The Kingdom of Aragon emerged in 1035, DC. While its origins lie as a Pyrenean country with its center in Jaca (9th century AD), the Kingdom would expand to include the Iberian Peninsula over the following centuries and ultimately beyond1. Between the 11th and 12th centuries, its kings reconquered territories of the Peninsula from Muslim influence, including the cities of Huesca (1096) and Zaragoza (1118) while gaining control of the Ebro Valley1. In 1150, the young future queen of the Kingdom of Aragon (Princess Petronila) married the Count of Barcelona. From this dynastic union, the House of Aragon would dominate, by conquest or matrimonial alliance, in addition to Aragon and the countries of Catalonia, several kingdoms and territories such as Valencia, Mallorca (Balearic Islands), Naples, Sicily, Athens and Neopatria (the last two in the territory that is now Greece). Together, this region was known as the Crown of Aragon1-2.
Archaeological and historical documents note three prominent pantheons of the early House of Aragon. Traversing the Aragonese domain, these pantheons are widely acknowledged as the primitive burial sites of the royal family. The Aragon family pedigree (early members) is illustrated in Figure 1. The patriarch of the House of Aragon, Ramiro I, established the monastery of San Juan de la Peña (Jaca, Aragon) as the royal pantheon within which he and his descendants were to be buried (Figure 2). Historical evidence supports the burial of King Ramiro I, along with other early members of the royal family such as King Sancho Ramírez and King Pedro I, within the tombs housed in the Royal Pantheon of San Juan de la Peña (Figure 3)1.
Figure 1. The family pedigree of the first members of the Royal House of Aragon (adapted from Martínez et al., 2018)1. Many Kings of Aragon were also Kings of Navarre (Pamplona)1. The putative remains of those shaded grey were examined. The remains of two young children were also analyzed as these skeletons were found buried alongside the royals. The remains of a four-year-old male were found in Countess Sancha’s sarcophagus, and the clavicle of very young individual was found in the sarcophagus of King Ramiro II “The Monk.” Those individuals shaded a darker grey, were analyzed by mitochondrial DNA sequencing.
Figure 2. Photograph of the Royal Monastery of San Juan de la Peña (courtesy of José Luis Solano)3. The Royal Monastery of San Juan de la Peña houses the primitive Royal Pantheon of Aragon Kings/ House of Aragon.
Figure 3. Map of Aragonese territory depicting the burial locations and movement of remains for the Royal House of Aragon early members according to historical documents1.
While it is believed that many (but not all) of the first members of the royal family were buried at San Juan de la Peña, recent archaeological evidence has emerged proposing the putative identities of a group of female remains exhumed at the monastery of Benedictinas de Jaca3. An intricately styled sarcophagus containing female remains attributed to the daughter of King Ramiro I, Countess Sancha, and perhaps those of her sister, Urraca, was found in the convent. Historical records depict Countess Sancha as an important political and religious leader in her time, unfrequent in a woman from the early medieval period. She joined the monastery Santa María de la Santa Cruz de la Serós (Figure 4), devoting her life to the convent. The Countess’ status and influence were reflected in the ornate design of her Romanesque styled sarcophagus1,3,4,5 (Figure 5). It is documented that Urraca embraced the religious habit and became abbess of that convent. Concordance among historical data and findings derived from forensic anthropology and archaeology studies (e.g., radiocarbon 14C dating and stable isotope analysis) lead forensic archaeologists to suggest that one female skeleton, housed in the sarcophagus, could pertain to Countess Sancha and that the other remains could be that of Urraca.
Figure 4. Photograph of the Monastery of Santa María de Santa Cruz de la Serós (courtesy of José Luis Solano).
Figure 5. Photograph of Countess Sancha’s sarcophagus. It is considered a masterpiece of Romanesque funerary medieval art (courtesy of Begoña Martínez-Jarreta)3.
Minimal to no evidence of Teresa, their other sister, survived or presently exists1,3,4. One skeleton exhumed from the same sarcophagus and dated to be from the appropriate time period, was determined to be that of a 70 to 80-year-old female. These remains have been suggested to be those of Sancha de Aibar, mother of the first King of Aragon and grandmother to Countess Sancha (Figure 6). Historical records support that Sancha de Aibar was a generous benefactor of the Santa María de la Santa Cruz de la Serós convent. The sarcophagus containing the putative remains of Countess Sancha, Sancha de Aibar, and possibly Urraca were moved from their original location at the monastery Santa María de la Santa Cruz de la Serós to Benedictinas de Jaca in 1622 (Figures 3 and 7)1.
Figure 6. Photograph of a skull exhumed in the sarcophagus of Countess Sancha found at the monastery of Benedictinas de Jaca. The remains are of a 70 to 80-year-old female individual, attributed to Sancha de Aibar (courtesy of Begoña Martínez-Jarreta)3.
Figure 7. Photograph of the Monastery of Benedictinas de Jaca where the sarcophagus of Countess Sancha is currently located (courtesy of José Luis Solano)3.
