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DOI: 10.1126/science.1141395, 1890 (2007);316Science

et al.Tom D. Dillehay,in Northern PeruPreceramic Adoption of Peanut, Squash, and Cotton

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Preceramic Adoption of Peanut,Squash, and Cotton in Northern PeruTom D. Dillehay,1* Jack Rossen,2 Thomas C. Andres,3 David E. Williams4

The early development of agriculture in the New World has been assumed to involve earlyfarming in settlements in the Andes, but the record has been sparse. Peanut ( Arachis sp.), squash

(Cucurbita moschata), and cotton (Gossypium barbadense) macrofossils were excavated fromarchaeological sites on the western slopes of the northern Peruvian Andes. Direct radiocarbondating indicated that these plants grew between 9240 and 5500 14C years before the present.These and other plants were recovered from multiple locations in a tropical dry forest valley,including household clusters, permanent architectural structures, garden plots, irrigation canals,hoes, and storage structures. These data provide evidence for early use of peanut and squash in thehuman diet and of cotton for industrial purposes and indicate that horticultural economies in partsof the Andes took root by about 10,000 years ago.

R esearch on the origins and dispersal of agriculture around the world has con-centrated on the environments and pe-

riods in which plants were first domesticatedfrom indigenous wild species. Less concern has

been given to the adoption of cultivars and their uneven use and development, the movement of populations practicing cultivation into areaswhere it was previously unknown, and thewider cultural contexts within which these pro-cesses occurred. In the Andes, potatoes, corn,squash, beans, manioc, cotton, and chili peppershave long been considered the primary “founder crops” by at least 5000 14C years before the present (yr B.P.) (1, 2). Here we report evidencefor radiocarbon-dated human cultivation of squash (9240 and 7660 yr B.P.), peanut (7840yr B.P.), quinoa (8000 and 7500 yr B.P.), andcotton (5490 yr B.P.) in the form of macro-

botanical remains recovered from sealed housefloors and hearths in buried preceramic sites in atropical dry forest of the Ñanchoc Valley, atributary of the Zaña Valley located at 500 mabove sea level, on the lower western slopes of the Andes in northern Peru (Fig. 1). Evidenceof other crops (manioc, unidentified tubers,and fruits), circular and later rectangular houses,storage units, stone hoes, ground stone bowlsand pallets, furrowed garden plots, small-scaleirrigation canals, and earthen mounds datingto the same period have been found nearby(3 – 9).

Before the adoption of crops and the devel-opment of new farming technologies, huntersand gatherers lived in semi-sedentary, dispersedencampments between 10,800 and 9000 yr B.P.(9). From 9000 to 7000 yr B.P., people formedmore tightly bound and organized commu-nities, living 200 to 400 m apart near springs

and along the banks of small streams on widealluvial fans 1 to 3 km from the valley floor where they gardened, continued gathering andhunting, and engaged in down-the-line ex-change of ideas and products with horticultur-

alists living in distant coastal, highland, andtropical forest areas.We recovered macrobotanical specimens

from late Paiján (10,000 to 9000 yr B.P.), LasPircas (9000 to 7000 yr B.P.), and TierraBlanca (7000 to 4500 yr B.P.) houses, includingsquash (Cucurbita moschata), a morphologicallywild peanut ( Arachis sp.), cotton (Gossypiumbarbadense), a quinoa-like chenopod (Cheno- podium sp. cf. quinua), manioc ( Manihot sp.),an edible malphigiaceous fruit ( Bunchosia sp.),and unidentified tubers and fruits (Figs. 2 to 5)(6 ) (see SOM Text, section 1, and table S1 for the number of remains recovered in sites).

Accelerator mass spectrometry (AMS) radio-carbon dates processed nearly two decades agofor charred and uncharred squash, peanut,cotton, and coca remains recovered from housesranged widely from 11,650 14C yr B.P. to 200years into the future (6 ). Despite the widevariation in dates, the plant remains werethought to represent early crop cultivation inthe Andes, because they were embedded in buried house floors and hearths beneath grinding

stones and in excavated stone-lined storage unitsand were directly associated with 14C datesranging between 7800 and 5800 yr B.P. onwood charcoal from sealed hearths (Table 1).Most of these remains display morphologicaltraits that do not correspond to those of anymodern varieties, and most of these plants arenot native to the lower western Andean slopesof northern Peru. New AMS dates on macrobotanical squash, peanut, and cotton remains

