In 1985, a frozen mummy was found in Cerro Aconcagua (Argentina). Archaeological studies identified the mummy as a seven-year-old Inca sacrifice victim who lived >500 years ago, at the time of the expansion of the Inca Empire towards the southern cone. The sequence of its entire mitogenome was obtained. After querying a large worldwide database of mitogenomes (>28,000) we found that the Inca haplotype belonged to a branch of haplogroup C1b (C1bi) that has not yet been identified in modern Native Americans. The expansion of C1b into the Americas, as estimated using 203 C1b mitogenomes, dates to the initial Paleoindian settlements (~18.3 thousand years ago [kya]); however, its internal variation differs between Mesoamerica and South America. By querying large databases of control region haplotypes (>150,000), we found only a few C1bi members in Peru and Bolivia (e.g. Aymaras), including one haplotype retrieved from ancient DNA of an individual belonging to the Wari Empire (Peruvian Andes). Overall, the results suggest that the profile of the mummy represents a very rare sub-clade that arose 14.3 (5–23.6) kya and could have been more frequent in the past. A Peruvian Inca origin for present-day C1bi haplotypes would satisfy both the genetic and paleo-anthropological findings.Introduction
In the summer of 1985 a group of mountaineers discovered a frozen mummy only partially unearthed at an altitude of 5,300 meters in the southwestern edge of the Aconcagua Mountain (“Cerro”), at the base of the Pirámide Mountain, in the Argentinean province of Mendoza. Instead of excavating the body, the mountaineers returned to their localities and informed specialists about this find. Excavations were subsequently carried out by professional archaeologists. In the ritual burial, archaeologists identified a very well preserved seven-year-old boy wrapped in numerous textiles and surrounded by six statuettes1. A range of archaeological and anthropological studies identified the mummy as the victim of an Inca sacrifice, a ritual known as “capacocha”, occurring approximately 500 years ago1. The ceremony (also known as “capac hucha”) involved the ritual sacrifice of children and it is interpreted today as one of several strategies used by the Inca state to integrate and control its vast civilization. The sacrificial rites involved children of great physical beauty and health in honor of the gods; the rituals were performed during or after important events (death of an emperor, the birth of a royal son, a victory in battle or an annual or biennial event in the Inca calendar), or in response to catastrophes (earthquakes, volcanic eruptions and epidemics)2,3. The children were selected from different locations throughout the Inca territories and taken to the high mountaintops for capacocha.
The Inca constituted the largest (about 12 million people) and one of the most complex civilizations in pre-Columbian America. Its political, administrative and military center was located in Cusco (modern-day Peru). The Inca arose in the highlands of Peru in the early 13th century. From 1438 to 1533 they conquered or peacefully assimilated a large portion of western South America, including present-day Peru, a large part of Ecuador, south-central Bolivia, southern Colombia, northwest Argentina and north-central/north Chile. The Aconcagua mummy dates to this period of expansion southwards and was found close to the southernmost edge of the Inca expansion. There is abundant archaeological evidence supporting the practice of sacrifice within Inca society4. The last emperor of the Incas (Atahuallpa) was executed in 1533 by the soldiers of the Spanish conqueror Francisco Pizarro, marking the end of 300 years of Inca civilization.
The analysis of ancient DNA (aDNA) flourished in the last decade with the arrival of new sequencing technologies5,6,7,8. However, relatively few genetic studies have been carried out on mummies9,10,11,12,13,14,15. Ermini et al.10 analyzed the Tyrolean Iceman, a 46-year-old man who lived in the Neolithic-Copper Age transition in Central Europe about 5 kya; this represented the first complete mitochondrial genome sequence of a prehistoric European. To the best of our knowledge, there are only a few studies carried out on Native American mummies; all of them only targeted the mitochondrial DNA (mtDNA) first hypervariable region (HVS-I). For instance, Monsalve et al.9 analyzed the Kwäday Dän Ts’ìchi ancient remains of a man found in a melting glacier in British Columbia (Canada); the authors could characterize his mtDNA as belonging to haplogroup A and found matches in populations from across the whole American continent. Wilson et al.16 analyzed samples from another Inca child sacrifice employing a multidisciplinary approach. These authors reported the mtDNA HVS-I and a few mtDNA SNPs from seven samples, allowing them to broadly allocate these haplotypes to Native American haplogroups C and D. The HVS-I segment of the remains of the mummy “Juanita” (also known as “Lady of Ampato” or “Inca Ice Maiden”) was published in GenBank (Acc. No. EF660742 and EF660743); this was identified as another Inca sacrifice victim: a 12–14-year-old child who lived in Mount Ampato (near Arequipa, Peru) about 500 years ago; its mtDNA haplotype could be allocated to haplogroup A (its sequence motif is widespread across the whole American continent).
