Nuragic Sardinians are still among us, and the Etruscans too. Two genealogical studies

Guido Barbujani

Dip. Biologia ed Evoluzione Università di Ferrarag.barbujani@unife.it

UCLA, April 8, 2009

1. The Etruscans do not resemble most modern Tuscans

A bit of history

Etruscan a non-Indo-European languageDocumented from the end of VIII century BCEtruscan cities independent statesCommon culture and language, but never a political unitMaximum territory expansion: VI century BCMilitary defeats, Roman assimilation in the II century BC

Dionysius of Halicarnassus: the Etruscans an Italic populationHerodotus: the Etruscans seamen from Lydia, escaping famine

V

A

S

PM

T

C

Rom e

Adria (17, 5), Volterra (6, 3),Castelfranco di Sotto (2, 1),Castelluccio di Pienza (1, 1),Magliano and Marsiliana (25, 6)Tarquinia (18, 5), Capua (8, 6)

80 bone samples from 8 Etruscan necropoleis

27 individuals, 22 different haplotypes, h=0.946Tuscans: 49 individuals from Francalacci et al. (1996)

Shared sequences between the Etruscans and modern populations

23 3

35

2

1

31

52

22

33

1

3

7

4

2

2

2

4

Genetic distances (Fst x 1000) between the Etruscans and

modern populations

3680 90

7048

74

118

5550

37

47

4176

6051

57

261

69

65

41

62

73

71

2. Testing hypotheses by serial coalescent simulation

Reconstructing (proceeding backwards in time) the maternal genealogy of a sample

Two possibilities: either each individual has a different mom

Or two individuals have the same mom (coalesence)

Coalescence probability a function of population size N and sample size n

Past

Present

N = 10N constant

GenealogiesMRCA

N = 10N constantn = 69 generations

Mutation

Mutation(sequences are arbitrary, their differences

are not)

1

00000

00001

00010

00101

101011 2 3 3 4 5

01010

Il Modello: Serial Simcoal

Serial coalescence

N=20Modern sample (n=5)0

100

Time(generations)

Ancient sample (n=2)

Anderson C.N.K., Ramakrishnan U., Chan Y.L. e Hadly E.A. (2005) Bioinformatics

INPUT Population size Population genealogy Population growth rate Migration matrix Mutation model and rate Sample sizes and ages

OUTPUT N haplotypes Haplotype diversity Nucleotide diversity Mismatch distribution Haplotype sharing

Etruscans Tuscans Murlo

Sample size 27 49 86

Haplotype n 22 40 60

Haplotype diversity 0.946 0.949 0.960

Nucleotide diversity 0.011 0.014 0.012

Avg. mismatch 3.91 5.03 4.50

Haplotype sharing 0.09 0.14

Fst0.024 0.028

Observed population statistics

Consistency criterion: overlap between the 95% confidence intervals of observed and simulated statistics

median observed value

simulated values

The posterior probability (two-tailed) of a simulated statistic is represented by the gray area in the

graph

Two ways to combine the results: 1 estimate a joint posterior probability for all statistics; 2. count the number of statistics with P<0.05.

Simulation parameters

• Population sizes: Etruscans: 292,00012 = 25,000 Tuscans: 3,500,00012 = 300,000

• Growth rate: Nt=N0ert r=1/100 ln 300,000/25,000 = 0.025

• Mutation rate: 1 mutation per million years per nucleotide 360 nucleotides, 25 years per generation, 2 0.0045

• 360 nucleotides

• Transition bias: 0.94

Etruscans and Tuscans a single population?

Nf=25,000

Nf=25,000

r = 0

Model 1: Small population, constant size

• Allele sharing: 4.2% (1.4-8.1) OK

• Hapl. diversity:

- Etruscans: > Obs.

- Tuscans: > Obs.

0

100

Generations

Tuscans

Etruscans

Etruscans and Tuscans a single population?

Nf=300,000

Nf=25,000

r = -0.025

Model 3: Expanding population

• Allele sharing: 5.0% (1.3-9.1) OK

• Hapl. diversity:

- Etruscans: > Obs.

- Tuscans: > Obs

0

100

Generations

Tuscans

Etruscans

Only models in which modern Tuscans and Etruscans belong to distinct genealogies are consistent with the data (2<31)

Interpretations, doubts

• Unless mutation rate is much higher than currently believed, the Etruscans left very few modern mitochondrial descendants in Tuscany (Belle et al. 2006)

• Did they all go extinct?• Was the sample studied only representative of a social elite?• Did massive immigration dilute a component of Etruscan origin

in the Tuscans’ mtDNA gene pool?

Postmortem DNA modifications and/or technical problems affected the Etruscan mtDNA sequences (Achilli et al. 2007)

The similarity between the modern Tuscans and the Near East/Turkey suggests that the Etruscans came from there (Achilli et al. 2007)

No evidence of sequence errors in the Etruscan dataset

61 tooth samples from Middle-Age Tuscany Guimaraes et al., submitted

Joint analysis of11 Etruscan sequences27 Medieval sequences (900-1300 A.D.), from 6 cemeteries

322 (Achilli et al.) and 49 (Francalacci et al.) modern Tuscan sequences

Murlo, Volterra, Casentino

Only the model in which medieval Tuscans and Etruscans belong to the same genealogy and modern Tuscans don’t is consistent

with the data (Guimaraes et al., submitted)

