopulation structure within emperor penguins does existand argue usingBy larger sample comprise atleast four metapopulations, and that the SeaRoss is clearlydistinct a metapopulation. emperor penguins are (10 using 4 Articlestronghold for the species, is isolated Two previous studies have differed in their conclusions, particularly whether the Sea,Ross a major quality seaon ice boundariescan be difficult when efforts a Understanding Abstract Running title: Emperor penguin metapopulations –

16 percolony)from eightcolonies around Antarctica. In . ,5 An understanding emperorof penguin population structureis therefore urgentlyneeded. nd 96 genome96 , and somepopulations mayin be jeopardy estimates of extinction riskfor threatened species. However,

the boundariesof breeding populationsof is great importancefor conservation - wide single nucleotidepolymorphisms (SNPs), characterised in110 individuals using largeSNP datasets, and arguethat panmictic sizes

andthorough a assessment of the lim ogists and ogists managers face

aroundentire the continent population structure is subtle. Emperor penguinshighly are reliant

or not.or We

assessed

as climate changealter in the detection and interpretation of

emperor penguin population structure , wefind that emperor penguins

contrast subtlestructure shouldtaken be into

itations differentof analytical to

determining these a ,

previous until accurate s sea ice extent and conclusion that

estimates

subtle

This article isprotected rights by All copyright. reserved. Acceptedthreatened species that are emblematic of these challenges, as evidenced by the vastly different response to future demographic linkage ofpopulation dynamics, which ultimately will affect a population’s very lar seem a subtle distinction, it is an important one from a on the number ofdispersal events among populations Hanski 1997; Lowe & Allendorf 201 growth rate, survival and birth rate, must be affected by immigration or For populations to be demographically connected, demographictheir rates, such as population between distinctions between interpreted evidence as for panmixi where the population structure is subtle This can produce misleading results this limitationif not is understood, particularly in cases minor a the allele frequencies in the dataset,Article and for some data types, including biallelic SNPs with rare commonly used methods for inferring population structure, are mathematically constrained by frequencies) are used when markers with low mutation rates (such biallelic as SNPs with low minor allele the truenumber of populations when gene flow is andhigh, performance is further impeded Gaggiotti 2006 when detecting and interpreting subtlepopulation structure Allendorf 2010; Palsbøll connectivity in order to develop meaningful conservation strategies It is common practice to use genetics to delimit breeding populations and assess population Introduction ge dispersal rates lleles, the maximum value that genetic genetic and demographic connectivity ) . Firstly, clu

climate ecological and evolutionary populations (Waples & Ga

et al. resulting in change. stering methods

2007)

Emperor penguins ggiotti 2006). Secondly, measures of a 0) . However, there areseveral key challenges to overcome .

genetic homogeneity do not necessarily indicate It is also crucial in such studies to recognise the . Genetic connectivity, on the other hand, depends solely F ST ,

because small values of

can can take is much lessthan 1 have been toshown (Lowe & All ( (Lowe & Allendorf 2010) conservation Aptenodytes forsteri

endorf 2010; Palsbøll (Lowe & Allendorf 2010; (Waples & Gaggiotti 2006), and perform poorly at F ST

(Funk perspective, because even c an be erroneously (Jakobsson emigration F ST

) et , one ofthe most are an iconic, al. .

2012; Lowe & While this may

et al.

et al. identifying (Gilpin & Waples

2007

2013) ) . .

& This article isprotected rights by All copyright. reserved. shifts may be particularly relevant for potentially adaptive alleles facilitat compensating for low survival orbirth rates of natives of aspecies in relationship between demographic rates and local d climate, where local vital rates are dependent climate known to decouple correla assumption of isolation is likely to haveled to erroneous projections, because dispersal is modelled as an isolated breeding unit, no with exchange ofindividuals among locations A major limitation global decline in emperor the year 2100 (colonylocal 2009 assessments for duration 2012) breeding colonies on sea changing E wide panmixia demographic isolation of breeding colonies (e.g. population structures that have been proposed for the species, ranging from complete Accepted Articlemperor penguins ,

2014) and have been shown to be sensitive to fluctuations in ice extentsea and seasonal es (Ainley Antarctic

gene flow among breeding sites, replenish . -

Using specific)

at two thirds ofthe colonies examined, concordant with a minimum of 19% several key ways (Cristofari the species

et al.

of are likely under threat sea sea ice conditions a sea a sea ice dependent demographic model paired with projected changes in the

2010; Fretwell sea ice sea ice conditions

Jenouvrier ice

penguin numbers et al. tions between population trajectories and climatic variables even ; and 3)

under future climate change scenarios have begun at the majority of

2016). : 1)

dispersal enables range shifts immigrants

et al.

et al. (Collins

emperor penguins

Jenouvrier (201 (Ainley

2014; Trathan .

(Tavecchia et al. 4) their can can promote population stability by Barbraud & forecasting study

et al.

2013; Vaughan known colony locations ing et al

(Lowe & Allendorf 2010) 2010; Jenouvrier

the gene p

. et al.

in the short term, as there is some (2014) et al.

ispersal Weimerskirch 2001) to species

2016)

2011)

(Walther predicted declines of>

ool ofa population newith et al. was thatwas each colony was . In addition to altering the can increase . In light of this threat, risk

2013)

et al.

(Fretwell

2009 .

(Jenouvrier Thes

; 2) 2002)

the ,

dispersal e birds 2014)

resilience et al. . Range . The

50%

from

et al. form - w, by

This article isprotected rights by All copyright. reserved. individual emperor penguins is known to be high, with at afteryears traditional breeding grounds were lost evidence for Weimerskirch 2001; Jenouvrier emperor penguins forward into almost every published ofobservedstudy and projected climate change impacts on grounds is close to 100% emperor penguins at onbased a banding study of 38species ofAntarctic seabirds, including a single colony of philopatric and colonies should be considered as isolated breeding units. This assumption was There is a long ecosystem penguin distributions and patterns of connectivity in order to achieve their stated goal of Conservation of 2012; Trathan many oftheir prey species are,particularly Antarctic krill ( (Funk monitoring ofpopulation trajectories and for implementing meaningful management plans Delimiting the and therefore crucial that connectivity conditions become evidence

AcceptedPointe Géologie Article ris

et al. k assessments for the species. that -

based management. 2012; Palsbøll six instancessix of either colony relocation or establishment of anew colony over they

- geographic boun et al. held assumptionheld in the scientific literature

Antarctic Marine may may be may may shift their c (A unfavourable

2015) badi Pointe Géologie ”

et al. . Southern Ocean managing bodies, such as the partly (Weimerskirch et al.

2016; Ainley daries ofbreeding populations alsois essential foraccurate

2007) et al. explained

(Ancel Living Resources (CCAMLR)

among colony sites is incorporated olony sites and establish new colonies local as sea ice

2009,

. that concluded that “the

While emperor penguins are not harvested by fisheries,

et al.

by temporary emigration. 2012

et al.

2014;

1985) ,

2014) 2010; Barbraud , a Fretwell nd suggested . This . This assumption has been carried juvenil . However, that emperor penguins are strictly Euphausia superba

et al. , require an understanding of es es fidelity rate to thehatching shown toshown

that

2014; et al. LaRue

In addition, mobility of

into modelling studies reduction

2011; Barbraud & LaRue Commission for the et al travel than more ) .

( et al. (2015) gave Nicol s s in colony size

2015)

et al.

It is five

This article isprotected rights by All copyright. reserved. species consists ofone single, panmictic population around Antarctica. Unfortunately, because concluded that emperor penguins are not genetically differentiate al investigation of genetic dif Multiple independent loci across from the genome uniparentally onbased mitochondrial DNA and therefore represents the genetic structure of asingle, two genetically distinct populations of genetically differentiated thosefrom elsewhere on the continent, suggesting there are at least (Taylor & Dizon 1996) frequencies, and genetic analyses will require high power to detect p large effective population sizes it can take many generations for genetic drift to alterallele genet ancestral population, and insufficient time has passed forthese colonies to d possiblealso that this pattern reflects that allextant arecolonies derived from a common breeding population upon frequent dispersal of individuals among colonies that are successfully recruited into the the Weddell Sea significant genetic differentiation across A study ofvariation in emperor penguin mitochondrial DNA (mtDNA) found no statistically are demographically unlinked should be interrogated. exchange among emperor penguin colonies seems colonies 7,000 km in just eight months

Accepted. (2016) in a follow Article ically,

(Kooyman

even if - inh (Younger erited, locus they

et al. - up study ofemperor penguin population structure, and the authors . Younger are not currently exchanging migrants

1996; Wienecke

et al. ferentiation among colonies. This approach was used by Cristofari arrival.

. (Thiebot

2015b) et al

However, given the nature of thegenetic marker used, it is . (2015b) also found that penguins from the Ross Sea were emperor penguins.

. et al.

This genetic homogeneity could be the result ca.

et al.

