Mammoth and Woolly Rhinoceros, Metagenomics of
KeywordsMicrobial Community Taxonomic Assignment Cellulolytic Bacterium Intestinal Microbiomes Clostridium Beijerinckii
Metagenomics is the study of metagenome, a composite of the animal genes and genes present in the genomes of microorganisms colonizing their bodies. More narrow definition of the term metagenome is limited to genomes of only microbial community (microbiome).
The subject of this entry is to outline the information available on the composition of the gut microbiomes of two extinct herbivorous animals – woolly mammoth (Mammuthus primigenius) and woolly rhinoceros (Coelodonta antiquitatis). The microbiome of an intestinal tract plays important role in the animal nutrition and overall health. In particular, herbivores have evolved to maintain microbial consortia that coordinate relatively rapid rates of degradation of complex plant carbohydrates under anaerobic conditions (Flint 1997). The coevolution of herbivorous mammalian lineages and their gut microbes involved enlargement of the foregut or hindgut to increase the gut retention times required for fermentation and selection of appropriate microbial communities (Ley et al. 2008). Comparison of the intestinal microbiomes of the present-day animals and their extinct relatives may provide information about the diet of the latter and the evolution of microbial communities.
Permafrost-preserved animals provide unique opportunity to study the genomes and microbiomes of extinct species. Several studies were focused on nuclear and mitochondrial genomes of woolly mammoth and woolly rhinoceros (e.g., Poinar et al. 2006; Miller et al. 2008); they are out of scope of this entry. The discoveries of permafrost-preserved mammals, especially the ones with intact intestines protected from the environment, are exceedingly rare events, and, to the best of our knowledge, only a single molecular study of their endogenous microbiomes has been reported (Mardanov et al. 2012).
Composition of the Gut Microbiomes of Mammoth and Woolly Rhinoceros
The mammoth Lyuba, who died at the age of 1 month, was found in Western Siberia (Russia) in 2007 and studied by the team of Dr. Tikhonov at Zoological Institute of the Russian Academy of Sciences and his collaborators (Kosintsev et al. 2010; Van Geel et al. 2011; Fisher et al. 2012). Although its geological age is around 40,000 years, it is the best preserved mammoth found to date. Lyuba is an unweaned calf; its intestinal tract was intact and contained milk and fecal matter, presumably of her mother’s (Van Geel et al. 2011). The 39,000-year-old woolly rhinoceros is an adult female found in 2007 near the settlement of Cherskiy, Eastern Siberia (Boeskorov et al. 2009). Her intestines contained the partly undigested composite plant material. The above intestinal samples were used for collection of bacterial fraction and isolation of microbial community DNA.
The intestinal microbiome of the mammoth Lyuba had a completely different content. Representatives of two bacterial phyla were identified – Proteobacteria (81 % of all clones) and Actinobacteria (18 %) – while less than 0.5 % of sequences were assigned to Firmicutes, TM7, and Bacteroidetes (Fig. 1). The majority of microorganisms (71 %) represented the family Pseudomonadaceae, widely distributed and metabolically diverse group of Gammaproteobacteria. Although the Pseudomonas spp. are usually not the major components of the animal intestinal microbiomes (Ley et al. 2008), some species are common bacterial contributors to spoilage of fluid milk products (Cousin 1982). The presence of indigested mother’s milk in Lyuba’s stomach and intestines may explain the prevalence of pseudomonads. Another particular characteristic of Lyuba’s intestinal microbiome is almost complete absence of Firmicutes, while this phylum dominates the rhino’s microbiome. It may reflect the differences in the diets of the animals (milk vs plant biomass).
Overall, the data presented by (Mardanov et al. 2012) provides the first insight into the composition of the intestinal microbiomes of the representatives of the Pleistocene megafauna. Some bacterial lineages found in microbiomes of present-day herbivores were also found in the woolly rhinoceros microbiome, while some important groups (e.g., Ruminococcus spp.) were absent. It is possible that such specialized microorganisms appeared and became widespread late in the evolution of herbivores and/or was associated with human activities. It is also possible that their absence in the particular sample reflects the specific diet of this woolly rhinoceros. For instance, the fraction of Ruminococcus spp. is higher in sheep consuming the starch-containing diet than in consuming grass (Larue et al. 2005). Analysis of a larger number of microbiome samples of permafrost-preserved animals is required to clarify these issues. Another factor that should be taken into account is the possibility of selective proliferation of particular microorganisms after the death of the animal, which might explain the high proportion of Pseudomonas spp. in the intestinal microbiome of mammoth Lyuba.
