Contents lists available at ScienceDirect Forensic Science International journal homepage: www.elsevier.com/locate/forsciint Commentary Genetic characterization of a collection of Tsantsas from Ecuadorian museums Verónica Baquero-Méndez a,1 , Karla E. Rojas-López a,1 , Juan Esteban Zurita a , María Mercerdes Cobo a,b , Consuelo Fernández-Salvador c , María Patricia Ordóñez c , María de Lourdes Torres a, ⁎ a Laboratorio de Biotecnologi ́ a Vegetal, Colegio de Ciencias Bioló gicas y Ambientales, Universidad San Francisco de Quito, 170901 Quito, Pichincha, Ecuador b Department of Paediatrics, University of Oxford, OX39DU Oxford, United Kingdom c Escuela de Antropología, Colegio de Ciencias Sociales y Humanidades, Universidad San Francisco de Quito, 170901 Quito, Pichincha, Ecuador article info Article history: Received 13 February 2021 Received in revised form 17 May 2021 Accepted 11 June 2021 Available online 16 June 2021 Keywords: Tsantsas Mitochondrial DNA HVR-1 region Sex determination Native American populations mtDNA haplogroup abstract Tsantsas are shrunken human heads originally made for ceremonial purposes by Amazonian indigenous groups of the Shuar and Achuar family, previously called Jivaroan tribes. A significant demand of these objects during the first half of the 20th century led to the manufacture of counterfeit shrunken heads for commercial purposes. For museums where these collections are held, as well as for the indigenous groups who claim their ownership, it is important to identify the origin and authenticity of these tsantsas. We hypothesized that a collection of 14 tsantsas from 3 different museum collections in Ecuador are human and aimed to characterize their sex and potential origin. We amplified the amelogenin gene and performed a high resolution melting analysis to determine their human origin and characterize their sex. We also analyzed a fragment (16209–16402) from the HVR-1 region to identify the mtDNA haplogroups present in the tsantsa collection. Our exploratory results show that all the tsantsas are human and that the collection is comprised of 13 males and 1 female. A total of seven mtDNA haplogroups were found among the tsantsa collection using the mtDNA EMPOP database. These results show a predominance of the Amerindian mtDNA haplogroups B, C and D. Additional principal component analysis, genetic distance tree and hap- lotype network analyses suggest a relationship between the tsantsa specimens and Native American groups. © 2021 Elsevier B.V. All rights reserved. 1. Introduction Tsantsas are reduced human heads -soft tissue only- made by indigenous peoples of the Shuar and Achuar families, located be- tween the borders of eastern Ecuador and northern Perú [1,2]. There is some discussion today regarding the believe system behind tsantsa manufacturing, nevertheless, the predominant narrative has been that these indigenous peoples believe that an individual´s dead was caused by enemy groups or individuals, and therefore needed to be revenged [3]. To prevent further death, the soul of the enemy was contained in the person´s head, which underwent a sacred ritual to remove the skull, and reform the skin, sewing the eyelids and mouth shut. This was done so that the soul could no longer hear or speak evil to and about its captors. Among the modern-day Shuar groups, there is a believe that tsantsas could also be made from clan leaders, that died from natural causes. The soul would have been contained in a tsantsa and its power used positively for the community. This was a way of showing respect for them [4]. The process of making a tsantsa involved cutting vertically from the top of the head to the base of the skull (bregma to opisthocra- nium), in order to remove the skull but keep the structure of the face, ears and neck. The ritual has been described in several sources [5–7], but the specific steps to create a tsantsa remain unknown even to the descendants of the shamans who took part in these ri- tuals. It is believed that the removed skin was submerged in boiling water three times at intervals of 30 min to 2 h. The lips and eyelids were then sewn, sometimes having removed the eyeballs by way of sharpened wooden pins, though sometimes leaving them in. The remaining skin was filled with stones, ash and sand as to create a firm background for the now shrunken head. Afterwards, the skin https://doi.org/10.1016/j.forsciint.2021.110879 0379-0738/© 2021 Elsevier B.V. All rights reserved. ]] ]] ]]]]]] ⁎ Corresponding author. E-mail address: ltorres@usfq.edu.ec (M.d.L. Torres). 1 Authors contributed equally to this work. Forensic Science International 325 (2021) 110879