8,000 km of coastline, from the Adélie Land Coast to

2013)

2010) possible sh ,

often in the vicinity of many different . ould allowould for a more sensi

Given these observations, genetic A caveat ofthat study that was it

and the assumption that colonies

(Wright 1931). For species with d among colonies and that the opulation differentiation rift apart tive

of was was et

This article isprotected rights by All copyright. reserved. Acceptedconclusions about genetic structure in an iconic species, we which decision makers might view two apparently similar studies that prov than a single panmictic population of emperor penguins. Recognising the likely frustration with genetic Ross Sea and otherAntarctic populations. Furthermore, we conclude that subtle yet significant (Cristofari other recently published studySNP restriction site 110 individuals genotyped for expanded ourprevious mitochondrial DNA anal around Antarctica In this westudy change through aunified evolutionary trajectory” Articleconclusion that “a reductions in sea ice include colonies in West Antarctica, the region of Antarctica experiencing t population of emperor penguins 2014) largest breeding colonies of both emperor penguin species and the Ross Sea contains genetically distinct populations ofboth emperor differentiation reported in You of the small sample size in the Ross Sea, Cristofari Adélie

differences revealed among colonies are indicative of multiple metapopulations rather nguins. Furthermore, it is the only region a with predicted stable or increasing

et al. (Ritchie

associated DNA sequencing (RAD

2016) further (Ritchie s a s single geneticpopulation, emperor penguins will respond to climate using

et al. (Stammerjohn , our investigate

a 2004) et al. robust robust sampling design

SNP SNP

2004; Younger 4,596 nger

penguins, acted as an age refugium ice for both Antarctic data (Jenouvrier , which genotyped the same totalnumber of individuals,

genetic et al et al.

confirmed the existence ofgenetic differences between the high high coverage . (2015b). representsThis a major limitation, because

2012; Vaughan (Fretwell

connectivity among emperor penguin colonies

et al.

et al. yses - s

(Cristofar et al including the Ross Sea 2014)

eq) 2015b) genome

et al. (Younger

(Baird . (2016) not could adequately explore the

. Finally, Cristofari et al.

2012) ,

and is currently home to the world’s - i advocate applying the wide

et al. et al.

2013) et al.

and SNPs

2016) 2008)

2015b) . As a result, their Adélie

(Younger generated using

. . is premature. To To achieve this, we In contrast to the he dramatic most

et al

(Lynch & LaRue ide contrasting with a with dataset of . (2016) did not

et al.

2015b)

This article isprotected rights by All copyright. reserved. Halley Bay in 2012. Whole chick carcasses were collected at Auster in 1993 and 1994. Blood locations). Skin biopsies from the foot or back were collected from dead adults and chicks at Pointe Géologie in 2010, and Amanda from B Muscle biopsies from the pectoral region of chick carcasses w AntarcticaEast (Figure 1). Ros continental and regional scales, two colonies from each of three major geographic regions (the around Antarctica were genotyped for this study. To assess genetic differentiation at both A total of110 individuals S year atappearssea to be highly variable (Jenouvrier ( Weimerskirch 2001; Forcada & Trathan 2009 success produce asingle egg per year, with no possibility of relaying an if egg is lost, and breeding is Emperor penguins are long species Study methods and Materials breeding populations identified here should be considered as separ precautionary principle such that, in the absence of definitive evidence forpanmixia, the Jenouvrier Accepted and RAD extraction DNA ampling, Article s Sea,s Weddell Sea and Prydz Bay) were included, along with an additional two colonies in typically

five to five six et al.

ranges from 0.60 2005; years Mougin & van Beveren 1979) ( 1

- and 0 lived seabirds –

generation length

individuals to

0.85 - s , but may low as fall as 0.02

eq that ay in 2012 and 2013 (see Figure 1 forcolony

) per colony . Adult survival typicalis

exhibit delayed breeding is

≥ 14 years et al.

whilst the probability ofsurviving the first )

2005) from eight emperor penguin colonies

ere collected at Fold Island and

(Forcada & Trathan 2009) .

ate units for management.

( Barbraud & ly high at0. ;

the average age offirst breeding 8 0 –

0.95 . Pairs

This article isprotected rights by All copyright. reserved. bp. Gel electrophoresis Acceptedsamples were then pooled into multiplexed libraries, an restriction enzyme, followed by ligation to barcoded P1 adapters. The individually barcoded Etter Edinburgh ( RAD a 1%gel and DNA contamination was measured using a Nanodrop (ThermoFisher Scientific). Qubit (ThermoFisher Scientific). The presence ofhigh molecular weight DNA confirmedwas on RNase A (QIAGEN) or 1 μL step was extended to 32 hrs. RNA contamination was reduced by treating samples with 1μL an(plus additional 10 μL 1 M dithiothreitol for skin samples from the foot) and the incubation and the incubation time was extended to 3 4 hrs; following modifications to the digestion step: 30 μL proteinase Kwas added to blood samples Genomic Article Survey. additional ethical approvals were received from the University of Oxford Division. Ethical approval of each ofthese permits was granted by the permitting institution and Commonwealth Office, the US National Science Foundation and the Australian Antarctic Younger alter allele frequencies throughout the colony. population structur (Forcada & Trathan 2009) and does so not present a problemfor estimating contemporary 1993 samples were collected fro -

. s et al. This This sampling interval represents less than two generations for emperor penguins eq, in

et al DNA

( cluding library preparation, was performed by Edinburgh Genomics, University of 2011) https://genomics.ed.ac.uk/ .

(2015b) (gDNA) . For each individual, 250 e, because any gene flow during this interval would not have had time to was extracted using QIAGEN DNEasy blood and tissue with kits the . Sampling was conducted under permits from the ForeignUK and

was usedwas to size m Gouldm Bay in 2013 Riboshredder (Epicentre). DNA concentration measuredwas with a ). The method used follows - select fragments. The NEBQuick Blunting used Kit was

ng ofgDNA wasdigested thewith SbfI

For detailed methods ofblood sampling see

and the from Ross Sea colonies in 1992 and 0 μL proteinase Kwas added to tissue samples d sheared into fragments of 300

Gonen and British Antarctic

et al.

( 2014) - HF (NEB)

after

–

400

This article isprotected rights by All copyright. reserved. pstacks cstacks methodolog Our ( http://dx.doi.org/10.5061/dryad.7c0q8 custom python script ( multiple alignments, more than 5 mismatches and/or more than 2 indels were removed using a (Li 2013) to the emperor penguin reference genome ( mismatch were excluded from further analysis. The remaining paired reads were then aligned pair had a low quality score, uncalled bas bases as sequence quality can decrease at the end of the read. Read pairs where either ofthe v1.35 demultiplexed, trimmed and usingcleaned process_radtags from the Overall readquality and the presence of adapters were assessed using FastQC. were Reads and filtering calling SNP Bioinformatics, of other species (v4mode chemistry). were sequenced using 125 base paired libr increased via enrichment PCR, followed by purification Ampurewith beads. The enriched to blunt the libraries, followed by A Acceptedhttp://broadinstitute.github.io/picard) Article aries were checked for size and concentration using Qubit and a qPCR assay. libraries The SNP calling

(Catchen

– we specified a minimum depth of readssix mapping to thesame location (

sstacks . Terminal alignments were prevented using a clipping penalty of100. Reads with y for king penguins

et al.

methodology followed the general guidelines of Benestan (for other studies) –

rxstacks

2011,

The individuals were sequenced across eight libraries alo filter.py available – 2013)

cstacks cstacks (Clucas . readsThe were truncated to 113bp to exclude four terminal .

- tailing and ligation to P2 adapters (IDT). Yields were –

sstacks -

end reads on an Illumina HiSeq 2500 in outputhigh et al. . ). PCR duplicates were removed using Picardtools from the Dr

es and/ores a barcode or cut http://gigadb.org/dataset/

2016) –

populations . usedWe yad Digital R ) with the following options: in

the Stacks pipeline ( epository - site withsite thanmore one Stacks software pipeline 100005 et al

. (2016) and our ) using bwa ng with samples pstacks - m 6)m and –

- mem

This article isprotected rights by All copyright. reserved. Acceptedat detecting loci genuinely under selection in a rang discard them. employs a Bayesian most population genetic analyses. We used BayeScan v2.1 underLoci directional or balancing selection violate theassumption of neutrality that is used in population genomic and vcftools. usedWe PGDSpider v 50% ofthe populations when genome, and c 100X using vcftools v0.1.13 population to be included ( ( dataset write_random_snp); a locus must have been present in allpopulations to be inc RAD Articlesections ofthe genome being merged into asingle locus( ( errors a with alleleminor frequency (MAF) < 0.01,which are likely to be theresult of sequencing removed from further a were removed from individuals ( were removed with aconservative confidence limit of0.25( many haplotypes to be biologically possible such as individuals were used to thebuild catalog in sequencing lanes, and a lower bound of 0.0041 (the highest sequencing error rate recorded by phiX s employed the bounded SNP model with asignificance level of α= 0.05, an upper bound of 0.1 - tag was ch -- - min_maf 0.01); loci with aheterozygosity > which0.5, could be the result of paralo p 8); and a locus must have been genotyped in at 80%least of individuals per It has been onfirmed osen osen at random -- model_type bounded

F analyses. ST

outlier test, to identify loci that might be under selection and hence nalysis ( that none were out of Hardy Weinberg equilibrium (HWE) in thanmore

demonstrated -

r 0.8). We (Danecek

< 0.01using the to remove tightly linked SNPs from the dataset ( – - ln_lim – 2.0.8.2 prune_haplo), and loci with a mean log likelihood <

et al. verified

- (Lotterhos & Whitlock 2014) 10). In the --

(Lischer & Excoffier 2012)

bound_low 0.0041 2011) cstacks

that no SNPs hada mean coverage greater than adegenet , to a

populations e ofdemographic scenarios, but witha ; in from repetitive regions or paralogous loci) void void SNPs from repetitive regions of the rxstacks --

