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Annals of Human Biology, January–February 2010; 37(1): 86–107
ORIGINAL ARTICLE
The Andalusian population from Huelva reveals a high
diversification of Y-DNA paternal lineages from
haplogroup E: Identifying human male movements within
the Mediterranean space
B. AMBROSIO
1
, J. M. DUGOUJON
2
, C. HERNÁNDEZ
1
,
D. DE LA FUENTE
1
, A. GONZÁLEZ-MARTÍN
1
, C. A. FORTES-LIMA
1
,
A. NOVELLETTO
3
, J. N. RODRÍGUEZ
4
& R. CALDERÓN
1
1
Departamento de Zoología y Antropología Física, Facultad de Biología, Universidad Complutense,
Madrid 28040, Spain,
2
Laboratoire d’Anthropologie, FRE 2960, Centre National de la Recherche
Scientifique (CNRS), Université Paul Sabatier, Toulouse 31073, France,
3
Dipartimento di Biologia,
Università ‘Tor Vergata’, Rome 00133, Italy, and
4
Servicio de Hematología, Hospital Juan Ramón
Jiménez, Huelva, 21005, Spain
(Received 22 April 2009; accepted 28 July 2009)
Abstract
Background: Gene flow among human populations is generally interpreted in terms of complex
patterns, with the observed gene frequencies being the consequence of the entire genetic and
demographic histories of the population.
Aims: This study performs a high-resolution analysis of the Y-chromosome haplogroup E in Western
Andalusians (Huelva province). The genetic information presented here provides new insights into
migration processes that took place throughout the Mediterranean space and tries to evaluate its impact
on 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 the
Y-chromosome Alu polymorphism (YAP). The group of YAP (+) Andalusians was genotyped for 16
Y-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 our
sample. The distribution of M81 frequencies in Iberia seems to be not concordant with the regions
where Islamic rule was most intense and long-lasting. The study also showed that most of M78 derived
allele (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 by M34 mutation, which is quite frequent
in Jews, was detected as well.
Conclusions: The haplogroup E among Western Andalusians revealed a complex admixture of genetic
markers 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, Universidad
Complutense, Ciudad Universitaria, 28040 Madrid, Spain. E-mail: rcalfer@bio.ucm.es
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 other
areas of the Iberian Peninsula.
Keywords: Y-SNPs,genealogical history,Mediterranean gene pool,Iberia,human migrations,source
populations
Introduction
Andalusia, a large and relatively densely populated region of southern Spain, has a long
history shaped by migrations from different parts of the world at different times, including a
relatively recent long Islamic settlement. Despite its implications for the peopling of the
Iberian Peninsula and its relevance to the evolution of modern Homo sapiens, the genetic
composition of the Andalusian people has never been studied in depth.
Within Andalusia, the westernmost province of Huelva is of particular interest, due to its
geographic position. It is located on the western fringe of Europe, bordering Portugal and
the Atlantic Ocean, and is also near the Strait of Gibraltar, which has served as both a
genetic barrier and corridor at different times. Its population is of moderate size and has
experienced a slow demographic growth. Furthermore, gene flow from contemporary
immigrants has been minimal, and the main features of the autochthonous population
were preserved.
The Y chromosome contains the largest non-recombining region (NRY) of the human
genome. It is a haploid locus harbouring a great deal of information that has found wide
applications 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 could
be 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 human
population (de Knijff 2000; Hammer et al. 2000; Underhill et al. 2001, Jobling and
Tyler-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 characteristics
of SNPs becomes even more evident if we consider that the NRY region, just like the
mitochondrial DNA (mtDNA) genome, has a small effective population size (approximately
one fourth that of autosomes) (Hartl and Clark 1989; Hammer et al. 1997) which enhances
the 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, and Western Asian populations, all of which have
knownhistorical relationships within theMediterranean area(Hammer etal. 1997;Rosser etal.
2000; Scozzari et al. 2001; Cruciani et al. 2002, 2004, 2007; Semino et al. 2004; Cinnio
glu et al.
2004; Roewer et al. 2005; Torroni et al. 2006 among others). Research into these topics is
providing interesting insights into demographic changes, migratory patterns, and admixture
episodes that have occurred during recent human evolution.
The polymorphic presence of an Alu element in the Y chromosome defines a deep-rooting
clade –containing haplogroups D and E –of the phylogenetic tree of the Y-chromosome
haplogroups (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 one
of the most mutationally diverse of the 20 major Y-chromosome clades. These particularities
make 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 can
therefore be used for comparison purposes. While haplogroup D seems to be confined to
Asia, 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 moderately
so 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 associated
with 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 the
Andalusian population of Huelva and to compare the lineages observed with those in other
Iberian, European, North African, and more distant Mediterranean populations. The
genetic information presented here will provide a reliable background against which to
discuss new insights into migration processes that took place throughout the Mediterranean
space –mainly during the period ranging from the protohistoric era, when the Tartessian
civilization flourished (from before 11th to 5th centuries BC), to the time of the rise and fall
of Islamic rule –and to thus evaluate the impact of these processes on the current genetic
composition 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 and
nurses from Hospital Juan Ramón Jiménez in Huelva city, accompanied by researchers (RC
and BA) from Universidad Complutense de Madrid. Participants were asked about the
origins of their parents and grandparents and their genetic relationships with other donors
contributing to this study. The sampling strategy was designed to be as representative as
possible and to include individuals from throughout the province (not including the city of
Huelva) and municipalities whose population size had remained more or less constant over
the last 2 centuries (http://www.ine.es). The municipalities chosen for sampling were
Aracena, 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 to
collect blood samples from 302 unrelated, healthy autochthonous males and females from
45 demographic units located throughout the province. Written informed consent was
obtained from all individuals prior to their participation. Additional information on the
geography, history, demography, and archaeology of the Andalusian province of Huelva and
its 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 males
described above were initially analysed for the presence/absence of the Y-chromosome Alu
polymorphism (YAP) following the recommendations of Hammer et al. (1997). Following a
descendent hierarchical order, we performed a high-resolution search of the Y-chromosome
binary 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 for
the 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 internal
lineages: 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 (+) Andalusian
males were V32, V27, V22, M107, M165, M281 and V6.
