Piperno, 2008. Starch Grains on Human Teeth

8/12/2019 Piperno, 2008. Starch Grains on Human Teeth

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Starch grains on human teeth reveal early broad cropdiet in northern PeruDolores R. Pipernoa,b,1 and Tom D. Dillehayc,d

aArchaeobiology Program, Department of Anthropology, Smithsonian National Museum of Natural History, Washington, DC 20560; bSmithsonian TropicalResearch Institute, Apartado Postal 0843-03092, Balboa, Republic of Panama; cDepartment of Anthropology, Vanderbilt University, Nashville, TN 37265;and dDepartamento de Antropologa, Universidad Austral de Chile, Valdivia, Chile

Edited by Joyce Marcus, University of Michigan, Ann Arbor, MI, and approved October 22, 2008 (received for review September 3, 2008)

Previous research indicates that the Nanchoc Valley in northernPeru was an important locus of early and middle Holocene humansettlement, and that between 9200 and 5500 14C yr B.P. the valleyinhabitants adopted major crop plants such as squash (Cucurbitamoschata), peanuts (Arachis sp.), and cotton (Gossypium barba-dense). We report here an examination of starch grains preservedin the calculus of human teeth from these sites that provides directevidence for the early consumption of cultivated squash andpeanuts along with two other major food plants not previouslydetected. Starch from the seeds ofPhaseolusandInga feuillei, theflesh of Cucurbita moschata fruits, and the nuts of Arachis wasroutinely present on numerous teeth that date to between 8210

and 6970 14

C yr B.P. Early plant diets appear to have been diverseand stable through time and were rich in cultivated foods typicalof later Andean agriculture. Our data provide early archaeologicalevidence for Phaseolus beans and I. feuillei, an importanttree crop,and indicate that effective food production systems that contrib-uted significant dietary inputs were present in the Nanchoc regionby 8000 14C yr B.P. Starch grain studies of dental remains documentplants and edible parts of them not normally preserved in archae-ological records and can assume primary roles as direct indicatorsof ancient human diets and agriculture.

early diets South America food production

T

he Late Pleistocene to Middle Holocene Nanchoc prece-

ramic culture is known from 46 sites in the N

anchoc Valley,a tributar y of the Zana Valley located at 500 m above sea levelon the lower western slopes of the Andes (17) (Fig. 1).Recovered from hearths and floors in 13 excavated housestructures dating from 10,100 to 5500 14C yr B.P. (11,200 to6000 calendar years ago or cal. B.P.) were the macrobotanicalremains of a variety of plant cultivars along with gathered wildplants and the bones of various large and small animal species,providing evidence for a broad spectrum and mixed subsistenceeconomy in a tropical dry forest setting (13, 6). Between 10,000and 7000 14C yr B.P. during the Late Paijan and Las Pircasphases, crop production was practiced close to small, permanent,circular houses. Associated with the plant remains during thesubsequent Tierra Blanca phase between 7000 and 5000 14C yrB.P. were stone hoes, larger grinding stones, plots for planting

crops, small-scale irrigation canals and public mounds (siteCA-09 04, Fig. 1), and rectangular houses (13, 6).

We examined 39 human teeth from Las Pircas occupations atfour different sites [Table 1, supporting information (SI) TableS1]. The teeth were mostly isolated remains from adults andsubadults that were securely embedded within intact and sealedhouse floors and directly associated w ith radiocarbon datesmade on human bone, macrobotanical plant remains, and woodcharcoal ranging from 8210 180 to 6970 60 14C yr B.P.(Methods; Table 1; Tables S1 and S2; see ref. 1 for a morecomplete list of 14C dates from the sites). Three teeth weresubmitted to Beta Analytic Inc. for accelerator mass spectrom-eter (AMS) radiocarbon dating but insufficient protein re-mained for an age determination (Table 1). The isolated teeth

come from an estimated minimum of six to eight individuals,probably from both sexes; three complete individuals are alsorepresented (Table 1).

We cannot state with much precision how much of an indi-viduals lifetime the tooth calculus that was studied represents.Little information is available on the rate of calculus formationbut because it accumulates over an individuals life if notremoved, at least several years of diet is probably represented foreach tooth studied. To remove dental calculus and examine andidentify the starch in it we used nondestructive methods devel-oped by ourselves and large modern reference collectionshoused in D.R.P.s laboratory (MethodsandSI; we also searched

for phytoliths in the calculus preparations and found none, notsurprising in view of the fact that the plants evidenced on theteeth and many others that were potentially available in the studyregion do not produce phytoliths in their edible parts).

Results

Most teeth yielded some starch grains and 13 contained them insignificant quantities. Table 1 contains the kinds and number ofgrains retrieved from these specimens. TableS1 containsdata onthe teeth with fewer grains. Ubiquitous and consistently commonover the 1,000-year period represented are four taxa,Phaseolus,Arachis, Cucurbita moschata, and Inga feuillei, indicating theywere major dietar y sources (Table 1) (Fig. 2 ae). We cannotdetermine conclusively whether the Phaseolus starch is from adomesticated bean or whether it has a greater affinity to P.

lunatusorP.vulgaris(Fig. 1aandbfor archaeologicalPhaseolus;Fig. S1 A and B for modern comparisons). It is generallyconsidered that single domestications of the Andean lima andcommon bean occurred, respectively, in southern Ecuador/northern Peru and southern Peru/Bolivia (812). Three speciesof wild Phaseolus closely related to domesticated species, all of

which were analyzed here, grow in Peru: P. augusti, P. pachyr-rhizoides, and wild P. vulgaris. The first two probably should bemerged taxonomically and are thought to be the limas wildancestor (9, 12).

In most teeth samples, average and maximum Phaseolusgrainsize is considerably larger than in wild common bean, which canbe excluded from representation on this basis (Table 2 andTableS3 for comparative modern data). However, there is a broadrange of size overlap between wild and domesticated lima bean

starch, and size values for all but one of the teeth fall within thisoverlap zone. In sample 19, dated to 6970 14C yr B.P., averagelength and width ofPhaseolusgrains exceed those of all modern

wild beans studied. Given the size similarities between wild and

Author contributions: D.R.P. and T.D.D. designed research; D.R.P. performed research;

D.R.P. and T.D.D. analyzed data; and D.R.P. and T.D.D. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/cgi/content/full/

0808752105/DCSupplemental .

