Annals of Human Biology

ISSN: 0301-4460 (Print) 1464-5033 (Online) Journal homepage: http://www.tandfonline.com/loi/iahb20

Mitochondrial DNA structure of an isolated

Tunisian Berber population and its relationship
with Mediterranean populations

Nizar Ben Halim, Sana Hsouna, Khaled Lasram, Mariem Chargui, Laaroussi
Khemira, Rachid Saidane, Sonia Abdelhak & Rym Kefi

To cite this article: Nizar Ben Halim, Sana Hsouna, Khaled Lasram, Mariem Chargui, Laaroussi
Khemira, Rachid Saidane, Sonia Abdelhak & Rym Kefi (2018) Mitochondrial DNA structure of an
isolated Tunisian Berber population and its relationship with Mediterranean populations, Annals of
Human Biology, 45:1, 86-97, DOI: 10.1080/03014460.2017.1414875

To link to this article: https://doi.org/10.1080/03014460.2017.1414875

View supplementary material Published online: 30 Jan 2018.

Submit your article to this journal Article views: 128

View related articles View Crossmark data

Citing articles: 1 View citing articles

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, 86–97
https://doi.org/10.1080/03014460.2017.1414875

RESEARCH PAPER

Mitochondrial DNA structure of an isolated Tunisian Berber population and its

relationship with Mediterranean populations
Nizar Ben Halima,b
, Sana Hsounaa,b
, Khaled Lasrama,b, Mariem Charguia,b, Laaroussi Khemirac,
Rachid Saidanec, Sonia Abdelhaka,b and Rym Kefia,b
a
Laboratory of Biomedical Genomics and Oncogenetics, Institut Pasteur in Tunis, Tunis, Tunisia; bUniversity of Tunis El Manar, Tunis, Tunisia;
Association de Sauvegarde de la Nature et de Protection de l’Environnement
a Douiret (ASNAPED), Tunis, Tunisia
c

ABSTRACT ARTICLE HISTORY

Background: Douiret is an isolated Berber population from South-Eastern Tunisia. The strong geo- Received 25 July 2017
graphic and cultural isolation characterising this population might have contributed to remarkable Revised 27 November 2017
endogamy and consanguinity, which were practiced for several centuries. Accepted 29 November 2017
Aim: The objective of this study is to evaluate the mitochondrial DNA (mtDNA) genetic structure of
Douiret and to compare it to other Mediterranean populations with a special focus on major hap- KEYWORDS
logroup T. Douiret; North Africa;
Subjects and methods: Genomic DNA was extracted from blood samples of 58 unrelated individuals endogamy; haplogroup T;
collected from the different patrilineal lineages of the population. The hypervariable region 1 of the phylogeny
mtDNA was amplified and sequenced. For comparative analyses, additional HVS1 sequences (n ¼ 4857)
were compiled from previous studies.
Results: The maternal background of the studied sample from Douiret was mainly of Eurasian origin
(74%) followed by Sub-Saharan (17%) and North African (3%) lineages. Douiret harbours the highest
frequency of haplogroup T in the Mediterranean region, assigned to the unique subclade T1a (38%).
Phylogenetic analysis showed an outlier position of Douiret at the Mediterranean level.
Conclusions: The genetic structure of Douiret highlights the presence of founders, most likely of Near/
Middle Eastern origin, who conquered this area during the Middle/Late Upper Palaeolithic and
Neolithic dispersals.

Introduction
Douiret is a Berber (Amazigh) population located in Southern
Tunisia (in the governorate of Tataouine) (Figure 1). The
Berber community of Douiret is part of the Ouerghemma tri-
bal confederacy of South Eastern Tunisia and shapes with
other nearby Berber villages the group of the Djebalia
(Macquart, 1906).
The original village belongs to the Berber fortified hilltop
villages of the Jebel Demmer (Demmer mountains), a moun-
tainous area located between Jebel Matmata (Matmata
mountains) and Jebel Nefoussa (Nefoussa mountains) in
Libya, bounding the vast plain of “Jeffara” and the Saharan
plateau of “Dahar” (Figure 1). Douiret inhabitants have taken
the fortress or “Ksar” as a refuge from Arabic nomad tribes
raids (Louis, 1975).
The ancestry of Douiret is believed to be traced back to a
founding father with the name of Ghazi Ben Douaieb Bou
Kenana, who migrated to the region more than 600 years
ago—possibly coming from the Moroccan region of Tafilalet
(Louis, 1975). However, it is very likely that Douiret was cre-
ated by founders allied to Hafsid central authority in Tunis at
the time of the Hafsid Sultan “Abu Faris Abd al Aziz al
Mutawakkil” (1394–1434 AD) who tried to secure the south-
ern regions of the country as well as the caravan route and
to impose the Sunni dogma and Maliki legislation to Ibadis
southern populations (Renouard & Robert, 1942). These
founders are probably Moroccan Berbers originating from the
Almohad conquest of the country. They have settled in the
area by buying large properties and marrying women from
different autochthonous tribes (Bni Maaguel, Chenini, etc … ).
Moreover, since its foundation, the population of Douiret has
selectively isolated itself through several cultural barriers.
Indeed, due to their beliefs in communal living and in order
to conserve their ethnic identity and living habits, the com-
munity members remained isolated for hundreds of years by
largely mating among themselves along with a very limited
level of external migration flows (Ben Halim, 2006; Louis,
1975). Economically, Douiret has been an important caravan
stop between the city of Gabes to the North and the Libyan
city of Ghadames to the South, constituting one of the main
platforms of the trans-Saharan slave trade. The abolition of
the slave trade and slavery in 1841 contributed to the

CONTACT Rym Kefi rym.kefi@pasteur.rns.tn, kefi_rym@yahoo.fr Laboratory of Biomedical Genomics and Oncogenetics, Institut Pasteur in Tunis, BP 74, 13,
place Pasteur, Tunis 1002, Tunisia

These authors contributed equally to the paper.
Supplemental data for this article can be accessed here.
ß 2017 Informa UK Limited, trading as Taylor & Francis Group

