Molecular Coproscopy: Dung and Diet of the Extinct Ground Sloth Nothrotheriops shastensis

S. Pääbo, Sci. Am. 269, 86 (Nov. 1993);

___, Higuchi R. G., Wilson A. C., J. Biol. Chem. 264, 9709 (1989).

Höss M., Kohn M., Knauer F., Schröder W., Pääbo S., Nature 359, 199 (1992);

Kohn M., Knauer F., Stoffella A., Schröder W., Pääbo S., Mol. Ecol. 4, 95 (1995);

; M. Kohn and R. K. Wayne, Trends Ecol. Evol. 12, 223 (1997).

Martin P., Sabels B., Shutler D., Am. J. Sci. 259, 102 (1961);

; R. Hansen, Paleobiology4, 302 (1978).

M. R. Harrington, Gypsum Cave, Nevada, Southwest Museum Papers (Southwest Museum, Los Angeles, CA, 1933), vol. 8; J. D. Laudermilk and P. A. Munz, Carnegie Inst. Washington Publ. 453, 31 (1934).

Long A., Martin P. S., Science 186, 638 (1974).

Höss M., Dilling A., Currant A., Pääbo S., Proc. Natl. Acad. Sci. U.S.A. 93, 181 (1996).

Stankiewicz B. A., Mastalerz M., Kruge M. A., van Bergen P. F., Sadowska A., New Phytol. 135, 375 (1997);

; M. M. Mulder, J. B. M. Pureveen, J. Boon, A. T. Martiniez, J. Anal. Appl. Pyrol. 19, 175 (1991);

Saiz-Jimenez C., de Leeuw J. W., Org. Geochem. 6, 417 (1984);

; P. F. van Bergen et al., Geochim. Cosmochim. Acta 58, 3823 (1994).

R. P. Evershed et al., Science 278, 432 (1997).

Mauron J., Prog. Food Nutr. Sci. 5, 5 (1981).

H. Weenen et al., Am. Chem. Soc. Symp. Ser.543, 142 (1994).

Bucala R., Model P., Cerami A., Proc. Natl. Acad. Sci. U.S.A. 81, 105 (1984).

Lee A., Cerami A., Mutat. Res. 179, 151 (1987);

Papoulis A., Al-Abed Y., Bucala R., Biochemistry 34, 648 (1995).

S. Pääbo, in PCR Protocols and Applications: A Laboratory Manual, M. A. Innis, D. H. Gelfand, J. J. Sninsky, T. J. White, Eds. (Academic Press, San Diego, CA, 1990), pp. 159–166.

S. Vasan et al., Nature 382, 275 (1996).

PTB (C₁₁H₁₀NOSBr) was synthesized as described (14). The melting point of the product was 224° to 227°C (expected, 223° to 223.5°C) and its elemental composition was 4.88% N, 46.46% C, and 3.86% H (expected, 4.93% N, 46.49% C, and 3.55% S). The coprolite was teased apart and about 5 g was ground under liquid nitrogen in a freezer/mill 6700 bone grinder (Spex Industries, Edison, NJ). To samples of 0.1 g of powder, 1 ml of buffer [as in

Pääbo S., Proc. Natl. Acad. Sci. U.S.A. 86, 1939 (1989);

], and 20 ml of proteinase K (10 mg/ml) was added and the sample was incubated under agitation for 48 hours at 37°C. Two samples received 10 and 100 μl of a 0.1 M PTB solution in 10 mM sodium phosphate buffer (pH 7.4) before (Fig. 2, lanes 3 and 4) and another two after (lanes 5 and 6) the organic extraction, whereas two received only the phosphate buffer (lanes 7 and 8). Samples were phenol extracted as described (20) and concentrated to about 200 ml with Centricon-30 microconcentrators (Amicon, Beverly, MA). All but one extraction (Fig. 2, lane 7) were purified [as in

Höss M., Pääbo S., Nucleic Acids Res. 21, 3913 (1993);

] except that only one L2 wash was performed and the silica pellet was washed twice with ice-cold New Wash (Bio 101, La Jolla, CA). Mock extractions with and without PTB (Fig. 2, lanes 9 and 10) were performed alongside all extractions. It may be noteworthy that eight extractions from Pleistocene bone samples and one 2300-year-old Egyptian animal mummy using PTB have to date failed to yield amplification products. It may be that Maillard products are particularly prevalent in coprolites.

