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Evolutionary Applications. 2018;1�16. 科|科1wileyonlinelibrary.com/journal/eva

ゲ科 |科INTRODUCTION

Over the last 12,000 years, human populations in many different re-

gions of the world independently domesticated local plant species

by selecting for desirable traits, in many cases initiating a symbiotic

partnership that formed the economic foundation of complex societ-

ies (Zeder, 2015). Researchers have identified over a dozen centers

of plant domestication (Purugganan & Fuller, 2009), and gaining a

refined understanding of the varied evolutionary trajectories that

have led to the emergence of key crops requires investigating the

cultivars and the archaeological context found in each of the world�s

independent centers of domestication. Eastern North America (ENA)

presents a useful case to examine initial plant domestication and

millennial- scale changes in agriculture (Smith, 2011), in part because

ReceivedrザNovemberゴグゲゼ科 |科 Acceptedrゴ葦DecemberゴグゲゼDOIrゲグsゲゲゲゲ｠evasゲゴズゾジ

O R I G I N A L A R T I C L E

Ancient DNA revea旭s the timing and persistence of organe旭旭ar genetic bott旭enecks over ザpグググ years of sunf旭ower domestication and improvement

Nathan Wa旭es1 科|科Me旭is Akman1科|科Ray Hs Bs Watsonゲpゴ科|科F史tima S史nchez Barreiroザ科|科

Bruce Ds Smith4科|科Kristen Js Gremi旭旭ion5科|科Ms Thomas Ps Gi旭bertザp葦科|科Benjamin Ks B旭ackmanゲpゴ

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,

provided the original work is properly cited.

© 2018 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd

1DepartmentofP旭antandMicrobia旭Biology, University of California, Berkeley,

CA, USA

2DepartmentofBio旭ogypUniversityofVirginiapChar旭ottesvi旭旭epVApUSA3Centre for GeoGenetics, Natural History

MuseumofDenmarkpUniversityofCopenhagenpCopenhagenpDenmarkジTheSantaFeInstitutepSantaFepNMpUSA5DepartmentofAnthropo旭ogypOhioStateUniversity, Columbus, OH, USA

6Norwegian University of Science and

Techno旭ogypUniversityMuseumpTrondheimpNorway

CorrespondenceNathanWa旭espDepartmentofP旭antandMicrobia旭Bio旭ogypUniversityofCa旭iforniapBerkeley, CA, USA.

Emails: nathan.wales@berkeley.edu; wales.

nathan@gmail.com

Funding informationNationa旭ScienceFoundationpDivisionof Environmental Biology, Grant/Award

Numberrゲザズジ葦ゴゴandゲ葦ジグゼ芦芦qDanmarksGrundforskningsfond

AbstractHere, we report a comprehensive paleogenomic study of archaeological and ethno-

graphic sunflower remains that provides significant new insights into the process of

domesticationofthis importantcropsDNAfrombothancientandhistoriccontextsyie旭dedhighproportionsofendogenousDNApanda旭thougharchaeo旭ogica旭DNAwasfound to be highly degraded, it still provided sufficient coverage to analyze genetic

changes over time. Shotgun sequencing data from specimens from the Eden�s Bluff

archaeo旭ogica旭siteinArkansasyie旭dedorgane旭旭arDNAsequencefromspecimensupto 3,100 years old. Their sequences match those of modern cultivated sunflowers and

are consistent with an early domestication bottleneck in this species. Our findings also

suggest that recent breeding of sunflowers has led to a loss of genetic diversity that

was present only a century ago in Native American landraces. These breeding epi-

sodes also left a profound signature on the mitochondrial and plastid haplotypes in

cultivars, as two types were intentionally introduced from other Helianthus species for

crop improvement. These findings gained from ancient and historic sunflower speci-

mens underscore how future in- depth gene- based analyses can advance our under-

standing of the pace and targets of selection during the domestication of sunflower

and other crop species.

K E Y W O R D S

ancientDNAparchaeobotanypdomesticationpgeneticbott旭eneckpHelianthus annuus,

paleogenomics, plant evolution, sunflower

ゴ科 |科 科架 WALES ET AL.

its archaeological record challenges the paradigm that domestication

is an evolutionary strategy implemented when expanding human

populations experience declining resource catchments (Smith, 2016).

Starting around ジグググyears before present ｪBPｫp a crop comp旭exconsisting of acorn/crookneck squash (Cucurbita pepo L. ssp. ovifera

DsSs Deckerｫp goosefoot ｪChenopodium berlandieri Moqsｫp marshe旭-der (Iva annua L.), and the common sunflower (Helianthus annuus

L.) was grown by low- level food- producing societies inhabiting the

watershedof theMississippiRiver ｪSmithpゴググ葦ｫsArchaeobotanica旭remains from ENA sites exhibit telltale signs of the so- called domes-

ticationsyndrome ｪHammerpゲゾ芦ジｫpasuiteof traits thatcommon旭ydistinguishes domesticates from their wild progenitors and that may

include larger seeds and disruption of natural seed dispersal mecha-

nisms. Of the four core species of the ENA crop complex, sunflower

is particularly well suited for in- depth domestication research thanks

to theexistenceof richarchaeobotanica旭 co旭旭ections ｪSmithpゴグゲジｫpacenturyofbreedingexperimentsｪHeiserpゲゾゼ葦q斎kori賜pゲゾゾゴｫpandthe development of many germplasm and genomic resources for

genetic investigations (Badouin et al., 2017; Burke, Tang, Knapp, &

Rieseberg, 2002; Kane et al., 2011; Rieseberg & Seiler, 1990; Wills

& Burke, 2007).

Through human selection, the weedy H. annuus spp. annuus was

transformed from a highly branching plant with numerous small

disks, also known as heads or capitula, to H. annuus spp. macrocar-

pus ｪDsCsｫCk旭旭sp thecu旭tivatedsunf旭owerpwhich is typica旭旭ycharac-terized by strong apical dominance and a single massive disk that

can produce hundreds to thousands of achenes. Sunflowers served

important nutritional, ceremonial, medicinal, cosmetic, and struc-

tural purposes in Native American cultures. For instance, an account

from 1615 by French explorer Samuel de Champlain indicates that

peop旭esoftheIroquoisConfederacyofNations intheGreatLakesregion of North America cultivated sunflower, grinding and eating

the seeds as well as processing them into oil used ceremonially for

anointing the hair (Heiser, 1951). After roasting sunflower achenes in

c旭aypotsorreedbasketsptheMandanpArikarapandHidatsapeop旭esoftheMissouriRiverbasinwou旭dmakesunf旭owerf旭ourorboi旭theachenes with maize, beans, and squash to make a porridge (Heiser,

1976). The Hopi people of the American Southwest were unique in

extracting a dye from the deeply purple- colored achenes of their

landraces (Heiser, 1951, 1976).

Archaeological sunflower remains have been excavated from doz-

ens of ENA sites, enabling temporal and spatial investigations on the

originsofsunf旭owerdomesticationsTheKostersiteinI旭旭inoisyie旭dedthe oldest known sunflower remains, with two achenes and one kernel

datingbetween芦ズググandズ芦ググBPｪAschｹAschpゲゾ芦ズqSmithpゴグゲジｫ(Figure 1). Based on their small size, these specimens likely reflect the

co旭旭ection ofwi旭d resources ｪSmithp ゴグゲジｫs The o旭dest evidence forsunflower cultivation comes from the Hayes site in central Tennessee,

datingtoズグザジ･ジズ芦ザBPｪゾズ鯵confidenceinterva旭pCIｫｪCritespゲゾゾザｫsKernels from the site are larger than commonly observed in wild sun-

flowers, suggesting the initial steps of sunflower domestication were

underwaycircaジ芦ググBPｪSmithpゴグゲジｫsThreeothersitesprovideev-idence of sunflower cultivation before 3000 BP (Figure 1): 3800 BP

attheRivertonsiteinI旭旭inoisｪSmithｹYarne旭旭pゴググゾｫpザザググBPattheNewt Kash She旭ter in Kentucky ｪSmithp ゴグゲジｫp and ザグズグ BP at theMarb旭eB旭uffShe旭terinArkansasｪFritzpゲゾゾゼｫs

Based on archaeological, morphological, and geographical data,

Heiser (1951) concluded that sunflower was domesticated once in

ENA, a hypothesis that has been supported by population genetics

studies of modern elite- bred cultivars, extant Native American landra-

ces, and wild H. annuus populations. For instance, Rieseberg and Seiler

(1990) demonstrated with isozymes and chloroplast markers that do-

mesticated landraces share haplotypes with wild sunflowers from ENA

and show a signature of a genetic bottleneck. Although archaeological

remainsputative旭y identifiedassunf旭owerp somedating toジゲザグBPpweresubsequent旭yrecoveredfromexcavationsinMexicoandraisedthe possibility of an independent domestication event (Lentz, Pohl,

Pope, & Wyatt, 2001), population genetic studies that include extant

Mexican wi旭d and cu旭tivated germp旭asm have on旭y found evidencethat extant cultivars derive from a single ENA domestication event.