Figure 1. Geographical position of Douiret.
decline of the region at the end of the 19th century (Louis,
1975). The Douiret population has progressively decreased
following the migration of its inhabitants to the urban areas,
mainly to the capital Tunis. A continued increase of immi-
grants from Douiret in Paris (France) was also recognised dur-
ing the twentieth century. A very strong rate of marital
endogamy has been revealed even in both exodus groups
(Capital Tunis, emigrants in Paris) with rates of 87 and 86%,
respectively (Ben Halim, 2006). Currently, Douiret inhabitants,
whose number is estimated according to the 2014 Statistics
Tunisia Census data (http://www.ins.nat.tn/sites/default/files/
publication/pdf/vol%201%20rgph%202014%20site%20%281%
29.pdf) at 2021, are occupying three localities in the region
of origin: the villages of Douiret (607), Ras El Oued (390) and
Bir Thlathine (1024). Interestingly, Douiret remained among
the few berberophone communities in Tunisia.
The genealogy of Douiret is also kept in an ancient manu-
script that dates back about four centuries (1617); the
genealogy is made of 111 families grouped into eight clans
or patrilineal lineages: Ouled Abid, Ouled Bouzid, Ouled
Abdelkrim, Ouled Belgacem, Ouled Zaghdene, Ouled Saber,
Ouled Taleb and Ouled Hamed (Ben Halim, 2006; Ch^alon-sur-
Saone, 1900; Louis, 1975). All these lineages claim to be dir-
ect descendants of the original Moroccan group with a rela-
tively well-defined filiation between the distinctive common
ancestors (Figure 2, Supplementary Appendix A).
Previous studies considered Chenini and Douiret as a one
pooled sample (Chenini-Douiret) (Coudray et al., 2009;
Ennafaa et al., 2011; Fadhlaoui-Zid et al., 2004, 2010, 2011;
Kefi et al., 2015; Khodjet-El-Khil et al., 2008). The compilation
of classical genetic markers (GM allotypes, HLA, mtDNA)
showed that Chenini-Douiret, taken as a pooled group, is
very little diversified and genetically very divergent from the
other North-African populations, even those that are cultur-
ally and geographically close (Berber populations of Southern
ANNALS OF HUMAN BIOLOGY 87
Tunisia) (Fadhlaoui-Zid et al., 2009). In a recent study using
the mtDNA uniparental maker, we revealed that Chenini-
Douiret has an outlier position within the Mediterranean
region (Kefi et al., 2015). However, despite their geographical
proximity and their belonging to the same tribal group of
Djebalia, the populations of Douiret and Chenini have differ-
ent ethnic origins. While Chenini is a very ancient autoch-
thonous community belonging to the Berbers of Louata, with
origins dating back more than 1000 years ago, Douiret is a
more recent confederation between male Moroccan founders
whose origins trace back to the Almohad conquest of Tunisia
and female founders from different autochthonous tribes, in
addition to some branches probably of Arab descent
(Macquart, 1906).
Given that the population of Douiret had witnessed sev-
eral major demographic events in the history of the settle-
ment of Tunisia such as the Almohade conquest, the
cohabitation and ethnic admixture between Arab Banu Hilal
invaders and local populations as well as the sub-Saharan
migration flow linked to the trans-Saharan slave trade; we
focussed only on this population and we proposed to ana-
lyse its mitochondrial genetic background by comparing it to
that of the neighbouring population of “Chenini”. We also
proposed to investigate the possible genetic contributions of
the different events cited above. Hence, this study is the first
to evaluate the matrilineal genetic background of the
Douiret population in the context of the Mediterranean
region with a special focus on major haplogroup T.
Materials and methods
Ethics statement
This study is a part of an international collaborative project
MEDIGENE (FP7–279171-1) which was approved by the
Ethical Committee of the Pasteur Institute in Tunisia (Tunis,
88 N. BEN HALIM ET AL.
Figure 2. Classification into haplogroups and tribes of the studied sample of 58 Tunisians from Douiret. See Supplementary Appendix A for more details on codes
and sequence information.
Tunisia- Registration number IRB00005445, FWA00010074)
under the reference IPT/LR11–05/Etude 04/2013. All partici-
pants gave written informed consent. This study was con-
ducted according to the principles of the Declaration of
Helsinki.
Samples
DNA was extracted from blood samples obtained from 58
volunteers, using salting-out method as described previously
(Miller et al., 1988). This sample was selected from an initial
sample of 209 individuals from the study population who
accepted to participate in this investigation. The selection
was performed according to the following criteria: (i) since
most members of the population are more or less related,
we tried to avoid selecting closely related participants by
choosing those who were all beyond second degree related
based on three-generation genealogical data; (ii) being repre-
sentative of the eight clans making up the study population;
(iii) having as much homogeneous distribution of samples as
possible across the different clans (Ouled Abid (7), Ouled
Bouzid (7), Ouled Abdelkrim (7), Ouled Belgacem (9), Ouled
Zaghdene (8), Ouled Saber (7), Ouled Taleb (7), and Ouled
Hamed (6)); and (iv) collecting as many families as possible
(45/111) to maximise the representativeness of the sampling
(Figure 2, Supplementary Appendix A).
For comparative analyses, we also extended analyses to
HVS1 sequences (n ¼ 4857) belonging to 54 other Tunisian
and Mediterranean populations (Table 1).
Genetic analysis
The HVS1 region of the mtDNA was amplified and sequenced
using procedures previously described by Stevanovitch et al.
(2004). Successfully amplified products were sequenced using
ABI 3130 (Applied Biosystems Life Technologies SAS, Saint
Aubin, France). Sequences were analysed using Seqscape
software (V2.7) (Applied Biosystems, Life Technologies SAS)
and the revised Cambridge Reference Sequence (rCRS) as ref-
erence (Andrews et al., 1999). Haplogroup assignment was
performed following van Oven and Kayser (2009). Generated
sequences were available in GenBank under the following
accession numbers: KJ187916–KJ187926; KJ187928–KJ187945;
and KP081700–KP081728. Results are detailed in
Supplementary Appendix A.
Statistical and phylogenetic analyses
The Median network of HVS1 sequences belonging to T hap-
logroup was constructed with the median-joining algorithm
(Bandelt et al., 1999), using the Network 4.6.1.1 software
(Copenhagen, Denmark; http://www.fluxus-engineering.com/)
(Saillard et al., 2000).
Computation of diversity parameters (number of different
sequences (K), haplotype diversity (H), average number of
pairwise differences (MNP)) and genetic distances (FST
matrix), was performed using the Arlequin software (V.3.5)
(Berne, Switzerland) (Excoffier et al., 2005). We used the stat-
istical package for the social sciences (SPSS, version 13.0,
Chicago, IL) for the multidimensional scaling (MDS) method.
All the statistical and phylogenetic analyses performed on
the study sample were applied for the HVS1 sequence range
16 069–16 383. For comparisons with other Tunisian and
Mediterranean populations, we analysed the HVS1 region
from the position 16 085–16 365.
Demographic events and changes in effective population
size through time were analysed according to two different
methods: (i) potential departures from a null hypothesis of
the mutation-drift equilibrium and constant population size
were estimated by computing the Tajima’s D test for select-
ive neutrality (Tajima, 1989), where negative values of
Tajima’s D statistic might be indicative of recent demo-
graphic expansions; and (ii) the distribution of pairwise differ-
ences (mismatch distribution) where we tested the
 ANNALS OF HUMAN BIOLOGY 89

