| in Mexico ‘ i Oss Multiple lines of evidence for the origin of domesticated chili pepper, Capsicum annuum, Kraig H. Kraft™', Cecil H. Brown®, Gary P. Nabhan‘, Eike Luedeling®, José de Jess Luna Ruiz*, Geo Coppens d'Eeckenbrugge’, Robert J. Hiimans®, and Paul Gepts* “oepartment of Pant Sionces, Section of Crop id Ecosystem Scinees, Univers of California, Davis, CA 95646-87 partment of Anthropology, Norther inci ivory, Pensacol, FL 32503634; Southwest Center, University of Atzon, Tucson, AZ 857210165; Mord ajotoresy Conte, NaI, Kenya; “Cento de Cencles Agropecuars, Univeniad norms de Aguascalientes, Aguascllantes, Metco CP 2013 “Cente te cooperation Intemationale en Recherche Agronomique pour fe Développement. Units Miate de fecnerche 515 Centre @tzologie Fonetionnels ee Gvoutve, Cmpus {entre National de la Recherche Scentique, Montpelier Cedex 5, Francs; and Department of Emvcnmetal sccnce and Poy, Unventy of Cao Datls, ca 85616 Eadie by Dolores R. Fire, Smithzonian National Museum of Natural Hitery and Smithsanlan Topical Research istut, Fara, Wesington, DC, end sporoved December 42013 (rectved for reve September 6, 2013), ‘The study of crop origins has traditionally involved identifying geographic areas of high morphological diversity, sampling popula ‘ions of wild progenitor species, and the archaeological retrieval of ‘macroremains. Recent investigations have addediidentification of plant microremains (phytolith, pollen, and starch grains), bio= chemical and molecular genetic approaches, and dating through “MC accelerator mass spectrometry. We investigate the origin of ‘domesticated chili pepper, Capsicum annuum, by combining two approaches, species distribution modeling and paleobiolinguis- tics, with microsatellite genetic data and archaeobotanical data. ‘The combination of these four lines of evidence yields consensus ‘models indicating that domestication of C. annuum could have ‘occurred in one or both of two areas of Mexico: northeastern ‘Mexico and centrabeast Mexico. Genetic evidence shows more support for the more northern location, but jointly all four lines of evidence support central-east Mexico, where preceramic mac. Yoremains of chili pepper have been recovered! in the Valley of TTehuacén. Located just tothe east of this valley Is the center of phylogenetic diversity of Proto-Otomanguean, a language spo- kken in mid-Holocene times and the oldest protolanguage for which a word for chili pepper reconstructs based on historical linguistics. For many crops, especialy those that do not have a strong archaeobotanical record or phylogeographic pattern it is difficult to precisely identity the time and place oftheir origin, Our results for chill pepper show that expressing all data in sim: iar distance terms allows for combining contrasting lines of evi- ‘dence and locating the regions) where cultvatign and domestication fof a crop began, he analysis of plant macroremains, morphological variation in crop varities, and identification of wild progenitor species determined through thei abiliy to hybziize wih the crop) constitute traditional methods for studying erop origins (I, 2) Currently, analysis of microremains sugh as starch grins, ac celerator mass spectrometry (AMS) "C radiocarbon dating, along with biochemical and molecular genetic analyses of wid and domesticated popalatons are also sed to date and locate seographic areas of domestication (4). ‘This set of approaches is extended here with two additional methods, species distribution modeling and paleobiolinguistcs, integrating these in a comprehensive study of the origin of do rmesticated chili peppes, Caps annum L, the world’s most widely grown spice. C. annuum: is one of five domesticated pepper species, which also include Capsicum badeatum Ly Capsicin chinense Jacq, Capsicum futescons L, and Capsictrt pubescens Ruiz & Pav. The ~30 specios of Capsicum are all native tothe Americas (5). Comparing karyotypes of wild and domesticated Carmo (vat. glabrusculim and var, annuum respectively), Fickergil (6) idenifed Mexico as the general rm pras oreo. 107e, 1208098111 region of domestication ofthis