The Andalusian population from Huelva reveals a high diversification of Y-DNA paternal lineages from...

Annals of Human Biology, January–February 2010; 37(1): 86–107

ORIGINAL ARTICLE

The Andalusian population from Huelva reveals a highdiversification of Y-DNA paternal lineages fromhaplogroup E: Identifying human male movements withinthe Mediterranean space

B. AMBROSIO1, J. M. DUGOUJON2, C. HERNÁNDEZ1,D. DE LA FUENTE1, A. GONZÁLEZ-MARTÍN1, C. A. FORTES-LIMA1,A. NOVELLETTO3, J. N. RODRÍGUEZ4 & R. CALDERÓN1

1Departamento de Zoología y Antropología Física, Facultad de Biología, Universidad Complutense,Madrid 28040, Spain, 2Laboratoire d’Anthropologie, FRE 2960, Centre National de la RechercheScientifique (CNRS), Université Paul Sabatier, Toulouse 31073, France, 3Dipartimento di Biologia,Università ‘Tor Vergata’, Rome 00133, Italy, and 4Servicio de Hematología, Hospital Juan RamónJiménez, Huelva, 21005, Spain

(Received 22 April 2009; accepted 28 July 2009)

AbstractBackground: Gene flow among human populations is generally interpreted in terms of complexpatterns, with the observed gene frequencies being the consequence of the entire genetic anddemographic histories of the population.Aims: This study performs a high-resolution analysis of the Y-chromosome haplogroup E in WesternAndalusians (Huelva province). The genetic information presented here provides new insights intomigration processes that took place throughout the Mediterranean space and tries to evaluate its impacton the current genetic composition of the most southwestern population of Spain.Subjects and methods: 167 unrelated males were previously typed for the presence/absence of theY-chromosome Alu polymorphism (YAP). The group of YAP (+) Andalusians was genotyped for 16Y-SNPs and also characterized for 16 Y-STR loci.Results: The distribution of E-M81 haplogroup, a Berber marker, was found at a frequency of 3% in oursample. The distribution of M81 frequencies in Iberia seems to be not concordant with the regionswhere Islamic rule was most intense and long-lasting. The study also showed that most of M78 derivedallele (6.6%) led to the V13* subhaplogroup. We also found the most basal and rare paragroup M78*and others with V12 and V65 mutations. The lineage defined byM34 mutation, which is quite frequentin Jews, was detected as well.Conclusions: The haplogroup E among Western Andalusians revealed a complex admixture of geneticmarkers from the Mediterranean space, with interesting signatures of populations from the Middle East

Correspondence: Prof. Rosario Calderón, Departamento de Zoología y Antropología Física, Facultad de Biología, UniversidadComplutense, Ciudad Universitaria, 28040 Madrid, Spain. E-mail: [email protected]

ISSN 0301-4460 print/ISSN 1464-5033 online � 2009 Informa UK Ltd.DOI: 10.3109/03014460903229155

and the Balkan Peninsula and a surprisingly low influence by Berber populations compared to otherareas of the Iberian Peninsula.

Keywords: Y-SNPs, genealogical history, Mediterranean gene pool, Iberia, human migrations, sourcepopulations

Introduction

Andalusia, a large and relatively densely populated region of southern Spain, has a longhistory shaped by migrations from different parts of the world at different times, including arelatively recent long Islamic settlement. Despite its implications for the peopling of theIberian Peninsula and its relevance to the evolution of modern Homo sapiens, the geneticcomposition of the Andalusian people has never been studied in depth.

Within Andalusia, the westernmost province of Huelva is of particular interest, due to itsgeographic position. It is located on the western fringe of Europe, bordering Portugal andthe Atlantic Ocean, and is also near the Strait of Gibraltar, which has served as both agenetic barrier and corridor at different times. Its population is of moderate size and hasexperienced a slow demographic growth. Furthermore, gene flow from contemporaryimmigrants has been minimal, and the main features of the autochthonous populationwere preserved.

The Y chromosome contains the largest non-recombining region (NRY) of the humangenome. It is a haploid locus harbouring a great deal of information that has found wideapplications in a range of fields such as human evolutionary, forensic, and medical genetics(Underhill et al. 2000; Shastry 2002; Jobling and Tyler-Smith 2003; Novelletto 2007; Camp-bell and Tishkoff 2008). Current knowledge of the Y-tree topology of the NRY region couldbe qualified as both refined and complex, and much has come from the ongoing identi-fication of new single-nucleotide polymorphisms (SNPs) and lineages in the humanpopulation (de Knijff 2000; Hammer et al. 2000; Underhill et al. 2001, Jobling andTyler-Smith 2003; Cruciani et al. 2004, 2006, 2007; Behar et al. 2006; Sims et al.2007; Karafet et al. 2008). The importance of the molecular and evolutionary characteristicsof SNPs becomes even more evident if we consider that the NRY region, just like themitochondrial DNA (mtDNA) genome, has a small effective population size (approximatelyone fourth that of autosomes) (Hartl and Clark 1989; Hammer et al. 1997) which enhancesthe signal of inter-population divergence.

Many recent studies on Y chromosome and mtDNA variation have largely focused on in-depth analyses of European, North African, andWestern Asian populations, all of which haveknownhistorical relationshipswithin theMediterraneanarea (Hammeretal.1997;Rosseretal.2000;Scozzari et al. 2001;Cruciani et al. 2002,2004, 2007;Seminoet al. 2004;Cinnio�glu et al.2004; Roewer et al. 2005; Torroni et al. 2006 among others). Research into these topics isproviding interesting insights into demographic changes, migratory patterns, and admixtureepisodes that have occurred during recent human evolution.

The polymorphic presence of an Alu element in the Y chromosome defines a deep-rootingclade – containing haplogroups D and E – of the phylogenetic tree of the Y-chromosomehaplogroups (see the human Y-chromosomal haplogroup tree at http://ycc.biosci.arizona.edu/ (Y-Chromosomal Consortium)). In a recently published paper, Karafet et al. (2008)reported that haplogroup E is characterized by a high number of mutations and is indeed oneof the most mutationally diverse of the 20 major Y-chromosome clades. These particularitiesmake it especially apt for investigating recent human migrations. Furthermore, many

Haplogroup E in Western Andalusia 87

populations around the world have already been studied in search of this haplogroup and cantherefore be used for comparison purposes. While haplogroup D seems to be confined toAsia, haplogroup E (mainly E1b1b1, formerly E3b, lineages), with its strong phylogeo-graphic structure, is more varied and appears to be highly frequent in Africa and moderatelyso in southern Europe and in other regions of the Mediterranean, including the Levant(Hammer et al. 1997, 2000; Underhill and Roseman 2001; Weale et al. 2003; Cruciani et al.2004, 2007; Sims et al. 2007). E1b1a (formerly E3a) lineages, in contrast, are associatedwith sub-Saharan Africa, and the Iberian Peninsula is one of the regions in the Mediter-ranean area in which the two major monophyletic E subclades, E1b1b1 and E1b1a, exist,albeit with varying frequencies between populations (Cruciani et al. 2004; Semino et al.2004; Beleza et al. 2005; Neto et al. 2007).

