Abstract
Coprolites are fossil scats and provide indirect witness of the activity of past animals of a given area, whether or not fossil bones of these animals are present in the site. The shape, size, inclusions and geo- and bio-chemical composition are criteria for identification of the animal that left the coprolite. Unit II from Azokh 1 has yielded two complete undamaged coprolites one of which contained partially digested fossil bones. Taphonomic and taxonomic indications from this coprolite could not conclusively identify the origin of the coprolites. Analysis of targeted mitochondrial DNA, performed on one of the coprolites, has provided evidence for the presence of hyena DNA, but this finding was not supported by further investigation using next-generation high throughput sequencing. The most parsimonious interpretation of the results of the genetic analyses is that the highly sensitive PCR assay reveals contamination of the coprolite with minute amounts of modern brown hyena DNA presumably originating from brown hyena scats sampled recently in South Africa.
Резюме
Следы активности травоядных и плотоядных животных главным образом распознаются по отпечаткам конечностей и экскрементам. Стоянки с хорошей сохранностью древних останков могут содержать копролиты (окаменелые экскременты животных) и следы троп травоядных и плотоядных, как это наблюдается в плио-плейстоценовой стоянке Летоли (Танзания). Наиболее часто, однако, встречаются копролиты плотоядных (среди них, главным образом, гиен), чем травоядных. Первые грызут и поедают кости, включая тем самым фосфат кальция в органические остатки фекалий, в то время как последние поедают растительные волокна и семена, которые разлагаются намного легче. Форма, размер, включения, гео- и биохимический состав являются основными критериями для идентификации животного, оставившего эти фекалии. В подразделении II из Азох 1 найдены два неповрежденных копролита. Тафономические и таксономические признаки не были достаточно убедительными для надежного установления их происхождения. При проведении сайт-специфичной реакции полимеразной цепи (РПЦ) в одном из копролитов обнаружены последовательности митохондриальной ДНК бурой гиены (Hyaena brunnea). Последующее секвенирование не выявило значительного присутствия эндогенной ДНК хищника; в основном были найдены бактериальные последовательности со следами человеческой ДНК – возможно, по причине контаминации. Наибоее простым объяснением результатов генетического анализа является то, что чувствительный метод РПЦ идентифицирует контаминацию копролитов ничтожно малым количеством ДНК бурой гиены, привнесенным, возможно, из современных экскрементов данного вида, собранных в Южной Африке. Высокопроизводительное секвенирование не обнаружило эндогенной ДНК хищника. В целом, несохранность эндогенной ДНК характерна для всех биологических останков в Азохской пещере, проанализированных до настоящего времени, поскольку мы не смогли найти данный субстрат и в многочисленных костях пещерного медведя.
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Keywords
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 look for evidence of gastric digestion. This bone fragment was analyzed, along with 15 bones from modern hyena scats as controls, by means of a FEI Inspect Low Vacuum scanning electron microscope (SEM), which is housed at the Museo Nacional de Ciencias Naturales. Observations were done in backscattered electron mode, combined with secondary electron emission mode, at 30 kV, 0.6–0.33 Torr. This type of SEM enabled us to analyze specimens directly with no coating or any other pre-treatment.
Chemical Analysis of the Coprolites
Both coprolites from Azokh 1 Unit II were chemically analyzed at the MNCN laboratories. Sample labeled 5153A corresponds to the outer layer of the coprolite with sediment attached to the surface (residue from pollen cleaning) and 5153B contains exclusively the inner part of the coprolite (also paleogenetically analyzed here). Coprolite 5246 was taken as a whole and the analyzed sample is a mixture of the outer and inner layers of the coprolite. A modern hyena scat (2160) from Burungi (Tanzania) was analyzed as control. All samples were ground to a fine powder using an agate pestle and mortar to be chemically analyzed. These samples were analyzed for X-Ray Diffraction (XRD, Philips PW-1830) and X-Ray Fluorescence measurements (XRF, Philips PW-1404) to obtain their mineral and element compositions respectively.
