Abstract The kings of the Spanish Habsburg dynasty (1516–1700) frequently married close relatives in such a way that uncle-niece, first cousins and other consanguineous unions were prevalent in that dynasty. In the historical literature, it has been suggested that inbreeding was a major cause responsible for the extinction of the dynasty when the king Charles II, physically and mentally disabled, died in 1700 and no children were born from his two marriages, but this hypothesis has not been examined from a genetic perspective. In this article, this hypothesis is checked by computing the inbreeding coefficient (F) of the Spanish Habsburg kings from an extended pedigree up to 16 generations in depth and involving more than 3,000 individuals. The inbreeding coefficient of the Spanish Habsburg kings increased strongly along generations from 0.025 for king Philip I, the founder of the dynasty, to 0.254 for Charles II and several members of the dynasty had inbreeding coefficients higher than 0.20. In addition to inbreeding due to unions between close relatives, ancestral inbreeding from multiple remote ancestors makes a substantial contribution to the inbreeding coefficient of most kings. A statistically significant inbreeding depression for survival to 10 years is detected in the progenies of the Spanish Habsburg kings. The results indicate that inbreeding at the level of first cousin (F = 0.0625) exerted an adverse effect on survival of 17.8%±12.3. It is speculated that the simultaneous occurrence in Charles II (F = 0.254) of two different genetic disorders: combined pituitary hormone deficiency and distal renal tubular acidosis, determined by recessive alleles at two unlinked loci, could explain most of the complex clinical profile of this king, including his impotence/infertility which in last instance led to the extinction of the dynasty.

Citation: Alvarez G, Ceballos FC, Quinteiro C (2009) The Role of Inbreeding in the Extinction of a European Royal Dynasty. PLoS ONE 4(4): e5174. https://doi.org/10.1371/journal.pone.0005174 Editor: Marc Bauchet, Max Planck Institute for Evolutionary Anthropology, Germany Received: December 4, 2008; Accepted: March 13, 2009; Published: April 15, 2009 Copyright: © 2009 Alvarez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing interests: The authors have declared that no competing interests exist.

Introduction The Habsburg dynasty (also known as the House of Austria) was one of the sovereign dynasties of Europe. The Spanish branch of this dynasty ruled over the Spanish kingdoms from 1516 to 1700. Under Habsburg rule, Spain reached the zenith of its influence and power in Europe and the world-wide Spanish Empire reached its apogee. The last king of the Spanish Habsburg dynasty was Charles II. He was physically disabled, mentally retarded and disfigured. He proved impotent since no children were born from his two marriages. When Charles II died in 1700 the line of the Spanish Habsburgs died with him and a new dynasty- the French Bourbons- was installed in Spain. In the historical literature it is frequently speculated that strong preference for consanguineous marriages within the Spanish Habsburg line contributed to its extinction. In order to keep their heritage in their own hands, the Spanish Habsburgs began to intermarry more and more frequently among themselves and the result, in a few generations, was a fatal inbreeding that brought the male line of the Spanish Habsburgs to extinction [1]–[5]. This hypothesis of inbreeding, the consequence of the mating of relatives, as a major factor responsible for the extinction of the Spanish dynasty is very suggestive but is based on historical data which have not been examined from a genetic perspective. This is precisely the main goal of the present study. Three lines of evidence support the inbreeding hypothesis. First of all, the incidence of consanguineous marriages in the Spanish Habsburgs was remarkable (Table 1). The Spanish Habsburg dynasty was founded by Philip the Fair (Philip I), son of the Holy Roman Emperor Maximilian I, by marrying Joanna the Mad (Joanna I of Castile and Aragon) daughter of the Catholic kings, Ferdinand of Aragon and Elizabeth of Castile and during a period of approximately two hundred years a total of 11 marriages were contracted by the Spanish Habsburg kings. Most of these marriages were consanguineous unions: two uncle-niece marriages (Philip II with his niece Anna of Austria and Philip IV with his niece Mariana of Austria), one double first cousin marriage (Philip II with his first wife Mary of Portugal), one first cousin marriage (Charles I, Holy Roman Emperor as Charles V, with Isabella of Portugal), two first cousins once removed marriages, one second cousin marriage and two third cousin marriages. In total, 9 (81.8%) of 11 marriages were consanguineous unions in a degree of third cousins or closer. Second, king Charles II suffered from many different disorders during his life and some of them might be the result of the increase in homozygosity due to inbreeding since the progeny of closely consanguineous couples has an increased incidence of disorders and, in general, detrimental health effects caused by the expression of rare, deleterious recessive alleles inherited from common ancestors [6]–[8]. Third, infant and child mortality was very high in the Spanish Habsburg families. From 1527 to 1661, when Philip II and Charles II were born respectively, the Spanish royal families had 34 children, 10 (29.4%) of them died before 1 year, and 17 (50.0%) of these children died before 10 years [5]. These figures are clearly higher than the mortality rates registered for contemporary Spanish villages which include families belonging to a wide range of social classes and where, for example, infantile mortalities were about 20% [5]. These data suggest that inbreeding depression for infant and child survival could be occurring in the Spanish Habsburg families as a consequence of prolonged consanguineous marriages. It must be emphasized, however, that all these evidences supporting the hypothesis of inbreeding as an important factor in the extinction of the Spanish Habsburg lineage are indirect and, therefore, they are not conclusive. Thus, it has not been demonstrated that the physical and mental disabilities suffered by the king Charles II were caused by the expression of detrimental recessive alleles inherited from common ancestors. These detrimental health effects could be due to either environmental causes or genetic effects which are not associated with consanguinity and the same argument is applicable for the high mortalities experienced by the Spanish Habsburg families. With respect to the high incidence of consanguineous marriages in the Spanish Habsburg dynasty it is necessary to take into account that, from a Western perspective, marriages between close biological relatives is generally regarded with suspicion and distaste, reflecting historical and religious prejudice. However, contrary to widespread opinion in Western countries, consanguinity is widely preferential in present large human populations of Asia and Africa where consanguineous marriages currently account for approximately 20–50% of all unions (in the computation of these percentages those unions contracted between individuals biologically related as second cousins or closer are categorized as consanguineous) [9]–[11]. The highest levels of inbreeding in major populations have been found in urban Pondicherry (South India) and among army families in Pakistan where 54.9% and 77.1% of marriages are consanguineous, respectively [12]–[13]. In Pondicherry 20.2% of marriages are uncle-niece and 31.3% first cousins, whereas in the Pakistan study 62.5% of marriages are between first cousins. Therefore, in the light of these data, the incidence of consanguineous marriages in the Spanish Habsburg dynasty does not seem so extreme when is considered in a general context of inbreeding in human populations. PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Table 1. The Spanish Habsburg kings and their marriages. https://doi.org/10.1371/journal.pone.0005174.t001