The aim of this study was to perform a high-resolution analysis of haplogroup E in theAndalusian population of Huelva and to compare the lineages observed with those in otherIberian, European, North African, and more distant Mediterranean populations. Thegenetic information presented here will provide a reliable background against which todiscuss new insights into migration processes that took place throughout the Mediterraneanspace – mainly during the period ranging from the protohistoric era, when the Tartessiancivilization flourished (from before 11th to 5th centuries BC), to the time of the rise and fallof Islamic rule – and to thus evaluate the impact of these processes on the current geneticcomposition of the most southwestern population of Spain.

Materials and methods

Population samples and geographical sampling

Blood samples were collected by venepuncture into EDTA tubes by a group of doctors andnurses from Hospital Juan Ramón Jiménez in Huelva city, accompanied by researchers (RCand BA) from Universidad Complutense de Madrid. Participants were asked about theorigins of their parents and grandparents and their genetic relationships with other donorscontributing to this study. The sampling strategy was designed to be as representative aspossible and to include individuals from throughout the province (not including the city ofHuelva) and municipalities whose population size had remained more or less constant overthe last 2 centuries (http://www.ine.es). The municipalities chosen for sampling wereAracena, El Repilado, El Cerro del Andévalo, La Puebla de Guzmán, Valverde del Camino,Villablanca, and Niebla (Figure 1). Between 2004 and 2007, seven field trips were made tocollect blood samples from 302 unrelated, healthy autochthonous males and females from45 demographic units located throughout the province. Written informed consent wasobtained from all individuals prior to their participation. Additional information on thegeography, history, demography, and archaeology of the Andalusian province of Huelva andits population can be found in Calderón et al. (2006) and references therein.

Laboratory analysis: Polymorphisms and haplotyping

Genomic DNA was extracted using a standard proteinase-K digestion followed by phenol–chloroform extraction and ethanol with some modifications. A total of 167 unrelated malesdescribed above were initially analysed for the presence/absence of the Y-chromosome Alupolymorphism (YAP) following the recommendations of Hammer et al. (1997). Following adescendent hierarchical order, we performed a high-resolution search of the Y-chromosomebinary E1b1b1 haplogroups, first characterizing the following Y-SNPs: M96, M35, M78,

88 B. Ambrosio et al.

M81, M123, M281 and V6. Individuals with the M78 derived state were also genotyped forthe binary markers V12, V13, V22, V27, V32 and V65 described in Cruciani et al. (2006,2007). Those individuals being positive for the M81 mutation were genotyped for internallineages: M107 and M165. Samples identified as M123 were also genotyped for M34.Binary markers that were genotyped but not detected in our sample of YAP (+) Andalusianmales were V32, V27, V22, M107, M165, M281 and V6.

To genotype the Y binary markers, we followed standard protocols already described inthe literature using the following methods: Polymerase-chain reaction (PCR)-restrictionfragment length polymorphism (RFLP) analysis, SNP multiplexing, and direct sequencing.Markers such as M96 and M35 were first amplified by duplex PCR and then by single baseextension using the SNaPshot multiplex kit (Applied Biosystems, Foster City, CA, USA)described by Brion et al. (2005). M78, V12, V13, V22, V27 and V32 were amplified usingpreviously published primers (Underhill et al. 2001, Cruciani et al. 2006) and theircorresponding allelic states were diagnosed by means of the restriction enzymes AciI(also for V13), BsgI, MmeI, PvuII and MnlI, respectively following the protocol givenin Cruciani et al. (2006). Other markers such as V65, M81, M107, M165, M123, M34,M281 and V6 were genotyped with published primers (Underhill et al. 2001, Cruciani et al.2004) by sequencing both strands (BigDye Terminator kit v.3.1) using an ABI Prism 3730DNA analyzer (Applied Biosystems).

Y chromosomes identified by the presence of biallelic polymorphisms (mutations) or SNPsare called haplogroups or subhaplogroups if they are defined by a terminal mutation within agiven haplogroup. We used the nomenclature system proposed by Karafet et al. (2008) andadopted by theYChromosomalConsortium tonamehaplogroups/subhaplogroupsdefinedby

N

S

ARACENA

EL REPILADO

EL CERRO DEL ANDÉVALO

Gua

dian

a riv

er

PUEBLA DE GUZMÀN

VILLABLANCA

Atlantic ocean

Po

rtu

gal

VALVERDE DEL CAMINO

NIEBLA

HUELVA

0 10 20 30 40 50 km

SEVILLA

France

Spain

Andalusia

Mediterranean sea

Por

tuga

l

Tint

o riv

er

Gua

dalq

uivi

r riv

er

Odi

el ri

ver

Figure 1. The geographic distribution of the municipalities sampled within the Huelva province (Andalusia, Spain).


the presence of a binary polymorphism. We also consulted information provided by theInternational Society of Genetic Genealogy (http://www.isogg.org/tree/).

Y-microsatellite (Y-STR) markers

All the samples from our group of YAP(+) Andalusian males were also characterized for 16short tandem repeat (STR) loci using the AmpFlSTRYfiler PCR amplification kit (AppliedBiosystems). The loci were DYS456[(AGAT)n], DYS389I/II[(TCTG)n(TCTA)n], DYS390[(TCTA)n(TCTG)n], DYS458[(GAAA)n], DYS19[(TAGA)n], DYS385a/b[(GAAA)n],DYS393[(AGAT)n], DYS391[(TCTA)n], DYS439[(AGAT)n], DYS635(Y GATA C4)[(TATC)n], DYS392[(TAT)n], Y GATA H4[(TAGA)n], DYS438[(TTTTC)n], DYS437[(TCTA)n], DYS448[(AGAGAT)n]. Alleles were detected using 5¢-labelled fluorescentprimers, an ABI3100 capillary sequencer (Applied Biosystems), internal size standards,and GeneMapper fragment analysis. In accordance with the recommendations of theInternational Society of Forensic Genetics (ISFG) (Gill et al. 2001), Y-STR alleles werenamed according to the number of variable repeats included. Alleles at DYS389II wereconsidered after subtracting the variation at DYS389I (Cooper et al. 1996).

Data analysis

Haplogroup and haplotype diversity, as described by Nei (1987, p. 187), as well as samplingvariancewere estimatedusingARLEQUINsoftware (version3.01) (Excoffier et al. 2005).Thecombination of alleles at multiple SNPs defines a NRY haplogroup of alleles whereas thecombinationatmultipleY-STRsonasingleYchromosomedefinesaY-STRhaplotype(deKnijff2000).E-M78andE-M81andtheirassociatedmicrosatellitehaplotypeswithfrequenciesof‡5ina set ofMediterranean population samples taken from the literature – four fromNorthernAfrica(Egypt (n = 2), Algeria, andTunisia); three fromSouthernEurope (Italy, Portugal and the studysample) and one fromWestern Eurasia (Turkey) – were used to infer mutational relationshipsbetween haplotype sequences based on fiveY-STR loci (DYS19,DYS390,DYS391,DYS392,DYS393). The phylogenetic pattern was visualized using the Reduced Median (RM) networkalgorithm (NETWORK 4.5 program; http://www.fluxus-engineering.com/) (Bandelt et al.1999). Microsatellites were weighted proportionally to the inverse of the repeat variance foreach haplogroup to reduce network reticulations.

