MEC1106.fm Page 1975 Saturday, November 11, 2000 2:34 PM Molecular Ecology (2000) 9, 1975–1984 ABlackwell Science, Ltdmolecular analysis of ground sloth diet through the last glaciation M. HOFREITER,* H. N. POINAR,* W. G. SPAULDING,† K. BAUER,‡ P. S. MARTIN,§ G. POSSNERT¶ and S. PÄÄBO* *Max Planck Institute for Evolutionary Anthropology, Inselstr. 22, D-04103 Leipzig, Germany, †Dames & Moore, 7115 Amigo Street, Suite 110, Las Vegas, NV 89119, USA, ‡Zoological Institute, University of Munich, Luisenstr. 14, D-80333 Munich, Germany, §Desert Laboratory, Department of Geoscience, University of Arizona, Tucson, AZ, 85721, USA, ¶Ångström Laboratory, Division of Ion Physics, PO Box 534, S-75121 Uppsala, Sweden Abstract DNA was extracted from five coprolites, excavated in Gypsum Cave, Nevada and radio- carbon dated to approximately 11 000, 20 000 and 28 500 years bp. All coprolites contained mitochondrial DNA sequences identical to a DNA sequence determined from a bone of the extinct ground sloth Nothrotheriops shastensis. A 157-bp fragment of the chloroplast gene for the large subunit of the ribulosebisphosphate carboxylase (rbcL) was amplified from the boluses and several hundred clones were sequenced. In addition, the same DNA fragment was sequenced from 99 plant species that occur in the vicinity of Gypsum Cave today. When these were compared to the DNA sequences in GenBank, 69 were correctly (two incorrectly) assigned to taxonomic orders. The plant sequences from the five coprolites as well as from one previously studied coprolite were compared to rbcL sequences in GenBank and the contemporary plant species. Thirteen families or orders of plants that formed part of the diet of the Shasta ground sloth could be identified, showing that the ground sloth was feeding on trees as well as herbs and grasses. The plants in the boluses further indicate that the climate 11 000 years BP was dryer than 20 000 and 28 500 years BP. However, the sloths seem to have visited water sources more frequently at 11 000 BP than at earlier times. Keywords: ancient DNA, climate change, coprolites, diet, sloth, vegetation change Received 11 March 2000; revision received 10 July 2000; accepted 10 July 2000 sloth Nothrotheriops shastensis (Hansen 1978; Thompson Introduction et al. 1980). In addition to a large number of plants that At the end of the last glaciation, some 11 000 years ago, are rare in the coprolites, these studies have identified at least 33 genera and more than 50 species of large mormon tea (Ephedra nevadensis), globemallow (Sphaeralcea mammals disappeared from the North American con- ambigua) and saltbushes (Atriplex spp.) as major parts of tinent (Martin 1984). In addition to bones and rare soft the ground sloth diet. Recently, we showed that coprolites tissue remains, several of these animals left large deposits contain DNA that can be released upon the addition of a of dung behind, particularly in caves that they inhabited chemical compound, N-phenacyl thiazolium bromide or visited regularly. These remains are valuable because (PTB), during the extraction procedure (Poinar et al. 1998). they offer insights into the diet of these creatures, the This compound breaks crosslinks between reducing sugars behaviour of the animals, and the flora during the late and primary amines (Vasan et al. 1996), formed by the Pleistocene. Maillard reaction (Ledl & Schleicher 1992). This reaction Coprolites from the extinct megafauna of the Americas has affected a coprolite from Gypsum Cave (Poinar et al. have been studied for over 60 years (Laudermilk & Munz 1998) and is likely to have affected many ancient tissues 1934). In particular, several macro- and microscopic studies (Pääbo 1990). PTB treatment allowed DNA sequences have been performed on the dung of the extinct ground both from the animal that deposited the coprolite and from the plants it had ingested to be determined. This Correspondence: S. Pääbo. Fax: + 49(0)341 9952555; E-mail: may greatly improve the understanding of the ecology pää[email protected] and behaviour of extinct animals because many plants are © 2000 Blackwell Science Ltd MEC1106.fm Page 1976 Saturday, November 11, 2000 2:34 PM 1976 M. HOFREITER ET AL. hard or impossible to identify morphologically after DNA from the Nothrotheriops shastensis bone was mastication and passage through the gastrointestinal extracted as follows: 0.2 g of ground bone was incubated tract. Here we report the molecular analysis of six late at 37 °C for 24 h in 1.4 mL of extraction buffer (0.5 m Pleistocene coprolites from Gypsum Cave, Nevada (36°12′ N EDTA, 5% N-lauryl sarcosine, 1% polyvinylpyrolidine, latitude, 114°39′ W longitude, 580 m a.s.l). 