The present study represents the first attempt at reporting the entire mtDNA of a Native American mummy, with the additional interest that this mummy represents a member of the Inca civilization. The main aim is to shed light on the genetic variation existing at the time of the Inca civilization and to interpret this variation in the light of the patterns observed in present-day populations.
Results and Discussion
DNA was extracted from a small piece of lung of the mummy (Fig. 1). The haplotype of the mummy has 51 variants with respect to the rCRS17 (Table 1). The variation observed in the mummy’s mitogenome fits perfectly within the phylogeny of one of the most typical Native American haplogroups, C1b. Moreover, this haplogroup represents one of the most frequent branches within C1 (together with C1c and C1d) and was recently identified as one in more than fifteen mtDNA American founders18,19,20,21.
The Aconcagua mummy.
The inset shows a picture of a portion of dissected lung from the mummy. A small piece of 350 mg was used for DNA extraction. The photo of the mummy has been taken from49 and it is reproduced here with the permission of the University of Cuyo Publisher (Argentina).
A total of 201 C1b mitogenomes could be compiled from the literature (although two of them lacking information on the control region). The phylogeny of C1b was reconstructed and updated (see full phylogeny in Figure S1 and its skeleton in Fig. 2) using the latest version available in Phylotree Build 16 (http://www.phylotree.org), which allowed dating the Time of the Most Recent Common Ancestor (TMRCA) at different C1b ancestral nodes. In this phylogenetic analysis, 23 new minor sub-clades could be identified, all of them characterized by at least two different mitogenomes. C1b is approximately 18.3 (16.2–20.4) kya (Table 2; Fig. 2), thus supporting previous assessments indicating that C1b most likely arose relatively early, either in Beringia or at a very initial stage of the Paleoindian southward migration19,22. In agreement with previous findings18,23, the fact that C1b is only slightly older in Mesoamerica than in South America (Table 2) confirms that the southward expansion of this clade was very rapid18. While some C1b sub-clades were exclusively observed in Mesoamerica or in South America, a few of them were found in both territories.
Skeleton of the global C1b phylogeny.
The C1b1i clade represented by the haplotype found in the Inca mummy is also located in the phylogeny. There is one mitogenome (JX413043) that belongs to haplogroup C1b13b sampled in a Spanish individual, although born in Talagante (Chile); therefore we labeled it here as originating in America. TMRCA are indicated above haplogroup labels. An asterisk to the right of the haplogroup labels identifies sub-clades that were newly identified in this study, compared to the last version of Phylotree (Build 16). The position of the revised Cambridge reference sequence (rCRS) is indicated for reading sequence motifs17. Mitochondrial DNA variants are indicated along the branches of the phylogenetic tree. Mutations are transitions unless a suffix A, C, G, or T indicates a transversion. Other suffixes indicate insertions (+), synonymous substitution (s), mutational changes in tRNA (−t), mutational changes in rRNA (−r), noncoding variant located in the mtDNA coding region (−nc) and an amino acid replacement (indicated in round brackets). Variants underlined represent recurrent mutations in this tree, while a prefix ‘@’ indicates a back mutation. Mutational hotspot variants at positions 16182, 16183 and 16519, as well as variation around position 310 and length or point heteroplasmies were not considered for the phylogenetic reconstruction. The numbers in small squares attached above haplogroup labels indicate the number of occurrences (mitogenomes) of the corresponding haplogroups available in the public domain (literature and/or GenBank); the color of the squares indicates their geographic origin according to the legend inset. More details on the geographic or ethnic origin of all the mitogenomes used in this network are provided in Supplemental Data Table S1. The bottom-right inset shows a network of HVS-I sequences that potentially belong to haplogroup C1bi (left) and a map of South America showing their geographic location (right). The map was built using a blank map based on GPS coordinates and the SAGA v. 2.1.1 (http://www.saga-gis.org/; see methods).
