Archaeogenomic evidence from the southwestern USpoints to a pre-Hispanic scarlet macaw breeding colonyRichard J. Georgea, Stephen Plogb,1, Adam S. Watsonc, Kari L. Schmidtd,e, Brendan J. Culletona,f, Thomas K. Harpera,Patricia A. Gilmang, Steven A. LeBlanch, George Amatoe, Peter Whiteleyc, Logan Kistleri, and Douglas J. Kennetta,f,1

aDepartment of Anthropology, The Pennsylvania State University, University Park, PA 16802; bDepartment of Anthropology, University of Virginia,Charlottesville, VA 22904; cDivision of Anthropology, American Museum of Natural History, New York, NY 10024; dDepartment of Ecology, Evolution andEnvironmental Biology, Columbia University, New York, NY 10027; eSackler Institute for Comparative Genomics, American Museum of Natural History, NewYork, NY 10024; fInstitutes for Energy and the Environment, The Pennsylvania State University, University Park, PA 16802; gDepartment of Anthropology,University of Oklahoma, Norman, OK 73019; hPeabody Museum of American Archaeology and Ethnology, Harvard University, Cambridge, MA 02138;and iDepartment of Anthropology, Smithsonian Institution, Washington, DC 20560

Edited by James A. Brown, Northwestern University, Evanston, IL, and approved July 13, 2018 (received for review April 4, 2018)

Hundreds of scarlet macaw (Ara macao cyanoptera) skeletons havebeen recovered from archaeological contexts in the southwesternUnited States and northwestern Mexico (SW/NW). The location ofthese skeletons,>1,000 km outside their Neotropical endemic range,has suggested a far-reaching pre-Hispanic acquisition network. Clearevidence for scarlet macaw breeding within this network is onlyknown from the settlement of Paquimé in NW dating between1250 and 1450 CE. Although some scholars have speculated on theprobable existence of earlier breeding centers in the SW/NW region,there has been no supporting evidence. In this study, we performedan ancient DNA analysis of scarlet macaws recovered from archae-ological sites in Chaco Canyon and the contemporaneous Mimbresarea of New Mexico. All samples were directly radiocarbon datedbetween 900 and 1200 CE. We reconstructed complete or near-complete mitochondrial genome sequences of 14 scarlet macawsfrom five different sites. We observed remarkably low genetic di-versity in this sample, consistent with breeding of a small founderpopulation translocated outside their natural range. Phylogeo-graphic comparisons of our ancient DNA mitogenomes with mito-chondrial sequences from macaws collected during the last 200years from their endemic Neotropical range identified genetic affin-ity between the ancient macaws and a single rare haplogroup(Haplo6) observed only among wild macaws in Mexico and northernGuatemala. Our results suggest that people at an undiscovered pre-Hispanic settlement dating between 900 and 1200 CE managed amacaw breeding colony outside their endemic range and distributedthese symbolically important birds through the SW.

scarlet macaws | American Southwest | prehistoric aviculture |archaeology | ancient DNA

Archaeogenomic research is revolutionizing our understand-ing of the past, including the origin, structure, and move-

ment of human populations (1–3), the processes of plant andanimal domestication (4–13), our biological adaptation to novelenvironments (1, 14–16), and sociopolitical systems among an-cient people (17). Here, we expand the use of these techniques toexamine the acquisition of exotic birds in two pre-Hispanic Na-tive American societies, specifically, the translocation of scarletmacaws from their northern endemic range in NeotropicalMexico to the southwestern United States between 900 and 1200CE and their apparent breeding by people at intermediate lo-cations through this ∼300-y period.The appearance of scarlet macaws (Ara macao cyanoptera) in

some parts of the southwestern United States and northwesternMexico (SW/NW) between 900 and 1200 CE co-occurs with theemergence of larger settlements in Chaco and in the Mimbresregion than was typical in the broader SW (excluding the Ho-hokam region; Fig. 1), increased interaction with Mesoamericaand California (18), and the emergence of more complex soci-eties in parts of the SW (17, 19–23). The immense costs involvedin procuring macaws over long geographical and social distances,

along with other exogenous items and products including cacao,copper bells, marine shell/bracelets, and distinct ceramic vessels,may have both contributed to and were products of emergentsociopolitical complexity in Chaco Canyon (19, 21).Some scholars have hypothesized that scarlet macaws were di-

rectly acquired from lowland tropical regions between 900 and1200 CE via long-distance treks to the northern extent of theirrange in the Mexican Gulf Coast state of Tamaulipas, and possiblyto more distant ranges in the Isthmus of Tehuantepec and CentralAmerica (24). Others have suggested that these birds passed fromone community to the next fromMesoamerica to the SW/NW (e.g.,refs. 25–27). Alternatively, here we test the hypothesis proposed byCrown (28), that lengthy trips were mitigated by the presence ofintermediate breeding centers in the area between the northernendemic range of scarlet macaws and the SW/NW.Such centers may have raised generations of captive macaws and

maintained a stock of age-specific macaws for transport throughoutthe SW/NW, as occurred at the later (1250–1450 CE) settlement ofPaquimé in the Mexican state of Chihuahua. Paquimé was an

Significance

Archaeogenomic analysis of scarlet macaw bones demon-strates that the genetic diversity of these birds acquired bypeople in the southwestern United States (SW) between 900and 1200 CE was exceedingly low. Only one mitochondrial DNAhaplogroup (Haplo6) is present of the five historically knownhaplogroups in the lowland forests of Mexico and CentralAmerica. Phylogenetic analyses indicate the ancient macawlineage in the SW shared genetic affinities with this wild line-age. These data support the hypothesis that a translocatedbreeding colony of scarlet macaws belonging to only onehaplogroup existed some distance north of their endemicrange, and SW peoples continuously acquired these birds fromthis unknown location for nearly 3 centuries, as no evidencecurrently exists for macaw breeding in SW.