50 mm DTT and 140 µg/mL proteinase-K). After addi- tion of 150 µL of 100 mm PTB and 150 µL of 8% CTAB the incubation was continued for another 72 h. The Materials and methods sample was centrifuged briefly, the supernatant extracted twice with chloroform:isoamylalcohol (24:1) and DNA Dating recovered by binding to silica as described (Höss & The five coprolites were radiocarbon dated in the Pääbo 1993; Poinar et al. 1998). Finally, the silica pellet Ångström laboratory, Uppsala, Sweden. Ages and laboratory was dried for 5 min at 56 °C and eluted with 130 µL TE numbers were: #1, 11 005 ± 100 years bp (Ua-12506); for 8 min at 56 °C. PCR was performed as described #2, 11 080 ± 90 years bp (Ua-12509); #3, 19 500 ± 205 years bp (Höss & Pääbo 1993) using a hot-start protocol. (Ua-13223); #5, 27 810 ± 455 years bp (Ua-12508); and Primers used were: 16S6 5′-TTTCGGTTGGGGCGACCT- #6, 29 205 years bp (Ua-13224). The date for #4 is in Poinar CGGAG-3′; 16S7 5′-TTGCGCTGTTATCCCTAGGGTAAC- et al. 1998. 3′; 16SNS1 5′-CCTCCGAACGACTATGCGCCCA-3′; rbcL Z1aF 5′-ATGTCACCACCAACAGAGACTAAAGC-3′; rbcL 19bR 5′-CTTCTTCAGGTGGAACTCCAG-3′; and hp2R DNA extraction, PCR and sequencing 5′-CGTCCTTTGTAACGATCAAG-3′. DNA was extracted (Rogers & Bendich 1985) from 112 PCR products for the 16S6–16S7 (Höss et al. 1996a) frag- herbarium specimens of plants from the Gypsum ments were reamplified with the primer pair 16SNS1 and Cave area (for a list of the plant species see http:// 16S7 and sequenced directly, using the primer 16S7. The rbcL www.eva.mpg.de/analysis/index.html). Briefly, 100 mg fragments from the coprolites were amplified and ream- of plant tissue were placed in an Eppendorf tube together plified using the primer pair Z1aF (3′ end at position 54 983 with 500 µL extraction buffer (1% cetyltrimethyl ammo- in the Arabidopsis thaliana chloroplast genome, GenBank nium bromide [CTAB], 10 mm EDTA, 50 mm Tris-HCl NC 000932.1) and 19bR (3′ end at pos. 55 094). The frag- (pH 8.0), 0.7 m NaCl, 50 mm 2-mercaptoethanol), shaken ments were made blunt-ended by treatment with T4-DNA for 2 h at 75 °C and chloroform extracted twice. The polymerase and cloned in pUC18 (Amersham Pharmacia aqueous phase was transferred to a new tube, 500 µL of Biotech, Germany). For each specimen, three independent precipitation buffer (1% CTAB, 10 mm EDTA, 50 mm amplifications of the rbcL fragment were performed, and Tris-HCl) was added, and the DNA was precipitated the sequences of 73 or more clones were determined. by centrifugation at 5700 g at room temperature. DNA PCR products from herbarium specimen were sequenced pellets were resuspended in 100 µL TEN buffer (10 mm directly, using the primer hp2R (3′ end at position 55 186), Tris-HCl, 1 mm EDTA, 1 m NaCl) and precipitated by the ‘Thermo Sequenase Kit’ (Amersham Pharmacia addition of 200 µL of 100% ethanol. The DNA was Biotech, Germany) and an ALF Sequencer (Amersham resuspended in 100 µL TE buffer (1 mm Tris-HCl, pH 8.0, Pharmacia Biotech, Germany). 0.1 mm EDTA) and 2 µL were used for 30 cycles of poly- merase chain reaction (PCR) with primers rbcL Z1aF and DNA sequence analysis hp2R, using an annealing temperature of 55 °C, AmpliTaq (Perkin Elmer, USA) and the reagents recommended by Sequences of the rbcL fragment were aligned and grouped the supplier. Ninety-nine samples yielded amplifiable DNA. into clusters by eye. All unique sequences were compared DNA from coprolites was extracted as described with the approximately 3600 rbcL sequences in GenBank (Poinar et al. 1998) with minor modifications. Coprolite by means of the blast program (Altschul et al. 1997) and pieces were ground to a fine powder under liquid nitrogen family(ies) and order(s) of the closest matches were noted. with a pestle and mortar. To 0.2 g of powder, 1.4 mL of Consensus sequences were also compared to the sequences extraction buffer (0.1 m Tris-HCl (pH 8.0), 2 mm EDTA, determined from herbarium specimens. All herbarium 0.7 m NaCl, 1% SDS, 50 mm DTT, 0.2 mg/mL proteinase- sequences were compared to sequences in GenBank K) were added. Samples were incubated for 24 h at 37 °C by means of the blast program, and the number of on a rotary shaker and 100 µL of a 100-mm PTB solution mismatches to the most similar sequence in the database were added. The incubation was continued for another were noted. In two cases, the length of the GenBank 72 h. The samples were extracted twice with chloroform sequences showing the highest similarity to the fragment and DNA was recovered by binding to silica (Höss & determined was only 108 bp, and in 11 cases it was 106 bp. Pääbo 1993; Poinar et al. 1998). In these cases, comparisons were limited to these lengths. © 2000 Blackwell Science Ltd, Molecular Ecology, 9, 1975–1984 MEC1106.fm Page 1977 Saturday, November 11, 2000 2:34 PM MOLECULAR COPROSCOPY THROUGH TIME 1977 Fig.