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A grave in my garden. Genetic identification of
Spanish civil war victims buried in two mass
graves in Espinosa de los...
Article in Forensic Science International Genetics Supplement Series · September 2015
DOI: 10.1016/j.fsigss.2015.09.133
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G Model
FSIGSS 1130 No. of Pages 3
Forensic Science International: Genetics Supplement Series xxx (2015) xxx–xxx
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ScienceDirect
Forensic Science International: Genetics Supplement Series
journal homepage:
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A grave in my garden. Genetic identification of Spanish civil war victims
buried in two mass graves in Espinosa de los Monteros (Burgos, Spain)
Carolina Núñez
a
, Miriam Baeta
a
, Leire Palencia-Madrid
a
, Lourdes Herrasti
b
,
Francisco Etxeberria
b
, Marian M. de Pancorbo
a,
*
a
b
BIOMICs Research Group, Lascaray Ikergunea Center of Research, University of the Basque Country UPV/EHU, Vitoria-Gasteiz, Spain
Departamento de Antropología, Sociedad de Ciencias Aranzadi, Alto de Zorroaga, 20014 Donostia-San Sebastián, Spain
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 30 July 2015
Accepted 18 September 2015
Available online xxx
Keywords:
Spanish Civil War
Mass grave
Genetic identification
We report the genetic study of 13 victims of the Spanish Civil war buried in two mass graves in Espinosa
de los Monteros (Burgos, Spain). The grave with the highest number of individuals (grave 1;
N
= 9) was
found in the garden of a private house of the village. The other grave (grave 2) was only 200 m away and
the remains of four women were unexpectedly found. Skeletal remains, mainly teeth, were better
preserved in graves 1 than 2. Mean DNA extraction yield was 0.17 ng/
m
l. In order to carry out the genetic
identification of the remains, buccal swabs from 11 family relatives were available. The choice of genetic
markers analyzed relied on the different levels of biological kinship to be elucidated. For 75% of the post-
mortem samples,
!12
autosomal STR profiles were obtained. Y chromosome STRs, as well as
mitochondrial DNA control region, were also analyzed in order to study paternal and maternal lineages,
respectively. Besides, X chromosome STRs were typed in one case to verify a paternal grandmother–
granddaughter relationship. We successfully identified 9 of 13 individuals buried in the two mass graves
of Espinosa de los Monteros, 6 in grave 1 and 3 in grave 2. The search of further relatives’ samples would
be of interest to perform more comparative analyses, and eventually do not leave any of the victims
without a name.
ã
2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
In the last decade, DNA analyses have played a decisive role in
the identification of victims found in mass graves from the Spanish
Civil War and posterior dictatorship
[1–2].
About 300 of the more
than 2000 mass graves located along the Spanish territory have
been excavated, with approximately 5000 bodies found
[3].
Most
of these burial sites were placed in isolated or outskirts areas such
as forest pits, wells, caverns, or hills. However, due to the long time
elapsed since the end of the conflict and the urban development;
some burial sites have disappeared, or are now engulfed beneath
the concrete, roads or edifications. Indeed, a garden of a private
house in the village of Espinosa de los Monteros (Burgos, Spain),
was recently uncovered as a hideout of an eight meters square
grave, which included nine different skeletons. Additionally,
another grave was unexpectedly discovered only 200 m away
with the remains of four women. Here we present the results of the
genetic analysis of the 13 skeletal remains found in Espinosa de los
Monteros’ graves and the comparison with alleged family
members in order to establish the identity of the victims.
2. Material and methods
A total of 13 human remains (nine teeth and four fragments of
femur) recovered from two graves of the Spanish Civil War and
posterior dictatorship, located in Espinosa de los Monteros (Burgos,
Spain), were analyzed (grave 1
N
= 9; grave 2
N
= 4). Recommenda-
tions for work with ancient DNA (aDNA) were followed
[4].
