http://informahealthcare.com/ahbISSN: 0301-4460 (print), 1464-5033 (electronic)

Ann Hum Biol, Early Online: 1–8! 2013 Informa UK Ltd. DOI: 10.3109/03014460.2013.824025

ORIGINAL ARTICLE

An investigation of the genetic diversity of the Kerkennah islands andMahdia (Tunisia) using biparental markers

Sabeh Frigi1, Amel Ben Ammar El Gaaied1, and Lotfi Cherni1,2

1Laboratory of Molecular Genetics, Immunology and Human Pathology at the Faculty of Sciences of Tunis, University El Manar, 2092 Tunis, Tunisia

and 2Biotechnology High Institute of Monastir, Department of Biology, University of Monastir, 5000 Monastir, Tunisia

Abstract

Background: Kerkennah is one of the main inhabited islands of Tunisia. The origin of thepopulation of Kerkennah has not been established and no well-defined ethnic groups havebeen identified nor are genetic studies available. Mahdia, a Tunisian coastal city, has a longhistory dating back to ancient times.Aim: To discover the genetic diversity of the two studied populations and analyse theirrelationships with other Mediterranean populations.Subject and methods: Seven human-specific Alu insertion polymorphisms were typed in 99individuals born in Kerkennah and Mahdia.Results: A neighbour-joining tree and MDS multidimensional scaling analysis showed that theseTunisian populations are scattered amongst North African and Europeans populations,indicating their high genetic diversity and mosaic aspect. The important finding of this studywas the proximity of Kerkennah to Moroccans. Hence, the actual gene pool of this insularpopulation may descend from the ancestral population known to be of Moroccan origin.Concerning Mahdia, its closeness to Eurasian populations and some Tunisian groups reflected ahigh Eurasian genetic component for North African populations and confirmed theirheterogeneity.Conclusion: The strategic location of the two studied populations and their fortifications haveallowed them to play a leading role in the Mediterranean basin.

Keywords

Alu insertion polymorphism, insularpopulation, Kerkennah, Mahdia, Tunisia

History

Received 31 October 2012Revised 2 June 2013Accepted 17 June 2013Published online 20 August 2013

Introduction

Uniparental markers (mtDNA and Y chromosome) have been

extensively used to characterize populations in terms of

diversity and origin. However, a full picture of the histories of

populations requires studies of markers in the recombining

parts of nuclear DNA, namely the autosomes (Branco et al.,

2006; Kidd et al., 2000). Polymorphic Alu insertions repre-

sent an important source of nuclear genetic variability. Their

distinctive features make them a good tool for studying the

genetic differentiations of human populations worldwide.

Their use is advantageous, as they are identical by descent,

widely dispersed throughout the human genome, subject to

very limited amounts of gene conversion, rapidly and easily

genotyped and selectively neutral (Batzer et al., 1996; Comas

et al., 2000). The discriminative properties of these markers

have been shown not only for geographically distinct popu-

lations but also for neighbouring populations such as Tunisian

populations (Cherni et al., 2011; Frigi et al., 2010a). This

feature may be related to the settlement history of this

country. We analysed seven Alu insertion polymorphisms in

two coastal areas of Tunisia: Kerkennah and Mahdia.

Kerkennah is a group of islands lying off the east coast

of Tunisia in the Gulf of Gabes. The archipelago has an

area of 160 km2 and a population of 14 400. On the ruins

of the Amazigh, a Berber civilization, which was fed by the

Phoenician presence, the Romans built their own world,

deeply rooted in cities and urbanism. They created Cercina

(antique Kerkennah) to be a sea port linked to Thyna and

Hadramut. In Kerkennah the Romans created an important

centre for both land and sea economies. Archeological

discoveries have shown that the huge rock (now called ‘‘the

stone of Al Baw’’ by Kerkenians) was a base built to hold a

minaret to show sailors the safe route. The story of Islamic

archaeology in Kerkennah starts with the Ottomans in the

al-Hsar Tower (Fehri, 2009).

