Genomic identification and complete mitochondrial recovery of a Late Holocene porcupine (Erethizon dorsatum) mummy from Yukon permafrost

TL;DR

Scientists discovered a 3000-year-old mummified porcupine in Yukon permafrost and sequenced its complete mitochondrial genome.

Contribution

The first complete ancient mitochondrial genome of Erethizon dorsatum and confirmation of its presence in Yukon 3000 years ago.

Findings

The specimen is the first known mummified porcupine from ancient North America.

The mitochondrial genome is the first complete ancient mitogenome for Erethizon dorsatum.

The porcupine's presence in Yukon 3000 years ago supports dispersal after the Last Glacial Period.

Abstract

We identified a 3000-year-old specimen from the Traditional Territory of the Tr’ondëk Hwëch’in in central Yukon Territory, Canada as the first known mummified remains of an ancient North American porcupine (Erethizon dorsatum), known as “Ts’ey” in the Hän language, using genetic analysis and metagenomic validation. Our analysis of the sample yielded the first-ever complete ancient mitochondrial genome for (E. dorsatum) and only the second full mitogenome for the species. Its Holocene age is considerably younger than the Pleistocene megafauna typically recovered in the Yukon permafrost, demonstrating the potential for these deposits to preserve specimens from interglacial periods. Crucially, this finding confirms the presence of porcupines in the region 3000 years ago, in line with the hypothesis that this species only dispersed into Yukon and Alaska following the establishment of boreal forests after the Last Glacial Period.

Figures

Mummy collection site and dating. (A) Map with the location of the site in the Yukon Territory, Canada where specimen YG 77.11 was recovered. (B) Photo of the mummified specimen YG 77.11 taken in July 2024, before sampling (both sides of the sagittal axis). (C) Calibrated radiocarbon age of specimen YG 77.11.

Species identification via mitogenomes and metagenomics. (A) Iterative mapping results against 18 candidate mitogenomes for species identification. (B) Metagenomic classification of reads against a mammalian full genome reference database. In top panel, fraction of the total reads classified within Mammalia. In bottom-left panel, majority of mammalian reads are classified within the order Rodentia and suborder Hystricomorpha. In bottom-right panel, the majority of Rodentia reads are classified as *Erethizon dorsatum* at the species level.

Mitochondrial phylogeny. (A) Mitogenome reconstruction of sample YG 77.11 depicting per site quality, depth and presence of variants corresponding. The outer circle represents quality per base covered by nucleotides. Inner green spikes represent the depth of coverage (average 32X), which correspond to the total number of mapped reads covering each base pair. The red dots scattered within the circle represent variant sites, bases where the Yukon mummy differs from the reference genome, which include substitutions (SNPs, n=440) as well as insertions and deletions (indels, n=6). (B) Maximum likel

Full text

Introduction

Since the earliest discoveries of frozen woolly mammoths *(Mammuthus primigenius)*from Siberia during the early 19th Century to today, discoveries of ancient mummified animals preserved in permafrost always attract fascination^1–4^.. Over the last two centuries, many examples of permafrost preserved mammal carcasses of extinct and extant fauna have been found during industrial mining activity, or as a result of natural erosion from permafrost melting due to climate change in recent decades^5–10^.

Well-preserved and nearly complete carcasses of iconic Pleistocene (2.6mya to 0.01mya) woolly mammoths have drawn worldwide interest. Examples include the 40-kya-old “Dima” in 1977^11^, the 42-kya-old mammoth calf known as “Lyuba” (2007)^5^, the 39-kya-old juvenile mammoth called “Yuka” (2010), all three found in Siberia, and the more recent discovery of North America’s best-preserved mammoth calf called “Nun Cho Ga” (2022) in the Klondike (Canada)^12^.

Other notable permafrost mummies include the steppe bison (Bison priscus) known as Blue Babe from Alaska^1^, the cave lions (Panthera spelaea) known as “Sparta and Boris”^8^, the grey wolf (Canis lupus) pup called “Zhùr”^7^, the wolf-dog (Canis sp.) pup known as “Digit”^9^, saber-toothed cats (Homotherium sp.)^13^, but also birds^6^ and other species covering much of the extinct ecological diversity of the Pleistocene^10,14^.