Author contributions: R.J.G., S.P., and D.J.K. designed research; R.J.G. performed research;R.J.G. and L.K. contributed new reagents/analytic tools; R.J.G., B.J.C., and T.K.H. analyzeddata; R.J.G., S.P., A.S.W., K.L.S., B.J.C., T.K.H., P.A.G., S.A.L., G.A., P.W., L.K., and D.J.K.wrote the paper; and S.P., A.S.W., K.L.S., P.A.G., S.A.L., G.A., and P.W. provided historicaland archaeological context.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.

Data deposition: The sequences reported in this paper have been deposited in the NCBISequence Read Archive (BioProject ID: PRJNA477839), Dryad Digital Repository (https://doi.org/10.5061/dryad.sv74pj2), and GenBank database (accession nos. MH400234–MH400248).1To whom correspondence may be addressed. Email: plog@virgina.edu or djk23@psu.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1805856115/-/DCSupplemental.

Published online August 13, 2018.

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unusually large settlement with some characteristics (e.g., pueblosof contiguous rooms) of the SW and other attributes (e.g., ballcourts) that were more typical of Mesoamerica (29, 30). Alsopresent were macaw pens in public plazas, eggshells, and skeletalremains of >300 scarlet macaws ranging in age between nestlingsand breeding age individuals. Oxygen isotopes support the con-clusion that a majority of the scarlet macaws at Paquimé were bredin northern Mexico outside their endemic range (31).The highest concentrations of scarlet macaws in the SW before

the 13th century and before the construction of Paquimé have beenrecovered from archaeological contexts in Chaco Canyon (n = 35)in northwestern New Mexico, the Mimbres region (n > 10) ofsouthwestern New Mexico, Wupatki in north-central Arizona (n >22, see ref. 28), and the Hohokam region of southern Arizona (21,23). Directly radiocarbon-dated scarlet macaw bones from ChacoCanyon and the Mimbres region indicate an increased rate of theacquisition of these birds in the SW between 900 and 1200 CEcompared with sparse evidence between 200 and 900 CE in theHohokam region (23) (Fig. 2). However, archaeological excava-tions have not revealed pre-Paquimé breeding centers (e.g., eggshells, possible nesting boxes, nestlings and breeding age birds allrecovered from the same settlement) in the SW/NW. At bothPueblo Bonito in Chaco Canyon and Wupatki, for example, whichhave relatively large numbers of scarlet macaws, analysis revealedonly one bird at each site that was possibly of breeding age. Weshould emphasize, however, that the intensity of archaeologicalresearch has been more limited in northern Mexico compared withadjacent areas of the SW and central Mexico. Archaeologists thushave generally assumed that during this period, wild scarlet macawswere acquired directly or passed from community to communityfrom their endemic range to the SW/NW.The presence of scarlet macaw skeletons at archaeological

sites in Mesoamerica and the SW/NW is not surprising. Thesignificance of macaws as metaphorical markers of human socialidentities is widespread among living and historic Amerindiansof the tropics, perhaps most famously the Amazonian Bororo(e.g., refs. 32 and 33), and in some other parts of the New World.In the Late Preclassic period (400 BCE–200 CE) in southernMesoamerica, depictions of high-status individuals adorned withscarlet macaw feathers suggest they were markers of prestige (34).Imagery of macaws and macaw feathers on carved stone monu-ments, painted murals, and polychrome pottery showing, forexample, macaws resting on elite headdresses continued into the

Classic period (250–900 CE) among the Maya and suggests theimportance of scarlet macaws in cosmology and religion (34).Among historic Pueblo groups in the SW, European explorers in

the 16th century and later ethnographers commented on the sig-nificance of scarlet macaws and their feathers in Pueblo society(e.g., refs. 21 and 28). The long-term presence of a high-statusParrot/Macaw clan among the Hopi, the prominent role of ma-caws and their eggs in mythology and ritual representations atAcoma and Isleta pueblos (e.g., ref. 35), and the continuing, wide-spread use of macaw feathers in ritual dress in contemporary Puebloceremonies demonstrate the significance of macaws. The impor-tance of macaw feathers also is demonstrated by contemporary ef-forts to supply Pueblos with bird feathers, including macaws (36). Incurrent, historic, and likely pre-Hispanic Pueblo cosmology, color-direction symbolism is very important. Macaws are the bird equiv-alent to reddish Spondylus (spp.) shell and the color red, oftenmarking the southern cardinal point, just as western bluebirds (Sialiamexicana) and turquoise mark the opposing cardinal direction (e.g.,ref. 37). It is thus almost certainly no coincidence that both scarletmacaws and turquoise are unusually abundant in Chaco Canyon.Throughout much of the SW/NW, the occurrence of scarlet

macaws is both temporally and geographically discontinuous, withunusual frequencies in areas such as Chaco Canyon or Mimbresthat are also characterized by other rare features (e.g., the re-markable representational images on Mimbres pottery and theunusually large great houses of Chaco). A previous study suggestedthat scarlet macaws served a central role in creating and main-taining the sociopolitical economic foundations of Chacoan society

Fig. 1. Map showing the locations of archaeological sites in the SW/NW referenced in this study. The historic range of wild scarlet macaws in the Americas isshown in red.