2008 by The National Academy of Sciences of the USA

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domesticated lima bean starch, we prefer to exercise caution atthis time with the interpretation of this single tooth showing adomesticated grain size. Starch from wild and domesticated

Phaseolus species is too similar in morphology to attempt aspecies-specific identification on this basis (MethodsandFig. S1A and B).

The ecology of wild Phaseolus does strongly suggest that thestarch grains represent cultivars. Wild lima and common beansare found today in dry and thorny or humid forest at elevationsbetween 1,800 and 3,000 m above sea level (asl) (9). TheNanchoc sites, located at 500 m asl in dry tropical forest, areconsiderably outside of the elevational range and native habitatsof these plants. The previous earliest record for Phaseolus inSouth America was the domesticated P. lunatus from Chilca,southern coastal Peru, dated to 5600 14C yr B.P. (ca. 6400 cal.

B.P.) (13). The earliest common bean remains, from GuitarreroCave in central highland Peru (13), presently date to ca.4600 14C

yr B.P. (ca.5000 cal. B.P.). Considering this evidence along w ith

the greater proximity of the Nanchoc region to the probablehearth of lima domestication, it may be more likely that thePhaseolus starch is from a lima rather than a common bean.

Previous work identified the early presence of a domesticatedsquash species, Cucurbita moschata, which likely is native tolowland northern South America, possibly Colombia. Distinc-tive, elliptical-shaped seeds dark brown in color that are uniqueto C. moschata were recovered from sites CA-0977 and CA-0927 where they were directly dated to 9240 50 14C yr B.P.and 7660 40 14C yr B.P., respectively (1). Starch grain dataprovide an independent line of evidence for this crop andbroaden our understanding of how Cucurbita was consumed

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Table 1. Types and Numbers of Starch Grains Recovered from the anchoc Teeth

Sample/14C Age

Phaseolus Arachis Cucurbita moschata Inga* Legume Globular Bell U #1, 2, 3, 4, 5, 6 Other Total

n n (xSize; range) n (xSize; range) n n n n n n n

Site CA-0977 Microns Microns

2A, 6970 60 HB 14 3 (8.7; 612) 11 (8.3; 612) 26 5 0 0 1 - - - - - 11(1) 71 I

29, 6970 60 HB 55 2 (6; 57) 2 (7; 59) 4 13 5 0 5 - - - - - 10(5) 91 M

19, 6970 60 HB 24 0 1 (12) 1 0 2 0 - - - - - - 7(4) 35 M

68, #1, 7840 40 PH 93 0 4 (9.8; 811) 0 1 2 0 2 - - - - - 12(2) 114 M

68, #2, 7840 40 PH 37 4 (77 ) 2 (13; 1115) 2 13 9 1 - - - - - - 12(8) 80 M

7, #2, 7840 40 PH 3 5 (10.2; 714) 0 0 0 1 0 - 2 - - - - 2 13 M

50, #1, 7840 40 PH 14 41 (11.9; 718) 0 2 0 See note 0 - - - - - - 41(8) 98 M

50, #4, 7840 40 PH 22 6 (9; 613) 2 (66) 14 1 12 1 15 2 2 - - - 13(3) 90 M

56, #4 7840 40 PH 5 2 (13; 817) 0 0 0 0 0 - - - - - - 11(9) 18 M

**59, 7840 40 PH 23 3 (13.3;1215) 0 0 4 0 0 1 2 - 1 - 1 4 42 M

58, #1, 7840 40 PH 22 1 (12) 0 0 0 0 0 - 2 - 2 2 - 4 27 M

58, #3 7840 40 PH 3 3 (8.7; 89) 4 (8.5; 611) 14 0 7 1 - 2 - 2 2 - 17 55 M

Site CA-0928

1A 8210 180 WC 4 1 (9) 9 (9.8; 712) 1 0 0 0 - - - - - - 9 15M

Note:14C dates from CA-0977 are derived from materials (HB human bone, PH charred peanut hull) directly associated with teeth from hearths sealed

in individual intact floors (ca. 3 cm thick) of buried house structures (seeTable S2and refs. 1, 6 for more details). For CA-0928, the associated date is on wood

charcoal.Teeth were submitted to Beta Analytic Inc. for AMS radiocarbon dating but insufficient protein remained for an age determination. *Identified as

Inga feuillei. ThecategoryLegume represents ovalgrains thatoccur in Phaseolus andalso in other Fabaceae genera. Globular grains are consistent withthose

inArachis and Inga andalsooccurin a variety of non-legume taxa. **Three grainstypical of underground organsoccurred. In sample50, #1 many of the Other

grains occurred in clumps and were difficult to describe in detail but they were globular in shape as in peanut and/or pacay. Bell grains that occurred in small

numbers on some teeth are consistent with manioc but a positive identification is not possible. Numbers in parentheses in the Other category represent

gelatinized grains that were too damaged from heat to identify. Letters in bold after Total nindicate type of tooth (I incisor, M molar). Teeth 50 and 58

are from sub-adults. Others are indeterminate as to the approximate age of the individual. Tooth 1A is from a complete skeleton.

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during early periods. Many teeth contained distinctive bell-

shaped grains diagnostic of the flesh ofC. moschatafruits (Fig.2c; Fig. S2a) (seeds from the genus have no starch). In mor-phology and size the grains are identical to those found in theflesh of traditional modern fruits ofC. moschata and they areunlike those of other Cucurbita species, including C. sororia, a

wild taxon closely related to C. moschata (14) (Methods; SI).Cucurbita flesh produces significantly less starch than legumeseeds, thus it is likely to be underrepresented in the records. Theevidence firmly indicates that at Nanchoc early domesticatedCucurbita was routinely consumed and not used primarily as autilitarian or industrial plant (e.g., as containers and fishnetfloats). Moreover, as all wild Cucurbita species have extremelybitter, nonedible flesh, human selection for the major domesti-cation gene that codes for nonbitter fruits by 8000 14C yr B.P. isimplied.

