When the Gene Pool Shrinks: What DNA Reveals About Inbreeding
It is well known that prolonged inbreeding can have serious consequences for a population. The reason is relatively simple. All of us carry some harmful recessive genetic variants without knowing it. Usually this causes no problem because we possess another, functioning copy of the gene.
If two unrelated people have children, they are relatively unlikely to carry exactly the same rare harmful variant. Close relatives, however, have inherited much of their DNA from the same ancestors. They are therefore more likely to carry the same recessive variant and more likely to pass two copies of it to a child.
Perhaps the easiest way to understand this is through the inbreeding coefficient, usually written as F. This expresses the probability that the two copies of a genetic region inherited by a child are identical by descent from the same ancestor.
The Habsburg experiment
Perhaps the consequences of this process are seen most dramatically in some of the royal families of Europe, where marriage between close relatives was repeatedly used to preserve territory, wealth and dynastic power.
The Spanish Habsburgs provide an extraordinary example. Genealogical research has calculated an inbreeding coefficient of 21.8% for Philip III of Spain and 25.4% for Charles II of Spain. Charles II therefore reached approximately the theoretical inbreeding level expected in the child of a brother and sister, even though his own parents were actually uncle and niece. The explanation is that both of his parents were themselves descended from generations of interrelated Habsburg ancestors.
This distinction is important. Charles did not acquire his extraordinary inbreeding level from one close marriage alone. It was the accumulated effect of repeated marriages between relatives over many generations.
One outward characteristic associated with the dynasty was the famous “Habsburg jaw”, or mandibular prognathism, in which the lower jaw projects prominently forward. A modern study comparing Habsburg portraits with their pedigrees found a significant relationship between the degree of mandibular prognathism and the calculated level of inbreeding.
Charles II also suffered from an extraordinary collection of health problems. It is impossible centuries later to prove that every one of them was caused by inbreeding, but his extreme level of consanguinity almost certainly increased his genetic vulnerability.
Modern medical reviews of Charles II describe developmental delay, dysarthria, skeletal deformities, recurrent infections, epilepsy and infertility among his many problems.
Tutankhamun: another royal example
A similar story may be seen in ancient Egypt.
DNA analysis published in 2010 concluded that the two mummies identified as Tutankhamun's parents were brother and sister. If so, his theoretical pedigree inbreeding coefficient would have been approximately 25% — strikingly close to the calculated 25.4% of Charles II.
Tutankhamun certainly suffered from physical problems. CT and genetic investigations identified abnormalities including problems with his feet and avascular bone necrosis, and malaria DNA was also detected. However, we should be careful about saying that these disorders were caused by inbreeding. His sibling parentage would have increased the probability of harmful recessive variants becoming homozygous, but it is not possible to attribute each of his conditions directly to consanguinity.
There is also an important difference between Tutankhamun and the ancient individuals whose complete genomes can now be examined. The famous Egyptian mummy study used genetic markers to establish relationships; it did not provide the sort of modern high-coverage whole-genome measurement of runs of homozygosity that can now be obtained from some ancient remains.
Ancient DNA allows us to see the process directly
Modern ancient-DNA techniques have transformed this subject. Instead of relying only upon a known family tree and calculating what the level of inbreeding should have been, scientists can examine an individual's genome for runs of homozygosity, usually abbreviated to ROH.
These are long stretches of DNA where the copies inherited from the mother and father are unusually similar because both ultimately came from shared ancestors.
The length and number of these stretches are particularly informative. Very long runs tend to indicate recent close-relative mating, while large numbers of shorter runs can reveal something rather different: a population that remained small and isolated for many generations.
That distinction becomes particularly interesting here in Malta.
The extraordinary genomes from Xagħra Circle
On Gozo, Malta's neighbouring island, the Xagħra Circle contained the remains of hundreds of people who lived during Malta's remarkable Neolithic Temple Period.
Researchers attempted to recover ancient DNA from several individuals. Three — Xaghra5, Xaghra6 and Xaghra9 — produced sufficiently good genome-wide DNA for detailed analysis. The researchers found that all three genomes contained unusually high levels of runs of homozygosity, indicating substantial inbreeding in their ancestry.
Xaghra9 was exceptional.
The researchers estimated that 19.12% of Xaghra9's genome was contained within runs of homozygosity. At the time the study was published, his was the second most extreme level of long ROH reported from prehistoric human remains, exceeded by the extraordinary individual buried at Newgrange in Ireland.
