Jou nal o A chaeological Science: Repo s 46 (2022) 103706
A ailable online 11 No embe 2022
2352-409X/© 2022 The Au ho s. Published by Else ie L d. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-
nc-nd/4.0/).
The domes ic dog ha li ed ~17,000 yea s ago in he Lowe Magdalenian
o E alla si e (Basque Coun y): A adiome ic and gene ic analysis
Mon se a He ella
a
,
1
, Asie San-Juan-N´
o
a
,
1
, Alo˜
na Aldaso o-Zabala
a
, Ko o Ma iezku ena
b
,
Jesús Al una
b
,
c
, Concepci´
on de-la-Rua
a
,
c
,
*
a
Uni e si y o he Basque Coun y (UPV/EHU), Facul y o Science and Technology, Dep. o Gene ics, Physical An h opology and Animal Physiology, Bizkaia, Spain
b
A kaios Ike ke ak, A chaeozoology Labo a o y, Donos ia/San Sebas i´
an, Spain
c
Jakiunde/Academy o Sciences, A s and Le e s, Donos ia/San Sebas i´
an, Spain
ARTICLE INFO
Keywo ds:
Domes ica ion
Wol
Dog
Ch onology
Lowe Magdalenian
Ancien DNA
Basque Coun y
ABSTRACT
Dogs a e known o be he i s species domes ica ed by humans, al hough he geog aphic and empo al o igin o
his p ocess is s ill unde deba e in di e en ields o knowledge. In he p esen s udy, we examined a hume us
om a canid eco e ed in he Lowe Magdalenian le el o he si e o E alla (Zes oa, Gipuzkoa, Basque Coun y,
Spain), combining mo phology, adioca bon da ing and gene ics. Ou esul s con i m he iden i ica ion o his
specimen as Canis lupus amilia is, disca ding miss-iden i ica ion wi h a dhole (Cuon alpinus) h ough gene ic
analyses o cy och ome b gene and m DNA haplog oup. The di ec AMS
14
C da ing (17,410–17,096 cal. BP)
indica ed ha he E alla specimen ep esen s one o he ea lies domes ica ed dogs in Eu ope, in he Lowe
Can ab ian Magdalenian pe iod. We discuss ou esul s in he ligh o he deba e o he o igin o dogs, conduc ing
a c i ical e iew o he da ings o si es o Eu asia ha ha e p o ided emains o Paleoli hic and Mesoli hic dogs,
including he so-called “dog-like wol es”.
1. In oduc ion
The dog (Canis lupus amilia is), he mos ancien ly domes ica ed
animal om i s ag io ype, he g ay wol (Canis lupus), appea ed in he
Uppe Paleoli hic. Ne e heless, when and whe e i was domes ica ed
a e s ill unsol ed ques ions. The mos ancien a chaeological emains o
clea ly domes ica ed dogs we e ound in dis an edges o Eu asia. They
li ed in Eu ope du ing he Magdalenian (Ab i le Mo in, F ance,
15,114–14,237 cal. BP) and he Epig a e ian (G o a Paglicci, I aly,
14,372–13,759 cal. BP), in he Nea Eas du ing he Keba an and
Na u ian cul u es (Keba a, Is ael, c. 12,500–12,000 BP) and in he Fa
Eas du ing he Peiligang cul u e (Jiahu, China, 9,000–7,800 cal. BP)
(Boudadi-Maligne e al., 2012; Boschin e al., 2020; Tche no and Valla,
1997; Gakuha i e al., 2015, espec i ely) (Fig. 1, N◦2, 3 and 4).
In addi ion, he dog was no only he i s domes ica ed animal, bu
also he closes one o human beings. Bu ials o humans wi h dogs da e
om as ea ly as 12,290–12,050 cal. BC (14,240–14,000 cal. BP), du ing
he La e Magdalenian, in Bonn-Obe kassel (Ge many, Eu ope) (Janssens
e al., 2018); c. 11,500–11,000 BP, du ing he la e Na u ian, in Eynan/
Mallaha and Hayonim Te ace (Is ael, Nea Eas ) (Tche no and Valla,
1997); and 7,414–7,273 cal. BP du ing he J¯
omon cul u e, in Kami-
ku oiwa (Fa Eas , Japan) (Gakuha i e al., 2015). These da a, in he
con ex o paleopa hological and symbolic s udies, sugges ha dogs and
humans had a unique bond (Tche no and Valla, 1997; Janssens e al.,
2016) (Fig. 1, N◦5, 6 and 7).
Since he 1990s, gene icis s ha e a emp ed o answe ques ions
abou dog domes ica ion aised by a chaeological indings (Vil`
a e al.,
1997). Rega ding he geog aphical o igin o dog domes ica ion, mul iple
si es ha e been p oposed. S udies o m DNA haplog oup di e si y in
con empo a y dogs and wol es poin o China (Sa olainen e al., 2002;
Pang e al., 2009; Wang e al., 2016), nuclea DNA ma ke s poin o
Cen al Asia (Shannon e al., 2015) and whole genome sequencing
poin s o he Nea Eas (F eedman e al., 2014).
Wi h espec o he empo al ame, nowadays da es be ween 17,000
and 10,000 yea s ago a e he mos ecen da es p oposed o he dog
lineage di e gence ime, bo h wi h m DNA (Sa olainen e al., 2002) and
* Co esponding au ho a : Uni e si y o he Basque Coun y (UPV/EHU), Facul y o Science and Technology, Dep. o Gene ics, Physical An h opology and Animal
Physiology, Bizkaia, Spain.
E-mail add ess: [email p o ec ed] (C. de-la-Rua).
1
These au ho s con ibu ed equally.
Con en s lis s a ailable a ScienceDi ec
Jou nal o A chaeological Science: Repo s
jou nal homepage: www.else ie .com/loca e/jas ep
h ps://doi.o g/10.1016/j.jas ep.2022.103706
Recei ed 19 July 2022; Recei ed in e ised o m 21 Oc obe 2022; Accep ed 21 Oc obe 2022
Jou nal o A chaeological Science: Repo s 46 (2022) 103706
2
high-quali y genomes o con empo a y dogs (F eedman e al., 2014).
Ne e heless, an unce ain y in hese da es could b ing hem back o
40,000–30,000 yea s ago (Sa olainen e al., 2002; F eedman e al.,
2014). In ac , we should also ake in o accoun ha he gene ic di e -
gence o wol es’ and dogs’ lineages does no ha e o concu wi h ha o
domes ica ion. P oposed da es as ecen as 17,000–10,000 would coin-
cide in ime wi h he i s dog a chaeological e idence, which seems
unlikely.
Paleogene ics con adic s some o hese conclusions om cu en
canid popula ion s udies. Acco ding o Thalmann e al. (2013), he o -
igins o mos dog lineages can be aced back o ancien Eu opean
wol es, sugges ing a Eu opean o igin o wol domes ica ion. Howe e ,
some au ho s sugges ed a dual o igin in Eu ope and Asia (F an z e al.
2016; Be gs ¨
om e al., 2022). Fu he mo e, wi h he con ibu ion o
ancien canids` DNA, he mu a ion a e o wol es was ecalib a ed,
p oposing ha he di e gence be ween dogs and wol es ook place
some ime be ween 40,000 and 27,000 yea s ago (Skoglund e al., 2015;
Bo igu´
e e al., 2017). These paleogene ic indings suppo ha wol -dog
lineages’ di e gence age di e s om he age o he i s domes ica ion
e en .