Another plant evidenced through previous research is thepeanut. Hulls from Arachissp. recovered from CA-0977 weredirectly dated to 7840 40 14C yr B.P. (1). The hulls were notexact morphological matches for modernA. hypogaea but neitherdid they conform to a known w ild species. They almost certainlyrepresent cultivars because they occur far outside the naturaldistribution of the genus in southern South America (15). Peanutnuts were not preserved in the macrobotanical records; however,starch grains diagnostic ofArachisnuts (hulls contain no starch)are present on many teeth (Table 1; Fig. 2d;Fig. S2b). In theirmorphological and size characteristics they are matches formodern Arachis hypogaea(compare peanut data in Table 1 withthat for modern varieties in Table S3). We also studied twopeanut hulls retrieved from sites CA-0977 and CA-0950 that

date to 8000 to 7800 14C yr B.P. and 7000 14C yr B.P.,

respectively, to ascertain whether any starch grains from decayednuts survived in them. These specimens also yielded grains likethose in A. hypogaea.

We examined a diploid wild species, A. williamsi, which isclosely related to A. hypogaea. It has starch grains with a meanlength of 6.6 m and a maximum length of between 4 and 10 m.This length is much smaller than grain size on the teeth and indifferent varieties of modern A. hypogaea, where maximumlength was 18 m and average length was from 9.5 to 10.1 m(Table S3). Grains from A. williamsi also lack surface decora-tions found in A. hypogaea. We also studied three wildArachisspecies,A.burchelli,A. marginata, andA. retusa, which are froma different taxonomic section of the genus that is more distantlyrelated to A. hypogaea. They all exhibited markedly differentstarch morphological attributes (bell shaped instead of globular)

when compared with A. hypogaea,A.williamsi, and the archae-ological grains, providing more evidence that the Nanchocpeanuts were closely related to A. hypogaea. This may be a case

where different traits indicative of domestication, such as hullmorphology and nut starch grain qualities in these peanutexamples, were not transformed from their wild states simulta-neously or at the same rate by early human selection pressure,resulting in early cultivars that have a mixture of wild- anddomesticated-type features. Analysis of the direct wild progen-itors of the peanut, thought to be A. ipaensisand A. duranensisor A. helodes (15, 16), w ill shed more light on this issue.

Inga feuillei (pacay) is an important tree crop that was wellused on the Peruvian coast by 4500 14C yr B.P (1719). It isprobably native to the lowland eastern slopes of the Andes and

Fig.2. (a) AnovalstarchgrainfromPhaseolus witha longitudinalfissure andlamellaefrom tooth 29.(b) Left, anovalstarchgrainfromPhaseoluswitha raggedlongitudinal fissure and lamellae. Right, a round starch grain from Phaseolus with a stellated fissure and lamellae concentrated near the edge. Both grains are

from tooth 50, no. 4. (c) Bell-shaped grains from teeth 1A (Left) and 2A (Right) with pleats (unique kinds of lamellae) and pressure facets from Cucurbita

moschata fruit flesh. (d) A cluster of four starch grains from peanut nuts from tooth 50, no. 1. The grains are globular in shape and have projections/knobs at

the edge, slight fissures, and tiny starch grains attached at the periphery. (e) Oval (Left) and elliptical (Right) starch grains with white longitudinal fissures from

Inga feuilleifrom samples 50, no. 4 and 58, no. 3.

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it can be grown at much higher elevations, which made itimportant to the Inca (20). Pacay has large edible pods with asweet, white pulp. Starch identified from this plant derives fromthe seeds; the pulp does not contain starch (Fig. 2e; Fig. S2c).This is clear evidence for early tree crop exploitation in theNanchoc region.

A variety of other starch grains occurred in lower quantitiesand just a few of them appear to be from underground organs(Table 1). Three grains were consistent with those from maniocroots although a positive identification is not possible from thegrain types present (21). Macrobotanical remains of manioc

were retrieved from the sites (6). Low overall frequencies ofstarch from underground organs suggest they were infrequentlyconsumed. Other grains that occurred in low numbers, includingsix types of unknown but distinctive-looking starch (U nos. 1 6),are conceivably from foods such as cacti and tree fruits that arenot represented in our modern collections. It also should bepointed out that some potentially important food sources that

were well used during the later pre-Columbian period in thestudy region do not produce starch or have nondiagnostic grains,thus their dietary contribution cannot be studied. They includedomesticated tree fruits of variousAnnona species (guanabana,custard apple) and guava (Psidiu m guajava), other fruits(Bunchosia armeniaca and hartwegiana; Solanum quitoense ornaranjilla), and the lupine bean (Lupinus mutabilis).

Discussion

Starch grain data from dental remains can inform a number ofimportant issues concerning early human diets and the transitionfrom hunting and gathering to agriculture. It has long beendiscussed, for example, whether the earliest plants taken undercultivation in some areas of the world were dietary items or usedfor nonfood purposes. In the Americas, at least four differentspecies ofCucurbitanative to North America, Mesoamerica, andSouth America are now known to have been among the first cropplants in these regions, and it has recently been proposed thatearly cultivation and diffusion ofCucurbita pepo ssp. ovifera in

the eastern United States were motivated much more by theutility of the plant for its nonfood uses than by its role in the diet(22,cf. ref. 23). In contrast, the Nanchoc evidence clearly showsthat C. moschata was commonly eaten by some of the firstfarmers in Peru.