(Jombart & Ahmed 2011)

(Foll & Gaggiot max_obs_het 0.5); a single SNP per

-- conf_lim 0.25), excess haplotypes -- bound_high 0.1

module, we removed any SNPs confounded loci (loci with too

that BayeScan powerfulis prepare ti 2008)ti luded luded in the final --

-- file alpha 0.05); all , which pikes in the

s package in R

for - 10 were n gous This article isprotected rights by All copyright. reserved. effectively showing their assignment probability to each cluster. From the results ofour Acceptedestimates the proportion of ancestry each individual shareswith ancestral populations, Bayesian clustering algorithm in the program Structure v2.3.4 determining cannot be d detecting whether there is any structure ( Criterion ( (Meirmans 2012) Analysis ofMolecular Variance (AMOVA) based method of taking the average. number of principal components to retain w Mawson Coast, Weddell Sea and Amanda Bay/Pointe Géolog on the individuals when they were grouped by colony and by geographic region (Ross Sea, However, the optimal number of clusters suggeste function), to avoid method can benefit from groups being defined by successive Article(DAPC) visualizedWe population structure with a and populations individuals of Clustering Whitlock 2014; Storey & Tibshirani 2003) expect one in ten loci to be afalse under selection were detected. deemedWe q parameter to five (for every fi positivefalse rate not was a concern. We conservatively the priorset odds of neutrality accompanying (Jombart BIC efined for

the true value of )

high falsehigh and pseudo

et al.

using simulated a a priori As well clustering as individuals, we clustered populations using the K

2010)

= 1, but thepseudo - positive rate. As our aim towas achieve a neutral set ofloci, a high

assignment of individuals to groups based on sampling locations - F statistic

using the ve loci one expectedis to be under selection) to ensure allloci K

(Meirmans 2012) - positive neutral locus that wronglyis discarded nnealing. Wecompared results with the

(Caliński & Harabasz 1974) adegenet K . Discriminant Analysisof

F statistic is known to perform better > 1 rather than as as found by running - values of< 0.1 tobe asignificant result, we so can

(Jombart & Ahmed 2011) . Finally, individuals were clustered using the d by K - means wasmeans one, and we ranso DAPC K

K - ie).

means clustering in Genodive K = 1) because the pseudo

- (Pritchard means In each case, the optimal optim.a.score . The BIC is useful for Principal

clustering (

et al.

package in R. Bayesian Inference Components

20 times and20 times 2000) than the BIC find.clusters

(Lotter

which - F statistic This hos &

at This article isprotected rights by All copyright. reserved. Accepted dive estimatedalso (Carvajal significance level was adjusted for multiple testing using Sequential Goodness ofFit (SGoF+) Tienderen 2004) Weddell Sea and Amanda Bay/Pointe Géologie), betw We differentiation Population 2004) individual assignments to ancestral populations were plotted using distruct v1.1 between the replicate runs at each value of Articlefiles for CLUMPP runs with different values of structure when itis supplied as prior information. Supplying sampling locations can be useful for detecting ge50,000 each value of we setso lambda to 0.40 for allsubsequent runs. We then varied allowing lambda to vary (inferlamda program initially with model with correlated allele frequencies was deemed the most appropriate model. ran We the population differentiation analysis, which showed that diff

calculated R een all pairs sity v1.9.89 .

- nerations ofeach runburnin.as Wedid this both and with without sampling locations Rodriguez & de Uña K

the Weir and Cocker

by bootstrapping over loci ten times a with random seed, for150,000 generations, discarding the first

. Significance was estimated using 5,000permutations the of data, and

(Jakobsson & Rosenberg 2007) of colonies, and between the geographic regions (Ross Sea, Mawson Coast, (Keenan subtle K

= 1, for 100,000 generations, discarding the first 50,000 burnin, as . Structure Harvester web v0.6.94 et al K -

Alvarez 2011) using theusing Evanno method .

2013) h am = 1). From this initial run lambda was estimated to be 0.40, .

unbiased estimator of with 1 K .

and to a Confidence intervals for the

, . We used CLUMPP to check for multimodality using Genodive v2.0b27 000 bootstrap re verage across the ten replicates. The

(Evanno erentiation low,was the admixture

(Earl 2012) F ST K

plicates (Weir & Cockerham 1984) et al.

from onefrom to eight, running

2005)

was to used compare

(Meirmans & Van ,

F using the R package ST

estimates were and to prepare

(Rosenberg

the

This article isprotected rights by All copyright. reserved. not contribute to Figure 1). generatedreads per library, althou barcodes and ambiguous RAD cut were retained after filtering averageThe number ofreads per library was14 million Genotyping Results visualize theentire posterior distribution of trees as a cloudogram. interest, here thetree, is estimated via theposterior distribution. We used DensiTree v2.0.1 to using a Bayesian method implementedas in BEAST that is the uncertainty in th also inspected the posterior distributions ofparameters for convergence. The advantage of v the firstwith 10% of generations discarded as burnin. We checked for convergence using Tracer rather than estimating them as part of the MCMC. The MCMC was run for 5 million generations datasets of 3,221 and 3,23 Within the reduced dataset we repeated the analysis twice, different with individuals, to ensure the results were replicable. selected two random individuals (i.e. four haplotypes) pe markers (like SNPs) to infer species trees. It is highly computationally demanding, hence we v.2.4.0 founded, we used a species tree approach in the SNAPP add estimateTo the evolutionary relationships among the colonies and the order in which they were Phylogeography Accepted 1.6 Article

(Rambaut & Drummond 2007)

(Bouckaert T he

differences

any of the

et al.

2014)

7 SNPs. We calculated the mutation rates (u and v) from the dataset in read depth

any that loci were no longer polymorphic were removed, leaving population structure for . SNAPP uses a coalescent approach with unlinked biallelic

reads ofreads low quality, adapter contamination, ambiguous - sites. gh the number of per reads colony did differ (Supplementary

and found ESSs > 4,000, which is more than sufficient. We

There were no significant differences in the number of and the library to

we observed in ourdata

(range 11 r colony to include in the analysis, and

which individuals were

- on

(Bryant –

17 million), 97% of which

et al.

(described below)

2012) e parameter of assigned

to BEAST

did . This article isprotected rights by All copyright. reserved. locationwith priors optimal number of clusters in Structure, the F For Structure, the population differen switching solutions. This was a result of analysis did yielded different results: hierarchical. twoThe different clusters, suggesting that differentiation among emperor pengu populationsgenetic individuals performed with Structure AMOVA and individuals populations of Clustering colonies (Table 2). indices (expected and observed heterozygosity, and nucleotide diversity) were similar across all individuals per colony ( repeated a subset ofanalyses (pairwise confirm that 16 (Table 2 of 33X (Benestan identified 423,479 SNPs, ofwhich we retained 4,596 after After r Acceptedigure Article . The number of individuals successfully genotyped at each colony varied between 10 and eads were aligned to theemperor penguin reference genome, S 3 - ) both with and without location priors. based

(Jakobsson & Rosenberg 2007) et al. ). not converge on a sin unequal sample numbers Population structure analyses were performed using allindividuals, and to K

2016) - means clustering ofpopulations highest posterior mean log

tiation subtle is .

and However,

(Table 1). The coverage of these SNPs ranged from 11 results K K

= 6

= 3

genuine multimodality between runs, as opposed to label without priors

for the both yieldedmethods possibilitiesseveral for the number of K provided in - means summary statistics for assessing the number of clusters gle result, different with

and

pseudo (Pritchard

among colonies

the MCMC F ST

( , DAPC and Structure) S S upplementary upplementary Evanno method , - -

consistent the with lik F statistic

The alternative method elihood was achieved at

et al. (Meirmans 2012) therefore becomes

2000)

were not influencing the results

and following standard replicate F

I nformation). igure

K (Evanno both separated

= 6 in colonies is subtle and/or using a random subset of K

2 for the - )

means runs , which and

et al. tacks trapped in local optima.

Bayesian for assessing th

K arri Gene BIC

pseudo = 1 (S 2005)

(Catchen can occur when

colonies into ving at distinct – . The . The Structure

filtering steps

tic diversitytic 56X, with56X, a mean upplementary , selected clustering of - F statistic

et al.

we e

2013)

K 10

= 3

This article isprotected rights by All copyright. reserved. clu between groups (Supplementary Figure 5b). The same pattern wasobserved with individuals per colony (Supplementary Figure 5a) differentiation and when individuals were grouped by geographic region (Figure 3). distinguished the Ross Sea colonies when individuals were grouped by their colony oforigin inset). the databefore first few Ahmed 2011) by I were included in the analysis (Supplementa entirely consistent whether the full dataset was used, or whether only 10 individuals per colony population is clearly genetically distinct from allother colonies (Figure 2). resolution of population structure (Figure 2). In every scenario from the finest l visual inspection of the Structure plots for scenario 32.2% ofthe total variance was explained (r colonies colonies), 2) followin the results does not allow and BIC results, respectively. It should be noted that the Evanno method for analysing Structure ndividuals

a Acceptedstering through Article K

successive - means

However g populations were defined: 1) a

(Pointe Géologie, principal evel of genetic structure that can be discerned. (as opposed(as to populations) a pseudo )

Mawson Coast

was diminished but still visible when the analysis was repeated 10 with as the K K ,

Discriminant Analy - - means clustering means procedure (find.clusters components ofthe PCA (Supplementary Figure 6). - F statistic minimum value for theBIC was K

= 1 Amanda Bay, Go

to be For tested. the three population scenario, suggested by both

population

and the Evanno method with loca were sis ofsis principal could not be separated into

(Fold Island Ross Sea unable to explain much of thevariation Principal K uld Bay and Halley Bay) ry Figure

= 2 to components analysis (

from the

population (Cape Roget and Cape Washington

K and when lost individuals were shuffled Components (DAPC)

= 6 (Figure 2),itis apparent that

2 4 and Auster colonies), foun

= 0.322) ).