To genotype the Y binary markers, we followed standard protocols already described in
the literature using the following methods: Polymerase-chain reaction (PCR)-restriction
fragment 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 base
extension 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 using
previously published primers (Underhill et al. 2001, Cruciani et al. 2006) and their
corresponding allelic states were diagnosed by means of the restriction enzymes AciI
(also for V13), BsgI, MmeI, PvuII and MnlI, respectively following the protocol given
in 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 3730
DNA analyzer (Applied Biosystems).
Y chromosomes identified by the presence of biallelic polymorphisms (mutations) or SNPs
are called haplogroups or subhaplogroups if they are defined by a terminal mutation within a
given haplogroup. We used the nomenclature system proposed by Karafet et al. (2008) and
adopted by the Y Chromosomal Consortium to name haplogroups/subhaplogroup s defined by
N
S
ARACENA
EL REPILADO
EL CERRO DEL ANDÉVALO
Guadiana river
PUEBLA DE GUZMÀN
VILLABLANCA
Atlantic ocean
Portugal
VALVERDE DEL CAMINO
NIEBLA
HUELVA
01020 3040 50km
SEVILLA
France
Spain
Andalusia
Mediterranean sea
Portugal
Tinto river
Guadalquivir river
Odiel river
Figure 1. The geographic distribution of the municipalities sampled within the Huelva province (Andalusia, Spain).
Haplogroup E in Western Andalusia 89
the presence of a binary polymorphism. We also consulted information provided by the
International 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 16
short tandem repeat (STR) loci using the AmpFlSTRYfiler PCR amplification kit (Applied
Biosystems). 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 fluorescent
primers, an ABI3100 capillary sequencer (Applied Biosystems), internal size standards,
and GeneMapper fragment analysis. In accordance with the recommendations of the
International Society of Forensic Genetics (ISFG) (Gill et al. 2001), Y-STR alleles were
named according to the number of variable repeats included. Alleles at DYS389II were
considered 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 sampling
variance were estimated using ARLEQUIN software (versio n3.01) (Excoffier et al. 2005). The
combination of alleles at multiple SNPs defines a NRY haplogroup of alleles whereas the
combinationatmultipleY-STRsona singleY chromosomedefinesa Y-STRhaplotype(deKnijff
2000).E-M78 andE-M81 and theirassociatedmicrosatellitehaplotypeswith frequenciesof ‡5in
a set of Mediterranean population samples taken from the literature –four from Northern Africa
(Egypt (n=2),Algeria, and Tunisia); three from Southern Europe (Italy, Portugal and the study
sample) and one from Western Eurasia (Turkey) –were used to infer mutational relationships
between haplotype sequences based on five Y-STR loci (DYS19, DYS390, DYS391, DYS392,
DYS393). The phylogenetic pattern was visualized using the Reduced Median (RM) network
algorithm (NETWORK 4.5 program; http://www.fluxus-engineering.com/) (Bandelt et al.
1999). Microsatellites were weighted proportionally to the inverse of the repeat variance for
each haplogroup to reduce network reticulations.
The geographical variation for E subhaplogroups was analysed by hierarchical cluster
analysis (HCA) using the statistical program SPAD (Système Portable Pour L’Analyse de
Donnés; Lebart et al. 1984). HCA was performed on the basis of Euclidean distances and
Ward’s linkage algorithm, and analysis of variance was used to evaluate distances between
clusters. 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 Western
Asia. 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 lineages
with interesting geographic variation patterns. Populations were denoted by the first two
letters 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 of
the 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 the
published studies on the subject.
90 B. Ambrosio et al.
Table I. Population frequencies of Y-chromosome haplogroup E and its subclades.