Overall the evidence indicates that byca.8000 14C yr B.P. (ca.8600 cal. B.P.) an effective farming system employing a range ofseed, vegetable, tree, and root crops provided balanced, nutri-tious, and stable diets to the inhabitants of the tropical westernslopes of the Peruvian Andes. The ubiquity of plants at Nanchocthat would come to typify later agricultural systems suggests thatregional dietary patterns began to emerge not long after foodproduction and crop dispersals began. The multifaceted botan-ical, settlement (sedentary and seminucleated communities),

Table 2. Phaseolusstarch grain morphology and size on the anchoc teeth

Site and

specimen Age

Oval, no damage Oval damaged

Round with

lamellae

Round with

fissures

Length, m Width, m n Length, m Width, m n Diameter, m n Diameter, m n

CA-0977

19 6970 60 39 (3253) 29 (2339) 5 37 (2852) 30 (2051) 15 30 (2930) 2 14 (1117) 2

29, #1 6970 60 29 (1850) 23 (936) 5 27 (1947) 21 (831) 19 24 (2029) 4 19 (1523) 2

27, #2 6970 60 44 38 1 0 0 0

10, #1 6970 60 20 (1921) 16 (1417) 2 0 02A 6970 60 35 (2641) 25 (2132) 7 24 (1931) 17 (1520) 4 0 23 (1332) 3

29, #2 6970 60 0 29 (2632) 2 0 0

29, #3 6970 60 0 34 27 1 0 0

68, #1 7840 40 29 (1944) 25 (1737) 13 29 (1641) 24 (1336) 17 27 (2036) 7 15 1

68, #2 7840 40 29 (2339) 23 (1733) 9 29 (1646) 24 (1242) 18 21 (1526) 9 22 1

7, #1 7840 40 0 39 (3643) 26 (2427) 3

7, #2 7840 40 27 24 1 28 22 1 0 26 1

50, #1 7840 40 33 (2739) 23 (2226) 3 23 (1827) 21 (1624) 7 41 1 14 (1016) 3

50, #4 7840 40 34 (2141) 25 (1532) 4 28 (1842) 21 (1329) 12 28 (2629) 2 15 (1219) 4

50, #2 7840 40 20 1 22 18 2 26 1 18 (1621) 3

56, #4 7840 40 0 36 (3045) 27 (2431) 4 31 1 0

59 7840 40 0 32 (2252) 24 (1744) 20 47 1 0

58 7840 40 29 (1748) 24 (1241) 19 0 0

58, #1 7840 40 0 29 (1748) 24 (1241) 19 16 (1417) 2 37 1

58, #3 7840 40 28 (2729) 23 (2223) 2 0 37 1 0

CA-0952

117 8080 70 0 0 19 1 0

113 7920 120 27 16 (1319) 2 0

15 7920 120 46 32 1 31 (2441) 24 (1431) 5 36 1 22 1

3A 7920 120 24 18 1 34 29 1 0 0

CA-0928

1A 8210 180 38 22 1 29 (2236) 21 (2021) 2 33 1 10 1

Phaseolus oval grainsin the no damage category exhibit no signs of damagefromheat or other sources. Oval grainsin thedamaged categoryexhibit heat

damagesuchas altered or no extinction crosses anddamagedfissures(Figs. S3 adandS4 ad). Round withlamellae grains are thosewithoutfissuresand with

lamellae covering the entire grain. Round with fissures are the smaller round grains that have a variety of fissure types, including stellate, and are without

lamellae or have lamellae concentrated in a thin, dense band at the periphery of the grain. The presence and proportions of these different grain types on the

teethwere also matches forthegenusPhaseolus andno other legumetaxon.In thesizecategories, thefirstnumber isthe mean andthenumbersin parentheses

are size ranges for the grains.

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landscape (irrigation canals and furrowed fields), and artifactualdata (e.g., stone hoes, grinding stones, storage pits) (17) showthat Nanchoc societies cannot be characterized as casual foodproducers who were deriving only negligible dietary inputs f romcultivated plants. The evidence from here and some other

well-studied regions in the Neotropics (17, 24, 25, 26) insteadindicates an early Holocene development of food productiontogether with an emergence not long afterwardat 8000 to7000 14C yr B.P.of subsistence economies that included sig-

nificant amounts of cultivated and domesticated products. Thus,although large sedentary and nucleated villages do not occur inthe Americas until after 5000 14C yr B.P., earlier food productionin some areas was practiced by people who, while still integratingplanting with foraging, were committed farmers who had takenthe first steps along the pathway to full-fledged agriculture.

A routine cultivation (a persistent cycle of sowing and har-vesting) of plants that provide stable and significant dietar yinputs, but that do not acquire all or even some of the phenotypictraits typical of the domestication syndrome for hundreds tothousands of years (known as predomestication cultivation), is apattern increasingly being documented at the beginning ofagriculture in the Old World (27, 28). The issue has received lessattention thus far in the New World, in part because earlymacrobotanical remains from the tropics are rare, but somepotential examples exist from highland Mexico (25). The peanutevidence from Nanchoc points to an instance of predomestica-tion cultivation (hulls lack features of modern A. hypogaea)possibly together with a time-transgressive appearance of dif-ferent domesticated traits in this plant (starch grains are matchesfor modernA. hypogaea and not wildArachis studied), suggestingthat domestication in some major New World crops may alsohave been a protracted and complex process. Possible instancesof predomestication cultivation should be sought more w idely inthe New World using multifaceted archaeobotanical data setsand allied paleoecological evidence when possible. Much moreevidence is needed but it is possible that as in the Old World,traditional, domestication-based c oncepts of agricultural ori-ginsthose that posit that a suite of fully domesticated plants

formed the basis for the first effective food production systemsand initial spread of cropsmay have to be adjusted.

In conclusion, the Nanchoc evidence elucidates in a numberof ways early agricultural lifeways in the Andean region. While,on the present evidence, most of the Central Andes between10,000 and 8000 C14 yr B.P. were occ upied by a wide varietyof maritime and terrestrial foragers that were still organized inrelatively small, mobile groups, some populations such as thosein the Nanchoc Valley were adopting more sedentary life stylesand becoming farmers. The incorporation into subsistenceeconomies of domesticated plants and other cultigens duringthis period did not immediately lead to a full-scale agriculturaleconomy, as Nanchoc people continued to hunt, fish, andgather wild plants. Managing a wide range of food sources

across several closely juxtaposed ecological zones may havehelped to reduce subsistence risk. By 6500 to 6000 C14 yearsago, the emergence of a planned aggregated community andsmall-scale public monuments in the area are evident, an-chored by more intensive food production systems that nowincluded irrigation canals and crop field systems (2, 5). Thesedevelopments, in turn, created new ways of life, new percep-tions of public and private spaces, and new relationshipsbetween members of a community, which represent some ofthe initial pulses toward the rise of Andean civilization (5).More complex ideological, technological, and subsistence ad-

vances that took place after 5500 14C yr B.P. in Peru (1819),including a shift to agricultural dependence, are probablydirect extensions of the Nanchoc developments.

