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DNA from Pre-Clovis Human Coprolites in Oregon, North America

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Science  09 May 2008:
Vol. 320, Issue 5877, pp. 786-789
DOI: 10.1126/science.1154116

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Abstract

The timing of the first human migration into the Americas and its relation to the appearance of the Clovis technological complex in North America at about 11,000 to 10,800 radiocarbon years before the present (14C years B.P.) remains contentious. We establish that humans were present at Paisley 5 Mile Point Caves, in south-central Oregon, by 12,300 14C years B.P., through the recovery of human mitochondrial DNA (mtDNA) from coprolites, directly dated by accelerator mass spectrometry. The mtDNA corresponds to Native American founding haplogroups A2 and B2. The dates of the coprolites are >1000 14C years earlier than currently accepted dates for the Clovis complex.

The timing, route, and origin of the first human migration into the Americas remain uncertain. Some archaeological (1) and genetic [reviewed by (2)] evidence has been used to argue for a settlement by 30,000 years ago (ka) (calendar years) or even earlier, but both lines of evidence remain controversial. The most widely accepted dates of occupation relate to the Clovis complex, ∼11,000 to 10,800 14C years before the present (yr B.P.) (13.2–13.1 to 12.9–12.8 ka), a distinct technology that appears to have originated and spread throughout North America in as little as 200 to 300 years (3).

The oldest directly dated human osteological remains from the Americas are no more than 11,000 14C yr B.P. (∼12.9 ka) (3, 4) and appear to be congruent with the “Clovis-first” model of colonization (5, 6). However, this theory is complicated by Monte Verde, in southern Chile, which contains artifacts dated to ∼12,500 14C yr B.P. (∼13.9 to 13.8 ka) that exhibit little technological connection to Clovis (7). Although a number of pre-Clovis occupation sites have been reported from North America (8), their age and cultural origins remain controversial, primarily because of the lack of directly dated human remains or artifacts (9).

Here we present evidence for human presence in North America before the Clovis complex, through the identification and genetic profiling of coprolites directly dated to 12,300 14C yr B.P. (∼14.27 to 14.0 ka) at the Paisley 5 Mile Point Caves in south-central Oregon (Fig. 1A). The Paisley Caves are wave-cut shelters located on the highest shoreline of pluvial Lake Chewaucan, which once filled the Summer Lake–Chewaucan–Lake Abert basins (Fig. 1A). As the lake level fell since the last glacial maximum (10, 11), the caves began filling with aeolian-transported silt and sand, gravel, roof spall, and organic material (bones, coprolites, plant remains, and artifacts) deposited by humans and animals. Sheltered from moisture, these extremely dry deposits contain perishable human artifacts: manufactured threads of sinew and plant fibers, hide, basketry, cordage, rope, and wooden pegs, as well as animal bones and diverse kinds of feces, in an unbroken stratigraphic sequence spanning the late Pleistocene and Holocene (12). Stone tool and debitage assemblages are small, suggesting that site occupations were generally brief. Pleistocene assemblages contain few chronologically diagnostic artifacts. The few projectile point fragments recovered are morphologically consistent with lanceolate, Western Stemmed, and foliate types common in the Younger Dryas (10,200 to 10,700 14C yr B.P.) and early Holocene archaeological sites of the region. However, the stratigraphic distribution of artifacts and radiocarbon dating of likely butchered bones suggest that the site may have been occupied as early as 12,400 and 12,000 14C yr B.P.

Fig. 1.

Geographical and stereographical information on Paisley 5 Mile Point Caves. (A) The location of Paisley 5 Mile Point Caves in Oregon and location of Oregon in the United States (inset). (B) Horizontal, vertical, and stratigraphic distribution of five of the human coprolites. Sample 1374-PC-1/2A-28 (Table 1) was excavated from another cave (Cave 1) and thus is not shown. Also indicated are a dated camelid astragalus, a horse phalange, and sample 8, a coprolite found out of context, indicating some stratigraphic disturbance. For further details, see SOM text.

Fourteen coprolites recovered from the lowest levels of Paisley Caves are morphologically human (based on size, shape, constituents, and color). In initial screening using a multiplex polymerase chain reaction (PCR) and minisequencing assay, all 14 were positive for human mitochondrial DNA (mtDNA) (13). This result is not surprising, because the coprolites had not been excavated under sterile conditions and could be expected to contain DNA derived from the excavation team (14, 15). Although all of the coprolites contained single-nucleotide polymorphisms (SNPs) diagnostic of European populations (which is consistent with contamination), independently generated cloned and pyrosequenced PCR products from six samples reproducibly yielded SNPs diagnostic of Native American founding mtDNA haplogroups (Hgs) A2 and B2, (Table 1; supporting online material (SOM) text; fig. S1; and tables S1, S5, S7, and S9) (16). The absence of PCR-amplifiable Native American mtDNA in the remaining eight samples may be due to differences in DNA survival across specimens or the possibility that these coprolites are nonhuman; or, what is more likely, the ratio of contaminant to endogenous DNA was too great for endogenous mtDNA detection using our techniques.

Table 1.

Results of mtDNA, nongenetic analysis and AMS dating of the six coprolites identified as of Native American origin.