Fig. 2. Summed probability distribution of all directly AMS radiocarbondated scarlet macaw samples from the SW compared with the directly datedsamples with complete mitogenomes (highlighted in blue). These data areshown relative to the known date range of scarlet macaw breeding atPaquimé in northern Mexico (black bar).

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(21). Macaw plumes fade and lose their vivid colors quickly, andthe effort required to obtain macaws is consistent with other luxuryitems sought after as markers of status in pre-Hispanic times (38).To infer the geographic source of scarlet macaws and to ex-

plore the character of acquisition networks before the 13thcentury, we examined the mitogenomic variability and pop-ulation structure of 14 scarlet macaws from five archaeologicalsites located in either Chaco Canyon (n = 11) or the Mimbresarea (n = 3) of New Mexico (SI Appendix, Table S1 and DatasetS1). We compare our results with phylogeographic data availablefor wild scarlet macaws collected within the last 200 y from theirendemic range in Mexico and Central America (39) to describegenetic relationships among the archaeological macaws and toinvestigate the geographic extent of socioeconomic interactionand possible long-distance acquisition in the SW/NW.

Archaeogenomic AnalysisWe initially extracted ancient DNA from the bones of 20 scarletmacaws excavated from two sites in Chaco Canyon (n = 14 ma-caws), three sites in the Mimbres region (n = 5 macaws), and to addsome spatial and temporal variation, one site (Arroyo Hondo; n =1 macaw) in north central New Mexico (SI Appendix, Table S1 andDataset S1). None of the scarlet macaws studied was associatedwith human burials. We prepared massively parallel sequencinglibraries from each extract, but before sequencing we used a DNAcapture approach (40) to enrich our libraries for scarlet macawmitochondrial DNA fragments, using biotinylated RNA baitscomplementary to the reference scarlet macaw mitochondrial ge-nome sequence (GenBank accession: CM002021.1; ref. 41). Post-capture libraries were then sequenced using the Illumina HiSeq2500 and Illumina NextSeq 500 platforms (SI Appendix, Table S2).Three of the 20 samples yielded no recoverable endogenous DNA.For 17 of the 20 ancient macaws included in this study, we re-

covered 133–32,607 nonredundant sequence reads that mapped tothe reference scarlet macaw mitochondrial genome from SouthAmerica (A. m. macao; GenBank accession: CM002021.1; ref. 41).We were able to reconstruct complete or nearly complete mito-genome sequences from 14 of these individuals. After maskingnucleotides at the end of reads subject to ancient DNA damage,we assembled consensus sequences using a minimum of 2× non-redundant reads covering each nucleotide position and 80% siteidentity (91–99.94% of the reference mitogenome represented:sevenfold to 218-fold average sequence coverage per nucleotide).The observed misincorporation pattern for each sample was con-sistent with that expected from ancient DNA damage (refs. 42 and43 and SI Appendix, Fig. S1), serving as a strong marker of au-thenticity. The three remaining mitogenomes were considerablyless complete and were only included in subsequent analyses toascertain whether the partial mitogenomes are assignable to areference haplogroup (9.15–65% of the reference mitogenome;see SI Appendix). All 14 samples with complete or near-completemitogenomes were directly radiocarbon dated to between 900 and1200 CE (Fig. 2, SI Appendix, Table S1, and Dataset S2).All the complete and nearly complete mitogenome sequences

belonged to haplogroup 6 (Haplo6) and were assigned by com-paring 23 unique modern A. m. cyanoptera reference haplotypesconsisting of multiple mtDNA gene segments from Schmidt (39)(SI Appendix, Fig. S3; base pairs analyzed = 3,128 from the 12S,16S, COI, cytb, control region). Samples collected from wild scarletmacaws in Mexico and Central America clustered within the evo-lutionarily distinct cyanoptera lineage consisting of five haplogroups(Haplo1, Haplo2, Haplo3, Haplo5, Haplo6; Dataset S3). Rangingfurther south in Costa Rica, Panama, and South America, the A. m.macao lineage consists of two haplogroups (Haplo4 and Haplo7;see refs. 39 and 44). We did not include A. m. macao haplotypes inthe majority of our analyses, given evidence of geographic isolationbetween lineages in lower Central America. However, the completemitogenome reference sample used here from an exemplar of A. m.