Appa en ly in ag eemen wi h such da es p oposed by paleogene i-
cis s (40,000–27,000 yea s BP), some emains o e y ancien canids
(40,000–15,000 yea s BP), called “dog-like wol es”, ha e been
desc ibed and iden i ied by means o classic mo phome y. Some s udies
sugges ha hese may be p o odogs. The “dog-like wol es” om
Wes e n Eu ope come om he ollowing si es: Goye (Belgium), 31,890
+240/-220 BP, Au ignacian (Ge monp ´
e e al., 2009; Ge monp ´
e e al.,
2012); Pˇ
edmos í (Czech Republic), c. 27,000–26,000 BP, G a e ian
(Ge monp ´
e e al., 2012; P assack e al., 2020; Gale a e al., 2020); and
he si e o Chau e ca e (F ance), c. 26,000 BP, whe e no emains o he
han dog-like oo p in s we e ound (Ga cia, 2005) (Fig. 1, N◦8, 9 and
10). Some o he “dog-like wol es” om Sibe ia a e: Razboinichya (Al ai,
Sou h Sibe ia, Russia), 33,000 cal. BP (O odo e al., 2011) and Ulakhan
Sula (Yaku ia, No heas Sibe ia, Russia), 13,925 ±70 BP (17,200 cal.
BP ap ox.) (Ge monp ´
e e al., 2017). Those om Eas Eu opean plains
a e: Kos enki-8 (Russia) c. 25,000–22,000 BP, G a e ian (Ge monp ´
e
e al., 2015a); Elisee ichi-I (Russia), c. 17,000–13,000 BP, Epig a e ian
(Sablin and Khlopache , 2002); and Mezhe ich and Mezin (Uk aine), c.
15,000–14,500 BP, Epig a e ian (Ge monp ´
e e al., 2009) (Fig. 1, N◦
11, 12, 13 and 14).
Con on ing hese classic mo phome y s udies, some new
app oaches conclude ha “dog-like wol es” a e clus e ed wi hin he
a iabili y o he wol , which mus ha e been g ea e in he pas (Bou-
dadi-Maligne and Esca guel, 2014; D ake e al., 2015; Janssens e al.,
2019). Some au ho s ha e p oposed ha hose ancien canids may ha e
been jus a mo pho ype o eco ype o wol es in he Uppe Paleoli hic
(Ge monp ´
e e al., 2015b; P assack e al., 2021). Thus, hey could no be
con iden ly placed wi hin he Paleoli hic dogs (Janssens e al., 2021a,
2021b). . This means ha o da e, he Magdalenian emains om Ab i le
Mo in (F ance) and Bonn-Obe kassel (Ge many) we e he oldes dog
emains iden i ied as domes ica ed dogs (Boudadi-Maligne e al., 2012;
Janssens e al., 2018).
In his s udy, we econside ed a canid hume us eco e ed in he
a chaeological exca a ions di ec ed by J. Al una in he E alla ca e
(Gipuzkoa, Basque Coun y) (Al una e al., 1985) (Fig. 2). The ca e is
loca ed in he No heas e n egion o he Basque Coun y, 10 km in a
s aigh line om he Can ab ian Sea and 460 m abo e he sea le el. I s
maximum leng h is 20 m and has wo en ances, one a each end. The
exca a ions a ec ed 30 m
2
om he 90 m
2
o he ca e. The si e p esen s
a solid s a ig aphy, wi h well-de ined and cha ac e ized le els ( om
oldes o mos ecen : le el VII o le el I). Le el VII is di ec ly o e he
bed ock o he ca e, and, oge he wi h le el VI, i is comple ely s e ile.
Le el V, whe e he canid hume us was ound, belongs o he Lowe
Can ab ian Magdalenian and is co e ed by le el IV, which, despi e i s
a chaeological s e ili y, p o ided he emains o se en moun ain goa s
(Cap a py enaica), which we e in ana omical posi ion, indica ing ha
hey died in he ca e, p obably due o a ligh ning s ike. Le els III and II
belong o he La e Magdalenian, and le el I is cons i u ed by he cu en
opsoil. Thus, he impo ance o he canid om his le el V lies in he ac
ha he le el is pe ec ly isola ed, wi h no elemen s om he o he
le els. Tha is, i was ound be ween wo s e ile le els, showing he
absence o con amina ion wi h o he cul u al le els.
Le el V p o ided a ich li hic and bone indus y, ypical o he Lowe
Magdalenian, as well as o namen al objec s and o he deco a ed i ems
(e.g., assegai poin s, cha ac e is ic o he Can ab ian egion in his
pe iod) (Balde´
on, 1985). This le el was laid du ing a e y cold phase
(D yas I), as has been shown by pollen, sedimen ological and a chae-
ozoological analyses (Al una e al., 1985). Mo eo e , he animal-based
die o humans o E alla in his pe iod indica es a p edominance o
Cap a py enaica (84%) o e Ce us elaphus (7.0%) (Al una and Ma -
iezku ena, 1985).
In addi ion, he canid hume us was ound in he lowe pa o le el V.
Fig. 1. A chaeological si es om Wes Eu asia
ci ed in his a icle. S a s: Magdalenian, Epi-
g a e ian and Epipaleoli hic dogs. Ci cles: Pu a-
i e Magdalenian dogs. T iangles: Mesoli hic
dogs. Squa es: Le an ine P eneoli hic dogs. Pen-
agons: Dog-like wol es. Whi e: Canids whose
gene ic in o ma ion is a ailable. Blue: E alla
dog. 1, E alla; 2, Ab i le Mo in; 3, G o a
Paglicci; 4, Keba a; 5, Bonn-Obe kassel; 6,
Eynan/Mallaha; 7, Hayonim Te ace; 8, Goye ; 9,
Pˇ
edmos í; 10, Chau e ; 11, Kos enki-8; 12,
Elisee ichi-I; 13, Mezhe ich; 14, Mezin; 15,
An on Koba; 16, Knieg o e; 17, Hau e i e-
Champ ´
e ey es; 18, Teu elsb ücke; 19, ¨
Olkni z;
20, Le Closeau; 21, Kessle loch; 22, G o e-ab i
du Moulin; 23, Pon d’Ambon; 24, Mon espan;
25, Ka s ein; 26, Palegaw a; 27, Cuina Tu cului;
28, Muge; 29, Icoana; 30, Os o ul Co bului; 31,
Vale Boi; 32, Poças S. Ben o; 33, Romanelli; 34,
Gni sh¨
ohle; 35, Hohle Fels. (Fo in e p e a ion o
he e e ences o colou in his igu e legend, he
eade is e e ed o he web e sion o his
a icle.)
M. He ella e al.
Jou nal o A chaeological Science: Repo s 46 (2022) 103706
3
Speci ically, he si e was exca a ed in ine s a ig aphical uni s, known
as beds. Le el V had eigh beds, comp ising om bed 17 (in i s uppe
pa , bo de ing wi h le el IV) o bed 24 (in i s lowe pa , bo de ing wi h
le el VI). The canid hume us was ound in bed 22.
Th ee da ings o Le el V we e pe o med by Teledyne Iso opes (New
Je sey) on di e en se s o bones sca e ed h oughou he en i e le el
(Al una e al., 1984; Al una, 1985). The alues p o ided we e 15,740 ±
240, 16,200 ±240 and 16,270 ±240 BP (uncalib a ed da es), which a e
in line wi h he Lowe Magdalenian da ings o o he si es in he Basque
Coun y and he Can ab ian egion (Ma iezku ena, 1979, Ma -
iezku ena, 1990).
In 1985, Al una and Ma iezku ena classi ied his bone as Canis sp.
(Fig. 2), and compa ed i wi h 22 p ehis o ic wol hume i om he
Basque Coun y and Eu ope, as well as wi h p esen - ime wol hume i. I
was conside ably smalle han e e y o he hume us. They also
compa ed i wi h ano he canid ha exis ed in ha ime in he Basque
Coun y: Cuon alpinus (dhole); al hough he measu emen s we e e y
simila , he mo phology o he piece was close o he dog han o he
dhole. In 1994, Al una, a e showing he piece o p es igious a chae-
ozoologis s o ha ime, such as G. Nobis, R. Musil, A. Gau ie , F. Poplin
and M. Teiche , among o he s, conside ed ha i was a dog (Al una,
1994). Howe e , his de e mina ion was no absolu ely ce ain. La e ,
Vigne (2005) examined his canid bone, classi ying i as a dog based on
i s mo phology. Mo eo e , e iewing Al una’s pape (1985), Vigne
calcula ed he calib a ed alues o he h ee da ings o le el V in E alla,
and p oposed an app oxima e uppe da e o 17,500–17,000 cal. BC (c.