PCR amplifications were performed [as in

Höss M., Pääbo S., Nucleic Acids Res. 21, 3913 (1993);

] using the following primers for the 12S rRNA gene and the chloroplast rbcL gene: 12ss, 5'-AATTTCGTGCCAGCCACCGCGGTCA-3'; 12st, 5'-AAGCTGTTGCTAGTAGTACTCTGGC-3'; 12sa, 5'-CTGGGATTAGATACCCCACTAT-3'; 12so, 5'-GTCGATTATAGGACAGGTTCCTCTA- 3'; 12sd, 5'-TAAAGGACTTGGCGGTGCTTCAC-3'; 12sn, 5'-CCATTTCATAGGCTACACCTTGACC-3'; 12shp, 5'-GCACAATTATTACTCATAAGC-3'; 12sb, 5'-TGACTGCAGAGGGTGACGGGCGGTGTGT-3'; rbcL Z1, 5'-ATGTCACCACAAACAGAGACTAAAGCAAGT-3'; rbcL19, 5'-AGATTCCGCAGCCACTGCAGCCCCTGCTTC-3'; rbcL h1aR, 5'-GAGGAGTTACTCGGAATGCTGCC-3'; rbcL h1aF, 5'-GGCAGCATTCCGAGTAACTCCTC-3'; rbcL h2aR, 5'-CGTCCTTTGTAACGATCAAG- 3'.

M. Höss, thesis, Ludwig-Maximilians University, Munich, Germany (1995).

O. Handt, M. Höss, M. Krings, S. Pääbo, Experientia 50, 524 (1994).

DNA sequences for 12S rRNA are available at.

Determination of DNA sequences from two or more amplifications is necessary to detect polymerase errors in the first cycles of the PCR because the amplification may start from a single or a few DNA molecules [M. Krings et al., Cell 90, 19 (1997)]. We consider reproduction of results in a second laboratory in order to exclude laboratory-specific contaminations to be necessary for human remains [O. Handt et al., Science 264, 1775 (1994); M. Krings et al., Cell90, 19 (1997)]. For animal remains, contaminations with human DNA are more easily detected and thus reproduction in different laboratories may be necessary only if extraordinary results are obtained. In this case, we believe that the sequence is authentic because it has been partially determined from five different boluses and found to be identical, and the phylogenetic analysis (Fig. 3) shows it to be related but not identical to a Mylodon sequence previously determined in our laboratory (6) and subsequently reproduced in another laboratory [

Taylor P., Mol. Biol. Evol. 13, 2839 (1996);

]. Furthermore, control extracts performed in parallel with the coprolite extractions yielded no amplification products and no work on these boluses had previously been performed in our laboratory. Finally, nuclear insertions are unlikely sources of the sequences because multiple primers yield the same sequence [M. Krings et al., Cell 90, (1997)].

Strimmer K., von Haeseler A., Mol. Biol. Evol. 13, 964 (1996).

Höss M., Jaruga P., Zastawny T. H., Dizdaroglu M., Pääbo S., Nucleic Acids Res. 24, 1304 (1996);

Handt O., Krings M., Ward R., Pääbo S., Am. J. Hum. Genet. 59, 368 (1996).

Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J., J. Mol. Biol. 215, 403 (1990).

Thorne R. T., Bot. Rev. 58, 225 (1992).

Pääbo S., Irwin D. M., Wilson A. C., J. Biol. Chem. 265, 4718 (1990).

H. N. Poinar et al., unpublished data.

B. A. Stankiewicz et al., Science276, 1541 (1997).

J. C. Hickman, Ed., The Jepson Manual, Higher Plants of California (Univ. of California Press, Berkeley, CA, 1993).

W. G. Spaulding, In Packrat Middens: The Last 40,000 Years of Biotic Change, J. L. Betancourt, T. R. Van Devender, P. S. Martin, Eds. (Univ. of Arizona Press, Tucson, AZ, 1990), chap. 9.

We thank J. Bada, V. Börner, D. Caccese, G. Eglinton, A. Greenwood, M. Höss, G. Poinar Jr., U. Schultheiss, W. Storch, L. Vigilant, and H. Zischler for help and discussions; the Natural History Museum of Los Angeles County, the Natural History Museum (London) (P. Andrews), and Northern Arizona University, Flagstaff (J. Mead), for coprolite samples; and the Deutsche Forschungsgemeinschaft (S.P.) for financial support. B.A.S., H.B., and R.P.E. thank J. F. Carter and A. Gledhill for technical assistance with the mass spectrometry and head space analysis, which were performed under Natural Environment Research Council grant GST/02/1027 to D. E. G. Briggs and R.P.E.