Over the last few years, the recovery and the analyses of genomes of ancient modern humans, Neandertals, and Denisovans have changed our understanding of the origins, the movements, and the relatedness of archaic and modern human populations. How- ever, in many cases endogenous DNA represents such a small fraction of the DNA extracted from specimens that sequencing of the complete ancient genomes is economically infeasible. Thus, to date, only three Neandertal genomes have been sequenced to high coverage [1-3]. Even though Neandertal genome sequences of low coverage [4] can be used to reconstruct various aspects of Neandertal genetic history, many analyses, for example estimation of population size and levels of inbreeding, rely on the reliable diploid genotypes. Recent studies have shown that certain skeletal elements, such as the inner part of the petrous bone and the ce- mentum layer in teeth [5 and references therein], may preserve DNA better over time. There is also evidence that the preservation of endogenous DNA may vary substantially even within a few millimeters distance in a single specimen [2, 4]. Due to the value and scarcity of ancient hominin remains, it is critical that the smallest possible amount of destructive sampling is involved in the recovery of genetic material. A usual sampling strategy typically involves taking around 50 mg of powder from a single location of a given bone or tooth. We investigated here whether taking multiple smaller samples in a step-wise manner of the Neandertal specimens from the Mezmaiskaya Cave in Russia and the Troisième caverne of Goyet in Belgium may improve the yield of an- cient human DNA. We removed between 8.5 and 27.2 mg of bone powder from a Mezmaiskaya 1 rib fragment, between 2.5 and 35.1 mg from a Mezmaiskaya 2 skull fragment, and between 5.8 and 53.8 mg from the Goyet Q56-1 femur fragment, amounting to between 15 and 38 powder subsets per specimen and an average input of 16.6 mg of powder per extraction. Importantly, to minimize the impact of contamination, we treated each powder aliquot with 0.5\% sodium hypochlorite solution prior to DNA extraction. The DNA extracts from the same specimen varied by several orders of magnitude in their proportion of endogenous DNA (between 0.07\% and 54.7\%), their content of nuclear genomes (between 0.01 and 78-fold coverage), as well as in the levels of present-day human contamination (0.2-50.3\%). There was no significant correlation between the amount of powder used for the extraction and the overall amount of the endogenous DNA or the levels of present-day human DNA contamination. Thus, these results indicate that ancient DNA preservation varies greatly within one specimen and that the removal of multiple, small sub-samples instead of one larger sample, here coupled with a decontamination procedure, can drastically improve the likelihood of isolating large enough amounts of DNA to make whole genome sequencing feasible. This approach allowed us to identify extracts with exceptionally high endogenous DNA content and low levels of present-day human DNA contamination (2\%), enabling us to generate three additional high-coverage Neandertal genomes. The high-quality genome sequences of multiple Neandertals form a unique reference resource for the scientific community and are valuable for analyses that require reliable diploid genotypes and haplotype information. For example, these data open new opportunities to investigate Neandertal population history, to identify genetic variants that arose uniquely on the Neandertal lineage and might have changed through time, and to determine those that may underlie archaic-specific traits or adaptations.
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RBINS Staff Publications 2019
The Aktulagay section in Kazakhstan provides an expanded northern Tethyan record of the middle Ypresian (calcareous nannoplankton zones NP11-13, ~ 54-50 Ma), including the Early Eocene Climatic Optimum (EECO). The marl sequence features a series of sapropel beds, observed throughout the Peri-Tethys, indicative of the basin-wide occurrence of episodic hypoxic events. In order to unravel the paleoenvironmental evolution at Aktulagay during this period of global warming, we investigated the benthic foraminiferal record by means of a detailed multivariate analysis of the > 63 μm fraction, as well as through stable isotopic (C, O) investigations on excellently preserved benthic foraminiferal specimens. The Alashen Formation (NP11 to lower NP12; ~ 54 to 52.5 Ma), in the lower part of the sequence, contains a diverse assemblage of deep outer neritic (~ 200-250 m) benthic foraminifera, with common Pulsiphonina prima and Paralabamina lunata. The sea-floor conditions are interpreted as initially (54 Ma) well-ventilated and oligo- to mesotrophic, gradually changing to more eutrophic and oxygen-limited, culminating in more permanent low-oxygen conditions and eutrophy in the sapropel-bearing Aktulagay B1 unit (middle NP12; ~ 52.5-52 Ma). The latter conclusion is corroborated by the dominance of Anomalinoides acutus and Bulimina aksuatica and the lower diversity. Also the upward migration of endobenthic species to the sediment-water interface, as suggested by rising δ13Cendobenthic values, supports this interpretation. A transgression, which flooded lowlands, might have caused this development. In the Aktulagay B2 unit (top NP12-NP13; ~ 52-50 Ma), benthic foraminiferal assemblages dominated by Epistominella minuta suggest an oligotrophic environment, with transient pulses of phytodetritus and moderate ventilation. The Aktulagay B2 unit coincides with the peak temperature interval of the EECO, as indicated by its position close to the base of NP13 and rising δ13Cepibenthic values. Large river plumes, episodically reaching the area, in a monsoonal climatic context, might explain this basin development. Although it is not unlikely that some of the observed patterns are related to long-term climate change, it can currently not be excluded that changing paleogeography and variable connections to the Tethys, Atlantic and the Arctic Ocean were responsible for the long-term period with dysoxia and anoxia during deposition of the sapropel beds at the Peri-Tethyan seafloor. The evolution of the basin as observed in Aktulagay shows similarities to the evolution of the North Sea Basin as observed in Denmark, suggesting that these basins were connected during the Early Eocene.
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