19,000 cal. BP). These da es ha e been inco ec ly ep oduced in some
pape s, maybe because hey we e conside ed oo old o a domes ica ed
dog (Pionnie -Capi an e al., 2011; Boudadi-Maligne e al., 2012; Jans-
sens e al., 2018, among o he s).
In he p esen s udy, we examined he hume us o he canid om he
Lowe Magdalenian le el o he si e o E alla, and we conduc ed a
c i ical e iew o he Eu opean si es ha ha e p o ided emains o
Paleoli hic and Mesoli hic dogs. In he Basque Coun y, no o he emains
ha e been classi ied as domes ic Canis in Magdalenian le els o da e.
The e a e some emains om he Azilian
2
, such as he dog om he si e
o An on Koba (O˜
na i, Gipuzkoa) (A menda iz, 1997; Al una and Ma -
iezku ena, 2013) (Fig. 1, N◦15). O he emains o C. l. amilia is
a ibu ed o Mesoli hic le els in si es o he Basque Coun y ha e been
ce ainly de e mined, al hough hei belonging o hose le els is un-
ce ain (Al una, 1994). This is he eason why hey a e no included in
he p esen wo k. Mo eo e , his is he i s s udy o include a di ec AMS
14
C da ing o his hume us and a gene ic analysis o species iden i i-
ca ion, in e p e ing hese esul s in he con ex o he exis ing da a abou
he domes ica ion o he wol in Eu ope.
2. Ma e ials and me hods
2.1. Radioca bon da ing
A sample o 115 mg o bone powde om he E alla hume us was
sen o he ¨
Angs ¨
om Labo a o y a Uppsala Uni e si y (Sweden) o
AMS
14
C da ing. The sample was s i ed a 10 ◦C in 0.8 M HCl o 30 min
o emo e apa i e. Then, dis illed wa e main ained a pH 3 was added o
he insoluble ac ion, which was hea ed while s i ing (6–8 h, 90 ◦C).
Fig. 2. E alla hume us. a) An e io iew. b) Pos e io iew. c) Medial iew. d) La e al iew.
2
Azilian: Pos magdalenian cul u e o he Epipaleoli hic and Mesoli hic pe-
iods in he F anco-Can ab ian egion. I is named a e Mas d’Azil ca e (Py -
enees, F ance).
M. He ella e al.
Jou nal o A chaeological Science: Repo s 46 (2022) 103706
4
The soluble pa , e e ed o as ac ion D, con ained mos o he o ganic
pa s ( he “collagen”) o he o iginal bone. The ac ion o be
14
C-da ed
was combus ed o CO
2
, which was g aphi ised h ough a Fe-ca alys
eac ion p io o he accele a o de e mina ion. In he p esen in es i-
ga ion, ac ion D was da ed.
2.2. Sampling and DNA ex ac ion
A small su ace o he E alla hume us (on he dis al me aphysis) was
p e iously decon amina ed by mechanical ab asion wi h a den al ile, in
o de o elimina e possible con amina ing exogenous DNA. Then, 135
mg o bone powde we e aken using a den is d ill.
DNA was ob ained om he bone powde in a single ex ac ion
p ocess using he silica-based me hod (Dabney e al. 2013), ollowing
he modi ica ions p oposed by S ensson e al. (2021), who used silica
spin-column con aining a olume ex ende om a High P e-Vi al
Nucleic Acid La ge Volume ki (Roche) (Yang e al., 1998; Glocke and
Meye , 2017). The o al olume o DNA ex ac was 75 ml (elu ed in
bu e EB, QIAGEN) and wo ex ac ion blanks we e p ocessed as
con ols.
The ampli ica ion o each mi ochond ial DNA agmen (D-loop and
cy och ome b gene) was unde aken in independen PCRs. In case o
posi i e ampli ica ion and absence o con amina ion, he ampli ica ions
we e pu i ied by ExoSAP-IT (USB Co po a ion), wi h subsequen
sequencing in an ABI310 au oma ic sequence using chemis y based on
Big-Dye 1.1 (Li e Technology). The ob ained esul s we e edi ed wi h
BioEdi so wa e (h ps://www.mbio.ncsu.edu/BioEdi /bioedi .h ml)
and he sequences we e aligned manually.
The DNA ex ac ion and ampli ica ion we e pe o med in he ancien
DNA labo a o y o he Uni e si y o he Basque Coun y (Spain). P o-
cessing o he samples in ol ed he applica ion o a se ies o s ic
c i e ia de ailed in P¨
a¨
abo e al. (2004) and Gilbe e al. (2005) in o de
o au hen ica e he esul s. To p e en ancien DNA con amina ion, he
ex ac ion and p epa a ion o he PCR we e unde aken in a posi i e-
p essu e s e ile chambe , physically sepa a ed om he labo a o y
whe e pos -PCR p ocesses we e ca ied ou . All he wo k su aces we e
egula ly cleaned wi h sodium hypochlo i e and i adia ed wi h UV ligh
(254 nm). Sui able disposable clo hing was wo n (lab co e all, mask,
glo es and cap). Con amina ion con ols we e applied in bo h he
ex ac ion and ampli ica ion p ocesses.
2.3. Gene ic analysis
The sequence o cy och ome b (cy b) gene is commonly used o
iden i y he alloca ion o a species (I win e al., 1991; Kuwayama and
Ozawa, 2000; Lud e al., 2004). In ou case, i was used o di e en ia e
Canis om Cuon, which is ano he close genus o canids, as well as o
asce ain he iden i ica ion o he species wi hin he genus Canis. To his
end, a sequence o ~200 bp was analysed h ough he sequencing o wo
o e lapping agmen s o ~120 bp o he cy b gene, ollowing he con-
di ions desc ibed in He ella e al. (2012).
Since he cy b sequence o Canis lupus amilia is (domes ic dog) p e-
sen s a small numbe o polymo phisms wi h espec o ha o Canis
lupus (wol ) (Linac e and Tobe, 2009), we e i ied he species ha he
E alla hume us belongs o, h ough he sequencing o a agmen o
181 bp om he D-loop egion o i s m DNA, be ween nucleo ide posi-
ions 15495 and 15676, ollowing he p oposal om Pi es e al. (2006,
2017, 2019), among o he au ho s. Bo h analyses, i.e., he sequencing o
he agmen s om cy b and om he D-loop, we e ca ied ou in ip-
lica e. The ob ained D-loop sequence also allowed de e mining he
mi ochond ial haplog oup o he canid (F egel e al., 2015).
A Median-Joining Ne wo k was cons uc ed using he sequences
(nps. 15,495–15,900) om he p esen s udy and o he published da a
om Paleoli hic and Mesoli hic dogs in Eu ope (Pionnie -Capi an, 2010)
using he Ne wo k 4.6.0.0 so wa e (h ps://www. luxus-enginee ing.
com).
3. Resul s
3.1. Ch onology: Radioca bon da ing o he hume us om he E alla si e
The di ec AMS
14
C da ing o he E alla hume us (Ua-56946) was
14,221 ±48 BP; 17,410–17,096 cal. BP (95.4%) (Table 1 and Fig. 3).
The da ing o he canid om he E alla ca e co esponds o he
Magdalenian pe iod; his is in line wi h he s a ig aphic con ex in
which i was ound, which was a chaeologically de ined as a Lowe
Can ab ian Magdalenian le el. The di ec da ing o he hume us educes
he empo al ange a ibu ed o i by he a chaeological le el, which
was es ablished based on he da ing o emains o animals sca e ed
h oughou he en i e le el (Al una, 1985) (see In oduc ion).
3.2. Mo phome ic analysis o he hume us om he E alla si e
The hume us eco e ed om he E alla si e p esen ed ex ao dina y
conse a ion, al hough he uppe end, which co esponds o he hu-
me al head, is missing (Fig. 2). A 3D econs uc ion o he hume us was
ca ied ou , in o de o p ese e he aluable bone and p o ide a digi al
ep oduc ion o i ha enables i s mo phome ic analysis ( Supplemen-
a y da a 1). The main measu emen s o he E alla hume us a e: min-
imum diaphysis wid h (14 mm), dis al b ead h (Bd) (34.5 mm) and
dis al hickness (23.5 mm) (Al una and Ma iezku ena, 1985). We
highligh dis al b ead h, since i is he os eome ic me hodology in e -
na ionally used in mac omammals (D iesch, 1976) (Fig. 4).