Paleogenetics and Paleogenomics
The intact half of the coprolite 5153 that was not used for the plant microfossil studies, has been subject to a paleogenetic and paleogenomic analysis. The pre-PCR experiments were carried out in the high containment laboratory of the Institut Jacques Monod (http://www.ijm.fr/ijm/plates-formes/pole-paleogenomique/), the post-PCR experiments in a series of separated laboratories of the Institut Jacques Monod designed to minimize carry-over contamination as described previously (Bennett et al. 2014). The surface of the coprolite was removed to reduce contamination with exogenous, environmental DNA and the quantity of inhibitors that can be enriched on the surface. Removal of the outer layer was performed with a sterile scalpel and 583 mg recovered from the inner part of the coprolite with a slowly moving drill. The powder was extracted in 10 ml extraction buffer (0.5 M EDTA pH 8.0, 0.25 M potassium dihydrogenphosphate, 0.14 M beta-mercaptoethanol) and purified over silica columns (Qiagen) as described (Charruau et al. 2010). The total DNA quantity (measured on a Qubit® 2,0 Fluorometer and comprising environmental and endogenous DNA) was 0.93 ng/µl. The purified DNA was amplified via quantitative real-time PCR (Pruvost and Geigl 2004).
Several procedures to prevent contamination were implemented in the protocol, such as elimination of contamination due to carry-over (Pruvost et al. 2005) and to reagents (Champlot et al. 2010). The extract strongly inhibited the polymerase in the polymerase chain reaction (PCR) with a 5 cycle delay at 10% reaction volume, 2.8 cycle delay at 5% reaction volume, and 0.1 cycle delay at 2.5%. Two PCR primer pairs targeting a 111 bp and a 84 bp (Bon et al. 2012) fragment of the mitochondrial cytochrome B region of the hyaenidae were used. When the primer sequences are removed from the PCR product sequences, the 84 bp and the 111 bp leave 43 bp and 64 bp, respectively, of informative sequence. Moreover, two primer pairs were designed that targeted 103 and 106 bp regions of the hypervariable region of Ursus spelaeus. In addition, a third primer pair was designed to amplify an 88 bp region of the NADH dehydrogenase 2 gene (ND2) with equal efficiency between both Ursus and Hyaenidae, the 36 bp internal sequence of which would differentiate between the two.
When using the hyena-specific primers, PCR products of 84 bp and 111 bp were obtained from the extracts at 2.5%, 5%, and 10% of reaction volume (20 µl total volume reactions), despite the inhibition with the larger extract volumes. A single 88 bp product was amplified using the universal bear-hyena primers. No product was obtained when using the bear-specific primer pairs. The PCR products were directly sequenced after purification.
In order to compare the obtained sequences with those of the three extant hyena species, DNA from the hair of two male brown hyenas from the Zoo “Fauverie du Mont Faron”, France, was extracted as described previously (Charruau et al. 2010) and analyzed using the same primers and PCR conditions as described above, but in a laboratory of the “Institut Jacques Monod” where modern DNA is analyzed. For next-generation high throughput sequencing, a library was prepared in the high-containment laboratory using the double-stranded DNA procedure as described in Bennett et al. (2014). The size-selected library was then amplified and sequenced on an Illumina MiSeq with paired-end 100 bp-long reads, using the manufacturer’s workflow.
Results
Bone and Coprolite Morphometry
The two complete and undamaged coprolites (5153 and 5246) measure 50 × 49 × 33 mm and 48 × 47 × 30 mm (major axis by minor axis by length) respectively. Coprolite sizes were compared to modern and fossil scats of spotted hyena (Crocuta crocuta) and brown hyena (Hyaena brunnea) by Fernández-Jalvo et al. (2010a). We added the raw values of modern spotted hyena scats from Colchester Zoo and hyena coprolites from European sites (Larkin et al. 2000; Pesquero et al. 2011), as well as more measurements from hyena and other carnivore coprolites from Laetoli (Tanzania) measured by Harrison (2011) see Fig. 12.1a.
The fossil bone fragment found in the residues from sawing and cleaning the coprolite 5153 is 5 mm long and, therefore, taxonomically unidentifiable. The small piece of fossil bone shows signs of moderate digestion (Andrews 1990), with slightly rounded edges (Fig. 12.2a) and a smooth surface (Fig. 12.2b), and in addition there is heavy diagenetic cracking on the bone surface (Fig. 12.2c). In contrast, bones contained in modern hyena scats (both spotted and brown hyenas) have higher degrees of rounding (Fig. 12.2d), enlarged bone porosity (Fig. 12.2e) and a characteristic “torn-like” damaged surface (Fig. 12.2f).