are now available from the same previouslydated house floors and hearths and from new buried floor contexts. Dates are 9240 ± 50 yrB.P. [Beta 179512: 10,403 to 10,163 calibrated(Cal) yr B.P.] and 7660 ± 40 yr B.P. (Beta219589: 8535 to 8342 Cal yr B.P.) on squashseeds from sites CA-09-77 and CA-09-27respectively; 7840 ± 40 (Beta 219588: 8640 to8435 Cal yr B.P.) on a peanut hull from siteCA-09-77; and 5490 ± 40 yr B.P. (Beta 183279:6278 to 5948 Cal yr B.P.) on cotton fibers fromCA-09-71. These new dates conform to thestandard radiocarbon dates derived from woodcharcoal in hearths and floors and directly

associated with the same plant species that previously produced erratic dates (see SOMText, section 2, and table S2). In addition to thenew dates, research carried out by botanical andother experts over the past two decades on thecharacteristics and distributions of modern wildand domesticated species of Cucurbita, ArachisGossypium, and Chenopodium allows the inter- pretation of these remains to be more precise.

The peanut was long thought to be amongthe later cultivated plants of the Andes, andone that is particularly suited to the lowlandtropical forests and savannahs where it was prized as a high-protein complement to starchy

manioc-based diets (1, 2). The peanut ’

s centerof origin is believed to be in an area east of theAndes comprising southeastern Bolivia, north-western Argentina, northern Paraguay, and thewestern Mato Grosso region of Brazil (10 – 12)It has not been found in other middle pre-ceramic archaeological contexts of southwest-ern Ecuador, Colombia, or the Amazon basin.The Las Pircas and Tierra Blanca peanuts areelliptically shaped, fibrous remains of fruits (6 )

1Department of Anthropology, Vanderbilt University,Nashville, TN 37221, USA. 2Department of Anthropology,Ithaca College, Ithaca, NY 14850, USA. 3The CucurbitNetwork, New York, NY 10458, USA. 4U.S. Department ofAgriculture, Washington, DC 20250, USA.

*To whom correspondence should be addressed. E-mail:[email protected]

Fig. 1. Location map of study areain north Peru.

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that appear to correspond morphologically to awild species, a situation that could be expectedduring the early stages of domestication. Thesites at which they were recovered are far removed from the known range of wild Arachis.

Early and middle preceramic squash phyto-liths have been recovered from the Las VegasPhase in southwestern Ecuador (10,000 to 7000yr B.P.) and the Colombian Amazon (9300 to8000 yr B.P.) (1, 2, 13). In both cases, phytolith

size indicates the presence of domesticatedspecies. The newly dated squash seeds fromthe late Paiján and Las Pircas sites have similar dates and are the earliest macrofossil remainsof Cucurbita recovered from an archaeologicalcontext. These small seeds (6 to 7 mm long, 2.5to 4 mm wide) have a uniform dark browncolor, prominent raised seed margins, and anelliptical shape. Seeds of this size, shape, andcolor have been found in fruits of modern tra-ditional landraces of C. moschata from lowlandnorthern Colombia (14). No other species of Cucurbita resemble these traits (15). The color alone is unique to this species in a genus com-

posed of about 14 species, including 5 domes-ticates. There is no evidence to suggest that postdepositional processes discolored lighter-colored seeds that are typical of other squashspecies and cultivars of C. moschata. Despitetheir small size for a domesticated squash, thearchaeological seeds were mature, because the

brown seed coat does not develop until near the end of maturation. The wild ancestor of thismajor domesticated squash has not yet beenfound, but lowland northern South America,especially Colombia, has been proposed as itsarea of origin on the basis of molecular data andoccurrence of modern, primitive-looking land-races (13 – 17 ). Fruits of C. moschata with dark brown seeds are also most prevalent today inthis region. It thus appears that the peanut spec-

imens and the Ñanchoc squash specimens rep-resent early cultigens dispersed at an early dateto northwestern Peru from their respective areasof origin.

Wild populations of Gossypium barbadenseare found on the coastal plains of southwesternEcuador and northwestern Peru (1), where their domestication likely occurred (18). Archaeolog-ical cotton has been found in the Valdivia strataof the Real Alto site in Ecuador (19) and inthe late preceramic sites of the Ancon-Chillonarea of central coastal Peru. Between 4500 and3500 yr B.P., this plant anchored what has beencalled the Cotton Preceramic Phase of Peru.

Cotton was initially used for fishing nets, and probably for hunting nets, storage bags, andclothing (20). Cotton is absent from the earlier Las Pircas Phase in the Ñanchoc Valley, but com- plete cotton bolls were recovered from housefloors of the later Tierra Blanca Phase. Duringthis same period, gourds ( Lagenaria) were

probably used for industrial containers and for the consumption of their seeds (21).