Table 1. Data collected from 55 Mediterranean populations. Table 1. Continued

Sample Sample
Populations Code size References Populations Code size References
North Africa France
Tunisia French FRE 109 Dubut et al. (2004)
North Centre Corsica COR 47 Varesi et al. (2000)
Qalaat El Andalous QAL 29 Cherni et al. (2009) Southern Corsica COB 53 Falchi et al. (2006)
Capital Tunis CTU 98 Cherni et al. (2009); Italy
Plaza et al. (2003) South Italy ITS 86 Francalacci et al. (1996);
El Alia ELA 48 Cherni et al. (2009) Richards et al. (2000)
Zriba ZRI 35 Cherni et al. (2009) Sardinianb SAR 303 Di Rienzo and Wilson (1991);
Slouguia SLO 28 Cherni et al. (2009) Falchi et al. (2006)
Testour TES 50 Cherni et al. (2009) Sicilian SIC 169 Cali et al. (2001);
Kesra KES 43 Cherni et al. (2009) Richards et al. (2000)
South Tuscan TUE 61 Falchi et al. (2006)
Skira SKI 20 Cherni et al. (2009) Total Eurasian sequences 2738
Jerba Arabs JEA 29 Loueslati et al. (2006) Total 4915
Jerba Berbers JEB 30 Loueslati et al. (2006) a
Chenini-Douiret CHO 53 Fadhlaoui-Zid et al. (2004) Fezzan group consists of the pooled Fezzan (Al Awaynat) and Fezzan (Tahala)
Sened SEN 55 Fadhlaoui-Zid et al. (2004) samples (Ottoni et al., 2009).
b
Bou Omrane OMB 40 Ennafaa et al. (2011) Sardinian group consists of the pooled Central Sardinia, Northern Sardinia,
Bou Sa^ad SAB 40 Ennafaa et al. (2011) Sardinian (Trexenta), Sardinian (San Pietro island) and Sardinian (Sant’ Antioco
Matmata MAT 53 Fadhlaoui-Zid et al. (2004) island) groups (Di Rienzo & Wilson, 1991; Falchi et al., 2006).
Douiret DOU 58 Present study
Total Tunisians 709
Libya
Fezzana FAL 129 Ottoni et al. (2009) distribution of all pairwise haplotype differences and calcu-
Egypt lated the goodness-of-fit of the estimated distribution to that
Upper Egypt EGY 102 Stevanovitch et al. (2004) predicted by a sudden expansion model using 1000 com-
Gurna GUR 34 Stevanovitch et al. (2004)
Siwa Berbers SIB 78 Coudray et al. (2009) puter simulations (Schneider & Excoffier, 1999). Mismatch dis-
Alexandria ALX 277 Saunier et al. (2009) tributions were graphically displayed in Microsoft Excel 2007.
Algeria Mismatch distributions tend to be unimodal in populations
Algerians ALG 47 Plaza et al. (2003)
Algerian Mozabites MOZ 85 Corte-Real et al. (1996)
that have undergone population expansion or multimodal,
Morocco random and rough in populations that have experienced
Southern Moroccan (Berbers) MBS 50 Brakez et al. (2001) long-term stability (Rogers & Harpending, 1992; Slatkin &
Northern Moroccan (Berbers) MBN 60 Plaza et al. (2003)
MoroccanArabs MOA 50 Plaza et al. (2003) Hudson, 1991). The significance of the observed data to the
Saharawi SAH 56 Plaza et al. (2003) predicted distribution modelled for sudden expansion growth
Marrakech MAR 52 Falchi et al. (2006) was assessed by using a sum of squares differences (SSD)
Asni Berbers ASB 53 Coudray et al. (2009)
Bouhria Berbers BOB 70 Coudray et al. (2009) method and raggedness index (rg) (Excoffier, 2004;
Figuig Berbers FIB 94 Coudray et al. (2009) Harpending, 1994). Significant differences in the sum of the
Total North African sequences 1946
square deviations (pSSD < 0.05) and raggedness index
Near East (prg < 0.05) between the observed and simulated mismatch
Palestine–Israel
Palestinian-Israeli PAL 117 Richards et al. (2000) distributions indicated that the population was at a non-
Druze DRU 45 Macaulay et al. (1999) expansion phase (Rogers & Harpending, 1992; Slatkin &
Syria Hudson, 1991).
Syrian SYR 69 Richards et al. (2000)
Total Near Eastern populations 231
Europe Results
Greece
Greeks GRE 184 Villems (2011) Diversity parameters
Northern Greeks GNG 319 Irwin et al. (2008)
Cyprus The study of the diversity parameters in Douiret showed a
Cypriots GRC 91 Irwin et al. (2008)
reduced heterogeneity at the haplotype level compared to
Turkey
Turks TUR 213 Richards et al. (2000) other Tunisian populations, mainly Northern ones as well as
Spain some Western Mediterranean populations (Egypt, Greece and
Andalusian AND 158 Plaza et al. (2003) Cyprus), reflected by the low value of haplotype diversity
Andalusia (Granada Province) AGP 66 Falchi et al. (2006) (H ¼ 0.933 ± 0.015) as well as the low mean number of pair-
Catalan CAT 162 Plaza et al. (2003)
Galician GAL 374 Alvarez-Iglesias et al. (2009); wise differences (MNP ¼ 6.294 ± 3.030). However, this diversity
Salas et al. (1998) is similar or slightly higher than that observed in the other
Basque BAS 45 Bertranpetit et al. (1995)
Majorcan MAJ 112 Falchi et al. (2006);
North African populations (Libya, Algeria and Morocco) as
Picornell et al. (2005) well as among the Northern Mediterranean ones (France,
Minorcan MIN 46 Picornell et al. (2005) Italy and Spain) (Table 2).
Valencian VAL 42 Picornell et al. (2005)
Ibizan IBI 50 Picornell et al. (2005) Sequence comparison of the HVS1 region from Douiret
Chuetas CHU 48 Picornell et al. (2005) leads to the identification of 22 different haplotypes, 10 of
(continued) which were unique and 12 were shared by more than two
90 N. BEN HALIM ET AL.