Chemical Analyses of Coprolites and Modern Scat
The XRD diagrams provide information of the mineral content in the sample and crystalline traits. Broadness of the peaks indicates low crystalline structure of the mineral content (Kolska Horwitz and Goldberg 1989; LeGeros 1994; Mulla et al. 2012). Diagrams of XRD shown in Fig. 12.3 have broad and irregular curves for the coprolites and modern scat and indicate that none of these samples are highly crystalline.
Amorphous phases obtained by XRD (Table 12.1) mainly refer to poor-crystallized minerals, but it may also be influenced by organic matter or volatile content. Azokh samples have higher amorphous content, especially sample 5153B (17.1% inner coprolite), than the modern scat (Table 12.1). The Loss on Ignition (LOI, Table 12.2) is the weight loss before and after heating. LOI values are also closely correlated to the organic matter and volatiles content in the sample (Heiri et al. 2001). Chemical analysis by XRD of the recent scat has yielded only hydroxyapatite from bones ingested by modern hyenas; amorphous phases are absent and the LOI is relatively high. Azokh coprolites have a low LOI compared to modern scats and moderately high amounts of amorphous phase (especially sample 5153B with 17.1% inner coprolite). Samples 5153A and 5246 have also calcium phosphate content as well as other minerals (feldspars and micas) from the surrounding sediment attached to the coprolites, but sample 5153B (cleaned inner layer of the coprolite) mainly contains hydroxyapatite (72.9%) and quartz (10.1%). However, influence of neo-formed minerals from the diagenetic processes cannot be fully discarded (see discussion).
Fluorescence results are displayed in Table 12.2. The XRF analysis has yielded similar results in all samples (fossil and modern scats). Some differences are observed with regard to silica and some elements, such as aluminum or potassium (components of feldspars) that are more abundant in Azokh coprolites. Trace elements, such as strontium, are more abundant in modern scat. The low value of Loss on Ignition (LOI) from Azokh coprolites (below 16.2%) indicates low proportions of organic materials and/or volatiles.
Paleogenetic Analysis of the Coprolite
In five out of eighteen attempts, PCR amplification of the coprolite extract yielded products of the 84 bp long mitochondrial DNA fragments of the cytochrome B. The longer, 111 bp fragment, however, yielded only non-specific amplifications of modern human contaminants. Depending on primer specificity, amplification of human sequences is an expected result, due to the low copy number and size degradation characteristic of targeted ancient DNA, as well as the ubiquity of modern human DNA in reagents and samples, particularly those that have not been aseptically excavated. One of nine attempts to amplify the 88 bp universal bear/hyena sequence of the ND2 gene was successful.
The sequences obtained for the 84 bp hyena-specific fragment were unambiguous and identical. Their comparison with the mitochondrial cytochrome B sequences in GenBank (NCBI Blast search) showed that the closest match was the cytochrome B sequence of the hyena (Rohland et al. 2005), rather than that of the cave bear (Krause et al. 2008). As a precaution, the sequences obtained were also compared with bovine (Bos taurus) and human sequences, the DNA of which can often contaminate ancient DNA analysis through reagents and handling. These sequences showed no similarity to either of these potential contaminants. The sequence from the ND2 gene fragment amplified with bear/hyena universal primers was also determined to be hyena sequence. Since the informative sequences of the coprolite obtained with the 84 bp CytB fragment most closely matched the brown hyena (Hyaena brunnea) and no ND2 sequences for brown hyena were available in Genbank, we subsequently determined the sequence of the analyzed ND2 gene fragment from modern brown hyena using hair samples from two brown hyenas from a zoo (Fauverie du Mont Faron, France). We also amplified and sequenced the two CytB fragments from these individuals, and their sequences were identical to the brown hyena sequences deposited in Genbank. Alignments of both the CytB and ND2 fragments were concatenated, compared to those of extant bears, the extinct cave bear Ursus spelaeus, the cheetah (Acinonyx jubatus), the tiger (Panthera tigris), and to the different extant hyena species and the extinct cave hyena (Fig. 12.4a). A maximal likelihood phylogenetic tree was constructed using PHYML (Guindon & Gascuel 2003) (Fig. 12.4b).
