A genetic survey for cave hyena diversity and food consumption. Insight from DNA analysis of coprolites from cave sites in the south of France. BXL_BFC

To gain information on the diet and genetic diversity of the cave hyena, we performed DNA analyses on coprolites from six cave sites in southern France, including four caves from Ardeche (Chateaubourg, Grotte‑aux‑Ours cave ; Soyons, Neron and Trou‑du‑Renard caves ; Le Figuier) and two caves from Aude (Tournal, La Crouzade). Eight coprolites from these six sites were analysed using high‑throughput shotgun DNA sequencing, with 60 to 250 million DNA reads being obtained for each sample. Mining the dataset for mitochondrial DNA reads for 35 animal species, including human, we found that hyena DNA was the most abundant one in all samples, hence validating the opinion that they correspond to cave hyena coprolites. Mitochondrial DNA reads for other species strongly suggest the presence of several large animals in the cave hyena diet, including the horse and the bison (Neron), the extinct cave bear (Trou‑du‑Renard), the reindeer (Tournal) and the woolly rhinoceros (Le Figuier). The reasons why the diet could not be identified from the other coprolites are discussed. Phylogenetic analysis of the hyena mitochondrial genomes sequences reconstructed from DNA reads discloses the presence at Neron of a specimen of haplogroup B, which has not been recorded previously in France. All other coprolites display mitochondrial genomes belonging to haplogroup A. AMS radiocarbon dating analysis of three coprolites yielded ages dating back to the Aurignacian (Chateaubourg, 39,670‑38,000 cal. BP) or the Gravettian ages (Soyons : Néron, 30,890‑30,180 cal. BP ; Trou‑du‑Renard, 25,800‑25,290 cal. BP), indicating the possible replacement of mitochondrial haplogroup B by haplogroup A in Soyons. The mitochondrial haplogroup A was shown previously to include both extinct cave hyena and extant spotted hyena specimens. With a large number of new cave hyena mitochondrial genomes sequences for haplogroup A, our studies show that ancient and extant hyenas are more intermingled than previously suspected in this haplogroup. Analysis of nuclear genome data revealed phylogenetic relationships between cave and extant spotted hyenas different than those conveyed by mitochondrial genomes. Specifically, the nuclear genomes of extinct and extant spotted hyenas robustly delineate distinct monophyletic clades. In the cave hyena clade, the poor support to the grouping of the specimens according to their mitochondrial haplogroups likely reflects the effects of both incomplete lineage sorting and gene flow.