Fig. 4 shows ha he dis al b ead h (Bd) o he E alla hume us (34.5
mm) is ou side o he ange o a ia ion o bo h Magdalenian and
Mesoli hic wol es o he Basque Coun y (42.4 ±1.26 mm) and Wü m
wol es o Eu ope (42.1 ±2.24 mm); howe e , i is wi hin he a iabili y
ange o a sample o Mesoli hic dogs o No he n Eu ope (32 ±4.63
mm).
Rega ding he mo phome ic di e en ia ion be ween Canis and
Cuon, i is almos impe cep ible a he le el o he hume us. Al hough a
mo e de eloped medial epicondyle in he medio-dis al di ec ion o he
Cuon has been desc ibed (Pionnie -Capi an, M. e al., 2011), in he case
o he E alla hume us, no dis inc ion was obse ed in ela ion o hese
mo phological ea u e.
3.3. Species iden i ica ion
The m DNA eco e ed om he sample o E alla was quan i ied
h ough SYBRG een RT-qPCR, using he p ime s o he cy b gene
(He ella e al., 2012), ob aining an a e age o 1,689 copies/µl o
ex ac , which is a highe alue han 1000 copies/µl, as indica ed by he
aDNA au hen ica ion c i e ia (P¨
a¨
abo e al., 2004; Gilbe e al., 2005).
Then, di e en ampli ica ions we e conduc ed ollowing he p o ocol
desc ibed in He ella e al. (2012), in o de o ob ain he sequence o a
agmen o ~200 bp o he cy och ome b gene. Nega i e con ols and
ex ac ion blanks we e sys ema ically used o moni o possible con-
amina ions. Nei he PCRs blanks no nega i e con ols ga e posi i e
esul s. Sequences om h ee independen PCRs om he same DNA
ex ac o E alla we e ob ained. The h ee sequences showed he same
polymo physms. The sequence ob ained ( Supplemen a y da a 2) was
hen included in he BLAST (NCBI) and Unip o da abases (Bou e e al.,
2016). This sequence was iden i ied as Canis lupus amilia is (99.6%
p obabili y) and no Cuon alpinus (dhole) (67.5%). The esul implies
ha he E alla hume us canno be a dhole.
In o de o con i m he iden i ica ion o he species and ob ain
Table 1
AMS
14
C di ec da ing o he E alla hume us.
Lab numbe δ
13
C‰
V-PDB
14
C age BP 95.4% p obabili y
cal. BC
95.4% p obabili y
cal. BP
Ua-56946 −17.8 14,221 ±48 15,460–15,146 17,410–17,096
M. He ella e al.
Jou nal o A chaeological Science: Repo s 46 (2022) 103706
5
in o ma ion o he mi ochond ial haplog oup ha he E alla dog be-
longs o, a egion o 181 bp o he mi ochond ial D-loop was sequenced (
Supplemen a y da a 3). Fo m DNA da a, he ob ained sequences we e
compa ed by alignmen o he e e ence sequence (Kim e al., 1998)
(GenBank accession en y: NC_002008), and he polymo phism was
numbe ed acco ding o Pe ei a e al. (2004).
The sequence om he D-loop agmen was ob ained by means o six
PCRs ( h ee o each agmen ), de ec ing he same mu a ions in all o
hem. The e was no ambigui y in he iden i ica ion o polymo phisms,
which we e T15611C, T15639G ( ans e sion) and T15650C. These
polymo phisms co espond o he haplog oup C om C. l. amilia is,
ollowing he in o ma ion p o ided by F egel e al. (2015). These au-
ho s p oposed a nomencla u e o he phylogeny o he mi ogenome o
C. l. amilia is, conside ing he da a o he mi ogenomes o ancien and
mode n wol and dog. The analysis o he D-loop easse s he E alla
hume us assigna ion o C. l. amilia is.
3.4.
14
C da ings o Magdalenian and Epipaleoli hic si es whe e dog
emains ha e been eco e ed
A e iew o
14
C da ings o Magdalenian, Epig a e ian and Epi-
paleoli hic Eu opean si es (Fig. 1) was ca ied ou in o de o
con ex ualise he da ing ob ained in he E alla hume us and hose om
o he ancien dogs (Table 2). We also inco po a ed calib a ed alues ha
we e calcula ed in he p esen s udy, wi h he aim o enabling he
compa ison o he da ings among si es, since we obse ed ha some
au ho s p o ide uncalib a ed da ings, o he s show BP calib a ed da ings
and o he au ho s p esen BC calib a ed da ings. Fu he mo e, he
calib a ed da ings we e no always done using he same calib a ion
cu e, and hus hey we e no uly compa able (Reime e al., 2013,
2020). The alues calib a ed in his s udy, using he OxCal4.4 so wa e
wi h he In Cal20 cu e (Reime e al., 2020), a e highligh ed wi h (*) in
Table 2. In addi ion, o calcula e cal. BP om cal. BC da es, he co e-
sponding +1950 yea s we e added (**).
Among he 15 si es o Eu ope wi h canid emains men ioned in his
e iew, six ha e a di ec
14
C da ing on dog bone, eigh on o he emains
o he le el in which he canids appea ed and only one o he si es
(Mon espan) lacks
14
C da ings due o he insu icien amoun o collagen
(Pionnie -Capi an e al., 2011) (Table 2). Mos o he Canis we e
con i med mo phologically as Canis lupus amilia is; howe e , h ee si es
ha e bones ha we e only a ibu ed o Canis lupus amilia is, bu ha e
no been con i med as such. In he o he si es, he Canis lupus amilia is
was gene ically o mo phologically di e en ia ed om Cuon genus.
Ne e heless, in G o a Paglicci, only wo bones ou o wel e (a ibia
om le el 5a and a me a a sal, di ec ly da ed, om le el 4c) we e
mo phologically and/o gene ically di e en ia ed om Cuon. In addi-
ion, some emains ha e a complex his o y o da ing, e-da ing, cali-
b a ion, e c. This in o ma ion is shown in Table 2, whe e he included
e e ences p o ide he de ails o each si e.
Table 2 p esen s he di ec da ing o he E alla dog hume us con-
duc ed in his s udy (Ua-56946), along wi h he p e iously published
da ings o le el V, whe e his canid appea ed (Al una, 1985). In he
Basque Coun y, he e a e di e en emains o dogs ha ha e been
mo phologically con i med, al hough hei a ibu ion o Azilian le els
is unce ain (Al una, 1994). An on Koba (O˜
na i, Gipuzkoa) is he only
si e, o da e, wi h a ce ain a ibu ion o an Azilian le el da ed be ween
11,800 ±330 and 11,700 ±180 BP (A menda iz, 1997; Al una and
Ma iezku ena, 2013) (Table 2 and Fig. 1). This bone has no been
gene ically analysed ye , due o he es ic ions imposed by he Cul u al
He i age Depa men o he Basque Go e nmen , e e ed o he mini-
mum weigh o each bone emain (5 g) and he ep esen a ion o he
species in pa icula in he a chaeological le el.
The Magdalenian si e o Ab i le Mo in (F ance) and he Epig a e ian
le el 4c o G o a Paglicci ha e he oldes di ec da ings o da e, a e
ha o E alla, which a e close o he Magdalenian si es o Kessle loch
Fig. 3. Calib a ion p obabili y dis ibu ion o he adiome ic da ings
conduc ed on bone collagen o he E alla hume us (Reime e al., 2013),
OxCal So wa e 4.4.4 B onk Ramsey (2021) using In Cal20 cu e.