Table 2. Indices of molecular diversity among the study population and 54 Mediterranean populations (Kefi et al., 2015).
Populations Code Sample size K S H ± SD ¶ ± SD MNP ± SD
Qalaat El Andalous QAL 29 16 24 0.933 ± 0.028 0.017 ± 0.010 4.884 ± 2.451
Capital Tunis CTU 98 80 169 0.993 ± 0.003 0.037 ± 0.019 10.367 ± 4.769
El Alia ELA 48 29 150 0.963 ± 0.016 0.076 ± 0.038 21.420 ± 9.615
Zriba ZRI 35 17 150 0.926 ± 0.028 0.062 ± 0.031 17.408 ± 7.925
Slouguia SLO 28 21 147 0.973 ± 0.018 0.068 ± 0.035 19.256 ± 8.788
Testour TES 50 36 187 0.958 ± 0.021 0.078 ± 0.039 21.933 ± 9.830
Kesra KES 43 29 46 0.959 ± 0.020 0.023 ± 0.012 6.445 ± 3.111
South
Skira SKI 20 14 144 0.937 ± 0.043 0.086 ± 0.044 24.259 ± 11.130
Jerba Arabs JEA 29 23 38 0.978 ± 0.017 0.020 ± 0.011 5.656 ± 2.793
Jerba Berbers JEB 30 26 40 0.984 ± 0.016 0.016 ± 0.009 4.549 ± 2.300
Chenini-Douiret CHO 53 23 148 0.939 ± 0.017 0.243 ± 0.118 68.244 ± 29.886
Sened SEN 55 39 132 0.977 ± 0.010 0.053 ± 0.027 15.033 ± 6.825
Bou Omrane OMB 40 14 22 0.853 ± 0.045 0.015 ± 0.008 4.233 ± 2.145
Bou Sa^ad SAB 40 16 36 0.897 ± 0.033 0.033 ± 0.017 9.275 ± 4.353
Matmata MAT 53 34 158 0.956 ± 0.019 0.044 ± 0.022 12.290 ± 5.639
Douiret (Present study) DOU 58 22 35 0.933 ± 0.015 0.175 ± 0.160 6.294 ± 3.030
Fezzana FAL 129 98 32 1.000 ± 0.004 0.015 ± 0.008 4.109 ± 2.077
Upper Egypt EGY 102 87 80 0.995 ± 0.003 0.028 ± 0.014 7.816 ± 3.668
Gurna GUR 34 29 57 0.989 ± 0.010 0.031 ± 0.016 8.670 ± 4.104
Siwa Berbers SIB 78 78 181 1.000 ± 0.002 0.071 ± 0.035 20.064 ± 8.964
Alexandria ALX 277 277 210 1.000 ± 0.001 0.154 ± 0.074 43.203 ± 18.794
Algerians ALG 47 24 44 0.940 ± 0.018 0.019 ± 0.010 5.219 ± 2.570
Algerian Mozabites MOZ 85 30 36 0.943 ± 0.010 0.017 ± 0.009 4.856 ± 2.392
Southern Moroccan (Berbers) MBS 50 34 35 0.961 ± 0.018 0.015 ± 0.009 4.313 ± 2.171
Northern Moroccan (Berbers) MBN 60 38 50 0.963 ± 0.015 0.016 ± 0.009 4.514 ± 2.253
Moroccan Arabs MOA 50 46 60 0.994 ± 0.007 0.024 ± 0.013 6.747 ± 3.234
Saharawi SAH 56 41 45 0.976 ± 0.012 0.020 ± 0.011 5.500 ± 2.685
Marrakech MAR 52 52 43 1.000 ± 0.004 0.019 ± 0.010 5.215 ± 2.564
Asni Berbers ASB 53 53 152 1.000 ± 0.004 0.071 ± 0.035 19.993 ± 8.980
Bouhria Berbers BOB 70 70 148 1.000 ± 0.002 0.039 ± 0.020 10.871 ± 5.004
Figuig Berbers FIB 94 94 46 1.000 ± 0.002 0.020 ± 0.011 5.666 ± 2.741
Palestinian-Israeli PAL 117 100 90 0.994 ± 0.003 0.021 ± 0.011 5.799 ± 2.794
Druze DRU 45 23 28 0.940 ± 0.019 0.016 ± 0.009 4.355 ± 2.194
Syrian SYR 69 56 70 0.988 ± 0.007 0.021 ± 0.011 5.433 ± 2.648
Greeks GRE 184 87 73 0.967 ± 0.007 0.016 ± 0.009 4.368 ± 2.169
Northern Greeks GNG 319 319 207 1.000 ± 0.001 0.163 ± 0.078 45.828 ± 19.909
Cypriots GRC 91 91 172 1.000 ± 0.002 0.036 ± 0.018 10.008 ± 4.618
Turks TUR 213 152 94 0.988 ± 0.004 0.018 ± 0.010 4.973 ± 2.428
Andalusian AND 158 141 74 0.994 ± 0.003 0.015 ± 0.008 4.091 ± 2.050
Andalusia (Granada Province) AGP 66 66 45 1.000 ± 0.003 0.015 ± 0.008 4.075 ± 2.058
Catalan CAT 162 138 172 0.990 ± 0.004 0.025 ± 0.013 7.157 ± 3.374
Galician GAL 374 335 121 0.996 ± 0.002 0.015 ± 0.008 4.244 ± 2.110
Basque BAS 45 27 29 0.949 ± 0.002 0.011 ± 0.006 3.106 ± 1.643
Majorcan MAJ 112 98 64 0.994 ± 0.003 0.016 ± 0.009 4.444 ± 2.208
Minorcan MIN 46 35 48 0.987 ± 0.007 0.016 ± 0.009 4.596 ± 2.298
Valencian VAL 42 39 44 0.995 ± 0.007 0.015 ± 0.008 4.167 ± 2.113
Ibizan IBI 50 19 23 0.939 ± 0.014 0.012 ± 0.007 3.484 ± 1.806
Chuetas CHU 48 28 37 0.938 ± 0.025 0.016 ± 0.009 4.596 ± 2.296
French FRE 109 62 57 0.963 ± 0.011 0.014 ± 0.008 3.921 ± 1.981
Central Corsica COR 47 31 36 0.943 ± 0.027 0.013 ± 0.007 3.557 ± 1.841
Southern Corsica COB 53 53 37 1.000 ± 0.004 0.013 ± 0.007 3.655 ± 1.880
South Italy ITS 86 68 71 0.962 ± 0.016 0.017 ± 0.009 4.715 ± 2.330
Sardinianb SAR 303 46 40 0.986 ± 0.009 0.014 ± 0.008 3.856 ± 1.979
Sicilian SIC 169 94 83 0.933 ± 0.017 0.014 ± 0.008 3.842 ± 1.941
Tuscan TUE 61 61 48 1.000 ± 0.003 0.013 ± 0.007 3.526 ± 1.819
n: number of individuals; K: number of different sequences; S: number of variable positions; H: haplotype diversity; ¶: nucleotide diversity; MNP: mean number of
pairwise differences; SD: standard deviation.
a
Fezzan group consists of the pooled Fezzan (Al Awaynat) and Fezzan (Tahala) samples (Ottoni et al., 2009).
b
Sardinian group consists of the pooled Central Sardinia, Northern Sardinia, Sardinian (Trexenta), Sardinian (San Pietro island) and Sardinian (Sant’ Antioco island)
groups (Di Rienzo & Wilson, 1991; Falchi et al., 2006).

individuals compared to the remaining Tunisian and The 22 haplotypes screened were assigned to 10 major
Mediterranean populations. haplogroups; the most frequent haplogroup T (38%) was fol-
lowed by L3, J, H and HV (9–14%), whereas L1, L2, K, U and
M1 contributed slightly to the mtDNA genetic background of
Genetic structure of Douiret Douiret (2–3%) (Figures 3 and 4, Supplementary Appendix B).
This study showed a stronger amount of Eurasian hap- Our results showed that the Eurasian haplogroup T
logroups (74%) compared to Sub-Saharan (17%) and North reaches its highest value in Douiret compared to other
African (3%) lineages that were less frequent or rare in Tunisian and Mediterranean populations (Figure 4,
Douiret. Supplementary Appendix B).