It can be seen from both the alignment and the tree that the DNA sequences recovered from the coprolite using the highly sensitive targeted PCR approach clearly belong to Hyaena brunnea and can be unambiguously distinguished from the other hyena species as well as from other Feliformia. The Ursidae sequences are even more distantly related.
To explore the possibility that the coprolite could contain DNA sequences from other organisms that were not targeted with the directed PCR approach used, we constructed a library from the total DNA extracted from the coprolite and sequenced a subset of this library using the Illumina Miseq platform. High throughput sequencing is an ideal approach to analyze the DNA composition of environmental samples (Shokralla et al. 2012), including feces (Murray et al. 2011). Shotgun next-generation sequencing was performed allowing the DNA molecule present in the extract to be randomly sequenced. This approach has two advantages: first, it provides an unbiased view of the DNA sequence composition of a fossil bone extract; second, it provides sequence information of very short DNA sequences that are too short to be analyzed with the targeted PCR approach. The disadvantage of this approach is that it does not discriminate between environmental and endogenous DNA and it can generate only minute amounts of sequencing data from endogenous DNA because of the pervasive nature of environmental DNA contamination. We analyzed the sequences of 619,848 fragments from a subset of the library of the Azokh coprolite (Fig. 12.5a). Of these, only 81,063 (13.7%) sequencing reads could be uniquely mapped to sequences present in databases (Fig. 12.5b). The vast majority of these uniquely mapped sequences, i.e. 95.6%, are of bacterial, archaeal or viral origin. The sequencing reads were also mapped to the human, cat, dog, and cow genomes, as well as the cave bear and striped hyena mitochondrial genomes. None of these attempted mappings revealed any appreciable presence of mammalian DNA, apart from the low level human sequences (0.4%), which are expected background contaminants with standard excavation techniques and non-decontaminated reagents. This result argues in favor of poor DNA preservation since the extract contains no detectable endogenous DNA from any of the likely scat producers, and indeed, no vertebrate DNA at all was detected (at least not in the 619,848 reads analyzed) beyond trace sequences that cannot be excluded from common biological reagent and handling contaminants. It is concluded that if any endogenous sequences are still preserved in this sample, they are too rare to be detected using a global approach without prior enrichment. In contrast to the targeted PCR approach, which is highly sensitive to longer, targeted sequencing reads, no sequencing reads indicating the presence of Hyaena brunnea were obtained via next-generation sequencing. It is noteworthy that the human mitochondrial DNA sequence obtained with the PCR approach does not match any individual working in the paleogenomics laboratory in Paris indicating that human contamination occurred most likely upstream to this analysis, or was introduced by reagents.
Discussion
Bone and Coprolite Morphometry
Digestion observed in the fossil bone fragment from coprolite 5153 is no higher than moderate in Andrews’ (1990) classification. Although this author did not include hyenas, bears or any other omnivorous predators in his study, experimental work on these predators (Denys et al. 1995; Matthews 2000, 2006; Mondini 2002; Montalvo et al. 2007) indicates that they produce highly digested bones, showing a “torn-like” damaged surfaces when exposed to strong gastric acids (Andrews and Fernández-Jalvo 1998). Effects of digestion on bones ingested and regurgitated by Crocuta crocuta show rounding of the broken edges of digested bone fragments and the characteristic “torn-like” damaged surface (Fernández-Jalvo et al. 2010b). Similar damage is observed on bones digested and excreted by Hyaena brunnea in Fig. 12.2d–f, but it is absent in the smooth surface bone from 5153 (Fig. 12.2b). Since we did not have bone specimens ingested by striped hyena (Hyaena hyaena), we could not analyze the effects of its digestion under high magnification electron microscopy (3,000×). Lower grades of digestion in the only bone that could be studied from the coprolite of Azokh1 Unit II could be pointing to an animal with weaker gastric action than hyenas, but differences in the degree of bone digestion may occur simultaneously during digestion depending on the position that the bone had in the stomach (Andrews 1990). The absence of a “torn-like” damaged surface is, however, unexpected, but further studies are needed using bones digested by Hyaena hyaena at high magnification electronic microscopy.
