Human population phylogenetic studies using mithochondrial DNA

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

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

3. Introduction The main aim of Human population genetics is to find answers concerning: • 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. Mitochondrial DNA mitochondria Eukariotic cellule D-Loop: 16569 bp circular DNA HVS I and HVS II segments maternal inheritance high mutation rate compared to nuclear DNA absence of recombination an important tool for Human population genetics

7. Mitochondrial DNA Studies history: Brown and Wallace in 1970: Pioneers in mtDNA investigation • 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 ) ,

8. on sequence analysis of the mtDNA control region • 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)

9. Sub-Saharan African individuals present the most variable mtDNA sequences 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

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 . Define related groups of mtDNA called haplogroups 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

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

13. H Individual 2: HVS1 Polymorphism + RFLP 162 G - 7025 Alu I HVS1 Polymorphism 16126C, 16294T, 16296T, 16304C + RFLP (+13366 BamH1) Haplogroup: T 2 Haplotype Examples : 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. Example I:Mitochondrial DNA diversity of the prehistoric population from Taforalt (12,000 years- Morocco). Dr Rym KEFI and Dr Eliane BERAUD-COLOMB U600 INSERM-FRE2059 CNRS Laboratoire d'Immunologie, Hôpital de Sainte-Marguerite- Marseille- France

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

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

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

21. Afalou Taforalt Columnata 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

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

23. Genetic data 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: • Ancestral indigenous component: U6- (Paleolithic: 45.000 years) • Eurasiatic component: T, H, U, J…(Neolithic?: 9000 years) • Sub-Saharan component : L (Historic?)

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). 28 burials 200 skeletons The cave of Taforalt in Morocco 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 42,8% (9/21) H ou U 19% (4/21) H 14,2% (3/21)JT 2 individuals (9,5%) U6 2 individuals (9,5%) V 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. • Genetic structure of Taforalt: • Eurasiatic Component : • H, U, JT, V: 90,5% • North African component: • U6: 9,5%

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

30. Example 2: Phylogenetic and Neandertal enigma 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. Homo Sapiens sapiens Homo neandertalensis • Neandertal is he our ancestor? • Did he belongs to another species? • Did he contribute in our genetic inheritance? or • did he disappear without leaving any trace in our genome?

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. Ancient bone DNA 377 bp Neandertal sequence was aligned with CRS (Cambridge reference sequence). The alignment shows 27 differences (24 transitions, 2 tranversions, 1 deletion)

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

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

36. Thank you Marseille