The geographical variation for E subhaplogroups was analysed by hierarchical clusteranalysis (HCA) using the statistical program SPAD (Système Portable Pour L’Analyse deDonnés; Lebart et al. 1984). HCA was performed on the basis of Euclidean distances andWard’s linkage algorithm, and analysis of variance was used to evaluate distances betweenclusters. We included data sets from 73 population samples in the literature: 52 from Europe(17 from the Iberian mainland and two from Iberian islands), 13 from North Africa(including eight from Morocco), four from the Middle East, and four from WesternAsia. Genetic information was based on population frequencies of the following E lineages:E-M35*, E-M78, E-M81, E-M123+E-M34 and E*(xE1b1b1), which contained lineageswith interesting geographic variation patterns. Populations were denoted by the first twoletters used in country code top-level domains for Internet addresses (http://www.iana.org/cctld/). The sample size for each population sample selected was ‡ 20. In the present state ofthe literature, sample sizes of several interesting populations are lower than 50 individuals,and thus the standard deviations can be high in relation to the sample frequencies. However,those samples have been included in Table I in accordance with the great majority of thepublished studies on the subject.


Tab

leI.

Pop

ulationfreq

uenc

iesof

Y-chrom

osom

eha

plog

roup

Ean

ditssubc

lade

s.

HgE

Frequ

ency

ofE

subh

aplogrou

p(%

)

Pop

ulations

ACRN

Region

nNo.

%E-M

35*

E-M

78E-M

81E-M

123+

E-M

34E*(xE-M

35)

Referen

ces

1.Spa

nish

Basqu

es1

ESB1

IB48

12.10

2.10

Sem

inoet

al.20

042.

Spa

nish

Basqu

es2

ESB2

IB55

23.60

3.60

Crucian

iet

al.20

043.

Spa

nish

Basqu

es3

ESB3

IB45

12.20

2.20

Und

erhillet

al.20

004.

Spa

nish

Basqu

es(G

uipú

zcoa

)ESBG

IB74

11.35

1.35

Alonsoet

al.20

055.

Pasiego

sESPA

IB56

2442

.90

1.80

41.10

Crucian

iet

al.20

046.

Can

tabrians

ESCB

IB70

912

.90

8.60

4.30

Floreset

al.20

047.

Asturians

ESAT

IB90

1213

.30

10.00

2.20

1.10

Crucian

iet

al.20

048.

Catalan

sESCN

IB33

26.00

3.00

3.00

Sem

inoet

al.20

049.

And

alusians

(Hue

lva)

ESAH

IB16

720

11.98

1.20

6.59

2.99

1.20

Present

stud

y10

.And

alusians

(Cordo

ba)

ESAC

IB27

311

.10

3.70

7.40

Floreset

al.20

0411

.And

alusians

(Seville)

ESAS

IB15

511

7.00

0.60

4.50

1.30

0.60

Floreset

al.20

0412

.And

alusians

1ESA1

IB76

79.20

3.90

5.30

Sem

inoet

al.20

0413

.And

alusians

2ESA2

IB62

46.40

1.60

3.20

1.60

Crucian

iet

al.20

0414

.Ten

erife

ESTN

IB17

833

18.60

3.40

10.70

3.90

0.60

Floreset

al.20

0315

.M

ainlan

dPortugal

PTM

LIB

657

8212

.50

0.90

4.10

5.60

1.20

0.70

Netoet

al.20

0716

.NorthernPortugu

ese1

PTN1

IB50

714

.00

2.00

4.00

4.00

4.00

Crucian

iet

al.20

0417

.NorthernPortugu

ese2

PTN2

IB10

916

14.60

6.40

5.50

2.70

0.90

Floreset

al.20

0418

.Sou

thernPortugu

ese

PTSU

IB49

918

.30

4.10

12.20

2.00

Crucian

iet

al.20

0419

.AzoresIsland

sPTAZ

IB31

942

12.90

1.30

6.10

3.70

0.90

0.90

Netoet

al.20

0720

.Frenc

hFR

WEU

857

8.20

4.70

3.50

Crucian

iet

al.20

0421

.Frenc

hBearnais

FRBN

WEU

271

3.70

3.70

Sem

inoet

al.20

0422

.Frenc

hBasqu

esFRB1

WEU

450

0.00

Sem

inoet

al.20

0423

.Corsicans

FRCO

WEU

140

96.40

0.70

4.30

1.40

Crucian

iet

al.20

0424

.Sardinian

s1

ITSD1

WEU

367

3710

.00

1.10

3.50

0.30

3.50

1.60

Crucian

iet

al.20

0425

.Sardinian

s2

ITSD2

WEU

139

75.00

0.70

2.90

1.40

Sem

inoet

al.20

0426

.Dan

ish

DK

WEU

351

2.90

2.90

Crucian

iet

al.20

0427

.Dutch

NL

WEU

340

0.00

Sem

inoet

al.20

0428

.NorthernItalians

ITN

CEU

678

12.00

9.00

1.50

1.50

Crucian

iet

al.20

0429

.North-C

entral

Italians

ITNC

CEU

566

10.70

10.70

Sem

inoet

al.20

04


Tab

leI(C

ontin

ued)

HgE

Frequ

ency

ofE

subh

aplogrou

p(%

)

Pop

ulations

ACRN

Region

nNo.

%E-M

35*

E-M

78E-M

81E-M

123+

E-M

34E*(xE-M

35)

Referen

ces

30.Cen

tral

Italians

ITC

CEU

8912

13.40

11.20

2.20

Crucian

iet

al.20

0431

.Sou

thernItalians

ITS

CEU

8712

13.80

11.50

2.30

Crucian

iet

al.20

0432

.Sou

thernItalians

(Calab

ria1)

ITCL1

CEU

8018

22.70

1.30

16.30

1.30

2.50

1.30

Sem

inoet

al.20

0433

.Sou

thernItalians

(Calab

ria2)

ITCL2

CEU

6816

23.50

1.50

5.90

13.20

2.90

Sem

inoet

al.20

0434

.Sou

thernItalians

(Apu

lia)

ITAP

CEU

8612

13.90

11.60

2.30

Sem

inoet

al.20

0435

.Sicilian

s1

ITSY1

CEU

136

2921

.30

14.00

0.70

6.60

Crucian

iet

al.20

0436

.Sicilian

s2

ITSY2

CEU

5515

27.30

5.50

12.70

5.50

3.60

Sem

inoet

al.20

0437

.M

ainlan

dCroatia

HRML

EEU

108

65.60

5.60

Peri� cic

etal.20

0538

.Herzego

vinian

sBA

EEU

141

128.50

8.50

Peri� cic

etal.20

0539

.Serbian

sRS

EEU

113

2421

.25

20.35

0.90

Peri� cic

etal.20

0540

.Maced

onians

MK

EEU

7919

24.06

24.06

Peri� cic

etal.20

0541

.Polish1

PL1

EEU

994

4.00

4.00

Sem

inoet

al.20

0442

.Polish2

PL2

EEU

381

2.60

2.60

Crucian

iet

al.20

0443

.Hun

garian

sHU

EEU

535

9.40

7.50

1.90

Sem

inoet

al.20

0444

.Eston

ians

EE

EEU

744

5.50

1.40

4.10

Crucian

iet

al.20

0445

.Russian

sRU

EEU

420

0.00

Crucian

iet

al.20

0446

.Ukrainian

UA

EEU

938

8.60

7.50

1.10

Sem

inoet

al.20

0447

.Geo

rgian

GE

EEU

410

0.00

Sem

inoet

al.20

0448

.Balkarian

(Sou

thernCau

casus)