A wide variety of more recent animal mummies (Holocene - 0.01mya to present) have also been found in some other regions of the world, namely South America and Egypt^15^ but there are also sporadic examples in Greenland and even the Sahara^16,17^. In contrast with specimens recovered from the permafrost, most of these deliberate mummies were made to last and linked to particular cultural practices in history.

For example, it has traditionally been believed that the ancient Egyptians maintained farms specifically dedicated to breeding the African sacred ibis (Threskiornis aethiopicus) to be used as mummified offerings^18^. Consequently, it was unclear how representative these mummies were of past wild populations, or of ancient wildlife more generally, when considering other species. However, this view has recently been challenged by genetic evidence derived from ancient DNA analyses^19^. This case illustrates the importance of animal mummies and the valuable insights they can offer into the populations of the past^20^.

South America on the other hand also offers more examples of mummies buried intentionally as goods or ritual offerings (dogs, parrots and llamas among others). These mummies are usually a byproduct of arid climatic conditions rather than deliberate manufacture^15^.

However, outside these special regions, only areas with permafrost are likely to yield natural mummies. Oftentimes, permafrost mummies are so well-preserved that represent one of the best opportunities to recover unprecedented genomic information^21^ and provide a direct insight into extinct genomes and ecosystems.

Complete or near complete mummified bodies offer scientists a unique snapshot into morphological features of ancient animals in ways that would be near impossible from fossil bones and teeth alone. In other instances, smaller fragments of mummified flesh and skin are unearthed and recovered from permafrost and accessioned into fossil collections but do not often receive the same level of scientific or popular attention^1^. While isolated pieces of mummified skin, hair, or muscle tissue may not preserve much of the animal’s morphology of interest to paleontologists, they still retain genetic material that can be used for species identification and improve understanding of the biology and evolutionary history of these specimens.

In the Klondike goldfields of west-central Yukon Territory (Canada), industrial mining activities that erode permafrost have exposed vast quantities of Quaternary (2.6mya to present) sediment since the onset of the Gold Rush in the late 19th century, providing a remarkable record of Pleistocene paleoenvironments in Beringia and the fauna of the Last Glacial period^22^. The Klondike goldfields mining operations encounter remains of Pleistocene animals (bones, teeth, antlers, tusks, horns) routinely and sometimes yield frozen mummies of animals^7^. However, it is also worth mentioning that the overwhelming majority of natural mummies derive from glacial periods, with few exceptions (caves, bogs, high altitude)^3^, due to the limitations imposed on permafrost preservation capacity by thawing cycles. Therefore, potential biases in frozen fauna must also be considered^1^.

In this work, we studied a mysterious and previously unidentified mummified specimen that was recovered from permafrost exposed by gold miners in the Klondike goldfields. The mummified specimen (YG 77.11) was recovered from a gold mine fossil locality where fossils from typical late Quaternary large mammals were also collected, including woolly mammoths (Mammuthus primigenius), Dall sheep (Ovis dalli), caribou (Rangifer tarandus), steppe bison (Bison priscus) and horse (Equus). The mummified specimen represents a piece of dried skin with flesh and coarse hairs about 30 cm x 20 cm in size that was originally found in 1998. The sample location is a site known as Homestake Gulch ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$63^{\circ }$$\end{document} 56’11” N, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$139^{\circ }$$\end{document} 13’39” W), which is an active gold mine located a couple of kilometres upstream from its confluence with the right limit of Upper Bonanza Creek in the Klondike. This piece of mummified flesh lacked clear morphological features to permit a reliable taxonomic identification. Our work aimed to determine the taxonomic identity of this cryptic piece of mummified mammal skin and hair through genomic analysis and determine its age through radiocarbon dating to place it in context with other mummified animal remains known from the region^1,4^.

Results

Radiocarbon age of the specimen

We obtained two radiocarbon dates from the Homestake Gulch (Fig. 1A) specimen YG 77.11 (Fig. 1B) as replicates (VIE-1482, VIE-1483) (Fig. 1C; Supplementary Table S1). Both dates are concordant and indicate ages of 2,845±25 and 2,790±25 years BP (Fig. 1C). The unmodelled ages at 95.4% probability encompassed by the two replicates ranged between 3,060 and 2,790 years BP (Supplementary Table S1).Fig. 1. Mummy collection site and dating. (A) Map with the location of the site in the Yukon Territory, Canada where specimen YG 77.11 was recovered. (B) Photo of the mummified specimen YG 77.11 taken in July 2024, before sampling (both sides of the sagittal axis). (C) Calibrated radiocarbon age of specimen YG 77.11.