One carbonized specimen of a large-seededchenopod (1.9 mm diameter) was recoveredfrom the house floor of CA-09-27 and placed bydirect association with dated hearths betweenabout 7500 and 8000 yr B.P. Its size and quad-

rilateral cross-section closely resemble those of quinoa (Chenopodium quinua), but ridges onthe specimen are a minor morphological differ-ence from herbarium specimens. Thirty similar specimens, both carbonized and desiccated,

were recovered from several later Tierra Blancasites. Large-seeded chenopods like quinoa arethought to have been domesticated in the LakeJunin and Lake Titicaca regions of southernPeru and the Bolivian highlands by 4000 yr B.P.(22). The highland origins and ecology of qui-noa contrast with the lower elevation, tropicaldry forest association, and northern location ofthe Ñanchoc sites. Quinoa has several character-istics that make it an unusual cultigen. The seed

bitterness, a saponin coating, must be removed by washing before preparation for consump-tion, but the bitterness is an advantage in stor-age, because rodents and insects do not infestthe seeds. Seed fragility also is a crucial at-tribute of quinoa. Seeds do not usually remainviable for more than 1 year, and quinoa must be planted every year, or it may be lost to aregion. Even a small of amount of quinoa in anarchaeological site thus may represent a long period of local cultivation.

There is no evidence to indicate that the Ñanchoc Valley was a domestication center forany of these major economic plants. Thus, the

adoption of peanut, squash, cotton, quinoa, andother crops suggests that these plants must have been cultivated elsewhere earlier than 9200 yrB.P. for squash (1, 2), 8000 yr B.P. for peanut5500 yr B.P. for cotton, and about 7500 yr B.P.for quinoa, after which groups of down-the-line local traders or mobile horticulturalists brought them into the valley. Between ~9200and 5500 yr B.P., the Ñanchoc communities passed from advanced Paiján foragers with a broad-spectrum economy, to Las Pircas horticulturalists primarily dependent upon seasonarainfall to grow a few crops, to Tierra Blancaincipient agriculturalists managing irrigated wa-

ter and growing a wide variety of crops. Asso-ciated with these changes are demographicarchitectural, and technological developmentsindicative of more complex social groupings.

The new dates confirm the internal logic ofthe archaeology of the Las Pircas and TierraBlanca phases, with their nucleated household patterns and relocation closer to the valley floorThe adoption and cultivation of both food andindustrial crops between 9000 and 5500 yr B.Pwere aspects of wider cultural processes tha

Fig. 2. Close-up of a fragment of a peanut hull( Arachis sp.) recovered from a buried housefloor at site CA-09-77.

Fig. 3. Close-up of two dark brown squash seed(C . moschata) fragments recovered from a buriedhouse floor at CA-09-27.

Fig. 4. Close-up of carbonized quinoa seed(Chenopodium quinoa) recovered from a buriedhouse floor in CA-09-77.

Fig. 5. Close-up of cotton boll recovered from aburied house floor at CA-09-71.

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included sedentism, artificial water management systems, mound-building, appearance of exoticartifacts, and probably ritually sanctioned crop production, the latter suggested by the presenceof rock crystals and other exotics in mounds, buried garden plots, and canals (7 , 8). These processes in Ñanchoc [and other areas of theAndes (23)] also served as catalysts for rapidsocial changes that eventually contributed to the

development of intensified agriculture, institu-tionalized political power, and towns in both theAndean highlands and on the coast between5500 and 4000 yr B.P. (24). The Ñanchoc dataindicate that agriculture played a more important and earlier role in the development of Andeancivilization than previously understood, espe-cially within suitable, low-elevation mountainenvironments.

The distribution of structures, canals, andfurrowed fields in the study area indicates thatearly agriculture was associated with manage-ment decisions made to socially aggregate people in the context of regulated crop production beyond the individual household level to emergeas creative agricultural communities. Our datashow that public ritual and probably ceremo-nialism were manifested to an unprecedenteddegree between 7000 and 6000 yr B.P. in the

form of small mounds associated with lime production probably for coca leaf consump-tion (4, 7 ), which also led to increased socialcohesion among local households. The moundsat site CA-09-04 were intermittently modified andused for two millennia, suggesting that publicritual coevolved with agriculture and wider com-munity developments (7 ).

