Annals of Human Biology

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The genetic landscape of Mediterranean North African populations through complete mtDNA sequences

Neus Font-Porterias, Neus Solé-Morata, Gerard Serra-Vidal, Asmahan Bekada, Karima Fadhlaoui-Zid, Pierre Zalloua, Francesc Calafell & David Comas

To cite this article: Neus Font-Porterias, Neus Solé-Morata, Gerard Serra-Vidal, Asmahan Bekada, Karima Fadhlaoui-Zid, Pierre Zalloua, Francesc Calafell & David Comas (2018) The genetic landscape of Mediterranean North African populations through complete mtDNA sequences, Annals of Human Biology, 45:1, 98-104, DOI: 10.1080/03014460.2017.1413133 To link to this article: https://doi.org/10.1080/03014460.2017.1413133

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Published online: 30 Jan 2018.

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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iahb20 ANNALS OF HUMAN BIOLOGY, 2018 VOL. 45, NO. 1, 98–104 https://doi.org/10.1080/03014460.2017.1413133

RESEARCH PAPER The genetic landscape of Mediterranean North African populations through complete mtDNA sequences

Neus Font-Porteriasa, Neus Sole-Morata a, Gerard Serra-Vidala, Asmahan Bekadab, Karima Fadhlaoui-Zidc, Pierre Zallouad, Francesc Calafella and David Comasa aDepartament de Ciencies Experimentals i de la Salut, Institute of Evolutionary Biology (CSIC-UPF), Universitat Pompeu Fabra, Barcelona, Spain; bDepartement de Biotechnologie, Faculte des Sciences de la Nature et de la Vie, Universite Oran 1 (Ahmad Ben Bella), Oran, ; cLaboratoire de Genetique, Immunologie et Pathologies Humaines, Faculte des Sciences de Tunis, Campus Univesritaire El Manar II, Universite El Manar, Tunis, Tunisia; dSchool of Medicine, The Lebanese American University, Chouran, Beirut, Lebanon

ABSTRACT ARTICLE HISTORY Background: The genetic composition of human North African populations is an amalgam of different Received 25 July 2017 ancestral components coming from the , , south-Saharan Africa and autochthonous Revised 23 November 2017 to . This complex genetic pattern is the result of migrations and admixtures in the region Accepted 30 November 2017 since Palaeolithic times. Aims: The objective of the present study is to refine knowledge of the population history of North KEYWORDS African populations through the analysis of complete mitochondrial sequences. ; North Subjects and methods: This study has sequenced complete mitochondrial DNAs (mtDNAs) in several Africa; mitochondrial DNA; North African and neighbouring individuals. Results: The mtDNA classification and phylogeny shows a high genetic diversity in the region as a result of continuous admixture. The phylogenetic analysis allowed us to identify a new haplogroup characterised by positions 10 101 C and 146 C (H1v2), a sub-branch of H1v, which is restricted to North Africa and whose origins are estimated as 4000 years ago. Conclusions: The analysis of the complete mtDNA genome has allowed for the identification of a North African sub-lineage that might be ignored by the analysis of partial mtDNA control region sequences, highlighting the phylogeographic relevance of mtDNA complete sequence analysis.

Introduction was one of the last major demographic events that occurred North Africa has traditionally been a geographically strategic in the region (Fadhlaoui-Zid et al., 2011). region surrounded by two natural borders: the Sahara Desert Despite the amount of knowledge about North African in the south and the Mediterranean Sea in the north, which populations obtained from palaeoarchaeological data, more have contributed to the isolation of this area. However, studies are needed to investigate the recent history of this changes in the climate conditions have promoted contacts region (Arauna et al., 2017). Indeed, genetic studies point out with south-Saharan Africa, Europe and the Middle East, that modern North Africans constitute a heterogeneous resulting in complex demographic events (Drake et al., 2011; group, whose ancestry is a result of at least three admixture Osborne et al., 2008). Archaeological studies report the suc- events from populations outside Africa: a “back-to-Africa” cession of different cultures and civilisations: the Aterian gene flow (12,000 ya), a gene flow (1400 ya) and lithic industry from 145,000 to 40,000 years ago (ya) migrations from south-Saharan Africa resulting from the slave (Nespoulet et al., 2008), which was replaced by the trade (1200 ya) (Arauna et al., 2017; Henn et al., 2012). culture during the Genome-wide autosomal studies reveal a gradient of likely (22,000–9000 ya) (Newman & Rogers, 1995); and the Capsian autochthonous Maghrebi ancestry that is reduced from West culture (10,000–6000 ya) (Rahmani, 2004). The tran- to East and an opposite cline of Near East ancestry with a sition, 5500 ya, progressively spread from the Near East to westward decrease (Henn et al., 2012). However, geographic North Africa, favouring the increase of social complexity, distance and genetic diversity were not found to be corre- which may have ultimately led to the formation of the ances- lated in North African populations, probably because of tors of the autochthonous Berber groups (Fadhlaoui-Zid heterogeneous or unbalanced admixture (Arauna et al., et al., 2011). In historic times, different populations succes- 2017). Y-chromosome data show an east–west cline from the sively settled in North Africa: Phoenicians (Zalloua et al., Near East compatible with a Neolithic demic expansion 2008), Romans, Vandals and Byzantines. Finally, the Arabic (Arredi et al., 2004), as well as a bidirectional gene flow from expansion linked to the spread of Islam in the 7th century North Africa to Iberia (Bosch et al., 2001).