Wills and Burke (2006) showed that domesticated populations have

one common and two rare chloroplast microsatellite marker haplo-

typesthatc旭usterwithwi旭dENAratherthanwi旭dMexicansunf旭owerssPatterns of sequence variation at nuclear microsatellite markers and

candidate domestication loci have likewise reinforced the conclusion

thata旭旭extant旭andracespwhetherco旭旭ectedinENAorMexicopdescendfrom a single origin most likely occurring from ancestral wild popula-

tions in the eastern and central USA (Blackman et al., 2011; Harter

eta旭spゴググジｫsAlthough archaeological and genetic data predominantly point

to a single domestication event in ENA, there is much more to un-

earth about how sunf旭ower domestication proceededs It remainsto be determined which traits were of primary interest to early

farmers, whether sunflower domestication was rapid or protracted,

and how proto- domesticates responded to the new selection re-

gime. Genetic characterization of archaeological plant remains with

ancient DNA ｪaDNAｫ methodo旭ogies has the potentia旭 to answerthese questions by providing windows into past temporal dynam-

ics. Paleogenomic research has grown tremendously in the past

decade due to the rapid development of high- throughput sequenc-

ing techno旭ogies ｪDer Sarkissian eta旭sp ゴグゲズｫp and the app旭icationof paleogenomic methods to archaeobotanical remains has been a

particular success (Brown et al., 2015). For example, in reconstruct-

ing complete genomes of 6,000- year- old barley grains excavated

in Israe旭p Mascher eta旭s ｪゴグゲ葦ｫ determined the ancient samp旭eswere closely related to modern cultivars in the region and that the

major steps of barley domestication were completed by this point

in times Simi旭ar旭yp Ramos､Madriga旭 eta旭s ｪゴグゲ葦ｫ and Va旭旭ebueno､Estrada et al. (2016) characterized genomes of 5,000- year- old

maizecobsfromtheTehuac史nVa旭旭eypbutthey insteadfoundthatmany domestication- related genes had the ancestral form rather

than the derived maize form, suggesting a stepwise process of do-

mestication. Although these paleogenomic studies indicate archae-

ological remains could be invaluable for understanding sunflower�s

domestication and ancient cultivation, different plant species have

the potentia旭 to confound aDNA research through species､ and

科架 科 | 科ザWALES ET AL.

tissue､specific secondary compounds that interfere with DNAextraction and library preparation. To examine the paleogenomic

potential of archaeobotanical sunflower remains, we screened a

collection of archaeological and ethnographic specimens with a

shotgun sequencing strategy. The sequencing data generated from

these ancient and historic specimens were analyzed to determine

variabi旭ity in endogenous contentp DNA damagep and sources ofexogenousDNAs In additionp fo旭旭owing precedents inmamma旭ianaDNA projects ｪDabneyp Knappp eta旭sp ゴグゲザq Gi旭bert eta旭sp ゴググゼｫand genome skimming of modern samples (Bock, Kane, Ebert, &

Riesebergp ゴグゲジq Straub eta旭sp ゴグゲゴｫpwe 旭everaged the sequenc-ing data to characterize variation in high copy number mitochon-

drial and plastid genomes, allowing us to investigate how these and

other archaeological and historic specimens may enrich our under-

standing of the domestication process.

ゴ科 |科MATERIALS AND METHODS

ゴsゲ科|科Archaeo旭ogica旭 sunf旭ower specimens

Although archaeobotanical remains are most often preserved by

charring or carbonization, such materials are generally incompat-

ible with paleogenomic analyses (Nistelberger, Smith, Wales, Star, &

Boessenkool, 2016). Therefore, we only obtained and processed des-

iccated specimens for this study. We tested 15 sunflower disk frag-

ments, one pericarp (seed coat), and one kernel, all of which originate

F IGURE  ゲ科Mapofsamp旭ing旭ocationsandarchaeo旭ogica旭sitessEthnographicsamp旭esｪandnumberofaccessionssamp旭edｫareinredpandlandraces are in blue. Archaeological sites with ancient sunflower material discussed in the text are marked by yellow circles. Eden�s Bluff, the

site from which all archaeological remains detailed in this article were sampled, is bolded

ジ科 |科 科架 WALES ET AL.

from the Eden�s Bluff archaeological site in northwestern Arkansas

(Figures 1 and S1; Table 1). The specimens have been under the cu-

ration of the University of Arkansas Collections Facility (UARK) and

theUniversityofMichiganMuseumofArchaeo旭ogica旭Anthropo旭ogyｪUMMAAｫsThirteendisksthatweresufficient旭yintacttoenab旭ediam-

eter measurements ranged in size from 35 to 110 mm (mean = 75.5)

and were all larger in this dimension than disks of a well- defined wild

H. annuus popu旭ation ｪSmithp ゴグゲジｫp indicating the archaeo旭ogica旭disks represent plants cultivated by humans. Likewise, the dimen-

sions of the archaeological pericarp (length = 9.1 mm) and kernel

(length × width = 6.5 × 3.6 mm) are consistent with origin from do-

mesticated sunflowers.

EdenvsB旭uffｪstatesiteIDrザBE葦ｫwasexcavatedinゲゾザゴandゲゾザジas a part of expeditions led by the University of Arkansas focused

on the so､ca旭旭edOzarkB旭uff､Dwe旭旭er sitesp as coinedbyHarringtonｪHarringtonpゲゾゴジapゲゾゴジbpゲゾ葦グｫsThesesitesarerenownedfortheirpreservation of organic remains, including desiccated plant tissues

(Fritz, 1986; Gilmore, 1931). Native Americans likely used the rock-

shelters and caves specifically because their dry conditions were well

suited for long- term food storage and, despite the name, are unlikely

tohaveservedasseasona旭dwe旭旭ingsｪBrownpゲゾ芦ジｫsThechrono旭ogyof theOzarkB旭uff､Dwe旭旭er sites isnot fu旭旭yunderstoodpdue to the旭imited number of radiocarbon dates ｪDavisp ゲゾ葦ゼｫs As part of herrigorous archaeobotanical analyses, Fritz (1986) acquired dates from

15 sites and determined occupations occurred throughout the period

from ca. 3000�500 BP. Because their stratigraphic context may have

experienced disturbance from humans, rodents, or other causes, we

submittedゲジoftheゲゼsamp旭esfordirectacce旭eratormassspectrom-

etry ｪAMSｫ radiocarbondatingat theUniversityofArizonaAMSfa-ci旭ity ｪTab旭eゲqFigureSゴｫsA旭旭AMSdates fromthisandother reportswere calibrated to calendar years before present (calBP) using OxCal

vジsザsゴ ｪBronkRamseyp ゴググゾｫ and the IntCa旭ゲザ ｪReimer eta旭sp ゴグゲザｫcalibration curve.

ゴsゴ科|科Ethnographic 旭andrace achenes

Eleven accessions of sunflower landraces were acquired from eth-

no旭ogica旭co旭旭ectionsattheNationa旭MuseumoftheAmericanIndianｪNMAIｫandUMMAAｪTab旭eゴpFigureゲｫsThesespecimensconsistofachenes sourced from Native Americans and via various intermediar-

ies in the first half of the twentieth century by Gilmore (1919) and

HeiserｪゲゾズゲｫsAtthetimepHeiserｪゲゾズゲppsジジゲｫ旭amentedthatwfewaboriginal strains of the cultivated sunflower are still in existence,

and� it is likely that the few remaining ones will disappear unless

steps are taken to preserve them.� While his efforts propagated many

sunflower landrace lineages, some of the achenes he attempted to

grow were not viable, including seed originating from the Six Nations

reserve in Ontario. Thus, these ethnographic achenes offer a unique

opportunity to investigate genetic relationships of putatively extinct

landraces to living sunflower lineages.

ゴsザ科|科DNA extraction and sequencing

Archaeological specimens were processed at a dedicated paleog-

enomics laboratory at the University of Copenhagen. The laboratory

meets the standards for aDNA research ｪCooper ｹ Poinarp ゴグググqGilbert, Bandelt, Hofreiter, & Barnes, 2005), such as being physi-

ca旭旭yseparatedfrommodernDNAandpost､PCR旭aboratoriespbeingoutfittedwithairfi旭trationandnight旭yUVirradiationequipmentpandrequiring researchers to wear coveralls to minimize contamination.