Fig. 4. Indi idual dis al b ead h o he E alla hu-
me us compa ed wi h ha obse ed in La e Paleo-
li hic and Mesoli hic dogs and wol es. Measu es gi en
in mm. Mesoli hic dogs om No h Eu ope (Mesoli hic
dog NE), Magdalenian and Mesoli hic wol es om he
Basque Coun y (Mag-Mesoli hic wol es BC), and Wü m
wol es om Eu ope (F ance and Belgium) (Chaix, 2000;
Pionnie -Capi an e al., 2011; Ge monp ´
e e al., 2021).
Box and whiske plo s ep esen he mean ±1 (box) and
1.5 (whiske ) s anda d de ia ions.
M. He ella e al.
Jou nal o A chaeological Science: Repo s 46 (2022) 103706
6
Table 2
14
C da ings o he Magdalenian, Epig a e ian and Epipaleoli hic le els o si es in Eu ope ha con ain emains a ibu ed o dog.
Si e Le el Code Da ed sample
14
C Uncal. BP Cal. BC Cal. BP
G o a Paglicci
[Ea ly Epig a e ian]
(Fig. 1, N◦3)
17b G N-14874 – 16,890 ±160
(8)
18,881–18,058
(*)
20,831–20,008
(**)
20,789–19,979
(8)
G o a Paglicci
[E ol ed Epig a e ian]
(Fig. 1, N◦3)
10 F-68 – 15,320 ±250
(8)
17,192–16,231
(*)
19,142–18,181
(**)
19,112–17,988
(8)
8 F-66 – 15,460 ±220
(8)
17,242–16,307
(*)
19,192–18,257
(**)
19,245–18,220
(8)
E alla
[Lowe Magdalenian]
(Fig. 1, N◦1)
5 Ua-56946 Dog hume us 14,221 ± 48
(1)
15,460–15,146
(1)
17,410–17,096
(1)
5 I-12868 Animal emains (se e al) 16,270 ±240
(2)
18,334–17,121
(*)
20,284–19,071
(**)
5 I-12551 Animal emains (se e al) 16,200 ±240
(2)
18,231–17,030
(*)
20,181–18,980
(**)
5 I-12540 Animal emains (se e al) 15,740 ±240
(2)
17,643–16,400
(*)
19,593–18,350
(**)
G o a Paglicci
[Final Epig a e ian]
(Fig. 1, N◦3)
7 F-65 – 14,820 ±210
(8)
16,696–15,518
(*)
18,646–17,468
(**)
18,541–17,551
(8)
5bc F-96 – 13,590 ±200
(8)
15,074–13,906
(*)
17,024–15,856
(**)
17,021–15,841
(8)
5c Fi2147
Fi2148
Wol hume us 12,570 ±60
(9)
13,225–12,556
(*)
15,175–14,506
(**)
15,200–14,450
(9)
4c OxA-26316 Dog hi d me a a sal 12,175 ± 55
(9)
12,361–11,910
(*)
14,311–13,860
(**)
14,372–13,759
(9)
Rw Fi2146 Canid mandible 11,300 ± 800
(9)
13,859–9,256
(*)
15,809–11,206
(**)
15,800–11,200
(9)
Knieg o e
[Uppe Magdalenian]
(Fig. 1, N◦16)
8 Low OxA-4852 Ho se lumba e eb a 13,520 ±130
(15)
14,799–13,942
(*)
16,749–15,892
(**)
8 Upp OxA-4832 Reindee scapula 13,310 ±110
(15)
14,376–13,737
(*)
16,356–15,687
(**)
8 Upp OxA-4846 Ho se emu 13,190 ±130
(15)
14,298–13,496
(*)
16,248–15,446
(**)
8 Mid OxA-4850 Alopex lagopus ibia 13,160 ±140
(15)
14,261–13,404
(*)
16,211–15,354
(**)
8 Mid OxA-4848 Ho se me a a sal 13,150 ±130
(15)
14,222–13,412
(*)
16,172–15,362
(**)
8 Upp OxA-4845 Reindee ibia 13,120 ±130
(15)
14,171–13,371
(*)
16,121–15,321
(**)
8 Mid OxA-4849 Saiga a a ica 13,130 ±120
(15)
14,160–13,401
(*)
16,110–15,351
(**)
Hau e i e-Champ ´
e ey es
[Magdalenian]
(Fig. 1, N◦17)
UZ-2285 Cha coal 13,050 ±160
(15, 21)
14,162–13,229
(*)
16,112–15,179
(**)
UZ-2283 Cha coal 12,950 ±110
(15)
13,858–13,223
(*)
15,808–15,173
(**)
UZ-2286 Cha coal 12,870 ±140
(15)
13,842–13,035
(*)
15,792–14,985
(**)
UZ-2282 Cha coal 12,830 ±160
(15)
13,891–12,667
(*)
15,841–14,617
(**)
UZ-2171 Cha coal 12,730 ±140
(15)
13,700–12,546
(*)
15,650–14,496
(**)
UZ-2175 Cha coal 12,630 ±130
(15)
13,472–12,365
(*)
15,422–14,315
(**)
UZ-2172 Cha coal 12,620 ±150
(15)
13,531–12,320
(*)
15,481–14,270
(**)
UZ-2177 Cha coal 12,600 ±150
(15)
13,465–12,266
(*)
15,415–14,216
(**)
UZ-2173 Cha coal 12,540 ±140
(15)
13,319–12,238
(*)
15,269–14,188
(**)
UZ-2174 Cha coal 12,510 ±130
(15, 21)
13,251–12,230
(*)
15,201–14,180
(**)
UZ-2287 Cha coal 12,500 ±150
(15)
13,274–12,176
(*)
15,224–14,126
(**)
Teu elsb ücke
[Magdalenian]
(Fig. 1, N◦18)
2 OxA-5723 Cap a ibex calcaneum 13,080 ±140
(15)
14,141–13,311
(*)
16,091–15,261
(**)
2 OxA-5724 A iodac yl adiocubi us 12,940 ±140
(15)
13,945–13,141
(*)
15,895–15,091
(**)
1 OxA-5725 Cap a ibex ibia 12,900 ±130
(15)
13,853–13,111
(*)
15,803–15,061
(**)
2 OxA-5722 Ho se phalanx 2 12,860 ±130
(15)
13,801–13,051
(*)
15,751–15,001
(**)
3 OxA-5726 Reindee hume us 12,640 ±130
(15)
13,515–12,374
(*)
15,465–14,324
(**)
3 OxA-5727 Ho se mandible 10,040 ±120
(15)
10,019–9,277
(*)
11,969–11,227
(**)
¨
Olkni z
[Magdalenian]
(Fig. 1, N◦19)
OxA-5713 Ho se 1s phalanx 12,740 ±120
(15)
13,665–12,655
(*)
15,615–14,605
(**)
OxA-5714 Reindee maxila 12,620 ±120
(15)
13,408–12,366
(*)
15,358–14,316
(**)
OxA-5709 Ho se me aca pus 12,270 ±120
(15)
12,965–11,916
(*)
14,915–13,866
(**)
OxA-5712 Reindee adius 12,270 ±110
(15)
12,930–11,924
(*)
14,880–13,874
(**)
OxA-5710 Ho se 3 d me a a sus 12,080 ±110
(15)
12,361–11,662
(*)
14,311–13,612
(**)
OxA-5711 Ho se me aca pus 12,050 ±110
(15)
12,250–11,65
(*)
14,200–13,607
(**)
Ab i le Mo in
[Uppe Magdalenian]
(Fig. 1, N◦2)
A-I OxA-23627 Dog uppe I3 12,540 ± 55
(10)
13,182–12,508
(*)
15,132–14,458
(**)
15,114–14,237
(10)
A-III OxA-23628 Dog ca nassial 12,450 ± 55
(10)
13,012–12,347
(*)
14,962–14,297
(**)
15,005–14,155
(10)
Le Closeau
[Azilian]
(Fig. 1, N◦20)
G A-18860 Ho se long bone 12,510 ±80
(7, 18)
13,157–12,363
(*)
15,107–14,313
(**)
15,138–14,513
(18)
G A-18815 Bo id Phalanx 12,480 ±70
(7, 18)
13,084–12,342
(*)
15,034–14,292
(**)
15,090–14,473
(18)
AA-41881 Wild boa 12,423 ±67
(7, 18)
13,001–12,266
(*)
14,951–14,216
(**)
14,999–14,319
(18)
G A-11665 Ce id emu 12,360 ±60
(7, 18)
12,896–12,167
(*)
14,846–14,117
(**)
14,894–14,218
(18)
G A-18816 Wild boa 12,350 ±70
(7, 18)
12,897–12,148
(*)
14,847–14,098
(**)
14,884–14,189
(18)
G A-11664 Ho se ibia 12,350 ±60
(7, 18)
12,892–12,153
(*)
14,842–14,103
(**)
14,877–14,194
(18)
G A-18819 Ce id igh adius 12,340 ±70
(7, 18)
12,894–12,138
(*)
14,844–14,088
(**)
14,867–14,163
(18)
AA-41882 Lion igh Me aca pal V 12,248 ±66
(7, 18)
12,859–12080
(*)
14,809–14,030
(**)
14,596–14,055
(18)
OxA-5680 Uniden i ied bone 12,090 ±90
(7, 18)
12,255–11,817
(*)
14,205–13,767
(**)
14,367–13,855
(18)
OxA-6338 Uniden i ied bone 12,050 ±100
(7, 18)
12,221–11,662
(*)
(con inued on nex page)