Figure 3. Mitochondrial DNA genetic structure of the Douiret population. EL, Eurasian lineages: H, HV, J, K, T and U; SL, Sub-Saharan lineages: L1, L2 and L3; NAL,
North African lineages: M1.
The comparisons of the observed haplogroup frequencies
among our sample (DOU) to those reported within the
“Chenini and Douiret” cohort (CHO) (Fadhlaoui-Zid et al.,
2004), showed a significant difference in the distribution of
the Eurasian haplogroup K, being rare in DOU (2%) and fre-
quent in CHO (15%) (p < .05).
The median joining network of the T haplogroup was con-
structed with 423 sequences from Tunisian and other
Mediterranean populations in which 22 sequences are from
Douiret (9 DOU0006, 9 DOU0007, 1 DOU0013, 1 DOU0146, 1
DOU0125 and 1 DOU0159). The network mainly displays the
presence of two nodes: T1a and T2b (Figure 5). The main T
lineages found in Douiret belong to the T1a subclade,
whereas T1b and T2 lineages were absent in Douiret. The
median joining network showed no specific node for the
samples of Douiret.
The haplotype DOU0013 is derived from DOU0159 by the
accumulation of the substitution T16189C. DOU0013
belonged to the largest node T1a haplogroup composed by
83 sequences from North Africa (n ¼ 45), the Near East (n ¼ 4)
and Europe (n ¼ 34). Haplotype DOU0007 was derived from
the former node T1a in addition to the deletion 192delC.
Haplotype DOU0007 occupied the same node as sequences
from Tunisia and Egypt. Haplotypes DOU0006 and DOU0146
are clustered in the same node with Tunisians, Egyptians,
Spaniards and Italians and descent from the node DOU0125
by the accumulation of the T16189C substitution. The haplo-
type DOU0159 is linked to DOU0125 by the occurrence of
the C16294T transition. Finally, DOU0125 was found with one
Tunisian sequence (1 JEA).
The second large node T2b contains 61 sequences
from North Africa (n ¼ 12), the Near East (n ¼ 2) and
Europe (n ¼ 47). In contrast, T1b, T2a, T2c, T2e and T2f were
less frequent in the Mediterranean region (Supplementary
Appendix E).
Relationships of Douiret with Tunisian and
Mediterranean populations
The FST matrix relating the 55 Tunisian and Mediterranean
populations was reported in Supplementary Appendices C
ANNALS OF HUMAN BIOLOGY 91
and D. Computation of the genetic distances between popu-
lation pairs revealed that Tunisians from Bou Sa^ad are the
most separated sample from Douiret in all the considered
populations (FST ¼ 0.21313).
A clear and significant genetic differentiation was found
between Douiret Berbers and all the studied Mediterranean
populations (Supplementary Appendices C and D). This differ-
entiation was reflected by the outlier position of Douiret
within the MDS plot (Figure 6).
According to the MDS plot (Figure 6), the population of
Douiret is an outlier such as the populations of Bou Omrane
(SAB), Mozabites (MOZ) and Chenini-Douiret (CHO).
Interestingly, the FST matrix underlined a clear and signifi-
cant differentiation between the present study Douiret
sample (DOU) and the pooled sample of the study of
Fadhalaoui-Zid et al. (2004) (Chenini and Douiret) (CHO)
(Supplementary Appendix D).
Demographic analysis
The shape of the mismatch distribution analyses between
pairs of HVS1 sequences of the study isolate, based on sum
of squared deviations (0.01066, pSSD ¼ 0.24) and raggedness
index (0.01653, Prg ¼ 0.36), showed an almost unimodal
curve for the population of Douiret, indicating a model of
sudden expansion (p(sim  obs) > 0.05) (Figure 7). The Fu’s Fs
parameter, which is based on the probability of recovering a
number of haplotypes greater than or equal to the observed
number in a sample drawn from a stationary population with
the same mean number of pairwise differences as the
observed sample, showed a negative value in our sample
(–4.17), also indicating a sudden expansion in the population
at various geographical locations. On the other hand, the
Tajima’s D parameter has a negative value for the sample
data set (D ¼ –0.76) indicative of a population expansion,
despite a non-significant p-value (p ¼ .23).
Discussion
The present study evaluates the mitochondrial DNA genetic
structure of Douiret, an isolated Berber community from
 92
N. BEN HALIM ET AL.

Figure 4. Haplogroup frequencies in Douiret and Mediterranean comparing populations. (A) Douiret vs Northern Tunisian, (B) Douiret vs Southern Tunisian, (C) Douiret vs North African populations, (D) Douiret vs European
populations, (E) Douiret vs Middle Eastern populations. See Table 1 for more details on codes and reference information.

