A century of polar bear history, written in DNA.

Inside a remarkable collection

By the shores of the Neva River, in the heart of Saint Petersburg, stands the Zoological Institute of the Russian Academy of Sciences. Its collection contains around 60 million items representing roughly 25% of the world’s fauna.

The polar bear collection reaches back to the heroic age of polar exploration in the mid-1800s. Most specimens date from the 1930s–1940s, when the Russian North was being explored under the Northern Sea Route development plan. The catalogue records donations by explorers including Alexander von Bunge, Leonid Starokadomsky and Boris Vilkitsky.

We were privileged to work within this institution for the following months.

Sampling the archive

The collection mainly consists of complete bear skulls, sometimes accompanied by skeletons and skins. Skeletal remains can preserve DNA for hundreds of thousands of years; cranial base bone and teeth are especially valuable.

Teeth act like sealed boxes, with a lower risk of modern or environmental contamination. We therefore use single-rooted incisors and premolars. Each tooth is carefully removed with dental instruments supplied by MARKA dental clinic, barcoded and photographed together with its skull and catalogue information.

The collection contains more than 450 skulls, including specimens from Indigenous sacred sites and hunting trophies collected before the 1956 hunting ban. We chose a representative subset across geography and time.

Building a clean workflow

Extracting small quantities of degraded DNA from skeletal tissue is difficult. Short fragments must be separated from organic compounds that inhibit the enzymatic steps preceding sequencing, while characteristic lesions can introduce errors during DNA replication.

Ancient DNA is normally handled in costly clean rooms that require complete cleaning after every sample. The Historical Genetics Lab at MIPT uses an alternative: each procedure takes place in a clean glove box. The boxes are easier to operate, reduce contamination and make a safe processing “conveyor” possible.

With the current protocol and facility, one researcher can extract DNA from six samples per day while maintaining quality and avoiding cross-contamination.

From tooth to DNA library

The process begins by removing dust, glue and chemicals from museum teeth. Samples are sonicated, treated with UV light and sterilised, then cut with dental drills. Small fragments are milled into powder, weighed and transferred to storage tubes.

DNA extraction and sequencing-library construction are performed in clean boxes. By the end of this phase, more than 100 samples of different origins and ages had been extracted. Two samples were selected for shallow whole-genome sequencing to evaluate contamination and the value of low-concentration libraries.

Modern samples for comparison

The project compares historical and modern polar bear DNA. Our collaborators at the Marine Mammal Research and Expedition Centre supplied teeth and tissue samples — blood, muscle and skin — collected primarily from living animals across the Russian Arctic.

First sequencing results

The first sequencing run tested our library protocol, measured contamination and examined whether low-concentration samples could provide useful results without enrichment.

We sequenced ZUM002, collected on Novaya Zemlya in 1883, and ZUM323, collected in the Kara Sea in 1930. After filtering low-quality reads, 62.2% of ZUM002 fragments and 98.6% of ZUM323 fragments aligned to the polar bear genome. At 3× or greater coverage, they represented 9% and 21% of the genome respectively.

A preliminary comparison with ten Canadian polar bear samples revealed a clear distinction between the Russian historical specimens and published data — an encouraging early result.

Testing the hyRAD approach

Museum samples are invaluable but often contain degraded DNA. The hyRAD protocol was designed to sequence reduced-genome subsets in non-model organisms using fresh-sample ddRAD fragments as capture probes for historical libraries.

This can recover large sets of homologous loci without prior genome information, but the experimental protocol is demanding. We followed the approach of Suchan et al., with modifications developed for our material.

Three hundred extractions completed

DNA extraction was completed for all 252 historical museum teeth and 48 modern samples — muscle, whole blood, skin biopsies and teeth collected between 1975 and 2022 from Novaya Zemlya to the Chukchi Peninsula. The modern material was provided by Dr Andrei Boltunov of the Marine Mammal Research and Expedition Centre.

Quality control

Every historical and modern extraction underwent quality control for DNA concentration, degradation and purity.

Changing course to low-coverage genomes

hyRAD combines ddRAD probes from fresh samples, historical DNA libraries and hybridisation capture. After repeated attempts to establish reliable ddRAD probe conditions, we decided to discontinue this route.

Instead, we adopted low-coverage whole-genome sequencing (lcWGS). hyRAD can deliver high coverage at a targeted set of loci across many samples; lcWGS surveys many more loci distributed throughout the genome, although at lower read depth and for fewer samples.

Selecting libraries for sequencing

From 252 historical extractions, 131 samples were selected for library preparation based on DNA yield, geography and collection date. Concentration, fragment size and quality were measured; libraries containing primer or adapter dimers were purified with magnetic beads. In total, 118 libraries met the quality and quantity requirements for sequencing.

Ninety-six libraries sequenced

The historical libraries were sent to Genetico in Moscow. A second quality-control pass confirmed that all 118 were ready; 96 were then selected to balance fragment size, geography and collection date.

Sequencing was performed on an Illumina NovaSeq 6000 with S2 chemistry in paired-end 2×100 mode.

Reading damage, contamination and population history

We began by measuring exogenous bacterial and archaeal contamination against endogenous polar bear DNA. Results varied among samples, with older specimens generally showing more exogenous DNA and less polar bear DNA.

Reads were aligned to the polar bear reference genome. The sequences showed the expected signatures of historical DNA: fragmentation through depurination and post-mortem cytosine deamination.

Population-genetic analyses are now using these alignments to reconstruct past population structure and genetic diversity.