Kerkennah is divided into six islands of which only two

are inhabited: Gharbi – also called Mellita, the name of the

Berber village existing there, and Chergui or Great

Kerkennah. Mellita was the first human settlement on the

island, where social development began with the arrival of the

Phoenicians and their inter-mixing with the Amazigh (the

natives). Mellita is characterized by its rich archaeological

heritage. Thus, we find rocks that were engraved to make the

pillars of Roman houses and the remains of some mosaics and

sauna baths. In Chergui – Chergui means the Eastern in

Arabic – and Kraten, there are caves which no doubt served as

Correspondence: Dr Sabeh Frigi, Laboratory of Molecular Genetics,Immunology and Human Pathology at the Faculty of Sciences of Tunis,University El Manar, 2092 Tunis, Tunisia. Email: sabehfrigi@yahoo.fr

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hiding places, as described by historians, which the natives

used to resort to whenever they were attacked from Sicily,

Malta or elsewhere in the Christian world.

The origin of the Kerkennah population is not established

and no well-defined ethnic groups are identified nor are

genetic studies available for this purpose. Hence, two

hypotheses are possible for its origin: Kerkennah probably

comes from a mixing of populations or from a clearly defined

ethnic origin. For a time during history, Kerkennah was a

prison receiving prisoners from different areas of the country.

Mahdia lies on the Mediterranean coast of the country.

It has an important history dating back to ancient times:

it was settled firstly by Phoenicians, then Carthaginians and

Romans. Mahdia, which was also known as Jemma,

Aphrodisium and Cap Africa, was the capital of the Fatimid

Arabic Empire. In fact, Mahdia was founded by the Fatimids

under the Caliph Abdallah al-Mahdi in 921 and made the

capital city of Ifriqiya, by Caliph Abdallah El Fatimi. It was

chosen as the capital because of its proximity to the sea and

the promontory on which an important military settlement had

existed since the time of the Phoenicians (Favreau, 1995).

Mahdia was considered as one of the most important

economic, cultural, commercial and military centres in the

southern Mediterranean.

Our first objective was to research and discuss the genetic

polymorphism of the Kerkennah islands and Mahdia using

molecular data. As secondary objectives we aimed to evaluate

their general position with regard to other Tunisians and to

other North African groups. Establishing their position in a

general Mediterranean context would add further information

that contributes toward clarifying how North Africa was

populated within the framework of population movements in

the Mediterranean area.

Materials and methods

Population samples

Blood samples were collected from 50 individuals belonging

to the islands of Kerkennah and 49 from Mahdia (Figure 1).

All sampled individuals were unrelated, healthy donors and

signed an informed consent form.

Alu genotyping

Seven human-specific Alu insertion polymorphisms: ACE,

APO-A1, F13B, TPA-25, PV92, B65 and D1, respectively

located on chromosomes 17, 11, 1, 8, 16, 11 and 3, were typed

in each sample, using previously described primers (Acrot

et al., 1995; Batzer et al., 1996; Garcia-Obregon et al., 2006).

The PCR amplification conditions were performed as already

described (Stoneking et al., 1997).

Statistical analysis

Allele frequencies were calculated by direct counting. Hardy–

Weinberg equilibrium was assessed by an exact test (Guo &

Thompson, 1992) provided by the Arlequin program v 2.0

package (Schneider et al., 2000). The statistical significance

of allelic and genotypic differentiation between loci and

populations was estimated by the GENEPOP web version

program (Raymond & Rousset, 1995). FST genetic distances

were computed between pairs of populations by means of the

DISPAN software (Ota, 1993) and the distance matrix was

used to construct a neighbour-joining (NJ) tree with PHYLIP

3.63 (Felsenstein, 1993). Neighbour-joining tree robustness

was assessed by bootstrap analysis (Felsenstein, 1985); every

occurrence of a particular cluster was recorded and given as a

percentage of the 1000 bootstrap trees drawn from the

previously bootstrapped matrix distances. Genetic relation-

ships among populations were depicted by a non-metric

multidimensional scaling (MDS) analysis (Kruskal, 1964)

based on the R-matrix (Harpending & Jenkins, 1973).