At d

adegenet at K

= 5 and K

in the

= 1. This could be because the tion priors in Structure, the .

Under the three

genetically distinct package in R ( K The power to detect this PCA K K

= 2 to -

means analysis = 6 (Jombart )

These patterns were used to transform and

there is no further K

= 6 the Ross Sea 3) all other

(Figure 3,

et al. Jombart & unsupervised - cluster K

2010)

= 4 is = 4 is groups Upon

This article isprotected rights by All copyright. reserved. twoAntarctic East Acceptedintervals nowas genetic differentiation (Figure 4 Of note that was within geographic regions (Weddell Mawson Sea, Coast and Ross Sea) there 2). Table sub statistically significant, frequencies differentiation minor allele frequencies, and in such cases small It should be noted that small values of (range 0 see Supplementary Table 1 for Cockerham 1984) ArticlePairwise differentiation Population subtle. some ofthe methods indicates that differentiation among these four genetic populations is differentiation from one another. The selection of Weddell Sea and Amanda Bay/ the most differentiate emperor penguins, each consisting ofat least two sampled breeding colonies. Ross The Sea is Overall, our clustering results suggest the existence ofat least four genetic populations of - sampled, thepatterns ofpairwise di

.002

). genetic

D ≥ espite the

–

0.01

0.006) (Jakobsson

differentiation, as measured by , was statistically sig , averaging 0.078across all SNPs, which is the likely cause of thesmall, yet colonies , consistent with theclustering results that indicated subtle differentiation d population overall, followed by the Mawson Coast population, then the ca F ST . 3,200 km between them, there was no differentiation between the

et al. values observed.values

of Amanda Bay and Point

Pointe Géologie F

2013) ST

confidence intervals . In this study we select nificant F

ST and see Supplementary Table fferentiation

are expected fordatasets ofbiallelic SNPs with low When 10 individuals per colony were randomly

for 17 outof28 pairs of colonies (Figure 4

populations, which show the least amount of Weir & Cockerham’s F K ST

= 1the as most likely number ofclusters by should not beshould interpreted a lackas of were Géologie ). valuesThe of

largely unchanged (Supplementary ed SNPs with minor allele

( F ST

= - 0.001, F

3 significant ST

for

estimator F p ST

= 0.813,Figure 4),

confidence

F ST

(Weir &

were small

and

. This article isprotected rights by All copyright. reserved. Acceptedthere penguins Some flow, whilst colonies within appear each to be panmictic because of highrates ofdispersal. four distinct metapopulations. These metapopulations ar this species and provides evidence thatemperor penguins in Antarctica are composed of atleast colonies spanning Our findings oflow Discussion differentiation among colonies the evolutionary relationships among the colonies well approach formed a diffuse posteriorThe distribution resulting oftrees from our SNAPP ArticlePhylogeograph of emperor penguins, corresponding with major geographical regions. our pairwise 0.005 (Figure 5). Th these four geographic regions was statistically significant, with Antarctica (Figure 4). Every comparison in a pairwise Sea, colonies in the Weddell Sea, colonies fr differentiation corresponding with the major geographic regions e.g. consistent thewith clustering results. At large spatial scales there was significant genetic - supported topology. lack The of awell

i clustering analyses s ones globally panmictic population. However, scrutiny , and i F ST

f th

analyses analyses further support the conclusion that there arefour genetic populations y e more thanmore

, yet statistically significant, genetic differentiation among empero s e Ross was Sea the most differentiated from all other populations. Overall, e

result struggled to detect thanmore cloud ( s

10,000 km of theAntarctic coastline were not scrutinised further it .

Supplementary Figure 7 - om the Mawson Coast and colonies from East supported topology pr

and F

further reflectsfurther the lack of strong genetic ST a single genetic cluster of emperor

e variously connected through gene analysis that grouped colonies into ) indicating

might

(Bryant of data F ST

refutes claims ofpanmixi

events from us determining values rangingvalues 0.002 from to be easy to conclude that

among et al. is crucial, because

that there no was single,

2012) colonies in the Ross

species r penguin tree

in This article isprotected rights by All copyright. reserved. satellite tag in the direction of Tracking offledglings from Pointe Géologie (140°E) showed several individuals emperor penguins are no Po satellite tracking offledgling and penguinsmoulting Our finding offour metapopulations i on geographic proximity alone. that assumptions should not be about made the connectivity of emperor penguin colonies based significantly differentiated the from Fold genetically differentiated thfrom of genetic differentiation locatedAuster, 600 km apart colonies ofthe Mawson Coast, which are separated by only well 190 as km, as Amanda Bay and located 300 km apart, are genetically indistinguishable, are as the Fold Island and Auster 550 kmapart genetically connected. Antarctic Coast (Figure 6 penguins within our sampling range arethe Ross Sea, Mawson Coast, Weddel supported by ourpairwise ge et al. may be the case foremperor penguins populations when ge clustering methods are known to perform poorly at estimating the number of genetic netic netic Acceptednganis 2008, 2016; Article

2015b) differentiation

failed . By examining

–

are not genetically differentiated.

(Thiebot Amanda Bay with(76°E), one individual reaching far as as 88°E ne flow is high Thiebot

The two colonies in the Weddell Sea among emperor penguin colonies c are less predictable t tiedto breeding colonies and hence have the opportunity to disperse. ). Over short distances, i.e. less than 600 km, colonies appear to be

F et al. multiple ST . Where colonies are separated by more than 600 km the patterns

comparisons et al.

2013). Notably, none ofthePointe Géologie fledglings travelled ose

2013 (Waples & Gaggiotti 2006)

values of ca s s supported by

( . 3,200 kmaway Kooyman & Ponganis 2016; ; Island Wienecke

and DAPC ; for example, Amanda Bay K

colony just 790 kmaway. We therefore urge we were able to ascertain a

Likewise, thetwo colonies in the Ross Sea,

et al. (Kooyman the limited the limited . The four genetic populations ofemperor at

using Structure,

2004) Pointe Géologie

–

Gould Bay and Halley Bay , which we reasonably expect

et al. . During these life stages evidence LaRue

1996

penguins are

a available , ,

nd was this

et al. 2000 whereas clear l an Sea

travelling west 2015; Younger ;

pattern of Kooyman & before from they are d East

not

further , located

the

This article isprotected rights by All copyright. reserved. Acceptedregion SNPs revealed further population subdivision within the East Antarctic and Weddell Sea Sea found that penguins in the Ross Sea were distinct from those in East In our first study of emperor penguin genetic connectivity, based on mtDNA sequences, we research. and likely that they utilise the same habitat (Wienecke (Wienecke Glacier were tracked to a moulting loca may also share moult habitat breeding individuals (Kooyman & Ponganis 2016). penguins et al. Article other during the period,moult and none of thetracked individuals left the Ross (KooymanSea (Kooyman Ross Sea, adults from remain in the region. In further support of the Ross Sea’s status as a distinct metapopulation, Kooyman & Po to travel in a trajectory consistent among emperor penguin colonies. Fledglings from western Ross Sea colonies have been shown homogenous suggests a potential role for oceanograph the influence oftheAntarctic Coastal Current. Our f towardeast the Ross Sea. Thiebot (Younger non

2000). s , with , with - breeding distribution on population structure is a promising avenue forfuture

have been tracked to a breeding at individual colony sites in the Ross Sea,

et al. et al.

Cape Cape Washington and Cape Roget, along with afurther three colonies in the western It has also been thatshown there largeis

three et al. nganis 2008)

2000). Individuals from these five colonies almost certainly encountere 2004). While moulting Fold Island individuals have not been tracked, it is very

2015b) distinct populationgenetic , afinding that is . This . This circulation pattern encourage could Ross Sea penguins to –

moulting individuals from Auster and a nearby colony at Taylor common areamoult at the end ofthe breeding season

et al with thewith circulation of theRoss Gyre (Kooyman . (2013) attributed theprolonged westward trajectory to tion in close proximity to theFold Island colony

supported s resolveds among the six colonies studied, which

et al. Similarly, the Mawson Coast metapopulation inding that these aresites genetically

annual variability in the number of here here y

2004). in shaping patterns ofconnectivity

by genome which may indicate The The influence of oceanography Antarctica and t - wide SNPs dispersal

. et al.

