A molecular analysis of ground sloth diet through the last glaciation

Authors: M. Hofreiter, H.N. Poinar, W.G. Spaulding, K. Bauer, P.S. Martin, G. Possnert and S. Pääbo
Journal: Molecular Ecology (2000) 9, 1975–1984

DNA was extracted from five coprolites, excavated in Gypsum Cave, Nevada and radiocarbon 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.

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Protein preservation and DNA retrieval from ancient tissues

Authors: H.N. Poinar and B.A.Stankiewicz
Journal: Proc. Natl. Acad. Sci. USA – Vol. 96, pp. 8426–8431 (July 1999)

The retrieval of DNA from fossils remains controversial. To substantiate claims of DNA recovery, one needs additional information on the preservation of ther
molecules within the same sample. Flash pyrolysis with GC and MS was used to assess the quality of protein preservation in 11 archaeological and paleontological remains, some of which have yielded ancient DNA sequences authenticated via a number of criteria and some of which have consistently failed to yield any meaningful DNA. Several samples, including the Neanderthal-type specimen from which DNA sequences were recently reported, yielded abundant pyrolysis products assigned to 2,5-diketopiperazines of prolinecontaining dipeptides. The relative amounts of these products provide a good index of the amount of peptide hydrolysis and DNA preservation. Of these samples, four stem from arctic or subarctic regions, emphasizing the importance of cooler temperatures for the preservation of macromolecules. Flash pyrolysis with GC and MS offers a rapid and effective method for assessing fossils for the possibility of DNA preservation.

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

Authors: H.N. Poinar, M. Hofreiter, W.G. Spaulding, P.S. Martin, B.A. Stankiewicz, H. Bland, R.P. Evershed, Go. Possnert, S. Pääbo
Journal: Science – Vol 281 (July 1998)

DNA from excrements can be ampliÆed by means of the polymerase chain reaction. However, this has not been possible with ancient feces. Cross-links between reducing sugars and amino groups were shown to exist in a Pleistocene coprolite from Gypsum Cave, Nevada. A chemical agent, N-enacylthiazolium bromide, that cleaves such cross-links made it possible to amplify DNA sequences. Analyses of these DNA sequences showed that the coprolite is derived from an extinct sloth, presumably the Shasta ground sloth Nothrotheriops shastensis. Plant DNA sequences from seven groups of plants were identified in the coprolite. The plant assemblage that formed part of the sloth’s diet exists today at elevations about 800 meters higher than the cave.

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Bacillus DNA in Fossil Bees: an Ancient Symbiosis?

Authors: R.J. Cano, M.K. Borucki, M. Hibgy-Schweitzer, H.N. Poinar, G.0. Poinar, and K.J. Pollard
Journal: Applied and environmental Microbiology -Vol 60 – No.6 (June 1994)

We report here the isolation of DNA from abdominal tissue of four extinct stingless bees (Proplebeia dominicana) in Dominican amber, PCR amplification of a 546-bp fragment of the 16S rRNA gene from Bacillus spp., and their corresponding nucleotide sequences. These sequences were used in basic local alignment search tool searches of nonredundant nucleic acid data bases, and the highest scores were obtained with 16S rRNA sequences from Bacillus spp. Phylogenetic inference analysis by the maximum-likelihood method revealed close phylogenetic relationships of the four presumed ancient Bacillus sequences with Bacillus pumilus, B. firmus, B. subtilis, and B. circulans. These four extant Bacillus spp. are commonly isolated from abdominal tissue of stingless bees. The close phylogenetic association of the extracted DNA sequences with these bee colonizers suggests that a similar bee-Bacillus association existed in the extinct species P. dominicana.

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