Previous analyses of the hominins fossils from Sima de los Huesos ("pit of bones") in 2013 showed that their maternally inherited mitochondrial DNA was distantly related to Denisovans, extinct relatives of Neanderthals in Asia.
This was unexpected since their skeletal remains carry Neanderthal-derived features.
Researchers at Max Planck Institute for Evolutionary Anthropology in Germany have since worked on sequencing nuclear DNA from fossils from the cave, a challenging task as the extremely old DNA is degraded to very short fragments.
The results now show that the Sima de los Huesos hominins were indeed early Neanderthals. They may have acquired different mitochondrial genomes later, perhaps as the result of gene flow from Africa.
Until now it has been unclear how the 28 fossils of individuals found at the Sima de los Huesos site in Northern Spain were related to Neanderthals and Denisovans who lived until about 40,000 years ago.
A previous report based on analyses of mitochondrial DNA from one of the specimens suggested a distant relationship to Denisovans, which is in contrast to other archaeological evidence, including morphological features that the Sima de los Huesos hominins shared with Neanderthals.
"Sima de los Huesos is currently the only non-permafrost site that allow us to study DNA sequences from the Middle Pleistocene, the time period preceding 125,000 years ago," said Matthias Meyer from Max Planck Institute for Evolutionary Anthropology.
"We have hoped for many years that advances in molecular analysis techniques would one day aid our investigation of this unique assembly of fossils," said Juan-Luis Arsuaga from the Complutense University in Spain.
"We have thus removed some of the specimens with clean instruments and left them embedded in clay to minimise alterations of the material that might take place after excavation," he said.
The nuclear DNA sequences recovered from two specimens secured in this way show that they belong to the Neanderthal evolutionary lineage and are more closely related to Neanderthals than to Denisovans.
This finding indicates that the population divergence between Denisovans and Neanderthals had already occurred by 430,000 years ago when the Sima de los Huesos hominins lived.
"These results provide important anchor points in the timeline of human evolution. They are consistent with a rather early divergence of 550,000 to 750,000 years ago of the modern human lineage from archaic humans," said Svante Paabo from Max Planck Institute for Evolutionary Anthropology.
The findings were published in the journal Nature.