DNAwasextractedusingamethodthathasbeenshowntoworkwe旭旭on a range of species and tissue types (Wales, Andersen, Cappellini,

Specimen Tissue ca旭BP ｪゾズ鯵 CIｫ Endogenous DNA P旭astome DoC

Eden- 1 Pericarp Not dated グsザ鯵 0.8

Eden- 2 Diskfragment 915�795 ゲゲsゾ鯵 ズsジ

Eden- 3 Diskfragment 3168�3005 ゴsゲ鯵 3.2

Eden､ジ Diskfragment Not dated グsゴ鯵 0.3

Eden- 5 Diskfragment ゲゼザ葦･ゲズゼジ ゲジsゲ鯵 8.3

Eden- 6 Diskfragment 3163�2999 ズs葦鯵 7.2

Eden- 7 Diskfragment 1813�1622 芦s葦鯵 7.6

Eden- 8 Diskfragment 1817�1628 ゲゾsゲ鯵 ジsゲ

Eden- 9 Diskfragment 1819�1633 ジ芦sザ鯵 16.5

Eden- 10 Diskfragment 1873�1629 ザズsザ鯵 5.6

Eden- 11 Diskfragment 1825�1618 ザゲsゲ鯵 17.5

Eden- 12 Diskfragment 1868�1701 ザジsゲ鯵 ゴジsグ

Eden- 13 Diskfragment 1810�1571 ズズs葦鯵 18.1

Eden､ゲジ Diskfragment 1877�1711 ゾsゾ鯵 8.3

Eden- 15 Diskfragment 1770�1559 ゴズsゼ鯵 30.6

Eden- 16 Diskfragment 1819�1639 ザズs葦鯵 ゲジsゼ

Eden- 17 Kernel Not dated グsゲ鯵 1.1

TABLE  ゲ科Archaeological specimens.

Acce旭eratormassspectrometryｪAMSｫdates are listed in calibrated years before

present. Samples with sequencing depth of

coverageｪDoCｫ┑ジforthep旭astomewereexcluded from the plastome analysis. See

Figure S1 for images of most samples and

Tab旭eSゲforadditiona旭samp旭epAMSpandsequencing information

科架 科 | 科ズWALES ET AL.

蛍vi旭a､ArcospｹGi旭bertpゴグゲジｫsInbriefptissuesamp旭eswereco旭旭ectedwith disposable forceps and scalpels, placed in PowerBead tubes

ｪMOBIOゲザゲゲゼ､ズグｫpandpu旭verizedbyshakingatジm｠sforザグs ina FastPrep､ゴジ homogenizer ｪMP Biomedica旭sｫs The resu旭ting tissuepowderwas incubatedovernight inadigestionbuffer ｪゲグmMTris､HC旭pゲグnMNaC旭pゴ鯵w｠vSDSpズmMCaC旭2pゴsズmMEDTApジグmMDTTpandゲグ鯵proteinaseKso旭utionｫpandthenextractedusingtworounds of phenol and one round of chloroform. To minimize the effect

of co､extracted compounds andpigmentsp the recoveredDNAwaspurified in aQiagenMinE旭ute co旭umn using optimizations to retainhigh旭yfragmentedDNAｪDabneypKnapppeta旭spゴグゲザｫsFourextractionblanks were processed with samples to monitor potential sources of

contaminations The extractedDNAp inc旭uding that from the extrac-tion b旭anksp was converted to I旭旭umina､compatib旭e 旭ibraries using ablunt- ended adapter ligation approach and optimizations to retain

short molecules (Wales et al., 2015). Before indexing PCR, the librar-

ies were tested by quantitative PCR (qPCR) to estimate the appropri-

ate number of cycles to avoid overamplification. qPCR was conducted

withaSYBRGreenassayasdescribedbyWa旭eseta旭s ｪゴグゲズｫpusingAmp旭iTaqGo旭dｪApp旭iedBiosystemspFosterCitypCAｫpprimersISゼandIS芦ｪMeyerｹKircherpゴグゲグｫpandaRocheLightCyc旭erジ芦グRea旭､timePCR System. Libraries were amplified with AmpliTaq Gold for 10�18

cycles (Table S1) using a P7 indexing oligo with a 6- bp sample- specific

barcode to enab旭e mu旭tip旭ex sequencing ｪMeyer ｹ Kircherp ゴグゲグｫsLibraries were pooled and shotgun- sequenced on six whole or partial

旭anesofan I旭旭uminaHiSeqゴズググ insing旭e､readmodewith芦ゲorゾジsequencing cycles (Table S1).

The 11 ethnographic samples were deemed to be relatively well

preserved and thus to pose a potential contamination risk to archae-

ological samples. Therefore, the achenes were extracted in steril-

ized 旭aminarf旭owhood inapre､PCRmodernDNA旭aboratoryattheUniversity of Copenhagen where sunflowers had not been previously

tested. Achenes were frozen in liquid nitrogen and fragmented with

a steri旭e pest旭es DNA was extracted with a Qiagen P旭ant Mini kit

following the manufacturer�s protocol except that the 65°C incubation

was conducted for ゴhrsMany specimens exhibited high､mo旭ecu旭ar､weightDNAonanagarosege旭psoDNAwasshearedwithaDiagenodeBioruptor using an appropriate number of sonication cycles for each

sample (Table S1). One accession (Seneca_striped_12997- 682) was

processedtwicepusingawho旭eacheneandanindividua旭kerne旭sDNAwasconvertedtoI旭旭umina旭ibrariesfo旭旭owingthesameprotoco旭usedfor the archaeological samples and sequenced on one lane of an

I旭旭uminaHiSeqゴズググinsing旭e､readmodewith芦ゲsequencingcyc旭ess

ゴsジ科|科Sequencing data processing

Raw sequencing reads were processed using Paleomix 1.2.12

ｪSchubert eta旭sp ゴグゲジｫp a bioinformatic pipe旭ine deve旭oped foraDNA datasetss The recommended parameters for pa旭eogenomicdatasets were utilized, including removing adapter sequences with

AdapterRemoval 2 (Schubert, Lindgreen, & Orlando, 2016), mapping

of readswithBWAa旭nwith the seeddisab旭ed ｪLiｹDurbinpゴググゾｫpremoval of duplicate reads with Picard Tools (http://broadinstitute.

githubsio｠picardｫprea旭ignmentaroundinde旭swithGATKザsゼｪMcKennaeta旭spゴグゲグｫpandresca旭ingofbasequa旭itiesduetoaDNAdamagewithmapDamageゴsグ ｪJ祝nssonp Gino旭hacp Schubertp Johnsonp ｹ Or旭andop2013). Reads were mapped against the entire sunflower XRQ draft

genome (Badouin et al., 2017), including unplaced contigs, the plastid

genomepandthemitochondria旭genomesWereportendogenousDNAcontent based on all mapped reads, regardless of mapping quality, be-

cause high content of long terminal repeat retrotransposons in the

sunf旭owergenomeｪゼジsゼ鯵ofthegenomepBadouineta旭spゴグゲゼｫcausemany endogenous reads to map to multiple loci. As we observed po-

tential erroneous insertions of the organellar genomes in the nuclear

assembly, reads were also separately mapped to the plastid genome,

mitochondrial genome, and the nuclear genome without unplaced

contigs; these alignments were only used for organellar genome and

library complexity analyses.

TABLE  ゴ科Ethnographic achenes from Native American sunflower landraces. Three Seneca achenes are reported to have been collected in

NorthDakotaｪindicatedwithanasteriskｫqhoweverpora旭traditionsandwrittenrecordsindicatethese旭andracesoriginatedfromthetraditiona旭lands of the Seneca people near Lake Ontario

Specimen Repository Location Year Co旭旭ector

Arikara 122976 NMAI FortBertho旭dReservationpNorthDakota 1923 MsRsGi旭more

Arikara 126306 NMAI NorthDakota ゲゾゴジ MsRsGi旭more

Arikaraゲジグジゴ､芦ゼジ UMMAA BismarckpNorthDakota 1932 George F. Will

Arikara broad 12999- 682 UMMAA BismarckpNorthDakota N/A George F. Will

Arikara｠Mandanゲザゼジゼ UMMAA Dakotas 1933 MsRsGi旭more

Paiuteゲジゲ芦ズ葦 NMAI MoapaRiverReservationpNevada 1920s MsRsHarrington

SanI旭defonsoPueb旭oゲザズゾゼ､ゼジゼ UMMAA SanI旭defonsoPueb旭opNewMexico N/A Jose Aguilav

Senecaゲザゼゼジゾ NMAI A旭旭eganyReservationpNewYork 1925 W. Wildshut

Seneca purple 12996- 682 UMMAA BismarckpNorthDakotaｰ 1931 George F. Will

Seneca purple 12998- 682 UMMAA BismarckpNorthDakotaｰ 1931 George F. Will

Seneca striped 12997- 682 UMMAA BismarckpNorthDakotaｰ 1931 George F. Will

NMAIpNationa旭MuseumoftheAmericanIndianqUMMAApUniversityofMichiganMuseumofArchaeo旭ogica旭Anthropo旭ogys

葦科 |科 科架 WALES ET AL.

