Tiny DNA fragments reveal two extinct Galápagos tortoise lineages

Less than 1% of a genome might look like too little to reconstruct an animal’s past. In old Galápagos giant tortoise bones, however, those scattered fragments were enough to place two extinct lineages back onto the islands’ evolutionary family tree.

A Yale-led team combined several computational tools to analyze ultra-low-coverage genomes from historical museum specimens. The approach identified distinct extinct tortoise lineages from San Cristóbal and Santa Fe, even when sequencing recovered only a tiny fraction of their nuclear DNA.

The work shows how specimens once considered too degraded for conventional genomic analysis can still preserve useful evolutionary information. It may also help conservation programs search living hybrid tortoises for surviving pieces of genomes from extinct island populations.

Ancient and historical DNA breaks apart over time. Contamination and chemical damage can further complicate the signal, while most positions in an extremely low-coverage genome may have no readable DNA at all.

The researchers worked with dried bones from five focal museum specimens: three Historical San Cristóbal tortoises and two from Santa Fe. Their nuclear genome coverage ranged from only 0.008× to 0.139×. In some specimens, just 0.7% of sequenced DNA actually came from the tortoise.

Yet even those incomplete samples retained millions of observations. Individual low-coverage genomes contained between 6.9 million and 153.2 million called genotypes.

“The challenge of this study was to reconstruct the genetic relationships of extinct Galapagos giant tortoises, knowing that the DNA we were able to obtain from museum specimens was going to be highly degraded and fragmented and have a high proportion of contamination,” said lead author Alexander Ochoa.

Instead of building one family tree that mixed incomplete specimens directly with high-quality genomes, the team first constructed a reference tree. It used 39 contemporary tortoises, seven higher-quality historical samples and about 1.1 billion genomic sites.

Researchers then used EPA-ng to determine where each damaged specimen best fit on that established tree. They repeatedly drew random subsets of the genetic sites actually observed in each sample and performed 100 placements per ultra-low-coverage genome.

The strategy mattered. When the researchers directly combined the heavily incomplete genomes with the reference samples, the Historical San Cristóbal tortoises clustered on unusually long branches in a position that conflicted with the more reliable analysis.

That result showed how missing information can create misleading similarities. The preferred method limits the comparison to observed positions, then checks the placement against independent population-genetic analyses.

The three Historical San Cristóbal specimens repeatedly clustered near another set of museum tortoises whose original geographic records were uncertain. Those specimens, called UGO, had previously been difficult to place confidently.

Additional analyses made the relationship especially strong. Admixfrog assigned at least 99.86% of 500-kilobase genomic segments from each Historical San Cristóbal tortoise to the UGO group. NGSadmix assigned more than 99.9% of their genomic variation to UGO.

The researchers concluded that the UGO specimens were probably members of the same extinct Historical San Cristóbal lineage.

That lineage was distinct from tortoises living on San Cristóbal today. Earlier mitochondrial estimates place the divergence between the historical and contemporary lineages at roughly 0.72 million years ago.

One specimen, CAS_8133, carried a more complicated signal. Most of its nuclear DNA matched the extinct Historical San Cristóbal and UGO group, but its mitochondrial DNA matched the contemporary San Cristóbal lineage. The researchers suggest the populations may once have come back into contact.

Santa Fe presented a different historical puzzle. Because only incomplete remains had been found there, including charred bones, researchers could not rule out the possibility that sailors transported tortoises from another island and butchered them on Santa Fe.

Nuclear DNA argued against that explanation.

All 100 placement tests for each Santa Fe specimen landed on the branch leading to Española tortoises, showing a close relationship. But principal component analysis placed the Santa Fe samples in their own genetic group rather than directly among Española animals.

The team therefore concluded that the Santa Fe tortoises represented a distinct evolutionary lineage rather than recently transported Española individuals.

Their mitochondrial DNA was less straightforward. It grouped loosely with Historical San Cristóbal, UGO and Pinta tortoises, raising the possibility of a more complicated colonization history. Resolving that history would require better nuclear genomes from additional Santa Fe specimens, which the researchers note may not exist.

The analysis strongly supports Historical San Cristóbal and Santa Fe as genetically distinct lineages, but the researchers did not formally classify either as a separate species.

That would require dedicated species-delimitation analyses and a broader taxonomic investigation.

The distinction matters because genomic placement answers one question, evolutionary relationship, without automatically settling another, formal taxonomy.

The study instead demonstrates how much information can survive inside samples that conventional analyses might discard. Even at 0.008× coverage, carefully filtered fragments could recover meaningful relationships when placed against high-quality genomic references.

For Galápagos conservation, identifying extinct lineages provides a clearer genetic target for breeding programs. Sailors historically moved tortoises between islands, and some displaced animals survived and bred with local populations.

As a result, pieces of genomes from extinct populations can remain inside living hybrids. Genetic screening could help identify animals carrying ancestry from lost lineages and guide pairings intended to increase that ancestry across future generations.

“In this regard, captive breeding programs that mate these hybrids may be able to recover the genomes of the extinct lineages in future generations,” Ochoa said.

The computational approach reaches beyond giant tortoises. Natural history collections contain many specimens whose DNA is fragmented, contaminated or available only in tiny amounts. Treating those samples as unusable can erase genetic evidence from populations that no longer exist.

By extracting reliable signals instead of trying to reconstruct every missing genotype, the new method provides another way to connect museum collections with modern conservation genetics. In the Galápagos, a few scattered pieces of DNA have already restored two lost branches of the tortoise family tree.

These studies show how museum DNA, whole genomes and genetic ancestry have reshaped understanding and restoration of Galápagos giant tortoises.

A new lineage of Galapagos giant tortoises identified from museum samples: Historical mitochondrial DNA revealed a previously unrecognized San Cristóbal lineage and established the foundation for later nuclear-genome work on those museum specimens. (Heredity, 2022)

The Galapagos giant tortoise Chelonoidis phantasticus is not extinct: Whole-genome comparisons showed that a tortoise found alive on Fernandina in 2019 belongs to the same distinct lineage as the island’s historical 1906 specimen. (Communications Biology, 2022)

Synteny Enabled Upgrade of the Galapagos Giant Tortoise Genome Improves Inferences of Runs of Homozygosity: Researchers improved the extinct Pinta tortoise reference genome without new sequencing, demonstrating another computational route for extracting better conservation information from limited genomic resources. (Ecology and Evolution, 2025)

Research findings are available online in the journal Proceedings of the Royal Society B Biological Sciences.

The original story "Tiny DNA fragments reveal two extinct Galápagos tortoise lineages" is published in The Brighter Side of News.

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