DNA
extraction was performed as described in
[5]
with minimal
modifications. DNA was quantified using Quantifiler
TM
Human
DNA Quantification kit (AB/LT/TFS: Applied Biosystems
TM
, Life
Technologies, ThermoFisher Scientific, Waltham, MA, USA).
Buccal swabs of 11 relatives were obtained under informed
consent, following the ethical standards of the Helsinki Declara-
tion. DNA was extracted using Gentra Puregene System (Qiagen)
and quantified by NanoDrop spectrophotometer (Thermo Fisher
Scientific, Wilmington, DE).
Austosomal STRs amplification was performed using
AmpFlSTR
1
NGM SElect
TM
PCR Amplification kit (NGMSE, AB/
* Corresponding author at: BIOMICs Research Group, University of the Basque
Country UPV/EHU, Avda. Miguel de Unamuno, 3, 01006 Vitoria-Gasteiz, Spain.
Fax: +34 945 01 4458.
E-mail address:
marian.mdepancorbo@ehu.es
(M.M. de Pancorbo).
http://dx.doi.org/10.1016/j.fsigss.2015.09.133
1875-1768/
ã
2015 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: C. Núñez, et al., A grave in my garden. Genetic identification of Spanish civil war victims buried in two mass
graves in Espinosa de los Monteros (Burgos, Spain), Forensic Sci. Int. Gene. Suppl. (2015),
http://dx.doi.org/10.1016/j.fsigss.2015.09.133
G Model
FSIGSS 1130 No. of Pages 3
e2
C. Núñez et al. / Forensic Science International: Genetics Supplement Series xxx (2015) xxx–xxx
Table 1
Individuals for each grave, kinship relationship regarding to the living relative, genetic markers analyzed in the present study and total LR values. N/A: not analyzed; Ø: no
p
genetic result; X: no match to the living relative; : favorable for identification.
Grave
Grave 1
Individual
IND01
IND02
IND03
IND04
IND05
IND06
IND07
IND08
IND09
IND01
IND02
IND03
IND04
Relationship
Unknown
Paternal grandfather
Unknown
Maternal uncle
Paternal uncle
Father
Unknown
Father
Brother
Unknown
Paternal grandmother
Maternal grandmother
Mother
Autosomal STRs
X
p
Ø
p
p
p
X
p
Ø
Ø
p
N/A
Ø
Y-STRs
X
p
N/A
X
p
p
X
p
Ø
N/A
N/A
N/A
N/A
X-STRs
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
p
N/A
N/A
mtDNA
X
N/A
X
p
N/A
N/A
X
X
p
N/A
N/A
p
p
Total LR
–
6.6
Â
10
7
–
1.1
Â10
5
1.2
Â
10
3a
2.5
Â
10
17
–
5.7
Â
10
6
6.6
Â
10
2a
–
3.4
Â
10
4
2.2
Â
10
4
2.5
Â
10
3a
Grave 2
a
Anthropology evidences were crucial when LR values were not sufficiently high in these cases.
LT/TFS) or MiniFiler
TM
PCR Amplification Kit (Minifiler, AB/LT/TFS)
in skeletal samples; and I-DNASE21
[6]
system in reference
samples. For Y-STRs analysis, AmpFISTR Yfiler PCR Amplification
Kit (YFiler, AB/LT/TFS) or PowerPlex
1
Y23 System (PPY23, Promega
Corporation, WI, USA) kits were used. X-STRs were studied with
the GHEP-ISFG decaplex
[7].
MtDNA hypervariable segments I and
II (HVS-I and HVS-II) in the skeletal samples were analyzed, and
the complete D-loop was analyzed in reference samples as
described in
[8].
Electrophoresis was conducted on an ABI Prism
3130 Genetic Analyzer (AB/LT/TFS). GeneMapper
1
Software v.4.0
(AB/LT/TFS) was used for allele designation. MtDNA sequences
were edited as
[8].
Statistical kinship calculations based on Likelihood ratios (LRs)
for autosomal STRs were carried out using GFF v.2.55
[9],
using
allele frequencies of the Spanish population (GHEP Collaborative
Exercise, 2014), and for X-STRs an Iberian frequency database was
applied
[10].
