Abstract Preceramic human skeletal remains preserved in submerged caves near Tulum in the Mexican state of Quintana Roo, Mexico, reveal conflicting results regarding 14C dating. Here we use U-series techniques for dating a stalagmite overgrowing the pelvis of a human skeleton discovered in the submerged Chan Hol cave. The oldest closed system U/Th age comes from around 21 mm above the pelvis defining the terminus ante quem for the pelvis to 11311±370 y BP. However, the skeleton might be considerable older, probably as old as 13 ky BP as indicated by the speleothem stable isotope data. The Chan Hol individual confirms a late Pleistocene settling of Mesoamerica and represents one of the oldest human osteological remains in America.

Citation: Stinnesbeck W, Becker J, Hering F, Frey E, González AG, Fohlmeister J, et al. (2017) The earliest settlers of Mesoamerica date back to the late Pleistocene. PLoS ONE 12(8): e0183345. https://doi.org/10.1371/journal.pone.0183345 Editor: Michael D. Petraglia, Max Planck Institute for the Science of Human History, GERMANY Received: March 31, 2017; Accepted: August 2, 2017; Published: August 30, 2017 Copyright: © 2017 Stinnesbeck et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: All the funding or sources of support received during this specific study have been presented. This financial support was granted to us by the Internationales Büro of the German Bundesministerium für Bildung und Forschung (BMBF project 01DN119) and the Deutsche Forschungsgemeinschaft (DFG project STI 128/28-1). MD acknowledges support by the Irish Research Council (IRC) by a Government of Ireland Postdoctoral Fellowship (GOIPD/2015/789). BMBF and DFG financed our field work in Mexico and provided funds for laboratory work. Author Michael Deininger received a salary from the IRC during part of this study. Other than that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Discussion The Chan Hol II individual was discovered at about 1240 meters away from the nearest modern entrance, the Chan Hol sinkhole. The skeleton disarticulated slightly during the final stages of decomposition and, probably again, during the early to middle Holocene flooding of the cave, but most bones still lie close to their original anatomical position. Even small bones, like auditory ossicles, hyoid, or ungual phalanges are present. The person must thus have died in the cave at a time when the cave floor was dry [21]. The decay of the carcass occurred in-situ. Growth of the CH-7 stalagmite began after the decay of the Chan Hol II individual was complete. This interpretation is consistent with the macroscopic sequence of basal-most stalagmite laminae. At that time, the drip point was located near the margin of the pelvis (Fig 5). The calcite layer precipitated by the lateral run-off dripping water embraced the pelvic bone from laterally to ventrally, with its ventral surface coalescent with the stalagmite, due to flow extension below the pelvis. This close overgrowth could not have taken place with bones covered by soft tissue. The porous tufa-like layer conforming the basis of the stalagmite must therefore have formed at that time, when the cave was dry and the pelvis completely exposed on the cave floor for an unknown amount of time. No data are at hand to define the amount of time that elapsed between the death of the individual and initial growth of the CH-7 stalagmite, nor the lapse needed for maceration and decay of this individual under the environmental conditions prevailing in the cave during the YD. Corpses decaying in caves are mostly decomposed by fungi and insects. Both are not able to move bones [66]. According to [66], a 25 kg kangaroo carcass, deposited in a cave in southern Australia, is completely decayed after a little more than 1000 days. For the complete decay of a human carcass with a body mass of 60 kg one would expect a minimum decay time of 2000 days as an estimate. Our U/Th datum of 11311±370 y BP at 72 mm of the CH-7 stalagmite and even the 13 ky BP age assignment resulting from the speleothem stable isotope record must therefore be regarded as minimum ages of the Chan Hol II skeleton. Validation of our age assessment has been done by comparison of our CH-7 stable isotope data with different independently dated climate records (Fig 7). We stress, that no age correlation or wiggle matching has been carried out to any of the records. All we did was to apply the linear age model from the closed system U/Th dates to the lower part of the stalagmite. The comparison of our CH-7 stable isotope record with other climate records indicates that the Chan Hol speleothem indeed covers the Younger Dryas time interval (Fig 7). In terms of amplitude and absolute value, the Yucatan δ18O record fits well the δ18O signal of speleothem records of New Mexico [51–52] and Arizona [53] that have been demonstrated to record the global climate signal of the YD. The American speleothem δ18O records have further been interpreted to reflect changes in the contribution, intensity and source of winter versus summer precipitation, the latter being fed from the Caribbean, and these changes have been linked to changes in the positioning of the polar jet stream related to the still northerly expansion of ice sheets causing modulation of winter storm tracks across the continent [52–53]. On the other hand, the relationship between the Pink Panther cave oxygen isotope record and solar forcing has been explained through changes in the Walker circulation and the Pacific Decadal Oscillation and El Niño–Southern Oscillation systems [51] but shows a significant similarity to Northern Hemisphere records. Finally, the climate signals at Peten Itza and Cariaco Basin have been discussed to reflect swings in the position of the ITCZ [57, 58]. In the end, these climate components are all linked to a complex system [67] and likely influenced our Chan Hol record. However, it is beyond the scope of this paper to disentangle the different components of this complex climate system. This deserves a thorough discussion in a separate paper. The focus of the current paper is on the dating of the Chan Hol II skeleton and we can confidentially state that with the U/Th dates and the stable isotope record at hand we can approximate the age of the Chan Hol II individual to ~13 ky BP.