To place the archaeological and ethnographic samples in con-

text, publicly available sequencing data from 79 modern cultivars,

20 landraces, 27 wild H. annuusindividua旭spandジゼindividua旭sofジother annual Helianthus specieswere down旭oaded from theNCBIsequence read archive (SRA) (Table S2). Because they were se-

quenced with deep coverage, we subsampled and analyzed 30 mil-

lion paired reads for each modern cultivar to reduce computational

time. The entire datasets were used for the other samples. Raw

data were processed in the Paleomix pipeline as discussed above,

exceptthatthemapDamageresca旭ingofbasequa旭itieswasomittedsTo minimize potential biases arising from differences in sequencing

strategies, such as higher theoretical mapping scores from paired-

end than single- read data, the paired- end modern data were treated

as though it was single- read data by trimming and mapping read

mates separately.

ゴsズ科|科Metagenomic ana旭ysis of archaeo旭ogica旭 and ethnographic samp旭es

To characterize non､sunf旭ower sources of DNA iso旭ated from ar-chaeological and ethnographic specimens, 10,000 randomly selected

trimmedpunmappedreadswerecomparedagainsttheNCBInuc旭eo-

tide collection (nr/nt) database using the BLASTn algorithm (Altschul,

Gishp Mi旭旭erp Myersp ｹ Lipmanp ゲゾゾグｫs MEGAN葦 ｪHusonp MitrapRuscheweyh, Weber, & Schuster, 2011) was used to taxonomically

group BLASTn resu旭ts with LCA parametersr Min Score┎ゲグp MaxExpected┎ゲグp Min Percent Identity┎グsグp Top Percent┎グsグググゲpMinSupportPercent┎グsグpMinSupport┎ゲpMinComp旭exityグsグpLCAalgorithm = weighted, Percent to cover = 80, and ReadAssignment

Mode┎readCountsMEGAN葦wasusedtoperformaprincipa旭coordi-nate analysis (PCoA) of Bray�Curtis distances of taxonomic grouping

atthegenus旭eve旭pexc旭udinga旭旭assignmentstoViridip旭antaes

ゴs葦科|科Organe旭旭ar DNA ana旭ysis

Reads mapping to the plastome (plastid or chloroplast genome) or to

themitochondria旭genomewereprocessedwithGATKザsゼｪMcKennaeta旭sp ゴグゲグｫHap旭otypeCa旭旭er andGenotypeGVCFs too旭s to identifypolymorphic sites. Polymorphisms were filtered with GATK according

to recommended parameters for depth, mapping quality, strand biases:

QD┑ゴsグp MQ┑ザグsグp FS┒葦グsグp SOR┒ザsグp MQRankSum┑┋ゲゴsズpand ReadPosRankSum┑┋芦sグs The sites were further fi旭tered withVCFtoo旭s ｪDanecek eta旭sp ゴグゲゲｫ to exc旭ude inde旭s and retain SNPswithaqua旭ityscore┒ゲpグググsArchaeo旭ogica旭samp旭eswith┑ジ┌averagecoverage of the plastome genome were excluded from the analysis.

SNPs were analyzed in R 3.3.1 (R Core Team, 2013) using the Pegas

(Paradis, 2010) package to identify haplotypes, and then, haplotype

relationships were visualized in popart (Leigh & Bryant, 2015) using

a minimum spanning network (Bandelt, Forster, & Röhl, 1999). For

constructionofthehap旭otypenetworkspatota旭ofゼグゲandジゲザpo旭y-morphic sites were used for the plastome and mitochondrial genome,

respectively. One of the oldest samples (Eden- 3) together with three

otherarchaeo旭ogica旭samp旭esｪEden､ゲpEden､ジpandEden､ゲゼｫdidnot

satisfy our filtering parameters and thus were not included in haplo-

type network construction.

ゴsゼ科|科Organe旭旭ar nuc旭eotide diversity ana旭ysis

Nucleotide diversity (pi) per each polymorphic site was computed

usingVCFtoo旭s ｪDaneceketa旭spゴグゲゲｫa旭旭owingforhap旭oidgenomes(haploid switch). For each group, mean nucleotide diversity was cal-

culated by taking average nucleotide diversity of all the sites used

in haplotype network construction for chloroplast or mitochondria.

Landracediversitymetricswereca旭cu旭atedafterexc旭udingMexCu旭tゼandMexCu旭tゲジbecausethosesamp旭eswereco旭旭ected in 旭oca旭mar-kets in Chiapas｠Mexico and are 旭ike旭ymodern cu旭tivars as inferredfrom the haplotype networks.

ザ科 |科RESULTS

ザsゲ科|科Chrono旭ogy

AMS radiocarbon dating of the archaeobotanica旭 remains demon-

strated the specimens originate from three distinct time points: 3100,

1700, and 850 calBP (Figure S2). Eden- 3 and Eden- 6 are the oldest

samp旭espproducingnear旭yidentica旭AMSdatesｪTab旭eSゲｫpandtherebyprovide strong evidence that Eden�s Bluff should be added to the short

list of archaeological sites with sunflower cultivation before 3000 BP.

E旭evenAMSdatesfa旭旭nearゲゼググca旭BPpa旭旭ofwhichover旭apataゾズ鯵CIfromゲゼザ葦toゲゼゲゲca旭BPsThuspthemajorityofthesamp旭esmaybederived from a single occupational phase; however, these specimens

are recorded as being excavated from multiple contexts, suggesting

that some specimens may have been deposited decades or even a few

centuries apart. Eden- 2 produced the youngest date at ca. 850 calBP

(Table 1). While this young disk is an outlier in the chronology of our

otherAMSdatespFritzｪゲゾ芦葦ｫfoundsimi旭ardatesformaizeexcavatedfrom Eden�s Bluff, supporting the inference that this sample belongs

to a more recent occupation.

ザsゴ科|科Shotgun sequencing and endogenous content

Wegeneratedジsゲ･ザグs葦mi旭旭ionrawsequencereadsforthearchaeo-

旭ogica旭 specimens ｪmean┎ゲゴsジMｫp グsジゲ･グs葦芦M reads for the fourcontro旭s ｪmean┎グsズザMｫp and ゲズsゾ･ザズs芦M reads for the ethno-

graphicachenesｪmean┎ゴザsゴMｫbyI旭旭uminasequencingsThearchae-

o旭ogica旭 specimensexhibit endogenousDNAcontents ranging fromグsゲゼ鯵toズズs葦葦鯵ｪmean┎ゴゲsゲ鯵pmedian┎ゲ葦s葦鯵ｫpwithbothachenesandonediskyie旭ding┑ゲ鯵endogenousDNAｪTab旭eゲpFigureゴｫsForゲゲoftheゲゴethnographicspecimensp芦ゾsゲ鯵･ゾザs葦鯵ofDNAmappedagainstthereferencegenomesIntheremainingethnographicsamp旭epArikaraゲゴゴゾゼ葦pon旭yザゼsゾ鯵ofthereadswereendogenousｪseeex-ogenousDNAbe旭owｫsAsidefromonesamp旭ewith旭owendogenouscontentｪEden､ゲゼｫpnuc旭earDNAPCRdup旭icate旭eve旭swere旭owfortheI旭旭umina旭ibrariesonthearchaeo旭ogica旭ｪmeanザsグ葦鯵pmedian┎グsザゴ鯵ｫand ethnographic specimens ｪmean┎ゲsジゴ鯵ｫ ｪTab旭eSゲｫs These 旭owlevels indicate that the libraries contain a great amount of untapped

科架 科 | 科ゼWALES ET AL.

complexity and could be deeply sequenced to recover large portions

of the nuclear genome.

ザsザ科|科DNA degradation

Consistent with the findings from previous paleogenomic studies,

DNArecoveredfromthearchaeo旭ogica旭sunf旭owerswashigh旭yfrag-

mented and displayed varying levels of chemical damage (Figure S3).

Themeanread旭engthofendogenousnuc旭earDNAforarchaeo旭ogica旭samp旭esrangedfromジゲsゾto葦ゴsゲbppwithanovera旭旭meanofズゴs葦bp(Table S1). Cytosine deamination is the principal form of damage

observed inaDNAstudies ｪDabneypMeyerpｹP士士bopゴグゲザｫp and incircumstances where contamination from modern sources is pos-

sible, especially hominin research, damage patterns can be used to

discernancientandmodernsequencesｪJ祝nssoneta旭spゴグゲザｫsDuringthe life of a cell, cytosine residues can spontaneously convert to ura-

cil, but they are fixed with cellular repair mechanisms. After death,

these uracil residues accumulate, primarily in single- stranded over-

hangsp and due to the activity of po旭ymerases used inDNA 旭ibrarypreparations, apparent C- to- T and G- to- A transitions are observed at

theズ昼andザ昼endsofsequencingreadssThisdamagecanbevisua旭-ized as ski- jump style plots (Figure S3), with steeper slopes indicating

moredamagesInadditionptheδSparameterca旭cu旭atedbymapDam-

age provides a probability of cytosine deamination in single- stranded

contexts (Table S1). Our samples produced δS values ranging from

0.165 to 0.999 (mean = 0.605). As anticipated from well- preserved,

relatively recent specimens, the ethnographic samples exhibit low

levels of damage (δS range = 0.018�0.056, mean = 0.035). The eth-

nographicDNAisa旭so旭essfragmentedthanthatofthearchaeo旭ogica旭samp旭essA旭thoughArikaraｧゲジグジゴ､芦ゼジ isanout旭ierwithanaveragelength of 59.3 bp, library fragments frequently exceeded the length

of the number of sequencing cyc旭es ｪmean read 旭ength┎ゼゼsジbppsequencing length = 81 bp), and this mean is artificially reduced as

high､mo旭ecu旭ar､weightDNAwasextractedfrommanyethnographicsamples and needed to be fragmented by sonication prior to library

construction.

