Mitochondrial DNA is used to study human population genetics and evolution. The document discusses two examples: 1) Analysis of mitochondrial DNA from 31 bone remains from the Taforalt cave in Morocco dated to 12,000 years ago found both Eurasian and North African components, showing genetic continuity with modern Moroccan populations. 2) Analysis of Neanderthal mitochondrial DNA found it to be distinct from modern human DNA, indicating Neanderthals were a separate species and did not directly contribute to the modern human gene pool.

1. Human population phylogenetic
studies using mithochondrial DNA
Dr Rym KEFI
MIGOD- Institut Pasteur -Tunis

2. Plan:
I- Introduction
II- Example: Phylogenetic and Neandertal enigma.
II- Example I: Mitochondrial DNA diversity of the prehistoric
population from Taforalt (12,000 years- Morocco).

3. The main aim of Human population genetics is to find
answers concerning:
Introduction
 Differentiation in single population (Bertranpetit et al
1995; Ann Hum Genet)

4. the migration patterns in certain geographic areas.

5.  human evolution, and
the spread of modern
humans (Richards et al 1996,
Am J Hum Genet )

6. mitochondria
Eukariotic cellule
16569 bp circular DNA
D-Loop:
HVS I
and HVS II segments
Mitochondrial DNA
maternal inheritance
high mutation rate compared
to nuclear DNA
absence of recombination
an important tool for
Human population genetics

7. The studies of mtDNA polymorphism in human populations
were based:
 initially on restriction enzyme (RFLP) analysis :
Low resolution restriction and high resolution
restriction mapping (Horai et al 1984, Johnson et al 1983, Horai et al 1984,
Cann et al 1987, Torroni et al 1993 ) ,
Brown and Wallace in 1970: Pioneers in mtDNA investigation
Mitochondrial DNA Studies history:

8. on combined method using sequence analysis and restriction
mapping (Bertranpetit et al 1995, Vigilant et al 1991, Richards et al 1996,
Richards et al 1998, Macaulay 1999, Torroni et al 2001, Maca-Meyer et al 2001,
Salas et al 2002)
 on sequence analysis of the mtDNA control region

9. Mitochondrial Eve
Cann et al; Nature 1987
 147 individuals from five
geographic populations: Europe,
Africa, Asia, Australia, New
Guinea
have been analysed by high-
resolution restriction mapping
Sub-Saharan African
individuals present the most
variable mtDNA sequences

10. Different mtDNA lineages have been diverged from an
ancestral women originated in Africa.
Mitochondrial Eve

11. RFLPs studies of mtDNA from a wide range of Human
populations have revealed a number of stable polymorphic
sites in the mtDNA coding region .
Mutations observed in both mtDNA coding region and
control region in modern human populations have
occurred on these pre-existing haplogroups
Define the individual mtDNA type or haplotypes
Define related groups of mtDNA called haplogroups

12.  Alignment of sequences with mtDNA reference (CRS)
using “Blast 2 sequences”
16126C
16294T
16296T
16304C
Haplotype and Haplogroup
+ RFLP analysis

13. Examples :
HVS1 Polymorphism
16126C, 16294T, 16296T, 16304C
+ RFLP
(+13366 BamH1)
Haplotype Haplogroup: T 2
Individual 2:
HVS1 Polymorphism + RFLP
162 G - 7025 Alu I
H
Individual 1:

14. Mitochondrial haplogroups

15. The phylogenetic relationships between haplotypes were
inferred in the first studies by
 Maximum parsimony tree
Then by Neighbor- joining trees
Later by Median joining network.
Trees were based on distance data calculated from
 Nucleotide sequence or
RFLPs data or
Nucleotide sequence combined with RFLPs data.

16. Macaulay et al, 1999; ,
Am J Hum Genet,

17. Dr Rym KEFI and Dr Eliane BERAUD-COLOMB
U600 INSERM-FRE2059 CNRS Laboratoire d'Immunologie, Hôpital de
Sainte-Marguerite- Marseille- France
Example I: Mitochondrial DNA diversity of the
prehistoric population from Taforalt (12,000 years-
Morocco).

18. Knowledge of the settlement of Northern Africa region
Study of molecular diversity
of modern Human
populations
Study of archaeological specimens
and their environment

19. Anthropologic data
Transition from Homo erectus
towards Homo sapiens archaic
From 40.000 years to 20.000 years: Homo sapiens sapiens
(Dar Es Soltan, Temara, Maroc)
Sidi Abderrahman: 200.000 years
Aîn hanech, Salé: 160.000 years
Djebel Irhoud: 100.000 years
(Morocco)
Homo erectus old of 700.000 years BP
(site of Ternifine in Algeria).
Aterian industry

20. Epipaleolitic period : 20.000 years to 10.000 years BP
Ibero-Maurusian
man
Ibero-Maurusian industry

21. Face basse et large
Forte arcade
sourcilière
Orbites rectangulaires
Pommettes saillantes
Mâchoire massive
squelette robuste
avulsion des incisives
(Ferembach 1962-Camps 1989)
Homme de
Mechta El -Arbi
Taforalt
Afalou
Columnata

22. Ibero-maurusian man
1-Europian origin? (Vallois 1969, Ferembach 1985)
2- Near East origin ? (Vandermeersch 1978)
4- North African origin ? (Camps 1989,
Dutour 1995)
3- Subsaharian origin? (Ferembach 1976)
Origins ???