RUBK

EEU

391

2.60

2.60

Sem

inoet

al.20

0449

.Bulgarian

sBG

EEU

116

2521

.60

20.70

0.90

Crucian

iet

al.20

0450

.Alban

ians

AL1

EEU

4411

25.00

25.00

Sem

inoet

al.20

0451

.NorthernGreeks(M

aced

onia)

GRMA

EEU

5912

20.30

18.60

1.70

Sem

inoet

al.20

0452

.Greeks

GR

EEU

8420

23.80

21.40

2.40

Sem

inoet

al.20

0453

.Turkish

(Edirne)

TRED

EAS

528

15.38

11.54

3.85

Cinnio� g

luet

al.20

0454

.Turkish

(Kars)

TRKA

EAS

8212

14.63

8.54

6.10

Cinnio� g

luet

al.20

0455

.Turkish

(Kon

ya)

TRKO

EAS

908

8.89

1.11

1.11

6.67

Cinnio� g

luet

al.20

0456

.Turkish

(Istan

bul)

TRIS

EAS

8113

16.05

7.41

4.94

3.70

Cinnio� g

luet

al.20

0457

.Iraq

iIQ

MDE

218

209.20

5.50

2.80

0.90

Sem

inoet

al.20

0458

.Leb

anese

LB

MDE

428

19.10

11.90

2.40

4.80

Sem

inoet

al.20

04


Tab

leI(C

ontin

ued)

HgE

Frequ

ency

ofE

subh

aplogrou

p(%

)

Pop

ulations

ACRN

Region

nNo.

%E-M

35*

E-M

78E-M

81E-M

123+

E-M

34E*(xE-M

35)

Referen

ces

59.Ashkena

zim

Jewish

ILA

MDE

7714

18.20

1.30

5.20

11.70

Sem

inoet

al.20

0460

.Sep

hardim

Jewish

ILS

MDE

4012

30.00

2.50

12.50

5.00

10.00

Sem

inoet

al.20

0461

.M

oroc

can(A

rabs)1

MAA1

NAF

5439

72.30

38.90

31.50

1.90

Crucian

iet

al.20

0462

.M

oroc

can(A

rabs)2

MAA2

NAF

4937

75.50

42.90

32.60

Sem

inoet

al.20

0463

.M

oroc

can(A

rabs)3

MAA3

NAF

4432

72.80

2.30

11.40

52.30

6.80

Sem

inoet

al.20

0464

.M

oyen

Atlas

(Berbe

rs)

MABA

NAF

6960

86.90

10.10

71.00

5.80

Crucian

iet

al.20

0465

.M

arrakesh

(Berbe

rs)

MABM

NAF

2926

89.50

3.40

6.90

72.40

3.40

3.40

Crucian

iet

al.20

0466

.M

oroc

co(B

erbe

rs)

MAB

NAF

6455

85.90

10.90

68.70

6.30

Sem

inoet

al.20

0467

.M

oroc

co(N

orth-

Cen

tral

Berbe

rs)

MABN

NAF

6355

87.30

7.90

1.60

65.10

12.70

Sem

inoet

al.20

04

68.M

oroc

co(Sou

thern

Berbe

rs)

MABS

NAF

4035

87.50

7.50

12.50

65.00

2.50

Sem

inoet

al.20

04

69.Sah

araw

ish

EH

NAF

2924

82.70

75.90

6.80

Sem

inoet

al.20

0470

.Algerians

DZ

NAF

3221

65.60

3.10

6.30

53.10

3.10

Sem

inoet

al.20

0471

.Tun

isians

TN

NAF

5832

55.10

3.40

15.50

27.60

5.20

3.40

Sem

inoet

al.20

0472

.NorthernEgyptians

EGN

NAF

218

38.20

28.60

4.80

4.80

Crucian

iet

al.20

0473

.Sou

thernEgyptians

EGS

NAF

346

17.60

17.60

Crucian

iet

al.20

04


Results

The clade E emerged as the second most common haplogroup in autochthonous Anda-lusians from Huelva, with a high frequency (12%, 20/167 individuals) in comparison toother Spanish populations. The corresponding value in neighbouring southern Portugal is18% (Cruciani et al. 2004). Most of the Y chromosomes analysed in the Andalusianpopulation from Huelva belongs to the R1b lineage (R-P25), with an incidence of 59.9%.

The E-M35 (E1b1b1) haplogroup variations observed in Huelva are shown in Figure 2,and E subhaplogroup frequencies for the 73 populations selected for comparison in Table I.The other important basal monophyletic subclade within haplogroup E, E1b1a, which isparticularly frequent in sub-Saharan Africa (Underhill et al. 2000; Cruciani et al. 2002), wasnot observed in our population, although carriers of this subclade have been detected inmainland Portugal and the Azores Islands with higher frequencies (0.2–0.9% of total Ychromosomes) (Beleza et al. 2005; Gonçalves et al. 2005; Neto et al. 2007) than thosedetected in other European populations.

A comparative analysis of the E haplogroups detected in the Andalusian population withfrequencies reported for the population data set in Table I revealed a number of interestingfindings. The E-M81 clade (five occurrences) accounted for 3% of the total haplogroup Efrequency detected in Huelva, all belonging to the paragroup E-M81* (E1b1b1b* lineage);

Lineages

YCC 20021

D* D*

E*

E1b1b1*

E1b1b1a*

E1b1b1a1*

E1b1b1a1b*

E1b1b1a2*

E1b1b1a2a*

E1b1b1a3*

E1b1b1a4*

E1b1b1b*

E1b1b1b1

E1b1b1b2

E1b1b1c*

E1b1b1c1*

E1b1b1d

E3b3a*

E3b3*

E3b2b

E3b2a

E3b2*

E1b1b1e

20 (11.98)

0

0

0

0

0

0

0

0

1 (0.60)

2 (1.20)

2 (1.20)3

0

0

Hg frequency

5.(2.99)

7 (4.19)

1 (0.60)

2 (1.20)

E*

E3b*

E3b1*

V12V32

V27V13

V22

V65

M107

M165

M34

M123

M81

M78

M35

M96

YAP

M281

V6

1YCC 2002 (Genome Res. 2002 12:339-348)

2Karafet et al. 2008

(Genome Res. 2008 18:830-838)

3Relative frequencies

Karafet et al. 20082

Figure 2. Phylogeny of the clade E-M35 (E1b1b1) with the 16 binary markers used in the genetic characterization ofthe autochthonous population of the Huelva province.


this frequency is similar to the mean value observed in Basques (Alonso et al. 2005) andlower than that reported for the majority of Spanish (Adams et al. 2008; Capelli et al. 2009),French (3.5%) and Portuguese populations (12% in mainland Portugal (Cruciani et al.2004) and 4% in the Azores Islands (Neto et al. 2007)). Phylogeographic analysis of theE-M81 lineages in Mediterranean populations has shown these lineages to be remarkablyfrequent in Berbers (80% in Mozabites; 65–73% in Berbers from Morocco) (Cruciani et al.2004; Semino et al. 2004) although their frequency declines sharply towards the north east(Egypt ffi5%). E-M81 lineages are practically absent in Eastern Europe (Peri�cic et al. 2005)and uncommon in Italy, with the exception of Sicily (5.5%) (Semino et al. 2004) where therewas an Islamic occupation that lasted for over two centuries (878–1091 AD). Presuming thatthe presence of E-M81 in southwestern Europe is a signature of a North-African gene poolshared also by Berbers, these migrants seem to have left a much smaller genetic imprint inthe male gene pool of Huelva than in other parts of Spain.