The data amplify existing evidence and ar-guments on the development of domesticated plant production. The squash remains constitutemore evidence from another region that squashesand gourds were among the earliest cultivars inthe Americas (13, 25) and that a number of dif-

ferent squash species were undergoing manip-ulation and incipient domestication at about thesame time during the early Holocene in Meso-america and the northern half of South AmericaThe evidence also points to an early develop-ment of horticulture in more southerly regionsof tropical South America, where the peanut isthought to have its origin. There is evidencefor similarly early domestication of manioc tothe north of the Ñanchoc Valley in Colombiaand Panama (26 , 27 ). Our data also show thathorticulture and cultural complexity developedin the Americas nearly as early as it did inmany parts of the Old World. Early to middle

Holocene populations exploiting suitable en-vironments in both the Old World and NewWorld combined different suites of resourcesand technologies to affiliate into larger, moreadvanced communities that differentiated them-selves from others between 12,000 and 9000 yrB.P. (1, 2, 28, 29).

References and Notes1. D. Piperno, D. Pearsall, The Origins of Agriculture

in the Lowland Neotropics. (Academic Press, New York,

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Did Foragers Become Farmers? (Oxford Univ. Press,

New York, 2006).

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4. T. D. Dillehay, P. J. Netherly, J. Rossen, Am. Antiq. 54,733 (1989).

5. J. Rossen, thesis, Univ. of Kentucky (1991).

6. J. Rossen, T. D. Dillehay, D. Ugent, J. Archaeol. Sci. 23,

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(1997).

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U.S.A. 102, 17241 (2005).

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P. Netherly, Quat. Int. 109-110, 3 (2003).

10. K. M. Olsen, B. A. Schaal, Am. J. Bot. 88, 131 (2001).

11. A. Jarviset al., Plant Genet. Resour. Newsl. 131, 28 (2002)

12. M. E. Ferguson et al., Biodivers. Conserv. 14, 1777

(2005).

Table 1. Radiocarbon dates from selected preceramic sites in the Ñanchoc Valley. New AMS dateson archaeological macrobotanical remains are in bold.

Label no. Site/unit Radiocarbon

age (yr B.P. ± SD)

2S -Cal age

range (yr B.P.) Provenience

Late Paiján phase (10,000 –9000 yr B.P.)

Beta 154099 PV-19-122-1 9980 ± 80* 11710–11201 Wood charcoal in house floorBeta 12384 CA-09-27 9870 ± 120† 11703–10775 Wood charcoal in hearth on

house floorBeta 154124 PV-19-97-8 9520 ± 130* 11178–10304 Wood charcoal in hearth on

house floorBeta 179512 CA-09-77 9240 ± 50* 10403–10163 Charred/desiccated squash

seed in house floor

Las Pircas phase (9000 –7000 yr B.P.) ‡Beta 154126 PV-19-101-11 8470 ± 60* 9532–9288 Wood charcoal in house floorBeta 33526 CA-09-27 8410 ± 140† 9580–8996 Wood charcoal in house floorBeta 154125 PV-19-100-7 8270 ± 60* 9400–9015 Wood charcoal in house floorBeta 33524 CA-09-27 8260 ± 130† 9484–8772 Wood charcoal in house floorBeta 33523 CA-09-27 8210 ± 180† 9481–8606 Wood charcoal in house floorBeta 30781 CA-09-27 8080 ± 70† 9111–8636 Wood charcoal in house floorBeta 12385 CA-09-27-5 7950 ± 180† 9256–8378 Wood charcoal in house

middenBeta 12384 CA-09-27 7920 ± 120† 9002–8427 Wood charcoal in hearth on

house floorBeta 33525 CA-09-27 7850 ± 140† 9005–8370 Wood charcoal in house floorBeta 219588 CA-09-77 7840 ± 40* 8640–8435 Charred peanut hull in

house floor

UCR-2371 CA-09-04-A 7720 ± 100† 8691–8203 Wood charcoal in burnedfeature in house

Beta 30779 CA-09-27-5 7690 ± 70† 8587–8330 Wood charcoal in house floorBeta 219589 CA-09-27 7660 ± 40* 8535–8342 Desiccated/charred squash

seed in house floor

Beta 30778 CA-09-27-5 7630 ± 80† 8541–8199 Wood charcoal in house floorBeta 182962 CA-09-04 7520 ± 40† 8373-8189 Organic sediment in buried

mound surfaceBeta 15708 CA-09-04-B 7190 ± 130† 8274–7677 Wood charcoal in hearth on

house floor

Tierra Blanca phase (7000-4500 yr B.P.) ‡

Beta 154128 CA-09-04-B 6970 ± 90† 79330–7595 Wood charcoal in hearth onhouse floor