Figure 5. Phylogenetic network of Douiret and other Tunisian and Mediterranean sequences belonging to haplogroup T (present data and data from literature).
The size of the circles is proportional to the number of sequences. Numbers along links refer to nucleotide positions in HVS1 minus 16 000.
South-Eastern Tunisia. It also provides the phylogenetic rela-
tionships between Douiret and other Tunisian, North African,
Eurasian and Near Eastern populations belonging to the
Mediterranean region.
This study revealed reduced diversity parameters in
Douiret. Our findings are in accordance with previous studies,
showing that Berbers from the pooled population (Chenini-
Douiret) are very little diversified and genetically very diver-
gent (Fadhlaoui-Zid et al., 2004, 2009).
Investigation of unique sequences in the context of
Mediterranean populations showed that Douiret contains a
large number of unique sequences (10/22). This finding is in
accordance with a recent study in which we reported the
high amount of unique sequences in Tunisians (10.2%) com-
pared to Mediterranean populations (Kefi et al., 2015).
The mtDNA haplogroup composition of Douiret showed a
high level of West Eurasian sequences (74%), of which hap-
logroup T is the most common (38%). It is also noteworthy
that this frequency appeared to be the highest among
Mediterranean populations. Haplogroup T is believed to have
originated in the Near East (Richards et al., 1998). The phylo-
genetic network of sequences belonging to haplogroup T
showed that the sequence DOU0013 is the most shared
haplotype among the Mediterranean populations.
Furthermore, the two haplotypes, DOU0006 and DOU0007,
belonging to the T1a haplogroup, are the most frequent hap-
lotypes in Douiret (18/22). This sub-clade, T1a, might be the
result of a molecular instability at nucleotide positions 16292,
16296 and 16304 (Malyarchuk & Derenko, 1999).
ANNALS OF HUMAN BIOLOGY 93
Moreover, haplogroup H was the second most frequent lin-
eage in Douiret (14%). Among H lineages, only H1 was pre-
sent in our sample. In a previous study in which haplogroup
H was dissected, it was apparent that the sub-clade H1 high-
lights the influence of the Iberian Peninsula on Western North
Africa (Ennafaa et al., 2009). Indeed, H1 originated in the
Middle East and spread from there to Europe and North
Africa via the Iberian Peninsula. Coalescence age for H1
(11 ± 2 ky) in North Africa points to the possibility of a late
Palaeolithic settlement for this lineage (Ennafaa et al., 2009).
Moreover, Douiret encompassed high and similar frequencies
for both haplogroups HV and J (9%) (Figure 3, Supplementary
Appendix B). HV lineages were refined to sub-haplogroup
HV1, while haplogroup J was assigned to the sub-clade J1b.
Haplogroup HV, which is mainly found in the Middle East, is
also the most common and diverse in the Southern Caucasus
(Roostalu et al., 2007). The Near Eastern haplogroup J reflects
the spread of the Neolithic from the Levant into North Africa
(Coudray et al., 2009; Pereira et al., 2010). Other Eurasian hap-
logroups (K and U) were also observed in the Douiret gene
pool with lower frequencies (2%).
Indeed, founder events followed by long isolation proc-
esses from the rest of the Tunisian population (both endog-
amy and consanguinity practiced for several generations)
seem to have played a major role in shaping the scattered
pattern of haplotype and haplogroup frequencies that is
often restricted to the geographic mountainous area of
Demmer. This pattern may also be explained by the intense
genetic drift in haplotype frequencies, as a result of the
94 N. BEN HALIM ET AL.
Figure 6. Position of Douiret among 55 Mediterranean populations using MDS method. See Table 1 for more details on codes and reference information.
Figure 7. Mismatch distribution of Douiret population inferred from mtDNA
HVS1 sequences. Bars in lineage charts are observed data, line is predicted dis-
tribution under a model of rapid demographic expansion of the population;
x-axis: number of pairwise mismatches, y-axis; frequency.
reduced community size that did not exceed 3500 inhabi-
tants in the mid-19th century (Louis, 1975).
Mitochondrial DNA sequences shared between Douiret
and other Mediterranean populations highlight different
waves of migrations, mainly from the Near East. In fact, with
the exception of H1, which most likely spread from Iberia to
North Africa, all mtDNA haplogroups (T, HV, J, U and K) are
most likely of Middle Eastern origin and were introduced in
Douiret by later arrivals in the Middle/Late Upper Palaeolithic
and during the Neolithic dispersals (Richards et al., 2000;
Torroni et al., 1998).
On another level, the differentiation between our sample
Douiret (DOU) and the sample “Chenini and Douiret” (CHO)
(Fadhlaoui-Zid et al., 2004) highlighted by the haplogroup K
frequency (p < .05), the FST matrix and MDS plots (Figure 5),
might be explained by differences in the ethnic origin:
Berber of Zenata for Douiret vs Berber of Louata for Chenini
(Macquart, 1906). In addition, compared to Chenini, Douiret
displayed more heterogeneity in female ancestry, which,
according to oral population history and some family tree
manuscripts, trace back to several local Berber groups
of Jebel Demmer (Demmer mountains), Jebel Abiod
(Abiod mountains) (Bni Maaguel, Chenini and Sedra from
Louata, Bni Silin from Kutama, Bni Wassin from Zenata, Riga
from Houara, Ksar El Galaa, Meguedmine, … ) and Jebel
Nefoussa (Nefoussa mountains) in Libya (Wazzin) (Louis,
1975) (Figure 1).
Our results also showed the relatively low contribution
of the Sub-Saharan lineages in the genetic structure of
Douiret (17%) compared to the general Tunisian population
(44%) (Kefi et al., 2015). We found that only haplogroup L3
is frequent in Douiret, reaching a frequency of 12%. This is
somewhat surprising since, despite its current perception as
an isolated outpost, Douiret was once an important