CONTACT David Comas [email protected] Institut de Biologia Evolutiva (UPF-CSIC), Departament de Ciencies Experimentals i de la Salut, Universitat Pompeu Fabra, 08003, Barcelona, Catalonia, Spain Supplemental data for this article can be accessed here. ß 2017 Informa UK Limited, trading as Taylor & Francis Group ANNALS OF HUMAN BIOLOGY 99

Previous mtDNA studies report an extensively heteroge- mtDNA sequence analysis neous haplogroup distribution in North Africa, which sug- The quality of the sequences was assessed using the soft- gests that it is a highly admixed region (Fadhlaoui-Zid fastqc et al., 2011). Although the frequency of south-Saharan L ware and, then, to optimise the mapping process, haplogroups in North Africa is not negligible, most mtDNA each sample was split in multiple breaks every 1,000,000 fastqbreak lineages in North Africa have a Eurasian origin. U5, V, R0a, base pairs with . Sequenced paired-end reads were J1b and U3 haplogroups have been recently brought from mapped to the reference genome using the Burrows-Wheeler Europe and the Middle East. However, U6 and M1b are Alignment (BWA) tool with the non-default parameter -q 15 estimated to have existed in the region since Palaeolithic (Li & Durbin, 2009). The Cambridge Reference Sequence times (Gonzalez et al., 2007) and are found in high fre- (rCRS) revised by Andrews et al. (1999) was used as the mito- quencies, hence they are considered specific North African chondrial reference in the mapping step. To avoid the erro- lineages, even though they originated elsewhere. Moreover, neous increase of coverage, PCR duplicates were removed Genome Analysis Tool kit these two mtDNA haplogroups have an opposite frequency with the (GATK) software package. gradient in North Africa: U6 is more frequent in the West In addition, the average sequence coverage was also com- and M1 in the East (Olivieri et al., 2006; Pennarun et al., puted with the GATK software (McKenna et al., 2010) 2012). (Supplementary Figure 1, Supplementary Table 1). Initially, most mtDNA studies were based on a few restric- Once the reads were mapped, Samtools 1.3.1 (mpileup tion sites along the molecule and/or a few hundred bp of and vcfutils software) (Li et al., 2009) were used, with the sequence in the hypervariable segment 1 of the control non-default parameters -B, -A and -Q 30, only considering region. Recently, whole mtDNA genome sequencing has minimum base qualities of 30, to obtain the variant call for- become feasible and increasingly popular; it prevents ascer- mat (vcf) file and the FASTA file of each sample. tainment bias and provides a new source of genetic variants Finally, to identify the mitochondrial haplogroups of each to identify novel lineages (Richards & Macaulay, 2001). Taking sample, as well as their local and private mutations, hotspots into account the genetic heterogeneity present in North and expected variants, the FASTA files were uploaded to Africa, the present study aims to describe the maternal lin- Haplogrep (Kloss-Brandst€atter et al., 2011). Haplogroup call- eage landscape and diversity of this region. ing was based on phylotree build 17 (http://www.phylotree. org, 18 February 2016) (van Oven & Kayser, 2008) (Supplementary Table 2). The 19 complete mtDNA sequences Materials and methods have been deposited in Genbank with accession numbers MG182024-MG182042 (Supplementary Table 1). Sample collection and genome sequencing Additionally, a neighbour-joining tree containing all the We collected samples from 19 individuals: nine Arabic speak- samples was built with the Molecular Evolutionary Genetics ers (two each from Morocco, Algeria, Tunisia and Egypt and Analysis 6.0 (MEGA) software (Tamura et al., 2013) in order to one from ), six Berber speakers (two each from analyse the phylogenetic relationships among the mitochon- Morocco, Algeria and Tunisia), two and two . drial sequences. The number of segregating sites was com- Blood samples were collected from healthy volunteers with puted using DnaSP v0.5 software (Librado & Rozas, 2009). appropriate informed consent and DNA was extracted follow- The H1v median-joining phylogenetic network was con- ing standard conditions. The complete genome sequences of structed with Network 5.0.0.1 (www.fluxus-engineering.com) the 19 samples of the present study were obtained by and included eight mitochondrial coding region sequences: whole-genome shotgun paired-end sequencing (Illumina FJ460532 and JQ703465 (H1v), HM171271 (H1v1a), HiSeq 2000) to a mean coverage of 30. HM171273 (H1v1b) (Behar et al., 2012; Costa et al., 2009; We also included, in the set of North African samples, Ottoni et al., 2010) and HGDP01253 (Lippold et al., 2014), nine Mozabite individuals (M'zab region, northern Sahara in HGDP01258, HGDP01264 and HGDP01267 (Poznik et al., Algeria) (Lippold et al., 2014; Poznik et al., 2013), one 2013). Specific mitochondrial sites were subtracted from the Western Saharawi and one Arab-speaker Libyan (Lorente- network analysis: hotspots and an insertion in positions Galdos et al., unpublished work). Twelve additional external 941–942 to avoid variable mutation rates. The coalescence references were also included: one North Chad individual, age of haplogroup H1v was estimated considering only the one sample from South Chad and one individual from Kenya mitochondrial coding region (577–16,023) using a mutation (Lorente-Galdos et al., unpublished work), one Mandeka rate of one nucleotide substitution every 3533 years (Soares € (Prufer et al., 2013), one French, one Sardinian, one Dinka et al., 2009). from South , one Yoruba, one San, two Chinese and one individual from the Democratic Republic of Congo (MbutiPygmy) (Reich et al., 2010). A total of 42 mtDNA Results sequences were analysed, including 26 North African and 16 Sequence diversity and haplogroup identification external references (Supplementary Table 1). The project was reviewed and approved by the In 26 North African individuals, we found 23 different com- Institutional Review Board of the Comite Etic d’Investigacio plete mtDNA sequences (BTUN01 and BTUN02 share a haplo- Clınica-Institut Municipal d’Assistencia Sanitaria (CEIC-IMAS) in type, as do BMOZ02, BMOZ04 and BMOZ06), with 258 Barcelona (2016/6723/I). segregating sites. As shown in Supplementary Table 2, 100 N. FONT-PORTERIAS ET AL.

(A)

(B)

Figure 1. Haplogroup distribution in each North African region. (A) Colour panel of the haplogroups represented in (B) and the haplogroups of the 26 North African sequences. Relative frequencies are shown in parentheses. (B) Map of North African haplogroup frequencies in each country from the literature (Cherni et al., 2005; Coudray et al., 2009; Fadhlaoui-Zid et al., 2004; Krings et al., 1999; Macaulay et al., 1999; Ottoni et al., 2009; Plaza et al., 2003; Rando et al., 1998; Stevanovitch et al., 2004; Turchi et al., 2009). Since these reference frequencies are based in control region sequences, haplogroup V (which can only be distin- guished with coding region sites) is subsumed within HV0.