Species identification via mitochondrial DNA

Independent mapping was carried out against a set of 18 complete mitochondrial reference genomes of diverse species. These species were selected as potential candidates either due to their presence in the fossil record or extant distribution in the Americas (Fig. 2A). Given that the mummy is preserved with fur/hair, which is a mammal feature, we only considered mammals to compare against from this point onwards (Fig. 1B).Fig. 2. Species identification via mitogenomes and metagenomics. (A) Iterative mapping results against 18 candidate mitogenomes for species identification. (B) Metagenomic classification of reads against a mammalian full genome reference database. In top panel, fraction of the total reads classified within Mammalia. In bottom-left panel, majority of mammalian reads are classified within the order Rodentia and suborder Hystricomorpha. In bottom-right panel, the majority of Rodentia reads are classified as Erethizon dorsatum at the species level.

The results of the iterative mapping yielded two useful indices that were combined to create a ranking for the best mitochondrial candidate matching the mummified specimen. These indices were the mean mapping quality and the number of mapped reads after applying quality filters (MQ > 30,) (Fig. 2A, Supplementary Table S2).

The mitogenome with the most mapped reads (21,454 out of the 2,491,638 generated) was Erethizon dorsatum, commonly known as the North American porcupine or “Ts’ey” in the Indigenous Hän language of the Tr’ondëk Hwëch’in whose Traditional Territory is in this region (Fig. 2A). The sequences mapped to this reference mitogenome also displayed the highest mean mapping quality (>35). The second highest number of mapped reads (7,879) corresponded to the reference mitogenome of Coendou insidiosus (Fig. 2A). The species C. insidiosus is also known as the Bahia porcupine, another New World porcupine species from South America and endemic in the Atlantic Forest of Brazil. Coendou is the closest living genus to Erethizon. However, the mean mapping quality for C. insidiosus dropped noticeably (<30). The rest of the species in the list, rodents or otherwise, displayed fewer than 1,300 mapped reads with a consistently low mean mapping quality (<10) (Fig. 2A). Following the result for the best match (E. dorsatum), we generated a consensus FASTA using majority base call weighted by the base qualities for the mitochondrial genome of specimen YG 77.11 for further phylogenetic analyses.

The breadth of coverage of this ancient mitogenome is 98%, the 2% missing corresponds with part of the hypervariable region (Fig. 3A). In total we identify 440 substitutions compared to the reference after excluding indels and heteroplasmies. This corresponds to 2.6% of the total mitogenome (16,828bp in CM051845.1) (Supplementary Fig. S2).

The phylogenetic affinities of the Yukon mummy mitogenome (Fig. 3A) were inferred using Maximum Likelihood (Fig. 3B). The phylogenetic tree placed the Yukon mummy within the Erethizontidae family (New World porcupines), in the Hystricomorpha order, alongside E. dorsatum, with bootstrap support of 100% (Fig. 3B). To gain further insight into the position of YG 77.11 within the variation of E. dorsatum, we extracted the 12S ribosomal RNA gene region of the mitochondria and aligned it to the reference (CM051845) and three other publicly available sequences (AF520694, AY012118, U12450) of this gene (12S) in the species (Supplementary Fig. S3). We find that YG 77.11 belongs to a different sub-clade or lineage (proposed nomenclature Clade Y, for Yukon-like) than that of the reference genome (proposed nomenclature Clade M, for Minnesota-like). These two sub-clades could potentially represent a East and West phylogeographic division of the population based on the information available. However, in addition to YG 77.11 and the reference, only AF520694 has associated geographic metadata. See Supplementary Fig. S3 for details.Fig. 3. Mitochondrial phylogeny. (A) Mitogenome reconstruction of sample YG 77.11 depicting per site quality, depth and presence of variants corresponding. The outer circle represents quality per base covered by nucleotides. Inner green spikes represent the depth of coverage (average 32X), which correspond to the total number of mapped reads covering each base pair. The red dots scattered within the circle represent variant sites, bases where the Yukon mummy differs from the reference genome, which include substitutions (SNPs, n=440) as well as insertions and deletions (indels, n=6). (B) Maximum likelihood tree with full mitochondrial genomes showing YG 77.11 clustering with Erethizon dorsatum.