We compiled data obtained for the seven polymorphic loci

from previously published papers (Stoneking et al., 1997,

France, Greek Cypriots and Turk Cypriots; Comas et al.,

2000, Basque, Catalonia, Andalusia, NorthMorocco, West

Morocco, South East Morocco, Sahara, Algeria and Tunisia;

Romualdi et al., 2002, Germany and Syria; Garcia-Obregon

et al., 2006, Valencia; Santovito et al., 2007, Genova; Bahri

et al., 2008, North-South-Centre Tunisia; Frigi et al., 2010a,

Sejnane and Takrouna; Cherni et al., 2011, Libya, Thala,

Zarzis, Smar and Bou Salem).

In order to ascertain the proportion of genetic variance

attributable to differences within or between populations,

genetic variance was hierarchically apportioned through the

analysis of molecular variance (AMOVA) (Excoffier et al.,

1992) performed with the Arlequin program v 2.0 package

(Schneider et al., 2000). In this statistical analysis, a

permutation procedure allows testing of significance of the

fixation indices, which measure the relative contribution of

genetic variation among populations within groups and

among groups, respectively.

Results

The frequency distribution of seven Alu polymorphisms was

determined in a sample set comprising 99 individuals from

the Kerkennah islands and Mahdia (Table 1). All Alu

insertions were polymorphic in both populations, with APO

being the closest to fixation. The least frequent insertions

were ACE and PV92 in Kerkennah and Mahdia, respectively.

B65 and F13B showed the highest values of heterozygosity

in Kerkennah, whereas B65 was only the highest in Mahdia.

The data also showed that all genotype distributions were in

agreement with Hardy–Weinberg equilibrium after applying

the Bonferroni correction. We observed a wide range of

Alu insertion frequencies, from 0.179 (ACE) to 0.810 (APO)

(Table 1). Nevertheless, when we focused on genotypic

differentiation between populations, which gave us the

differences in the genotypes distribution locus-by-locus, the

estimation using GENEPOP software showed significant

differences between the populations of Kerkennah and

Mahdia (p50.05). Alu frequency comparisons, checked

through the exact test of population differentiation, yielded

significant differences between our two samples for D1

(p¼ 0.034), ACE (p¼ 0.024) and F13B (p¼ 0.006) and also

across the seven markers considered (p¼ 0.011). We also

used the exact test to compare our two samples with

other Tunisian groups. For the Kerkennah group, the

exact test indicated a marked differentiation from Zarzis

and Thala populations. In fact, the comparison of Kerkennah

2 S. Frigi et al. Ann Hum Biol, Early Online: 1–8

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with the Thala population showed seven statistically signifi-

cant differences (0.015p50.05) over a total of 17 cases,

while that with the Takrouna population showed only two

statistically significant differences over a total of 17 possible

tests. On the other hand, comparisons of the Mahdia group

with other Tunisian populations showed fewer differences

from Bou Salem (four significant cases over a total of 17,

p¼ 0.031) than from the Zarzis population (eight out of 17

significant cases, p50.001).

We also used the exact test to compare our two samples

with North-African populations on one hand and Eurasian

populations on the other. For the Mahdia population, the

exact test indicated a more marked differentiation from the

North African than from the Eurasian populations. Indeed,

Figure 1. Geographical location of the eight Tunisian population groups analysed in the present study. Studied populations are Mahdia and Kerkennahislands.