macao originating from South America is Haplo4 (39). There arecurrently no published modern or historically collected A. m. cya-noptera complete mitogenomes available.The nucleotide sequences of our 14 complete or near-complete

ancient macaw mitogenomes are remarkably similar to each otherand belong to four separate haplotypes (Haplo6a1, Haplo6a2,Haplo6a3, Haplo6a4). The primary haplotype (Haplo6a1) includes10 of the 14 individuals, and each sequence is identical across allbase pairs, whereas the remaining four mitogenomes differ fromthe primary haplotype and each other at only two to five nucleo-tides across 15,584 and 16,982 positions (99.9–100% similarity; Fig.3A). In contrast, the ancient mitogenome haplotypes differ fromthe South American reference scarlet macaw mitogenome at 102–105 positions (98.8% similarity). The 10 Haplo6a1 sequences wererecovered across four archaeological sites — Pueblo Bonito (n =6), Pueblo del Arroyo (n = 2), the Mitchell Site (n = 1), and theWind Mountain site (n = 1) — and they represent the entire timespan of our sample based on the radiocarbon dates, including theearliest and latest dated individuals and multiple other individualswithin the ∼300-y span (Fig. 3B and SI Appendix, Table S2).Haplo6a2 (n = 1) and Haplo6a3 (n = 2) were only recovered atPueblo Bonito after 1045 CE, and Haplo6a4 (n = 1) was foundonly in the Mimbres area at Old Town 1020 CE.We separately aligned the three additional specimens (PB74 and

PB80A from Chaco Canyon, and BA660 from Arroyo Hondo)with partial mitogenomes to the 14 complete or near-completesequences to determine the degree of haplotype similarity amongthese samples (SI Appendix, Fig. S2). Although the two partialsequences from Pueblo Bonito (PB74 and PB80A) were 100%identical to Haplo6a1 across 3,580 and 11,006 alignable positions,only three of the five positions used to distinguish the ancienthaplotypes were present in PB80A, and only one of the five po-sitions was present in PB74. None of these positions was present inthe BA660 sequence found at Arroyo Hondo across 1,523 align-able positions. We then aligned 16 ancient macaws (14 complete ornear-complete and two partial mitogenomes) to the 23 uniquemodern A. m. cyanoptera reference haplotypes. We found that twoof the three partial mitogenomes cluster within the Haplo6 cladeacross all alignable reference mtDNA gene segments (base pairsanalyzed = 3,128; SI Appendix, Fig. S3 and Table S2). Althoughcoverage was too low to accurately define the ancient haplotype,the two partial mitogenome sequences from Pueblo Bonito shareidentical segments across conserved genes used to identify theA. m. cyanoptera lineage and known variable positions used todefine Haplo6. These data indicate that these two partial mito-genomes share a high degree of genetic similarity with our 14complete or nearly complete ancient scarlet macaw mitogenomes.There has been one previously published ancient DNA study of

scarlet macaws from three archaeological sites in the SW (45). In thatstudy, sequence data from a 396-bp fragment of the mitochondrialgenome (12s gene) of four scarlet macaws were reconstructed usingPCR-based ancient DNA amplification and Sanger sequencingmethods (45). We aligned each of these sequences and modernreferences to our 14 complete or near-complete mitogenomes (SIAppendix, Table S4 and Dataset S4). The 12s gene mtDNA se-quences of two ancient macaws (Grasshopper Ruin 2 in Arizona andCameron Creek in the Mimbres region) are identical to those fromour study across all alignable positions. One of the previously studiedancient macaws (Grasshopper Ruin 1 in Arizona) differs by a singlenucleotide from our sequences, and the fourth macaw (SalmonRuins, a site related to those in Chaco Canyon) has differences atfour positions (SI Appendix, Fig. S5A). Importantly, all but one ofthese differences could potentially be explained by unmasked C to Tor G to A mismatches attributed to ancient DNA damage (SI Ap-pendix, Fig. S5B). After masking for potential DNA degradation,three of the four ancient macaws from the previous study clusterwithin a region of the 12s gene consistent with Haplo6. However,Haplo3 cannot be ruled out when examining the 12s region alone.

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After assigning the ancient scarlet macaws to the cyanopteralineage, we next investigated the potential source populations bycomparing the 14 complete or near-complete ancient mitoge-nomes alongside previously published sequence data from themitochondrial control region (885 bp) of 84 modern A. m. cya-noptera samples collected from across its distribution in Mexicoand upper Central America; Fig. 4 and Dataset S4). Among the 98total (n = 14 ancient; n = 84 modern) macaw control region se-quences, there were 24 unique haplotypes. A previous study ofscarlet macaw genetic diversity and population substructure re-covered two populations within the cyanoptera lineage (39). Thenorthern population extends across Mexico, Guatemala, andBelize (Haplo1, Haplo2, Haplo5, and Haplo6; Fig. 4A), whereasthe southern population is found in Honduras, Nicaragua, ElSalvador, and northern Costa Rica (Haplo2, Haplo3, Haplo5). The14 ancient macaws with complete mitochondrial control regionsgroup together in Haplo6 with three exemplars from the northernpopulation: Isthmus of Tehuantepec in Mexico (n = 2) and northernGuatemala (n = 1; see SI Appendix, Fig. S4 for network analysis).To estimate the probability that the complete or nearly complete