Kolska Horwitz (1990) observed signs of digestion on large mammal bones from recent striped hyena scats as well as coprolites from Kebara and Fazel 6 Levant sites. This author provided a size range of bone chips and splinters from 17 to 3 mm (62% smaller than 2 mm long). The small bone fragments found in the Azokh coprolite (6 and 5 mm long) fall within this range. However, higher abundance of bone splinters and larger pieces than the bones in the Azokh coprolites have been found in hyena scats/coprolites (Kolska Horwitz and Goldberg 1989; Kolska Horwitz 1990). Similarly, other carnivore feces referred to by Binford (1980), Haynes (1980), Maguire et al. (1980), Payne and Munson (1985) contain small sized bone splinters showing heavier signs of digestion. This is not the case with the small piece of fossil bone from 5153, in which digestion is moderate.
Kolska Horwitz and Goldberg (1989) indicate the breadth measurement (maximum diameter) of the scat may distinguish between spotted and brown hyena, with that of the spotted hyena being significantly wider. Crocuta crocuta is the largest extant hyaenid with a weight ranging between 45 and 85 kg. Hyaena brunnea weighs on average around 45 kg with exceptional cases reaching up to 72.6 kg (Roberts 1954), and Hyaena hyaena weighs between 30 and 35 kg. The major and minor diameters are the most consistent measurements, because length depends on the number of segments attached (see Fig. 12.1b). Most authors, however, refer to the length and maximum diameter (major axis) and we have used these dimensions to include most coprolite and modern scat measurements available. The results of these measurements are plotted in Fig. 12.1a. Unfortunately, other papers display results of coprolite measurements as average and range values of length and width which cannot be included here.
The larger Azokh coprolites (Fernández-Jalvo et al. 2010a) are still smaller than three of the coprolites from the West Runton Freshwater Bed site in Norfolk (WRFB-UK) measured by Larkin et al. (2000). Parfitt and Larkin (2010) mention an Early Pleistocene site (Untermassfeld, Germany, Keiler 2001) where the dimensions of the coprolites are even larger than those from Norfolk. The exceptionally large coprolites from Untermassfeld, which exceed the dimensions of modern and fossil hyena scats are considered by Keiler (2001) to have been produced by adults, whilst the smaller and more abundant coprolites derive from hyena pups. However, this would suggest that all coprolites found in other sites (see Table 12.1) are produced by pups and this would need further study. In addition, the size of modern brown hyena scats is also subject to individual diversity (E.M.G. and personnel from the “Fauverie du Mont Faron”, personal observation). On the other hand, the shape of the large coprolites from Norfolk (see Fig. 2b in Larkin et al. 2000) and modern bear (Ursus arctos) scats are alike, both being segmented (Fig. 12.6), and, there is fossil evidence of the presence of Ursus sp. at West Runton (see bear taxonomic discussion in Lewis et al. 2010). Therefore, coprolite morphometry is indicative, but is not conclusive.
In this respect, there is the contention that Ursus spelaeus cannot produce the coprolites because hibernation and fasting would limit bear scats in caves (Nelson et al. 1973; Fernández et al. 2001; Grandal d’Anglade and Fernández-Mosquera 2008). However, we find evidence in the site that bears were not only hibernating in Azokh 1, but living for longer periods in the cave (Marin-Monfort et al. 2016, see also discussion on Ursus spelaeus diet in that chapter). The fossil bones recovered from Unit II show no characteristic crushed bone hyena traits, nor do they have extensive gnawing or intensive bone digestion (Marin-Monfort et al. 2016). Bone splinters and breakage linked to chewing is low in Azokh 1. Chewing affected only 6.4% of the total number of fossils of Azokh1 and 7.24% in Unit II. These values seem too low for hyena (Skinner et al. 1998; Pickering 2002; Pokines and Peterhans 2007; Pobiner 2008; Diedrich 2012). The maximum size of tooth marks recorded on bones from Unit II is much larger than those of (known) hyenas, or even lions (Selvaggio and Wilder 2001; Domínguez-Rodrigo and Piqueras 2003; Pobiner 2008; Delaney-Rivera et al. 2009, see discussion in Marin-Monfort et al. 2016).