pepper, Loaiza-Figueroa et al. (7) ‘sed allozyie similarity to identity putative wild ancestral pope ulations for eli pepper in a larger collection of wild and do- restcated populations. They narrowed the likely domestication area fo te easter Mesican sates of Tanalipas, Nuevo Le6n, San Luis Rotos, Veracruz, and Hidalgo. Since these inves: tigations, others have sought to determine genetic relationships among-vld and domesticated populations of el pepper (8, 9). ‘The oldest macroremains unambiguously identied as Caps cum pepper were retrieved from preceramie strata of dy eaves in two sites of Mosieo: Puebla (Febuacén Valley es. 10,11) and ‘Tamaulipas (Ocampo eaves ref. 12) Fig. LA), These were found ‘with macroremains of maizs (Zea mays) squash (Cucurbita spp), and other species used by humans, all of which, at both sites, vwere indirectly dated through associations in archacolopeal Strata, suggesting a rough date forthe cil pepper macroremains ‘of around 9000-7000 B.P. (13), Subsequeny emains of maize ftom Tehuacin were dated direely by AMS and found to be more recent, 600 y calibrated BLP, (14). AMS dating applied to boitle gourd and squash from Ocampo aso yielded more recent ages, 6400-6000 y calibrated BLP. (15). Whereas no AMS dates | Significance { i | The novelty ofthe information of this manuscript resides in the | addition of species distribution modeling and paleobiolin- | guisties date, combined with genetic and existing archae- botanical data, to trace back the geographic origin of a crop, namely domesticated pepper, Capsicum annuum. Furthermore, ‘the utilization of a geographic framework of reference for the | four types of data has allowed us to combine these indepen- dent data types into a single hypothesis about the origin of this crop. Our results suggest that food crops in Mexico had ‘2 multiregional origin with chili pepper originating in cen- tral-east Mexico, maize in the Balsas River Basin and com- mon bean in the Lerma Santiago River Basin, resembling similar finds for the Fertile Crescent and China. Aor canbutons KHX. CH, ASL, RAH, and RG dep ser KI, Ch LAR, Gd, Ri and 8. prtarmad ana RMI eae ELS AIL it ane. coma now reapentsanststole Ki, Cd. Ee 1M, anand date and KALE, CRs GPa Ele ASULN. Ges cond Pace te per ‘The sth dedare no oni of nee. Tsar r= RAS Dic Sin, ‘Prant dees: Conmtes on SsaubtyAseznet, Pads PA 1087 2ro whom crspondne shoul be afiend Eal pgrpettose Thani ena suprring fomatononne st wir gneopfokamwpntea Toyipas 03351 IMO CSuenena PAS Ey Eon | 1066 1 ‘aencutronat ‘Sones| x a ' [A) Archaeology B) Ecology oes o2C Jas ]oaCJos Ces Cer Eos mes mo Fig. 1. Possible area of Meco for Capsicum annuum demestiation based on (A) archaeological, (0) paleoclimatic mi-Holocen, (Cig and (0) ‘genetic data, In adtion tothe strangth af evdenes (between Dard 1), the mapr zh: (A) Location ofthe olde achacologiearemane of chile Romero ave, Ocampo, Taraulioas; Covcatlan Cave, Tehuacin Vale, Pudba.() Location ofthe homeland of Proto-Otemanguean (otted cee) and ofthe four _bgroup of arront Otomangueon languages ee Fig 55 legend and Tabet fora more detalled view ofthe subgroup detibuion, see i, $3} Open cles represent approximate lations of protolanguages with reconstructed werd for cl. (2) Open cede indcets the location ofthe vd chil smples vied in tha geneti distance analysis (9). For explanation of valu, se Mateos and Method. have been recorded for the Tehuacén and Ocampo remains of chili pepper, remains from Guils Naquitz and Sivia’s Caves in the atid eastern valley of Oaxaca state were dated indirectly by ‘AMS to 1400-500 B.P. (16). Rock shelters in the soasonally dry tropical forest of the Central Balsas watershed (state of Guer~ e10) have produced phytoliths and starch grain residue for do- restated maize and squash (Cucurbita sp.) dated by association to around 9000 BP. (17). However, no femains of Capsicum pepper have been found at that site. Species distribution modeling (SDM) can be used to predict areas that are environmentally suitable for a species from the sites where itis known to occur (18). In SDM, locations of the known current distribution of a species