Coprolites as a source of information on the genome and diet of the cave hyena

Abstract

We performed high-throughput sequencing of DNA from fossilized faeces to evaluate this material as a source of information on the genome and diet of Pleistocene carnivores. We analysed coprolites derived from the extinct cave hyena (Crocuta crocuta spelaea), and sequenced 90 million DNA fragments from two specimens. The DNA reads enabled a reconstruction of the cave hyena mitochondrial genome with up to a 158-fold coverage. This genome, and those sequenced from extant spotted (Crocuta crocuta) and striped (Hyaena hyaena) hyena specimens, allows for the establishment of a robust phylogeny that supports a close relationship between the cave and the spotted hyena. We also demonstrate that high-throughput sequencing yields data for cave hyena multi-copy and single-copy nuclear genes, and that about 50 per cent of the coprolite DNA can be ascribed to this species. Analysing the data for additional species to indicate the cave hyena diet, we retrieved abundant sequences for the red deer (Cervus elaphus), and characterized its mitochondrial genome with up to a 3.8-fold coverage. In conclusion, we have demonstrated the presence of abundant ancient DNA in the coprolites surveyed. Shotgun sequencing of this material yielded a wealth of DNA sequences for a Pleistocene carnivore and allowed unbiased identification of diet.

Figure 1.

Selecting coprolites. (a) Coumère cave coprolite. (b) PCR amplification of a fragment of the cave hyena cytb gene. Amplification (33 PCR cycles) was carried out on 0.04 to 2.5% of each DNA extract. (c) Production of libraries of DNA fragments for high-throughput sequencing. Coprolite and mock extracts ligated to oligonucleotide adapters were amplified using 12 PCR cycles. The upper band (specific for coprolite samples) was recovered for Illumina sequencing.

Figure 2.

Cave hyena mitochondrial genome. (a) Number of reads for each position of the cave hyena mitochondrial genome for CC8 (top) and CC9 (bottom) coprolites. (b) Phylogenetic analysis of the cave hyena with complete mitochondrial genome sequences. Tree construction was performed by Bayesian phylogenetic inference using Caniformia sequences as an outgroup. The posterior probability and bootstrap values that support the nodes are indicated for Bayesian phylogenetic inference and maximum-likelihood analysis, respectively. Coloured characters indicate mitochondrial genomes provided by this study. The scale indicates the genetic distance.

Figure 3.

Cave hyena nuclear sequence data for CC8 coprolite. (a) Number of reads for each position of the 18S gene sequence. (b) Individual sequence coverage of 14 cave hyena nuclear genes. The dark column indicates the mean read depth ± s.e.m. for the 14 genes.

Figure 4.

Identifying Cervus elaphus DNA in coprolites. (a) Number of reads that display a perfect match to the indicated mitochondrial genomes. (b) Phylogenetic analysis of the Cervidae mitochondrial sequences retrieved from cave hyena coprolites. Tree construction was performed by Bayesian phylogenetic inference using Bovidae sequences for delineating an outgroup. The posterior probability and bootstrap values that support the nodes are indicated for Bayesian phylogenetic inference and maximum-likelihood analysis, respectively. Yellow bars, CC9 coprolite; orange bars, CC8 coprolite.