Conclusions Speleothem (U/Th) age data indicate that the Chan Hol underwater cave south of Tulum, state of Quintana Roo, Mexico, was accessed by humans during the Younger Dryas period, i.e. during the late Pleistocene. This is indicated by a minimum speleothem age of 11311±370 y BP of a stalagmite encrusting and overgrowing the pelvic bone of an almost articulated human individual in this cave. The age was measured at 72 mm depth from the top of the CH-7 stalagmite, at about 21 mm above the pelvis and 33 mm above the base of the stalagmite, while ages in the immediate bone vicinity are altered due to uranium dissolution. 11311±370 y BP is thus a minimum age for the skeleton. Based on a linear growth model and extension of growth rates from the well-dated upper part of the CH-7 stalagmite to its lower portion and base, the age of the Chan Hol II human rises to ~13 ky BP. The Chan Hol II skeleton thus represents one of the oldest directly dated osteological heritage of a human from the American continent. Age of the Chan Hol II human equals that of other skeletons in the Tulum cave system (e.g. Naia, Najaron), thus emphasizing the importance of these caves for early human settlement in the Americas [20, 21, 25].

Methods The CH-7 stalagmite consists of only calcite (no aragonite was detected), as was confirmed by 25 measurements using Raman spectroscopy techniques at the Institute of Earth Sciences at Mainz University, Germany (Figs 5 and 8). For Raman spectroscopy a Horiba Jobin Yvon was used that was connected to an Olympus BX41 microscope using a Nd-YAK laser at a wavelength of 532.12 nm (hole = 400 μm; slit = 100 μm). PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Fig 8. Raman spectrum of CH-7. Values measured are compared to Raman spectra of reference calcite and aragonite (RRUFF database; [68]. Comparison shows that CH-7 consists of calcite. https://doi.org/10.1371/journal.pone.0183345.g008 U/Th dating Samples for U/Th-dating were drilled from stalagmite CH-7 using a Dremel Fortifex precision tool, 1 mm in diameter. The samples were taken along laminar growth layers to minimize mixing of material of different age and thus age uncertainties. Individual sample thickness is typically 2 mm (in growth direction), with an individual sample weight of between 100 and 150 μg. All samples (carbonate powder) were prepared for measurements in the clean laboratories at the Institute of Earth Sciences and Institute of Environmental Physics, both Heidelberg University, by wet-column chemistry using UETVA® resin. All samples were spiked using a Th-U multi-spike. U- and Th-isotopes were analyzed using ICP-MS (Thermo Finnigan Neptune Plus and iCAP (RQ), respectively) at the Institute of Environmental Physics at Heidelberg University. Details for sample preparation and U- and Th-isotope analysis are documented in [69]. Ages were calculated using the half-lives of both elements as determined by [70]. Detrital correction was performed using a bulk Earth value of 3.8 ± 1.9. Age uncertainties are quoted at the 2-σ level and do not include half-life uncertainties. The reference year for all ages given in the study is 1950 AD (i.e. 0 BP). U/Th ages and the growth of the CH-7 stalagmite U/Th ages in the upper 72 mm of the CH-7 stalagmite are approximately consistent with the macroscopic sequence of individual laminae growing onto each other (Fig 4). The basal-most layer was dated to 11363±304 y BP. In this layer, the drip point (the highest point of each stalagmite layer) is located at about 20 mm lateral to the pelvis (Fig 4). The layer embraces the bone from lateral to ventral and its ventral