Past domestication of T.

cacao in Latin America

revealed by paleogenomics and analyses of
methylxanthines

Claire Lanaud, Hélène Vignes, José Utge, Gilles Valette, Mariella Garcia
Caputi, Natalia Angarita, Bénédicte Rhoné , Olivier Fouet, Nilesh Gaikwad,
Terry Powis, Ann Cyphers, Francisco Valdez, Sonia Zarrillo, S. Quirino Olivera
Nuñez, Camilla Speller, Francisco Romano, Scott Raymond, R. Gaston Loor
Solorzano, Xavier Argout

FRANCE EQUATEUR COLOMBIE USA CANADA MEXICO

2022 International Symposium on
Cocoa Research (ISCR), Montpellier, France
 Diversity and classification of T. cacao
Phylogenic tree constructed
with 54 SSR CRIOLLO Colombia
CAQUETA

Ecuador Ecuador
Mexico Belize Genetic groups repartition CURARAY NACIONAL

Panama
Peru MORONA

Colombia
CONTAMANA Peru
NANAY
Ecuador
PURUS
IQUITOS
Brazil
Brazil MARANON
Peru
Fr GUYANA AMELONADO
Peru
Fr. Guiana
Brazil

2022 International Symposium on

Cocoa Research (ISCR), Montpellier, France
 Hypotheses made on the origin of oldest varieties
domestication
1) Introduction
of Nacional
genotypes
from South
Ecuadorian
Amazonia
CRIOLLO
?
?
2) Trinitario • •••
• •
• • ••
• • • • • ••
•• • • • • •• •
(Criollo x •
• •• • • • • •• • • •
• • • • •
•

• •
• ••
Amelonado) • ••
• EXEMPLE
introduced NACIONAL
D’ IMAGE
from Venezuela
one century
ago ? AMELONADO

2022 International Symposium on

Cocoa Research (ISCR), Montpellier, France
 Chronology of ceramics from different cultures
collaborations established with archaeologists and
anthropologists from different Latin American countries and
biochemists to analyse ceramic food residues
SAN JACINTO
PUERTO HORMIGA
CALIMA-LLAMA
BARLOVENTO
SAN AUGUSTIN
NARINO
Colombia LETICIA
ARARACUARA
LA TOLITA 379 ceramic items
ZENU
VALDIVIA analysed from 19
MACHALILLA
Ecuador CHORRERA different culltures:
BAHIA
JAMA COAQUE • directly excavated
Per u JAEN MONTEGRANDE
• in the museum
Belize
Mexico OLMEC
MAYA storage rooms
Panama
→ Methylxanthines
-4000 -3000 -2000 -1000 0 1000
→ Ancien DNA
Years BC Years AD
2022 International Symposium on Cocoa Research (ISCR), Montpellier, France
 Methylxanthine Analyses
332 analysed samples from South America
• 121 are positive for theobromine caffein ECUADOR caffein COLOMBIA
20000
• 129 are positive to Caffeine 9000

• 25 are positive for theophylline 8000 18000

16000
7000

→ Positive for methylxanthine in 15 cultures, and 6000

14000

among them the oldest (Valdivia-San Jacinto) 12000

5000
10000
4000
THEOPHYLLINE THEOBROMINE CAFFEINE 8000
3000
6000

2000
4000
Standard 1000 2000
15ng/ml
0 0
0 1000 2000 3000 4000 5000 0 2000 4000 6000 8000
Theobromine Theobromine

Valdivia
sample

2022 International Symposium on Cocoa Research (ISCR), Montpellier, France

 Use of molecular tools to access cocoa
ancient DNA (aDNA)
PUERTO HORMIGA
MX114_direct

80
Single−end read length distribution Single−end read length per strand

1) COLLECT of ceramic residues

+ strand
40
− strand

2) EXTRACTION of ancient DNA (very 60

30

subplus$Occurences
Occurences

Occurences
40
20

(archéo. excavations, museums) degradated) – libraries construction

20
10

0 0

28
38
48
58
68
78
88
98
108
118
128
138
148
158
168
178
188
198
208
218
228
238
248

28
38
48
58
68
78
88

98
108
118
128

138
148
158
168
178
188
198
208
Read length Read length
subplus$Length

C>T G>A

c(0, cumsum(subplus[, mut]/sum(subplus[, mut])))

cumsum(subplus[, mut]/sum(subplus[, mut])))

of 170 items 1

0.9
+ strand
− strand
+ strand
− strand
1

0.9

Cumulative frequencies

Cumulative frequencies
0.8 0.8

0.7 0.7

0.6 0.6

0.5 0.5

0.4 0.4

0.3 0.3

0.2 0.2

0.1 0.1

0 0

Experimentations made in a white

c(0,
0

10

20

30

40

50

60

70

10

20

30

40

50

60

70
Read position Read position
Index Index

Posterior prediction inter vals

room to avoid external contamination 0.075

Substitution rate
by modern DNA.(MNHN)
Subs. type
0.050
C−>T
G−>A
Others

0.025

3) SEQUENCING and BIOINFORMATIC analyses (mapping on the 1 3 5 7 9 11

Relative position
−11 −9 −7 −5 −3 −1

cocoa genome and blast against international databases (NCBI) Signature of aDNA

4) GENETIC analyses Identification of SNP markers in the aDNA sequences, based on the SNP pool
obtained after resequencing 185 modern cocoa genotypes (Pangenome project)