are compiled; values for” climatic predictor variables at these locations and a large set of random (background) locations are extracted from sptil databases; and the climatic values are used to fit a model that catimates the similarity of the limatc,n any location to climatic conditions at Kowa occurrence locations, using a machinc- [earning algorithm such as Maxnt (19) The model is then used to predict the climatic suitability fora species across a area of igorest. This prediction can be made using curent climate data, but the model canals be “transferred” in time, by using past ot future climate data simulated by global climate modcls (GCM). ‘This approach. has been used for many purpose, including predic the effect of climate change on the geographic distribu tion of erop wild relatives (20) and to successfully Toeate un- kagwa Capsicum populations (21. ‘Grop origins can also be studied using paleobiolingustcs (PBL), which employs the comparative method of historical linguistics to roconstruct the biodiversity known to human groups of the remote, unrecorded past (22-24), By comparing words for 4 species in modern languages terms for plants and animals in 2of6 | smusnasorfegldol0.073pmas1308533111 ancestral languages can be retrieved. The presence of words for ‘species in an ancesteal language isan indication of the species? significance to speakers of thal language (25, 26), if not their status as domesticated plants. PBL usos Automated Similarity ‘Judgment Program (ASIP) chronology for estimating the latest date at which a protolanguage was spoken based on lexical similarity (27). Lexical similarity found among relate languages is calibrated with historical, epigraphic, and archaeological vergence dates for $2 language groups. In addition, the general area in which an ancestral language was spoken, ie, the tolanguage homeland, can be approximately determined by lo- ‘eating the area where its modesn descendant languages are found to be most diverse (28). Tn this paper, we complement existing archaeobotanical data with ecological, paleobiolinguistic, and molecular diversity data to identify the region of initial intensification of human interest in chili pepper that led to crop domestication. The novelty of our ‘approach resides in the addition of SDM and PBL to this type of analysis and the expression of all nes of evidence in comparable spatially explicit units (distance (o the area of origin) that allows for their integration into a single prediction. Results ‘Archaeological Evidence. "The remains from Tehuacén andl Ocampo constitute at present the oldest macrobolanical evidence for preceramie ci pepper in the New World, Although these chili Specimens cannot be identified as cultivated or domesticated, their archaeological association with domesticated remains of important crops, such as maize and squash, is strongly suggestive of ancient intensive human interaction with chil in these areas. Based on this evidence, we assumed that the nearer a place may, be tocither ofthese sites, the more likely the location was part of raft tathe region where the crop was first grown and domesticated Fig. 14). Ecological Evidence. Wild cil popper (C: annuone vax. glabrivseuum), the ancestor of domesticated C. annuum: (6), sa perensial sheub that produces dozens of erect, globular, pea-sized fruits. The fruits are consumed and dispersed by frugivorous birds, wl pass the seed through their digestive system. Generally found in the northern half of Mexico, the wild chili pepper is asociated with a nurse plant—often a hackberry (Celis palida Torey), (Prosopis sp), or colurmnar cacti. As one moves fur- ther southwards, wild chili pepper is found more frequently in human-disturbed landscapes—fence rows, home. gardens, and roadsides (28). Based on our own collecting localities and those of herbarium specimens and gene bank accessions (25), we est tate that wild gil peppers grow currently in envitonments with amedian annual average temperature of 4 °C and between 20°C «and 26 °C for 90% ofthe locations. The coldest locations with ‘known wild pepper populations are mostly in the central