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Human ancient DNA studies are extremely prone to contamination that may occur during sampling or subsequent laboratory analyses (14, 15). To exclude laboratory-based contamination, the Hg A and B results were independently confirmed with several different DNA typing and sequencing methods (13) in multiple laboratories (in Copenhagen, Denmark; Uppsala, Sweden; and Leipzig, Germany) (Table 1). Furthermore, to ensure that the Hg A and B results were not the result of contamination during the excavation, we mitotyped the 55 individuals (students, instructors, and visitors) who had been present at the site during the two-season excavation (13). Additionally, we mitotyped the 12 researchers present in the principal DNA laboratory (13). This was performed regardless of whether an individual played an active role in the excavation or in the genetic analysis of the coprolites. For example, although 55 people had been present at the archaeological site, only 14 were actively involved in the excavation of the six coprolites reported here, and only two of those could have come in contact with all six specimens. The results show that none of the individuals tested are the sources of the Hg A and B mtDNA (13) (table S4). Because the coprolites had been stored in sealed plastic bags from the time of excavation until the genetic analyses were undertaken, it is difficult to explain the results by other sources of post-excavation contamination. To provide additional confirmation for a human origin of the coprolites, we submitted part of the four oldest samples (as material allowed) for protein residue (crossover immunoelectrophoresis) and reconstitution analyses (trisodium phosphate solution analyses) (13). The results of these tests confirmed the results of the morphological and genetic analyses that the coprolites are of human origin (Table 1 and SOM text).

The leaching of DNA from younger to older stratigraphic layers may be a problem in relatively wet, temperate cave settings (17). Although it is unlikely that leaching would be able to provide crossover immunoelectrophoresis false positives, because of the relatively large amounts of proteins needed, we conducted two additional tests for DNA leaching. Wood rat (Neotoma lepida) fecal pellets are a major constituent of the strata in the Paisley 5 Mile Point Caves and were found in direct contact with the six coprolites in question (table S9). Thus, we screened the six coprolites for Neotoma mtDNA (13). To account for any possible differences in Neotoma species inhabiting the caves in the past, we used primers designed to amplify mtDNA from all members of the genus. Control Neotoma fecal samples from the caves gave positive results for Neotoma DNA (table S6). However, all six coprolites testing positive for human DNA tested negative for Neotoma DNA. We additionally screened 14 control sediment samples and two long bones morphologically identified as Spermophilus lateralis (golden-mantled ground squirrel), recovered from around the coprolites, for the Hg A and B SNPs (13) (table S6). Although 8 out of the 16 samples were positive for human mtDNA (presumably derived from the excavation team), none of the samples were positive for the Native American SNPs (table S6). Additionally, primers specific for S. lateralis gave positive results for the two long bones, demonstrating that DNA survived in noncoprolite specimens from the Paisley Caves for long times also. The results strongly suggest that leaching of DNA is not a concern in the Paisley Caves and are in agreement with empirical and theoretical evidence suggesting that substantial amounts of liquid water are required to move free DNA molecules between strata (17, 18).

Three of the six coprolites also contained canid 16S mtDNA with high similarity to red fox (Vulpes vulpes, one substitution difference), coyote (Canis latrans, one substitution difference), or domestic dog or wolf (Canis familiaris or C. lupus, 100% match) (Table 1) (13). In light of the nongenetic tests showing a human origin of the coprolites and the findings of diverse canid bones in the strata, the most likely explanations for these results are that humans may have eaten canids or that canids living in the caves during periods of nonhuman occupation urinated directly onto human feces.

Accelerator mass spectrometry (AMS) radiocarbon dating of bone collagen from a camelid astragalus, recovered in stratigraphic association (stratum LU1b) with three of the oldest human coprolites, produced an age of 12,300 14C yr B.P. (Fig. 1B). To ensure reliable ages of the coprolites, the five specimens from the deepest layers were submitted for direct dating by AMS at two independent laboratories; Beta Analytic (Florida, USA) and the Oxford Radiocarbon Accelerator Unit (University of Oxford, UK). Although each laboratory used different methodologies (13), all specimens except one (sample 1294-PC-5/6B-40) produced consistent dates, ranging from approximately 1300 to 12,300 14C yr B.P., and three of the coprolites pre-dated 11,000 14C yr B.P. (Table 1 and Fig. 2). Thus, our data show that humans were present in North America before the Clovis complex. Analyses of complete Native American mtDNA genomes imply that the Hgs A2 and B2 originated at 13.9 ± 2.0 and 16.5 ± 2.7 ka, respectively (16). The coprolites at Paisley may thus derive from among the earliest members of these Hgs. The Paisley Caves contain only small pre-Clovis lithic tool assemblages, and thus the cultural and technological associations of the early site occupants and their relationship to the later Clovis technology are uncertain.

Fig. 2.

Calibrated radiocarbon determinations from the four oldest coprolites excavated at Paisley 5 Mile Point Caves (bottom section), obtained using INTCAL04 (19) and the OxCal4.0 software (20). Mean dates for Clovis sites reported by Waters and Stafford (3) are also included for comparison (top section). The calibrated results for Paisley Caves shows that the oldest are ∼1000 14C years older than the earliest Clovis dates. Beta, Beta Analytic; OxA, Oxford Radiocarbon Accelerator Unit.

Supporting Online Material

www.sciencemag.org/cgi/content/full/1154116/DC1

Materials and Methods

SOM Text

Figs. S1 to S5

Tables S1 to S9

Cloned DNA Sequence Alignments

References and Notes

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