The BSP of C1b (Fig. 3) overall points to one major episode of constant population growth within America that starts very early during the initial Paleo-Indian settlement into the American continent and their spread southwards. Approximately 9 kya there is a progressive decrease of effective population size that last until about 5 kya. Then the BSP indicates a new episode of constant population growth broadly coinciding with the beginning of the Archaic period, that is, with the increasingly intensive gathering of a wide range of resources and the decline of the hunting lifestyle. This progressive growth expands during the Formative and the Classic period (therefore including the initial periods of development of the Inca civilization). Only at very recent times, the BSP seems to suggest a final episode of population reduction, fitting with the arrival of Europeans.
BSP indicating the median of the hypothetical effective population size through time based on data from the C1b mitogenomes.
The maximum time is the median posterior estimate of the genealogy root-height.
Figure 4 shows that C1b mitogenomes were found around two main geographic locations, one in Mesoamerica and the other one around Peru and extending southwards from here along the Pacific coast. Figure 4 also shows a phylogenetic skeleton of the main C1b branches in the American chronology context. The observed pattern of geographic variation is compatible with the following broad migration scenario for C1b carriers: (i) early and rapid spread of C1b across the full American continent during the period of initial continental settlement; (ii) important population isolation of the Mesoamerican and the South American gene pools for long periods, as indicated by the presence of very old and very young clades exclusively found in each of these two sub-continental regions; and (iii) sporadic gene exchange between both sub-continental regions, as suggested by the existence of a few clades that are present today in Mesoamerica and South America; these clades have ages ranging from 15 kya (C1b3) to 2 kya (C1b2) ago. The main ancient American civilizations, such as the Maya and the Inca, could have contributed to the gene flow between the main continental regions24.
(A) ML phylogeny and TMRCA of the main C1b clades analyzed in the present study and the American chronology (LGM: Last Glacial Maximum). (B) Spatial-frequency distribution of haplogroup C1b. The map was built as indicated in Figure 2 and based on control region information. Note there are two main peaks of haplogroup C1b frequencies, one centered in Mexico and another one in Peru. In addition, there is a third peak in Puerto Rico (n = 23); this high frequency on the island projects over the North-East of South America (i.e. Venezuela and North of Brazil) where in reality C1b is virtually absent. The map was built using a blank map based on GPS coordinates and the SAGA v. 2.1.1 (see methods).
The haplotype of the Inca mummy belongs to a new clade that branches off from the root of haplogroup C1b, thus providing additional support for the authenticity of the haplotype (see M&M section). This new clade, C1bi (where ”i” stands for ‘Inca’), has 10 private mutations (Table 1). All private mutations were checked very carefully and replicated in confirmatory sequence analysis. Although there is no way to date C1bi using only one mitogenome, the amount of variation accumulated in the mummy’s haplotype is compatible with an old age, at least as old as other old branches within C1b that have accumulated a similar amount of variation. Figure S2 shows the number of mutations accumulated from the root of haplogroup C1b to all the tips of the phylogeny (Figure S1) and their relative frequency; the ten mutations observed in the mummy’s branch fall within expected values. The HVS-1 motif (Table 3) was used to search for members of C1bi in public haplotype databases (>170,000 partial mtDNA sequences). Only four samples belonging to C1b share the transition at position 16124 (Table 3; Fig. 2). A tentative dating can be carried out using these few control region haplotypes that fall within C1bi (Table 3). The TMRCA estimated from these haplotypes (based on ρ) is 14.3 (5.0-24.0) kya, which is consistent with the suggestion that C1bi constitutes an old clade. At the same time, the phylogeographic patterns of C1bi control region haplotypes point to a distribution of this lineage constrained to South America. Moreover, these patterns fit well with the maximum extension of the Inca Empire around 1525, when the mandate of Huayna Capac (eleventh “Sapa” of the Inca Empire) ended. Within C1b, there is a different sub-clade that shows very similar characteristics to C1bi, namely, C1b13. The TMRCA of this sub-clade is 11.8 (8.6–15.1) kya; it is virtually absent from North-Central America and its geographic location is mainly centered in Chile. C1b13 most likely arose in the Southern Cone region and differentiated locally soon after human arrival, during the tribalization and linguistic differentiation process25. The geographic distribution of C1b13 also fits well with the expansion of the Inca Civilization into the northern territories of Chile although its age is much older, thus suggesting that perhaps only some (still un-sampled) sub-lineages of C1bi might be related to the Inca’s timeline (as it occurs with other C1b sub-clades; Fig. 4).