mitochondrial coverages of the 14 macaws could have been in-dependently captured from the wild and individually transporteddirectly to the SW/NW, we performed a permutation analysis withthe modern/historic wild macaw haplogroup data. Specifically, weconsidered the wild scarlet macaw haplogroups from the Gulfcoast region of the Isthmus of Tehuantepec, Mexico, the closestregion to the SW/NW in our current dataset with Haplo6 wildmacaws (Fig. 4B). We then drew with replacement an artificialsample of 14 individual sequences from three wild macaw hap-logroups in this region known during the last 200 y and recordedhow many times an individual with Haplo6 was in the artificialsample. We repeated this artificial sampling 10,000 times and thencompared the frequency distribution of the number of individualswith Haplo6 per permutation to the observed result for the ancientmacaws (in which all 14 individuals have Haplo6) as an empirical Pvalue. Of 10,000 permutations, there was a maximum of 11 indi-viduals with Haplo6 (mean = 4.67; SD = 1.75; P < 0.001; Fig. 4C),indicating very low probability that the archaeological macaws

could represent a population sample drawn randomly from thetested wild location. We repeated this permutation analysis usingvarious modern/historic sampling regions, always obtaining equiv-alent results (SI Appendix, Fig. S6 and Table S5).

Discussion and ConclusionsArchaeogenomic analyses of scarlet macaws were used to helpresolve long-standing questions regarding the origins and acquisi-tion of these exotic birds at SW archaeological sites. The early 900–1200 CE presence of these macaws, far outside of their endemicNeotropical range, along with studies demonstrating the exchangeof cacao, marine shell, and copper bells (20, 22, 46–48) over similarperiods, indicate significant and long-standing interactions betweenMesoamerican societies and the native peoples of the SW/NW.Wild scarlet macaws have occurred historically over vast portions

of the lowland Neotropics of Mexico, Central America, and SouthAmerica. Archaeologists working in the SW/NW have previouslyhypothesized that the birds in archaeological sites originated in thenorthernmost extent of this range, along the Gulf Coast of Mexico(21). Our archaeogenomic results indicate that the original breed-ing stock came from this general region. Specifically, only a singlemitochondrial haplogroup, Haplo6, was identified among our ar-chaeological samples from Chaco Canyon and the Mimbres region.Meanwhile, Haplo6 was observed in only three of 84 modernmacaws (39): two individuals from the Gulf Coast/Isthmus ofTehuantepec region and a third individual from northern Guate-mala. Haplo6 co-occurs with other haplogroups in both these areas.We currently do not have comparative wild scarlet macaw samples

from the very northernmost extent of their range in northernVeracruz/southern Tamaulipas. However, given the species behavior,it is highly unlikely that northern Veracruz/southern Tamaulipasrepresents an isolated subpopulation with a disproportionately highabundance of Haplo6. Scarlet macaws, similar to other large parrots,exhibit a high capacity for dispersal, with known long-distance sea-sonal migrations. Although exact distances traveled by individualmacaws are not well understood because of the logistical challengesof radio/satellite telemetry, documented changes in relative abun-dances suggest movements up to several hundred kilometers (49, 50).

Fig. 3. (A) Mitogenome phylogeny of the relationships among ancient scarlet macaw samples obtained from archaeological sites in the southwestern USfrom this study (light blue) and the reference scarlet macaw mitogenome sequence and those from closely related extant Ara species (black) (SI Appendix,Table S3). The minimum-spanning network was created with all missing positions masked (base pairs analyzed = 15,584). All phylogenetically informativepositions were retained in the ancient samples. (B) Calibrated AMS 14C dates for 14 scarlet macaws from Chaco Canyon [Pueblo Bonito (n = 9), Pueblo delArroyo (n = 2)] and the Mimbres region [Mitchell (n = 1), Old Town (n = 1), and Wind Mountain (n = 1)], with an unpartitioned Bayesian consensus treecreated using the Markov chain Monte Carlo method (HKY with four gamma-distributed rate categories) and 10 million generations, enforcing a strictmolecular clock parameter in BEAST 2 (55). The phylogeny represents all alignable positions with gaps and missing positions included in the analysis. Eachsample represents the 67.2% and 95.4% probability distributions for calibrated dates.

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It is also important to note that Haplo6 occurs at a low fre-quency within the modern dataset — three of 84 cyanopterasamples cluster within Haplo6 — whereas all ancient scarlet ma-caws cluster within this haplogroup. Our permutation analysissuggests that the low diversity observed in the ancient SW macawsample was unlikely to represent a random sampling of individualwild macaws. Moreover, 71% of the SW macaws shared the exactsame mitogenome sequence, increasing the likelihood these an-cient macaws share a matrilineal pedigree. These observations areinconsistent with models proposing direct acquisition or thepassing of individual macaws from community to community frommultiple distant source areas in Mexico and Central America (27).The logistical challenges of transporting scarlet macaws (eggs,chicks, juveniles, or adults) also lends support to the need ofhuman-mediated propagation of this animal population.All the SW scarlet macaws we sequenced predate the well-