Our study also showed that most of the Y chromosomes carrying the E-M78-derived allelewere further classified into the E-V13 subhaplogroup (E1b1b1a2). The frequency of E-V13 inour study populationwas relatively high (seven occurrences, 4.2%of total) in comparisonwithother Iberian populations. In North Africa, this subhaplogroup shows a mean frequency of4.5%, as reported by Cruciani et al. (2007) (see Table I), and is indeed most common inAlbanians (32.30%),Macedonians,Greeks, andBulgarians (15–18%) (Peri�cic et al. 2005). Inour analysis of the subclade E-M78, we also found 2 (1.2%) Y chromosomes in themost basaland rare paragroup, E-M78*, one (0.60%) in E-V12, and, interestingly, a single occurrence ofE-V65 (0.60%). The average frequency of E-M78* has been estimated at 0.08% (Crucianiet al. 2007) (see Table I) and the highest values have been registered in Egyptians fromGurna(5.9%), followed byMoroccan Arabs (3.6%) and Sardinians (0.27%). No occurrences of thissubhaplogroup were found in any of the other 81 populations included in the analysisby Cruciani et al. (2007). The subhaplogroup E-V12 has also been found in high frequenciesin Egyptians (ranging from 44% in the south to 6% in the north) and in Berbers (3.5%) andMoroccan Jews (2%). The surprisingly high frequency observed in French Basques (6%, oneoccurrence) seems to be due to the small size of the sample. The frequency of the sub-haplogroup E-V65 in the sample from Huelva coincides with the mean frequency reportedby Cruciani et al. (2007) in Table I. E-V65 is relatively frequent in Arabs from northMorocco(29%) and Libya (20%) but less common in other groups from Morocco, and in Libyans,Egyptians, Sardinians, and Sicilians.

Finally, E-M34 (E1b1b1c1*), a lineage internal to the E-M123 haplogroup, was found in1.2% of the Huelvan sample (two occurrences). The E-M34 lineage has been found atrelatively high frequencies in Jews (10–12%) and in a sample from Calabria (13%). Thecorresponding frequencies reported for Turkey and Tunisia are between 5% and 6%.

It is worth noting that two individuals (1.2% of total) in our study sample were found tocarry the derived state at M35 but not at all other known SNPs further downstream (http://www.familytreedna.com/public/E3b/). The deep paragroup E-M35* (E1b1b1*, formerlyE3b*) is rare in Europe (~0.4%) but present in high frequencies in East Africa (8%-17%)(Cruciani et al. 2004). Other frequencies reported for this paragroup are 8% for MoroccanBerbers, 5.5% for Sicilians, 3% for Algerians and Tunisians, 2.5% for Sephardic Jews, and1.3% for Ashkenazi Jews (see references in Table I of present study).

Frequency and structure of Y-STR haplotypes associated with each E binary markersdetected in the study Andalusian population sample is shown in Table II. The analysisinitially revealed a high haplogroup diversity for the E-M35 clade (h = 0.8211 ± 0.06) beingthe subclade E-M78 that yielding a rather high level of SNP h (0.5906 ± 0.15). The subcladeE-M78 also revealed a high level of internal Y-STR diversity (H = 0.9818 ± 0.05), with a


Tab

leII.Distributionof

Y-chrom

osom

eha

plotyp

esam

ongthesubh

aplogrou

psE

foun

din

Hue

lva(Spa

in).

Hap

lotype

sHap

logrou

psDYS19

DYS38

9IDYS38

9II

DYS39

0DYS39

1DYS39

2DYS39

3DYS38

5a,b

DYS43

8DYS43

9Frequ

ency

H1

E1b

1b1*

-M

3514

1330

229

1114

12,13

1011

1H2

E1b

1b1*

-M

3514

1330

229

1114

13,13

1011

1H3

E1b

1b1a

*-M

7814

1432

2410

1113

18,21

1013

1H4

E1b

1b1a

*-M

7815

1431

2511

1113

16,20

1012

1H5

E1b

1b1a

1*-V12

1313

3124

1111

1316

,17

1012

1H6

E1b

1b1a

2*-V13

1313

3024

1011

1217

,19

1012

1H7

E1b

1b1a

2*-V13

1313

3024

1011

1316

,17

1014

1H8

E1b

1b1a

2*-V13

1313

3024

1011

1316

,18

1012

1H9

E1b

1b1a

2*-V13

1313

3024

1011

1316

,18

1013

2H10

E1b

1b1a

2*-V13

1313

3025

1011

1316

,18

1014

1H11

E1b

1b1a

2*-V13

1313

3123

1011

1317

,18

1012

1H12

E1b

1b1a

4-V65

1312

2924

1111

1316

,17

1010

1H13

E1b

1b1b

*-M

8113

1430

249

1113

13,14

1010

4H14

E1b

1b1b

*-M

8114

1430

249

1113

13,14

1010

1H15

E1b

1b1c

1*-M

3413

1330

2411

1113

15,16

1013

1H16

E1b

1b1c

1*-M

3413

1331

2410

1113

16,16

1012

1


mean variance of 0.3933 ± 0.24. Conversely, the haplotype variability within the sistersubclade E-M81 was much lower (0.4000 ± 0.24): Four of the five males carrying the M81marker had identical Y-haplotypes: DYS389I(14)-DYS389II(30)-DYS390(24)-DYS391(9)-DYS392(11)-DYS393(13)-DYS385a/b(13,14)-DYS438(10)-DYS439(10). This find-ing was accompanied by a very low mean variance in allele size (0.0444 ± 0.06). WithinE-M81* the fifth haplotype differed only in one mutational step at the DYS19 locus (13- to14–repeat alleles). Analysis of variance at single Y-microsatellite loci showed that theDYS390 locus yielded the highest value (0.70); the corresponding figure for DYS19 was0.33, with the 13–repeat allele being the most frequent (15 of the 20 YAP+ chromosomesdetected in the study sample). An in-depth analysis of the relationship betweenhaplogroup E frequency and associated haplogroup/haplotype diversities within and betweenlarge geographic areas in the Mediterranean would provide interesting insights into pop-ulation demographic histories.

