Human population phylogenetic studies using mithochondrial DNA

Dr Rym KEFI

MIGOD- Institut Pasteur -Tunis

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).

The main aim of Human population genetics is to find answers concerning:

Introduction

Differentiation in single population (Bertranpetit et al 1995; Ann Hum Genet)

the migration patterns in certain geographic areas.

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

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

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:

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

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

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

Mitochondrial Eve

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

Alignment of sequences with mtDNA reference (CRS) using “Blast 2 sequences”

16126C

16294T

16296T

16304C

Haplotype and Haplogroup

+ RFLP analysis

Examples :

HVS1 Polymorphism16126C, 16294T, 16296T, 16304C

+ RFLP (+13366 BamH1)

Haplotype Haplogroup: T 2

Individual 2:

HVS1 Polymorphism + RFLP 162 G - 7025 Alu I

H

Individual 1:

Mitochondrial haplogroups

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.

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

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).

Knowledge of the settlement of Northern Africa region

Study of molecular diversity of modern Human populations

Study of archaeological specimens and their environment

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

Epipaleolitic period : 20.000 years to 10.000 years BP

Ibero-Maurusian man

Ibero-Maurusian industry

Face basse et largeForte arcade sourcilièreOrbites rectangulairesPommettes saillantesMâchoire massivesquelette robuste avulsion des incisives

(Ferembach 1962-Camps 1989)

Homme de Mechta El -Arbi

Taforalt

Afalou

Columnata

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 ???

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

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:

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

Ancient DNA was extracted from 31 bone remains from Taforalt

Phenol/ChloroformeExtraction

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)

Mitochondrial DNA diversity of Taforalt populationDébut et fin

de la séquenceTaf I 16054-16454 CRSTaf II 16054-16454 CRSTaf V 5 16054-16317 CRSTaf V 7 16081-16404 CRSTaf V 20 16054-16317 CRS H ou U ?Taf XVa 16054-16317 CRSTaf XV0 16054-16317 CRSTaf XVII 16054-16317 CRSTaf XIXa 16054-16317 CRSTaf XXI-6 16054-16317 CRSTaf XXV 16190-16317 CRSTaf 55-IB 16105-16317 16239 TTaf VI-10 16054-16317 16124T/C-16239T H ?Taf V 26 16054-16317 16204C-16226TTaf XVIa2-19 16054-16317 16189C-16261TTaf 55-I 16054-16454 16126C-16355TTaf V 18 16054-16317 16126C-16304C JTTaf XXV 3 16054-16317 16126 C Taf XXIV 16054-16317 16126C-16172C-

16174TU6

Taf VI9E 16054-16317 16172C-16174T U6Taf V 27 16054-16317 16298T/CTaf XIX 16054-16317 16179T-16298T/CTaf 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

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

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

Phylogenetic and Neandertal enigmaExample 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

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?

HomoSapienssapiens

Homo neandertalensis

Did he belongs to another species?

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 boneDNA

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.

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

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 sapiensHomo neandertalensis

Marseille