Species validation with a metagenomic approach

We used a metagenomic approach to validate the species identification using the combined sequencing effort. Of the total of circa 2 million sequences, 8.3% of the reads were correctly classified within the mammalian reference dataset (Fig. 2B). The grand majority of the classified reads fell within the Rodentia order (7% of the total and >90% of the classified reads). Within Rodentia, the majority of sequences belong to Hystricomorpha suborder, and within Hystricomorpha the majority were identified as Erethizon dorsatum (6% of the total and >80% of the reads classified as Rodentia) (Fig. 2B).

Within the minimal reads classified as non-Rodentia, we detected Artiodactyla (0.15% of the total), Metatheria (0.06% of the total), and Primates (0.16% of the total). The provenance of the Artiodactyla and Metatheria sequences can be easily explained by the fact that the sequencing was carried out in a shared lane that also contained species that belong to those categories. Most likely due to some low levels of index hopping despite being double indexed libraries.

Genetic sex inference

Sex determination in living porcupines is not an easy task^23^, luckily genetic sex determination using DNA is a relatively simpler task.

We inferred the genetic sex of the individual by evaluating the ratio of the number of reads mapping to the X and Y chromosomes, as well as the autosomes. While genetic sexing is usually conducted for porcupines using a diagnostic Zinc Finger Protein found on the Y-chromosome, primarily for veterinary purposes^23^, the generation of whole genome data allows us to apply genome-wide methods inferring genetic sex. We applied three different methods (see Supplementary Table 3): Ry^24^ the ratio of reads mapping to the X-Chromosome to a similarly-sized autosome (Supplementary Table S3), Chr5^25^, and the ratios of reads mapping to each of the sex chromosomes to the sum of the autosomes^26^. All three tests indicate the individual is genetically male. Additionally, as genetic sex estimation is notoriously difficult in living porcupines, due to their internal genitalia and defensive spines, this method could provide researchers and veterinarians another option for genetic sex estimation from low-coverage whole-genome data.

Preservation of the specimen and ancient DNA authentication

Following the identification of the mummy as a North American porcupine, we mapped all libraries to the available reference genome for the species (mEreDor1.pri). AMBER^27,28^ results validated the authenticity of the ancient DNA through the post-mortem damage patterns at the end of the reads (Supplementary Fig. S1). Endogenous DNA content was very poor in subsample SS001a for both extraction methods (<1%) but higher in the Dabney extracted subsamples SS001b and SS001c (8%) (Supplementary Table S2). The mean read length of the mapped reads was 44.8bp. The total number of mapped reads to the reference genome with BWA aln was similar to that obtained with Kraken2 (Supplementary Table S2). Despite the relatively low endogenous DNA content, the total number of uniquely mapped reads (complexity) for all three libraries was high, ranging between 90-93% (Supplementary Table S2).

Discussion

Using both mitogenomic mapping, metagenomic classification, and radiocarbon dating we have identified the mysterious mummy YG 77.11 as a male North American porcupine (Fig. 2) of the species Erethizon dorsatum or “Ts’ey” (in the Indigenous Hän language of the Tr’ondëk Hwëch’in) dating to about 3000 years old (Fig. 1). This result is notable because fossil records of porcupines are virtually absent from the region and little is known about their phylogenetic or biogeographic history in North America. Furthermore, such a young radiocarbon age was unexpected (Fig. 1C) since the locality where it was found has yielded many remains of extinct Pleistocene mammals and because there are very few examples of mummified mammals from the permafrost dated to the Holocene.

North American porcupines (Erethizon dorsatum) are large members of the rodent family (Family Rodentia) (Fig. 3) that are best known for their coat of coarse, hollow hairs or quills which are important for defense against predation. Seven subspecies are recognized from a large geographic range that covers coniferous forests, shrublands and other habitats across most of the continent, from northern Mexico to arctic Alaska^29^. While E. dorsatum is the only species of porcupine in North America, it has adapted to a very large geographic range and eco-climatic zones.