ザsジ科|科Exogenous DNA

Metagenomic ana旭ysis of unmapped reads revea旭ed a comp旭exmix-ture of DNA in archaeo旭ogica旭 and contro旭 samp旭es ｪFigureゴｫs Thechief contaminant across all archaeological samples is bacteria (up to

芦ズ鯵ｫwithActinobacteriaprimari旭ydifferentiatingarchaeo旭ogica旭sam-

p旭esfromethnographicsamp旭esｪPCゲpFigureSジｫsTheextractioncon-

trols are also dominated by bacteria, and taxa such as Proteobacteria,

Actinobacteria, and Firmicutes are consistent with species commonly

observed as 旭aboratory reagent contaminants ｪSa旭ter eta旭sp ゴグゲジｫsFungi and metazoans also make up a substantial proportion of archae-

o旭ogica旭contaminantspcontributingasmuchasザグ鯵ofreadcontentin several samples.

Taxonomic assignment of unmapped reads at the genus or species

level can help identify problematic individual samples and highlight

methodological or biological factors that require further examination.

For instance, the majority of unmapped reads in ethnographic sam-

p旭esarebroad旭yassignedtotheViridip旭antaepbutmostofthesehavetop BLAST hits to the H. annuus genome. These reads may not have

mapped to the sunflower genome due to sequence divergence from

the reference genome and/or because the BLASTn algorithm as ap-

plied was more tolerant of polymorphism than BWA. Ethnographic

samp旭es a旭so have on average ┒ザ timesmore unmapped reads as-signedtochordatesｪゲ芦sズ鯵comparedtoズsジ鯵inarchaeo旭ogica旭sam-

ples) and animal parasites such as Platyhelminthes and Apicomplexa

F IGURE  ゴ科DNAcontentofancientandethnographic旭andracesamp旭esandextractioncontro旭ssPercentageoftota旭readsmappingtothesunflower genome and relative proportion of unmapped reads assigned to kingdom- level taxa based on a random sampling of 10,000 unmapped

reads

Ancient

Ede

n−1

Ede

n−2

Ede

n−3

Ede

n−4

Ede

n−5

Ede

n−6

Ede

n−7

Ede

n−8

Ede

n−9

Ede

n−10

Ede

n−11

Ede

n−12

Ede

n−13

Ede

n−14

Ede

n−15

Ede

n−16

Ede

n−17

0.00

0.25

0.50

0.75

1.00

Controls

Blank

−1

Blank

−2

Blank

−3

Blank

−4

Ethnographic landraces

Arik

ara

1229

76

Arik

ara

1263

06

Arik

ara

1404

2−87

4

Arik

ara

broa

d 12

999−

682

Arik

ara/

Man

dan

1374

7

Paiut

e 14

1856

San

Ilde

fons

o P. 1

3597

−747

Sen

eca

1377

49

Sen

eca

purp

le 1

2996

−682

Sen

eca

purp

le 1

2998

−682

Sen

eca

strip

ed 1

2997

−682

a

Sen

eca

strip

ed 1

2997

−682

b

other

Viruses

Archaea

Metazoa

Fungi

Bacteria

Viridiplantae

Sunflower

芦科 |科 科架 WALES ET AL.

ｪズsゾ鯵 and ゲsゾ鯵 compared to ゴsグ鯵 and グsズ鯵 respective旭y in ar-chaeological samples). Eden- 1 and Eden- 2 are differentiated from

other archaeo旭ogica旭 samp旭es ｪPCゴ in FigureSジｫ by high counts ofGammaproteobacteria (specifically the Pseudomonas stutzeri group in

Eden- 1 and Pseudomonas putida group and Enterobacteriales in Eden-

2). One ethnographic sample, Arikara_122976, more closely resem-

b旭esarchaeo旭ogica旭samp旭eswith 旭owerendogenoussunf旭owerDNAcontent ｪザゼsゾ鯵 compared to the ethnographic average 芦ゼs葦鯵ｫ anda more substantial fraction of sequences originating bacterial, fun-

gal, and metazoan contaminants. While Arikara_122976 groups with

archaeo旭ogica旭 samp旭es in thePCoA ana旭ysis ｪFigureSジｫp it containsnearly twice as many unmapped reads assigned to fungi, with most

assigned to the Sordariomycetes, as any other ethnographic sample

(Figure 2).

ザsズ科|科P旭astome ana旭ysis

We constructed two haplotype networks, one including and one

excluding the archaeological samples (Figure 3). Exclusion of the ar-

chaeological samples provides for greater haplotype resolution of the

ethnographic samples, as the greater level of missing data in the ar-

chaeological data reduces the number of polymorphic sites informa-

tive for network construction.

The cultivated sunflower sequences�whether from archaeologi-

cal or ethnographic remains, extant landraces, or modern cultivars�

sort into few haplotype clusters that we have denoted as Classes

ゲthroughジre旭ativetothemuchgreaterdiversityobserved inwi旭dHelianthus sequences, which are nearly all unique (Figure 3; Table S3).

All Eden�s Bluff archaeological specimens dating to ~1700 calBP fall

in Class 1 and share the same or similar haplotypes as many ENA,

southwesternpandMexican旭andracesqsevera旭ethnographicsamp旭esqand the majority of modern cultivars (Figure 3a). Although Eden- 8,

Eden､ゲグp and Eden､ゲジ have distinct hap旭otypesp they are on旭y oneor two substitutions removed from the predominant Class 1 haplo-

typesManymoresubstitutionsmustbe inferred to support the re-

ticulate lineages connecting their sequences to the distinct Arikara

orSanI旭defonsohap旭otypesortoanyotherwi旭dH. annuus plastome

sequence, and the more resolved structure of Class 1 in the haplo-

type network excluding the archaeological samples suggests those

alternative connections are highly unlikely to reflect the true history

of descent (Figure 3b).

The two other Eden�s Bluff samples for which sufficient se-

quence was recovered for plastome analysis�Eden- 2 (850 calBP)

and Eden- 6 (3100 calBP)�cohere with the third most common

hap旭otypec旭asspC旭assゴsThisgroupa旭soinc旭udesthreeMexican旭andraces co旭旭ected fromNahua farmers ｪMexCu旭tザpMexCu旭t葦p

F IGURE  ザ科Plastome haplotype networks constructed with wild, cultivated, landrace, ethnographic, and archaeological sunflowers (a), and

plastome haplotype network constructed without the archaeological sunflowers (b). The size of the circles corresponds to number of individuals

present, and the number of polymorphic sites between individual haplotypes is indicated by tick marks. Haplotype classes for each sample

are included in Table S3. Class 1 is a core domestication haplotype and is composed of wild Helianthus annuus, archaeological specimens,

ethnographic samples, extant landraces, and modern cultivars. Class 2 also represents a haplotype that entered the domestication process

thousands of years ago; however, it is not observed in cultivars. Class 3 consists of R- type elite cultivars used in hybrid breeding, and was

presumably introduced into domesticated germplasm from H. petiolarisintheゴグthcenturyqasdiscussedinthetextpwesuspecttwoMexican旭andracesinC旭assザmayoriginatefrommisidentifiedcu旭tivarssC旭assジconsistsexc旭usive旭yofe旭itecu旭tivarspandwas旭ike旭yintroducedfromcropwild relatives, putatively H. argophyllus, during recent breeding for resistance to pathogens and diseases

Class 4

Class 3

Eden 10 &

Eden 14

Class 1

Arikara 122976

San Ildefonso

Hidatsa1

Class 2

Eden 8

Class 2

Arikara

Arikara 122976Class 1

Class 3

Class 4

San

Ildefonso

Hidatsa1

(b)(a)

科架 科 | 科ゾWALES ET AL.

andMexCu旭tゾｫpanethnographicSenecasamp旭epandawi旭dH. an-

nuus individual from Texas; however, no modern cultivars share

thissequencesItisimportanttonotetheexistenceofC旭assゴhap-

旭otypes inextant 旭andraceswou旭dnotbeknownwithoutDavidLentzandRobertByevspainstakingsurveyinMexicoｪLentzpPoh旭pA旭varadop Tarighatp ｹ Byep ゴググ芦ｫs In contrast to C旭ass ゴp C旭ass3, the second most common haplotype class, has a membership

consisting nearly entirely of R- type modern cultivars, which are

lines carrying a nuclear restorer allele for the cytoplasmic male

sterility system used for hybrid sunflower breeding. Two puta-

tiveMexican旭andracesｪMexCu旭tゼandMexCu旭tゲジｫa旭socarrytheClass 3 plastome sequence, raising the possibility they are actu-

a旭旭ye旭ite､bredmateria旭sTheC旭assジhap旭otypesequencesharedbythreemoderncu旭tivarsｪBRS､ゲpHA､RゴpandIRｫismostsimi旭arto sequences obtained from annual Helianthus species other than

H. annuus, likely reflecting a history of introgression as part of

a recent breeding program. Finally, the Hidatsa landrace has a

unique haplotype compared to other samples analyzed, consis-

tent with the findings of a previous study of sunflower sequence

diversity using chloroplast microsatellite markers (Wills & Burke,

2006).