Hg E Frequency of E subhaplogroup (%)
Populations ACRN Region nNo. % E-M35* E-M78 E-M81 E-M123+E-M34 E* (xE-M35) References
1. Spanish Basques 1 ESB1 IB 48 1 2.10 2.10 Semino et al. 2004
2. Spanish Basques 2 ESB2 IB 55 2 3.60 3.60 Cruciani et al. 2004
3. Spanish Basques 3 ESB3 IB 45 1 2.20 2.20 Underhill et al. 2000
4. Spanish Basques (Guipúzcoa) ESBG IB 74 1 1.35 1.35 Alonso et al. 2005
5. Pasiegos ESPA IB 56 24 42.90 1.80 41.10 Cruciani et al. 2004
6. Cantabrians ESCB IB 70 9 12.90 8.60 4.30 Flores et al. 2004
7. Asturians ESAT IB 90 12 13.30 10.00 2.20 1.10 Cruciani et al. 2004
8. Catalans ESCN IB 33 2 6.00 3.00 3.00 Semino et al. 2004
9. Andalusians (Huelva) ESAH IB 167 20 11.98 1.20 6.59 2.99 1.20 Present study
10. Andalusians (Cordoba) ESAC IB 27 3 11.10 3.70 7.40 Flores et al. 2004
11. Andalusians (Seville) ESAS IB 155 11 7.00 0.60 4.50 1.30 0.60 Flores et al. 2004
12. Andalusians 1 ESA1 IB 76 7 9.20 3.90 5.30 Semino et al. 2004
13. Andalusians 2 ESA2 IB 62 4 6.40 1.60 3.20 1.60 Cruciani et al. 2004
14. Tenerife ESTN IB 178 33 18.60 3.40 10.70 3.90 0.60 Flores et al. 2003
15. Mainland Portugal PTML IB 657 82 12.50 0.90 4.10 5.60 1.20 0.70 Neto et al. 2007
16. Northern Portuguese 1 PTN1 IB 50 7 14.00 2.00 4.00 4.00 4.00 Cruciani et al. 2004
17. Northern Portuguese 2 PTN2 IB 109 16 14.60 6.40 5.50 2.70 0.90 Flores et al. 2004
18. Southern Portuguese PTSU IB 49 9 18.30 4.10 12.20 2.00 Cruciani et al. 2004
19. Azores Islands PTAZ IB 319 42 12.90 1.30 6.10 3.70 0.90 0.90 Neto et al. 2007
20. French FR WEU 85 7 8.20 4.70 3.50 Cruciani et al. 2004
21. French Bearnais FRBN WEU 27 1 3.70 3.70 Semino et al. 2004
22. French Basques FRB1 WEU 45 0 0.00 Semino et al. 2004
23. Corsicans FRCO WEU 140 9 6.40 0.70 4.30 1.40 Cruciani et al. 2004
24. Sardinians 1 ITSD1 WEU 367 37 10.00 1.10 3.50 0.30 3.50 1.60 Cruciani et al. 2004
25. Sardinians 2 ITSD2 WEU 139 7 5.00 0.70 2.90 1.40 Semino et al. 2004
26. Danish DK WEU 35 1 2.90 2.90 Cruciani et al. 2004
27. Dutch NL WEU 34 0 0.00 Semino et al. 2004
28. Northern Italians ITN CEU 67 8 12.00 9.00 1.50 1.50 Cruciani et al. 2004
29. North-Central Italians ITNC CEU 56 6 10.70 10.70 Semino et al. 2004
Haplogroup E in Western Andalusia 91
Table I (Continued)
Hg E Frequency of E subhaplogroup (%)
Populations ACRN Region nNo. % E-M35* E-M78 E-M81 E-M123+E-M34 E* (xE-M35) References