MethodsStarch Grain Sampling of Teeth. Our sampling strategy used methods non-

destructive to the teeth and the starch (29). First, teeth were brushed with

a soft tooth brush and water to remove adherent soil and other particles.

A dentalpickwasusedto scrapeareas ofteethwithvisiblecalculus,and the

residue was transferred directly to a microscope slide where a few drops of

waterhad been placed. Onmanyteeththisprocedurewas done from three

to five separate times by sampling different areas of the specimen such as

thecrownsof molarsandthe gumlineandmakingnew microscope mounts

each time. The plaque could at times be seen to be yellowish in color when

it was transferred to the slide. Sampling was stopped when no additionalstarch grains were recovered. Before the coverslip was put on, one drop of

50% water/glycerine was added to the residue-water suspension to retard

drying and allow grains to be more easily rotated when encountered. This

light solution of glycerine does not alter starch morphology. The non-use

of chemicals ensures that starch grains will not suffer damage during and

after their removal from dental remains.

Archaeological peanut hulls wereexaminedby addinga fewdropsof water

tothe insideof thespecimensusinga pasteur pipette,gently washingthe hull

wall with the water, and transferring the liquid to a microscope slide.

Starch Grain Identification. We used a large modern reference collection

numbering500 economic and other plant species from50 differentfamilies

along with published sources (21, 24, 30 35). Our modern collection includes

all major and many now-minor crops and other plants known or thought to

have been used in Latin America/the Andean region during prehistory and

many wild species closely related to crop plants such as: (1) the wild progen-

itors of lima and common beans from South America and Mexico; (2) other

wild Fabaceae includingInga,Canavalia,Vigna, andMacropitilium; (3) wild

Arachis; and (4) wild Cucurbita native to southern Central America and South

America (C. maximassp.andreana, which is the wild ancestor of C. maxima

andC. argyrospermassp.sororia) (see also ref. 21 for further details on the

collection).C. sororia is closely relatedto theas yetundiscoveredwild ancestor

ofC. moschata(14).

We studieda diversecollection of modern,traditionallima andcommonbean

land races from Peru, Mexico, and other areas of Latin America. Our results

indicate thefollowing:(1) there is considerableoverlapin grain size of wild lima

beans, domesticated lima beans, and domesticatedP.vulgaris; (2) grain size in

wildcommon beans appearsto besignificantlysmaller thanin bothdomesticated

common and lima bean, and (3) there is considerable morphological similarity

among wildand domesticatedspeciesin Phaseolus, making species-specific iden-

tification difficult on this basis (Table S3;Fig. S1A and B).

Ourstarchkeys andclassification system emphasize shape, surface,and size

attributes known to be useful in identification (21, 24, 3035) (see also Figs.S1S5). To confirm identifications of Phaseolus,Arachis,C. moschata, and I.

feuillei we took advantage of the fact that each, along with genera and

species closely related to them, produce more than one type of starch grain,

resultingin taxon-unique starch population signatureswhen a multiplegrain

analysis is undertaken. Archaeological sample sizes from individual teeth

often permittedsuch an analysis,and these population signatures in addition

to the occurrence of diagnostic, individual grain types robustly indicate the

presence of these four major taxa(Figs. S1S5).

We encountered partiallyand completely gelatinized grains, presumably a

resultof cooking, on many of theteeth. Some were identifiable because they

retained their shape and surface characteristics, and some still exhibited an

extinction cross. Recognition of these grains was based on cooking experi-

ments we carried out (below) and other research (3436).

Cooking Experiments.We cooked whole common and lima beans by placing

them in a test tube and then into a hot water bath for times ranging from 15to45 min.After15 minmany grains expandedto three timestheirnormalsize,

lost their lamellae and fissures, and exhibited no or damaged extinction

crosses. However, many retained their characteristic shape, size, and surface

attributes and their extinction crosses, showing no signs of damage(Fig. S3 a

and b). Additionally, a small proportion of damaged grains still exhibited the

characteristic oval to kidney Phaseolusshape and had lamellae and remnant

fissures. The majority of starch grains in samples that were cooked for 45 min

lost lamellae and fissures, but many retained the characteristic Phaseolus

shapes, andlamellae andremnantfissureswerestillvisibleon a few(Fig. S3c).

Most still polarized, exhibiting the diffuse extinction cross arms that are

characteristic of heat damage(Fig. S3d). Cooked grains that retain Phaseolus

shapes and have lamellae or remnant fissures are recognizable as Phaseolus,

and they routinely occurred on the teeth (Fig. S4ad).

We made comparisons of starch grain size in uncooked and cooked beans,

in the latter measuring only grains that were recognizable as Phaseolus

19626 www.pnas.orgcgidoi10.1073pnas.0808752105 Piperno and Dillehay
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(Table S3). Cooked grains were either marginally larger or smaller than

uncookedspecimens.Our analysisof roastedand unroastedpeanuts indicates

thatstarch fromroasted specimens doesnot suffer morphological damage or

size alteration, presumably because thehull offersa good deal of protection.

We expect we will achieve a better understanding of these issues when

experiments are carried out using cooking methods more analogous to pre-

historic techniques, such as pit roasting and hot rock boiling. Nonetheless, it

is clear that starch grains from cooked foods are surviving in an identifiable

formon ancient human teeth. Theyhave alsobeen recoveredfrom blackened

food residues in Early Formative ceramic cooking vessels from Ecuador and

stone tools from Ecuador and New Guinea (3436).