23. Ancestral indigenous component: U6- (Paleolithic: 45.000 years)
Eurasiatic component: T, H, U, J…(Neolithic?: 9000 years)
Sub-Saharan component : L (Historic?)
Former studies using Mitochondrial DNA (Côrte-Real et al.
1996; Rando et al. 1998; Comas et al. 2000; Brakez et al. 2001;
Esteban et al. 2004…) showed that the genetic structure of
North Africa is composed of 3 components:
Genetic data

24. to contribute to the knowledge of North Africa settlement
We proposed to analyse the mitochondrial DNA diversity of
the prehistoric population from Taforalt (13,000 years BP-
Morocco).
Aim:

25. The population of Taforalt (13,000 years
BP- Morocco).
The cave of Taforalt in Morocco
28 burials
200 skeletons
The cave of Taforalt is Located at 55 km in the North-
West of Oujda

26. Ancient DNA was extracted from 31 bone remains from Taforalt
Phenol/Chloroforme
Extraction
Dissolution of
bone powders
ADN
Hypervariable segment 1 (HVS1) of control region (D-Loop) was
amplified by PCR and sequenced (R. Kefi et al; C.R.Palévol 2003)

27. Mitochondrial DNA diversity of Taforalt population
Début et fin
de la séquence
Taf I 16054-16454 CRS
Taf II 16054-16454 CRS
Taf V5 16054-16317 CRS
Taf V7 16081-16404 CRS
Taf V20 16054-16317 CRS H ou U ?
Taf XVa 16054-16317 CRS
Taf XV0 16054-16317 CRS
Taf XVII 16054-16317 CRS
Taf XIXa 16054-16317 CRS
Taf XXI-6 16054-16317 CRS
Taf XXV 16190-16317 CRS
Taf 55-IB 16105-16317 16239 T
Taf VI-10 16054-16317 16124T/C-16239T H ?
Taf V26 16054-16317 16204C-16226T
Taf XVIa2-19 16054-16317 16189C-16261T
Taf 55-I 16054-16454 16126C-16355T
Taf V18 16054-16317 16126C-16304C JT
Taf XXV3 16054-16317 16126 C
Taf XXIV 16054-16317 16126C-16172C-
16174T
U6
Taf VI9E 16054-16317 16172C-16174T U6
Taf V27 16054-16317 16298T/C
Taf XIX 16054-16317 16179T-16298T/C
Taf VIII 16054-16317 16223T L3, M, ou N ?
Spécimens Polymorphismes Haplogroupes
V
Genetic structure of Taforalt:
Eurasiatic Component :
H, U, JT, V: 90,5%
North African component:
U6: 9,5%
42,8% (9/21) H ou U
14,2% (3/21) JT
2 individuals (9,5%) U6
In modern Human population,
JT is presents only in:
 1,6% Berbers from the North
of Morocco
 1,8% of Sicilians,
1,6% of Italians.
19% (4/21) H
2 individuals (9,5%) V

28. The genetic inheritance of Taforalt population (12,000
years) is composed of:
 Eurasiatic component (J/T, H, U et V)
 North African component (U6).
Similarities between Taforalt and Moroccan populations
(Berbers from the North of Morocco) Underline a genetic
continuity

29. Ibero-maurusian Origin
4- local origin ? (Camps 1989, Dutour 1995)
3- Sub-Saharan origin? (Ferembach 1976)
1-European origin? (Vallois 1969, Ferembach 1985)
2- Near East origin ? (Vandermeersch 1978)
Kefi et al 2005 Anthropologie ; Xliii/1: 55-64

30. Phylogenetic and Neandertal enigma
Example 2:
Neandertal lived in Europe and west Asia
between 150.000 and 30.000 years (Grimaud–Hervé
et al 2001, Klein et al 2003)
Neandertal has specific morphological
characters (lengthened Cranium, presence of
Taurus on orbits , big cranial capacity...) which
distinguish him from the anatomically modern
man
Neandertal coexisted with anatomically modern man, before
disappearing 30.000 years ago

31. Many interrogations about the role of Neandertal
in the Human evolution.
 Neandertal is he our ancestor?
 Did he contribute in our genetic inheritance? or
 did he disappear without leaving any trace in
our genome?
Homo
Sapiens
sapiens
Homo
neandertalensis
 Did he belongs to another species?

32. Krings and collaborators (Krings et al. 1997, Cell) studied
for the first time ancient DNA extracted from Neandertal
humerus. Neandertal was discovered in West Germany.
377 bp Neandertal sequence was aligned with CRS
(Cambridge reference sequence). The alignment shows 27
differences (24 transitions, 2 tranversions, 1 deletion)
Ancient bone
DNA

33. Neandertal sequence was compared to 994 mt DNA
sequences from the five continents.
The difference between the Modern Man and Neandertal
is higher than the intra specific diversity in Modern
Human specie.

34. indicates that Neandertal position is distinct from
the group including all the Modern Human sequences.
NJ tree constructed with 986 modern Human mt DNA
sequences, 16 chimpanzee sequences and Neandertal
sequence

35. These results show that Neandertal is not the
ancestor of the modern Human.
Homo sapiens sapiens and Homo neandertalensis
constitute two distinct species.
Homo sapiens sapiens
Homo neandertalensis

36. Marseille