The RM network of E-M78microsatellite haplotypes in a group of Mediterranean samplesavailable in the literature is shown in Figure 3. The population data set included a total of 36distinct haplotypes. The structure of the haplotype network is highly diversified, which is to beexpected given the haplogroup substructure that it includes. The most common node (68 of160 Y chromosomes) is represented by the modal haplotype 13-24-10-11-13 (for the lociDYS19-DYS390-DYS391-DYS392-DYS393, respectively), shared by different populationsinmainland Italy, Turkey, and Portugal and to a lesser extent by southern Egyptians, AlgerianArabs, and Andalusians from Huelva (four chromosomes) (for more details see the legendto Figure 3). This node bears the highest number of connections with other haplotypes (n = 6),strongly suggesting that it is the root, which is in turn the modal haplotype of the network(Crandall andTempleton1993).The longest lineage is composedof sevennodes, separatedbysinglemutational differences. Huelvan Y-haplotypes fall into three nodes (one specific copy ineach), which contain, in varying combinations, most of the populations noted above and otherneighbouring Mediterranean groups. Within this lineage, the third node (represented by theY-STRhaplotype14-24-10-11-13) is representedbymales fromnorthernand southernEgypt,Italy, and Andalusia. We also detected one singleton (haplotypes represented by a singleindividual) in the study sample. Haplotypes showing high frequencies are expected to be goodindicators of when a particular mutation originated, whereas those with low frequencies (rarehaplotypes), usually represented by a single individual, point to a recent evolutionary origin.Indeed they occur preferentially at the tips of networks (Golding 1987; Excoffier andLanganey1989). An RM E-M81 network for the same samples was also constructed but is not shownhere. The most common node for E-M81 (61 of 126 chromosomes) is represented by themodal haplotype 13-24-9-11-13; this coincided with the four identical Y chromosomesdetected in Huelva and is shared – in variable frequencies – by most (n = 8) of the populationsamples analysed.

Figure 4 portrays the results of HCA based on frequencies of the E-M35 haplogroup andits main subclades within the data set of populations shown in Table I. Factors I and IIaccount for 97.81% of the variance, with a predominance of factor I (83.83%). Themultivariate analysis formed six distinct clusters, with E-M78 and E-M81 haplogroupscontributing significantly to the pattern observed. With the aim of giving more support to thenumber of clusters (six) suggested by the dendrogram, it is interesting to note that whencomputing the Inertia decomposition on the first two factors, the quotient (Inertia inter-clusters/Inertia Total) = 0.9565. Thus, this amount is highly coherent with the number ofmajor ramifications showed by the tree, and it demonstrates that with six clusters a highpercentage (96%) of the data variation is explained. Clusters C1, C2, and C3 on the right sideof the plot are formed by European, Middle Eastern, and western Eurasian populations,


whereas clusters C4 and C6 on the upper left and lower sides of the bidimensional space, andC5 in an intermediate position, include populations almost exclusively from northern Africa.The genetic map clearly evidences the combined effect of interactions between differentevolutionary processes during the coalescence time of these populations for the major Ehaplogroup.

Andalusians fromHuelva (ESAH) are included inC2, which is distinguished by a highmeancluster value for the lineage E-M123+E-M34 with respect to the overall mean (test-value = 3.35, p < 0.001) and a low value for E-M81 (test-value = -2.15, p < 0.02). The 18populations that shape this cluster are Asturians from northern Spain, most (9 of 11) of theItalian population samples, three Turkish population samples, Bosnians, Hungarians, Ukrai-nians, Sephardic Jews, and Lebanese. Our Andalusian sample, thus, is grouped with popula-tions from the middle and eastern areas of the Mediterranean basin although the topology ofthe HCA plot also reveals a genetic affinity of Huelva with several populations located withincluster C1 such as Portuguese from the Azores Islands and northern mainland Portugal,

HuelvaItalyPortugalTurkeyAlgeriaNorth EgyptSouth EgyptTunisia

Figure 3. Reduced median (RM) microsatellite haplotype network for E-M78 haplogroup based on five Y-STRsloci (DYS19, DYS390, DYS391, DYS392, DYS393). Circles are proportional to the number of individuals sharingthat haplotype. Mediterranean populations used to construct the network have been taken from the literature:Tunisians, Egyptians from Northern and Southern, Arabs and Berbers from Algeria (Arredi et al. 2004); Turkish


Andalusians (in general), Andalusians from Cordoba, and French. The genetic topology ofHuelva closely agrees with the results of an earlier analysis of GM immunoglobulin allotypeson the same population sample (Calderón et al. 2006).

Cluster C1 is the most numerous cluster, and contains mostly European populations,including 15 of the 17 population samples analysed in Spain. Also included in this cluster are

-22.5

-4 0 4

EGN

8Factor 1 - 83.83%

Factor 1 - 83.83%

M78

MAA1

MAA2

C4

AL1

MK

GRBG

RS

GRMA

EGS

ITSY1

LB

ITC

ESAT

ESAH

PTAZ

ESASESB2

ESB3

ESCN

ESB1 ESBG

EGN

AL1

MK

GRBG

RSGRMEG

ITCL1

ITSY1

TREDLBILSITSY2

ITNESAITC

TRKAIT BATRIS

UA

HRMLFR

PTN2ESAH

C2

C3

M123/M34

PTMLPTAZ

PTNITSD1 IL

ITCL

ITSD

ESBGFRBESB1ESB2

ESB3ESAS

ESCBE*(xE-M35)

M35 ESAC ESA1C1

ESTNPTSU

TRKESAGE

ITA IT

TRKO

ESA3

RUNL

FRB1PL2

RUBK GEITSD2

ITSD1PTN1

PL1EEFRBN

FRESA1PTML

ESAC

ESCB

ESTNPTSU

7.5

0

-7.5

-15.0

Factor 2-13.98%

-60 -45 -30 -15 0

PTN2

ITCL1

ITSY2 ILS

C3

C2

C1

ITN

TREDITAP

ITSITNC

TRKABA

HUUA

ILATRIS

ITCL2HRMLIQ

FRCODK

MAA3

DZ

ESPA

TN

C5

MABS

MAB

MABA

MABM

M81

C6

EHMABN

Factor 2-13.98%

10

0

-10

-20

-30

a.

b.

Figure 4. (a) Hierarchical cluster analysis (HCA) of 73 European, Western Asian, Middle Eastern and NorthernAfrican populations based on Y-chromosomal diversity of E-M35 subhaplogroups (M35, M78, M81, M123+M34and E*(xE-M35)). (b) A zoom plot of clusters C1, C2 and C3. Population codes are as in Table I.


Turks from Konia, Ashkenazi Jews, and Iraqis. The populations in C1 are distinguished bylow frequencies of all the E-M35 lineages considered (test value range, -1.79 to -5.36; pvalue range, 0.037–0.000). Interestingly, haplogroup E is absent from other populations inthis cluster, such as the Georgians, the Russians, the Dutch, and the French Basques, and isvery infrequent in Spanish Basques, Danes, Poles, and people from the Balkans. Thesefindings suggest the existence of a prehistoric migration corridor following the arching plainsof Europe stretching from Caucasus to the Basque area, as has been postulated by Calderón(2000) and Calderón et al. (1998).

Most of the samples in cluster C3, which is characterized by high frequencies of the M78haplogroup (test value = 4.76, p < 0.001), are from the Balkan region (Greeks, Albanians,Serbs, Macedonians, and Bulgarians) but there were also two Egyptian samples and asample from Calabria (Italy), which historically formed part of Magna Graecia.

