Introduction

Azokh 1 has yielded several coprolites from Units II, III and V, but two complete and undamaged coprolites were found on the top of Unit II (dated by ESR to around 100 ka, see Appendix, ESR). These two entire and unbroken coprolites (5153 and 5246) were chosen to investigate micro-plant remains after failed attempts to obtain pollen grains from these sediments (see Scott et al. 2016). One of these complete coprolites contained two small pieces of fossil bone. Bone inclusions indicate a carnivorous (or omnivorous) diet of the animal that produced the coprolites.

Taphonomic and genetic analyses of this coprolite were carried out in order to investigate the species that produced it. Azokh 1 Cave has yielded a low number of hyenid fossil bones in Units Vu and Vm (older than Unit II), which were identified as Crocuta crocuta by Van der Made et al. (2016). So far, hyena fossil remains are absent in Unit II. Other potential carnivores recorded in the site, more specifically in Unit II, are leopards (Panthera pardus), wolves (Canis lupus) and foxes (Vulpes vulpes). Scats of other carnivores such as, panthers, wolves, foxes or lions have a different size and shape (Walker 1993; Macdonald and Barrett 1993; Stuart and Stuart 1994; Harrison 2011). Lion bones (Panthera leo) are absent in the whole sequence of Azokh 1 (Van der Made et al. 2016). The most abundant species of Carnivora recorded in Unit II, with 88.7% of fossils taxonomically identified, is the cave bear, Ursus spelaeus, but there have been many studies indicating that it was herbivorous (Kurtén 1976; Bocherens et al. 1994; Mazza et al. 1995; Fernández 1998; Mattson 1998; Vila Taboada et al. 1999, 2001; Fernández et al. 2001; Grandal d’Anglade and López-González 2005); and some suggesting it was at least partly carnivorous (Richards et al. 2008; Figueirido et al. 2009; Peigné et al. 2009). Therefore, taxonomic identification of species recorded in Unit II does not indicate the most obvious predator that produced the coprolite.

Coprolites can be very informative when analyzed paleogenetically. Indeed, animal scats contain huge amounts of cell debris shed from the mucous membrane of the intestinal tract (Albaugh et al. 1992). The gut epithelium has a high rate of renewal, which matches an equally high rate of shedding. Conservation biologists take advantage of this fact by genetically analyzing scats from rare and endangered species in order to reveal the identity of the animal that produced them (e.g., Dalen et al. 2004; Miotto et al. 2007; Shezad et al. 2012). In the scats of predators, DNA from prey species is highly degraded due to the acidic gastric juice and intestinal nucleases. As a consequence, almost all of the recovered DNA sequences come from the predator and the identity of the prey species can only be determined when the host’s DNA is masked, for example through blocking primers (Shezad et al. 2012). When DNA in coprolites is analyzed, as reported for example

for ground sloth, human, and hyena coprolites (Poinar et al. 1998; Gilbert et al. 2008; Bon et al. 2012, respectively), a similar situation is found. Indeed, in coprolites of cave hyenas roughly ten times more DNA of cave hyena than of reindeer was found using high throughput sequencing (Bon et al. 2012).

With the aim of investigating the animal that produced the coprolite from Azokh Unit II, a paleogenetic and paleogenomic analysis was performed in the core facility of paleogenomics and molecular taphonomy of the Institut Jacques Monod in Paris by amplifying targeted diagnostic sequences using qPCR and shotgun sequencing.

Materials and Methods

Coprolite/Scat Morphometry

The two complete coprolites recovered from Unit II (lab. no. 5153, AZ1’08 II-I50#12 and lab. no. 5246, AZ1’08 I-H49#4, Fig. 12.1), as well as a large number of modern and fossil hyena scats were measured using calipers. These comparative samples include modern and fossil African hyenas of brown and spotted hyena (L. Scott collection), coprolites from Laetoli (Tanzania), produced by hyena and by other carnivores (Harrison 2011), coprolites from European sites (West Runton Norfolk, Larkin et al. 2000; La Roma, Pesquero et al. 2011) as well as spotted hyena scats from Colchester Zoo (UK, Larkin et al. 2000) making a total of 216 coprolites and modern scats.

Ursus spelaeus is an extinct species and modern representatives of bear are different species the scats of which cannot be directly compared for morphometric or paleogenetic analyses.

Scats and coprolites are measured taking the maximum diameter of the transversal section. The second diameter is taken at right angles to the maximum diameter, but it is not strictly the minimum diameter. We then named ‘minor axis’ to the perpendicular diameter of the maximum dimension, which is here named ‘major axis’ as the reciprocal word of minor. Orthogonally to these axes is the length of the scat or coprolite.

Bone Observations

During plant microfossil extraction, one small fossil bone fragment (6 mm long) was found in one of the coprolites (5153) after HCl (10%) treatment. Another small piece of fossil bone (5 mm long) was found in the residues that came from sawing and cleaning the coprolite 5153. In order to avoid interpretations on the bone that could have been altered by HCl during pollen preparation, only the observations of the surface conditions of the latter bone fragment are considered here to