30. Central Italians ITC CEU 89 12 13.40 11.20 2.20 Cruciani et al. 2004
31. Southern Italians ITS CEU 87 12 13.80 11.50 2.30 Cruciani et al. 2004
32. Southern Italians (Calabria 1) ITCL1 CEU 80 18 22.70 1.30 16.30 1.30 2.50 1.30 Semino et al. 2004
33. Southern Italians (Calabria 2) ITCL2 CEU 68 16 23.50 1.50 5.90 13.20 2.90 Semino et al. 2004
34. Southern Italians (Apulia) ITAP CEU 86 12 13.90 11.60 2.30 Semino et al. 2004
35. Sicilians 1 ITSY1 CEU 136 29 21.30 14.00 0.70 6.60 Cruciani et al. 2004
36. Sicilians 2 ITSY2 CEU 55 15 27.30 5.50 12.70 5.50 3.60 Semino et al. 2004
37. Mainland Croatia HRML EEU 108 6 5.60 5.60 Peri
cic´ et al. 2005
38. Herzegovinians BA EEU 141 12 8.50 8.50 Peri
cic´ et al. 2005
39. Serbians RS EEU 113 24 21.25 20.35 0.90 Peri
cic´ et al. 2005
40. Macedonians MK EEU 79 19 24.06 24.06 Peri
cic´ et al. 2005
41. Polish 1 PL1 EEU 99 4 4.00 4.00 Semino et al. 2004
42. Polish 2 PL2 EEU 38 1 2.60 2.60 Cruciani et al. 2004
43. Hungarians HU EEU 53 5 9.40 7.50 1.90 Semino et al. 2004
44. Estonians EE EEU 74 4 5.50 1.40 4.10 Cruciani et al. 2004
45. Russians RU EEU 42 0 0.00 Cruciani et al. 2004
46. Ukrainian UA EEU 93 8 8.60 7.50 1.10 Semino et al. 2004
47. Georgian GE EEU 41 0 0.00 Semino et al. 2004
48. Balkarian (Southern Caucasus) RUBK EEU 39 1 2.60 2.60 Semino et al. 2004
49. Bulgarians BG EEU 116 25 21.60 20.70 0.90 Cruciani et al. 2004
50. Albanians AL1 EEU 44 11 25.00 25.00 Semino et al. 2004
51. Northern Greeks (Macedonia) GRMA EEU 59 12 20.30 18.60 1.70 Semino et al. 2004
52. Greeks GR EEU 84 20 23.80 21.40 2.40 Semino et al. 2004
53. Turkish (Edirne) TRED EAS 52 8 15.38 11.54 3.85 Cinnio
glu et al. 2004
54. Turkish (Kars) TRKA EAS 82 12 14.63 8.54 6.10 Cinnio
glu et al. 2004
55. Turkish (Konya) TRKO EAS 90 8 8.89 1.11 1.11 6.67 Cinnio
glu et al. 2004
56. Turkish (Istanbul) TRIS EAS 81 13 16.05 7.41 4.94 3.70 Cinnio
glu et al. 2004
57. Iraqi IQ MDE 218 20 9.20 5.50 2.80 0.90 Semino et al. 2004
58. Lebanese LB MDE 42 8 19.10 11.90 2.40 4.80 Semino et al. 2004
92 B. Ambrosio et al.
Table I (Continued)
Hg E Frequency of E subhaplogroup (%)
Populations ACRN Region nNo. % E-M35* E-M78 E-M81 E-M123+E-M34 E* (xE-M35) References
59. Ashkenazim Jewish ILA MDE 77 14 18.20 1.30 5.20 11.70 Semino et al. 2004
60. Sephardim Jewish ILS MDE 40 12 30.00 2.50 12.50 5.00 10.00 Semino et al. 2004
61. Moroccan (Arabs) 1 MAA1 NAF 54 39 72.30 38.90 31.50 1.90 Cruciani et al. 2004
62. Moroccan (Arabs) 2 MAA2 NAF 49 37 75.50 42.90 32.60 Semino et al. 2004
63. Moroccan (Arabs) 3 MAA3 NAF 44 32 72.80 2.30 11.40 52.30 6.80 Semino et al. 2004
64. Moyen Atlas (Berbers) MABA NAF 69 60 86.90 10.10 71.00 5.80 Cruciani et al. 2004
65. Marrakesh (Berbers) MABM NAF 29 26 89.50 3.40 6.90 72.40 3.40 3.40 Cruciani et al. 2004
66. Morocco (Berbers) MAB NAF 64 55 85.90 10.90 68.70 6.30 Semino et al. 2004
67. Morocco (North-
Central Berbers)
MABN NAF 63 55 87.30 7.90 1.60 65.10 12.70 Semino et al. 2004
68. Morocco (Southern
Berbers)
MABS NAF 40 35 87.50 7.50 12.50 65.00 2.50 Semino et al. 2004
69. Saharawish EH NAF 29 24 82.70 75.90 6.80 Semino et al. 2004
70. Algerians DZ NAF 32 21 65.60 3.10 6.30 53.10 3.10 Semino et al. 2004
71. Tunisians TN NAF 58 32 55.10 3.40 15.50 27.60 5.20 3.40 Semino et al. 2004
72. Northern Egyptians EGN NAF 21 8 38.20 28.60 4.80 4.80 Cruciani et al. 2004
73. Southern Egyptians EGS NAF 34 6 17.60 17.60 Cruciani et al. 2004
Haplogroup E in Western Andalusia 93
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 to
other Spanish populations. The corresponding value in neighbouring southern Portugal is
18% (Cruciani et al. 2004). Most of the Y chromosomes analysed in the Andalusian
population 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 is
particularly frequent in sub-Saharan Africa (Underhill et al. 2000; Cruciani et al. 2002), was
not observed in our population, although carriers of this subclade have been detected in
mainland Portugal and the Azores Islands with higher frequencies (0.2–0.9% of total Y
chromosomes) (Beleza et al. 2005; Gonçalves et al. 2005; Neto et al. 2007) than those
detected in other European populations.
A comparative analysis of the E haplogroups detected in the Andalusian population with
frequencies reported for the population data set in Table I revealed a number of interesting
findings. The E-M81 clade (five occurrences) accounted for 3% of the total haplogroup E
frequency 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*
V12
V32
V27
V13
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 of
the autochthonous population of the Huelva province.
94 B. Ambrosio et al.
this frequency is similar to the mean value observed in Basques (Alonso et al. 2005) and
lower 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 the
E-M81 lineages in Mediterranean populations has shown these lineages to be remarkably
frequent 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 there
was an Islamic occupation that lasted for over two centuries (878–1091 AD). Presuming that
the presence of E-M81 in southwestern Europe is a signature of a North-African gene pool
shared also by Berbers, these migrants seem to have left a much smaller genetic imprint in
the 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 allele
were further classified into the E-V13 subhaplogroup (E1b1b1a2). The frequency of E-V13 in
our study population was relatively high (seven occurrences, 4.2% of total) in comparison with
other Iberian populations. In North Africa, this subhaplogroup shows a mean frequency of
4.5%, as reported by Cruciani et al. (2007) (see Table I), and is indeed most common in
Albanians (32.30%), Macedonians, Greeks, and Bulgarians (15–18%) (Peri
cic´ et al. 2005). In
our analysis of the subclade E-M78, we also found 2 (1.2%) Y chromosomes in the most basal
and rare paragroup, E-M78*, one (0.60%) in E-V12, and, interestingly, a single occurrence of
E-V65 (0.60%). The average frequency of E-M78* has been estimated at 0.08% (Cruciani
et al. 2007) (see Table I) and the highest values have been registered in Egyptians from Gurna
(5.9%), followed by Moroccan Arabs (3.6%) and Sardinians (0.27%). No occurrences of this
subhaplogroup were found in any of the other 81 populations included in the analysis
by Cruciani et al. (2007). The subhaplogroup E-V12 has also been found in high frequencies
in Egyptians (ranging from 44% in the south to 6% in the north) and in Berbers (3.5%) and
Moroccan Jews (2%). The surprisingly high frequency observed in French Basques (6%, one
occurrence) 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 reported
by Cruciani et al. (2007) in Table I. E-V65 is relatively frequent in Arabs from north Morocco
(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 in
1.2% of the Huelvan sample (two occurrences). The E-M34 lineage has been found at
relatively high frequencies in Jews (10–12%) and in a sample from Calabria (13%). The
corresponding 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 to
carry 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*, formerly
E3b*) 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 Moroccan
Berbers, 5.5% for Sicilians, 3% for Algerians and Tunisians, 2.5% for Sephardic Jews, and
1.3% for Ashkenazi Jews (see references in Table I of present study).