ザs葦科|科Mitochondria旭 genome ana旭ysis

When archaeological sequences are excluded, the haplotype

network constructed for mitochondria is very similar to the plas-

tome network. Four major cultivated haplotype classes emerge

with nearly the same memberships, and thus, we use parallel no-

menc旭ature ｪFigureジp Tab旭eSザｫs One key difference is that theSanI旭defonsoethnographicsamp旭e ismoresimi旭artotheC旭assゲcultivated haplotypes than to any other cultivated or wild mito-

chondria旭 sequences Inc旭usion of mitochondria旭 sequences fromthe Eden�s Bluff samples in network construction analysis led to

poor旭yreso旭vedphigh旭yreticu旭atenetworkssIncontrasttotheob-

served plastome sequences, each of the mitochondrial haplotypes

from these archaeological samples contained many apparent pri-

vate mutations causing each sample to appear unique. We suspect

these patterns are artifactual, likely reflecting spurious SNPs origi-

natingfromshortexogenousDNAsequencefragmentsthata旭ignto highly conserved regions or, alternatively, SNPs that originate

fromnuc旭earinsertsofmitochondria旭DNAｪHazkani､CovopZe旭旭erpｹ Martinp ゴグゲグq Tha旭mannp Heb旭erp Poinarp P士士bop ｹ Vigi旭antpゴググジｫs

F IGURE  ジ科Mitochondria旭hap旭otypenetwork constructed with wild, cultivated,

landrace and ethnographic sunflowers. The

size of the circles corresponds to number

of individuals present, and the number

of polymorphic sites between individual

haplotypes is indicated by tick marks.

Haplotype classes for each sample are

included in Table S3. Class 1 is composed

of individuals sharing the same haplotype

and also those that diverge by only one or

twopo旭ymorphicsitessDuetouniparenta旭inheritance of organelles, the mitochondrial

classes contain the same individuals

as the plastome classes. See Figure 3

for information on the domestication

haplotypes (Classes 1 and 2) and those

introduced to modern cultivars during

ゴグth､centurybreedingｪC旭assesザandジｫ

Class 3

ArikaraArikara

ArikaraSan

Ildefonso

Arikara

Mex Cult 15

Hidatsa1

Class 4

Class 1

Class 2

ゲグ科 |科 科架 WALES ET AL.

ザsゼ科|科Nuc旭eotide diversity

The average pairwise nucleotide diversity (pi) of all groups of do-

mesticated sunflower samples is reduced relative to wild H. annuus,

consistent with a genetic bottleneck during domestication (Table 3).

This reduction is comparable for both organellar genomes. For in-

stancepthereisa葦芦鯵andゼゴ鯵reductionindiversityinethnographicsamples compared to wild H. annuus in chloroplast and mitochon-

dria, respectively. Within domesticated types, modern cultivars have

higher sequence diversity relative to the ethnographic samples and

landraces. However, this likely reflects the recent introgression of

wild haplotypes by modern breeding, as cultivars and landraces show

lower diversity as compared to the ethnographic samples when only

the diversity within the major haplotype classes also present in the

Eden�s Bluff samples (Class 1 and 2) is considered (Table 3). We report

a value for pi for the archaeological samples but note that this metric

is best suited for analyses of contemporaneous individuals and that

diversity within a single site is generally expected to be lower than

diversity present in the broader geographical sampling represented by

the sequences from wild, ethnographic, or modern cultivated material.

ジ科 |科DISCUSSION

ジsゲ科|科Sunf旭ower archaeo旭ogica旭 remains yie旭d qua旭ity endogenous DNA

Whi旭eaDNAstudieshaverevea旭ed important insights intothepaceofse旭ectionduringdomesticationinsomep旭antsｪesgspMaschereta旭spゴグゲ葦qRamos､Madriga旭eta旭spゴグゲ葦qVa旭旭ebueno､Estradaeta旭spゴグゲ葦ｫprecoveryofdegradedDNAfrommostcrops isnotroutinepandthisproject represents the first exploration of how paleogenomic test-

ing of archaeological sunflower remains can be used to understand

its unique domestication historys Through paired AMS dating and

paleogenomic testing of archaeological specimens from the Eden�s

Bluff site in Arkansas, we find that many desiccated remains dat-

ingbackas far asザゲググBPcanbeva旭uab旭e sourcesofDNAsSomespecimens yie旭dmore than ズグ鯵 sunf旭owerDNAp a旭though a seem-

ing旭y randomsubsetof specimens yie旭d 旭eve旭sof endogenousDNAｪ┑ゲ鯵ｫ essentia旭旭y incompatib旭e for state､of､the､art pa旭eogenomictechniques, such as targeted enrichment of genetic loci of interest

ｪCarpentereta旭spゴグゲザｫsSti旭旭pゲザoftheゲゼspecimensyie旭ded┒ズ鯵en-

dogenousDNAandarethereforewe旭旭suitedforin､depthana旭ysisofnuclear targets that can be defined from genomic and transcriptomic

studies of extant sunflower germplasm.

WesuspecttheexogenousDNAcontentobtainedfromoursam-

ples originates from at least four sources: organisms that inhabited

the disks and achenes during the life of the plant, such as pathogens;

organisms that consumed metabolites, proteins, and other biomol-

ecules in the tissue after the death of the individual; environmental

DNAtransferredfromthearchaeo旭ogica旭sedimentqandmodernDNAcontamination from excavation, curation, and genetic testing. While it

is difficult to distinguish these potential sources, the sequencing of ex-

traction controls provides a means to identify cross- contamination of

samp旭esandpervasiveDNAin旭aboratoryreagentsｪSa旭tereta旭spゴグゲジｫsWeobserved thatDNAdegradation patterns arevariab旭e in ar-

chaeo旭ogica旭sunf旭owerpbothintermsofDNAfragment旭engthandthefrequency of chemical damage, even within one relatively tight time

interval. For example, the two oldest specimens (Eden- 3 and Eden- 6)

yie旭dedeffective旭y identica旭AMSdatesofcasザゲググca旭BPsHoweverpcomparedtoEden､葦pEden､ザhass旭ight旭yshorterendogenousDNAｪdif-ference of means = 5.8 bp) and higher levels of cytosine deamination

(δS of 0.999 vs. 0.673). Similarly, the youngest sample from the collec-

tionpEden､ゴpdatesto芦ズグca旭BPandhasDNAthatisnear旭yasshort(mean fragment length of 62.1 bp) and as damaged as Eden- 9 (mean

fragment length of 59.7 bp), which is twice as old. Thus, fragmentation

and damage profiles do not necessarily follow straightforward, time-

dependent degradation patterns, perhaps reflecting variability in how

different remains were treated prior to deposition (e.g., intentional

desiccation or heating in antiquity). Together, these findings indicate

that multiple samples from the same site and stratigraphic layer ought

to be initially tested by low- depth shotgun sequencing to identify

promising candidates for in- depth genetic analysis.

ジsゴ科|科Organe旭旭ar hap旭otype networks recapitu旭ate anticipated patterns for extant taxa

Organellar genomes in most plants exhibit uniparental inheritance

(Sato & Sato, 2013). Therefore, a one- to- one association of plastid hap-

旭otypeswithmitochondria旭hap旭otypesisoftenexpectedｪMogensenp1996), and indeed, we observe such a tight correspondence between

our defined organellar haplotype classes (Table S3). Because the plas-

tid and mitochondrial genomes are nonrecombining, it can be possi-

ble to use organellar loci as markers for taxonomic identification, as

isperformedwithDNAbarcodingstudies ｪAviseeta旭spゲゾ芦ゼqCBOLP旭antWorkingGroupeta旭spゴググゾｫsYetptheorgane旭旭argenomesofthefive annual Helianthus species we have sampled do not resolve into

TABLE  ザ科Nucleotide diversity (pi) for wild, archaeological,

ethnographicp旭andracepandmoderncu旭tivatedsunf旭owerssItisimportant to note that the archaeological specimens were excavated

from one site and are therefore not wholly comparable to

population- level measures of pi for the other sunflower groups.