Y chromosome and mtDNA LRs were calculated based
on haplotype frequencies obtained in the YHRD and EMPOP
databases.
3. Results and discussion
A total of 13 post-mortem remains from two different graves
were studied. Macroscopically, most of the samples presented a
good state of preservation. Accordingly, DNA extraction was
successful in 11 out of 13 remains, with a mean nuclear human
DNA quantity of 0.17 ng/
m
l. No DNA was detected in the two femur
samples from grave 2, which presented the poorest macroscopic
appearance.
Different genetic markers were studied in the remains (Table
1),
depending on the different biological kinship to be elucidated. For
75% of the post-mortem samples analyzed,
!12
autosomal STR
profiles were obtained by using NGMSE kit. Mini-STRs (MiniFiler)
were studied in two samples with DNA quantities
<0.023
ng/
m
l,
without results. Y-STR genetic profiles (!7 Y-STRs) were obtained
for 75% of the post-mortem remains analyzed by using YFiler or
PPY23 kits. Successful mtDNA typing was obtained for all samples
analyzed. Finally, the X-STR decaplex panel was used in one case to
verify a paternal grandmother-granddaughter relationship.
We successfully identified 9 of 13 individuals buried in the two
mass graves of Espinosa de los Monteros. In grave 1, two victims
were identified by comparison to their living children. Individual 6
was identified as the alleged father of two siblings with a combined
LR value of 2.5
Â
10
17
. Fortunately, the mother of these children was
also potentially identified but in grave 2 (individual 4), with a LR
value for mtDNA of 2.5
Â
10
3
. In this case, autosomal STR results
were not obtained; consequently, the identification was
accomplished by combining mtDNA and anthropological results.
A father-son match was also established in grave 1 for individual 8
with a combined Y chromosome and autosomal STR LR value of
5.7
Â
10
6
. Moreover, a maternal lineage relationship was estab-
lished among individual 9 and a living brother with a LR of 6.6
Â
10
2
based on mtDNA.
The rest of positive kinship identifications consisted of second-
degree relationships (grandparent–grandchildren and uncle–
nephew/niece) with combined LR values of 1.2
Â
10
3
–6.6
Â
10
7
.
Finally, four individual’s identity remained unknown due to the
lack of genetic results and/or matches to the living relatives.
4. Conclusions
In summary, the kinship relationship of 9 out of 13 corpses and
10 relatives was favorably established. Our results emphasize the
necessity of analyzing different genetic markers in human remains
to improve kinship statistical calculations, especially when only
distant relatives are available. Furthermore, it is worth mentioning
that anthropology played a fundamental role in the identification
process, particularly when not enough genetic data was available
and low LR values were obtained. The search of further potential
relatives’ samples would be of interest to perform more
comparative analyses, and eventually do not leave any of the
victims without a name.
Conflict of interest statement
None.
Role of the funding source
Funds were provided by the Basque Government (Grupo
Consolidado IT833-13).
Acknowledgments
The authors are grateful to PhD Maite Alvarez for her technical
and human support provided by the DNA Bank Service (SGIker) of
the University of the Basque Country (UPV/EHU).
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Please cite this article in press as: C. Núñez, et al., A grave in my garden. Genetic identification of Spanish civil war victims buried in two mass
graves in Espinosa de los Monteros (Burgos, Spain), Forensic Sci. Int. Gene. Suppl. (2015),
http://dx.doi.org/10.1016/j.fsigss.2015.09.133
G Model
FSIGSS 1130 No. of Pages 3
C. Núñez et al. / Forensic Science International: Genetics Supplement Series xxx (2015) xxx–xxx
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Please cite this article in press as: C. Núñez, et al., A grave in my garden. Genetic identification of Spanish civil war victims buried in two mass
graves in Espinosa de los Monteros (Burgos, Spain), Forensic Sci. Int. Gene. Suppl. (2015),
http://dx.doi.org/10.1016/j.fsigss.2015.09.133

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