ACKNOWLEDGMENTS. Thisresearchwas supported by the NationalMuseumofNatural History, Washington, DC, Smithsonian Tropical Research Institute (Bal-boa, Panama), National Science Foundation, University of Kentucky, VanderbiltUniversity, Earthwatch, Guggenheim Foundation, National Geographic Society,and the Fulbright Commission. We thank the Instituto Nacional de Cultura ofPeru for permits to carry out the work and International Center for TropicalAgriculture (CIAT), Cali,Colombiaand DavidWilliams for providing many of themodern Phaseolus and peanut samples, respectively. We are grateful to ourcolleagues Jack Rossen, John Verano, and Sonia Guillen who provided unpub-lished data, and Patricia Netherly, Greg Maggard, and Kary Stackelbeck whoassisted with theresearch. We thank three anonymous reviewerswho providedhelpful comments on the manuscript.

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Supporting InformationPiperno and Dillehay 10.1073/pnas.0808752105

Fig. S1A. (a) Starch Grains from Modern DomesticatedPhaseolusBeans (P.vulgaris,a andP. lunatus,b). Grains in both species are simple and mostly oval tokidney-shaped with a centric hilum. They usually have a distinct longitudinal fissure that may be ragged. Lamellae are usually present and sometimes are mostevident near the edge of the grains. Large, round grains with lamellae covering the entire grain and without fissures can be present. Smaller, round grainscommonly occur that have a variety of fissure types, including stellate, and are without lamellae or have lamellae concentrated in a thin, dense band at theperiphery of the grain. These grains have a shiny surface. See Figs. S5aandbfor comparisons withCanavaliaandVigna.

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Fig. S1B. (b) Starch Grains from Modern WildPhaseolusBeans (P. augusti, a andP. pachyrrhizoides,b). Also studied wereP. bolivianusand wildP.vulgarisfrom Mexico. Grains in all species are simple with a centric hilum. Most are oval to kidney-shaped and they usually have a distinct longitudinal fissure that maybe ragged. Lamellae are usually present and sometimes they are most evident near the edge of the grains. These grain types from wild P.vulgarisappear to besmaller than in both wild anddomesticated lima bean. Large,round grainswithlamellaeand no fissures occur.Smaller, round grainscommonly occur that havea variety of types of fissures, including stellate, and are without lamellae or have lamellae that are concentrated in a thin, dense band at the periphery of thegrain. These grains have a shiny surface.

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Fig.S2. (a) StarchGrains from Modern Cucurbita moschata FruitFlesh. Grains are compoundand bell-shaped withdistinctive pleat-like lamellae thatare morepronounced at the distal edge. Many grains have distinctive small and deeply impressed pressure facets at the distal end. The facets may also have a distinctivethickened edge that appears as a cap-like structure. Hila are mostly eccentric and open, and most grains lack fissures. Grains range in length from four to 24

microns with an average size of from 6.7to 13 microns, dependingon thevariety.The closely related wild taxon C.sororia has bell-shaped grains (mean size 6.3microns; range 412) without pleats or other surface features characteristic of C.moschata. There appear to be species-specific morphological differences indomesticatedCucurbitafruit starch. For example,Cucurbita ficifoliahas large, irregular, cup-like types of grains with eccentric hila, lamellae, and facets. C.maxima haslarge bell-shaped forms witheccentrichila, small fissures,and sometimescup-likestructures at thehila. Alsopresent in thisspeciesare ellipticalgrainswithlamellae anda small, eccentricfissure.C. ecuadorensishas other types of grains characteristicof thistaxon. (b) Starch Grains fromModernArachis hypogaeaNuts. Grains are simple and usually globularin shape. Theyoften havedistinctive surface decorations consisting of an overall roughened appearance and/or thepresence of projections/knobs andsometimes short,fine lines at theedge. They mayalsohave a slightfissure.At times,small (2 m) starch grains are attachedat the periphery. (c) Starch Grains from ModernInga feuilleiSeeds. Grains are simple and most are globular to elliptical without lamellae and with distinctivewhite fissures. The fissures can be curved. They may also have a surface decoration consisting of faint and thin, randomly-oriented lines. The grains range from5 to 11 microns long. Small oval grains with distinctive white and thin longitudinal fissures occur that range in length from 14 to 21 microns. Starch frompacayis of a different morphology when compared with other Ingaspecies, including the well-usedInga edulis.



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Fig. S3. (ad) Starch Grains from Cooked ModernPhaseolus vulgarisBeans. The starch grains in image awere cooked for 15 min and those in image cwerecooked for 45 min. Imagesbanddare cross-polarized views of aandc, respectively.



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Fig. S4. (ad) CookedPhaseolusStarch Grains from the Nanchoc Teeth. Imageais from Tooth 58, #2;bfrom tooth 50, #4;canddfrom tooth 68, #1.



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Fig. S5. (a) Starch Grains from ModernCanavalia plagiospermaBeans. Grains are simple. Many are oval to kidney-shaped with lamellae and commonly with deep

andthick transverse fissures,or curved andirregularly-placed fissures. These types of grains are alsomore irregularin overall shape thanin Phaseolus. Oval grainswithlongitudinal fissures that dominate in Phaseolus arerare.Round, shiny grainswith various types of fissures andwithoutlamellaethat arecommon in Phaseolus werenot observed inCanavalia. Also studied were the wild speciesC.dictyota, andC.maritima. There appears to be little morphological or size difference between wildanddomesticatedCanavaliastarches(TableS3). (b)StarchGrainsfromModern VignaluteolaSeeds.Grainsaresimpleandmostareovalwithacentricoreccentrichilum.Lamellae are most evident near the edge of grains. Fissures are often transverse and placed closer to the edge of the grain rather than in the center. Grains withlongitudinal fissures are rare. Round grains without lamellae and small, non-descript fissures or no fissures occur. The fissure characteristics makethem differentiablefrom round grains that occur inPhaseolus. Also studied wasV.adenantha, which has the same types of grains asV.luteola.

1. Rossen, J. & Dillehay, T.D. Ancient cultigens or modern intrusions?: Evaluating plant remains in an Andean case study. Jour. Arch. Sci.23, 391407 (1996).2. Dillehay, T.D., Rossen, J., Andres, T.C, & Williams, D.E. Preceramic Adoption of Peanut, Squash, and Cotton in Northern Peru. Science316, 18901893 (2007).