However, he he Weddell

1996; d each

the

This article isprotected rights by All copyright. reserved. arguments for panmixia of emperor penguins in Cristofari Ho differenceThe between populations within each. degree Specifically, the emperor penguin species is comprised of multiple metapopulations, with some scenario, scenarios. Our genetic data, both h complete isolation to forecast population trends for the species under future climate change Gaggiotti 2006 studied, to complete isolation, in which there is no geneticexch mating is random (i.e. equally likely) amongst all pairs ofindividuals across the colonies demographic connectivity can be described a as spectrum ranging from panmixia, in which emperor penguins A using genome have higher levels ofgenetic differen of emperor penguins microsatellites, resolved similar levels of al. species, including Ad colonies, as exp indicates that SNPs are superior to mtDNA for were grouped as a single populationgenetic using mitochondrial DNA alone

recent study

2015a) Acceptedw might two similar data Article of connectivity among metapopulations, and very high connectivity between sub

as wouldas be expected for most species in the natural world and chinstraps -

wide SNPs. ). Whilst Cristofari of emperor p ected are fully“a panmictic species” é (Younger (Baird lies

our results and that of Cristofari

(Clucas (Clucas sets be differently so interp

enguin genetic connectivity used also et al.

et al.

2008) et al et al. et al. ere and in Younger

2015b) tiation than . (2016) proposed panmixia, Jenouvrier

2014; Freer 2014; Ritchie . Analyses of genetic . It

detecting fine is thereforeis

reported to date, differentiation as found in (Cristofari

et al.

genetic genetic connectivity et al. et al reted? attestWe that one ofthe main

2015) et al

. (2015b), support an intermediate 2004; et al

possible that other penguin species

- et al. scale . (2016) warrants interrogation.

. (2016) was the “very low” ange among colonies ( using either m Roeder

2016)

SNPs which could be detected genetic differentiation among (Waples & Gaggiotti 2006)

and concluded

(Figure 6 et al

within other the

(Figure 6 . 2001 t et al DNA or

mtDNA b). . (2014) used ; Younger Genetic or Waples & a,c)

penguin that analysis - .

This This

et . This article isprotected rights by All copyright. reserved. DNA becausethe rate lineageof sorting is expected to b lineages within emperor penguins (Fretwell population sizesin question sortingdriven is primarily by geneticdrift, andtherefore is directly proportional genealogically distinctpopula recovered evidence of distinctevolutionary lineages within emperor penguins. However,a lack of Neither SNAPPour species analysistree nor theneighbour penguins. 2016) SNPs. This chrysoptera recent study level and Fold that Island) represented stati 0.002 (between represent differentiation or homogeneity. As a comparison, we observed p Cristofari to yield small 2016) MertzWest colonies, to 0.0240 between Cape Washington and values of - Accepted Articlevalue associated with their AMOVA test, it so is (Pamilo Nei& 1988) after SGoF+ correction for multiple tests . , is smaller than the smallest However, as discussed above, biallelic SNP datasets with rare minor alleles a

et al. F

et al ST F

ST a

between pairs ofcolonies, ranging from 0.0080 between the proximate East and (Toews nd

F . (2016) did not report the significance value 2012) ST Pointe G Vermivora cyanoptera

values values , ameasure ofdifferentiation between two distinct bird species . Interestingly, our m

et al. . (Jakobsson

éologie and Halley Bay, Amanda Bay and Halley Bay, and Amanda Bay

2016) (Avise tions shouldtions not be misinterpretedpanmixia. as

(Younger compared two species ofparulid warblers, et al.et F

et al. ST

value reported by Cristofari , and found an stically significant genetic differentiation at the 1983) t DNA study revealed three distinct, well

2013)

etal. , which may be very large for emperor p . placeTo these findings in a broader context, a

, and are these not evidence of panmixia. 2015) e foure times fasterfor mitochondrial than nuclear unknown whether the reported values

. Thisapparent discrepancynot is surprising,

F levels oflevels their pairwise ST -

value net analysis Cristofariof

of 0.0045 based on 11.4 million Pointe G et al . éologie (2016) for emperor F

Stochastic lineage ST

Vermivora to values F - ST supported the effective (Cristofari

et alet values enguins

(Toews as low as re expected . (2016) α , nor the

= 0.05

et al.

This article isprotected rights by All copyright. reserved. Acceptedmoulting emigrate respectwith to their colony locations juveniles diverge among now isolated colonies, which have simply not been isolated for a sufficient tim it is also possible thatthe observed genetic similarities could be theresult of shared ancestry individuals genetic observed genetic patterns, we must consider two alternative scenarios. Firstly, t and effective conservation strategies species and growth are influenced by dispersal estimate Ideally, we could Article management unit recommend that the Ross emperorSea peng differentiationgenetic of theRoss Sea based on robustly called genome analysis ofpopulation structure included only four individuals from the Ross Sea, asample siz panmictic with allother emperor penguins across Antarctica ( their dataset,SNP Cristofari colonies and those elsewh Our original mtDNA study

differentiation risk assessments genetically

of ( (Kooyman locations Kooyman & Ponganis 2016;

demographic among breedi translate ourknowledge of thegenetic patterns among colonies into a (Funk

. (Kooyman Given the growing body ofevidence thatemperor penguins travel widely as

et al. between some colonies

et al.

(Jenouvrier connectivity, defined the as extent to which apopulation’ ng sites,suggesting that demographic linkage a possibility.is However, 1996; Thiebot

ere in East Antarctica (Figure 6 found a high degree of geneticdifferentiation between the Ross Sea et al

2012) et al (Cristofari . (2016) reported that Ross Sea emperor penguins were . 2000; .

(Funk et al. (Ancel LaRue (Lowe & Allendorf 2010)

et al.

Wienecke

2014; Tavecchia

et al. et al.

uins be conservatively considered a distinctas et al.

et al. 2013; Wienecke could be theresult of

2016) 2

2014; LaRue

012; Palsbøll 2015) et al . We have now conclusively validated the . 2004), , a)

and that multiple colonies share

et al. (Younger e thatprecludes any reasonable

Figure 6 et al.

et al.

we consider . This would enable 2016)

2010)

contemporary 2015)

2007)

et al. b). However, their study , population monitoring - wide SNPs and , exhibit dynamism

, 2015b)

. may temporarily

To interpretTo the

the former he lack lack he of . Based on s vitals rates dispersal of meaningful e to n ,

This article isprotected rights by All copyright. reserved. issues described above and, at any rate, only describe dispersal over evolutionary timescales, onbased coalescent methods, however, these noteWe that g for which the they are not estimation of that exchange migrants with the studied po Brumfield 2015; Harvey not is method is contingent on accurate estimation of evolutionary rates fo meaning SNP data may not be suitable forthis type of analysis. dispersal rates (P. Beerli have on contemporary connectivity. dispersal colonies. (Faubet few generations degree ofaccuracy. differentiation understanding ofemperor penguin demographic connectivity. An accurate estimate of the explanation for the genetic similarity found here. scenario Accepted enetic studies to date (Figure 6 Article currently

et al. coalescent , that

Coalescent methods, such as Migrate over evolutionary timescales,

(Beerli 2004; Slatkin 2005)

re are many unsampled colonies that lie between the colonies included in allthe Cristofari 2007) dispersal dispersal observed possible to accurately estimate evolutionary rates for RAD loci (Wilson & Rannala 2003) estimates. Low frequency SNPs are believed to be important for estimati BayesAss, which is –

a full order of magnitude greater

rates, specifically, populations appear to be exchanging when migrants et al is maintainin

et al. among colonies precl pers. comm. . (2016) rate ofdispersal It is alsoIt is unclear what effect different SNP calling pipelines may

2016) ).

. compoundTo these issues further, unsampled populations reported dispersal ), but arehard to distinguish from sequencing errors, g gene flow among populations, to be the likely most typically used to estimate rates of

making them inappropriate for . This is ,

is pulations have serious confounding effects on the

among colonies would substantially improve our unreliable where

- estimates udes the estimation of dispersal n

almost certainly the case for emperor penguins,

(Beerli 2009)

than observed among emperor penguin rates among emperor penguin colonies are likely to be affected by many ofthe

, are used to infer rates of

Unfortunately, F Furthermore, the coalescent ST

values are less than 0.05 r the loci in question estimating dispersal over the past the low

rates withrates any (Harvey &

and it

This article isprotected rights by All copyright. reserved. linked. incorrectly assume al. mayd be spanningwhole the Antarctica,of therea risk is that localised clima very differentconclusions. If emperor penguins are misdefined asa single demographic unit “ was overlookedCristofari by forecasts should take this structure into account. eight study colonies, and futu our dataset we have identified four genetic populatio most likely not demographically isolated, nor are they likely to be linked continent theseWith caveats acknowledged, we tentatively conclude 2016) reproductive success ofboth immigrants and residents from capture address this gap by combining genetic methods with dataon movement behav the extent to which int (Waples & Gaggiotti 2006) framework for determining the level of accurate estimate rather than contemporary connectivity of colonies. a

single global population sharedwith a demography”. erdependence Accepted2007; Taylor Article .

ownplayed - mark

et al.et (Lowe & Allendorf 2010) d of the number of emperor penguin migrants, yet as there is no generalized -

th emperor penguin colonies are demographically linked. in management plans for the species,resulting inlocal extinctions recapture methods, local demographic rates and measures ofthe