DOI: 10.3109/03014460.2013.824025 Investigation of the genetic diversity of Kerkennah islands 3

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this comparison showed eight statistically significant differ-

ences (0.015p50.05) over a total of 17 cases, while that

with Eurasians showed only three statistically significant

differences. On the other hand, the comparisons of Kerkennah

with North African and Eurasian populations showed the

opposite trend: fewer differences from the North Africans

(three significant cases over a total of 17, p¼ 0.021) than

from the Eurasian populations (eight out of 17 significant

cases, p50.001).

Genetic distance analyses also confirmed the differen-

tiation between Kerkennah and Mahdia. Fst genetic

distance gave an elevated value (0.193, p50.05).

Considering Tunisian groups Kerkennah showed a high

genetic distance from all Tunisian groups except with

Takrouna (0.041) and the North-South-Centre Tunisian

group (0.008). However it presented a low genetic distance

with the rest of North Africans essentially with Southeast

Moroccans (0.005). This result confirms the heterogeneity

of Tunisians.

In order to assess the relationship between the two

populations analysed in the present study and to compare

them with other world-wide populations previously reported

we compiled data obtained for the seven polymorphic loci

from previously published papers (Bahri et al., 2008; Comas

et al., 2000; Cherni et al., 2011; Frigi et al., 2010a; Garcia-

Obregon et al., 2006; Romualdi et al., 2002; Stoneking et al.,

1997; Santovito et al., 2007). We used both tree reconstruc-

tion and MDS analysis to investigate population relationships.

A neighbour-joining tree (Figure 2), depicting the population

relationships, shows that Tunisian populations are not clus-

tered together on the tree. As expected, the European

populations cluster relatively together, thus reflecting their

genetic closeness. The studied population of Kerkennah is

intermingled with North African populations such as Libya,

Sahara and Southeast Morocco, whereas Mahdia is grouped in

the pool formed by Bou Salem, Zarzis, Genova and the Berber

community of Sejnane. The branches with low bootstrap

values suggest that the members on the branch should not be

Figure 2. Neighbour-joining tree of population relationships.

Table 1. Alu insertion frequencies and heterozygosity (Het) in studied populations.

Loci B65 ACE D1 APO-A1 F13B PV92 TPA-25 All loci

KerkennahFrequency 0.479 0.179 0.190 0.650 0.510 0.300 0.409Het 0.499 0.293 0.307 0.455 0.499 0.420 0.483 0.422

MahdiaFrequency 0.449 0.349 0.320 0.810 0.339 0.260 0.439Het 0.494 0.454 0.435 0.307 0.448 0.384 0.492 0.430Average Het per locus 0.496 0.373 0.371 0.381 0.473 0.402 0.487 0.426

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divided into two separate groups as it appears. On every

iteration of bootstrapping the members of the low bootstrap

value branch jumped into other branches. This can happen

due to either an insufficient number of informative sites or

due to the presence of chimeric genes due to recombination or

gene flow.

MDS analysis (Figure 3) shows a similar pattern to that

displayed in the NJ tree. Tunisian populations are clearly

segregated along dimension I in the MDS representation. The

plot indicates that there are four distinct clusters of popula-

tions. The first cluster is composed of the majority of North

African populations. The second represents the Europeans

which are intermediate between North Africans and the mixed

third cluster (composed of Mahdia, Sejnane, Bou Salem,

Smar, Thala, Andalusia France and Syria). Zarzis and Genova

form the final group. It is important to note that the two

studied populations are not grouped together. Indeed,

Kerkennah is closest to North African populations, but

Mahdia is contiguous to some Tunisian populations, Syria

and some Western European populations (France and

Andalusia), which form the third cluster.

In order to clarify the position of Kerkennah and Mahdia,

we performed a MDS consisting only of North African

popultions (Figure 4). Results confirmed the previous results

(Figure 3). In fact, Tunisian populations featured a certain

dispersion along dimension I and the two studied populations

do not cluster together. It is worth mentioning the closeness of

Kerkennah to Southeast Morocco, in accordance with

historical studies. whereas Mahdia is grouped with Smar,

Bou Salem, Libya and Zarzis.

