The complete mitochondrial genome of the extinct Pleistocene horse (Equus cf. lenensis) from Kotelny Island (New Siberian Islands, Russia) and its phylogenetic assessment

Abstract The complete mitochondrial genome from the Pleistocene stallion horse (Equus cf. lenensis) which complete skull was found in 1901 on Kotelny Island (New Siberian Archipelago, Sakha Republic, Russia) is published in this paper. The mitochondrial DNA (mtDNA) is 16,584 base pairs (bp) in length and contained 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes. The overall base composition of the genome in descending order was 32.3% – A, 28.5% – C, 13.4% – G, 25.8% - T without a significant AT bias of 58.2%.

Lenskaya or Lena horse (Equus cf. lenensis Russanov, 1968) is extinct Equidae species, which roamed in the Northeastern Siberia during the Late Pleistocene. Presumably, this species lived up to the Middle Holocene in the Arctic zone of Yakutia (northern of 70 north latitude); the latest remains of a frozen mummy is dated by 5450-5310 cal BP (Boeskorov et al. 2014;. The investigated Lena horse specimen was collected in 1901 on the south coast of the Kotelny Island (New Siberian Archipelago, Sakha Republic, Russia) by Russian Polar Expedition of 1900-1902 that was led by the Russian Arctic explorer Eduard von Toll (1858-1902. The first description of the Late Pleistocene mammals collection from the Kotelny Island was conducted by M. V. Pavlova (Pavlova 1906). The specimen (part of the cranium in a good safe, that was belonged to a stallion by 5-8 years age old) is stored in the Mammal's Laboratory of the Borissiak Paleontological Institute, Russian Academy of Sciences (collection number PIN 301/1). The absolute age (AMS) of the sample established by collagen from the lower jaw bone is 21,105 ± 55 cal BP (IGAN/CIR UG6966). Morphological characters of the PIN 301/ 1 specimen allow to define it as Equus cf. lenensis.
Lena horse DNA was extracted from the bone powder at the ancient DNA facilities of the National Research Center "Kurchatov Institute" (Moscow, Russia), following the methodology described previously (Orlando et al. 2013).
DNA-library was prepared using an OvationV R Ultralow Library System V2 (NuGEN, Redwood City, CA). Amplified DNA library was quantified using a high-sensitivity chip on a 2100 Bioanalyser instrument (Agilent Technologies, Palo Alto, CA). To sequence, the DNA-library of the E. cf. lenensis specimen the S2 flowcell of Illumina Novaseq6000 genome analyzer (Illumina, San Diego, CA) was used with paired-end reads with 150 base pairs (bp) length. 307,010,901 Illumina paired-end reads were generated for DNA library for PIN 301/1 Pleistocene horse specimen. Obtained Illumina reads were processed through PALEOMIX pipeline , mapping was done against the E. caballus reference sequence (assembly EquCab3.0) using Bowtie 2 under the "very-sensitive" and "rescale" options. We used mapDamage2 (Jonsson et al. 2013), that implemented in PALEOMIX, to model postmortem DNA damage from nucleotide misincorporation patterns (Figure 1(A)). 5,732,968 reads were filtered by PALEOMIX then were used for de novo assembling of mitochondrial DNA sequence by a Norgal software package (Al-Nakeeb et al. 2017). The resulting consensus sequence was annotated using the MITOS (Bernt et al. 2013). The obtained annotation was then used to define partitions in the subsequent phylogenetic analysis.
This study represents the first mitochondrial DNA analysis of E. cf. lenensis phylogenetic position based on the available mitochondrial genomes of Equidae species for comparison. Our data show that Lena horse does not represent a separate mitochondrial lineage within Equus genus, also as previously published Middle and Late Pleistocene extinct horses from Siberia (Orlando et al. 2013;Librado et al. 2015).

Acknowledgments
This work was carried out using high-performance computing resources of federal center for collective usage at NRC "Kurchatov Institute", http:// computing.kiae.ru/. E. Mashchenko was supported by the Russian Academy of Sciences program: "The evolution of the organic world. The role and influence of planetary processes. Project: "Comparative analysis and synthesis of paleontological and molecular genetic data on the phylogeny of key groups of terrestrial vertebrates".

Disclosure statement
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.