2000) at emperor penguins at allcolonies around Antarctica are demographically . Furthermore, future

and even rateshigh ofdispersal donot guarantee demographic et alet re . (2016),who instead concluded that emperor penguins compris management plans, monitoring schemes, and dispersal . Therefore, it is currently impossible todetermine species

Thesubtle population structurewe describe here necessary to maintain demographic linkage Even itwere if possible to generate an ns ofemperor penguins (Figure 6

From a management point of viewthese are risk assessments will be confounded if (Lowe & Allendorf 2010; Tavecchia

that emperor penguin colonies are te change or fisheriesimpacts

Future studies could population iour, perhapsiour, - (Palsbøll wide. Within c) c) among

et al. it is et

This article isprotected rights by All copyright. reserved. conservation planning. range understanding ofemperor penguin population dynamics, behavio like a difference between synthesised the available information on the patterns ofconnectivity among colonies. Here we have assessed the geneticpopulation structure among emperor penguin colonies and widespread extinctions ( confounding conservation efforts and, in the worse case scenario, result in localised or Such conclusions could lead to thedefinition of inappropriately large management units, to demographic linkage being incorrectly inferred, this may be further misinterpreted by equating genetic connectivity and statistical tests not conducted. Ifany ofthese methodological oversights result in panmixi F co studies report and visualise only a single value of when using low mutation rate markers detect the true numbe example, the detected the if subtleties and limitations of theanalytical methods used are not respected. For have alimited ability to detect structure in the affected sampling regimes with small sample sizes or that omit large portions of aspecies’ range will There are keyseveral reasons why populationsubtle structure may be overlooked unique to emperor penguins, but difficulties that many ecologists molecular must g The ST nstraints on

Accepted Article values values inherent - subtle shift pote that may be

distinction limited challenges of detecting and interpreting subtlepopulation structure are not F ntial, population traject ST

as a resultas of

complete panmixia ability ofclustering methods ( r of geneticpopulations ( incorrectly interpreted panmixi as , but it is a distinction that has important implications for our As a research community i ( Palsbøll Gilpin Gilpin & Hanski 1997

allele frequencies

et al.et

2007; T and is wellis documented (Waples & Gaggiotti 2006),yet many ories and, most importantly, risk assessments and

distinct aylor ; K Lowe & Allendor )

(Jakobsson et al.2013) K

t is crucial in the presence of high gene flow and etal. Evanno . In another example, the meta

regions. Secondly, structure may not be 2000 populations with gene flowmay seem a

if theif constraints are

et al. ) that .

2005; Pritchard f 2010; Palsbøll we u r, ecology, adaptability, clearly delimit the extent of

can result in very small mathematical

misunderstood

et al. et al. . Firstly, rapple with.

2007 2000) The /or

) . to a

This article isprotected rights by All copyright. reserved. ofuse the University ofOxford Advanced Research (R8/H10/56),NERC MRC (MR/K001744/1) preparation and sequencing. International at Survey Francoise Amelineau W kindtheir donations of emperor penguin samples: the Expeditions Ltd (TH). the Charities Advisory Trust (TH) and by donations, public in particular through Quark (JY), Darwin The Initiative ( (JY & KM), the Natural Environment Research Council (GC), an Endeavour Research Fellowship a researchThis funded was by the Australian Antarctic Science Program Acknowledgements management units. precautionary approach would warrant that these metapopulations are considered as separate are connected by some level ofdispersal. demographicUntil co this study, w work colonies that have not been included in genetic studies to date,and including these properly informed decisions. communicated our knowledge, and that the

Holsworth Wildlife Research Endowment (JY), the World Sea Research and Rescue Foundation Acceptedienecke Article will at

Halley Bay further our understanding ofthe true links among emperor penguin populations. In and the Australian Antarctic Division e present

in our scientific reporting, that so managers and conservation planners can make

Gould Bay. Wewouldlike to thank

; andFilby Dick and

We We gratefully evidence for distinct metapopulations within emperor penguins, which

Pointe Géologie; DPLUS 002

subtleties and limitations ofpopulation genetic studies are fully

Edinburgh Genomics is partly supported through core grants from It is important to note thatthere are many emperor penguin

acknowledge the

T Hann Phil H), an NSF Creative Science Award and BBS

Trathan and ah McKeandah at Fold Island

the Computing (ARC) facility in carrying outthis RC (BB/J004243/1). We acknowledge

following people and organisations for

Kooyman Edinburgh Genomics assistants from

from , Amanda Bay and Auster; nnectivity can be determined, a Adventure Network t eam at

(Project 4184

the Ross Sea the

staff for lib

(DPP British Antarctic - in future 87

; – - Barb

15864), KM & rary ara the

JY) , This article isprotected rights by All copyright. reserved. AcceptedBouckaertR, Heled J,Kühnert D Benestan LM, Ferchaud AL, Hohenlohe PA Beerli (2009)P How to use MIGRATE or why are Markov chain MonteCarlo programs difficult to use. Beerli (2004)P Effect unsampledof populations on theestimation populationof sizesand migration Barbraud C, Weimerskirch H (2001) Emperor penguins and climate c Barbraud C,Gavrilo M, Mizin Y, Weimerskirch H (2011) Comparison of emperor penguin Baird NA, Etter PD, Atwood TS JC,Avise Shapira J, Daniel AquadroSW, CF, Lansman RA (1983) Mitochondrial DNA AncelA, Cristofari Fretwell R, PT ArticleAinley D, Russel J,JenouvrierS Abadi F,BarbraudGimenez C, (201 O References methods and interpretation of theecological patterns. anonymous reviewers, all of whom gave considered, insightfu Finally, we arevery grateful to Michelle LaRue, and Jerry Kooyman for helpful discussions about emperor penguin population dynamics work

( http://dx.doi.org/10.5281/zenodo.22558 analysis. 25 managed popu Population Genetics for Animal C betweenrates sampled populations. 186. Science, declines between Pointe Géologie and Haswell Island over the past 50 years. sequenced RAD markers. Evolution, differentiation during the speciation complement each other in Antarctic exploration. tropospherereaches 2 °Cabove preindustrial levels. theon survival juvenileof emperor penguins. , 2967

23 – PLoS Computational Biology, 2977.

1 , 461 , 38 lations: buildingconceptual a and practical framework.

- - 56. 468.

et al. et al.

et al. et al. PLoS ONE,PLoS

(2008) Rapid SNP discovery and genetic mapping using (2010)Antarctic penguin response to habitatchange as Earth’s 7

(2014) BEAST 2: a software platform for Bayesian evolutionary (2014) Emperors inhiding: ice when ) Integrated population modeling reveals theimpact climateof onservation,

et al.

3 Molecular Ecology,

, e3376. 10 process in Peromyscus.

(2016) Conservation genomics of natural and , e1003537.

Laura Benestan, ).

17 Global e would like t , 42

PLoS ONE, -

79. Ecological Monographs, Change Biology

13 l feedback on both our analytical

Bruce Deagle, and two , 827

9 , e100404. o thank Barbara Wienecke Molecular Biology and - hange. 836. - breakersand satellites ,

23 Molecular Ecology, Nature, , 1353

80 – 1359

, 49 411 Antarctic - 66. . . , 183

- This article isprotected rights by All copyright. reserved. AcceptedFoll M, Gaggiotti O (2008) A genome Faubet P, Waples RS,Gaggiotti (2007)OE Evaluating theperformance a of multilocus Bayesian Evanno G, Etter PD,Bassham S,Hohenlohe JohnsonPA, EA, Cresko WA (2011) SNP discovery and genotyping Earl DA (2012)STRUCTURE HARVESTER: a websiteand program for visualizing STRUCTURE output Danecek AutonP, A,Abecasis G D’Amen ZimmermannM, NE,Pearman PB (2013) Conservation phylogeographicof lineages under CristofariBertorelle R, Ancel G, A Collins KnuttiM, Arblaster R, J ArticleClucas GV, YoungerJL, Kao D Clucas GV, Dunn MJ, Dyke G Catchen J, Hohenlohe BasshamPA, S, Amores A,Cresko WA Catchen J, AmoresA, Hohenlohe CreskoPA, WA, Postlethwait JH (2011)Stacks: building and Carvajal Caliński Harabasz T, J(1974) Bryant BouD, - dominant and codominant markers:Bayesian a perspective. met softwareSTRUCTURE: simulation a study. Genetics evolutionaryfor genetics using sequencing. RAD In: and implementing theEvanno method. 2156 climatechange. inemperor the penguin. Press, Cambridge,United Kingdom and NewYork, NY, USA. Change Working Group Ito theFifth Assessment Reportof the Intergovernmental Panel on Climate and Irreversibility. In: Biology, remarkably little population geneticdifferentiation across their range. in Antarctic Peninsula penguins. population genomics. 182. genotyping de loci novo from short by sequential goodness fitof and q andtheory Methods, Molecular Biology and E directly from biallelicgenetic markers: bypassing gene trees inafull coalescent analysis. Rodriguez A,de Uña

RegnautS, Goudet J(2005)Detecting the number clustersof individualsof using the hod for theestimation of migration rates.

- 2158. ckaert R, Felsenstein J, Rosenberg NA, RoyChoudhuryA (2012) Inferring species trees

(eds. StockerTF, Qi 16

(eds. Orgogozo V, Rockman MV), pp. 157 , 211.

