Mitochondrial genomes of Pleistocene megafauna retrieved from recent sediment layers of two Siberian lakes

Reviewer #1 (Public Review):

This manuscript presents an important study that contributes to our understanding of the reliability of ancient environmental DNA (aeDNA) extracted from sediment cores. The authors address the potential biases and challenges associated with using aeDNA to infer past ecosystems, specifically focusing on the case of mammoths and woolly rhinoceroses in the Yamal peninsula, West Siberia.

The introduction provides an overview of the significance of sedimentary deposits as archives of past ecosystem changes and illustrates the remarkable insights gained from previous studies using aeDNA, highlighting its potential for reconstructing paleoecology, phylogeography, and understanding extirpation and extinction events of keystone taxa. The authors then report the detection of DNA and near complete mitochondrial genomes of multiple mammoth and woolly rhinoceros individuals in the sampled sediment cores (which are dated to the last few centuries). The authors then employed additional methods to confirm the presence of ancient DNA from mammoths in these sediment cores. Conventional PCR and Sanger sequencing of a mammoth COI fragment confirmed the amplification of mammoth DNA. Mammal metabarcoding and droplet digital PCR (ddPCR) further supported the detection of mammoth DNA in both cores.

The hybridisation enrichment experiment results showed high read counts assigned to Mammuthus, ranging from 2,852 to 72,919 reads per library in core LK-001. Negative controls did not produce any reads assigned to mammals, indicating the absence of contamination. The study also revealed the presence of woolly rhinoceros sequences in the sediment cores, with 12 out of 23 libraries producing more than 100 reads assigned to woolly rhinoceros. The total number of woolly rhinoceros reads was 2,737, and the cumulative mitogenome coverage reached 44%.

The authors carefully addressed the incongruity between the temporal occurrence of these extinct species and the presence of their DNA in recent sediments. They proposed several mechanisms that could explain the recovery of Pleistocene megafaunal DNA in the sediment cores. The minor amount of ancient DNA post mortem damage observed in the mammoth sequences indicates exceptional preservation, consistent with an origin from (recent) permafrost. The dynamics of permafrost thawing and redeposition in the study area provide a plausible explanation for the presence of ancient DNA in the sediments. The authors discuss potential mechanisms for the redistribution of Late Pleistocene material in the sediments, including thermo-denudation processes, methane emissions from degrading permafrost, and the formation of taliks and methane seepage. These processes can disturb the stratigraphy of lake sediments and potentially mix ancient material within the modern sediments. I believe the conclusions are supported by the data and the manuscript is well-written and clear to follow for the reader.

Reviewer #2 (Public Review):

Summary:
The authors report the successful retrieval of mitogenomes from extinct Pleistocene megafauna (woolly Mammoth and woolly rhino) from recent sediment cores from two close Siberian lakes. The cores are too recent to represent real time points of these two extinct species (known to have been extinct for several thousands of years) and therefore, the most plausible interpretation is that permafrost thawing and similar physical processes in the lakes have made surface old ancient DNA, maybe from nearby, deep-buried carcasses.

Strengths:
The pattern of postmortem damage at the end of the Mammoth DNA reads as well as the length distribution (reported in Figure 1) is expected for authentic ancient DNA extracts (besides the phylogenetic evidence). These results pose a question, in my view, on the general reliability of sedimentary DNA in similar contexts, especially in the absence of direct radiocarbon dating of associated remains and in the absence of an understanding of the local geo-physical dynamics. At the same time, the evidence reported here suggests that, at least in Siberian lakes, the sediments can preserve a rich ancient DNA record that it is worth surveying.

Weaknesses:
Although admittedly the work can represent two cases of environments with singular thermal conditions and geodynamics, it opens also the possibility of studying more lake sediments for trying to understand if these findings can be generalized.

Reviewer #3 (Public Review):

Summary:
In this study, the researchers used ancient environmental DNA (aeDNA) retrieved from sediment cores, from two lakes in the Arctic, on the Yamal peninsula, in Siberia. The dating of one of the cores, showed that the sediment layers were very recent (ranging between the years 2019 - 1895). From this core they sequenced 23 libraries which were enriched for mammal mitochondrial genomes. They found a high proportion of two species that have been extinct for thousands of years, the mammoth and the woolly rhinoceros. The highest proportion of mammoth reads were found in very young layer (~81 years old) and as this initial finding does not match the temporal occurrence of the species, they confirmed the identification with several other methods. Additionally, they applied a different dating method on some samples and found that the aging of the samples was not completely congruent. The authors suggest the that the presence of these two Pleistocene megafauna in such recent sediment layers is a consequence of physical processes, specific to the study site, and that the high quality of the aeDNA recovered is a result of permafrost preservation.

Strengths:
The strengths of the study are in the rigorous confirmation of the identification of the taxa with four different PCR and sequencing techniques being used, the initial enrichment panel, and then subsequent metabarcoding PCRs, and taxa specific PCR for COI and cytB. Along with the ancient DNA protocol applied, this is therefore very convincing that the DNA detected in the samples is indeed from the Pleistocene mammals. Additionally, two methods were used to age the sediment cores, and although the depth of the samples tested do not overlap, they give reasonable ages (apart from the anomalous sample) and all together these are robust results.

Weaknesses:
The paper could benefit from clearer aims in the introductions because as it stands the initial aim states that the authors are looking for Arctic mammal abundances through time. However, there are no results relating to general arctic mammal biodiversity presented, which leaves the reader wondering. Perhaps the focus of the study is more on identifying and dating the Pleistocene megafauna. Additionally, it is presented as an analysis on the two taxa, but it feels like the woolly rhinoceros does not receive the same treatment as the mammoth, as there are no additional molecular results, confirmation or figures relating to DNA from this taxa.

Overall the results support that there has been some movement of DNA throughout the sediment core which may impact the dating of the last occurrence of particular extinct taxa. As highlighted, though the geological processes by which this may have arisen are specific to this particular lake and may not be broadly relevant, therefore highlighting that knowledge of each system is important to understanding DNA distribution.