stopover in the sub-Saharan slave trade from Eastern,
Central and Western Africa to Maghreb. The presence of
diverse Sub-Saharan mitochondrial lineages in the mtDNA
background of Douiret (L1c3b, L2a1c, L3d5, L3h1 and L3i2)
may have resulted from a possible mixing with many
African families who have lived for a long time with Douiret
inhabitants.
It is noticeable that, among North African lineages, only
haplogroup M1 was present with low frequency (3%),
whereas U6 lineage was absent in the mtDNA gene pool of
Douiret. It seems that the presence of the haplogroup M1 in
Douiret signals an East African influence with possible minor
introductions from the Levant.
The observed genetic structure of Douiret was confirmed
at the phylogenetic analysis level. A clear genetic differenti-
ation was observed between Berbers from Douiret and
other Berber groups from Tunisia (Bou Omrane) and Algeria
(Mozabites), which harbour more North African lineages (U6
and M1). Likewise, our sample does not cluster with other
Berber communities from Tunisia like Sened, Bou Saad,
Matmata, Jerba Berbers and Kesra revealing that the
autochthonous inhabitants of Jebel Demmer, who make up
the main maternal genetic background of Douiret, bear a
particular mtDNA structure compared to other populations
with the same Amazigh culture. In addition, Douiret was
not connected to any European population (Figure 6). It
seems that the different European migratory waves that
have been experienced by Tunisia during its history
(Romans, Vandals, Byzantines, Normans, Turkish, etc … ) left
no traces on this isolated community of South Eastern
Tunisia. MDS plots also revealed the lack of affiliation
between our sample and Moroccan populations. This gives
evidence that migration from Morocco during Almohad and
Hafsid times (1159–1574 AD), which concerned the whole
country in general and Douiret in particular, could be exclu-
sively composed of males. On the other hand, given the
isolation of this community as shown by the high endog-
amy rates, it may be that genetic drift had a great role in
the present haplogroup composition, by eliminating some
lineages and enriching others. The position of our sample
in the plot is also distant from the groups of Arab descent
(Jerba Arabs, Moroccan Arabs). Thus, geographical proximity
between Douiret and the surrounding Hilalian Arab tribes
for nearly six centuries does not seem to be reflected in
terms of genetic exchange. This is most probably explained
by the intense reproductive isolation undergone by the
community during its history, as evidenced by the very
high rate of geographical and ethnical endogamy; 98.16%
(Ben Halim, 2006).
Regarding the demographic events experienced by the
study community during its history, the mismatch distribu-
tion analysis revealed the genetic signature of a population
expansion through the observed unimodal curve shape,
despite a very small second mode towards the beginning,
indicating that some minor population expansion might
have occurred at some stage in various geographical areas.
This could also be linked to other factors like inbreeding
and admixture that could affect the shape of mismatch dis-
tribution. In this same context, the observed shape of the
ANNALS OF HUMAN BIOLOGY 95
pairwise differences curve was also supported by Fu’s Fs
and Tajima’s D values that indicated a sudden demographic
population expansion from a limited number of founders.
Conclusions
This is the first study that has exclusively targeted the ana-
lysis of the mtDNA structure of the population of Douiret, a
Berber community that was witness to the population-
exchanges that took place in Tunisia between local popula-
tions and migrants from the West (Morocco and Western
Algeria) during the Almohad and Hafsid times. Phylogenetic
analysis revealed that Douiret is an outlier within the North
African and Mediterranean mtDNA genetic landscapes.
Almost three-quarters (74%) of Douiret mtDNA heritage was
comprised of Eurasian lineages (T, HV, J, U and K), among
which haplogroup T was the most common, with a fre-
quency (38%) that is the highest in Tunisia and in the other
considered Mediterranean populations. All these Eurasian lin-
eages are most likely of Near/Middle Eastern origin and were
introduced to this area by later arrivals in the Middle/Late
Upper Palaeolithic and Neolithic dispersals. The substantial
contribution and the persistence of genetic lineages from the
very first settlers in the Jebel Demmer area showed that the
mitochondrial gene pool of Douiret was not diluted by the
many historical invasions and migrations as a result of long
periods of population isolation. Furthermore, the origins and
diversity of the ancient Berber populations from the isolated
mountainous areas of South Eastern Tunisia seem to be quite
different from those installed further North and West.
Further studies on Y-chromosome lineages of Douiret and
both uniparental mtDNA and Y-chromosome genetic markers
of the neighbouring Berbers and Arab populations are
needed for better understanding of the ancient settlement
processes in this area in particular and in North Africa in
general.
Acknowledgements
The authors thank the three associations: “Association de Sauvegarde de
la Nature et de Protection de l’Environnement
a Douiret (ASNAPED)”,
“Association Twiza pour le Patrimoine, la Solidarite et le Developpement”
and “Association Tunisienne de la Culture Amazighe” for supporting this
research project. They would also like to express their sincere gratitude
to Mr Sami Boussetta for his help. They also thank all the volunteers that
donated their DNA, making this study possible.
Disclosure statement
The authors report no conflicts of interest. The authors alone are respon-
sible for the content and writing of the paper.
Funding
This work was supported by the Tunisian Ministry of Public Health, the
Ministry of Higher Education and Scientific Research under Grant [num-
ber LR11IPT05] and the European Commission under Grant: [Number:
FP7–279171-1, MEDIGENE Project].
96 N. BEN HALIM ET AL.
References
Alvarez-Iglesias V, Mosquera-Miguel A, Cerezo M, Quintans B, Zarrabeitia
MT, Cusco I, Lareu MV, et al. 2009. New population and phylogenetic
features of the internal variation within mitochondrial DNA macro-
haplogroup R0. PLoS One 4:e5112.
Andrews RM, Kubacka I, Chinnery PF, Lightowlers RN, Turnbull DM,
Howell N. 1999. Reanalysis and revision of the Cambridge reference
sequence for human mitochondrial DNA. Nat Genet 23:147.
Bandelt HJ, Forster P, Rohl A. 1999. Median-joining networks for inferring
intraspecific phylogenies. Mol Biol Evol 16:37–48.
Ben Halim N. 2006. Etude de la population de Douiret sous l’angle
l’endogamie et de la consanguinite. Diplo ^me de mast
ere, Faculte des
Sciences de Tunis, 134.
Bertranpetit J, Sala J, Calafell F, Underhill PA, Moral P, Comas D. 1995.
Human mitochondrial DNA variation and the origin of Basques. Ann
Hum Genet 59:63–81.
Brakez Z, Bosch E, Izaabel H, Akhayat O, Comas D, Bertranpetit J, Calafell
F. 2001. Human mitochondrial DNA sequence variation in the
Moroccan population of the Souss area. Ann Hum Biol 28:295–307.
Cali F, Le Roux MG, D’Anna R, Flugy A, De Leo G, Chiavetta V, Ayala GF,
et al. 2001. MtDNA control region and RFLP data for Sicily and France.
Int J Legal Med 114:229–231.
Ch^alon-sur-Saone. 1900. Secretariat general du gouvernement
tunisien:Nomenclature et repartition des tribus de Tunisie.
Cherni L, Fernandes V, Pereira JB, Costa MD, Goios A, Frigi S, Yacoubi-
Loueslati B, et al. 2009. Post-last glacial maximum expansion from
Iberia to North Africa revealed by fine characterization of mtDNA H
haplogroup in Tunisia. Am J Phys Anthropol 139:253–260.
Corte-Real HB, Macaulay VA, Richards MB, Hariti G, Issad MS, Cambon-
Thomsen A, Papiha S, et al. 1996. Genetic diversity in the Iberian
Peninsula determined from mitochondrial sequence analysis. Ann
Hum Genet 60:331–350.
Coudray C, Olivieri A, Achilli A, Pala M, Melhaoui M, Cherkaoui M,
El-Chennawi F, et al. 2009. The complex and diversified mitochondrial
gene pool of Berber populations. Ann Hum Genet 73:196–214.
Di Rienzo A, Wilson AC. 1991. Branching pattern in the evolutionary tree
for human mitochondrial DNA. Proc Natl Acad Sci USA 88:1597–1601.
Dubut V, Chollet L, Murail P, Cartault F, Beraud-Colomb E, Serre M,
Mogentale-Profizi N, et al. 2004. mtDNA polymorphisms in five French
groups: importance of regional sampling. Eur J Hum Genet
12:293–300.
Ennafaa H, Cabrera VM, Abu-Amero KK, Gonzalez AM, Amor MB,
Bouhaha R, Dzimiri N, et al. 2009. Mitochondrial DNA haplogroup H
structure in North Africa. BMC Genet 10:8.
Ennafaa H, Fregel R, Khodjet-El-Khil H, Gonzalez AM, Mahmoudi HA,
Cabrera VM, Larruga JM, et al. 2011. Mitochondrial DNA and
Y-chromosome microstructure in Tunisia. J Hum Genet 56:734–741.
Excoffier L. 2004. Patterns of DNA sequence diversity and genetic struc-
ture after a range expansion: lessons from the infinite-island model.
Mol Ecol 13:853–864.
Excoffier L, Laval G, Schneider S. 2005. Arlequin (version 3.0): an inte-
grated software package for population genetics data analysis. Evol
Bioinform 1:47–50.
Fadhlaoui-Zid K, Buhler S, Dridi A, Benammar El Gaaied A, Sanchez-
Mazas A. 2010. Polymorphism of HLA class II genes in Berbers from
Southern Tunisia. Tissue Antigens 76:416–420.
Fadhlaoui-Zid K, Mazas AS, Buhler S, Khodjet Ell khill H, Ben Amor M,
Comas D, Dugoujon JM, et al. 2009. Caracterisation genetique des iso-
lats berberophones du Sud tunisien par confrontation des resultats de
trois marqueurs polymorphes. Antropo 18:73–86.
Fadhlaoui-Zid K, Plaza S, Calafell F, Ben Amor M, Comas D, Bennamar El
gaaied A. 2004. Mitochondrial DNA heterogeneity in Tunisian Berbers.
Ann Hum Genet 68:222–233.
Fadhlaoui-Zid K, Rodriguez-Botigue L, Naoui N, Benammar-Elgaaied A,
Calafell F, Comas D. 2011. Mitochondrial DNA structure in North Africa
reveals a genetic discontinuity in the Nile Valley. Am J Phys Anthropol
145:107–117.
Falchi A, Giovannoni L, Calo CM, Piras IS, Moral P, Paoli G, Vona G, et al.
2006. Genetic history of some western Mediterranean human isolates
through mtDNA HVR1 polymorphisms. J Hum Genet 51:9–14.
Francalacci P, Bertranpetit J, Calafell F, Underhill PA. 1996. Sequence
diversity of the control region of mitochondrial DNA in Tuscany and
its implications for the peopling of Europe. Am J Phys Anthropol
100:443–460.
Harpending H. 1994. Signature of ancient population growth in a low-
resolution mitochondrial DNA mismatch distribution. Hum Biol
66:591–600.
Irwin J, Saunier J, Strouss K, Paintner C, Diegoli T, Sturk K, Kovatsi L,
et al. 2008. Mitochondrial control region sequences from northern
Greece and Greek Cypriots. Int J Legal Med 122:87–89.
Kefi R, Hsouna S, Ben Halim N, Lasram K, Romdhane L, Messai H,
Abdelhak S. 2015. Phylogeny and genetic structure of Tunisians and
their position within Mediterranean populations. Mitochondrial DNA
26:593–604.
Khodjet-El-Khil H, Fadhlaoui-Zid K, Gusmao L, Alves C, Benammar-
Elgaaied A, Amorim A. 2008. Substructure of a Tunisian Berber popu-
lation as inferred from 15 autosomal short tandem repeat loci. Hum
Biol 80:435–448.
Loueslati BY, Cherni L, Khodjet-Elkhil H, Ennafaa H, Pereira L, Amorim A,
Ben Ayed F, et al. 2006. Islands inside an island: reproductive isolates
on Jerba island. Am J Hum Biol 18:149–153.
Louis A. 1975. “Douiret: Etrange Cite Berb
ere”. Tunis, STD.
Macaulay V, Richards M, Hickey E, Vega E, Cruciani F, Guida V, Scozzari R,
et al. 1999. The emerging tree of West Eurasian mtDNAs: a synthesis
of control-region sequences and RFLPs. Am J Hum Genet 64:232–249.
Macquart E. 1906. Les Troglodytes de l'Extre^me-Sud Tunisien. Bulletins Et
Memoires De La Societe D'anthropologie De Paris 7:174–187.
Malyarchuk BA, Derenko MV. 1999. Molecular instability of the mitochon-
drial haplogroup T sequences at nucleotide positions 16292 and
16296. Ann Hum Genet 63:489–497.
Miller SA, Dykes DD, Polesky HF. 1988. A simple salting out procedure
for extracting DNA from human nucleated cells. Nucleic Acids Res
16:1215.
Ottoni C, Martinez-Labarga C, Loogvali EL, Pennarun E, Achilli A, De
Angelis F, Trucchi E, et al. 2009. First genetic insight into Libyan
Tuaregs: a maternal perspective. Ann Hum Genet 73:438–448.
Pereira L, Cerny V, Cerezo M, Silva NM, Hajek M, Vasikova A, Kujanova M,
et al. 2010. Linking the sub-Saharan and West Eurasian gene pools:
maternal and paternal heritage of the Tuareg nomads from the
African Sahel. Eur J Hum Genet 18:915–923.
Picornell A, Gomez-Barbeito L, Tomas C, Castro JA, Ramon MM. 2005.
Mitochondrial DNA HVRI variation in Balearic populations. Am J Phys
Anthropol 128:119–130.
Plaza S, Calafell F, Helal A, Bouzerna N, Lefranc G, Bertranpetit J, Comas
D, et al. 2003. Joining the pillars of Hercules: mtDNA sequences show
multidirectional gene flow in the western Mediterranean. Ann Hum
Genet 67:312–328.
Renouard Y, Robert B. 1942. La Berberie orientale sous les Hafsides des
origines
a la fin du XVe si
ecle, t. I, Bulletin Hispanique, 185–187.
Richards M, Macaulay V, Hickey E, Vega E, Sykes B, Guida V, Rengo C,
et al. 2000. Tracing European founder lineages in the Near Eastern
mtDNA pool. Am J Hum Genet 67:1251–1276.
Richards MB, Macaulay VA, Bandelt HJ, Sykes BC. 1998. Phylogeography
of mitochondrial DNA in western Europe. Ann Hum Genet
62:241–260.
Rogers AR, Harpending H. 1992. Population growth makes waves in the
distribution of pairwise genetic differences. Mol Biol Evol 9:552–569.
Roostalu U, Kutuev I, Loogvali EL, Metspalu E, Tambets K, Reidla M,
Khusnutdinova EK, et al. 2007. Origin and expansion of haplogroup
H, the dominant human mitochondrial DNA lineage in West Eurasia:
the near Eastern and Caucasian perspective. Mol Biol Evol
24:436–448.
Saillard J, Forster P, Lynnerup N, Bandelt HJ, Norby S. 2000. MtDNA vari-
ation among Greenland Eskimos: the edge of the Beringian expansion.
Am J Hum Genet 67:718–726.