M. He ella e al.
Jou nal o A chaeological Science: Repo s 46 (2022) 103706
7
and Bonn-Obe kassel. The si e o Kessle loch (Swi ze land) p o ides a
di ec da ing, whose BC calib a ion was pe o med by Napia ela and
Ue pmann (2012), al hough a di e en calib a ion (in cal. BP) was e-
po ed in he gene ic s udy o he dog (Baumann e al., 2021) (Table 2).
The Bonn-Obe kassel dog (Ge many) has been da ed by di e en au-
ho s (S ee , 2002; Higham e al., 2015). A e iew o his si e by
Janssens e al. (2018) p o ides a weigh ed mean da ing o
12,290–12,050 cal. BC. Finally, he six h di ec da ed dog is ha o he
Epipaleoli hic si e o Pon d’Ambon. O he Magdalenian and Epi-
paleoli hic si es ha e only indi ec da ings coming bo h om di e en
animals and cha coal emains.
The analysis o he di ec da ing o he E alla dog, in he con ex o
he dogs om he Uppe Paleoli hic and he Epipaleoli hic, indica es
ha i is he oldes published o da e. I belongs o he Lowe Can ab ian
Magdalenian, and i was ound in an a chaeological le el domina ed by
he cold clima e o he Lowe D yas I (Oldes D yas), whe eas he o he
men ioned dogs a e om he Uppe Magdalenian, he Final Epi-
g a e ian o he Epipaleoli hic, and hey belong o he in e s adial
Bølling/Alle ød and D yas III (Younge D yas).
3.5. A chaeogene ics
3.5.1. Median-Joining Ne wo k: m DNA sequences om Magdalenian and
Mesoli hic dogs
The mi ochond ial DNA da a o he Eu opean dogs om he
Magdalenian (E alla, Bonn-Obe kassel and Kessle loch), Epig a e ian
(G o a Paglicci), Epipaleoli hic (Pon d’Ambon, Ka s ein and Cuina
Tu cului) and Mesoli hic, show ha haplog oup C is he main hap-
log oup in hese pe iods, al hough some Mesoli hic dogs p esen o he
haplog oups, such as B in a dog o I aly and A in i e dogs o Po ugal
(Table 3).
To de e mine he ela ionship be ween he mi ochond ial hap-
log oups o he Magdalenian, Epipaleoli hic and Mesoli hic Eu opean
dogs, a Median-Joining Ne wo k (MJN) was pe o med (Table 3, Fig. 5).
Fo his analysis, we conside ed he in o ma ion o he nucleo ides
comp ised be ween posi ions 15495 and 15900 o he m DNA D-loop.
The Magdalenian, Epig a e ian and Epipaleoli hic dogs, including
ha om E alla (highligh ed in blue) (Fig. 5), p esen he diagnos ic
polymo phisms o mi ochond ial haplog oup C, hus hey sha e he
same node along wi h i e Mesoli hic Eu opean dogs: a dog om he
Cabeço da A uda si e (Muge, Po ugal) and ou om he Icoana si e
(Romania) (Table 3, Fig. 1, N◦3, 28 and 29). Haplog oup C includes wo
mo e haplo ypes co esponding o wo dogs om Romania [one om
Cuina Tu cului (Epipaleoli hic) and ano he om Os o ul Co bului
(Mesoli hic) (Fig. 1, N◦27 and 30)]. Mesoli hic dogs p esen a g ea e
mi ochond ial haplog oup di e si y, since, apa om haplog oup C,
haplog oup A was ound in i e dogs o Po uguese si es (Fig. 1, N◦28,
31 and 32) and haplog oup B in a dog om I aly (Romanelli ca e)
(Fig. 1, N◦33).
4. Discussion
In he analysis o he canid bone ound in he Lowe Magdalenian
le el o he E alla si e (Basque Coun y, Spain), Al una and Ma -
iezku ena (1985) concluded ha he mo phome ic da a excluded i
om being a wol , al hough i s size, simila o ha o a dhole, aised
doub s abou he iden i ica ion o i s species. Fou se s o bone emains
sca e ed h oughou le el V, whe e he canid hume us was ound,
p o ided alues be ween 16,270 ±240 and 15,740 ±240 BP
(20,223–18,536 cal. BP, calib a ion pe o med in he p esen s udy,
Table 2 (con inued)
Si e Le el Code Da ed sample
14
C Uncal. BP Cal. BC Cal. BP
14,171–13,612
(**)
14,327–13,813
(18)
An on Koba
[Azilian]
(Fig. 1, N◦15)
8 I-16236 Animal emains (se e al) 11,800 ±330
(3)
12,927–11,145
(*)
14,877–13,095
(**)
8 I-17479 Animal emains (se e al) 11,700 ±180
(3)
12,071–11,241
(*)
14,021–13,191
(**)
Kessle loch
[Magdalenian]
(Fig. 1, N◦21)
KIA-33350 Dog igh maxilla 12,225 ± 45
(17)
12,369–12,090
(*)
12,360 þ-210
(17)
14,319–14,040
(**)
14,286–13,975
(6)
Bonn-Obe kassel
[Magdalenian]
(Fig. 1, N◦5)
OxA-29869 Ulna sin. dog 12,390 ± 55
(14)
12,916–12,232
(*)
12,900–12,150
(16)
14,866–14,182
(**)
OxA-4793 12,270 ± 100
(19)
12,901–11,936
(*)
12,850–11,900
(16)
14,856–13,886
(**)
14,809–14,140
(21)
KIA-4162 Hume us dex . dog 12,210 ± 60
(19)
12,837–11,928
(*)
12,390–11,910
(16)
14,787–13,878
(**)
14,337–13,861
(21)
KIA-4161 Maxila dex . dog 12,110 ± 45
(19)
12,140–11,860
(*)
12,180–11,840
(16)
14,090–13,810
(**)
14,122–13,799
(21)
KIA-4163 Ulna dex . dog 11,620 ± 60
(19)
11,647–11,395
(*)
13,597–13,345
(**)
13,569–13,319
(21)
G o e-ab i du Moulin (T ouba )
[Azilian]
(22)
(Fig. 1, N◦22)
6 base Ly 5275 Cha coal 10,770 ±100
(5)
10,976–10,543
(*)
10,928–10,489
(13)
12,926–12,493
(**)
12,878–12,439
(13)
6 Ly-9968 Dee bone 10,225 ±45
(13)
10,471–9795
(*)
10,165–9,816
(13)
12,421–11,745
(**)
12,115–11,766
(13)
Pon d’Ambon
[Labo ian]
(11)
(Fig. 1, N◦23)
Gi A-99102 Dog cubi us 10,730 ± 100
(12)
10,941–10,536
(*)
12,891–12,486
(**)
12,952–12,451
(18)
Ka s ein
[Epipaleoli hic]
(Fig. 1, N◦25)
2 KN-4023 Lagopus 10,090 ±100
(4)
10,021–9,316
(*)
9,578 ±314
(20)
11,971–11,266
(**)
2 KN-4072 Rangi e 9,550 ±90
(4)
9,228–8,642
(*)
8,724 ±178
(20)
11,178–10,592
(**)
2 KN-4073 Rangi e 9,530 ±90
(4)
9,217–8,635
(*)
8,683 ±183
(20)
11,167–10,585
(**)
In bold: di ec da ings on dog bone emains. In i alics: si es and le els whe e Canis emains we e a ibu ed o C. l. amilia is. Supe sc ip b acke s indica e he
bibliog aphic e e ence: (1) his a icle; (2) Al una, 1985; (3) A menda iz 1997; (4) Baales e al., 1996; (5) Ba baza, 1996; (6) Baumann e al., 2021; (7) Bodu e al.,
2006; (8) Boschin e al., 2018; (9) Boschin e al., 2020; (10) Boudadi-Maligne e al., 2012; (11) Boudadi-Maligne e al., 2018; (12) C´
el´
e ie e al., 1999; (13) Fa Cheung,
2015; (14) Higham e al., 2015; (15) Housley e al., 1997; (16) Janssens e al., 2018; (17) Napia ela and Ue pmann, 2012; (18) Pionnie -Capi an e al., 2011; (19)
S ee , 2002; (20) S ee e al., 1994; (21) S ee e al., 2015; (22) Boudadi-Maligne e al.,2020. (*) Calib a ions calcula ed in his a icle wi h he In Cal 20 cu e. (**)
Da es in cal. BP calcula ed in his a icle.
