Abstract Accounts of woolly mammoths (Mammuthus primigenius) preserved so well in ice that their meat is still edible have a long history of intriguing the public and influencing paleontological thought on Quaternary extinctions and climate, with some scientists resorting to catastrophism to explain the instantaneous freezing necessary to preserve edible meat. Famously, members of The Explorers Club purportedly dined on frozen mammoth from Alaska, USA, in 1951. This event, well received by the press and general public, became an enduring legend for the Club and popularized the notorious annual tradition of serving rare and exotic food at Club dinners that continues to this day. The Yale Peabody Museum holds a sample of meat preserved from the 1951 meal, interestingly labeled as a South American giant ground sloth (Megatherium), not mammoth. We sequenced a fragment of the mitochondrial cytochrome-b gene and studied archival material to verify its identity, which if genuine, would extend the range of Megatherium over 600% and alter our views on ground sloth evolution. Our results indicate that the meat was not mammoth or Megatherium but green sea turtle (Chelonia mydas). The prehistoric dinner was likely an elaborate publicity stunt. Our study emphasizes the value of museums collecting and curating voucher specimens, particularly those used for evidence of extraordinary claims.

Citation: Glass JR, Davis M, Walsh TJ, Sargis EJ, Caccone A (2016) Was Frozen Mammoth or Giant Ground Sloth Served for Dinner at The Explorers Club? PLoS ONE 11(2): e0146825. https://doi.org/10.1371/journal.pone.0146825 Editor: Anthony Fiorillo, Perot Museum of Nature and Science, UNITED STATES Received: August 13, 2015; Accepted: December 18, 2015; Published: February 3, 2016 Copyright: © 2016 Glass 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: The sequence for YPM HERR 19475 is archived in GenBank (Accession No. KT276329) and the specimen is housed in the Yale University Peabody Museum of Natural History. All other data used for analysis are archived in GenBank and referenced within the paper. Funding: Funding was received by JRG from The Explorers Club Exploration Fund (https://explorers.org/expeditions/funding/expedition_grants) and by AC from the Yale Institute for Biospheric Studies (http://yibs.yale.edu). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: JRG and MD are members of The Explorers Club. The authors have declared that no competing interests exist.

Materials and Methods DNA Extraction The specimen of meat was originally designated BRCM 16925 before a transfer in 2001 from the Bruce Museum to the Yale Peabody Museum of Natural History (New Haven, CT, USA) where it gained the number YPM MAM 14399. The specimen is now permanently deposited in the Yale Peabody Museum with the designation YPM HERR 19475 and is accessible to outside researchers. The meat was never fixed in formalin and was initially stored in isopropyl alcohol before being transferred to ethanol when it arrived at the Peabody Museum. DNA extraction occurred at Yale University in a clean room with equipment reserved exclusively for aDNA analyses. We obtained five 250 mg samples of the cooked muscle tissue, which were air dried to remove all ethanol. We then mixed each sample with 3900 μL of EDTA digestion buffer (0.5 M, pH 8.0, UltraPure Invitrogen) and 100 μL of Proteinase K solution (20 mg/ml, Qiagen). Samples were incubated for 18 hours at 40°C, then for 12.5 hours at 61°C after an additional 40 μL of Proteinase K was added. After digestion, as an initial purification step, samples were centrifuged for 3 min at 4000 rpm and the supernatant was transferred to a Centricon YM 30 (MWCO 30,000; Millipore) filter device. We then centrifuged samples at 4000 rpm for a minimum of 30 min to achieve a final volume of 250 μL. Next, we went through a second stage of purification using the MinElute PCR Purification Kit (Qiagen), following manufacturer’s guidelines and eluting 60 μl of dH 2 0. Due to the degraded nature of the DNA, we subsequently concentrated the five samples down to two by executing the MinElute protocol again (Qiagen), also eluting 60 μl of dH 2 0. Sequencing We targeted a 308 base pair (bp) region of the cytochrome b (cytb) gene in the mitochondria using one pair of universal vertebrate primers from Kocher et al. [28]. The primers used were L14841 (5’-AAAAAGCTTCCATCCAACATCTCAGCATGATGAAA-3’) and H15149 (5’-AAACTGCAGCCCCTCAGAATGATATTTGTCCTCA-3’). PCR preparation was carried out in a laminar flow hood sterilized with UV light. DNA was amplified in 20 μL reactions consisting of Promega GoTaq Thermus aquaticus (Taq) DNA polymerase (ProMega) and a reaction buffer containing 1.5 mM MgCl 2 , 0.2 mM each dNTP, 0.25 mM forward and reverse primers, and 1 μg/ μL bovine serum albumin (BSA). The PCR protocol consisted of 35 cycles of an initial denaturization period of 2 minutes at 95°C, followed by 35 cycles of denaturization at 95°C (1 min), annealing at 52°C (1 min), and extension at 72°C (1 min). This was followed by a final extension at 72°C (5 minutes) and a standby temperature of 4°C. The PCR was carried out in an Eppendorf Mastercycler DNA Thermocycler. In addition to the two concentrated “aDNA” samples, a blank reaction was included to monitor for contamination, as was a control sample of American angler (Lophius americanus), with which the primers were previously optimized. Amplified PCR products were run on a 2% agarose gel to confirm amplification success and purified with a polyethylene glycol precipitation protocol. All sequences were edited and aligned using Geneious version 8.0.4 [29]. Identification We used the BLAST nucleotide search function to match our DNA sequence with those registered in GenBank/EMBL and provide a preliminary identification. We calculated pairwise distances between the meat sample, 14 Chelonia mydas samples, Mammuthus primigenius, and Nothrotheriops shastensis (the closest ground sloth species to Megatherium with published cytb data) in MEGA v6.06 using the Maximum Composite Likelihood model (Table 1) [30,31]. For this analysis, we trimmed sequences to a 371 bp alignment. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. List of GenBank accession numbers for specimens used in phylogenetic and pairwise distance analyses. https://doi.org/10.1371/journal.pone.0146825.t001 To determine the geographic origin of the specimen, we produced a phylogeny of Chelonia mydas using cytb sequences available on GenBank. Fourteen sequences with locality information were available on GenBank and were trimmed to a 369 bp alignment (Fig 4, Table 1). Bayesian analyses were used to estimate the phylogenetic tree, executed in MrBayes version 3.2.5 [32,33]. C. mydas cytb samples were analyzed as a single partition (1 million generations, nruns = 2, nchains = 4, burninfrac = .25) using the molecular evolutionary model HKY. We used Markov chain Monte Carlo methods to approximate posterior probabilities and parameter values, and used an invariant gamma rates unconstrained model with a rate parameter of 10.0. We monitored the average SD of the split frequencies between the two runs to assure convergence of the MrBayes runs, ensuring the SD value was < 0.01. The hawksbill sea turtle (Eretmochelys imbricata) and the loggerhead sea turtle (Caretta caretta), both in the family Cheloniidae, were used as outgroups to root the tree (Table 1). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 4. Phylogenetic tree of C. mydas and relatives. Phylogeny based on a 369 bp alignment of the mitochondrial cytochrome-b gene, inferred from Bayesian analyses. Specimens from the Pacific Ocean form a clade (green), whereas those with Atlantic or Mediterranean origins (blue) are unresolved. The YPM specimen is bolded. Node labels indicate posterior probability percentage support estimates. All branch lengths are in substitution units. https://doi.org/10.1371/journal.pone.0146825.g004