Possible Technical Problems Related to Analysis of the Ancient Microbiomes
The major problem in analysis of the ancient microbiomes is the possible ancient and/or modern microbial contamination of the permafrost-preserved samples from the environment material such as soil and water as well as human-related contamination in course of sample processing. Contamination issues are particularly important for ancient samples since most DNA extracted from fossil remains is truncated into fragments of very short length (usually 80–120 nucleotides) from the hydrolysis of the DNA backbone. This is much shorter than the length of 16S rRNA amplicons used for identification of microorganisms. For instance, the study of Mardanov et al. (2012) involved analysis of about 500 bp-long 16S fragment. Therefore, PCR amplification may enrich the amplicons derived from the longer fragments of modern contaminating DNA. It is always difficult to prove the indigenous nature of the revealed microbial community, and the appearance of known environmental or human-related microorganisms would be an alarming indication.
Taxonomic assignment of 16S reads may be also confounded by nucleotide transitions that normally happen in ancient DNA. Most frequent among such pitfalls is the deamination of cytosine to uracil resulting in C-to-T and G-to-A substitutions in sequencing data (Hofreiter et al. 2001). The rate of deamination is considerably higher for single-stranded DNA than for double-stranded. For highly degraded ancient DNA samples, local unpairing of double strand near the breaks may create a lot of C-to-T transitions (up to 40 %) within the nearest 5–7 nucleotides with a significant decrease of the transitions (to less than 5–7 %) in the “body” of DNA fragment (Overballe-Petersen et al. 2012). The deamination of cytosine may result in underestimation of similarity between the amplicons and reference 16S sequences. Therefore taxonomic assignment of the 16S reads may be complicated, especially at lower taxonomic ranks. On the other hand, the prevalence of C-to-T and G-to-A transitions over other types of dissimilarities between the 16S reads and the closely homologues reference sequence (derived from present day samples) could be a good indication of an ancient origin of the reads. Such dissimilarity patterns have been reported for the mammoth and elephant genomes (Poinar et al. 2006). The study of the gut microbiome of the mammoth (Mardanov et al. 2012) also revealed the bias toward C-to-T and G-to-A transitions in 16S rRNA sequences closely related to Pseudomonas (Ravin et al., unpublished data). However, the frequencies of C-to-T transitions in both studies were quite low (less than 1 %) and thus could not impair taxonomic assignment of 16S sequences.
The herbivorous animals have evolved to maintain microbial consortia that coordinate relatively rapid rates of degradation of complex plant carbohydrates under anaerobic conditions. Comparison of the intestinal microbiomes of the present-day animals and their extinct relatives may provide information about the diet of the latter and the evolution of microbial communities. The intestinal microbiomes of two permafrost-preserved extinct animals, unweaned calf woolly mammoth and adult woolly rhinoceros, were analyzed by pyrosequencing of 16S rRNA genes. The microbiome of the woolly rhinoceros was dominated by polysaccharide-degrading lineages of Clostridia, also found in the present-day herbivores, while Ruminococcus spp., most commonly isolated cellulolytic bacteria from herbivores intestines, were absent. Representatives of Bacteroidetes, typically the second after the Firmicutes major bacterial phylum in microbiomes of herbivores, were found in minor amounts. The intestinal microbiome of the young mammoth, unweaned calf, comprised mainly bacteria of the family Pseudomonadaceae, while Firmicutes and Bacteroidetes were present in minor amounts. The presence of undigested mother’s milk in mammoth stomach and intestines may explain the prevalence of pseudomonads, since some species of this group are common bacterial contributors to spoilage of fluid milk products. This data provides the first insight into the composition of the intestinal microbiomes of the representatives of the Pleistocene “megafauna.”
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