M. He ella e al.
Jou nal o A chaeological Science: Repo s 46 (2022) 103706
8
Table 2). This ch onology was olde han he da ings o he oldes dogs
iden i ied un il hen [Bonn-Obe kassel (Nobis, 1984), Knieg o e and
Teu elsb ücke (Musil, 1984)]. In 1994, in an a icle abou dogs in he
a chaeological si es o he No he n Ibe ian Peninsula, Al una insis ed
on he ce ain de e mina ion o his canid (Al una, 1994). La e , Vigne
(2005) ag eed wi h he iden i ica ion o Al una, indica ing ha he hu-
me us belonged o an indi idual o he genus Canis. The compa ison o
he dis al b ead h o he E alla hume us in he con ex o he La e
Paleoli hic and Mesoli hic dogs and wol es (Fig. 4) is also in line wi h
he p e iously men ioned e alua ions.
The di ec da ing o he E alla hume us h ough AMS
14
C
(17,410–17,096 cal. BP) (Lab. Uppsala) (p esen s udy) is in ag eemen
wi h he da ings o le el V, whe e his dog bone was ound (Table 2). The
ch onological disc epancy is due o he ac ha he da ing me hodol-
ogies used when he si e was s udied (Al una, 1985) we e less de eloped
han he cu en da ing me hodologies; u he mo e, hey equi ed la ge
amoun s o bone aken om he en i e le el. The p esen s udy ends he
unce ain y abou he age o he E alla dog, and alida es i in i s
ch onological con ex .
The gene ic analysis conduc ed in he E alla hume us indica es ha
he sequence o he cy och ome b gene (~200 bp) coincides wi h ha o
he genus Canis and no wi h ha o he genus Cuon. The p obabili y
alue was abo e 90% coincidence wi h he sequence o Canis, based on
he in o ma ion o he BLAST (NCBI) and Unip o da abases (Bou e
Table 3
Mi ochond ial haplog oup o Magdalenian, Epig a e ian, Epipaleoli hic and Mesoli hic dogs o Eu ope published o da e.
A chaeological si e Coun y Age cal. BP Labo a o y code Accession numbe Hg Sou ce
E alla Spain 17,410–17,096 Ua-56946 – C P esen s udy
Bonn-Obe kassel Ge many 14,809–13,319 NA KF661093 C Thalmann e al., 2013
G o a Paglicci
(Apulia)
I aly 14,372–13,759 OxA-26316 MH376892 C Boschin e al., 2020
Cuina Tu cului Romania *13,229 ENS, F ance d yad.8gp06 C Pionnie -Capi an, 2010
Kessle loch Swi ze land 14,286–13,975 MAMS-23874 KF661087 C Thalmann e al., 2013
Pon d’Ambon F ance 12,952–12,451 ENS, F ance d yad.8gp06 C Pionnie -Capi an, 2010
Ka s ein Ge many 12,378–11,256 NA KF661094 C Thalmann e al., 2013
Romanelli Ca e I aly 11,393–11,198 NA AY741668 B Ve ginelli e al., 2005
Icoana Romania *9,197 ENS, F ance d yad.8gp06 C Pionnie -Capi an, 2010
Icoana Romania *9,197 ENS, F ance d yad.8gp06 C Pionnie -Capi an, 2010
Icoana Romania *9,197 ENS, F ance d yad.8gp06 C Pionnie -Capi an, 2010
Icoana Romania *9,197 ENS, F ance d yad.8gp06 C Pionnie -Capi an, 2010
Os o ul Co bului Romania *8,921 ENS, F ance d yad.8gp06 C Pionnie -Capi an, 2010
Muge
(Cabeço da A uda)
Po ugal 7,570 Be a-152956 KY014676 C Pi es e al., 2019
Poças S. Ben o Po ugal 7,595 OxA-26094 KY014682 A Pi es e al., 2019
Muge
(Cabeço Amo ei a)
Po ugal 7,735 OxA-24571 KY014677 A Pi es e al., 2019
Muge
(Cabeço Amo ei a)
Po ugal 7,735 WK-36713 KY014683 A Pi es e al., 2019
Muge
(Cabeço Amo ei a)
Po ugal 7,755 Be a-448544 KY014675 A Pi es e al., 2019
Vale Boi (Alga e) Po ugal 7,903 Be a-448544 KY014652 A Pi es e al., 2019
(*): app oxima e da ings. The ollowing is indica ed: a chaeological si e, coun y, ch onology (acco ding o bibliog aphy), labo a o y code, accession numbe om
Genbank, mi ochond ial haplog oup (Hg) and sou ce o he gene ic s udy.
Fig. 5. Median-Joining Ne wo k o m DNA haplo ypes dis ibu ion o he Magdalenian/Epig a e ian/Epipaleoli hic (pink), Mesoli hic dogs (yellow) and
E alla dog (blue). Da a encompass m DNA D-loop (nps. 15495 o 15900). Posi ion 15639 is ans e sion. (Fo in e p e a ion o he e e ences o colou in his
igu e legend, he eade is e e ed o he web e sion o his a icle.)
M. He ella e al.
Jou nal o A chaeological Science: Repo s 46 (2022) 103706
9
e al., 2016). In addi ion o he gene ic in o ma ion ob ained om he
agmen o cy och ome b, he analysis o he mu a ions o a agmen o
181 bp o he m DNA D-loop (nps. 15495–15676) allowed assigning he
E alla hume us o he Canis lupus amilia is species wi h 99.6% p oba-
bili y and 98% iden i y (NCBI nucleo ide da abase). Simila ly, he
m DNA sequence eco e ed om he E alla hume us co esponds o
haplog oup C, which is he clade ha includes he Magdalenian Eu o-
pean dogs analysed o da e (Table 3) (Thalmann e al., 2013; F an z
e al., 2016). The e o e, he m DNA analysis indica es ha he hume us
o he E alla si e belongs o a dog (C. l. amilia is) o mi ochond ial
haplog oup C.