surface is coalescent with the stalagmite. The dripping water accumulated lateral of the bone and ran off diffusing laterally, with the flow expanding below the pelvis. To do so, the body must then have been completely decayed already. In a second step, calcium carbonate-rich water, dripping from the ceiling, accumulated next to the pelvis and enclosed the bone completely. The porous tufa-like layer conforming the base of the stalagmite and dated to >11363 y BP, must therefore have formed when the cave was dry and the carcass already completely skeletonized. This is concluded from the spongy carbonate crust that formed beneath the pelvis, at a time when this bone blank of soft tissue. C and O isotope analysis of CH-7 stalagmite Stable oxygen and carbon isotope samples were micro-milled and measured at the Institute for Earth Sciences (GeoZentrum Nordbayern), Friedrich-Albert-Universität Erlangen, Germany. A total of 117 data points was sampled along five transects, each along the growth axis of stalagmite CH-7 (Figs 4 and 6). A minimum of 0.05 to 0.1 mg CaCO 3 was analyzed to ensure precise measurement. Carbonate powders were reacted with 100% phosphoric acid at 70°C, using a Gasbench II connected to a ThermoFisher Delta V Plus mass spectrometer All values are reported in per mil relative to V-PDB through international standard NBS19. Reproducibility was monitored by international and in house laboratory standards and was 0.5‰ and 0.8‰ for δ13C and δ18O, respectively.

Supporting information S1 Table. Stable isotope (δ13O, δ18O) measurements of the CH-7 stalagmite from Chan Hol cave. https://doi.org/10.1371/journal.pone.0183345.s001 (DOCX)

Acknowledgments We acknowledge Valentina Cucchiara and Nick Poole (Liquid Jungle) and Thomas Spamberg for the use of underwater photographs of the ChanHol II site prior to the robbery of the skeleton and Ben McGeever (DiveXtras) for the permission to use diver propulsion vehicles, and we thank Carmen Rojas Sandoval and Adriana Velásquez (INAH, Tulum) for helpful discussions. Réne Eichstaedter is gratefully acknowledged for his expertise on MC-ICPMS and Th/U dating and Michael Joachimski for enabling stable isotope analysis. We gratefully acknowledge support of the project “Atlas Arqueológico Subacuático para el Registro, Estudio y Protección de los Cenotes en la Península de Yucatán” and “Estudio de los grupos humanos preceramicos de la costa oriental de Quintana Roo, México, a través de los contextos actualmente inundados” by the Instituto Nacional de Antropología e Historia (INAH). Michael Waters and one anonymous reviewer, as well as journal editor Michael Petraglia, are gratefully acknowledged for their many helpful comments and corrections to this manuscript. Material The Chan Hol II osteological material, including the CH-7 stalagmite encrusting pelvic remains, is housed at the Área de Prehistoria y Evolución of the Instituto de Investigaciones Antropológicas at the Universidad Nacional Autónoma de México (UNAM), in Mexico City. Here the material is publicly deposited and accessible by others in a permanent repository. Specimen numbers: Chan Hol II human: PQR2012-CHAN HOL-2; the Chan Hol stalagmite is housed under the number CH-7. The material was recovered from the Chan Hol cave sytem at 20°9.467' N, 87°34.165' W, 15 km southwest of Tulum, Quintana Roo, southern Mexico, and about 11.5 km from the coast line (Fig 1). Permits All necessary permits were obtained for the described study, which complied with all relevant regulations from Instituto Nacional de Antropología e Historia, Mexico (INAH permit number: C.A. 401.B (4)19.2011/36/1723).