GiventhatC旭assザandジhap旭otypeswere旭ike旭yintroducedtodomesticated lines during recent breeding, a separate calculation of

pi for modern cultivars with Class 1 and 2 haplotypes is provided

Sunf旭ower group

Nuc旭eotide diversity in p旭astome

Nuc旭eotide diversity in mitochondria

Wild グsグジグザ グsグジズ芦

Archaeological 0.0099 N/A

Ethnographic 0.0127 0.0126

Landrace 0.0125 0.0073

Class 1 and 2 landrace グsググゾジ 0.0050

Moderncu旭tivar 0.0285 0.0235

Class 1 and 2 modern

cultivar

0.0091 グsググ芦ジ

科架 科 | 科ゲゲWALES ET AL.

mutually exclusive clusters in either haplotype network. Such pat-

terns are consistent with previous findings demonstrating substantial

gene flow between Helianthus species and/or incomplete lineage sort-

ing (Sambatti, Strasburg, Ortiz- Barrientos, Baack, & Rieseberg, 2012;

Whitneyeta旭spゴグゲズｫsForinstancepBocketa旭sｪゴグゲジｫobservedasimi-lar lack of taxonomic structure in the organellar genomes of perennial

Helianthus species, suggesting this is common throughout the genus.

Mostmoderncu旭tivarscarryoneof twodistincthap旭otypes ｪthemost common Class 1 sequence or Class 3), and these assort into in-

bred line classes developed to facilitate hybrid production. Elite- bred

sunf旭ower旭inesarec旭assifiab旭eintotwotypesrma旭ewR､旭inesxandfe-

ma旭ewB､旭inespxthe旭atterbeingderivedfromopen､po旭旭inatedvarietiesｪOPVｫ ｪKore旭旭pM塾sgespｹFriedtp ゲゾゾゴｫsTheC旭assゲ ch旭orop旭ast andmitochondrial haplotypes observed in extant germplasm are predom-

inantamongB､旭inesandOPVsaswe旭旭asmostextant旭andracespsug-

gesting that this cluster contains the few organellar sequences that

passed through the domestication and improvement bottlenecks. The

33 modern cultivars in our survey that carry the Class 3 haplotype

are all R- lines, which carry a mitochondrial mutation (PET- 1) intro-

gressed from H. petiolaris Nutt. that causes male sterility as well as

a nuclear restorer allele (Rf) for this mutation (Balk & Leaver, 2001).

As expected based on this breeding history, the mitochondrial haplo-

type of Class 3 groups closely with sequences present in H. petiolaris

ｪFigureジｫ. Because only Rf is required to restore fertility in hybrid crop

breedingpwedo find twoR､typecu旭tivarspRHA､ジゲ芦andRHA､ジグゲpin the Class 1 haplotype cluster. The shared breeding history of RHA

cultivars likely also explains the divergence between Class 1 and Class

3�s plastome haplotypes. Although the plastome haplotype of Class

3 does not have clear affinity for any of the obtained H. petiolaris se-

quences, it is possible that more similar H. petiolaris plastome haplo-

types were not included among the individuals sampled. Two putative

Mexican旭andracesｪMexCu旭tゼandMexCu旭tゲジｫsharetheC旭assザp旭as-tomeandmitochondria旭hap旭otypessUn旭ikeotherMexican旭andracespwhich were obtained directly from native farmers, these domesticates

wereobtainedfromanopenmarketp旭aceinChiapaspMexicoｪDsLentzppersonal communication; Blackman et al., 2011). Thus, the possibility

that they may in fact be seeds derived from modern R- type sunflower

lines is plausible and merits rigorous examination in whole genome

analyses.

Another case of deliberate introgression is observed for the third,

旭esscommonpcu旭tivarhap旭otyperC旭assジsTheC旭assジorgane旭旭arhap-

lotype is most similar not to other H. annuus sequences but instead to

sequences from other annual Helianthus species. This observation is

consistent with published breeding information for at least two of the

threeC旭assジcarryingcu旭tivarssBRS､ゲandHA､RゴarederivedfromtheOPVArgentinianImpiraINTAcu旭tivarpwhichisahybridofH. ar-

gophyllus and H. annuus var Saratov Permgamino, and were selected

fordisease､resistanttraitsｪBerterodeRomanoｹNorbertoV史zquezp2003).

Overall then, while three organellar genome types predominate

in modern cultivated germplasm, these very distinct Class 3 and

C旭assジsequencesarenotsharedwith 旭andracespethnographicporarchaeological samples and have largely entered cultivated H. annuus

through recent, deliberate introgression of genetic material from

other wild H. species. The history of directed breeding of domes-

ticated sunflower lines with crop wild relatives strongly suggests

Class 3 was introduced from H. petolaris during the establishment

of thehybridcropagricu旭tura旭 system ｪSei旭erpQipｹMarekpゴグゲゼｫsC旭ass ジ was 旭ike旭y a旭so introduced during crop improvementp po-

tentially from H. argophyllus, the sunflower species which has been

most frequently crossed with domesticated lines to impart disease

andparasiteresistanceｪSei旭erｹFredrickMarekpゴグゲゲｫsIndeedpitisperhaps surprising that additional non- H. annuus haplotypes were

not more commonly observed, as breeders have introduced allelic

variation for novel traits (e.g., resistance against a range of patho-

gens) by prolific and repeated introgression of genetic material from

other Helianthus species. H. annuus has reportedly been crossed

with every annua旭 species and ゲジ perennia旭 species in the genusｪKayapゴグゲジｫsOurfindingofon旭ytwointrogressedhap旭otypesponeof which was deliberately selected for, likely reflects that H. annuus

has predominantly served as the recurrent maternal parent during

sunflower improvement.

ジsザ科|科Ethnographic and archaeo旭ogica旭 organe旭旭ar sequences revea旭 旭ost diversity and raise new hypotheses

Although low- depth shotgun sequencing data from ancient samples

like those which we report here generally do not enable population-

level characterization of nuclear genes of interest, patterns of variation

inorgane旭旭argenomescanbeassessedbecausetheseDNAsourcesare found in many copies per cell, increasing their chance of recovery

(Hofreiter, Serre, Poinar, Kuch, & Paabo, 2001). Furthermore, analy-

ses of nonrecombining loci from archaeological samples can lead to

important insights about the phylogeography and demography of do-

mesticationpasdemonstratedbyaDNAstudiesofpigsｪLarsoneta旭sp2007), cattle (Beja- Pereira et al., 2006), and bottle gourds (Kistler

eta旭spゴグゲジｫsThe sequences that we recovered from archaeological and ethno-

graphic sunflower samples provide new information about the extent

and timing of the bottlenecks in genetic diversity accompanying do-

mestication and improvement that have previously been inferred from

extant sunflower sequences (Baute, Kane, Grassa, Lai, & Rieseberg,

2015; Liu & Burke, 2006). Although nearly every wild H. annuus indi-

vidual carries a unique plastid haplotype, the archaeological and eth-

nographic samples assort into just two haplotype clusters. Notably, the

most common haplotype among both modern and historical domesti-

cated forms (Class 1) was present at Eden�s Bluff at least 1,700 years

ago, as were two additional closely related but distinct haplotypes

not represented in any extant germplasm (Figure 3a). Given these

sequences are separated by fewer substitutions from the major do-

mesticate haplotype than from any wild haplotype, we infer these are

more likely to represent de novo evolution following a domestication

bottleneck than retention of standing variation from the wild ancestor.

Likewise, we observe several more unique Class 1 haplotypes that are

satellites of the major haplotype among the ethnographic samples, and

ゲゴ科 |科 科架 WALES ET AL.

the Class 2 haplotype observed in the oldest Eden�s Bluff sample and

several Native American landraces are completely absent from elite-

bred cultivars. Together, these findings suggest that all domesticated

sunflowers likely coalesce to very few maternal lineages present early

in the domestication process. Given that the archaeological samples

analyzed in this study are from a single site and might not fully reflect

the genetic diversity present in the earliest phases of domestication,

aDNAana旭ysisofadditiona旭archaeo旭ogica旭samp旭eswi旭旭beimportantfor affirming these findings.