Mexican highlands, the warmest locations in the southern coastal regions of Mexico and Guatemala, The median annual rainfall of these locations is 907 mam, and between 495 and 2,253 mm for 9096 of {he locations, with the driest locations inthe northwestern part of tho distribution (eg, Baja California and the Sonoran Desert) and the wettest locations in southeastern Mesico. “The MaxEnt species distribution mode! ad an internal (rine ing) ft area under the curve (AUC) of the receiver operating characteristic (ROC) curve of 0.89. The average cAUC (bias corrected) obtained with fivefold eross-alidation was 0.80, which suggests that the mode hus very good predietive power (30). The {wo most important predictor variables (based on permutation : Table 1. Reconstructed word importance) were mean temperature of the coldest quarter (63%), followed by annual presipitation (14%). ‘Under the climate conditions of the mid-Holocene (about 6000 BP.) the regions predicted to be most suitable for wild chill pepper include areas along the western and eastern coasts of Mexico southeast Mexico and northern Guatemala (Fi. 13). The central highlands were clearly unsuitable for this species ating this period. The correlation coefficient between the pre= icted suitability forthe current eimate (Pig $1) andthe mid- Holocene climate was 0.92, Despite this overall similarity, there were important diferenees between these predictions, with areas in the southeast of Mexico more suitable and ateas in the northeast es suitable during the mid-Holocene (Fig. 82). Paleobiotingulsti Evidence. Brown (22) surveyed the reconstructed vocabularies of 30 protolanguages of Mesoamerica (southern half of Mexico and northern Central America) and abutting areas for terms for 41 different crops, including chili pepper. His survey presented for each protolanguage the estimated date it was spoken at the latest, making it possible to stratify reconstructed words for crops chronologically (Table 1) (27, 8). ‘Proto-Otomanguean is the oldest (~6300 BP.) protolanguage of the New World for which a word for chili pepper reconstructs (G1). All daughter languages of Proto-Otomanguean, as defined by Kaulinan (2), show reconstructed terms for chili pepper (Table 1). Given that estimated dates are to be understood as the latest dates at which ancestral languages were spoken, i is plausible that speakers of Proto-Otomanguean actually had a word for chili pepper hundreds, if not thousands, of years before ~6500 BLP. The oldest protolanguage of Table 1 not belonging to the Otomanguea family 55 Proto-Totozoquean (~4300 BLP), for which a term for chil pepper does not reconstruct. Non-Otomanguean Innguages for Reconstruction of torms for Capsicum'in selected protolanguages of Mesoamerica and abutting areas Location of modern Years before preent Protolnguoge! fori dlesendent languages ___ Genet fitation en Otemangoeen a Werco oromanguean 5976 Enstrn xomanguesn stato Mesco Comenguesn 508 Popolocan-tapotecn xi tomangiean 557 myo Miecan sens tomenguean Fase tec syst Hyak Hot? torangian t] aan Tetoroquean we Mesca ‘etoroquean | 4018 Uto-Aztecen NR US Southwest, Mexico, Uto-Aztecan “eval America, || sa ctopamean ston erica tamanguean xn Southern UtoActecan mm Mavic, Contra America Ureretecn |] ee Kowtanoan tt Us southwest Kowertanoan |) Shea Zapotec skins Dereo Stemengueen i 3036 ropelocen “hit tesco Ctemanguean || 3000 teen tt Cerra America tencan ie malpan” uma Central america sumalpan i] 3576 Northern Uto-Aztecan NR Us Southwest Utorartecan bas ChapanecMangue sotto Mesico Otomsnguean 2400 Sonoran stotokel Meee Uroratean ian ‘oven the Mee, Central Ameria Mayon i938 Chinantecan oe Mesce tomangueen tees Yuan mR Us southwest Yura 1397 humic mm 1s soutneat Utratecan 87 Take a US southwest Uteaztecen 1509 General Aztec sai Mesice Uteartecan ee ‘otenacan Mesco Totoroquean io? Meezoquean Mexico _Totemoguean TR, not reconsvuctable, "source for each language ae listed nS! Materials and Methods under Paleobotinguiss. "Explanation for phonetic representation of pepper words ae sted In Sf Metals and Methods under Paleobiolingustis. att PNAS Ea Ein | 3016