Materials and methods
Chapter 1: Autosomal markers
2. Maternal gene pool in Chuetas: Middle Eastern legacy and a novel sub-branching of the rare haplogroup R0a2m
The third chapter contains the genetic characterization of female lineages in the Chueta population based on mtDNA analyses.
Mitochondrial DNA has been widely studied to shed light on the origin and demographic history of human populations. Specifically, in Jewish populations, Ashkenazi maternal lineages have been well characterized (e.g. Behar et al., 2006; 2008b; Brandstätter et al., 2008; Costa et al., 2013), whereas Sephardic lineages studies are more limited (e.g. Behar et al., 2008b; Nogueiro et al., 2015a). These studies will enable a comparison with the Chueta results.
A preliminary study carried out only with mtDNA Hypervariable region I (HVRI) revealed that the Middle Eastern haplogroup pre-HV was the most prevalent (23%) in Chuetas (Picornell et al., 2005). In the present work, we studied the D-loop region of 104 individuals from the Chueta population, and 79 individuals from its host population (Majorca) in order to obtain a better picture of Chueta maternal lineages, and to scrutinize, on the one hand, the genetic footprints of Sephardim in this population and, on the other hand, the introgression of mtDNA lineages from their host population.
Moreover, to go further into the identification of different sub-haplogroups, and taking into account the complexity of haplogroup H classification, 51 SNPs were analysed to ensure the correct assignation of haplogroup H samples.
Once the Chuetas’ mtDNA lineages were identified in terms of the D-loop region, the complete genome of 16 molecules, belonging to 10 different haplogroups, were sequenced to compare them with other published sequences, and to infer a more detailed origin of these haplogroups.
This chapter is divided into two sections: first, preliminary results in D-loop published in Ferragut et al. (2015c); and, second, a manuscript in preparation with full mtDNA results.
147 Founding mothers of Chueta population
Ferragut JF1; Marques SL2; Ramon C1; Castro JA1; Amorim A2,3; Alvarez L2,3 and Picornell A1
1Institut Universitari d’Investigació en Ciències de la Salut (IUNICS) and Laboratori de Genètica, Departament de Biologia, Universitat de les Illes Balears, Palma de Mallorca, Illes Balears, Spain
2Institute of Pathology and Molecular Immunology of the University of Porto (IPATIMUP) and Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal
3Faculty of Sciences of the University of Porto, Porto, Portugal Corresponding author
Joana F. Ferragut (firstname.lastname@example.org). Ctra Valldemossa Km 7.5 07122 Palma (Balearic Islands, SPAIN) Telephone number: +34 971173143 Fax number: +34 971173184
mtDNA; Control Region; Jews; Chuetas; haplogroups Abstract
In the present work, we studied 109 unrelated Chueta individuals aiming to characterize their maternal gene pool composition. The entire mtDNA control region (1121 bp) was sequenced. The highest frequencies were found for haplogroups R0a and H, indicating a remarkable signature of Middle Eastern ancestry along with some degree of European admixture. These data confirm that Chuetas have been able to maintain some ancestral genetic identity. The significant differences with their host population should therefore be taken into account in forensic casework.
148 1. Introduction
The presence of Jews in the Balearic Islands is archaeologically documented since the 5th century AD, but the Jewish communities that can trace a historical continuity with today’s Chuetas date back to the Muslim period (10th-13th centuries). Although the Christian conquest of Majorca in 1229 guaranteed the survival of the Jewish population, social and religious pressures forced their conversion between 1391 and 1435. Many of these converted Jews were assimilated into the general population, but a good few families continued to live in the ghetto and keep a secret adherence to Judaism. This Crypto-Jewish community was persecuted by the Inquisition (15th-17th centuries) (Braunstein, 1936) and, although the last “Autos de Fe” in 1691 put an end to their hidden Jewish religious practices, these convicts and their descendants (called Chuetas) were socially isolated and discriminated by their Majorcan neighbours until the mid-twentieth century. Therefore, Chuetas were an inbred population with scarce intermarriage with the Majorcan host population (Laub and Laub, 1987). The family names known as Chuetas in Mallorca are Aguiló, Bonnin, Cortés, Fortesa, Fuster, Martí, Miró, Picó, Piña, Pomar, Segura, Tarongí, Valentí, Valleriola and Valls, which do not have any relationship with Judaism anywhere but in Majorca.