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Table S1. Summary of records for teeth containing little to no starch

Sample,tooth no. 14C age B.P.

Phaseolus,n

Arachis,n

Cucurbita,n

Inga,n

Legume,n

Globular,n

Unknowns,nOther,

n

Total,n

Toothtype1 2 3 4 5 6

CA-097729, #2 6970 60 HB 2 1 2 5 Molar29, #3 6970 60 HB 0 Molar3 6970 60 HB 1 1 Molar22 6970 60 HB 0 Molar

27 6970 60 HB 0 Molar40 6970 60 HB 0 Molar10, #1 6970 60 HB 2 1 4 7 Molar27, #2 6970 60 HB 1 1 Molar50, #3 7840 40 PH 1 1 Molar50, #5 7840 40 PH 1 1 2 Incisor50, #6 7840 40 PH 1 1 1 1 2 3 9 Molar56, #1 7840 40 PH 2 2 Molar56, #2 7840 40 PH 0 Molar56, #3 7840 40 PH 5 1 6 Molar56, #5 7840 40 PH 1 5 6 Molar59, #1 7840 40 PH 1 1 2 Molar60, #1 7840 40 PH 5 5 Molar60, #2 7840 40 PH 0 Incisor58, #2 7840 40 PH 1 3 1 1 1 2 9 Molar

58, #3 7840 40 PH 1 2 2 1 1 3 10 IncisorCA-0952

3A 7920 120 WC 2 1 2 5 Incisor15 7920 120 WC 8 2(2) 10 Incisor113 7920 120 WC 2 1 1 4 Incisor41 8080 70 WC 2 2 Molar117 8080 70 WC 1 1 Incisor

CA-09714A 7600* 3(1) 3 Incisor

Teeth nos. 27, 41, 50, 58, and 113 are from sub-adults, and nos. 15, 18, 40, 60, and 117 are from adults. Others are indeterminate as to the approximate ageof the individual. Teeth from CA-09-52 are from complete skeletons. *Estimated age is based on stratigraphic position in intact cultural layer located 25 cmbelow two radiocarbon dates on upper strata measured at 6210 18014C years B.P. (33523) and 6310 15014C years B.P. (33524) and on direct associationwith diagnostic stone tools [Rossen J, Dillehay TD (1996) Ancient cultigens or modern intrusions?: Evaluating plant remains in an Andean case study.J Arch Sci23:391-407].



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Table S2. Radiocarbon dates from selected preceramic sites in the anchoc valley and association with human teeth studied

Label Site/unitRadiocarbon

YBP2E-calibrated age

range B.P. Provenience/teeth association

Late Pajan Phase (100009000 YBP)

Beta 79512 CA-09-77-4 9240 50* 1040310163 Desiccated squash seed in house floorLas Pircas Phase (90007000 YBP)

Beta 33523 CA-09-28 8210 180* 95268648 Wood charcoal in house floor directly associatedwith tooth 1A.

Beta 8260 CA-09-29 8260 130* 95318795 Wood charcoal in house floor directly associatedwith tooth 1A

Beta 30781 CA-09-52-3 8080 70 91118636 Wood charcoal in house floor directly associatedwith human bones and teeth nos. 41, 117

Beta 12384 CA-09-52-3-3 7920 120 90028427 Wood charcoal in hearth on house floor directlyassociated with human bones and teeth nos.3A, 15, 113

Beta 33525 CA-09-52-3-3 7850 140 90028427 Wood charcoal in hearth on house floor directlyassociated with human bones and tooth no.113

Beta 219588 CA-09-77-3 7840 40* 86408435 Charred peanut hull in house floor directlyassociated with human bones and teeth nos.4A, 7, 50, 56, 58, 59, 60, 68

Beta 219776Beta 219745

CA-09-77-2 6970 60*7130 70*

7866762278257715

Human bone remains in house floor directlyassociated with teeth nos. 2A, 3, 10, 19, 22, 27,

29, 40

YBP, years before present. See Dillehay TD, et al.[(2007) Preceramic adoption of peanut, squash, and cotton in northern peru.Science316:18901893] fora more complete listing of carbon-14 dates from the anchoc Valley sites.*AMS radiocarbon dates from human bone, macrobotanical remains, and wood charcoal in house floors and hearths. Dates on macrobotanical remains are in

bold.Standard radiocarbon dates from wood charcoal in house floors, hearths, and middens.See Dillehay TD, et al. [(2007) Preceramic Adoption of Peanut, Squash, and Cotton in Northern Peru.Science316:18901893] for a more complete listing ofcarbon-14 dates from the sites.



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Table 3. Size of starch grains in modern Phaseolusand other legumes

Sample

Oval to kidney-shaped, m Round, with fissures, mRound, with lamellae,

m

xLength Range SD

xWidth Range SD

xDiameter Range SD

xDiameter Range SD

Phaseolus

Phaseolus lunatus, wild, South AmericaPhaseolus augusti

G40606 Junin, Peru 34 2058 9.2 25.6 1735 4.1 19.7 1524 4.0 29.5 2633 4.9G40649 Cuzco, Peru 30 1846 7.2 23.4 1638 6.3 17.2 1033 6.3 N.O.G40731 Cuzco, Peru 30.9 1749 6.6 23.9 1434 4.1 19.2 1024 5.2 N.O.G40775 Cuzco, Peru 33.7 1946 6.2 26 1537 4.7 19.5 1323 3.9 N.O.G40758 Azuay, Ecuador 28.5 2047 5.0 21.5 1528 2.8 17.9 1718 0.1 N.O.NMNH 3168433 Cuzco, Peru 31.2 1650* 7.2 24 1040 5.6 18 738 6.7 N.O.NMNH 3168434 Cuzco, Peru 26.8 1539 5.9 19.4 1028 4.3 18.9 926 4.7 19.8 1724 3.1NMNH 1022071 Huigra, Ecuador 22.9 1334 5.5 14.3 819 4.2 9.3 516 3.9 N.O.