Results We successfully amplified the cytb gene from the cooked muscle tissue. The two samples yielded identical sequences that were 363 bp long after editing (Genbank Accession No. KT276329, Table 1). A BLAST search produced 14 close matches belonging to Chelonia mydas, with low pairwise distance values ranging from 0.017 to 0.034 (Table 1). The mammoth M. primigenius and the ground sloth N. shastensis had much greater genetic distance values (0.364 and 0.341, respectively) (Table 1). We were unable to resolve the geographic origin of the YPM specimen with a phylogenetic analysis including the 14 other cytb fragments from Genbank belonging to C. mydas specimens with known geographic information (Fig 4, Table 1).

Discussion The genetic data indicate that the meat served at the 1951 ECAD was not prehistoric, but sea turtle, likely from the soup served during the same meal. We acknowledge it is possible that members of the Club actually consumed Megatherium or mammoth meat but that Dodge sent Howes a sample from the wrong dish; however, this seems unlikely. There is no archival evidence suggesting that Hubbard or Kosco discovered a frozen mammoth or ground sloth. Hubbard described other specimens he found in Alaska [34] but out of the hundreds of photographs he took while exploring Akutan and Unimak (a neighboring island sometimes listed as the Megatherium specimen’s locality), none feature anything resembling a carcass of a mammoth or ground sloth (D. Dominguez, pers. comm.). Kosco frequently explored uninhabited islands in the Pacific and often shared interesting discoveries of his voyages with his family or other members of The Explorers Club (W.G. Kosco, pers. comm.) [35]. However, we were unable to find any instance where he described frozen mammoth or ground sloth. Curiously, Arnold Hauerslev Haverlee, Club member and chef for several Explorers Club dinners, claimed he did cook mammoth meat at the 1951 ECAD [36]. When the Sportsmen’s Club of South Glastonbury, CT, USA, publicly contacted The Explorers Club in 1957 for assistance in finding mammoth for their own annual dinner, Haverlee guaranteed the sportsmen that for a $20,000 fee, he could find and cook mammoth meat [36]. Haverlee was nationally famous for his role as chef at the extravagant 50th ECAD in 1954 where he served oddities such as polar bear and fried termites to honorary guests Tenzing Norgay and Werner non Braun [37], but we could find no independent evidence that he was also the 1951 ECAD mammoth chef. Had he actually cooked prehistoric meat, it likely would have been mentioned in his biography [38] or in the Explorers Cookbook where he described recipes for his other exotic dishes [39]. Fellow Explorers Club member Coleman Shaler Williams provided a detailed recipe in the book for the fossil horse he claimed to have served at the 1969 ECAD (See S1 Appendix for the authenticity of this horse dish) [22]. It is likely that Haverlee’s description of cooking mammoth meat was all in jest, playing along with the Sportsmen’s Club tongue-in-cheek publicity stunt. The previous year, the South Glastonbury Sportsmen lamented that they had been unable to bag a pterodactyl for the planned main course [40]. Our archival research suggests that the prehistoric meat served at the 1951 ECAD was a jocular publicity stunt that mistakenly wound its way into fact [12,13]. Although there was one previous incident of a paleontologist mistaking a ground sloth for a sea turtle [41], it still seems odd that a skilled naturalist like Howes, as well as other Explorers Club members and journalists, continued to believe in the authenticity of the sloth meat even after Dodge admitted it was a playful prank. However, Dodge’s facetious admission of guilt [14], published in the Club’s Explorers Journal, was confusing to Club members and later researchers, and may not have been taken for the confession that it was [8,12,13]. The designation of Hubbard and Kosco as the collectors also gave the story an air of authenticity. Hubbard, a Jesuit priest, taught in the Geology Department at the University of Santa Clara and was famous for his expeditions to the volcanoes and ice fields of Alaska [42]. Kosco led several polar expeditions, earned an eponymous glacier in Antarctica, and collected specimens for the Smithsonian [43,44]. At the time of the 1951 ECAD, Kosco