Table 1. Frequencies of mitochondrial haplogroups of the 26 North African samples. Haplogroup Individuals (n ¼ 26) Frequency (%) Haplogroup Individuals (n ¼ 26) Frequency (%) Eurasian origin 18 72.6 South-Saharan origin 5 19.2 H1 6 23 L3 3 11.5 HV 1 3.8 L2 2 7.7 V 2 7.7 R0 1 3.8 North African origin 2 7.6 U3 1 3.8 U6 1 3.8 U5 1 3.8 M1 1 3.8 K1 3 11.5 J2 1 3.8 T1 1 3.8 T2 1 3.8 W1 1 3.8 we identified 22 different haplogroups in the whole dataset, mitochondrial haplogroups have an autochthonous North showing a high diversity of haplogroups. Within the 26 North African lineage (Table 1). These mtDNA haplogroup frequen- African samples, a total of 17 haplogroups were described cies agree with previous studies performed in the area based (H1, H14, H1v, HV0, J2, K1a, L2, L3, M1, R0, T1, T2, U3, U5b, on partial mtDNA sequences from Arab- and Berber-speaking U6a, V, W1), supporting a high diversity of mitochondrial individuals: Brakez et al. (2001) found in Moroccan samples sequences across all North African groups (Figure 1). that 68% of haplogroups had a Eurasian origin, 26% came To further describe the North African mtDNA lineages from the south-Saharan region, and 6% belonged to autoch- found in our analysis, haplogroups were classified in three thonous North African lineages; Fadhlaoui-Zid et al. (2004) general categories according to their geographical origin, as described Tunisian Berber sequences as 71% from Eurasia, suggested by Coudray et al. (2009): Eurasian (H, HV, R0, J, T, 26% with south-Saharan origin, and 3% from autochthonous U and W), south-Saharan (L0, L1, L2 and L3) and North North Africa; and Fadhlaoui-Zid et al. (2011) reported hap- African lineages (U6 and M1). The results show that 73% of logroup frequencies in Libya of 65% with a Eurasian origin, the samples carry Eurasian haplogroups, 19% of the individu- 28% from south-Saharan lineages and 7% from autochthon- als belong to south-Saharan lineages, and 8% of ous North Africa. ANNALS OF HUMAN BIOLOGY 101

Figure 2. Neighbour joining tree of the 42 samples based on mitochondrial sequences. Boxes are coloured by haplogroup. Figures along the branches are boot- strap robustness estimates.

No major differences were observed between Arab- and (the North African sequences and the external references). Berber-speaking individuals. The mtDNA characterisation of The phylogeny was rooted considering the Mbuti Pygmy Berber-speaking individuals included Western Eurasian hap- sample (MBU), a south-Saharan individual belonging to the logroups, south-Saharan L lineages, and autochthonous most ancestral mitochondrial haplogroup (L0a2b) of the 42 North African haplogroups. The 42 analysed samples include samples, bearing in mind that the root of human mitochon- 15 Berber individuals, whose haplogroup and lineage origin drial sequences is found between haplogroups L0 and match the frequencies described by Coudray et al. (2009). L1’2’3’4’5’6 (Behar et al., 2012)(Figure 2). Our analysis shows that 73.3% of Berber samples belong to We identified a novel mitochondrial lineage (designated Eurasian haplogroups, 13.3% to south-Saharan lineages and as H1v2) which belongs to a sub-branch of haplogroup H1v, 13.3% to North African haplogroups. From west to east, the according to the phylogenetic analysis (Figure 3). two from Morocco belong to M1 and U5, the eight Haplogroup H1v2 is defined by the motif T10101C-T146C, a Mozabites belong to H1, V, U6, U3 and L3, the two Zenata coding region mutation and a reversion to the ancestral state Berbers from Algeria carry HV and L2 haplogroups and the in the control region, respectively. This newly-identified lin- two Tunisian Berbers belong to K1. eage includes four Mozabite sequences from the HGDP pro- ject (HGDP01253, HGDP01258, HGDP01264, HGDP01267). An Identity by Descent (IBD) analysis, performed with PLINK 1.9 Phylogenetic analysis (Purcell et al., 2007) with 660,918 autosomal SNPs (Li et al., To deeply analyse the phylogenetic relationships among indi- 2008), shows that the IBD values between these Mozabite viduals and haplogroup distribution among populations, a individuals are not higher than with those from the rest of neighbour joining tree was built including the whole dataset the Mozabites with different mtDNA haplogroups. The 102 N. FONT-PORTERIAS ET AL.