Beta 34332 CA-09-15, QSN-1 6705 ± 75† 7656–7426 Wood charcoal in possibleCanal 4

Beta 181279 CA-09-71 5490 ± 60* 6278–5948 Cotton boll in house floor

Beta 154127 QSN-1 5380 ± 80* 6279–5936 Wood charcoal in Canal 3Beta 182966 CA-09-15, QSN-1 4390 ± 40* 5038–4833 Wood charcoal in Canal 2

*AMS radiocarbon dates from wood charcoal and macrobotanical remains excavated in house floors and hearths. Samples werestored and processed in a museum warehouse in Peru and not subjected to possible contamination from biological labeledradiocarbon material. See explanation in SOM Text, section 2. †Conventional radiocarbon dates from wood charcoal inhouse floors, hearths, and middens. ‡Prior publications listed the Las Pircas Phase dating between 8500 and 6500 yr B.P.and the Tierra Blanca Phase dating between 6500 and 4500 yr B.P. ( 3–9). Recent dates and new site traits have shifted eachphase 500 years back in time to 9000 to 7000 yr B.P. for Las Pircas and 7000 to 4500 yr B.P. for Tierra Blanca.

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(2000).

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the 8th EUCARPIA Meeting on Cucurbit Genetics and

Breeding, Czech Republic, A. Lebeda, H. S. Paris, Eds.

(Palacký University, Olomouc, Czech Republic, 2004),

pp. 13–118.

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Proc. Natl. Acad. Sci. U.S.A. 99, 535 (2002).

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110, 392 (2005).

19. J. E. Damp, D. M. Pearsall, Econ. Bot. 48, 163 (1994).

20. S. G. Stephens, M. E. Moseley, Am. Antiq. 39, 109

(1974).

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N. Tuross, Proc. Natl. Acad. Sci. U.S.A. 102, 18315

(2005).

22. M. C. Bruno, W. T. Whitehead, Latin Am. Antiq. 14, 339

(2003).

23. D. Bonavia, Rev. Hist. 47, 77 (1993–1995).

24. J. Haas, W. Creamer, Curr. Anthropol. 47, 745 (2006).

25. B. D. Smith, Science 276, 932 (1997).

26. D. R. Piperno, A. J. Ranere, I. Holst, P. K. Hansell, Nature

407, 894 (2000).

27. F. J. Aceituno, N. Castillo, Before Farming 2, 2 (2005).

28. D. Zohary, M. Hopf, The Domestication of Plants

in the Old World (Oxford Science Publications, Oxford,

ed. 3, 2000).

29. M. E. Kislev, A. Hartmann, O. Bar-Yosef, Science 312,

1372 (2006).

30. We thank the Instituto Nacional de Cultura, Lima,

the National Science Foundation, the Heinz Foundation,

the University of Kentucky, and Vanderbilt University

for supporting this research. We are grateful to

D. Bonavia, P. Netherly, and especially D. Piperno

for comments. We thank D. Hood of Beta Analytic and

G. Burr of the AMS lab at the University of Arizona for

discussing the 14C dates with us.

Supporting Online Materialwww.sciencemag.org/cgi/content/full/316/5833/1890/DC1

SOM Text

Tables S1 and S2

References

16 February 2007; accepted 9 May 2007

10.1126/science.1141395

Sponge Paleogenomics Revealsan Ancient Role for CarbonicAnhydrase in SkeletogenesisDaniel J. Jackson,1,2 Luciana Macis,1 Joachim Reitner,1 Bernard M. Degnan,2 Gert Wörheide1*

Sponges (phylum Porifera) were prolific reef-building organisms during the Paleozoic and Mesozoic~542 to 65 million years ago. These ancient animals inherited components of the first multicellularskeletogenic toolkit from the last common ancestor of the Metazoa. Using a paleogenomicsapproach, including gene- and protein-expression techniques and phylogenetic reconstruction, weshow that a molecular component of this toolkit was the precursor to the a-carbonic anhydrases (a-CAs), a gene family used by extant animals in a variety of fundamental physiological processes. Weused the coralline demosponge Astrosclera willeyana, a “living fossil” that has survived from theMesozoic, to provide insight into the evolution of the ability to biocalcify, and show that the a-CAfamily expanded from a single ancestral gene through several independent gene-duplicationevents in sponges and eumetazoans.