Cluster C4, which has the highest E-M78 frequencies (test value = 5.37, p < 0.001) can befound in the lower part of the plot. This cluster is made up of two Moroccan Arabpopulations who, in contrast to neighbouring Maghreb populations, would have originatedfrom somewhere in Arabia or the Fertile Crescent and had relatively little contact withBerber populations.

Cluster C5, on the contrary, is characterized by high frequencies of E-M81 and theparagroup E-M35* (test value > 2.5, p < 0.01) and is made up of three Maghrebi populations(Algerians, Tunisians, and Arabs from Morocco), and surprisingly, a sample from thePasiega region in Cantabria, northern Spain. The abnormal presence of this northernSpanish population in this cluster, which has no known historical or demographic justifi-cation, strongly suggests the need to repeat analyses in this population. While there is astrong Arab component in the north African populations in C5, it is not as strong as thatobserved in C4.

Finally, cluster C6 has the highest frequencies of E-M81, E-M35* and E*(xE-M35) (testvalue range, 3.93–7.19; p = 0.000). This cluster is formed by six populations, including fiveMoroccan Berbers populations and a Saharawi population. Cluster 6, therefore, is the groupthat best defines the current Berber population.

Discussion

The geographic position of Huelva, located at the southwestern fringe of Europe, and thefact that haplogroup E – the focus of the present study – appears to have originated in EastAfrica and is not frequent in this part of Europe prompt us to consider that the nativepopulation of Huelva received these genes from elsewhere. In this section, we will exploredifferent hypotheses that might shed some light on who these populations were and wherethey came from.

The times to the most recent common ancestors (TMRCA) of the main haplogroups inthe E clade observed in Huelva, E-M78 and E-M81, were estimated at 18.6–4.3 thousandyears ago (kya) (Cruciani et al. 2004, 2007). The most frequent haplogroups in Huelva,E-V13 and E-M81, emerged after the Younger Dryas (~12 kya), with E-M81 emerging closeto the beginning of the Neolithic age (5.6 kya). The estimated time of coalescence for E-V65is more recent (~4.5 kya), and would have coincided with the beginning of the Early BronzeAge in Iberia. The TMRCA for the paragroups E-M35* and E-M78* would be some timebefore the Last Glacial Maximum (~18 kya). It should be noted that alleles (mutations) mustbe present in high frequencies in order to be transferred effectively (i.e. in notablefrequencies) by migrants to other populations. This is why effective gene flow will onlyoccur quite some time after the TMRCA. Consequently, migration linked to the movement


of males with E-M35 lineages should be situated within a relatively recent evolutionaryperiod. In this context, it should be noted that TMRCA estimates are dependent on manyassumptions about mutational processes and population structures (Hein et al. 2005).

The wide but non-homogeneous spatial distribution pattern of E-M81 chromosomes inIberia does not seem to be concordant with the regions in which Islamic occupation wasmost intense and prolonged (Lopez-Davalillo 2000; Martinez-Ruiz et al. 2003). This wouldstrengthen the hypothesis that migratory movements took place betweenMaghreb and Iberiaprior to the Islamic occupation, and those other important movements within the peninsulaoccurred later (Calderón 2006). The Islamic occupation of western Andalusia lasted from711 to 1262 and 5 years later Portugal and Castile (Spain) agreed on the southern borderdividing their kingdoms. The Berbers, who arrived in several waves, were the mostnumerous of all the migrant populations that arrived in Iberia during Islamic rule. It hasbeen estimated that around one-third of the 300 000 Berbers that arrived during these yearsdid so in the eighth century (Mackay 1977), a time when the total population of thepeninsula has been estimated at between 6 and 7 million (Dupaquier 1997). The Berberstended to settle in the mountainous areas of the peninsula and, interestingly, most of themwere men of reproductive age, many of whom came with their wives, also Berbers in manycases. Some of the descendants of these early occupiers were to return to Maghreb between1264 and 1609. There are several relevant historical contexts preceding the Islamicoccupation that are likely to have had a considerable impact on population dynamics inthe Mediterranean area and in the Iberian Peninsula in particular. These were (i) theexistence of a Berber gene flow associated with the Carthaginian period, which included aportion of north African natives; (ii) the establishment of the Roman Empire on both sides ofthe Strait of Gibraltar, which considerably improved communication and safety throughoutthe Empire; and (iii) the considerable and lasting difference in population sizes betweenEurope and North Africa (always higher in Europe), which would explain the low NorthAfrican contribution to the gene pool in Europe, although the Berbers did leave a geneticimprint in numerous locations in Europe due to the remarkably high frequency of E-M81 inthis population.

The levels of E-V13* detected in our study population could perhaps be explained bypossible contact with populations that would have travelled by sea from areas under Greekcontrol during the protohistoric period, when the Kingdom of Tartessos gained strategicimportance thanks to its extraordinarily rich deposits of copper, silver, and tin. Abundantremains of the Tartessian culture have been found in the area of Huelva, in particular,remains corresponding to the period after the eight century BC marked by intense contactswith civilizations from the eastern Mediterranean (Almagro-Basch et al. 1974; Fernandez-Jurado et al. 1997). Herodotus, in 1.163, reports that ‘the Phocaean were the first of theGreeks who performed long voyages, and it was they who made the Greeks acquainted withthe Adriatic and with Tyrrhenia, with Iberia, and the city of Tartessos. The vessel which theyused in their voyages was the long penteconter (50–oared ship). On their arrival at Tartessus,the King Arganthonius took a liking to them. He begged the Phocaeans to quit Ionia andsettle in whatever part of his country they liked’ (Placido 1999). The existence of traderelations across the Mediterranean Sea therefore seems to be a more plausible explanationfor this gene flow into Huelva than the theory of population movements following the long,winding river waters connecting the southern Balkans and north-central Europe as Peri�cicet al. (2005) and others have suggested. Nonetheless, a small proportion of the E-V13Y-chromosomes found in Huelva might have arrived much later, through the Visigoths, whotravelled to Hispania after 411 AD from the northern region of the Black Sea and theBalkans.


The presence of Y chromosomes E-M78* and E-V12* might be due to an Egyptian geneflow, which is supported by historical evidence. Around 742 AD, a large army led by theSyrian Balch arrived in the Iberian Peninsula to suppress a Berber rebellion against theArabs. The army was made up of several contingents (chunds) from different Islamizedregions of the Levant and Egypt. Following the defeat of the Berbers, the troops wereseparated according to their regions of origin and sent to different parts of the south of thepeninsula. Part of the Egyptian contingent settled in the district of Beja (today a Portuguesetown bordering Huelva; Ajbar Machmuâ, translated into Spanish by Lafuente Alcántara.Madrid 1867; Ibn-Al-Jatib cited in Dozy, Recherches, I3, 78 Leyden 1881).