Global Ecology and Biogeography,

A dendrite method for cluster analysis. et al.et

- et al. 3 Alvarez J(2011)Assessing significancein high ClimateChange 2013: The Physical Science Basis. Contribution of

Molecular Ecology, et al. , 1

volution, et al. Nat

- et al.

27. (2014)A reversal of fortunes: climate change ‘winners’and ‘losers’

(2016) Dispersal in the sub n D, n D, PlattnerG

(2013) L - ure

scan method to identifyselected loci appropriate for both (2011) The variantcall formatand VCFtools.

(2016) Full circumpolar migration ensures evolutionaryunity

C Scientific R

29 ommun - - value estimation. read sequences. , 1917 ong Conservation Genetics R - term ClimateChange: Projections, Commitments

ications 22 - Molecular Ecology, - K 1932. eports, , et al. , 3124 Molecular Ecology,

- ), pp.), 1029

7 -

4 178. Humana Press. 22 3140. , 11842. Molecular Methodsfor Evolutionary , (2013)Stacks: ananalysis tool setfor G3: Genes, Genomes,Genetics, 5024. - PLoS , 93

Antarctic: king penguins show

- 104.

One,

Communicationsin Statistics Genetics, –

14 1136. CambridgeUniversity

esources, 6 , 2611

, e24700. 16 - throughputexperiments , 1149

- 180 BMC Evolutionary

2620.

4 , 977 Bioinformatics, -

, 359 1166.

- 993. - 361.

, 171 -

27 - , This article isprotected rights by All copyright. reserved. AcceptedJombart T, Devillard S, BallouxF (2010) Discriminantanalysis principalof components: a new Jombart Ahmed T, I (2011) adegenet 1.3 Jenouvrier S,Holland M, StroeveJ Jenouvrier HollanS, Jenouvrier S,Caswell H, Barbraud C Jakobsson M, Rosenberg NA (2007)CLUMPP: a cluster matching and permutation program for Jakobsson M, Edge MD, Rosenberg NA (2013)relationship The between F Harvey MG, Smith BT, Glenn Fair TC, ArticleHarvey MG, Brumfield RT(2015) Genomic variation inawidespread Neotropical bird ( S, Gonen Lowe NR,Cezard T Gilpin MEH, FunkWC, McKay JK, Hohenlohe AllendorfPA, FW (2012)Harnessing genomics for delineating Fretwell PT,Trathan PN, WieneckeB, Kooyman GL (2014) Emperor penguins breeding iceshelves. on Fretwell PT,LaRue MA, Morin P Freer J, Mable B, Clucas G For cada J, Trathan PN (2009) Penguin responses to climatechange in the Southern Ocean. method for theanalysis of genetically structured populations. Bioinformatics, penguin underclimate change. 18 population: analysis of coupled demographic and climate models. of Sciences, predictthe decline of an emperor penguin population. Bioinformati dealing label with switching and multimodality inanalysis populationof structure. frequentmost allele. 910 restriction site associated DNA sequencing for shallowsystematics. and Evolution, minutus derived from RAD sequencing. Press. conservation units. PLoS ONE, global, synoptic survey aof species from space. Biology, ( Change Biology Pygoscelis antarctica , 2756 - Hanski 924.

) reveals) divergence,population expansion, and geneflow. 38

- 2770.

, 1493

I 106 , e85285. A (1997) cs, d M, Stroeve J

, 27 23 , 1844

15, – , 305

, 1801 , 3070 etal. 1502. Trends in Ecology and Evolutio

1618 et al.et Metapopulation Biology: Ecology, Genetics, and E

Genetics, ) colonies in - - 1847. 316.

(2015) Limited genetic differentiation among chinstrap penguin

etal. - - 3071. 1806. -

1630. (2014) Linkage maps of the Atlanticsalmon ( et al.et

et al. et al.et cloth BC, Brumfield (2016) RT Sequencecapture versus

(2012) An emperor penguin population estimate: the first,

BMC G BMC 193

Nature ClimateChange,

(2014) Projected continent -

1: new1: fortools the analysis of genome (2012) Effects ofclimate change on an emperor penguin (2009) Demographic models and IPCC climateprojections the Scotia Arcand Western Antarctic Peninsula. , 515 enomi - 528. cs,

15 PLoS ONE, , 166. n,

, 489 Proceedings ofthe National Academy

4 , 715

7 - wide dec - , e33751. 496. - BMC G 718.

and thefrequency theof Molecular Phylogenetics Systematic B lines of the emperor Global Change Biology, enetics,

Salmo salarSalmo volution - wide SNP data.

11 Xenops .

, 94. Academic iology, ) genome Polar Polar Global

65 ,

This article isprotected rights by All copyright. reserved. RambautA, Drummond AJ (2007)Tracer v1. 4. AcceptedPritchard JK, Stephens M, Donnelly (2000)P Inferenceof population structure using multilocus Pamilo P, Nei M (1988) Relationships between gene andtrees species trees. Palsbøll PJ, Berube AllendorfM, FW(2007)Identification of management units using population S,Nicol Foster J, Kawaguchi S(2012) fisheryThe for Antarctic krill Mougin JL, van Beveren M (1 Meirmans VanPG, Tienderen (2004) PH GENOTYPE and GENODIVE: two programs for the analysis of Meirmans (2012)PG AMOVA H,Lynch LaRue M (2014)First global census theof Adélie penguin. Lowe WH, Allendorf FW (2010) What cangenetics tell usabout population connectivity? Lotterhos KE,Whitlock MC (2014)Evaluation of demographic history and neutral parameterization HEL,Lischer Excoffier (2012)L PGDSpider: anautomated dataconversion tool for connecting Article H(2013)Li Aligning sequencereads, clonesequences and assemblycontigs with BWA LaRue M, Kooyman G, Lynch H,Fretwell (2015)P Emigration in emperor penguins: implications for Kooyman GL, Ponganis PJ (2016) Rise and fall ofRoss emperorSea penguin colony populations: Kooyman GL, Ponganis PJ (2008) The initial journey ofjuvenile emperor penguins. Kooyman GL, Kooyman TG, Horning M, Kooyman CA(1996) Penguin dispersal afte Kooyman GL, Hunke Ackley EC, SF, van Dam RP, Robertson G (2000) Moult of the emperor Keenan K, McGinnity P, Cross Crozier TF, WW, & Prodöhl (2013) PA genotypedata. and E geneticdata. and Fisheries, Rendusde l’Acad Aptenodytes forsteri geneticdiversity asexualof organisms. 744 Ecology, theon performance F of populat arXiv:1303.3997preprint interpretation longof 2000 to 2012. Conservation: Marine and Freshwater Ecosystems, 383 277. penguin: travel, location, and habitat selection. Ecology and Evolution estimation of population genetics paramet , 397. - 750.

volution, ion genetics and genomics programs.

, 3038 Trendsin Ecology and E

Antarctic Science 5 Genetics, , 568 é , 30 - mie desmie Sciences, 3051. 979)Structure et dynamique dela population de manchots empereurs dela colonie l’archipel de de Pointe Géologie, TerreAdélie. - - - based clustering p of , - 40. t 583. 4 erm studies.

, 782 ST

155

outlier outlier tests.

, 945 - 788 , in press. . -

959. 289 Ecography, volution,

Molecular Ecology Notes,

Molecular Ecology, ,

157 opulation geneticdata.

ers and their associated errors. - 160 Bioinformatics,

38 , 11 . Marine Ecology Progress Series,

, 114

17 - 16.

, S37 - 120.

The Auk,

23 diveRsity: A -

S43. 28 , 2178 –

, 298 recent developments.

, 792 Journal ofHeredity,

- 131 2192. - 299. - n R package for the Molecular Biology , 457 794.

r fledging.

Methodsin

- 466. - MEM. Aquatic Comptes Molecular

204 Nature, arXiv

103 , 269 Fish , -

This article isprotected rights by All copyright. reserved. AcceptedWapl Walther G Vaughan Comiso DG, JC,Allison I Trathan PN, García Trathan PN, Fretwell PT, StonehouseB (2011)First recorded loss an of emperor penguin colony in Toews DP, Taylor SA, Thiebot J Taylor BL, Wade PR, De Master DP, Barlow J (2000) Incorporating uncertainty into management Article Taylor BL, Tavecchia G, Tenan S, Pradel R Storey JD, Tibshirani R (2003)Statistical significancefor gen Stammerjohn S, Massom R, Rind D, Martinson (2012)D Regions of rapid sea ice change: aninter Slatkin M (2005)Seeing ghosts: the effect unsampledof populations migrationon rates estimated Rosenberg NA (2004) DISTRUCT:program a forgraphical the display populationof structure. Roeder AD, Marshall RK, Mitchelson AJ Ritchie PA, Millar CD, Gibb GC, Baroni C, Lambert DM (2004) Ancient DNA enables timing theof es RS,es Gaggiotti O (2006)is What population? a An empirical evaluation of geneticsome Molecular Ecology, methods for identifying the number geneof pools and their degree connectivity.of 416 USA. pp. 317 the Intergovernmental Panel on Climate Change The Physical Science Basis. Contribution ofWorking Group theFifth Ito AssessmentReport of changeas critical threats to penguins. 6 the recent period A of hybridizingof warblers. Science, juvenile emperor penguins Aptenodytes forsteri during post models for marine mammals. conservation driven population. National Academyof Sciences, hemispheric seasonal comparison. sampledfor populations. Molecular Ecology Notes, among Adélie penguin colonies around Antarctica. Molecu Pleistocene origin and Holocene expansion of two Adéliepenguin lineages inAntarctica. - , e14738. B, Lescroël A, BarbraudBost C, C - R, E,Post Convey P Dizon , 389

lar Biology and E –

25 - 382. Cambridge University Press, Cambridge, United Kingdom and New York, NY, 395. AE (1996

‐ , 536 Borboroglu P, Boersma D VallenderR .