But Xaghra9 presents an interesting puzzle.
He was not a Spanish Habsburg with a documented family tradition of marriages between uncles, nieces and cousins. Nor does his genome look exactly like that of the straightforward child of one brother–sister union.
Instead, researchers found a mixture of long and shorter ROH. Their interpretation was that his extraordinary genome probably resulted from a combination of recent marriages between relatives and a population that had already been very small and genetically isolated for generations. They specifically cautioned against assigning Xaghra9 one precise parental relationship.
The other two Xagħra genomes reinforce that interpretation because, although less extreme than Xaghra9, they also contain unusually elevated homozygosity. One predates him by roughly four centuries. This suggests that the problem was not confined to one unfortunate family.
Just how small was the population?
The genetics offer an extraordinary glimpse of the size of this prehistoric breeding community.
Using one method, researchers estimated an effective population size of approximately 515 individuals, with a 95% confidence interval of 397–633. Another analysis gave a 30-generation average effective population size of only 382.
“Effective population size” does not mean that precisely 382 or 515 people were living on Gozo. It is a genetic concept referring approximately to the number of individuals contributing genes to subsequent generations. The actual population could have been larger.
Nevertheless, the result is striking. The researchers concluded that mating networks appear largely to have remained within the island, producing a genomic signature of an unusually small and restricted population over at least 400 years.
This makes Xaghra9 particularly interesting when compared with Charles II.
Charles II's 25.4% is a pedigree prediction based upon his known ancestry. Xaghra9's 19.12% is a measurement derived from his actual ancient genome. The numbers are therefore not precisely equivalent measurements, but both point towards an extraordinary degree of shared ancestry.
A society approaching its end
The story becomes even more intriguing because Xaghra9 and Xaghra5 lived close to a turning point in Maltese prehistory.
For more than a thousand years the inhabitants of Malta and Gozo had created one of prehistoric Europe's most extraordinary cultures. They built the enormous megalithic complexes at Ġgantija, Ħaġar Qim, Mnajdra and Tarxien, along with elaborate underground burial places.
Yet during the later third millennium BC this distinctive Temple culture disappeared.
There is unlikely to have been one simple cause. Archaeological and environmental evidence points towards a combination of pressures:
The ancient-DNA researchers themselves noted that Xaghra5 and Xaghra9 date from a period associated with a declining density of radiocarbon dates, worsening diet and nutritional status, increasing aridity and thinning soils.
Inbreeding therefore need not have caused the collapse of Malta's Temple culture. It may instead reveal something equally important: a population becoming dangerously small and isolated at precisely the time when environmental and nutritional pressures were increasing.
And then there is Newgrange
Perhaps the most extraordinary comparison comes from Ireland.
DNA was recovered from an adult man whose remains had been placed in the most elaborate recess of the great Newgrange passage tomb, constructed around 3200 BC. His genome revealed something remarkable.
His parents were first-degree relatives. Genetically, that means the relationship was consistent with either: brother and sister, or parent and child. Both relationships would normally produce an expected inbreeding coefficient of approximately 25% in their offspring.
Unlike Xaghra9, whose unusual combination of ROH probably reflects both recent relatedness and generations of life within a small isolated population, the Newgrange man's genome showed the unmistakable signature of an extremely close, first-degree incestuous union. The researchers described him as the adult son of such a union.
His burial place makes the discovery even more intriguing. He was not deposited in an insignificant grave but in the most prestigious part of one of prehistoric Europe's greatest monuments. Researchers have therefore suggested that he belonged to a powerful politico-religious elite in which such otherwise strongly prohibited relationships may have been deliberately permitted to preserve a sacred or dynastic bloodline. That interpretation is fascinating, although it cannot be proved from one individual alone.
So across thousands of years we can see very different routes leading towards the same genetic danger.
With Charles II, repeated dynastic marriage gradually accumulated relatedness generation after generation.
With Tutankhamun, the evidence points towards sibling parents within an Egyptian royal dynasty.
With Xaghra9, the genome tells of life within a tiny, isolated island breeding population, probably combined with relatively close marriages.
And at Newgrange, DNA reveals something more immediate and extreme: a man whose mother and father were themselves first-degree relatives.
It is extraordinary that thousands of years after these people died, their DNA can reveal not merely where their ancestors came from, but something as intimate as how closely related their parents were and how isolated the communities in which they lived had become.