attested scarlet macaw breeding activities evident at Paquimé,which was an important socioeconomic center from 1250 to1450 CE in northern Mexico (29–31, 51). We argue that the lowhaplotypic diversity shown here resulted from a pre-Hispanic cap-tive breeding program of a small founder population, with fledglingsand immature birds being transported to other communities in theSW/NW throughout a period perhaps as long as 300 y. The ra-diocarbon results from the SW scarlet macaws predate the breedingactivities at Paquimé, thus suggesting the existence of a previouslyunobserved captive breeding population being managed between900 and 1200 CE that served as the source for macaws in ChacoCanyon and the Mimbres region. Our results are consistent withthe hypothesis proposed by Crown (28), that lengthy trips weremitigated by the presence of intermediate breeding centers in the

area between the northern endemic range of scarlet macaws andthe SW/NW. The location of this hypothesized early breedingcolony is unknown, although additional research in the SW/NWmay uncover the archaeological remains of this site.

MethodsDetails for all ancient scarlet macaw radiocarbon dating and genomicmethodsused in this study are provided in SI Appendix. Ancient scarlet macaw samplesfrom Chaco Canyon were accessed through a submitted research proposal tothe Smithsonian Institution for accelerator mass spectrometer (AMS) 14C ra-diocarbon dating and ancient mitogenomic DNA analysis (SI Appendix, TableS1). Additional scarlet macaw samples from the Mimbres area at the Mitchell,Old Town, and Wind Mountain sites and the Arroyo Hondo macaw weresampled from existing collections. Schmidt (39) provided genetic data fromwild and historic scarlet macaw (A. m. cyanoptera) specimens. Detailed in-formation about ancient and reference macaw sequences are provided in SIAppendix and Datasets S1–S4.

Mitochondrial DNA from 20 ancient macaw samples were extracted andprocessed in the ancient DNA facility at Pennsylvania State University (SIAppendix, Table S2). Detailed information about laboratory procedures,sequencing, and bioinformatics are provided in the SI Appendix. In a dedi-cated clean facility, DNA extraction of bone samples followed a modifiedversion of ref. 52 and/or ref. 53 (detailed in SI Appendix). Double-strandedDNA libraries were constructed from DNA extracts following a publishedprotocol (54), and were then enriched for mitochondrial DNA fragments bybead capture in-solution biotinylated RNA bait hybridization (40), using theMycroarray MyBaits system (probe design: 140429 and 150610). Postcapturelibraries were sequenced at the Clinical Microarray Core, University of Cal-ifornia, Los Angeles. All computational methods and damage profiles are de-tailed in the SI Appendix, Fig. S1 and Dataset S2. Sequencing data are availablefrom the National Center for Biotechnology Information’s Sequence ReadArchive (BioProject ID: PRJNA477839), merged DNA assemblies are availablefrom Dryad Digital Repository at https://doi.org/10.5061/dryad.sv74pj2, and

Fig. 4. (A) Bayesian phylogeographic comparison among wild scarlet macaws specimens (n = 84) in Central America and ancient scarlet macaw samples (n =14) recovered in the southwestern US using 885 base pairs of the mtDNA control region (this study; ref. 39). The unpartitioned Bayesian consensus tree wasgenerated using model parameters (GTR + G + I) with four gamma-distributed rate categories and a Markov chain Monte Carlo and 10 million generations,enforcing a strict molecular clock parameter in BEAST 2 (ref. 55 and SI Appendix). Bayesian posterior probabilities are displayed above major nodes (>70%).(B) Map of the historic range of scarlet macaws in Central America and the wild specimens analyzed in ref. 39, with colors indicating haplotypes defined in A.(C) Results from a permutation analysis in which 10,000 sets of 14 simulated observations were randomly selected from three wild macaw references from theGulf of Mexico. The distribution of the number of Haplo6 individuals from each permutation were then compared with the observed result for the ancientmacaws (for which all 14 individuals have Haplogroup 6; P < 0.001). Similar results were obtained for all tested modern sample regions and combinationsunder this permutation scheme (SI Appendix, Fig. S6).

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mitogenome consensus sequences are available from GenBank (accessionnos. MH400234–MH400248; Dataset S2).

ACKNOWLEDGMENTS.We thank the Smithsonian Institution Department ofAnthropology for granting us permission to sample scarlet macaws fromPueblo Bonito and Pueblo del Arroyo, and we are grateful for the assistanceof Torben Rick and Esther Rimer. Thomas Holcomb of the Bureau of LandManagement in Las Cruces gave permission to sample the Old Townmacaws. Darrell Creel provided those samples, and Michael Cannon sentthe Mitchell site sample. Christine Szuter and Eric Kaldahl at the AmerindFoundation provided the Wind Mountain sample. Many thanks to GeorgePerry for his important contributions in designing and writing up this work.Patricia Crown, Dick Drennan, John Kantner, and Joyce Marcus, and twoanonymous reviewers provided valuable feedback on the manuscript. We

also thank members of the Human Paleoecology & Isotope GeochemistryLab for their assistance processing AMS 14C radiocarbon samples: LaurieEccles, Margaret Davis, Lindsay Simmins, and Matthew Veres. AMS 14C ra-diocarbon dates from this project were analyzed at the Penn State AMS 14Cfacility and the W. M. Keck Carbon Cycle Accelerator Mass SpectrometryLaboratory. We acknowledge and thank Ximin Li and Janice Yoshizawa atthe University of California, Los Angeles, Clinical Microarray. Data from thisproject were processed using the high-performance computing infrastruc-ture at the Pennsylvania State University Institute for Cyber Science Ad-vanced Cyber Infrastructure. This project was supported through grantsfrom the National Science Foundation (Archaeometry Program, BCS-1460367), research support from the Dean of Arts & Sciences, University ofVirginia, and the Pennsylvania State University.