King Alfonso I of Aragon, grandson to King Ramiro I and known as “the Battler,” was one of Aragon kings most notably recognized because of his military skills and his conquering of Iberian territory for the Aragon Kingdom. He ruled for 30 years before dying from a wound in combat (d.1134). Due to lacking children (descendants-heir), his brother, King Ramiro II “the Monk” (so named because he was a cleric), assumed the role as sovereign. The remains of King Alfonso I were buried at Castle of Montearagón (Figure 3) next to Huesca. King Ramiro II was buried in the Royal Pantheon of San Pedro el Viejo (Huesca, Aragon) immediately following his death in 1157. In 1845, due to a fire, the remains of King Alfonso I were relocated to the Royal Pantheon of San Pedro el Viejo (Figures 3 and 8). Subsequent tomb restorations of Kings Alfonso I and Ramiro II were well documented with inscriptions identifying the remains on their sarcophagi1,6,7.
Figure 8. Photographs of the Royal Pantheon of San Pedro el Viejo (left) and the sarcophagi of King Alfonso I “The Battler” (upper right) and King Ramiro II “The Monk” (lower right) located in the Chapel of San Bartolomé of this Romanesque monastery (photographs courtesy of Begoña Martínez-Jarreta)3.
The burial taphonomy of the three royal pantheons plays a significant role in assigning identities because it can alter archaeological interpretation of historical accounts as well as contributing potential challenges to scientific analyses. The pantheons were each subjected to interventions. Attempts were made to relocate and protect the remains throughout the centuries. The Royal Pantheon of San Juan de la Peña, the resting place of many of the early members of the royal family and their wives, suffered considerable damage or alterations from pillaging, fires in 1494 and 1675, and an important architectural restoration in 1770. San Juan de la Peña was excavated in 19851. Each of these events potentially introduced challenges such as the comingling, movement or the loss of remains as well as bone and DNA damage. The sarcophagi of the Royal Pantheon of San Pedro del Viejo (Kings Alfonso I and Ramiro II) and that of the Countess Sancha (then relocated to Benedictinas de Jaca) were opened in the 1990s with a second exhumation in 2008. In addition, skeletons were treated with a synthetic resin for preservative purposes; however, the negative effects on particular scientific analyses can only be speculated (e.g., radiocarbon dating) or remain unknown as in the case of modern genetic analyses1.
Overall, the remains of 20 individuals were exhumed from the three prominent pantheons believed to contain the burials of the Royal House of Aragon. Through collaborative efforts of archaeologists/anthropologists, historians, analytical chemists, and forensic geneticists, the putative identities of the members of the royal dynasty were attributed to the skeletal remains discovered within specified Spanish pantheons1,3-4,6-7. Radiocarbon dating and stable isotope testing combined with historical accounts of the time period were used to attempt to identify the remains believed to belong to the Medieval Royal House of Aragon. These methods and complementary analyses have been published 1,3-9.
In 2011, the skeletal remains of the individuals studied were reburied with the exception of those limited samples maintained for this study. The skeletal remains analyzed and presented herein were the most challenged of all of the samples. Only fragments of bone remained after all other analyses were performed and these samples were found, in particular, to be challenging for genetic analysis. Previous attempts had been made to genetically profile the remains using conventional short tandem repeat (STR) typing. In addition, the use of mini STRs were also pursued with minimal success as the DNA appeared to be highly degraded and/or damaged 3,5,8,9. DNA damage often yields highly fragmented (damaged) and low copy number (LCN) DNA template which may not be sufficient for conventional forensic genetic testing. Mitochondrial DNA (mtDNA) testing often is performed on skeletal remains due to its relatively high copy number and capacity to resist degradation. Preliminary genetic analyses are underway exploring both mitochondrial and nuclear DNA contained within these ancient bones (estimated range dated to be approximately 850-950 years old).
The nuclear genome of ancient remains typically is highly degraded and damaged, and thus genetic information often cannot be obtained. Current forensic methodologies are incapable of typing such low-quality samples. However, a novel one-step PCR technology, Reverse Complement PCR (RC-PCR), validated to target 27 human identity single nucleotide polymorphisms (SNPs) was used to attempt to analyze these highly degraded samples. The EasySeq™ NGS Reverse Complement-PCR kit (NimaGen B.V.; Nijmegen, Netherlands) was designed to amplify these 27 targeted SNPs within amplicons approximately 50 base pairs in length. The RC-PCR assay is highly sensitive and has the added feature of being a single, closed system which reduces chances of contamination and improves workflow10.
The RC-PCR technology appeared to enrich the identity SNPs from the remains of the Medieval Royal House of Aragon dating back approximately 950 years (Figure 9). Although the samples examined exhibited low read depth as to be expected, genotyping results were observed for most samples analyzed. Partial consensus profiles were obtained; however, an expanded identity SNP panel should be considered to improve upon identity attribution. Due to the rarity and precious nature of the DNA available from these remains, further genetic testing will not be pursued until a more expanded RC-PCR identity SNP panel is developed.