Chemical Analyses of the Coprolites
Results from XRD and XRF chemical analyses of coprolites obtained by other authors (Kolska Horwitz and Goldberg 1989; Larkin et al. 2000; Lewis 2011; Pesquero et al. 2011) are similar to those obtained here from Azokh coprolites (Tables 12.1 and 12.2, and Fig. 12.3). Percentages of the amorphous phase, however, are higher in Azokh coprolites than in other sites (see Pesquero et al. 2011). Most coprolites and modern hyena scats analyzed by Kolska Horwitz and Goldberg (1989) contained apatite minerals (except two, likely to have been produced by striped hyena). Poor crystallization and better crystallized apatites were indistinctly obtained from both modern and fossil specimens. Coprolites analyzed from West Runton Norfolk and Boxgrove sites give peaks of calcium phosphate in similar proportions to recent Crocuta crocuta scats from Colchester Zoo, all of which were chemically analyzed in Larkin et al. (2000) and Lewis (2011). Other elements (Fe, Mn, Al, Si) detected in these two British fossil sites are probably due to diagenetic alteration of the host sediment. Similarly, La Roma site (Pesquero et al. 2011) shows hydroxyapatite below 50%, with other minerals (calcite, quartz, gypsum and moscovite) formed in the calcareous marginal lake environment of this site. The sample from the interior of the coprolite (5153B-inner) contains hydroxyapatite (72.9%), and quartz (10.1%) and other minerals obtained from the other portions or coprolites from Azokh show the influence of the host sediment. Lewis (2011) observed a shift between the hydroxyapatite and fluorapatite phases in the range between 2q 44–54 region of the XRD coprolite diagrams. This shift was interpreted as result of diagenetic alterations frequently occurring in hydroxyapatite minerals. Some of the double peaks observed in this 2q region in Fig. 12.3 may suggest some influence of similar diagenetic alteration, though standard hydroxyapatite positions are better conformed in Azokh coprolites than in coprolites analyzed by Lewis (2011). Therefore, the diagenetic historical context has to be considered when analyzing the chemical composition of fossil materials.
Hydroxyapatite found in Azokh coprolites could in principle come from bones ingested by the animal that produced these coprolites. However, we cannot assert that hydroxyapatite identified in coprolites is exclusively the result of digested bones because Azokh Unit II has been intensively affected by diagenesis due to fluid percolation enriched in acidic bat guano. A wide variety of secondary minerals are associated to bat guano diagenesis in caves, and hydroxyapatite is the most common and stable neo-formed mineral, which has actually been formed in the geological materials of Unit II, such as stones and sediments (Tables 12.3 and 12.4). Hydroxyapatite together with quartz and tinsleyite have been identified as neo-formed (secondary) minerals in Azokh 1 (see Marin-Monfort et al. 2016, Table 10.10 and discussion in that chapter, and Murray et al. 2016). The peaks of samples analyzed in Fig. 12.7 are less broad than in coprolites, but still form short (except for the sediment) and irregularly shaped curves. As said above, the higher and thinner the peak of a mineral is, the better crystallized it is. Thus, the short, broad and irregular peaks suggest poor crystallinity in stones, which is abnormal and indicates the presence of abundant secondary minerals due to diagenesis. The percentages of amorphous phases in these highly diagenetically altered samples, are higher than in coprolites (up to 30%) and the LOI is variable with high values in fossil and sediment and low values in decayed stones.
In this context, the relative high abundance of amorphous phase in Azokh coprolites (above 7% up to 17.1%, Table 12.1), absent in modern scats, suggest the presence of poor-crystallized minerals rather than organic matter content, which may agree better with secondary neo-formed minerals during diagenesis. This is also in agreement with the bone diagenesis results obtained by Smith et al. (2016) who concluded that fossil materials from Azokh 1 (Units II and III) show typical ACH (Accelerated Collagen Hydrolysis), with only small amounts of collagen remaining and often extreme mineralogical changes. Similarly, no preserved DNA could be PCR-amplified from any of the numerous bones analyzed from various locations and layers in Azokh (Bessa-Correia and Geigl, unpublished results).