The considerable number of differences we observe between the mitochondrial reference genome (CM051845.1) and the mummified porcupine mitogenome (Supplementary Fig. S2) may reflect population structure across the seven recognized subspecies. The 2.6% mitochondrial divergence between the reference and YG 77.11 is a relatively high number for intra-species comparisons. In the case of rodents, it is above the average (1.5%)^30^ but not as extreme as seen in certain rodents and some other species (up to 4.7%)^30^. This high value could be an indicator of the early stages of speciation^31^. Given the vast range of E. dorsatum, which occupies most of North America, and the geographic distance between the Yukon specimen and the reference mitogenome from Minnesota, as well as the temporal separation, these factors may explain the observed excess of differences. It is likely that strong population structure exists within the species, supported by this apparent deep mitochondrial divergence.

However, little is known of phylogeographic patterns of E. dorsatum and the lack of other available full mitogenomes and phylogeographic metadata precludes further analysis in this study. Nevertheless, we propose two tentative subclades for this species in Supplementary Fig. S3 based on the scarce 12S available sequences.

The fossil history of ancient porcupines in North America can be traced to a northward dispersal from South America during the Late Pliocene Great American Biotic Interchange^32^. Fossils of porcupines (Erethizon) from Pleistocene localities, from Mexico to southern Alberta reveal their presence across a variety of biogeographic zones from the late Irvingtonian Land Mammal Age (1.8-0.25 mya) through to the Sangamonian interglaciation (0.125 mya)^33–35^. While fossils of Eurasian porcupine (Hystrix) are known from the Late Pleistocene from the Altai Mountains^36^, fossils of the North American genus Erethizon are absent in Siberia, indicating that North American porcupines did not disperse across the Pleistocene Bering Land Bridge to Asia. Fossils of porcupine from mountainous terrain of both western and eastern North America reveal that climatically driven environmental change during the Holocene led to the biogeographic range shifts and local extirpations in the last few thousand years in some areas^37,38^.

Available data are lacking to firmly establish when porcupines may have first colonized the northern latitudes of Yukon and Alaska. Given the ecological preferences of present day porcupines, we hypothesize that they could only successfully inhabit the region during interglacial times when spruce-dominated boreal forest became the main biome, rather than the open, treeless steppe-tundra of the cold glacial periods^1^. However, fossil remains of porcupine are totally absent from the abundant and highly diverse Pleistocene faunas of the Old Crow Basin, Klondike goldfields or Alaska^1,34,39^, suggesting that they may have been absent regionally for much of the Quaternary, including past Pleistocene interglaciations. The only other fossil evidence for ancient porcupines in the region comes from faecal remains that are radiocarbon dated 4000 to 5000 years ago that were collected from the floor of the Porcupine River Caves in Alaska^40^, and Tsi-tche-Han Cave, in northern Yukon^41^, supporting the chronology for their initial appearance during the Holocene. Palaeoecological evidence suggests that spruce-dominated boreal forests which would serve as ideal porcupine habitat became established in the central Yukon roughly 10 kya^42^. Fossils reveal the post-glacial boreal forest was rapidly colonized by mammals typically associated with today’s boreal forest, such as moose (Alces alces) and beaver (Castor canadensis)^43,44^. Altogether, available evidence suggests that North American porcupines were not long term residents of Beringia through successive interglacial and glacial cycles, but rather they became part of this contemporary boreal forest fauna during the Holocene by at least 5000 years ago after northward dispersal from southern habitats.

The arrival of porcupines to the boreal forest of Yukon (Fig. 1(A)) during the Holocene highlights a story of colonization of new habitats in the wake of climatic changes, megafaunal extinctions and environmental turnover at the end of the Pleistocene. It also exemplifies a rarer event, new species establishing and thriving alongside human populations within a similar timeframe^45^. With archaeological evidence clearly placing people in the Yukon during the end of the late Pleistocene^46^, the dramatic environmental turnover and megafaunal extinction associated with warming climates would have posed new challenges and opportunities for people. With the establishment of the boreal forest in the early Holocene, newly arriving animals from the south such as porcupines certainly took on cultural importance for people in the region for the first time.

Across the continent, porcupines are an iconic species that bear cultural importance to Yukon First Nations and Indigenous peoples of North America for food, medicine and art in the form of quillwork^47–49^. For the Tr’ondëk Hwëch’in, quills from porcupines or “Ts’ey” have been used to decorate clothes and other personal items since long before European trade goods became available within their traditional territory in the 1840s. In fact, porcupine quills were often dyed a variety of colors using berry juice or flowers and attached to clothing in a variety of ways and patterns used to show social status of people in the community^50^. This tradition of decorating moccasins, jackets, vests, baskets, boxes and jewellery with porcupine quills remains strong and continues as an important cultural practice for the Tr’ondëk Hwëch’in and other Yukon First Nations today^51^.