Genetic similarities and differences among Jewish populations and between Jews and their host people have been widely studied in an attempt to unravel what must be a complex system of interrelations. Chuetas are, together with the Crypto-Jewish communities in Portugal (from Bragança and Belmonte) (Nogueiro et al., 2015b), the only direct descendants of the original Sephardic population; hence they are an interesting population to be studied due to their particular history.
Monoparental genetic markers have been widely used to further the knowledge of factors that have shaped modern human population’s structure. Moreover, monoparental lineage studies enable to estimate the maternal and paternal contributions to the genetic pool of a particular population.
The aim of the present study was to study the mitochondrial DNA (mtDNA) haplogroups in a sample of Chueta individuals and to compare with other Jewish populations and with their host population, in order to investigate the founding maternal lineages in this population.
2. Material and methods
In the present work, we analysed 109 non-related Chueta individuals, with ancestors in the community until at least their third generation. Buccal cells collected on cytology brushes were obtained, under informed consent. DNA was extracted by standard phenol-chloroform method. Two mitochondrial DNA (mtDNA) fragments were amplified using
specific primers (L15997, H016, L16555, H639) and sequenced with ABI prism 3130 in order to obtain the entire Control Region (CR) (16024-576). Sequences were aligned against the revised Cambridge Reference Sequence (rCRS) using Genious software version 7.1.3 (Kearse et al., 2012). Haplogroups were classified following the updated mtDNA phylogeny, PhyloTree, mtDNA tree Build 16 (http://www.phylotree.org/) using Haplogrep tool (Kloss-Brandstätter et al., 2011) and assigned haplotypes were validated by EMPOP (EMP00672) (http://empop.org/) curators. Diversity parameters were estimated using the ARLEQUIN package version 3.5 (Excoffier and Lischer, 2010).
3. Results and Discussion
In the 109 individuals studied 50 different haplotypes were identified. The haplotype and nucleotide diversities of the complete CR were 0.952 and 0.010, respectively. Comparing with other Iberian Jewish population, the genetic diversity values were just slightly lower than in Bragança Crypto-Jews (Teixeira et al., 2011) and much higher than in the Portuguese Jewish community from Belmonte (Table 1), where a very strong founder maternal effect has been previously described (Behar et al., 2008b).
Table 1. Diversity parameters of HVRI (16024-16365bp) and HVRII (72-300bp) of the Chueta population and other Iberian populations with Jewish ancestry.
Diversity Chuetas (N=109) Bragança Jews (N= 57) Belmonte Jews (N=30)
K (%) 46 (42.20%) 35 (61.40%) 2 (6.67%)
S 75 61 6
Ĥ ± SD 0.950 ± 0.013 0.967 ± 0.012 0.129 ± 0.115
π ± SD 0.013 ± 0.007 0.014 ± 0.008 0.001 ±0.001
θK [95%CI] 29.477 [19.852, 43.505] 37.616 [22.328, 63.866] 0.279 [0.065, 1.097]
N, sample size; K, number of different haplotypes; S, number of polymorphic sites; Ĥ, gene diversity; π, nucleotide diversity; θK, theta estimator based on the number of different haplotypes.
High haplogroup (Hg) diversity was found in Chuetas (Figure 1), with 35 different haplogroups. The Middle Eastern haplogroup R0a was the most prevalent (19.3%), followed by the widespread European haplogroup H (16.5%).
In order to identify traits of Jewish ancestry, we took into account those Hgs with frequencies higher than 4% and significantly different frequencies to those found in the Majorcan host population (unpublished data). The haplogroups that stood out in the Chueta sample were: R0a, T1a, T2c1d, K1a1b1a, U1a1a and L3e2b, indicating a remarkable signature of Middle Eastern ancestry along with some degree of European and North African admixture. The haplogroup pattern in Chuetas pointed out that the most important Jewish putative founding lineage is R0a, found in other Jewish (especially North Africans) and Middle Eastern populations, like Druzes, Palestinian and Bedouins, but not in Portuguese Jewish populations.
Figure 1. Network showing the haplogroup distribution of the 109 mtDNA Control Regions studied in Chuetas.