T

he increased abundance of calcified struc-tures during the late Neoproterozoic and

the “

Cambrian Explosion”

(1) suggeststhat certain biotic (2) and/or abiotic factors (3)enabled the genesis of calcification strategieswithin many metazoan clades during this period(4). To what extent these ancestral lineages drewupon a shared skeletogenic toolkit remains aquestion in our understanding of the early evo-lution of metazoan life; does the surge in earlyCambrian calcification reflect a common inheri-tance of a key genetic toolkit, or did the ability to biocalcify (5) evolve independently in different lineages? Paleogenomics, the study of ancient genomes through the comparison of extant orga-nisms (6 ), attempts to address such questions.

A related milestone in the evolution of complex life was the evolution of the capacity tocatalyze the hydration of CO2. The chemical re-action [CO2 + H2OHCO3

− + H+ (7 )] functionsin processing metabolic wastes, regulating pH,fixing carbon, and transporting ions across organ-

ic membranes (8). The metalloenzyme carbonicanhydrase (CA) is pivotal to these processes by

catalyzing this reaction approximately 1 millionfold (9). Multiple duplications of the a-CA genewithin metazoan lineages, and a paucity of se-quence data from early branching metazoanlineages, have made the evolutionary origins of this family difficult to decipher (8, 10).

Coralline sponges are members of the earliest branching metazoan taxon (Porifera) to secrete aCaCO3 skeleton and were major contributorsto the first metazoan reef-building processes inthe early Paleozoic and Mesozoic eras (11). Astrosclera willeyana [Porifera, Demospongiae,Agelasida (12)] (Fig. 1A) is a coralline marinesponge with a stromatoporoid-like body-plan(13). In addition to primary glass spicules (acardinal skeletal feature of most Demospongiae),it constructs a secondary skeleton of solidaragonite (CaCO3) (Fig. 1, B and C), and thetaxon Astrosclera is present in the fossil recordfrom the late Triassic (14). These features make A. willeyana a valuable model for studies aimedat elucidating the early mechanisms of biocalci-fication. Here we provide evidence that a major molecular component of the calcification processin A. willeyana isan a-CA that was inherited as asingle-copy gene from the last common ancestor of the Metazoa (LCAM).

The CaCO3 skeleton of A. willeyana consistsof spherical-to-ovoid aragonitic elements(spherulites; Fig. 1, D to F) that are initially deposited inthe distal ectosome by large vesicle cells (LVCs)Spherulites gradually enlarge and fuse to form the“hypercalcified” basal skeleton (Fig. 1, B and C)(14). We isolated cell-free spherulites and the basal skeleton from A. willeyana and from thismaterial extracted the soluble organic matrix

(SOM). The N terminus of three predominantSOM protein bands (Fig. 1G) was sequenced byEdman degradation, and three full-length cDNAsequences that contained these N-terminal motifswere isolated (15). All three isoenzymes, namedAstrosclerin-1, -2, and -3, had clear homology tothe a-CAs and signal sequences indicative of anextracellular or membrane-bound localization ofthe mature protein (figs. S1 andS2). Phylogeneticanalysesof a representative set of metazoana-CAsequences placed Astrosclerin-1, -2, and -3 withAmq-CA1, -2, and -3 that were bioinformaticallyrecovered from the genome of another demo-sponge, Amphimedon queenslandica.These demo-

spongea

-CA sequencesconstitute the sister groupto all other metazoan a-CAs (Fig. 2) (16 ).Whole-mount in situ hybridization (WMISH)

reveals that Astrosclerin-1, -2,and -3 are expressedin LVCs within the ectosome (Fig. 1, H and I)Sections through decalcified WMISH tissue re-vealed a characteristic annulus morphology of Astrosclerin-positive cells, with the central vesicledevoid of the calcifiedspherulite (Fig. 1I,asterisk)

To confirm that the Astrosclerins possess CAactivity, we overexpressed isoenzymes 1, 2, and3 in Escherichia coli. Recombinant Astrosclerin-2 and -3 were expressed as soluble proteinswhereas Astrosclerin-1 formed inclusion bodies(fig. S3). Astrosclerin-1 was refolded under avariety of conditions, none of which yielded afunctional protein. Isoenzymes 2 and 3 were subjected to affinity chromatography. Astrosclerin-2did not bind to the column, presumably due tothe lack of a zinc-coordinating histidine residue(figs. S1 and S2). The activity of Astrosclerin-3was assayed with a modification of the Wilbur-Anderson assay (17 ) and was found to have com- parable activity to that of the highly active bovinea-CAII (Fig. 3).