Comparison with a ref. collection of 81 individuals On 170 analysed samples:

representing the diversity of the T. cacao species; • 92 are positive for cocoa aDNA
Structure and genetic distance. • 56 allowed genetic comparisons
2022 International Symposium on Cocoa Research (ISCR), Montpellier, France
 Structure analysis of cocoa residues in Ecuador and Peru
VALDIVIA Genotype membership proportion
MACHALILLA
FIIa-5.300 - 5.100 BP (Before Present) Mexico Belize Genetic groups repartition 1
2
3.600 - 3.000 BP Guiana
Wild Criollo 3
MX29 Caqueta
C 4
Nanay Curaray 5
C MX131 MX187
Panama
Amelonado Nacional
6
7
C C Colombia Iquitos Purus 8
Contamana 9
Maranon
1

FIII-4.950 - 4.600 BP Chorrera 1

Ecuador

MX30
Valdivia Brazil
MX6 MX36
C C C Machalilla Peru

MX42 MX33 MX38 MX228

3000 - LA TOLITA JAEN-PERU
C C C C CHORRERA 2500 BP
2600 BP – 3830 BP –
MX1 MX7 MX204
MX35 1600 BP 3590 BP
MX46 C C C
C C
MX5
C MX213 MX232
C C
F8-3800 - 3400 BP
MX41 MX43 MX44
C C C
 SAN JACINTO CALIMA LLAMA Colombia Genotype membership proportion
1

5750 à 4000 BP 3600 à 2100 BP Wild Criollo

2
3
Guiana Caqueta
C 4
Mexico Belize
5
MX75 MX93 Nanay Curaray
6
C C Puerto Hormiga Amelonado Nacional
7
Iquitos Purus 8
Panama
Contamana 9
Maranon
1
Calima llama Colombia 1
MX96
C San Augustin
PUERTO HORMIGA Ecuador

Leticia Brazil

5100 à 4550 BP
MX98 Peru

MX67 MX120 SAN AUGUTIN 3000 à 2000 BP LETICIA

C C MX99

C
MX21 MX61 2350 à 800 BP
C C
MX87
MX178
MX74 MX68
C C
MX129
C C
C ZENU 2200 à 1900 BP

MX69 MX20
MX73
MX22 C
C C
C
2022 International Symposium on
Cocoa Research (ISCR), Montpellier, France
 1
2

Central America Genotype

Guiana
Wild Criollo

Caqueta
C
3
4
Curaray 5

membership
Nanay
6
Amelonado Nacional

Mexico Belize proportion

7
Iquitos Purus 8
Contamana 9
Maranon
10

OLMEC 3800-3000 BP MAYA 2600-2300 BP 11

Mexico Belize

MX8 MX151

C C
Panama

Colombia

MX156
MX9 Ecuador

C C Brazil

Peru

Panama
MX225
2300 - 1750 BP
C
MX144 MX198
Gran Chiriqui 3500 - 1600 BP
C C
MX92 MX91
MX224
C C
C

2022 International Symposium on

Cocoa Research (ISCR), Montpellier, France
 Barlovento
Conclusion
PANAMA Puerto

Zenu
Hormiga
• Widespread use of cocoa in South America for over 5000 years, in
Ecuador as well as in Colombia, evidenced by ancient DNA and
COLOMBIE
La Tolita
Calima-Llama

San Augustin
methylxanthine presence.
Atacames
Jama-coaque Araracuara
Chorrera

Valdivia

PEROU
Leticia
• Large diversity and genetic mixing of introduced cocoa trees from
Jaen
different origins, sometimes distant, that reflects the numerous
Archaeological sites positive for cocoa
interactions between populations

• During Olmec and Maya occupation, Criollo was not the unique cultivated
variety, but genotypes related to Amelonado, Nacional and other genetic
2022 groups were also present, suggesting possible interactions with Ecuador where
International
Symposium these genotypes existed , and with Colombia where genotypes close to Criollo
on
Cocoa already existed for 5000 years .
Research
(ISCR),
Montpellier,
France
 Conclusion

• Thus, paleogenomic approaches have strongly challenged our first hypotheses

on cocoa domestication and help us to better understand and manage the genetic
resources available.

• All these results show the complex domestication history of cocoa varieties
probably linked to different migratory waves from the beginning of the Holocene
and to many trading exchanges within the Amazon and with the Pacific Coast.

• These movements and activities were associated to direct or undirect multiple

cocoa introductions from different origins, leading to hybrid forms, favorable to
the domestication of productive varieties adapted to new environment , and now
at the basis of the current cocoa varieties
2022 International Symposium onCocoa Research (ISCR), Montpellier, France
Archaeologists-anthropologists Acknowledgements
MAAC (Ecuador)
• Mariella Garcia Caputi and her team Geneticists
• Musée du quai Branly (France) UMR AGAP-CIRAD (France)
• IRD/INPC (France-Ecuador) - Francisco Valdez • Hélène Vignes
• Xavier Argout
University of British Columbia - (Canada) • Bénédicte Rhone
• Sonia Zarillo • Olivier Fouet
• Camilla Speller
• MNHN (France) – José Utge
University of Calgary - (Canada)
• Scott Raymond
Biochemists
Museo Nacional de Colombia- ICANH (Colombia) • UM (France) - Gilles Valette
• Natalia Angarita
• Francisco Romano • Steroidomics (USA)- Nilesh Gaikwad

University of Kenesaw – (USA)

• Terry Powis
Financial contribution of
UNAM – (Mexico) • ASICAMPE - (Peru)
• Ann Cypher Quirino Olivera Nuñez MUSE (Montpellier Université d’excellence)
2022 International Symposium on VALRHONA
Cocoa Research (ISCR), Montpellier, France
 Thank you for
your attention

2022 International Symposium on

Cocoa Research (ISCR), Montpellier, France