Salas A, Comas D, Lareu MV, Bertranpetit J, Carracedo A. 1998. mtDNA
analysis of the Galician population: a genetic edge of European vari-
ation. Eur J Hum Genet 6:365–375.
Saunier JL, Irwin JA, Strouss KM, Ragab H, Sturk KA, Parsons TJ. 2009.
Mitochondrial control region sequences from an Egyptian population
sample. Forensic Sci Int Genet 3:e97–103.
Schneider S, Excoffier L. 1999. Estimation of past demographic parame-
ters from the distribution of pairwise differences when the mutation
rates vary among sites: application to human mitochondrial DNA.
Genetics 152:1079–1089.
Slatkin M, Hudson RR. 1991. Pairwise comparisons of mitochondrial DNA
sequences in stable and exponentially growing populations. Genetics
129:555–562.
Stevanovitch A, Gilles A, Bouzaid E, Kefi R, Paris F, Gayraud RP, Spadoni
JL, et al. 2004. Mitochondrial DNA sequence diversity in a sedentary
population from Egypt. Ann Hum Genet 68:23–39.
ANNALS OF HUMAN BIOLOGY 97
Tajima F. 1989. Statistical method for testing the neutral
mutation hypothesis by DNA polymorphism. Genetics 123:
585–595.
Torroni A, Bandelt HJ, D’Urbano L, Lahermo P, Moral P, Sellitto D, Rengo
C, et al. 1998. mtDNA analysis reveals a major late Paleolithic popula-
tion expansion from southwestern to northeastern Europe. Am J Hum
Genet 62:1137–1152.
van Oven M, Kayser M. 2009. Updated comprehensive phylogenetic tree
of global human mitochondrial DNA variation. Hum Mutat
30:E386–E394.
Varesi L, Memmi M, Cristofari MC, Mameli GE, Calo CM, Vona G. 2000.
Mitochondrial control-region sequence variation in the Corsican popu-
lation, France. Am J Hum Biol 12:339–351.
Villems R. 2011. Homo sapiens mitochondrial DNA D-Loop HVR1sequence.
Accession numbers AJ274757–AJ274942. Available online at: http://
www.ncbi.nlm.nih.gov/nuccore (accessed January 8, 2014).