Model 1

0 C

E

M

Model 4

C

E

M

Model 2

C

E

M

Model 3

C

E

M

Model 5

C

E

M

Model 6

E

M

C

Model 7

E

M

C

3. Or maybe the Etruscans are still among us, hiding somewhere?

Excoffier et al. (2005) Genetics 169:1727-1738

Estimating parameters and comparing models by ABC (Approximate Bayesian Computations)

Parameters: priors and posterior distributions

Parameters Priors

Ne Modern Tuscans 50 000 – 500 000 | 10 000 – 70 000

μ 0.0003 – 0.0075

T estimated (bottleneck) 101 – 1500

Ne Generation 26 100 – 10 000

Ne Generation 27 10 000 – 100 000

Ne at split 100 – 2000

Ne Medieval Tuscans 10 000 – 50 000

Ne Etruscans 4000 – 21 000

Mod 1

Method Thresh. Mod 1 Mod 2 Mod 3

RL 50000 0.972 0.028 0.000

SR 100 0.990 0.010 0.000

Method Thresh. Mod 1 Mod 2 Mod 3

RL 50000 0.000 1 0.000

0.SR 100 0 1 0

Method Thresh. Mod 1 Mod 2 Mod 3

RL 50000 0.000 1 0.000

SR 100 0 1 0

Casentino

Murlo

Volterra

SR= Straightforward rejection; LR = Logistic regression

Mod 2 Mod 3

E M

C

27

26

a1 a2

E

M

27

26

a1 a2

Mod 1

C

E

M

27

26

Parameters: priors and posterior distributions

Parameters Priors

Ne Modern Tuscans 20 000 – 200 000

Ne Modern Tuscans from Casentino valley 10 000 – 70 000

μ 0.0003 – 0.0075

T estimated (bottleneck) 101 – 1500

Ne Generation 26 100 – 10 000

Ne Generation 27 10 000 – 100 000

Ne at split 100 – 2000

Ne Medieval Tuscans 10 000 – 50 000

SR= Straightforward rejection; LR = Logistic regression

Method Thresh. A B C D

RL 50000 0.000 0.003 0.912 0.056

RL 100 0 0 1 0

SR 100 0.023 0.011 0.966 0.000

Ca

E

M

Mu Vo

E

M

Mu VoCa

E

M

Mu VoCa

A B

C D

Ca Mu Vo

E

M

27

26

a1 a2

27

26

27

26

a1 a2

a1 a2 a1 a2

27

26

4. Nuragic Sardinians resemble some, but not all, modern Sardinians

A genetic map of Europe (Menozzi, Piazza, Cavalli-Sforza 1978)

53 tooth samples from 6 nuragic sites

Elimination of samples that do not comply with the strictest quality standards

10 different sequences in 23 nuragic individuals

h, haplotype diversity=0.83

Etruscans: 0.95Tuscans: 0.96Basques: 0.96Greeks: 0.98Sicilians: 0.96

Ogliastra 0.78Gallura 0.93

North Africa: 13.9 4

Near East: 10.7 6

Europe: 18.3 8

Iberians: 29.4 2Etruscans: 22.2 4

Shared sequences among Nuragic people and other modern and ancient populations

Gallura: 18.5 1Ogliastra: 54.6 4

Ogliastra

126

126

Ogliastra Gallura Gallura GalluraOgliastra Ogliastra

Model 2Model 1 Model 3

0

0

Model 4 Model 5 Model 6

Gallura Gallura GalluraOgliastra OgliastraLatium Latium Latium

Six models describing the genealogical relationships among Nuragic people and modern Sardinians

Parameters: priors and posterior distributions

Parameters Priors

Ne Ogliastra 500 – 20 000

Ne Gallura 1000 – 40 000

Ne split 100 – 6 000

Ne Ogliastra, Gallura at split 100 – 6 000

Ne Latium 400 000

Migration rate from Latium 0 – 0.01

T split (Ogliastra vs. Gallura) 127 – 1000 [1, 2, 4, 5] or 1- 125 [3, 6]

T split (Sardinia vs. Latium) 1000

μ 0.06 - 1.3 per million years per site

Observed summary statistics describing genetic variation in the Sardinia study

Bronze Age Ogliastra Gallura Latium

Haplotype number 10 26 21 36

N of segregating sites 10 22 31 45

Mean pairwise difference 1.39 2.49 4.42 4.07

Haplotype diversity 0.83 0.79 0.97 0.95

Tajima’s D -1.64 -0.97 -1.66 -2.02

Fst 0.0218

Haplotype sharing Ogliastra / Bronze Age = 0.400

Gallura / Bronze Age = 0.100

Ogliastra / Gallura = 0.095

Model 2Model 1 Model 3

Model 4 Model 5 Model 6

Posterior probabilities of the models, with and without immigration(best 50 000 simulations)

0.983 0.002 0.015

0.813 0.081 0.106

Model 4 beats Model 1 >77% of times

What happened in Italy between the Bronze-Age and now?

Many things.

Major demographic changes in the last few centuries documented by mtDNA in the Netherlands (Manni et al. 2002), in the British Isles (Töpf et al. 2007) and in Iceland (Helgason et al. 2008), but not in the Iberian peninsula (Sampietro et al. 2005).

Relatively recent immigration may have deeply changed the genetic structure of the population in part of Tuscany and in Gallura, but not Casentino and Ogliastra

In studies of admixture, genealogical continuity between past and present is no longer an inevitable assumption, but rather a testable hypothesis (only at the mtDNA level, at present).

David CaramelliGiorgio Bertorelle

Andrea Benazzo, Silvia Ghirotto

Loredana Castrì

Elise Belle

Many thanks to

Enza Colonna

Stefano Mona

Silvia Guimaraes

Erica Fumagalli