Frequency and structure of Y-STR haplotypes associated with each E binary markers
detected in the study Andalusian population sample is shown in Table II. The analysis
initially revealed a high haplogroup diversity for the E-M35 clade (h=0.8211 ±0.06) being
the subclade E-M78 that yielding a rather high level of SNP h(0.5906 ±0.15). The subclade
E-M78 also revealed a high level of internal Y-STR diversity (H=0.9818 ±0.05), with a
Haplogroup E in Western Andalusia 95
Table II. Distribution of Y-chromosome haplotypes among the subhaplogroups E found in Huelva (Spain).
Haplotypes Haplogroups DYS19 DYS389I DYS389II DYS390 DYS391 DYS392 DYS393 DYS385a,b DYS438 DYS439 Frequency
H1 E1b1b1*- M35 14 13 30 22 9 11 14 12,13 10 11 1
H2 E1b1b1*- M35 14 13 30 22 9 11 14 13,13 10 11 1
H3 E1b1b1a*- M78 14 14 32 24 10 11 13 18,21 10 13 1
H4 E1b1b1a*- M78 15 14 31 25 11 11 13 16,20 10 12 1
H5 E1b1b1a1*- V12 13 13 31 24 11 11 13 16,17 10 12 1
H6 E1b1b1a2*- V13 13 13 30 24 10 11 12 17,19 10 12 1
H7 E1b1b1a2*- V13 13 13 30 24 10 11 13 16,17 10 14 1
H8 E1b1b1a2*- V13 13 13 30 24 10 11 13 16,18 10 12 1
H9 E1b1b1a2*- V13 13 13 30 24 10 11 13 16,18 10 13 2
H10 E1b1b1a2*- V13 13 13 30 25 10 11 13 16,18 10 14 1
H11 E1b1b1a2*- V13 13 13 31 23 10 11 13 17,18 10 12 1
H12 E1b1b1a4- V65 13 12 29 24 11 11 13 16,17 10 10 1
H13 E1b1b1b*- M81 13 14 30 24 9 11 13 13,14 10 10 4
H14 E1b1b1b*- M81 14 14 30 24 9 11 13 13,14 10 10 1
H15 E1b1b1c1*- M34 13 13 30 24 11 11 13 15,16 10 13 1
H16 E1b1b1c1*- M34 13 13 31 24 10 11 13 16,16 10 12 1
96 B. Ambrosio et al.
mean variance of 0.3933 ±0.24. Conversely, the haplotype variability within the sister
subclade E-M81 was much lower (0.4000 ±0.24): Four of the five males carrying the M81
marker 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). Within
E-M81* the fifth haplotype differed only in one mutational step at the DYS19 locus (13- to
14–repeat alleles). Analysis of variance at single Y-microsatellite loci showed that the
DYS390 locus yielded the highest value (0.70); the corresponding figure for DYS19 was
0.33, with the 13–repeat allele being the most frequent (15 of the 20 YAP+ chromosomes
detected in the study sample). An in-depth analysis of the relationship between
haplogroup E frequency and associated haplogroup/haplotype diversities within and between
large geographic areas in the Mediterranean would provide interesting insights into pop-
ulation demographic histories.
The RM network of E-M78 microsatellite haplotypes in a group of Mediterranean samples
available in the literature is shown in Figure 3. The population data set included a total of 36
distinct haplotypes. The structure of the haplotype network is highly diversified, which is to be
expected given the haplogroup substructure that it includes. The most common node (68 of
160 Y chromosomes) is represented by the modal haplotype 13-24-10-11-13 (for the loci
DYS19-DYS390-DYS391-DYS392-DYS393, respectively), shared by different populations
in mainland Italy, Turkey, and Portugal and to a lesser extent by southern Egyptians, Algerian
Arabs, and Andalusians from Huelva (four chromosomes) (for more details see the legend
to 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 and Templeton 1993). The longest lineage is composed of seven nodes, separated by
single mutational differences. Huelvan Y-haplotypes fall into three nodes (one specific copy in
each), which contain, in varying combinations, most of the populations noted above and other
neighbouring Mediterranean groups. Within this lineage, the third node (represented by the
Y-STR haplotype 14-24-10-11-13) is represented by males from northern and southern Egypt,
Italy, and Andalusia. We also detected one singleton (haplotypes represented by a single
individual) in the study sample. Haplotypes showing high frequencies are expected to be good
indicators of when a particular mutation originated, whereas those with low frequencies (rare
haplotypes), usually represented by a single individual, point to a recent evolutionary origin.