Figure 9. An example of SNP results from the nuclear DNA of the alleged remains of Sancha of Aibar (Mother of the First King of Aragon) dating approximately 950 years ago. The Integrative Genomics Viewer (IGV, https://software.broadinstitute.org/software/igv/) was used to display the SNP results at rs1077376011.
Sequencing of the mtDNA control regions (HVI and HVII; 16,024-576) was performed using an early version of the ForenSeq™ mtDNA Control Region Solution kit (Verogen; San Diego, CA)12. The mtDNA profiles for samples pertaining to the putative King Alfonso I and Ramiro II were examined. All mtDNA profiles exhibited single base damage and sequence error; however, interpretable data were obtained. The mtDNA results did not support a maternal lineage relationship between the samples examined (Figure 10). Therefore, the mtDNA results are inconsistent with the brotherhood hypothesis between the skeletons that could be tentatively attributed to Kings Alfonso I and Ramiro II based on non-DNA evidence described in previous publications1. MtDNA findings support previous reports describing different 14C (radiocarbon dating) results obtained from the adult male skeleton recovered from the sarcophagus of King Ramiro II6. This individual radiocarbon dated much older than expected for Ramiro II. However, possible reservoir effects could influence the 14C analysis and thus could not rule out the remains being those of Ramiro II1. However, this new DNA evidence gives light and presents mtDNA findings that reinforce the notion that the attribution of this skeleton to King Ramiro II should be discarded. Alternatively, the two brothers (King Alfonso I and Ramiro II) may not be related maternally, contradicting the historical genealogy of the Royal House of Aragon, among other hypotheses.
Figure 10. Hypervariable region I (HVI) mtDNA sequence results from the DNA of the putative remains of King Alfonso I (top) and King Ramiro II (bottom). IGV was used to display the results11.
Additional genetic analyses, based on a larger SNP panel and massively parallel sequencing, could shed light on the identities of these human remains. DNA technologies and approaches such as those used in this study help overcome some of the limitations of highly compromised biological samples. This study adds genetic (nuclear DNA and/or mtDNA) evidence to data from archaeological studies and other complementary analyses (e.g., 14C, stable isotopes, historical records) to help identify remains of the Royal House of Aragon.
The authors would like to thank the University of Zaragoza (Spain) that directed the excavation and putative identification of the skeletal remains ultimately attributed to the Medieval Royal House of Aragon. Their exhaustive and systematic multivariant analyses to assign identity considerably aided the sampling of remains subjected to genetic analyses. Additionally, the authors would like to recognize Dr. Angie Ambers for her expertise and efforts in the extraction of DNA from the skeletal remains during her time at the University of North Texas Center for Human Identification (UNTCHI).
Martínez‐Jarreta, B., Sosa, C., Laliena, C., Budowle, B., Hedges, R.E.M. Stable isotope and radiocarbon dating of the remains of the Medieval Royal House of Aragon (Spain) shed light on their diets, life histories and identities. Archaeometry 60(2), 366-382 (2018).
Bisson, T. N., 1991, The medieval Crown of Aragon: a short history, Oxford University Press, Oxford.
Núñez, C., Baeta, M., Martínez-Jarreta, B. La ciencia forense a la búsqueda de respuestas: descifrando la historia de Sancha de Aibar. In: Martínez Jarreta, B. (coord.). Sancha de Aibar: una mujer necesaria en una encrucijada histórica. Zaragoza: Imprenta Féliz Arilla & Gobierno de Aragón, 39-88 (2018).
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Núñez, C., Sosa, C., Baeta, M., Geppert, M., Turnbough, M., Phillips, N., Casalod, Y., Bolea, M., Roby, R., Budowle, B., Martínez-Jarreta, B., Genetic analysis of 7 medieval skeletons from the Aragonese Pyrenees. Croatian Medical Journal, 52, 336–43 (2011).
Martínez-Jarreta, B. Nuevas claves para enigmas antiguos. La ciencia rinde homenaje a los Reyes de Aragón. Asociación de Obreros de San Pedro el Viejo (eds) Panteón Real de Aragón: San Pedro el Viejo. Gráficas Huesca SL-Huesca, 116-122 (2014).
Martínez-Jarreta, B. Los estudios antropológicos de los restos óseos conservados, Panteones Reales de Aragón. Zaragoza: Gobierno de Aragón & Fundación Ibercaja, 240-253 (2018).
Sosa, C., Baeta, M., Núñez, C., Casalod, Y., Luna, A., Martínez-Jarreta, B., Nuclear DNA typing from ancient teeth. American Journal of Forensic Medicine and Pathology, 33, 211–14 (2012).
Sosa, C., Vispe, E., Núñez, C., Baeta, M., Casalod, Y., Bolea, M., Hedges, R.E., Martínez-Jarreta, B. Association between ancient bone preservation and DNA yield: A multidisciplinary approach. American Journal of Physical Anthropology, 151(1), 102-9 (2013).
Kieser R., Buś M., King J., van der Vliet W., Theelen J., Budowle B. Reverse Complement PCR: A novel one-step PCR system for typing highly degraded DNA for human identification. Forensic Science International: Genetics 44, 102201 (2020).