The Holocene age of this porcupine (Fig. 1C) also holds significant palaeontological implications regarding the preservation of permafrost mummies. Most mummified remains from high latitude permafrost date to Pleistocene stadials or interstadial phases that were colder and drier than climatic conditions at present^4^. Mummified animals remains from Holocene permafrost are rare across the Holarctic, but do include recent spectacular examples of large mammals that survived the end Pleistocene extinctions such as steppe bison^52^, wild horse^53^ and brown bear^54^. The prevalence of Pleistocene age mummies compared to the limited examples from the Holocene are likely the result of differences in taphonomic conditions that are important for soft tissue mummification. Many Pleistocene age mummies are thought to be preserved through rapid burial by mass movements of sediments or entrapment after animals became mired in water-saturated mud^1^ or entombment in subterranean burrows or hibernacula^7,55^. By contrast, generally acidic soils in the present day and Holocene boreal forests are not conducive to preservation of bone or animal remains^56^ and few mummies exist of recent Holarctic animals^1^. While our porcupine specimen was not recovered and analysed in situ, and we do not know the particular stratigraphic or taphonomic context of its preservation, it is clear that some particular circumstances involving rapid burial, likely within Holocene organic rich sediments do may allow the successful preservation of soft tissue.

Research on this mysterious permafrost-preserved, Holocene mummy has shed light on the enigmatic history of North American porcupines in the region and the interesting possibility of finding other Holocene and interglacial mummies. The arrival of porcupines or “Ts’ey” in the far northwest latitudes of the subarctic during the Holocene signals the end point on a long biogeographic journey that started out over three million years during the Pliocene in South America^29^. From Southern Hemisphere roots, to successfully occupying a wide swath of habitats across the North American continent, porcupines have evolved and adapted through the millennia to become an animal of great cultural significance to the Tr’ondëk Hwëch’in of Yukon and Indigenous people wherever these animals are found. Additional research is needed to further resolve the history of colonization and dispersal given the almost complete lack of other palaeontological and genetic information about North American porcupines.

Methods

Sampling and radiocarbon dating

Records at the Yukon Government in Whitehorse indicate the specimen was donated to the Yukon Paleontology Program on September 1, 1999 and accessioned into the fossil collections with accession number YG 77.11. Given the presence of fossils from extinct Pleistocene fauna on the site and rarity of Holocene age mummified animals in the region, we started this research with the hypothesis that the mummified specimen was also Pleistocene in age.

We carried out sampling in the Whitehorse collection in July 2024 with permission granted by the Government of Yukon Paleontology Program. A sample of tissue from the mummified specimen (YG 77.11) was collected and labelled OC24-136. In the laboratory, the different tissues of the sample were further subsampled based on appearance (SS001a, SS001b, SS001c), ranging in weight from 93 to 215mg.

Tissue from the specimen (YG 77.11) was chemically pretreated in preparation for radiocarbon dating at Higham Lab (University of Vienna)^57,58^. Samples were measured at the Keck AMS facility (University of California at Irvine)^59,60^. Radiocarbon dates are reported here both in radiocarbon years Before Present (BP) (year 1950 CE) using the half-life of 5,568 years and calibrated calendric ages (Supplementary Table S1)^61^.

We conducted all laboratory activities at the Centre for Palaeogenetics (CPG), Stockholm University, Sweden. Analyses prior to DNA amplification, including tissue subsampling, DNA extraction, and library preparation, were carried out inside the ancient DNA (aDNA) laboratory. All post-PCR procedures, such as DNA amplification and magnetic bead cleaning, were performed in a separate building, in a designated post-PCR facility^62^ at Stockholm University. The facilities adhere to the technical requirements of aDNA laboratories^63^.