Paleogenetic and Paleogenomic Analyses
The paleogenetic analysis of the coprolite using a targeted, highly sensitive quantitative PCR approach revealed the presence of hyena DNA. The mitochondrial cytochrome B/ND2 gene sequences obtained matched those that were produced from modern brown hyena hair (Hyaena brunnea, formerly Parahyaena brunnea) rather than the extant spotted hyena (Crocuta crocuta) or the extinct cave hyena (Crocuta crocuta spelaea). Brown hyenas have never been recorded outside of Southern Africa, and it appears surprising and highly unlikely that the range of this species could have extended 100,000 years ago as far as to the Caucasus without any prior evidence of the past presence of this species on a wide geographical area (Rohland et al. 2005). Before proposing a profound reappraisal of the past distribution of brown hyenas, it is worthwhile to consider an alternative hypothesis: sample contamination.
There are several arguments in favor of contamination: first, the cytochrome B/ND2 sequences of the Azokh coprolite are identical to those of modern brown hyenas that presently show a reduced diversity of the cytochrome B gene. It would be surprising that the putative brown hyenas that would have lived in the Caucasus 100,000 years ago would have had a mitochondrial DNA identical to extant brown hyenas from South Africa. Indeed, a past population size that would cover such a wide geographical range should have a higher genetic diversity. Second, high throughput sequencing revealed that the coprolite contains essentially environmental DNA and traces of contaminating human DNA. Thus, the DNA from the scat producer is extremely rare, if present at all. The traces of human DNA having a higher mean fragment size than the bulk of DNA can most confidently be attributed to contamination. Indeed, the coprolite was identified as cave bear or hyena scat and originally intended to be solely subject to pollen and taphonomic analyses. Therefore, no contamination prevention procedures were applied. In contrast, the coprolite was extensively manipulated prior to the genetic analysis. In the high throughput next-generation sequencing data, there is no evidence of DNA from another species. This indicates that the hyena DNA sequences obtained in the PCR approach correspond to minute traces of DNA that can be detected only due to the high sensitivity that PCR can achieve when it is optimized. When downstream procedures of high sensitivity must be used, one must ensure that extreme precautions of contamination prevention are used at every stage of the analysis, especially upstream of the high sensitivity analyses, starting from sample collection in the field. These precautions were not applied to this sample prior to the paleogenetic analysis.
It is particularly striking that the hyena species that was identified via a genetic analysis is present only in Southern Africa where the sample was prepared for pollen analysis. Contamination occurred most likely at this stage of the analysis. Contamination in the paleogenomics laboratory in Paris is unlikely since contamination prevention is routinely practiced at all stages of sample analyses: strict physical separation of the different experimental steps in positive air pressure laboratories, as well as multiple contamination prevention procedures, carry-over contamination prevention and reagent decontamination that have been developed and optimized in the laboratory and are used without exception (Pruvost et al. 2004, 2005; Champlot et al. 2010). Furthermore, in the paleogenomics laboratory no hyena DNA of any species had ever been analyzed prior to this study and all genetic data from the coprolite were obtained prior to the analyses of modern hyena samples, which were performed later in the modern DNA laboratory.
The contamination of the coprolite may have occurred in South Africa, for the Azokh coprolite was sawed for pollen analysis in the same laboratory room in Bloemfontein in which 10 days before fresh brown hyena scats had been cleaned. Thus, in spite of careful preparation to avoid pollen contamination, residues might have contaminated the coprolite before it was returned to the sample bag immediately after sawing. We believe therefore that the most likely explanation for the presence of brown hyena DNA sequences in the Azokh coprolite is that it had been contaminated with modern brown hyena DNA through secondary contact (bench surface, saw etc.) or residues produced in the Bloemfontein laboratory. This explanation is more parsimonious than would be the reappraisal of past brown hyena distribution with a range extending up to Nagorno-Karabakh. The fact that, apart from brown hyena, no other carnivore sequences were obtained via the targeted PCR approach, and that no indication of the producer’s species was found in the genomic data set, taken together with our previous investigations of numerous cave bear bones from the Azokh cave from which no PCR product was obtained, argue in favor of poor endogenous DNA preservation in the fossil remains of the Azokh cave. Samples with poor DNA preservation, however, are particularly prone to produce artifactual results in paleogenetic studies. The present study highlights the importance of addressing the problems of contamination starting at the very early stages of sample collection during field work when a paleogenetic analysis of the samples is considered. Taken together with our previous demonstration of the importance of early sample treatment to favor optimal DNA preservation (Pruvost et al. 2007) our work reveals the importance of a close collaboration between molecular geneticists and archaeologists or paleontologists.