Conservation Biology, - 544.

Global Change Biology, 15 )

The needThe to estimate power to link genetics and

, 1419 ntarcticregional warming: implications for othercolonies.

et al.et etal. Current Biology, volution,

etal.

et al. Molecu 4 , 137

(2002) Ecological responses to recent climate change. - (2016) Climate 1439.

(2013) Observations: Cryosphere. In:

100 (2016) Plumagegenes and little elsedistinguish genomes the Conservation Bi et al.et - 138.

lar Ecology, 21 -

, 9440 Geophysical Research Letters, A (2013)Three , 240

(2001)Gene flow theon ice: geneticdifferentiation

etal 1 Conservation Biology 0

, 26 - - 661 248. . (2015) Pollution, habitatloss, fishing, and climate 9445.

22 , 2313 - driven ratesvital do notalways mean climate

14 , 3960 -

664

ology, , 67

(eds. StockerTF, Qin D, Plattner G - - 2318. . dimensional use of marinehabitats by

Molecular Ecology, - omewide studies. – 73. 3966.

, 1243

, - 29 natal dispersal. , 31 - 1252.

39 - 41. , L06501. ClimateChange 2013: Proceedingsof the

demography for , 1645 Antarctic

- 1656. PLoS ONE, Nature, - K , et al.

- -

This article isprotected rights by All copyright. reserved. samples, Acceptedanalyzing the the manuscript and participated in conc JY & Contributions Author http://dx.doi.org/ SNPfinal dataset available from the Dryad https://www.ncbi.nlm.nih.gov/bioproject/ IlluminaThe short reads are available Accessibility Data ArticleYoungerJL, Clucas GV, Kooyman G YoungerJ, Emmerson SouthwellL, C, LelliottP, Miller (2015a)K Proliferation of EastAntarctic Adélie WrightS (1931) Evolution in Mendelian populations. Wilson GA, Rannala B (2003)Bayesian inferenceof recent migration ratesusing multilocus Wienecke B, Raymond B, Robertson G (2010) Maiden journey of fledgling emperor penguins from Wienecke B, Kirkwood R, Robertson G (2004) Pre BS,Weir Cockerham CC (1984) Estimating F Weimerskirch H, Jouventin P, Mougin J, Stahl J, Van GC carried GC carried out molecular laboratory work, analyzed thedata, interpreted the data,drafted Biology, forced emperor penguins into refugia duringlast the glacial maximum. penguins inresponse to historical deglaciation. genotypes. the Mawso emperor penguins at the Mawson Coast. Evolution 33. dispersal ofseabirds breeding in French Austral and Antarctic Territories. participated in

data.

21 , is 38 , 2215 10.5061/dryad.4s7t3 n Coast, East Antarctica. Genetics, AR & KG available the from Dryad , 1358 - interpret 2226.

Digital R Digital - 1370.

163 participated in conceiving and designing the study. TH collected

, 1177

et al. ing epository

the data and conceiving and designi - 1191. from (2015b) To . eiving and designing the study. DK participated in

- Marine Ecology Progress Series, statistics for the analysis of population structure. 384210

the NCBI sequence read archive, http://dx.do Digital R Polar Biology, o o much good a of thing: seaice extent have may - moult foraging trips and moult locations of Genet

. Our custom python script (filter.py) BM (1985) Banding recoveries and the BMC Evolutionary Biology, epository ics, i.org/10.5061/dryad.7c0q8

, 97 27

, 83 - 159. - 91.

410

ng the study. KM Global Change , 269

15 - , 1 282. Emu, - 11.

and our

is , 22 - This article isprotected rights by All copyright. reserved. ( Figure 1.Map showing Figures n declare The authors read and approved the final manuscript. participated in conceiving and designing the study and in interpretation of the data. All authors AcceptedLaRue Article

et al.

201 5 ) . The colonies sampled in this study are labe the locations of o competing financial interests financial competing o all known emperor penguin colonies around Antarctica

l led.

This article isprotected rights by All copyright. reserved. Acceptedvertical bar, with thecolors showing the proportion ofancestry assigned to each ofthe clusters. averaged forthe ten Figure Article 2 . Structure results showing population structure from

replicate runs that were performed at each value of

= 2 to K

= 6. The results are K . Each . Each individual a is

This article isprotected rights by All copyright. reserved. Accepted components are shown in black on the inset graphs and were a) 27 and b) 23 grouped by a) colony and b) major geographic region Figure 3.Results from Article Discriminant Analysis ofPrincip . The . The al

Components numbers ofretained

when individuals are .

principal

This article isprotected rights by All copyright. reserved. Accepted correction for multiple tests are shown in bold. diagonal, with associated Figure Article 4. Genetic differentiation between allpairs of colonies. p - values above the diagonal. Significant

F ST

values p - values after SGoF+

are shown below the

This article isprotected rights by All copyright. reserved. Accepted Article below the diagonal, with associated Figure P A M o W m i a n R 5 w e a t o n d e . s s d d

o Genetic differentiation between all major geographic regions G s a e n

é S

B l C o e

S a o a l o e y

a g a

s & i

t e

W e 0 0 0 d * . . . d 0 0 0 * e 0 0 0 * l l 5 2 4

e a

M a w < 0 0 s 0 p * . . o . 0 0 * - 0 n 0 0 * values above the diagonal. 0

5 3 C

a s t

P A o m i n a t < 0 0 n e 0 * . . d

. 0 0 G * 0 a 0 0 * é 0

B 4 4

o 1

a l

o y g

& i e

R < < < o 0 0 0 * s

. . . s * 0 0 0

* S 0 0 0

e 1 1 1 a

. F ST

values arevalues shown This article isprotected rights by All copyright. reserved. Accepted Article

and Amanda (orange), theMawson Coast (purple), in the Ross (green),Sea Weddell Sea metapopulations ofemperor penguins that there are at least four and c)this study based on SNPs found continent (blue) (Cristofari single populatio fully panmictic species consisting ofa concluded that emperor penguins are a 2015b); b) a study based on SNPs AntarcticaEast (pink) (Younger and one spanning the Weddell Sea and populations, one in the Ross (green)Sea mitochondrial DNA stud coloured shading: a) our original populationswith indicated with e noted inside the triangle. the numberwith of individuals sampled colonies included in the study design, 2015). The coloured triangles indicate emperor penguin colonies (LaRue (pink). Grey triangles indicate allknown Figure mperor penguin population structure

6 . Our evolving understanding of

Bay/ n spanning the Antarctic Pointe Géologie y found two

et al . 2016); et al

et al . , . ,

This article isprotected rights by All copyright. reserved. the mean. of error standard H heterozygosity; N filtering. after only) retained sites SNPs (variant 4,596 of dataset the on based colony, each for indices diversity 2. Genetic Table Putativelyneutral Single SNPper RAD Genotyped80%> in Heterozygosity0.5 < Minorallelefrequency > 0.01 Afterrunning rxstacks Initialstacks catalog Filter the filters of each applying after SNPs retained of number 1. The Table AcceptedWashington Cape CapeRoget Géologie Pointe Bay Amanda Auster Island Fold Bay Halley Bay Gould Article

11 10 15 16 16 16 13 13 N

- individualsper population tag O

1 1 6 9 5 11 6 7 Alleles Private

–

observed heterozygosity; π heterozygosity; observed

0.1174 0.1151 0.1227 0.1243 0.1223 0.1231 0.1223 0.1218 (mean) H E

–

number of individuals successfully sequenced, H sequenced, successfully individuals of number

0.0186 0.0196 0.018 0.0173 0.0173 0.0177 0.0182 0.0178 (var) H

SNPsretained 4,596 4,600 9,857 30,125 30,441 64,320 423,479 0.002 0.0021 0.002 0.0019 0.0019 0.002 0.002 0.002 (stdErr) H E

–

nucleotide diversity; var diversity; nucleotide 0.1134 0.1071 0.1131 0.1171 0.1189 0.1112 0.1187 0.1133 (mean) H

0.0199 0.0201 0.0174 0.0171 0.0177 0.0164 0.0192 0.0176 (var) H O

0.0021 0.0021 0.0019 0.0019 0.002 0.0019 0.002 0.002 (stdErr) H O

–

0.1232 0.122 0.1269 0.1283 0.1264 0.1271 0.1265 0.1261 (mean) π variance; stdErr stdErr variance;

–

expected expected 0.0205 0.022 0.0192 0.0184 0.0185 0.0189 0.0194 0.0191 (var) π

0.0021 0.0022 0.002 0.002 0.002 0.002 0.0021 0.002 (stdErr) π –