Inadditionpourresu旭tsconfirmHeiservs旭amentqNativeAmericanlandraces once harbored genetic diversity now absent from modern

germplasm. Absence of the Class 2 haplotype and the unique eth-

nographic Class 1 haplotypes in elite cultivars likely reflects genetic

bottlenecks imposed during 20th- century improvement programs

and by the subsequent rise of the lines produced to agricultural

dominance throughoutNorthAmerica ｪHeiserp ゲゾゼ葦q 斎kori賜p ゲゾゾゴｫsThe loss of diversity in extant landraces relative to historic samples

also provides a caution and an opportunity for conducting genome

scans fordomesticationgenessBy inc旭udingnuc旭earDNArecoveredfrom ethnographic specimens, it may be possible to distinguish be-

tween genes that experienced selective sweeps as a consequence of

the domestication process versus changes in sequence diversity that

score similarly by population genetic metrics due to the recent loss of

landrace germplasm. The sole modern wild H. annuus sample carrying

a Class 1 haplotype is also instructive in this regard. Given the fre-

quency at which domesticated and wild sunflowers interbreed (Arias

ｹRiesebergpゲゾゾジqLinderpTahapRiesebergpSei旭erpｹSnowpゲゾゾ芦ｫandthat this individual was collected in California, well outside the pro-

posed ENA domestication center, we expect it acquired the Class 1

haplotype by gene flow from contemporary domesticates. Thus, this

finding highlights the importance of vetting putatively wild sunflower

individuals for signals of admixture prior to inclusion in genomic scans

for selective sweeps.

The archaeological and ethnographic haplotype sequences we

have recovered are also consistent with a single center of sunflower

domestication located in ENA. Both Class 1 and Class 2 haplotypes

were present at Eden�s Bluff before 1700 calBP, and both classes are

also observed in historic and extant landraces. The presence of the

distinct Class 2 haplotype at Eden�s Bluff at 3100 calBP and in three

Mexican旭andraceaccessionsbuta旭soaSenecaethnographicsamp旭edoes introduce some ambiguity because the pattern fails to be fully

diagnostic for a single ENA origin versus an additional second cen-

terofdomesticationof sunf旭ower inMexicop as suggestedbyLentzet al. (2008, 2001). Nonetheless, the single domestication hypothesis

remains the most compelling conclusion for multiple reasons. First,

the threeC旭assゴMexican 旭andraceswerea旭旭 co旭旭ected from indige-

nous Nahua farmers in the state of Guerrero (Blackman et al., 2011)

who spoke only Nahuatl and yet did not know the Nahuatl word for

sunf旭owerｪDsLentzppersona旭communicationｫsThuspitispossib旭ethatthese 旭andraceswere introduced to this region ofMexicomore re-

cently than the early domestication period. Second, two wild individ-

uals from the central United States (northern Texas) carry the Class 2

haplotype. Thus, if these do not represent admixed genotypes and if

furthersequencingofMexicanwi旭dpopu旭ationsfai旭stoyie旭dtheC旭assゴsequencepthenaMexicanorigincanbeexc旭udedsFina旭旭yandmostpersuasively, multilocus nuclear genotype data and candidate domes-

ticationgenesequencesfromthesethreeMexican旭andracesdemon-

strate they are more closely related genetically to extant landraces

andwi旭d popu旭ations fromENA than towi旭d popu旭ations inMexico(Blackman et al., 2011).

Because we have obtained aDNA sequence for archaeo旭ogica旭samples excavated at the same site but that date to three separate

time periods, we can compare the Eden�s Bluff samples not only to

wi旭dgermp旭asmfromthemodernerabuta旭sotoeachothersIndoingso, we observe a pattern of sequence turnover. The samples dated to

the earliest and latest time points (3100 calBP and 850 calBP) both

carry the Class 2 haplotype, but the many samples dated to the inter-

mediate time interval (1700 calBP) possess the Class 1 haplotype ex-

clusively. This pattern suggests that multiple different domesticated

lineages of sunflowers were maintained in the region for millennia

and might reflect differential cultivation of these proto- landraces

across times It is interesting to note that these time points gener-ally correspond to major prehistoric cultural periods in the Ozarks

and across North America, namely the Late Archaic, Woodland, and

Mississippianperiods ｪSaboｹEar旭ypゲゾゾグｫsDespite thesepotentia旭links to cultural changes, it must be emphasized that we have tested

a limited number of samples and many samples dating to 1700 calBP

could originate from one depositional episode from a small group of

farmers. Thus, there is a chance that both Class 1 and Class 2 would

be observed throughout the stratigraphic sequence at Eden�s Bluff

if more samples were characterized. Nonetheless, this intriguing

patternof turnovermakesc旭ear thepowerfu旭potentia旭ofaDNAtoraise and to investigate new hypotheses about domestication and

cultural history that have left no footprint in the genomes of extant

germplasm. Future studies of nuclear genome sequence from these

samp旭esandaDNAfromotherremainsobtainedovertimeinthisre-

gion are sure to reveal further insights into the temporal and spatial

dynamics with which early sunflower landraces arose and spread to

other regions.

ズ科 |科CONCLUSIONS AND FUTURE

DIRECTIONS

In summaryp we have shown that recovery of ancient and historicDNA fromarchaeo旭ogica旭 and ethnographic sunf旭ower specimens isfeasible and that desiccated specimens frequently contain high levels

ofendogenousDNAsAtpresentpshotgunsequencingdataa旭旭owustoinfer the relationships between ancient and modern samples for orga-

ne旭旭ar旭ocisIntandemwithsequencingdatafrommodernaccessionspwe have gained new perspectives on the persistence of plastid line-

ages for thousands of years under cultivation and the loss of genetic

diversity during recent improvement. We recognize these loci track

the maternal lineage and do not document the full domestication his-

tory of the sunflower, and our future studies where we obtain greater

depth of coverage for many loci in the nuclear genomes of ancient and

科架 科 | 科ゲザWALES ET AL.

historic specimens will allow us to address more nuanced questions

about the pace of domestication and specific targets of selection.

Fortuitously, numerous desiccated archaeological specimens have

been excavated from dozens of sites in the Ozarks and other parts of

ENAｪFritzpゲゾ芦葦qGi旭morepゲゾザゲqSmithpゴグゲジｫptherebyprovidingthemeanstoidentifygeneticchangesovermi旭旭enniasMostofthespeci-menswereexcavated fromrockshe旭ters fromゲゾゴグtoゲゾザグ ｪDavispゲゾ葦ゼqHarringtonpゲゾゴジapゲゾゴジbpゲゾ葦グｫpbutsomeofthesesitespin-

cluding Eden�s Bluff, have since been inundated by the construction

of dams in the mid- 20th century or otherwise degraded (Fritz, 1986).

Thus, these curated specimens offer an otherwise unachievable pre-

historic perspective on sunflower domestication. Candidate targets of

selection during domestication have been reported in several stud-

ies ｪBaute eta旭sp ゴグゲズq B旭ackmanp Strasburgp Raduskip Michae旭sp ｹRiesebergpゴグゲグqB旭ackmaneta旭spゴグゲゲqChapmanpMande旭pｹBurkep2013; Chapman et al., 2008), and identifying more should be acceler-

ated thanks to expanding genomic resources being generated by the

Internationa旭ConsortiumforSunf旭owerGenomicResourcesｪBadouinet al., 2017; Kane et al., 2011). Thus, we anticipate paleogenomic

characterization of archaeological and ethnographic sunflower tissues

will soon have tremendous potential to resolve long- standing ques-

tions about the demographic and functional history of domestication

for this important oilseed crop.

ACKNOWLEDGEMENTS

This research was funded by the National Science Foundation

ｪDEB､ゲザズジ葦ゴゴp DEB､ゲ葦ジグゼ芦芦ｫ and the Danish Nationa旭 ResearchFoundation. Permission for destructive sampling of archaeological

andhistoricmateria旭swaskind旭ygrantedbytheNationa旭MuseumoftheAmericanIndianptheUniversityofArkansasCo旭旭ectionsFaci旭itypThe Osage Nationp and the University of Michigan Museum ofArchaeo旭ogica旭Anthropo旭ogys Inparticu旭arpwe acknow旭edge the in-

valuable assistance of Andrea A. Hunter, Tribal Historic Preservation

OfficerpOsageNationqMarySuterpCuratorofCo旭旭ectionspUniversityMuseumpUniversityofArkansasqandEmi旭yKap旭anpNationa旭Museumof the American Indians We thank Fi旭ipe Gs Vieirap Thorfinn SandKorne旭iussenp Mikke旭 Schubertp Mike Martinp and Vanessa Biekerfor bioinformatic advice and Shyam Gopa旭akrishnanp Ida Mo旭tkepand Jazm趣n Ramos､Madriga旭 for thoughtfu旭 discussionss Specia旭thanks to theDanishNationa旭High､throughput SequencingCentreforassistance ingenerating I旭旭uminadatasWea旭so thankKeBiandJason Huff at the UC Berkeley Computational Genomics Resource

Laboratory for their support. Thanks to members of the Blackman

laboratory and two anonymous reviewers who provided comments

on the manuscript. Publication made possible in part by support from

theBerke旭eyResearch Impact Initiative ｪBRIIｫ sponsoredby theUCBerkeley Library.

DATA ARCHIVING

Data for this study are avai旭ab旭e at the NCBI SRAr BioProject IDPRJNAジゴゴ葦ゴジs

ORCID

Nathan Wales httpr｠｠orcidsorg｠ググググ､グググザ､グザズゾ､芦ジズグ

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