An analysis of molecular variance (AMOVA) shows that

when all North African populations were treated as single

groups [our two samples of Kerkennah and Mahdia and those

studied by Comas et al. (2000), Bahri et al. (2008), Frigi et al.

(2010a), Cherni et al. (2011): Mahdia, Kerkennah, Northern

Moroccans; Western Moroccans; South Eastern Moroccans;

Saharawis; Algerians; Tunisians, North-Centre-South

Tunisians; Sejnane, Takrouna, Libya, Smar, Thala, Bou

Salem, and Zarzis], 98.05% of the total genetic variance

was within populations and 1.95% was between populations

(Table 2). When considering the four groups defined by the

previous analysis, the fraction of genetic variance resulting

from differences among groups was 3.35% (p50.001),

whereas the remaining variance was found within populations

(95.50%). This data showed a remarkable heterogeneity

among the considered groups (Table 2).

Discussion

Alu insertions are widely distributed throughout the human

genome, constituting convenient markers to assess genetic

diversity between human populations. This study is a

contribution towards a better knowledge of the origin of an

insular Tunisian population, Kerkennah islands, in order to

improve our understanding of the settlement history of

Tunisia. We also investigated the genetic structure of another

Figure 3. Non-metric multidimensional scaling (MDS) applied to analyse the genetic relationships among 25 populations. Stress value is 0.211.N.Moro, North Morocco; SE.Moro, Southeast Morocco; W.Moro, West Morocco; Greek Cyp, Greek Cypriots; Turk Cyp, Turk Cypriots; NSC Tun,North-South-Centre Tunisians.

DOI: 10.3109/03014460.2013.824025 Investigation of the genetic diversity of Kerkennah islands 5

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coastal city used as a control: Mahdia. The choice of this

population as a control was made due to its rich history of

settlement and its mixture of different types of founders

settled in this territory, particularly the Fatimid. We avoided

the use of Sfax since this town is the closest to Kerkennah and

receives numerous migrants from these islands.

Starting from the fact that the markers analysed are

biallelic and that each allele is observed with a frequency

greater than 0.1, to enhance the size of the samples would not

change anything in the results. This would have been different

if we had analysed STRs that are multiallelic markers and

enhancing the samples could reveal rare alleles. With a

sample of 50 individuals, alleles with frequencies upper or

equal to 1% are revealed. Alleles with lesser frequencies

could not be determinant for the structure of a population.

Moreover, the Tunisian populations analysed are small, with

�10 000–20 000 inhabitants, and the samples collected from

each community may be considered as representative.

Starting from the size of the populations studied, the

number of family names should not exceed 100. Family

names are used to define if the subjects belonging to the

sample are not related.

In all the analyses performed, a differentiation appears

between the population of Kerkennah and Mahdia. Likewise,

the comparatively high heterozygosity in the two studied

populations (considering the seven Alu types) evidences the

potentially high rate of mixture of their gene pool. Such

findings seem to be strongly conditioned by the historical past

of these two populations, which has been in turn largely

determined by their geographical location in the

Mediterranean.

Tree reconstruction methods depict population relation-

ships as a series of bifurcations, which are commonly

interpreted as representing population splits, however, it’s

important to realize that clusters of populations in such trees

could arise from migration instead of from shared ancestry

(Stoneking et al., 1997). A neighbour-joining tree depicting

population relationships shows that Tunisian populations are

Figure 4. Non-metric multidimensional scaling (MDS) applied to analyse genetic relationships among North African populations. Stress value is 0.147.N.Moro, North Morocco; SE.Moro, Southeast Morocco; W.Moro, West Morocco; NSC Tun, North-South-Centre Tunisians.

Table 2. Results of the analyses of molecular variance for seven polymorphic Alu insertions.