1. Nielsen R, et al. (2017) Tracing the peopling of the world through genomics. Nature541:302–310.

2. Moreno-Mayar JV, et al. (2018) Terminal Pleistocene Alaskan genome reveals firstfounding population of Native Americans. Nature 553:203–207.

3. Skoglund P, et al. (2017) Reconstructing prehistoric African population structure. Cell171:59–71.e21.

4. Swarts K, et al. (2017) Genomic estimation of complex traits reveals ancient maizeadaptation to temperate North America. Science 357:512–515.

5. Speller CF, et al. (2010) Ancient mitochondrial DNA analysis reveals complexity ofindigenous North American Turkey domestication. Proc Natl Acad Sci USA 107:2807–2812.

6. Marshall FB, Dobney K, Denham T, Capriles JM (2014) Evaluating the roles of directedbreeding and gene flow in animal domestication. Proc Natl Acad Sci USA 111:6153–6158.

7. Larson G, et al. (2014) Current perspectives and the future of domestication studies.Proc Natl Acad Sci USA 111:6139–6146.

8. Manin A, et al. (2018) Diversity of management strategies in Mesoamerican turkeys:Archaeological, isotopic and genetic evidence. R Soc Open Sci 5:171613.

9. Kistler L, et al. (2015) Gourds and squashes (Cucurbita spp.) adapted to megafaunalextinction and ecological anachronism through domestication. Proc Natl Acad Sci USA112:15107–15112.

10. Librado P, et al. (2017) Ancient genomic changes associated with domestication of thehorse. Science 356:442–445.

11. da Fonseca RR, et al. (2015) The origin and evolution of maize in the SouthwesternUnited States. Nat Plants 1:14003.

12. Ramos-Madrigal J, et al. (2016) Genome sequence of a 5,310-year-old maize cobprovides insights into the early stages of maize domestication. Curr Biol 26:3195–3201.

13. Larson G, et al. (2010) Patterns of East Asian pig domestication, migration, andturnover revealed by modern and ancient DNA. Proc Natl Acad Sci USA 107:7686–7691.

14. Marciniak S, Perry GH (2017) Harnessing ancient genomes to study the history ofhuman adaptation. Nat Rev Genet 18:659–674.

15. Fehren-Schmitz L, Georges L (2016) Ancient DNA reveals selection acting on genesassociated with hypoxia response in pre-Columbian Peruvian highlanders in the last8500 years. Sci Rep 6:23485.

16. Jeong C, et al. (2016) Long-term genetic stability and a high-altitude East Asian originfor the peoples of the high valleys of the Himalayan arc. Proc Natl Acad Sci USA 113:7485–7490.

17. Kennett DJ, et al. (2017) Archaeogenomic evidence reveals prehistoric matrilinealdynasty. Nat Commun 8:14115.

18. Kennett DJ (2005) The Island Chumash: Behavioral Ecology of a Maritime Society(Univ California Press, Berkeley, CA).

19. Plog S, Heitman C (2010) Hierarchy and social inequality in the American Southwest,A.D. 800-1200. Proc Natl Acad Sci USA 107:19619–19626.

20. Crown PL, Hurst WJ (2009) Evidence of cacao use in the prehispanic AmericanSouthwest. Proc Natl Acad Sci USA 106:2110–2113.

21. Watson AS, et al. (2015) Early procurement of scarlet macaws and the emergence ofsocial complexity in Chaco Canyon, NM. Proc Natl Acad Sci USA 112:8238–8243.

22. Smith EM, Fauvelle M (2015) Regional interactions between California and theSouthwest: The Western Edge of the North American Continental system. AmAnthropol 117:710–721.

23. McKusick CR (2001) Southwest Birds of Sacrifice (WorldCat lists, Tucson, AZ), No. 31.24. Gilman PA, Thompson M, Wyckoff KC (2014) Ritual change and the distant: Meso-

american iconography, scarlet macaws, and great kivas in the Mimbres region ofSouthwestern New Mexico. Am Antiq 79:90–107.

25. Toll HW (1991) Material distributions and exchange in the Chaco system. Chaco andHohokam: Prehistoric Regional Systems in the American Southwest, eds Crown PL,Judge WJ (School of Am Res, Santa Fe, NM), pp 77–107.

26. Mathien FJ (2005) Culture and Ecology of Chaco Canyon and the San Juan Basin (NatlPark Service, Santa Fe, NM).

27. Creel D, McKusick C (1994) Prehistoric macaws and parrots in the Mimbres area, NewMexico. Am Antiq 59:510–524.

28. Crown PL (2016) Just macaws: A review for the US Southwest/Mexican Northwest.KIVA 82:331–363.

29. Di Peso CC (1974) Casas Grandes: A Fallen Trading Center of the Gran Chichimeca(Amerind Foundation, Dragoon, AZ), Vol 1–3.