The V65 marker detected should also be considered a signature of the Arabs. BecauseArabs also settled in Maghreb after the Islamic conquest of this region, the E-V65chromosomes in Huelva might have come directly from the Middle East, without necessarilyhaving passed through an intermediate North African reproductive stage. It has beenestimated that the first influx of Arabs into the Iberian Peninsula numbered 30 000–40000, a relatively small number and substantially inferior to that of the Berbers. This firstgroup of Arabs consisted of two rival tribes: The Qaysi, from the north of the ArabianPeninsula, who eventually settled in the province of Huelva, and the Yameni from the south.Arabs, unlike Berbers, settled mostly in cities, chose their wives from among the Visigothicnobility, avoiding marriage with Berbers. Many were rich and powerful and, interestingly,they practised polygamy, which would have had a multiplier effect on chromosome Ylineages. By way of example, the 10 emirs and caliphs that governed Cordoba between 756and 1013 had at least 143 sons (who did not die prematurely) who had male offspringranging from 40 to none (Vallvé 1977).

Following the Reconquista of western Andalusia, a considerable proportion of the Muslimpopulation, who had lived there for over 5 centuries, left these lands for Granada orMorocco. Later, during the 14th and 15th centuries, about 400 000 Muslims (»4.5% of thetotal population) abandoned the peninsula, and in 1609, with the passing of the decree toexpel Moriscos (Spanish Muslims), many were sent to North Africa (Morocco and Algeria),a region considerably less populated than the Iberian Peninsula (Lopez-Davalillo 2000).A portion of the present Moroccan and Algerian Arab and Berber male gene pool would thushave remained a long period of time in the Iberian Peninsula. As far as the E-M35 isconcerned, populations carrying this mutation from East Africa would presumably havemigrated to southwestern Andalusia after an intermediary settlement period alongsideMaghreb Berbers during the Islamic occupation. The frequencies of this mutation observedin Jewish populations could be the result of previous contact with populations from theMiddle East and West Africa or later links with Berbers in North Africa.

Finally, the Jewish settlements in Andalusia could explain the frequency of the E-M34subclade in Huelva. Tartessos is mentioned numerous times in ancient writing sources(see Myro 1999 for a list of citations), indicating the existence of close contact between bothextremes of the Mediterranean dating back many centuries. In Andalusia, there were Jewishcommunities at least as far back as the time of the Roman Empire (García Iglesias 1978).

Several requisites must be satisfied for a source population to contribute noticeably tofrequencies of a particular genetic marker in a recipient population. Firstly, the frequenciesof the marker in the source population and the size of the migrating population in relation tothat of the recipient population must be high enough in order not to excessively dilute thegene flow. When testing such a hypothesis, thus, it is necessary to analyse the demographicsizes of both populations and the relative and absolute frequencies of the marker in question.Because human genetic diversity mainly seems to consist of frequency clines (Serre andPääbo 2004), substantial initial differences in frequencies are generally found between


sources and recipients when populations are separated by large distances or abrupt barriers.Gene frequencies only reach high enough levels to produce an effective admixture (i.e. afrequency that lasts and is easy to detect) in the recipient population a long time after themutation event occurred in the source population. Occasional, motivated group migrationstherefore are more plausible and genetically effective than small persistent, gene flows.A recipient population can receive a particular gene directly from the source population orthrough intermediate populations which inherited it from the source group. In such a case,however, effective gene flow is seriously compromised by the time required for themovement to be completed and the mutation frequencies that would occur in the inter-mediate populations. This reduces the number of theoretically possible migrations to just afew plausible ones. Migrations that occur in several stages have several characteristics.Firstly, a considerable amount of time is required at each stage for admixture to occur and tohave an effect on the spread of gene in the next stage; and secondly, there is a progressivereduction of gene frequencies at each intermediate stage (population) governed by thedecreasing power law, fi = ki fo, where fo is the marker frequency in the original sourcepopulation, fi the marker frequency at each stage i, and k the roughly constant fraction ofmigrants at each stage. For example, with k = 0.25 and i = 3, f3 = 0.156 fo. Consequently, justa few stages are necessary to reduce gene frequency in the final recipient population to a levelof close to zero, regardless of the frequency of the marker in the original source population. Itwould therefore seem logical to reject hypotheses involving migrations that occur in severalstages because the final effect in the recipient population would be negligible.

Populations that have not experienced direct gene flow might have similar proportions of aparticular genetic marker if a source population, which might even be now extinct, had sentthe same proportion of migrants in two opposite directions but in what was considered asingle migratory movement. In such a case, an allele would have virtually travelled twice asfar as the distance covered by the migrants. The Mediterranean space is characterized byrelatively short distances and an absence of major geographic barriers (maximum sea spansare 3800 km from east to west and 900 km from north to south and the land distancefollowing the north African coastline is 5740 km) (Hofrichter 2004). This means that itcould be crossed without great difficulties by foot, horse, or rudimentary ships. The mainrestriction to human movement would have been the presence of hostile humans. Untilrecent times, sea travel would have been the most rapid and safest mode of transport.

Because we generally know less about ancient, prehistoric migratory processes than we doabout more recent processes, it is more difficult to reject hypotheses regarding earlierprocesses and discover caveats in later ones, particular when there are well documentedmigrations and information on what motivated the movement (trade, wars, matingsearches). Historic movements, however, must not be ignored, and relevant parametersrelating to known recent migrations must be estimated and added to hypotheses regardingthese movements.

Many of the genetic traits that characterize human populations everywhere are the result ofmigratory processes that have shaped the general peopling of the world. Basques, forexample, are different because they have not been effective gene sources and have aweak signature from East Asia (Calderón et al. 1998; Hellenthal et al. 2008) Berbers aredifferent because they lived in moderately small numbers in a long, narrow area bordered bythe sea and the desert and have a very high frequency of certain genetic markers (e.g.haplogroup E) which has been transmitted in low frequencies to many European popula-tions; and Andalusians from Huelva are different in that they have a considerable proportionof gene markers from the opposite Eastern edge of the Mediterranean.


Conclusions

Our analysis of the Y-chromosome haplogroup E in the native population of Huelva revealeda complex admixture of genetic markers from the Mediterranean space, with interestingsignatures of populations from the Middle East and the Balkan Peninsula and a surprisinglylow influence by Berber populations compared to other areas of the Iberian Peninsula. Theseparticular traits can, plausibly, be explained by protohistoric and other documentedhistorical movements against the backdrop of the Tartessian civilization, the rise and fallof the Roman Empire, and the different migrations associated with the expansion anddecline of Islam during the Middle Ages. We believe that an explanation based onprehistoric movements is less plausible. As a result of the magnitude of these migratorymovements, Huelva occupies a central position on the Mediterranean genetic map, despiteits location at the western edge of the Mediterranean Basin.

Acknowledgements

We express our sincere thanks to the people of Huelva who generously donated bloodsamples to contribute to this study, and also to Dr A. Fernández-Jurado from theHaematology Department and Dr E. Prado and Dr D. Fernández from the BloodTransfusion Center at Hospital Juan Ramón Jiménez in Huelva for their invaluable helpin organizing the fieldwork to collect samples, and to Dr P. Cuesta from ComplutenseComputer Center for his help with statistical analysis. This research was supported by grantsfrom the Spanish Ministry of Education and Science (Investigation ProjectsBOS2002-01677 and CGL2006-04749/BOS) awarded to RC and from the Italian Ministerodell’Istruzione, dell’Università e della Ricerca (MIUR-PRIN 2007) awarded to AN.

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