Methods Genealogical information of the Spanish Habsburg kings obtained from several sources [1]–[3], [14]–[15] was introduced into a data base which included more than 3,000 individuals along 16 parent-offspring generations (Fig. 1 shows a partial pedigree of the Spanish Habsburg kings represented by chains of descent). The data base was used for computing the inbreeding coefficient F (probability that an individual receives at a given locus two genes identical by descent due to the common ancestry between his parents) of the Spanish Habsburgs by means of the FSpeed computer programme [16]. PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 1. Pedigree of the Spanish Habsburg kings (in capital letters). https://doi.org/10.1371/journal.pone.0005174.g001 In order to evaluate the impact of inbreeding on survival in the Spanish Habsburg dynasty, mortality data of progenies of eight royal families were obtained from several sources [17]–[18]: the families of the Spanish Habsburg kings Philip I, Charles I, Philip III, two families of king Philip II with his third and fourth wives (Elizabeth of Valois and Anna of Austria) and the two families of king Philip IV. In addition, the family of Ferdinand of Aragon and Elizabeth of Castile, parents of Joanna I of Castile and Aragon, is also considered in order to increase the number of studied families. The families of the king Philip II with his first and second wives (Mary of Portugal and Mary I of England) are not considered since Mary of Portugal had only a single child (Charles) and there was no child in the marriage between Philip II and Mary I of England (Mary Tudor). Mortality data of progenies of the eight royal families were classified into three categories: late miscarriages and stillbirths, neonatal deaths (deaths in the first month of life) and deaths between month 1 and year 10. As a whole, there were 51 pregnancies in the eight families: 5 miscarriages and stillbirths, 6 neonatal deaths, 14 deaths between month 1 and year 10 and 26 survivals at age 10, and the mean number of pregnancies per family was 6.38±0.42. Multiple regression analyses and other statistical methods were performed by means of the SPSS 15.0 statistical software system.

Acknowledgments The authors are grateful to Carlos Morales for developing the data base for genealogical information. The authors are also grateful to Lorena Prieto for many useful comments on the European Royal dynasties.

Author Contributions Conceived and designed the experiments: GA FCC CQ. Performed the experiments: GA FCC CQ. Analyzed the data: GA FCC CQ. Wrote the paper: GA CQ.