Knoll (4) points out that phylogenetic recon-structions based on morphology indicate re

1Geoscience Centre Göttingen, Department of Geobiology,Goldschmidtstrasse 3, D-37077 Göttingen, Germany.2School of Integrative Biology, University of Queensland,Brisbane 4072, Australia.

*To whom correspondence should be addressed. E-mail:[email protected]

www.sciencemag.org SCIENCE VOL 316 29 JUNE 2007

REPO


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www.sciencemag.org/cgi/content/full/316/5833/1890/DC1

Supporting Online Material for

Preceramic Adoption of Peanut, Squash, and Cotton in Northern Peru

Tom D. Dillehay,* Jack Rossen, Thomas C. Andres, David E. Williams

*E-mail: [email protected]

Published 29 June 2007, Science 316, 1890 (2007)

DOI: 10.1126/science.1141395

This PDF file includes:

SOM Text

Tables S1 and S2References


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Supporting Online Materials

1. Extracting and Studying Macrobotanical Remains

Botanical remains from the Zaña and Ñanchoc Valley sites were recovered through waterflotation. A small plastic tank was assembled in the field and soil samples were floated in the Ñanchoc River. Window screen in the bottom of the tank captured heavy fraction (sinking)materials, while lighter (floating) materials passed through a pour spout and were captured infine gauze or paint strainers. Soil samples were taken from the middens of all excavationlevels and from all major features such as house floors, hearths, and storage units.Preservation of organic remains generally is not good in the forested alluvial fans of the Ñanchoc Valley.

During the 1987 field season, a total of 46 samples representing 690 liters of soil werecollected and floated for analysis. The 1989 and 1992 field seasons yielded 35 samplesmeasuring approximately 250 liters of sediment for flotation. Table 1 presents the number ofmacrobotanical remains recovered by species from excavated sites and discussed in the text.Initial identifications were conducted by Jack Rossen and Donald Ugent using a binocularlight microscope and for selected specimens, a scanning electron microscope at theUniversity of Kentucky was employed. Ugent’s herbarium at Southern Illinois Universitywas used to make direct comparisons. Many discussions with Ugent and later the third authorand fourth authors (Tom Andres and David Williams) and Dolores Piperno of theSmithsonian Institution were invaluable in compiling and interpreting the botanical remains.Ugent was an author of the 1996 article that presented the chronological dilemma of the plantremains that had intact contexts and ancient morphologies, yet produced erratic andunsatisfactory AMS dates in the late 1980s and early 1990s (S1).

In the Ñanchoc region, the semi-arid environment allows for desiccated plant remains whenhouse floors or overlying stones protected the materials from seasonal rains. The botanicalremains from the late Paijan and Las Pircas Phase sites were recovered from a hard-packedcircular structure floor, a small elliptical (ca. 1.3m by 1m) stone structure and, rarely, fromthe general midden. The Tierra Blanca Phase materials were recovered from very packedfloors of multi-room rectangular structures (ca. 3m by 4 to 5m). The remains from both time periods are either carbonized or desiccated, with desiccated materials usually located inisolated pockets beneath grinding stone fragments or fallen stones from the side walls of thehouses.

Despite a systematic and intensive water flotation program for recovering plant materialsfrom all site contexts, the low frequencies of plant material and their recovery from only afew contexts reflect the sporadic nature of preservation (Table S1). The contexts of thecultivated plant materials from Site CA-09-27 provide a particular case in point. At this site,36 squash seeds were recovered, 34 from a circular (3m diameter) structure floor and onesquash seed each was recovered from the elliptical structure and the general midden. Eight ofthe nine recovered peanuts are from the elliptical structure and the ninth was in


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the midden. A single specimen of carbonized quinoa-like chenopod and a hollowed outmanioc tuber, identified from starch grain analysis, are from the edge of the structure floor,where hard-pack dirt floor met softer general midden and where the structure walls and floormet.

Phytolith and starch grain analyses currently are underway on human teeth, grindingstones, and hearth sediments from both Las Pircas and Tierra Blanca sites.

Supporting Table 1. Frequencies of Primary Plants in Paijan Phase, Las Pircas Phase, andTierra Blanca Phase sites of the Ñanchoc Valley.#

_____________________________________________________________________Phase: Paijan Las Pircas Tierra Blanca

_____________________________________________________________________

Site #: CA-09-77 CA-09-27 CA-09-50 CA-09-71 CA-09-77 CA-09-80 _____________________________________________________________________ peanut Arachis sp. 9 2 8 5_____________________________________________________________________

squashCucurbita moschata 2 36 2

_____________________________________________________________________

quinoa-like chenopodChenopodium sp. cf. quinua 1 31 1

_____________________________________________________________________

cotton Gossypium barbadense 1 2

_____________________________________________________________________# Listed are only those species discussed in the text. All specimens were recovered from buried house floors.