Phaseolus pachyrrhizoides

G40541 Apurimac, Peru 32.6 1543 6.8 ND ND ND 26 2526 0.7 N.O.G40607 Junin, Peru 34 2256 7.2 27.7 1945 5.1 17.2 922 4.9 N.O.G40719 Apurimac, Peru 34.5 1752 8.3 25.8 1638 4.8 18.6 1623 2.9 30 G40717 Junin, Peru 32 2153 6.2 22.5 1532 4.2 19.6 1920 0.6 25.5 1932 9.2G40509 Cuzco, Peru 33.2 1749 7.7 24.4 1733 3.5 19.7 1624 2.1 21.7 1222 0.6NMNH 3168421Apurimac, Peru

25.6 1446 6.9 18.1 931 5.3 15 1119 5.7 21

Phaseolus bolivianus

G40759 Cochabamba Bolivia 29.1 1839 4.9 21.7 1527 2.9 16.1 1517 1.5 N.O.Phaseolus lunatus, wild, Mexico

G25234 Yucatan 31.9 2245 5.1 24.1 1834 3.7 22.4 30.8 G25218 Chiapas 33.6 2345 6.0 22.8 1431 3.3 15.2 1318 3.6 22.3

Phaseolus lunatus,DomesticatedG25306 La Libertad, Peru 35.3 2248* 6.3 23.9 1632 4.5 15.9 1020 4.5 N.O.G25300 Lima, Peru 35.9 2153 7.2 24.9 1517 4.2 22.3 1429 6.3 N.O.G25129 Loreto, Peru 33.7 1949 6.1 25.8 1738 4.7 18 1225 4.5 N.O.G25700 Cocle, Panama 35 2147 6.8 26.9 1734 4.1 24 1930 7.9 27 2632 2.8G25293 Guerrero, Mexico 35.6 1853 6.7 26.4 1539 5.1 25.3 921 3.8 N.O.

USA commercial 1 36.8 2349 6.9 ND ND ND 10.4 816 2.7 21 1432 7.9USA commercial 2 42.6 1764 8.5 ND ND ND 22.2 1227 2.7 29 2238 7.8Phaseolus vulgaris, wild Mexico

NMNH 2545524 Jalisco 19.1 1332 3.7 14.9 1123 2.3 13.1 1116 1.4 N.O.NMNH 2545541 Michoacan 24.7 1736 4.7 16.7 1123 2.8 15.8 1318 2.2 N.O.

Phaseolus vulgaris, wild South AmericaNMNH 3168404 Apurimac, Peru 26 1239 3.4 18.1 1024 2.7 16.7 1221 3.4 N.O.NMNH 3168412 Apurimac, Peru 22 1332 4.9 16 1221 2.5 12 1015 2.0 N.O.NMNH 3168406 Junin, Peru 29 1839 5.2 22 1528 2.8 18 1523 2.2 N.O.NMNH 1022229 Huigra, Ecuador 25 1940 4.9 19 1423 2.4 18 1519 1.7 N.O.

Phaseolus vulgarisdomesticatedG4740 Junin, Peru 31.1 1856 8.7 21.3 1526 2.9 17.4 1127 3.7 N.O.G111 Huanuco, Peru 33.5 1849 6.7 24 1432 3.7 17.7 1320 2.5 N.O.G1679 Lima, Peru 28 1944 5.6 21.8 1533 3.8 18.4 1521 1.9 22

USA commercial 1 38.1 2256 8.9 30.3 1850 7.4 16 1028 7.2 36 3242 5.5USA commercial 1, cooked 36.8 2356 9.0 29.5 1848 7.8 21 1326 5.6 33USA commercial 2 33.2 1460 8.7 24 1239 5.3 14.8 1218 2.8 26 2233 4.1USA commercial 3, Kidney 28.9 1943 5.7 22.7 1631 3.3 18.1 926 3.5 N.O.USA commercial 3, cooked 31.8 1747 8.4 24 1437 5.2 17.6 1321 2.4 N.O.

Peanutsxlength

mRange,m SD

Arachis williamsiDEW 1118, Beni,Bolivia

6.6 410 1.4

Arachis hypogaeaUSA commercial(roasted)

9.9 518 2.6



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Sample

Oval to kidney-shaped, m Round, with fissures, mRound, with lamellae,

m

xLength Range SD

xWidth Range SD

xDiameter Range SD

xDiameter Range SD

A. hypogaea Spanish peanuts, USAcommercial (raw)

9.5 618 2.1

A. hypogaea var.hirsutaDEW, Lima,Peru (roasted)

10.1 519 2.7

CanavaliaandVigna

Canavalia plagiospermaNMNH 111547Miami, Florida

42.3 2267 9.4 31.5 1347 7.1 N.O. 32.4 2244 8.9

Canavalia dictyotaNMNH 1801714Bolivar, Venezuela

41.6 2951 7.2 33.2 2239 5.8 N.O N.O

Canavalia maritimaNMNH 2440147Antioquia, Colombia

41.1 3160 7.6 32.1 2441 4.4 N.O. 39

Vigna adenathaNMNH 2678714 MatoGrosso, Brazil

29.1 1348 7.6 22.1 935 6.3 23.3 1835 7.9 N.O.

Vigna luteolaNMNH 3026998 Loreto,Peru

23.6 1340 4.8 15.4 823 2.9 12.4 817 2.9 N.O

N.O., notobserved; ND, notdetermined; DEW, from thecollections of David E. Williams.The category Round, fissures represents smaller, round grains witha shiny surface that have a variety of fissure types, including stellate, and are without lamellae or have lamellae concentrated in a thin, dense band at theperiphery of the grain. The category Round, lamellae represents large, round grains withlamellaecoveringthe entirety of the grain and no fissures. Accession

numbers beginning withthe letter G arefrom the CIATaccessions,Cali, Colombia. Accessionnumbers beginningwith NMNHare fromthe foldersof theNationalHerbarium, National Museum of Natural History, Washington DC. Specimens listed as USA commercialwere bought in markets in Falls Church, VA. The starchgrains fromArachisare globular in shape.*A 59-micron long grain was observed on extended scanning.Fissures on theseVignagrains are small and unlike inPhaseolus.

Documents

Piperno, 2008. Starch Grains on Human Teeth