Indeed they occur preferentially at the tips of networks (Golding 1987; Excoffier and Langaney
1989). An RM E-M81 network for the same samples was also constructed but is not shown
here. The most common node for E-M81 (61 of 126 chromosomes) is represented by the
modal haplotype 13-24-9-11-13; this coincided with the four identical Y chromosomes
detected in Huelva and is shared –in variable frequencies –by most (n=8) of the population
samples analysed.
Figure 4 portrays the results of HCA based on frequencies of the E-M35 haplogroup and
its main subclades within the data set of populations shown in Table I. Factors I and II
account for 97.81% of the variance, with a predominance of factor I (83.83%). The
multivariate analysis formed six distinct clusters, with E-M78 and E-M81 haplogroups
contributing significantly to the pattern observed. With the aim of giving more support to the
number of clusters (six) suggested by the dendrogram, it is interesting to note that when
computing 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 of
major ramifications showed by the tree, and it demonstrates that with six clusters a high
percentage (96%) of the data variation is explained. Clusters C1,C2, and C3 on the right side
of the plot are formed by European, Middle Eastern, and western Eurasian populations,
Haplogroup E in Western Andalusia 97
whereas clusters C4 and C6 on the upper left and lower sides of the bidimensional space, and
C5 in an intermediate position, include populations almost exclusively from northern Africa.
The genetic map clearly evidences the combined effect of interactions between different
evolutionary processes during the coalescence time of these populations for the major E
haplogroup.
Andalusians from Huelva (ESAH) are included in C2, which is distinguished by a high mean
cluster 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 18
populations that shape this cluster are Asturians from northern Spain, most (9 of 11) of the
Italian 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 of
the HCA plot also reveals a genetic affinity of Huelva with several populations located within
cluster C1 such as Portuguese from the Azores Islands and northern mainland Portugal,
Huelva
Italy
Portugal
Turkey
Algeria
North Egypt
South Egypt
Tunisia
Figure 3. Reduced median (RM) microsatellite haplotype network for E-M78 haplogroup based on five Y-STRs
loci (DYS19, DYS390, DYS391, DYS392, DYS393). Circles are proportional to the number of individuals sharing
that 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
98 B. Ambrosio et al.
Andalusians (in general), Andalusians from Cordoba, and French. The genetic topology of
Huelva closely agrees with the results of an earlier analysis of GM immunoglobulin allotypes
on 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
GR
BG
RS
GRMA
EGS
ITSY1
LB
ITC
ESAT
ESAH
PTAZ
ESAS ESB2
ESB3
ESCN
ESB1 ESBG
EGN
AL1
MK
GR
BG
RS
GRM
EG
ITCL1
ITSY1
TRED
LB
ILS
ITSY2
ITN
ESA
ITC
TRK
A
IT BA
TRIS
UA
HRML
FR
PTN2
ESAH
C2
C3
M123/M34
PTML
PTAZ
PTN
ITSD1 IL
ITCL
ITSD
ESBG
FRB
ESB1
ESB2
ESB3
ESAS
ESCB
E*(xE-M35)
M35 ESAC ESA1
C1
ESTN
PTSU
TRK
ESA
GE
ITA IT
TRKO
ESA3
RUNL
FRB1
PL2
RUBK GE
ITSD2
ITSD1
PTN1
PL1
EE
FRBN
FR
ESA1
PTML
ESAC
ESCB
ESTN
PTSU
7.5
0
-7.5
-15.0
Factor 2-13.98%
-60 -45 -30 -15 0
PTN2
ITCL1
ITSY2 ILS
C3
C2
C1
ITN
TRED
ITAP
ITS
ITNC
TRKA
BA
HUUA
ILA
TRIS
ITCL2
HRMLIQ
FRCO
DK
MAA3
DZ
ESPA
TN
C5
MABS
MAB
MABA
MABM
M81
C6
EH MABN
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 Northern
African populations based on Y-chromosomal diversity of E-M35 subhaplogroups (M35, M78, M81, M123+M34
and E*(xE-M35)). (b) A zoom plot of clusters C1, C2 and C3. Population codes are as in Table I.
Haplogroup E in Western Andalusia 99
Turks from Konia, Ashkenazi Jews, and Iraqis. The populations in C1 are distinguished by
low frequencies of all the E-M35 lineages considered (test value range, -1.79 to -5.36; p
value range, 0.037–0.000). Interestingly, haplogroup E is absent from other populations in
this cluster, such as the Georgians, the Russians, the Dutch, and the French Basques, and is
very infrequent in Spanish Basques, Danes, Poles, and people from the Balkans. These
findings suggest the existence of a prehistoric migration corridor following the arching plains
of 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 M78
haplogroup (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 a
sample 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 be
found in the lower part of the plot. This cluster is made up of two Moroccan Arab
populations who, in contrast to neighbouring Maghreb populations, would have originated
from somewhere in Arabia or the Fertile Crescent and had relatively little contact with
Berber populations.
Cluster C5, on the contrary, is characterized by high frequencies of E-M81 and the
paragroup 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 the
Pasiega region in Cantabria, northern Spain. The abnormal presence of this northern
Spanish 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 a
strong Arab component in the north African populations in C5, it is not as strong as that
observed in C4.
Finally, cluster C6 has the highest frequencies of E-M81, E-M35* and E*(xE-M35) (test
value range, 3.93–7.19; p=0.000). This cluster is formed by six populations, including five
Moroccan Berbers populations and a Saharawi population. Cluster 6, therefore, is the group
that best defines the current Berber population.