was living on Kodiak Island, Alaska, and arranged for the U.S. Navy to fly king crabs, table settings of arctic vegetation, and glacial ice (for chilling cocktails) back to New York for the dinner at the Roosevelt Hotel (W.G. Kosco, pers. comm.) [14]. Two well-known polar explorers finding a frozen mammoth or sloth on an island similar to those where fossil mammoths had already been unearthed must not have seemed extraordinary, especially for a Club that keeps a mutant, four tusked elephant in its trophy room and whose members discovered the North and South Poles. Although it remains unclear why later accounts identify the 1951 ECAD meat almost exclusively as mammoth instead of sloth [8,12,15], one Club member, Lieutenant Colonel Herbert Bishop Nichols, may bear some responsibility. Nichols, the first science editor for The Christian Science Monitor [45], published the most detailed, and likely influential, description of the meat four days after the 1951 ECAD and claimed it was mammoth [11]. It is unknown why he chose “mammoth” when other members explicitly told reporters they had eaten “sloth” [16,46] but the article is taken as proof by modern journalists that mammoth was actually served at the dinner [12,13]. Tall tales like The Explorers Club “mammoth” can have a lasting impact on scientific thought and the public’s perception of natural history. A fictitious account of one frozen carcass written for a German children’s book in 1859 is cited heavily in early scientific literature and is still reproduced as fact in some paleontological textbooks today [47,48]. A story in McClure’s Magazine recounting the hunting of a live mammoth later donated to the Smithsonian proved so popular that the museum had to issue a public statement denying the specimen’s existence and challenging donors to fund an expedition to seek out a real frozen mammoth with which to replace it [49]. More importantly, historical museum metadata such as collection localities are frequently aggregated and used as the basis for macroecological studies, but these data are susceptible to numerous, significant sources of error from careless labeling to purposeful fraud, and are rarely validated before downstream analyses [50]. The responsibility of maintaining accurate specimen records falls to the museum housing those specimens; however, our findings further emphasize the value of collecting and curating voucher specimens, even those with contentious metadata, as evidence for extraordinary claims. Presaging this study, Dodge ended his mea culpa about the mystery meat by writing, “Science in all its Divisions must form the Jury and decide the fate of the, shall we say, ‘Defendant’.”[14]. Had Howes not accessioned the “ground sloth” into his museum, the identity of the “Defendant” could never have been examined. Sending Howes his sample years before the structure of DNA had been discovered, Dodge probably never anticipated that one day the several nondescript pieces of meat he saved would finally lay to rest the myth of The Explorers Club “mammoth.”

Supporting Information S1 Appendix. The authenticity of the fossil horse purportedly served at the 1969 ECAD. https://doi.org/10.1371/journal.pone.0146825.s001 (DOCX)

Acknowledgments We are grateful to Kristof Zyskowski and Gregory Watkins-Colwell, Division of Vertebrate Zoology, Yale Peabody Museum of Natural History; Aris Parmakelis, Department of Ecology and Systematics, University of Athens; Danielle Edwards, University of California Merced, School of Natural Sciences; and Carol Mariani, Yale University, Department of Ecology and Evolutionary Biology, for their excellent advice and assistance. We also thank William G. Kosco; Daniel Ksepka, Bruce Museum; Lacey Flint, The Explorers Club Research Collections; Susan Bell, American Museum of Natural History, Division of Paleontology Archives; and Dennis Dominguez and Shelia Conway, Santa Clara University Archives and Special Collections, for assistance with archival collections. We appreciate the helpful comments of Louis L. Jacobs and two anonymous reviewers on previous versions of this manuscript.

Author Contributions Conceived and designed the experiments: JRG MD EJS AC. Performed the experiments: JRG MD. Analyzed the data: JRG MD. Contributed reagents/materials/analysis tools: TJW AC. Wrote the paper: JRG MD TJW EJS AC.