Recent genomic research conducted by Colossal Biosciences has revolutionized our understanding of dire wolf (Aenocyon dirus) evolution, revealing a more complex ancestry than previously thought. These insights not only informed the successful revival of dire wolves but also resolved long-standing questions about these iconic Ice Age predators.

Until recently, dire wolves were thought to represent a distinctly separate lineage from modern canids. A 2021 paper titled “Dire wolves were the last of an ancient New World canid lineage” suggested minimal genetic exchange with other wolf species. However, comprehensive new research based on higher-quality ancient DNA samples has revealed a more nuanced evolutionary history.

Colossal’s scientists extracted and deeply sequenced DNA from two dire wolf fossils: a tooth from Sheridan Pit, Ohio, approximately 13,000 years old, and an inner ear bone from American Falls, Idaho, around 72,000 years old. This yielded unprecedented coverage of the dire wolf genome—more than 500 times more genetic data than was previously available.

“Our novel approach to iteratively improve our ancient genome in the absence of a perfect reference sets a new standard for paleogenome reconstruction,” explained Dr. Beth Shapiro, Colossal’s Chief Science Officer. “Together with improved approaches to recover ancient DNA, these computational advances allowed us to resolve the evolutionary history of dire wolves and establish the genomic foundation for de-extinction.”

The analysis revealed that gray wolves are indeed the closest living relatives of dire wolves, with dire wolves and gray wolves sharing 99.5% of their DNA code. This close relationship made gray wolves the ideal donor species for the de-extinction process.

Perhaps most surprising, the research showed that dire wolves have a hybrid ancestry. The dire wolf lineage emerged between 3.5 and 2.5 million years ago through hybridization between two ancient canid lineages: an ancient member of the tribe Canini (possibly represented in the fossil record as Eucyon or Xenocyon) and a lineage that was part of the early diversification of wolf-like canids.

This discovery helps explain previous uncertainties about dire wolf origins. The hybrid ancestry created conflicting signals in the genetic data, making it difficult to place dire wolves definitively within the canid family tree without comprehensive genome sequencing.

The research team identified multiple genes undergoing positive selection in dire wolves, linked to skeletal, muscular, circulatory, and sensory adaptations that helped these predators thrive during the Pleistocene. They also discovered dire wolf-specific variants in key pigmentation genes, revealing that dire wolves had a white coat color—a trait impossible to determine from fossil remains alone, but consistent with adaptations to Ice Age environments.

This evolutionary history informed Colossal’s approach to reviving dire wolves. By understanding which genetic variants contributed to dire wolf-specific traits, scientists could target specific genes for editing. They focused on 14 genes with 20 distinct genetic variants that distinguish dire wolves from other canids, including genes influencing size, musculature, coat color, and other characteristics.

The research also revealed an intriguing pattern of genetic exchange between ancient canid populations. While earlier studies suggested dire wolves evolved in isolation from other canids, the new analysis indicates periods of interbreeding between ancestral dire wolf populations and the lineages that led to modern gray wolves.

“The dire wolf genome has protein-coding substitutions in three essential pigmentation genes: OCA2, SLC45A2, and MITF, which directly impact the function and development of melanocytes,” explains a Colossal report. These discoveries allowed scientists to prioritize specific genetic targets when creating the revived dire wolves.

This advanced understanding of dire wolf genomics has broader implications for conservation biology. The techniques developed to analyze degraded ancient DNA can be applied to modern conservation challenges, such as recovering genetic information from rare specimens of critically endangered species or understanding genetic adaptations that might help species survive in changing environments.

“Functional de-extinction uses the safest and most effective approach to bring back the lost phenotypes that make an extinct species unique,” said Dr. Shapiro. “We turn to ancient DNA to learn as much as we can about each species and, whenever possible, to link specific extinct DNA sequence variants to each key trait.”

The dire wolf genomic research has been published in a preprint paper titled “On the Ancestry and Evolution of the Extinct Dire Wolf,” making this valuable data available to the scientific community. The authors identified 80 genes evolving under diversifying selection in dire wolves, providing insight into the adaptive forces that shaped these remarkable predators.

By resolving long-standing questions about dire wolf evolution and ancestry, this research not only facilitated their revival but also enhanced our understanding of canid evolution more broadly. The discovery of the dire wolf’s hybrid origins adds to growing evidence of the importance of hybridization as an evolutionary force—a finding with implications for how we conceptualize species boundaries and conservation strategies in the present day.

As these de-extinct dire wolves continue to develop, they offer an unprecedented opportunity to observe how these ancient genetic variants manifest in living animals, potentially providing new insights into the biology and behavior of one of North America’s most iconic extinct predators.

Published by HOLR Magazine.