Discussion
The geographic position of Huelva, located at the southwestern fringe of Europe, and the
fact that haplogroup E –the focus of the present study –appears to have originated in East
Africa and is not frequent in this part of Europe prompt us to consider that the native
population of Huelva received these genes from elsewhere. In this section, we will explore
different hypotheses that might shed some light on who these populations were and where
they came from.
The times to the most recent common ancestors (TMRCA) of the main haplogroups in
the E clade observed in Huelva, E-M78 and E-M81, were estimated at 18.6–4.3 thousand
years 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 close
to the beginning of the Neolithic age (5.6 kya). The estimated time of coalescence for E-V65
is more recent (~4.5 kya), and would have coincided with the beginning of the Early Bronze
Age in Iberia. The TMRCA for the paragroups E-M35* and E-M78* would be some time
before the Last Glacial Maximum (~18 kya). It should be noted that alleles (mutations) must
be present in high frequencies in order to be transferred effectively (i.e. in notable
frequencies) by migrants to other populations. This is why effective gene flow will only
occur quite some time after the TMRCA. Consequently, migration linked to the movement
100 B. Ambrosio et al.
of males with E-M35 lineages should be situated within a relatively recent evolutionary
period. In this context, it should be noted that TMRCA estimates are dependent on many
assumptions about mutational processes and population structures (Hein et al. 2005).
The wide but non-homogeneous spatial distribution pattern of E-M81 chromosomes in
Iberia does not seem to be concordant with the regions in which Islamic occupation was
most intense and prolonged (Lopez-Davalillo 2000; Martinez-Ruiz et al. 2003). This would
strengthen the hypothesis that migratory movements took place between Maghreb and Iberia
prior to the Islamic occupation, and those other important movements within the peninsula
occurred later (Calderón 2006). The Islamic occupation of western Andalusia lasted from
711 to 1262 and 5 years later Portugal and Castile (Spain) agreed on the southern border
dividing their kingdoms. The Berbers, who arrived in several waves, were the most
numerous of all the migrant populations that arrived in Iberia during Islamic rule. It has
been estimated that around one-third of the 300 000 Berbers that arrived during these years
did so in the eighth century (Mackay 1977), a time when the total population of the
peninsula has been estimated at between 6 and 7 million (Dupaquier 1997). The Berbers
tended to settle in the mountainous areas of the peninsula and, interestingly, most of them
were men of reproductive age, many of whom came with their wives, also Berbers in many
cases. Some of the descendants of these early occupiers were to return to Maghreb between
1264 and 1609. There are several relevant historical contexts preceding the Islamic
occupation that are likely to have had a considerable impact on population dynamics in
the Mediterranean area and in the Iberian Peninsula in particular. These were (i) the
existence of a Berber gene flow associated with the Carthaginian period, which included a
portion of north African natives; (ii) the establishment of the Roman Empire on both sides of
the Strait of Gibraltar, which considerably improved communication and safety throughout
the Empire; and (iii) the considerable and lasting difference in population sizes between
Europe and North Africa (always higher in Europe), which would explain the low North
African contribution to the gene pool in Europe, although the Berbers did leave a genetic
imprint in numerous locations in Europe due to the remarkably high frequency of E-M81 in
this population.
The levels of E-V13* detected in our study population could perhaps be explained by
possible contact with populations that would have travelled by sea from areas under Greek
control during the protohistoric period, when the Kingdom of Tartessos gained strategic
importance thanks to its extraordinarily rich deposits of copper, silver, and tin. Abundant
remains 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 contacts
with 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 the
Greeks who performed long voyages, and it was they who made the Greeks acquainted with
the Adriatic and with Tyrrhenia, with Iberia, and the city of Tartessos. The vessel which they
used 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 and
settle in whatever part of his country they liked’(Placido 1999). The existence of trade
relations across the Mediterranean Sea therefore seems to be a more plausible explanation
for 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
cic´
et al. (2005) and others have suggested. Nonetheless, a small proportion of the E-V13
Y-chromosomes found in Huelva might have arrived much later, through the Visigoths, who
travelled to Hispania after 411 AD from the northern region of the Black Sea and the
Balkans.
Haplogroup E in Western Andalusia 101
The presence of Y chromosomes E-M78* and E-V12* might be due to an Egyptian gene
flow, which is supported by historical evidence. Around 742 AD, a large army led by the
Syrian Balch arrived in the Iberian Peninsula to suppress a Berber rebellion against the
Arabs. The army was made up of several contingents (chunds) from different Islamized
regions of the Levant and Egypt. Following the defeat of the Berbers, the troops were
separated according to their regions of origin and sent to different parts of the south of the
peninsula. Part of the Egyptian contingent settled in the district of Beja (today a Portuguese
town 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. Because
Arabs also settled in Maghreb after the Islamic conquest of this region, the E-V65
chromosomes in Huelva might have come directly from the Middle East, without necessarily
having passed through an intermediate North African reproductive stage. It has been
estimated that the first influx of Arabs into the Iberian Peninsula numbered 30 000–40
000, a relatively small number and substantially inferior to that of the Berbers. This first
group of Arabs consisted of two rival tribes: The Qaysi, from the north of the Arabian
Peninsula, 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 Visigothic
nobility, avoiding marriage with Berbers. Many were rich and powerful and, interestingly,
they practised polygamy, which would have had a multiplier effect on chromosome Y
lineages. By way of example, the 10 emirs and caliphs that governed Cordoba between 756
and 1013 had at least 143 sons (who did not die prematurely) who had male offspring
ranging from 40 to none (Vallvé 1977).