To da e, only ano he h ee Uppe Paleoli hic (Magdalenian and
Epig a e ian) dogs om he si es o Kessle loch (Swi ze land), Bonn-
Obe kassel (Ge many) and G o a Paglicci le el 4c (I aly) ha e been
iden i ied by a double c i e ion, i.e., mo phome ic and gene ic (Nobis,
1979; Napie ala and Ue pmann, 2012; Thalmann e al., 2013; Boschin
e al., 2020) (Table 2 and 3, Fig. 1), whe eas o he Magdalenian and
Epig a e ian dogs ha e been iden i ied only by he mo phome ic c i-
e ion (Ab i le Mo in, Hau e i e-Champ ´
e ey es, Le Closeau, Mon espan
and G o a Paglicci le el 5a: Bodu e al., 2006; Pionnie -Capi an e al.,
2011; Boudadi-Maligne e al., 2012; Boschin e al., 2020) (Table 2,
Fig. 1). Las ly, i is impo an o highligh ha some Magdalenian canids
ha e been a ibu ed o C. l. amilia is, al hough hey ha e no been
ce ainly con i med: ¨
Olkni z, Teu elsb ücke and Knieg o e (Musil 1985,
1980; 1974, espec i ely) (Table 2, Fig. 1). Ou s udy abou he E alla
bone allows including i wi hin he sca ce g oup o Magdalenian Eu o-
pean dogs iden i ied as C. l. amilia is h ough bo h c i e ia (mo pho-
me ic and gene ic). Thus, hei belonging o o he possible gene a is
disca ded, as well as hei inclusion wi hin he mo phome ic a iabili y
o he wol .
The da ing o he E alla hume us is he oldes among he da ings o
Paleoli hic dogs ound o da e, bo h he Magdalenian dogs [Ab i le
Mo in (15,114–14,237 cal. BP: Boudadi-Maligne e al., 2012), Kessle -
loch (14,286–13,975 cal. BP: Napie ala and Ue pmann, 2012; Baumann
e al. 2021), Bonn-Obe kassel (mean age 14,240–14,000 cal. BP: S ee ,
2002; S ee e al., 2015)] and he Epig a e ian dog o G o a Paglicci,
le el 4 (14,372–13,759 cal. BP: Boschin e al., 2020). Fu he mo e, he
ch onology o he E alla dog is olde o simila o ha o some speci-
mens o “dog-like wol es” (Elisee ichi I, c. 17,000–13,000 BP; Mezin
and Mezhe ic, c. 15,000–14,500; Ulakhan Sula , 17,200 cal. BP ap ox.)
(Sablin and Khlopache , 2002; Ge monp ´
e e al., 2009; Ge monp ´
e
e al., 2017) (Fig. 1). The coincidence in ime o he E alla dog wi h
some “dog-like wol es” opens new discussion lines abou he wol
domes ica ion p ocess, in which he so-called “dog-like wol es” may
ha e been a p edomes ica ion phase ha migh ha e occu ed in se e al
places o Eu asia in di e en imes. Howe e , some au ho s claim ha
“dog-like wol es” canno be he p edecesso s o dogs (O odo e al.,
2011; Mo ey and Jege , 2015), al hough his deba e should be
app oached wi h u he gene ic e idence.
The ch onology and da a o he m DNA o he E alla dog indica e
ha he e was a leas one dog lineage in Wes e n Eu ope ha belonged
o haplog oup C, al eady in he Lowe Magdalenian. Mos o he
Magdalenian and Mesoli hic dogs analysed o da e sha e he same
mi ochond ial haplog oup (Table 3, Fig. 5), excep o some Po uguese
Mesoli hic dogs ha ca y haplog oup A (Pi es e al., 2019). These e-
sul s sugges ha he e could ha e been a con inui y in he popula ion o
dogs o Magdalenian hun e -ga he e s in he Epipaleoli hic and
Mesoli hic.
Mo eo e , he p esen s udy abou he E alla dog may con ibu e o
he deba e on he da e o he wol domes ica ion o igin, since he e is no
ag eemen be ween he esul s o paleogene ics and he conclusions
in e ed om gene ic da a o mode n canids. Thus, some o he da es
p oposed o he di e gence o he dog lineage om he wol , such as
ha o 17,000–10,000 BP (Pang e al., 2009; F eedman e al., 2014,
among o he s udies) di e om hose in paleogene ic s udies, which
p opose da es be ween 41,500 and 36,900 BP (Bo igu´
e e al., 2017). In
his espec , he exis ence o domes ic specimens in he Lowe Magda-
lenian, as is he case o he E alla dog, p o es ha some in e ences
based in p esen -day gene ic da a o e da es ha a e oo ecen .
The e o e, his shows he impo ance o ha ing da a o ancien DNA o
calib a e mu a ion a es and make mo e accu a e es ima ions abou he
age o he dog lineage (Skoglund e al., 2015).
The o igin o clade C, which includes he Magdalenian dogs analysed
o da e, has been aced back o he pe iod be ween 24,000 and 16,000
BP in Eu ope by a paleogene ic s udy (Thalmann e al., 2013), which
coincides wi h he Las Glacial Maximum (LGM). Simila ly, some ge-
ne ic s udies sugges ha he popula ion o he dog lineage inc eased
a ound hose da es (Duleba e al., 2015). Mo eo e , i has been p oposed
ha he o iginal popula ion o Eu opean hun e -ga he e dogs would be
mos ly o haplog oup C (F an z e al., 2016; Olli ie e al., 2018).
The e o e, he E alla dog, om ~ 17,000 yea s cal. BP, belonging o
clade C, highligh s he impo ance o LGM in he o igins o wol
domes ica ion. The popula ion changes and he inc ease o densi y ha
ook place in he glacial e ugia. (S aus and Gonz´
alez Mo ales, 2012;
Maie , 2017) could ha e accele a ed a domes ica ion p ocess, which
may ha e begun ea lie , h ough he enhancemen o he in e ac ion
be ween humans and wild species du ing his s age o he clima e c isis.
In conclusion, he da a analysed o da e indica e ha , du ing he
Magdalenian, he domes ic dog was pa o he g oups o Wes e n Eu-
opean hun e -ga he e s. The dog om E alla (17,410–17,096 cal. BP)
is one o he oldes specimens iden i ied as Canis lupus amilia is, and i
sha es he mi ochond ial haplog oup C wi h he Magdalenian dogs
analysed o da e. These indings p o e ha his haplog oup exis ed in
Eu ope a leas since he Lowe Magdalenian, du ing he Ea ly D yas,
and lead us o conside a possible domes ica ion o he wol olde han
wha has been p oposed so a , a leas in Wes e n Eu ope.
CRediT au ho ship con ibu ion s a emen
Mon se a He ella: Concep ualiza ion, Me hodology, Fo mal
analysis, Resou ces, Supe ision, W i ing - o iginal d a . Asie San-
Juan-N´
o: Me hodology, Fo mal analysis, W i ing - o iginal d a . Alo˜
na
Aldaso o-Zabala: Me hodology, Fo mal analysis, W i ing – e iew &
edi ing. Ko o Ma iezku ena: Concep ualiza ion, Funding acquisi ion,
Resou ces, W i ing - o iginal d a . Jesús Al una: Concep ualiza ion,
Funding acquisi ion, Resou ces, Supe ision, W i ing - o iginal d a .
Concepci´
on de-la-Rua: Concep ualiza ion, Me hodology, Funding
acquisi ion, Fo mal analysis, Resou ces, Supe ision, W i ing - o iginal
d a , W i ing - e iew & edi ing.
Decla a ion o Compe ing In e es
The au ho s decla e ha hey ha e no known compe ing inancial
in e es s o pe sonal ela ionships ha could ha e appea ed o in luence
he wo k epo ed in his pape .
Da a a ailabili y
No da a was used o he esea ch desc ibed in he a icle.
Acknowledgmen s
This esea ch was suppo ed by a Basque Go e nmen G an o he
s udy o he E alla canid (C.R.) and o Resea ch G oups in he Basque
Uni e si y Educa ion Sys em (IT 1693-22). The awa d o a p edoc o al
ellowship o A.S.J.N. by he Spanish Go e nmen (FPU, Minis e io de
Ciencia Inno aci´
on y Uni e sidades) and a p e ious ellowship by he
Basque Go e nmen con ibu ed o his esea ch. We a e g a e ul o he
Basque Go e nmen (Cul u al He i age Depa men ) o g an ing us
pe mission o s udy he canid specimen and o he Cen o de Colecciones
Pa imoniales de Gipuzkoa (Go dailu) o g an ing us access o he
s udied a chaeozoological specimen. The au ho s would like o hank J.
M. He ella e al.