These data confirm that the Chuetas, due to their singular history, have kept not only the cultural memory of their Jewish origin through centuries but also a substantial degree of ancestral genetic signature. Also, some degree of the host admixture can be detected, as in other diaspora Jewish populations.
The significant differences observed between Chuetas and their host population should therefore be taken into account in forensic casework.
This work was partially supported by grant AAEE24/2014 from the Direcció General de R+D+I (Comunitat Autònoma de les Illes Balears) and European regional Development Fund (ERDF). SLM is supported by FCT (grant PTDC/ATP-DEM/4545/2012) and financed by the European Social Funds (COMPETE-FEDER).
Conflict of interest statement
The authors declare no conflict of interest.
Maternal gene pool in Chuetas: Middle Eastern legacy and a novel sub-branching of the rare haplogroup R0a2m
Key words: mitochondrial DNA, Chuetas, Middle Eastern, Haplotype, Haplogroup, Jewish.
The first mitochondrial DNA (mtDNA) studies in Jewish populations (Thomas et al., 2002;
Picornell et al., 2006; Brandstätter et al., 2008) revealed that most Jewish communities were founded by few women, that the process was independent and different in each geographical area, and that there were different rates of introgression from their corresponding host populations.
Most of the studies were mainly focused on the analysis of Ashkenazi Jews, a situation that has remained until today. As a major outcome, from the study of different parts of the mitochondrial genome, 4 women, together with several minor contributors, were identified as the founders of all the Ashkenazi Jews. However, there is some controversy regarding the identification of the origin of these 4 main contributors, with different studies claiming the Near East, while others suggest the Caucasus, Europe, or even the Far East through the Silk route (Behar et al., 2004b; 2006; Costa et al., 2013; Tian et al., 2015).
Behar et al. (2008b) widen the focus, analysing non-Ashkenazi communities, in order to investigate their demographic history, including how these were founded. Contrary to the Ashkenazim, they found no evidence for narrow founder effects for non-Ashkenazi populations: e.g. in Indian and Ethiopian communities an important local/host female contribution was found, while for North African and Iberian communities the contribution was admixed. These results led them to conclude that there are differences in the demographic history of the widespread communities resulting from the Jewish Diaspora in terms of maternal ancestries.
A specific group can be identified with its origin in Spain and Portugal: the Sephardic and Iberian Crypto-Jewish descendants – where the Crypto-Jewish phenomenon is defined as the secret adherence to Judaism while publicly professing another faith. Data from these populations still living in the Iberian Peninsula – which constitutes the original geographic source of Sephardic Jews – is very limited (Nogueiro et al., 2015b). The first study including Iberian Crypto-Jewish communities was performed by Behar et al. (2008b) on the Belmonte community, revealing a great founder effect in the community with extremely low diversity levels; unlike the analysis of the Bragança community, located in northeast Portugal, which found high levels of diversity, defining 5 haplogroups as founding lineages (Teixeira et al., 2011; Nogueiro et al., 2015a). For the descendants of Majorcan Jews, a
preliminary study was carried out (Picornell et al., 2005) using only the HVRI and a small number of samples, whose results also showed high rates of diversity in this community.
The presence of Jews in the Balearic Islands is archaeologically documented since the 5th century AD. Although the Christian conquest of Majorca in 1229 guaranteed the survival of the Jewish population, social and religious pressures forced their conversion between 1391 and 1435. Consequently, there were officially no more Jews in Majorca nearly 60 years before the Edict of Expulsion by the Catholic Kings in 1492. Many of these converted Jews were assimilated into the general population, but a good few families continued to live in the ghetto and keep a secret adherence to Judaism. This Crypto-Jewish community was persecuted by the Inquisition (15th-17th centuries) (Braunstein, 1936) and, although the last “Autos de Fe” in 1691 put an end to their hidden Jewish religious practices, these convicts and their descendants (called Chuetas) were socially isolated and discriminated by their Majorcan neighbours until the mid-twentieth century. Therefore, Chuetas were an inbred population with scarce intermarriage with the Majorcan host population (Laub and Laub, 1987; Porqueres, 2001).
In this study, we aim to evaluate the maternal genetic composition of the Majorcan Chueta population in order to infer its demographic history. Specifically, we attempt to identify and describe its founders and trace back their origins. Besides, we endeavour to determine the Majorcan host population’s influence on the Chueta population gene pool.
2. Material and methods