DNA extraction and library preparation

Three subsamples of the tissue (SS001a (D), SS001b (D), and SS001c (D)) were extracted following the Dabney protocol (D), a method designed to optimise DNA recovery from ancient and degraded short aDNA sequences^64,65^. Additionally, three more 20mg subsamples of tissue were extracted with the Qiagen Blood and Tissue kit (Q) to evaluate the potential recovery of longer endogenous DNA fragments. Unfortunately, of these three Qiagen kit extractions, only SS001a (Q) was successful; the other two could not be completed due to clogging of the membrane, likely due to tissue, protein or lipid residuals that could not be fully removed despite centrifugation and pelleting after thorough digestion. Double-stranded DNA (dsDNA) sequencing libraries^66^ were built and sequenced on the Illumina NovaSeqX platform. Libraries for the Yukon mummy (YG 77.11) SS001a (D), SS001b (D), SS001c (D), and SS001a (Q) were prepared according to the protocol without USER enzyme treatment. A quantity of 20 µL of the total 50 µL DNA extract was used to create each genomic library.

Data processing and bioinformatic analyses

The raw sequencing data were processed to trim adapter sequences with Cutadapt v.3.5^67^. Forward and reverse sequences were merged into a single read following the removal of polyX tails with Fastp v0.24.0^68^.

For mapping to various reference genomes (mEreDor1.pri and other mitochondrial sequences), we used BWA v0.7.18^69^ with “aln” algorithm and standard aDNA specifications (-l 16500 -n 0.01 -o 2)^70^. The resulting BAM files^71^ were indexed and further handled with SAMtools v1.20^72^. For evaluation of the alignment of sequencing data and mapping performance, the individual BAM alignment files were run on Qualimap^73,74^, both before and after applying quality filters using SAMtools.

To visualise aDNA damage patterns, fragment length distribution, and potential mapping biases, we used AMBER^27^. Based on the rate of mismatches per base-pair obtained with AMBER (Supplementary Fig. S1), a minimum read length threshold of 30 basepairs (bp) was established. We also excluded all reads with mapping quality (MQ) below 30 and removed all PCR duplicates using SAMtools. We determined the genetic sex of the specimen by evaluating the ratio of mapping reads between the X (GenBank ID: CM051842) and Y (GenBank ID: CM051843) chromosomes, using the Ry method^24^ and other metrics.

Mitochondrial phylogenetic analyses

We generated a list of 18 candidate mitochondrial reference genomes (see Fig. 1A), based on the known biodiversity of the region.

We performed BWA “aln” mapping of the data from data from all combined libraries against the candidates species in this set of reference mitogenomes to identify the specimen’s taxonomic affiliation based on the most significant alignments obtained. The best mapping stats corresponded to reference mitogenome CM051845 E. dorsatum.

The new consensus mitogenome was obtained with SAMtools consensus applied on the deduplicated and quality filtered BAM mapped to CM051845. The resulting mitochondrial sequence was further evaluated with BLAST (Basic Local Alignment Search Tool)^75^. The resulting consensus ancient mitogenome corresponds to accession number PV951521.

Prior to tree construction, the reconstructed mitogenome of YG 77.11 was aligned using MAFFT v7^76^ against mitochondrial genomes from 23 other rodent species representing multiple families, including Sciuridae, Castoridae, Hystricidae, Erethizontidae, Caviidae, Chinchillidae, and Echimyidae. The multiple alignment was subsequently analysed in IQ-TREE v2^77^, which identified the best-fit substitution model for ML inference as TIM2+F+I+G4 according to the Bayesian Information Criterion (BIC). The resulting phylogeny was exported in Newick format and visualised with iTOL v7^78^. The 12S phylogenetic tree was built with the same methodology with the alignment restricted to the five E. dorsatum sequences and the outgroup.

Finally, the circlize R package was used to generate a circular map of the Yukon specimen’s mitochondrial genome following variant calling with BCFtools v1.20^79^ and subsequent quality filtering.

Metagenomic analysis

We cross-validated our mapping output with large databases with a broad coverage of biodiversity. A microbial reference was constructed using 402,709 assemblies sourced from the Genome Taxonomy Database (GTDB) v214.0 by Jin et al.^80^ . This reference uses the default settings of Kraken2-build (Wood et al 2019), including 4,416 archaeal and 80,789 bacterial species clusters^81^. Initially, microbial reads were filtered out by retaining only those that remained unclassified against the GTDB database using Kraken2 v2.1.2^82^. Subsequently, these unclassified reads underwent further classification against a Kraken 2 mammalian database, which includes 742 assemblies of different species (retrieved in April, 2023). For each mammalian species, assemblies were selected based on the highest contig N50 when multiple assemblies of the same species were available^80^.

Supplementary Information

Supplementary Information.