Following the Reconquista of western Andalusia, a considerable proportion of the Muslim
population, who had lived there for over 5 centuries, left these lands for Granada or
Morocco. Later, during the 14th and 15th centuries, about 400 000 Muslims (»4.5% of the
total population) abandoned the peninsula, and in 1609, with the passing of the decree to
expel 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 thus
have remained a long period of time in the Iberian Peninsula. As far as the E-M35 is
concerned, populations carrying this mutation from East Africa would presumably have
migrated to southwestern Andalusia after an intermediary settlement period alongside
Maghreb Berbers during the Islamic occupation. The frequencies of this mutation observed
in Jewish populations could be the result of previous contact with populations from the
Middle 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-M34
subclade 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 both
extremes of the Mediterranean dating back many centuries. In Andalusia, there were Jewish
communities 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 to
frequencies of a particular genetic marker in a recipient population. Firstly, the frequencies
of the marker in the source population and the size of the migrating population in relation to
that of the recipient population must be high enough in order not to excessively dilute the
gene flow. When testing such a hypothesis, thus, it is necessary to analyse the demographic
sizes 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 and
Pääbo 2004), substantial initial differences in frequencies are generally found between
102 B. Ambrosio et al.
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. a
frequency that lasts and is easy to detect) in the recipient population a long time after the
mutation event occurred in the source population. Occasional, motivated group migrations
therefore are more plausible and genetically effective than small persistent, gene flows.
A recipient population can receive a particular gene directly from the source population or
through 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 the
movement 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 a
few 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 to
have an effect on the spread of gene in the next stage; and secondly, there is a progressive
reduction of gene frequencies at each intermediate stage (population) governed by the
decreasing power law, f
i
=k
i
f
o
, where f
o
is the marker frequency in the original source
population, f
i
the marker frequency at each stage i, and kthe roughly constant fraction of
migrants at each stage. For example, with k=0.25 and i=3, f
3
=0.156 f
o
. Consequently, just
a few stages are necessary to reduce gene frequency in the final recipient population to a level
of close to zero, regardless of the frequency of the marker in the original source population. It
would therefore seem logical to reject hypotheses involving migrations that occur in several
stages because the final effect in the recipient population would be negligible.
Populations that have not experienced direct gene flow might have similar proportions of a
particular genetic marker if a source population, which might even be now extinct, had sent
the same proportion of migrants in two opposite directions but in what was considered a
single migratory movement. In such a case, an allele would have virtually travelled twice as
far as the distance covered by the migrants. The Mediterranean space is characterized by
relatively short distances and an absence of major geographic barriers (maximum sea spans
are 3800 km from east to west and 900 km from north to south and the land distance
following the north African coastline is 5740 km) (Hofrichter 2004). This means that it
could be crossed without great difficulties by foot, horse, or rudimentary ships. The main
restriction to human movement would have been the presence of hostile humans. Until
recent 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 do
about more recent processes, it is more difficult to reject hypotheses regarding earlier
processes and discover caveats in later ones, particular when there are well documented
migrations and information on what motivated the movement (trade, wars, mating
searches). Historic movements, however, must not be ignored, and relevant parameters
relating to known recent migrations must be estimated and added to hypotheses regarding
these movements.
Many of the genetic traits that characterize human populations everywhere are the result of
migratory processes that have shaped the general peopling of the world. Basques, for
example, are different because they have not been effective gene sources and have a
weak signature from East Asia (Calderón et al. 1998; Hellenthal et al. 2008) Berbers are
different because they lived in moderately small numbers in a long, narrow area bordered by
the 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 proportion
of gene markers from the opposite Eastern edge of the Mediterranean.
Haplogroup E in Western Andalusia 103
Conclusions
Our analysis of the Y-chromosome haplogroup E in the native population of Huelva revealed
a complex admixture of genetic markers from the Mediterranean space, with interesting
signatures of populations from the Middle East and the Balkan Peninsula and a surprisingly
low influence by Berber populations compared to other areas of the Iberian Peninsula. These
particular traits can, plausibly, be explained by protohistoric and other documented
historical movements against the backdrop of the Tartessian civilization, the rise and fall
of the Roman Empire, and the different migrations associated with the expansion and
decline of Islam during the Middle Ages. We believe that an explanation based on
prehistoric movements is less plausible. As a result of the magnitude of these migratory
movements, Huelva occupies a central position on the Mediterranean genetic map, despite
its location at the western edge of the Mediterranean Basin.
Acknowledgements
We express our sincere thanks to the people of Huelva who generously donated blood
samples to contribute to this study, and also to Dr A. Fernández-Jurado from the
Haematology Department and Dr E. Prado and Dr D. Fernández from the Blood
Transfusion Center at Hospital Juan Ramón Jiménez in Huelva for their invaluable help
in organizing the fieldwork to collect samples, and to Dr P. Cuesta from Complutense
Computer Center for his help with statistical analysis. This research was supported by grants
from the Spanish Ministry of Education and Science (Investigation Projects
BOS2002-01677 and CGL2006-04749/BOS) awarded to RC and from the Italian Ministero
dell’Istruzione, dell’Università e della Ricerca (MIUR-PRIN 2007) awarded to AN.
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