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Researchers Sequence Mitochondrial Genome from 400,000-Year-Old Hominin

NEW YORK (GenomeWeb News) – Mitochondrial genome sequences from a 400,000-year-old hominin found in a cave in Spain indicate that the ancient individual belonged to a mitochondrial lineage most closely related to that of the Denisovans — archaic Neandertal relatives that roamed eastern Eurasia.

An international team, led by Max Planck Institute for Evolutionary Anthropology Director Svante Pääbo, used specialized DNA extraction and enrichment methods to sequence a nearly complete mitochondrial genome from the ancient individual's thigh bone — believed to be the oldest non-permafrost sample successfully sequenced so far.

"Our results show that we can now study DNA from human ancestors that are hundreds of thousands of years old," Pääbo said in a statement. "This opens prospects to study the genes of the ancestors of Neandertals and Denisovans. It is tremendously exciting."

As they reported online today in Nature, researchers' phylogenetic assessments at the mtDNA sequences suggested that the ancient individual belonged to a mitochondrial lineage that split from the Denisovans roughly 700,000 years ago.

More research is needed to understand the nature of this relationship, particularly since the remains of the newly sequenced hominin and other individuals in the same cave had notably Neandertal-like skeletal features.

The bone sample used for the study came from a collection of ancient hominin remains retrieved during the excavation of a Spanish cave site known as Sima de los Huesos, or the "pit of bones."

The dozens of hominin skeletons found there had physical features resembling Neandertals as well as fossils grouped together under the moniker Homo heidelbergensis, researchers noted. Previous estimates put their age at more than 300,000-years-old, placing them in the Middle Pleistocene period.

To characterize the Sima hominin's mitochondrial genome, they turned to the same silica-based DNA extraction and library amplification approaches that members of the team used for sequencing mtDNA from cave bear bones. As they reported in the Proceedings of the National Academy of Sciences in September, those samples came from the same cave and were also at least 300,000-years-old.

After using shallow Illumina MiSeq sequencing on a subset of DNA libraries to assess DNA preservation in the hominin sample, researchers turned to the HiSeq 2500 to sequence libraries specifically enriched for mtDNA.

The enrichment was originally done with probes designed to jibe with human mitochondrial sequences, the study authors noted. But once they saw the Denisovan-like mtDNA sequences being generated, they enriched DNA libraries using Denisovan-based probes as well.

With the help of cytosine deamination profiles and sequence lengths, the team weeded out apparent contaminants from the endogenous DNA sequences, which were also subjected to quality filtering as they were mapped to a mitochondrial reference genome.

With data at 16,302 consensus sites in the newly sequenced mitochondrial genome covered by at least three reads a piece — and to a depth of nearly 32-fold, on average — researchers went on to perform a phylogenetic analysis that compared Sima hominin sequences to mitochondrial sequences from Denisovans, Neandertals, humans, chimpanzees, and bonobos.

The researchers did additional phylogenetic assessment as well, using somewhat more conservatively selected consensus sequences from the Sima hominin data. In each case, they found that the Sima hominin was more closely related to the Denisovans than to the other hominins.

A calibration based on the subtle sequence changes in more than a dozen ancient mtDNA sequences suggested that the individual is likely around 400,000-years-old and belonged to a mitochondrial lineage that split from the Denisovans roughly 700,000 years ago.

The relationship is unexpected given the physical similarities between the Sima de los Huesos fossils and Neandertals as well as the more Eastern distribution of Denisovan sequences in current human populations.

The study's authors speculated that the mitochondrial ties have stemmed from a relationship between the Sima hominins and a common Neandertal and Denisovan ancestor, followed by differences in mitochondrial divergence patterns in the latter groups.

Still, they noted that the data available so far might be explained by several alternative scenarios and cautioned that the precise relationships between the ancient hominins remain murky in the absence of nuclear sequences from the Sima de los Huesos skeletons.

"Considering their age and Neandertal-like features, the Sima hominins were likely related to the population ancestral to both Neandertals and Denisovans," first author Matthias Meyer, with the Max Planck Institute for Evolutionary Anthropology, said in a statement.

"Another possibility is that gene flow from yet another group of hominins brought the Denisova-like mtDNA into the Sima hominins or their ancestors," Meyer said.

With one Sima hominin mitochondrial genome in hand, the researchers are planning to not only sequence mtDNA from more of the Sima hominin samples, but also to try their hand at characterizing some nuclear sequences, if possible.