30. Whalen ME, Minnis PE (2001) The Casas Grandes regional system: A late prehistoricpolity of northwestern Mexico. J World Prehist 15:313–364.

31. Somerville AD, Nelson BA, Knudson KJ (2010) Isotopic investigation of pre-hispanicmacaw breeding in Northwest Mexico. J Anthropol Archaeol 29:125–135.

32. Lévi-Strauss C (1969) The Raw and the Cooked: Introduction to a Science of Mythol-ogy, trans Weightman J, Weightman D (Harper and Row, New York), Vol 1.

33. Crocker JC (1977) My brother the parrot. The Social Use of Metaphor, eds Sapir DJ,Crocker JC (Univ of Pennsylvania Press, Philadelphia), pp 164–192.

34. Guernsey J (2006) Ritual and Power in Stone: The Political and Cosmological Significanceof Late Preclassic Izapan-Style Monuments (Univ Texas Press, Austin, TX).

35. Tyler HA (1991) Pueblo Birds and Myths (Northland, Flagstaff, AZ), 1979.36. Reyman JE (2008) Feathers and ceremonialism in the American southwest, past and

present. J West 47:16–22.37. Whiteley PM (2012) Turquoise and squash blossom. Turquoise in Mexico and North

America, eds King JCH, Carocci M, Cartwright C, McEwan C, Stacey R (Archetype/British Museum, London), pp 145–154.

38. Houston S, Brittenham C, Mesick C, Tokovinine A, Warinner CG (2009) VeiledBrightness: A History of Ancient Maya Color (Univ Texas Press, Austin, TX).

39. Schmidt K (2013) Spatial and temporal patterns of genetic variation in scarlet macaws(Ara macao): Implications for population management in La Selva Maya, CentralAmerica PhD dissertation (Proquest Dissertations Publishing, Columbia University,New York).

40. Gnirke A, et al. (2009) Solution hybrid selection with ultra-long oligonucleotides formassively parallel targeted sequencing. Nat Biotechnol 27:182–189.

41. Seabury CM, et al. (2013) A multi-platform draft de novo genome assembly andcomparative analysis for the scarlet macaw (Ara macao). PLoS One 8:e62415.

42. Briggs AW, et al. (2007) Patterns of damage in genomic DNA sequences from aNeandertal. Proc Natl Acad Sci USA 104:14616–14621.

43. Sawyer S, Krause J, Guschanski K, Savolainen V, Pääbo S (2012) Temporal patterns ofnucleotide misincorporations and DNA fragmentation in ancient DNA. PLoS One 7:e34131.

44. Wiedenfeld DA (1994) A new subspecies of scarlet macaw and its status and con-servation [Una nueva subespecie de la lapa roja y su estado de conservación]. OrnitolNeotrop 5:99–104.

45. Bullock PY, Cooper A (2002) Ancient DNA and macaw identification in the AmericanSouthwest: A progress report. Artifact 40:1–10.

46. Vokes AW, Gregory DA (2007) Exchange networks for exotic goods in the Southwestand Zuni’s place in them. Zuni Origins: Toward a New Synthesis of SouthwesternArchaeology, eds Gregory DA, Wilcox DR (Univ Arizona Press, Tucson, AZ), pp318–357.

47. Neitzel J (1989) The Chacoan regional system: Interpreting the evidence for socio-political complexity. The Sociopolitical Structure of Prehistoric Southwestern Societies,eds Upham S, Lightfoot K, Jewett R (Westview, Boulder, CO), pp 509–556.

48. Vargas VD (1995) Copper Bell Trade Patterns in the Prehispanic US Southwest andNorthwest Mexico (Arizona State Museum, Univ Arizona, Tucson, AZ), No. 187.

49. Renton K (2002) Seasonal variation in occurrence of macaws along a rainforest river.J Field Ornithol 73:15–19.

50. Karubian J, et al. (2005) Temporal and spatial patterns of macaw abundance in theEcuadorian Amazon. Condor 107:617–626.

51. Morales‐Arce AY, Snow MH, Kelley JH, Anne Katzenberg M (2017) Ancient mito-chondrial DNA and ancestry of Paquimé inhabitants, Casas Grandes (AD 1200–1450).Am J Phys Anthropol 163:616–626.

52. Rohland N (2012) DNA extraction of ancient animal hard tissue samples via adsorp-tion to silica particles. Methods Mol Biol 840:21–28.

53. Dabney J, et al. (2013) Complete mitochondrial genome sequence of a MiddlePleistocene cave bear reconstructed from ultrashort DNA fragments. Proc Natl AcadSci USA 110:15758–15763.

54. Meyer M, Kircher M (2010) Illumina sequencing library preparation for highly mul-tiplexed target capture and sequencing. Cold Spring Harb Protoc 2010:pdb.prot5448.

55. Bouckaert R, et al. (2014) BEAST 2: A software platform for Bayesian evolutionaryanalysis. PLOS Comput Biol 10:e1003537.

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