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2. Supporting Comments on Previous Erratic AMS Dates

Despite the contextual and morphological lines of evidence that suggested themacrobotanical samples excavated in 1987,1989, and 1992 were ancient and retrieved fromexcellent contexts in buried house floors and hearths, and the absence of evidence ofdisturbance or later intrusion at the sites, there were repeated problems with the earlier AMSdating of these remains. Between 1987 and 1995 various AMS dates placed the plants between 11,650 14C YBP (Table S2) and in the 1950’s early nuclear test era or in the future,despite the fact the Las Pircas and Tierra Blanca areas were uninhabited in historic times.Also, there was no collective memory among present-day Ñanchoc people of habitation orfarming in the area, which today is a dry thick forest. We have been very cautious in presenting these plant materials to the scientific community (S1), and decided to wait severalyears before dating the new samples using AMS methods again. Now, nearly two decadesafter initial excavation and dating of the primary plants, the new set of AMS dates reportedhere on the previously studied and newly excavated materials has confirmed the early tomiddle Holocene age of these ancient plant remains.

Why the earlier AMS dates were incorrect remains unanswered. We have maintained acontinuous dialogue with the dating laboratories to discuss potential contamination of thedated materials and the measures taken to mitigate the effects of any contaminates. At first,we thought that the dated materials were contaminated by natural causes, such as travertinedeposits in the area, but later dismissed this possibility. We now suspect that the buriedmacrobotanical remains from the Ñanchoc sites were contaminated by radiolabeled carbon 14 biological compounds that may have been present in a United States Customs laboratory in astorage facility in Miami where the samples were opened and retained for more than a monthin 1987 or in a biomedical forensic anthropology laboratory at the University of Kentuckywhere the dated samples were stored between 1988 and 1995. The authors have been told byDarden Hood, President of Beta Analytic, Inc. and by Dr. George Burr, the Arizona AMS

Laboratory at the University of Arizona that such compounds would significantly affect anyAMS dates on the order of magnitude measured in the erratic young Ñanchoc dates (TableS2). Several similar cases of unacceptable young AMS dates affected by biological labeled14C materials in radiocarbon and biomedical laboratories have been documented, with themost recent for archeological sites in Alaska (S2). We cannot explain why the coca leaf fromsite CA-09-77 resulted in the excessive and unacceptable early date of 11,650 +/- 400 14CYBP (Table S1). A second coca leaf recovered from beneath stones fallen from a side wall atsite CA-09-77 recently was dated by AMS methods at 7120 +/- 50 14 C YBP (Beta 226458:8010-7850 CAL), which agrees with prior dates processed for wood charcoal from hearthsand other features in an excavated house in this site (Table 1). There may be a cautionary talehere that there has been a reliance on the use of some AMS dates to act as judge and jury towinnow reliable data. We need to reestablish the fundamental primacy of context andassociation in archaeology and also to redate questionable archeological materials.


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Supporting Table 2: Erratic AMS Dates on Macrobotanical Remains from 1987-1992

Samples

Label No. Site/UnitRadiocarbon

Yr B.P.

2Σ-calibrated Age

Range B.P.Provenance

Beta 72409 CA-09-77 11,650 ± 400* Date beyondcalibration range

Charred/desiccated coca leaffragment in house floor

AA-5964 CA-09-27 250 ± 60* 444 to -1 B.P. Desiccated/charred squash

seed in house floor

Beta 39113CA-09-27 130 ± 65* 279 to -4 B.P.

Desiccated squash in housefloor

AA-6488CA-09-27 125 ± 90*

288 to -5 B.P. Carbonized chenopod in house

floorBeta 39112 CA-09-27 0-5 ± 1%* -200 B.P. Desiccated peanut in house

floor

* AMS radiocarbon dates from archeologically buried macrobotanical remains from site

CA-09-27 and CA-09-04. Dated materials from CA-09-27 were probably contaminated

by labeled radiocarbon materials.

Supplementary References Cited

1. Rossen, T.D. Dillehay, D.Ugent, J . Arch. Sci. 23, 391-407 (1996).

2. J.D. Reuther, S.C. Gerlach, Radiocarbon 47, 359-366 (2005).

dillehay-et-al-2007-preceramic-adoption-of-peanut-squah-and-coton-in-northern-peru-copia (4).pdf

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