H African coast being the nucleus for the dispersion of the H1 haplogroup (Ennafaa et al., 2009; Ottoni et al., 2010). Within

G3010A this region, new H1 sub-clades progressively appeared (H1w, H1x and H1v) (Ottoni et al., 2010). Our results show that the H1 H1v sub-branch includes Berber sequences from Tunisia (basal H1v), Tahala (H1v1a) and Al Awaynat (H1v1b), Tuareg

C10314T groups from Libya and the novel mitochondrial lineage H1v2 is supported by four Mozabite sequences from Algeria. Our H1v coalescence age estimation of H1v is found between 1900 and 6100 years, which agrees with previous time estimations FJ460532 Tunisia - Berber (Behar et al., 2012; Ottoni et al., 2010); and it might be corre- JQ703465 lated with an ancestral split of a North African nomadic population (Hernandez et al., 2017; Ottoni et al., 2010). T10101C T4313C One of the limitations of North African genetic studies is T146C(!) the non-homogeneous distribution of samples, focussing H1v1 H1v2 only in the north-western or north-eastern areas (Fadhlaoui- BMOZ02 Zid et al., 2011), so, in order to overcome this problem, our dataset includes individuals from the whole North African BMOZ04 region (Morocco, Algeria, Tunisia, Libya and Egypt), resulting T9148C G14560A BMOZ06 in a highly homogeneous and geographically well distributed BMOZ09 H1v1a H1v1b group of samples. Moreover, the identification of the novel HM171271 HM171273 lineage H1v2 was based on complete mitochondrial sequen- Libya -Tuareg Algeria - Mozabite ces. Indeed, it could not be described using only partial sequences (mainly HVS-I), as T146C mutation belongs to the Figure 3. Phylogeny of haplogroup H1v. Mitochondrial DNA variants are indi- HVS-II and T10101C to the coding region. cated along the branches. Mutations annotated with an exclamation mark (!) are reversions to an ancestral state (back mutations). Positions are relative to the rCRS. Sample names are shown in italics and ethnicity is displayed if avail- able. The novel mitochondrial lineage (H1v2) is highlighted. Conclusions The North African region presents great gene pool hetero- coalescence age of the H1v haplogroup estimated from the geneity due to its complex demographic history and back mitochondrial coding region is 3975 ± 2118 years. migrations. A proof of this variability comes from its maternal genetic landscape, which consists of a combination of the Discussion Middle Eastern, south-Saharan and Western European com- ponents as a result of the past events which occurred in this The mitochondrial analysis in the North African region shows area (Fadhlaoui-Zid et al., 2011; Gonzalez et al., 2007). Our the presence of haplogroups from three different origins: findings, in concordance with previous data (Brakez et al., Eurasian (H, HV, R0, J, T, U, W), south-Saharan (L) and autoch- 2001; Fadhlaoui-Zid et al., 2004; Gonzalez et al., 2007; Turchi thonous North African (M1 and U6). These results agree with et al., 2009), confirm that 10% of the mitochondrial sequen- the previously reported historical events that occurred in the ces are considered autochthonous from North Africa (U6 and region. First, human expansions from Europe and the Middle M1 haplogroups), 20% of the individuals belong to south- East during the Palaeolithic and the Neolithic contributed to Saharan L lineages and the remaining 70% have an Eurasian the presence of Eurasian haplogroups in North Africa (Plaza origin (H, HV, R0, J, T, U, W), whose dispersal caused the et al., 2003). Second, the appearance of south-Saharan L line- appearance of several sub-haplogroups, as is the case of the ages is attributed to the recent Arabic slave trade, starting in novel mitochondrial lineage H1v2. the 7th century CE, characterised by trans-Saharan migrations (Harich et al., 2010). Finally, it has been suggested that Acknowledgements autochthonous North African haplogroups have been present in the region since Palaeolithic times: the ancestor M1 has an We want to thank the thousands of volunteers who made this work Asian origin (Maca-Meyer et al., 2001; Olivieri et al., 2006) possible. and the U6 clade may have actually originated in South-East Europe (Hervella et al., 2016). Disclosure statement In North Africa, H1 is the most frequent West-European The authors report no conflicts of interest. The authors alone are respon- haplogroup (Coudray et al., 2009), with an east to west fre- sible for the content and writing of the paper. quency gradient (Hernandez et al., 2017). It has an estimated coalescence age of 8000–9000 years (Ottoni et al., 2010) and, as previously reported, this supports the post-glacial Funding radiation from the to the African continent Funding was provided by the Agencia Estatal de Investigacion and (Achilli et al., 2004; Roostalu et al., 2007), the Northwestern Fondo Europeo de Desarollo Regional (FEDER) (grants CGL2013–44351-P, ANNALS OF HUMAN BIOLOGY 103

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