Conclusions
-
1.
Two coprolites recovered from Azokh 1, Unit II have been studied.
-
2.
Their size and form are comparable to hyena scats, but there is no indication in the form of bone crushing or tooth marks that hyenas were present. No hyena fossils have been recovered from Unit II so far, but they are known in underlying deposits in Unit V.
-
3.
The most abundant species in this site is the cave bear (Ursus spelaeus), an extinct species whose dietary and living behaviors have been considered to be different (though still controversial) to modern bears.
-
4.
The much larger body size of U. spelaeus compared to the largest sized hyena recorded in the site, should have produced larger sized coprolites. Indeed, comparisons with hyena coprolites from other fossil sites show that bear coprolites are larger than the Azokh coprolites. Coprolite morphomotry has not been conclusive. However, the involvement of hyenas with no further taxonomic remains or taphonomic evidence of their presence, except for their coprolites, appears dubious.
-
5.
Chemical analyses of the coprolites by diffraction and fluorescence suggest the possibility of hyenas as the coprolite producer by the presence of the bone mineral (hydroxylapatite). However, hydroxyapatite is the most common and stable neo-formed mineral derived from bat guano diagenesis, which is very intense in Unit II, and has actually been identified in geological materials of Unit II, such as stones and sediments.
-
6.
Relatively high content of amorphous phases and low crystallinity in both geological (stones and sediments) and biological (coprolites) samples may agree with neo-formed minerals, excluding the influence of any organic content in Azokh materials. However, further investigations using other techniques and a higher number of samples are needed.
-
7.
The paleogenetic analysis of the coprolite yielded mitochondrial sequences identical to those of modern brown hyena (Hyaena brunnea) while the paleogenomic analysis did not reveal any indication for DNA sequences of a potential predator.
-
8.
Brown hyenas are today and for their known history restricted to southern Africa, and it is unlikely that their range ever extended into Eurasia. The most parsimonious explanation for this result is contamination of the coprolite from fresh brown hyena scats that were treated in the University of the Free State laboratory in Bloemfontein prior to the opening of the coprolite.
-
9.
In summary, none of the methods applied here has provided conclusive indication of the species that produced these coprolites. None of the results obtained could support or discard bears vs. hyenas as the taphonomic agent. Thus, the producer’s species cannot be defined at this time.
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Acknowledgements
We are grateful to the authorities of Nagorno-Karabakh for permissions to work on these specimens. We thank the Electron Microscopy Unit of the Museo Nacional de Ciencias Naturales for their careful and professional work, Teresa Sanz for pictures taken of the coprolites before processing and Pablo Silva for pictures of modern bears. The authors are also grateful to M.D. Pesquero for providing coprolite measurements from La Roma site. We thank Corinne Esser from the Zoo Fauverie du Mont Faron, France, for providing hair and scats of brown hyenas. The authors are grateful to comments from Mark Lewis, Nigel Larkin, the three anonymous reviewers and the editor in charge (Peter Andrews) who greatly improved this chapter.
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Bennett, E.A., Gorgé, O., Grange, T., Fernández-Jalvo, Y., Geigl, EM. (2016). Coprolites, Paleogenomics and Bone Content Analysis. In: Fernández-Jalvo, Y., King, T., Yepiskoposyan, L., Andrews, P. (eds) Azokh Cave and the Transcaucasian Corridor. Vertebrate Paleobiology and Paleoanthropology. Springer, Cham. https://doi.org/10.1007/978-3-319-24924-7_12
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