Groups% of varianceamong groups

% of variance amongpopulations within groups

% of variancewithin populations

All Tunisian populations – 1.95* 98.05Four groups defined by MDS analysis 3.35 1.15* 95.50

*p50.05.

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not clustered together on the tree, which would reflect their

genetic heterogeneity. An MDS plot (Figure 3) shows that

Tunisian populations are scattered amongst North African

and European populations, indicating their higher genetic

diversity and their mosaic aspect. Figure 4 shows a variable

linkage of the Tunisian DNA pool with surrounding North-

African populations. This picture reflects the complex

settlement history in the Maghreb (Frigi et al., 2010b),

associated with weak exchange rates between Tunisian

population communities. This result is confirmed by genetic

distance analyses. Based on the previous analyses, the

presence of four clusters was observed, which are coherent

with the genetic diversity that would be expected as a result

of historical dynamics and the exchange of populations. The

results of the AMOVA analysis support the reliability of the

four clusters defined by the MDS, given the statistical

significance of the between-group variation for the seven

markers. The important finding of this study is the affinity

and the proximity of Kerkennah to Southeast Moroccans.

The actual gene pool of this insular population could be the

descendant of the ancestral population known to be of

Moroccan origin. Our results, based on Alu markers, are in

accordance with historical data showing that this population

has a lengthy, but simple history. Natives of Tunisia and

Kerkennah originally settled there but, during the spread of

the Roman Empire, Kerkennah was used as a port and look-

out point by the Romans, to keep note of off-shore activity.

However, after the collapse of the Roman Empire,

Kerkennah reverted to the natives. To this day, Kerkennah

has been relatively untouched by modernization and remains

beautiful in its natural state. Historians report that this group

of islands was founded by ‘‘Sidi Ali Khanfir’’ coming from

Morocco (Favreau, 1995).

Concerning Mahdia, its closeness to Eurasian populations

and some Tunisian groups reflects a high Eurasian genetic

component for North African populations and confirms their

heterogeneity. In fact, in all the analyses performed a

differentiation appears between Mahdia and the majority of

North African populations. However, this distinction was not

observed when considering only North-African populations in

the MDS analysis (Figure 4) and not significant when we

performed the AMOVA, indicating that this population

behaves as North African but occupies an intermediate

position in the Euro-Mediterranean region. These results are

in line with an ancient Euro Mediterranean background that

has already been studied by historians. Indeed, the region of

Mahdia entered history with the Berbers, and then with the

Phoenicians. Subsequently under Roman rule, the agricultural

sector flourished, notably the olive tree groves. Their

successors were the Aghlabites, the first Arab dynasty settled

in this coastal city. In 1087 the town was attacked by raiding

ships from Genoa and Pisa who burned the Muslim fleet in

the harbour. This played a critical role in winning control of

the Western Mediterranean and allowing the First Crusade to

be supplied by sea (Thomas, 1887). The Zirid dynasty had its

residence here in the 11th century, but was brought to an end

by the Norman conquest of the city in 1148. In 1160 the city

came under Almohad rule (Fuller, 1987). The role of the

capital was taken over by Tunis in the 13th century during the

Hafsid Dynasty.

In sum, Tunisia is a significant region in the history of

modern humans in that it served and continues to serve as a

crossroad between sub-Saharan Africa, the Middle East and

Europe. In order to progress our knowledge of the settlement

history of Tunisia, we studied for the first time the genetic

diversity of an insular population and a Mediterranean coastal

city using biparental markers. Our data support the use of

Alu insertion polymorphisms to assess the origin and history

of populations. However, genetic study of mtDNA and

Y-chromosome markers is needed to better understand the

genetic structure and past history of Kerkennah and Mahdia.

Acknowledgements

This work was supported by the Tunisian Ministry of Higher Education,Scientific Research and Technology. We gratefully acknowledge theblood donors whose availability made this study possible. Thanks also tothe anonymous reviewers whose comments improved the presentation ofthis manuscript.

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