Genomic analysis of a novel Neanderthal from Mezmaiskaya Cave provides insights into the genetic relationships of Middle Palaeolithic populations

www.nature.com/scientificreports OPEN Genomic analysis of a novel Neanderthal from Mezmaiskaya Cave provides insights into the genetic relationships of Middle Palaeolithic populations Tatiana V. Andreeva1,2,3*, Andrey D. Manakhov1,2,3, Fedor E. Gusev1,2,3, Anton D. Patrikeev2, Lyubov V. Golovanova4, Vladimir B. Doronichev4, Ivan G. Shirobokov5 & Evgeny I. Rogaev1,2,3,6* The Mezmaiskaya cave is located on the North Caucasus near the border that divides Europe and Asia. Previously, fossil remains for two Neanderthals were reported from Mezmaiskaya Cave. A tooth from the third archaic hominin specimen (Mezmaiskaya 3) was retrieved from layer 3 in Mezmaiskaya Cave. We performed genome sequencing of Mezmaiskaya 3. Analysis of partial nuclear genome sequence revealed that it belongs to a Homo sapiens neanderthalensis female. Based on a high-coverage mitochondrial genome sequence, we demonstrated that the relationships of Mezmaiskaya 3 to Mezmaiskaya 1 and Stajnia S5000 individuals were closer than those to other Neanderthals. Our data demonstrate the close genetic connections between the early Middle Palaeolithic Neanderthals that were replaced by genetically distant later group in the same geographic areas. Based on mitochondrial DNA (mtDNA) data, we suggest that Mezmaiskaya 3 was the latest Neanderthal individual from the early Neanderthal’s branches. We proposed a hierarchical nomenclature for the mtDNA haplogroups of Neanderthals. In addition, we retrieved ancestral mtDNA mutations in presumably functional sites fixed in the Neanderthal clades, and also provided the first data showing mtDNA heteroplasmy in Neanderthal specimen. Since the late Marine Isotope Stage (MIS) 9, Neanderthals occupied regions in West Eurasia that were most affected by climatic fluctuations during the glacial and interglacial cycles of MIS 8–MIS 3. In Central Europe and Eastern Europe, the Micoquian stone-working bifacial tradition1 lasted from at least MIS 5 to the end of Middle Palaeolithic (MP) in MIS 3, and was widespread in Central and Eastern Europe2–4. Palaeogenetic research of Neanderthal mitochondrial DNA (mtDNA) identified at least two population turnovers that occurred during Neanderthal history and both were associated with the Micoquian. The earlier population turnover at ~ 90 ka was related to the replacement of the earlier Altai Neanderthals by western European Neanderthals, which was associated with the spread of the Eastern Micoquian to the Altai region5,6. The later population turnover occurred towards the end of Neanderthal history from 47 to 39 ka within the Eastern Micoquian Neanderthal population in the North Caucasus or among late Middle Palaeolithic (LMP) Neanderthal groups throughout Europe7. Palaeogenetic research also detected a great similarity between the mitochondrial genomes of two Neanderthals from geographically distant Eastern Micoquian contexts dated to MIS 5—Stajnia Cave (Poland) and Mezmaiskaya Cave (Russia)—and both of them fall outside of the mtDNA variation of the later European Neanderthals4. 1 Center for Genetics and Life Science, Sirius University of Science and Technology, Sochi, Russia 354340. 2Laboratory of Evolutionary Genomics, Department of Genomics and Human Genetics, Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia 119333. 3Faculty of Biology, Centre for Genetics and Genetic Technologies, Lomonosov Moscow State University, Moscow, Russia 119192. 4ANO Laboratory of Prehistory, St. Petersburg, Russia 190020. 5Peter the Great Museum of Anthropology and Ethnography (Kunstkamera), Russian Academy of Sciences, St. Petersburg, Russia 199034. 6Department of Psychiatry, University of Massachusetts Medical School, Worcester, MA 01604, USA. *email: ; ; Scientific Reports | (2022) 12:13016 | https://doi.org/10.1038/s41598-022-16164-9 1 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 1.  Eurasia map showing the locations of the Neanderthal samples used in the analysis. Mezmaiskaya Cave is typed in red. Neanderthal haplogroups (see Results, Supplementary Fig. S10) are indicated at each location by coloured sectors, and large haplogroups combine subsequent branches: NA2 ~ includes NA2 and NA2a; NM1 ~ includes NM1, NM1a, NM2; NL1 ~ includes NL1a1, NL1a2a, NL1a2b1, NL1a2b. NA ~ TeshikTash individual. The figure was generated using ggplot2 (https://ggplot2.tidyverse.org), ggspatial (https://github. com/paleolimbot/ggspatial), rnaturalearth (https://github.com/ropensci/rnaturalearth), rnaturalearthdata (https://github.com/ropensci/rnaturalearthdata), scatterpie (https://CRAN.R-project.org/package=scatterpie) and sf (https://github.com/r-spatial/sf) packages for R. The Mezmaiskaya Cave, located in Russia, is widely known as a Palaeolithic site that has recorded the longest MP and Upper Palaeolithic sequences in the North Caucasus area (Fig. 1, see SI, Supplementary Figs. S1–S3). In the MP deposits, two Neanderthal fossils were found in this cave, namely, a newborn skeleton (Mezmaiskaya 1) that was found in the lowermost Eastern Micoquian level (layer 3) and a juvenile (Mezmaiskaya 2) that was found in the uppermost Eastern Micoquian level (layer 2)4,7–13. Here, we present mtDNA and genome sequencing results on a new Eastern Micoquian Neanderthal individual (Mezmaiskaya 3) based on a milk incisor found in layer 3 at the Mezmaiskaya Cave. Results Anthropological definition of Mezmayskaya 3. Mezmaiskaya 3 tooth morphology suggested that it may belong to archaic hominins (Fig. 2). While the tooth crown is strongly worn, morphological features suggest that this is a milk right, upper central incisor (Rdi1). The crown of Mezmaiskaya 3 is completely formed, and the wear of the crown approximately corresponds to the wear of the central incisor No. 32 of the child from Renne Grotto at Arcy-sur-Cure, whose age was estimated at 5–6 years old14. Based on this comparison, we assumed that the Mezmaiskaya 3 individual was about the same age. Data generation and basic processing. DNA was extracted from the Mezmaiskaya 3 (Mez3) tooth (Supplementary Fig. S4) and converted into a single-stranded library for s equencing15. Almost half a billion raw DNA sequences were generated (Supplementary Table S1) by sequencing on the Illumina HiSeq 2000/2500. DNA fragments were mapped to the human reference genome (GRCh37) as well as to the revised Cambridge Reference Sequence (rCRS, NC_012920.1) of the human mitochondrial genome and the Neanderthal mitochondrial genome sequence (NC_011137.1) (see Materials and Methods for details, Supplementary Tables S2, S5). C-to-T base substitutions at the ends of the DNA sequences in the mapped sequences were related to the postmortem degradation pattern of ancient DNA (Supplementary Fig. S5). A total of 75 Mbp of nuclear genomic sequences with a signature of deamination mapped to the human genome were recovered (Supplementary Table S2). Based on the coverage of the X chromosome and autosomes, the M (...truncated)