Alina Guliaeva1*, Albert Protopopov2, Maksim Cheprasov3, Gavril Novgorodov3, Alexander Rakitko1, Fedor Sharko4 and Artem Nedoluzhko3,4
1ITMO University, Saint Petersburg, Russia
2Academy of Sciences of the Republic of Sakha, Yakutsk, Russia
3Mammoth Museum, North-Eastern Federal University, Yakutsk, Russia
4Laboratory of Palaeogenomics, European University, Saint Petersburg, Russia
auguliaeva [at] gmail.com
Abstract
Wolves (Canis lupus) served as key predators in Pleistocene ecosystems, regulating large herbivore populations across the mammoth steppe. Permafrost-preserved remains offer rare opportunities for direct genomic and metagenomic analysis of extinct fauna and their associated microbiota. We therefore aimed to reconstruct the phylogenetic position, gut microbiome composition, and diet of a Late Pleistocene wolf from Yakutia.
A well-preserved wolf carcass recovered in 2021 from the Tirekhtyakh River, Yakutia, radiocarbon dated to approximately 44 ka, was subjected to ancient DNA extraction from hair, skin, and gastrointestinal tract (GIT) contents using the GENECLEAN® Ancient DNA kit, followed by sequencing on the Illumina NovaSeq 6000 platform. Reads were processed in PALEOMIX, and the mitochondrial genome was constructed de novo using SPAdes, followed by annotation with MitoZ. A maximum-likelihood phylogenetic tree (IQ-TREE) was inferred from 138 mitogenomes of modern and ancient canids. GIT reads were assembled (metaSPAdes), binned (metabat2, comeBIN, maxbin2), refined with DAS Tool, quality-filtered (CheckM2), and taxonomically assigned (GTDB-Tk).
We reconstructed a complete mitochondrial genome (16,727 bp; mean coverage 195×). Phylogenetic analysis placed the Yakutian wolf within a strongly supported clade (bootstrap >95) of northeastern Siberian Pleistocene wolves (41–67 ka) that have no direct descendants among modern populations. From the GIT contents, we obtained 15 metagenome-assembled genomes (MAGs), including a high-quality MAG of a pathogenic bacteria Erysipelothrix (completeness 91.4%, contamination 1.2%). This MAG encodes type III secretion system components, virulence factors, antioxidant proteins, antimicrobial resistance determinants, and mobile genetic elements. Preliminary taxonomic profiling of GIT contents also revealed diverse eukaryotic taxa, providing initial insight into the diet of this Late Pleistocene predator.
This permafrost-preserved specimen recorded both the phylogenetic identity of an extinct wolf lineage and a snapshot of its gut microbiome and diet at the time of death. We additionally uncovered ancient origins of Erysipelothrix virulence and antimicrobial resistance, demonstrating that permafrost-preserved gastrointestinal material can recover ancient pathogen genomes with functional traits and extend the known timeline of antimicrobial resistance determinants to >40 ka.
Keywords: Pleistocene fauna, paleogenomics, canids, mitogenome