Insight into the Wild Origin, Migration and Domestication History of the Fine Flavour Nacional Theobroma cacao L. Variety from Ecuador
Randall P. Niedz, Editor
Ecuador’s economic history has been closely linked to Theobroma cacao L cultivation, and specifically to the native fine flavour Nacional cocoa variety. The original Nacional cocoa trees are presently in danger of extinction due to foreign germplasm introductions. In a previous work, a few non-introgressed Nacional types were identified as potential founders of the modern Ecuadorian cocoa population, but so far their origin could not be formally identified. In order to determine the putative centre of origin of Nacional and trace its domestication history, we used 80 simple sequence repeat (SSR) markers to analyse the relationships between these potential Nacional founders and 169 wild and cultivated cocoa accessions from South and Central America. The highest genetic similarity was observed between the Nacional pool and some wild genotypes from the southern Amazonian region of Ecuador, sampled along the Yacuambi, Nangaritza and Zamora rivers in Zamora Chinchipe province. This result was confirmed by a parentage analysis. Based on our results and on data about pre-Columbian civilization and Spanish colonization history of Ecuador, we determined, for the first time, the possible centre of origin and migration events of the Nacional variety from the Amazonian area until its arrival in the coastal provinces. As large unexplored forest areas still exist in the southern part of the Ecuadorian Amazonian region, our findings could provide clues as to where precious new genetic resources could be collected, and subsequently used to improve the flavour and disease resistance of modern Ecuadorian cocoa varieties.
Theobroma cacao L. is the most economically important species of the Theobroma genus which is a member of the Malvaceae family. It is a diploid perennial species (2n = 2× = 20) with a small genome ranging in size from 411 to 494 Mb . Most of the T. cacao accessions are self incompatible due to a gameto-sporophytic incompatibility system –. However, some varieties, domesticated long ago, as the varieties Criollo from central America, Comun from Brazil, or Nacional from Ecuador, and some of their hybrid forms are self compatible.
Traditionnaly, two main genetic groups have been defined to classify cocoa accessions, based on morphological traits and geographical origins : Criollo and Forastero. This classification also reflects the first commonly cultivated cocoa varieties: Criollo, first domesticated in Central America more than 2000 years ago, and a Lower Amazon Forastero variety (Amelonado type) domesticated in Brazil. Trinitario, a third group, corresponding to hybrids between Criollo and Forastero, was also recognised. However, the Forastero group also includes many other populations from all the Amazonian and Orinoco regions and presents a high diversity, as revealed by many genetic studies , , –. A new classification, identifying 10 genetic clusters, was more recently proposed .
The domestication of Criollo, originally cultivated by the Mayas in Central America was previously studied . The authors concluded that the genetic base of the ‘ancient’ Criollo variety, cultivated in Central America before foreign cocoa introductions occurred during the 18th century, was very narrow.
Cocoa cultivation in Ecuador is very ancient, dating back to the pre-Columbian age before the Spanish colonization of this territory. Pizarro, during his first voyage in 1526 along the South American coasts (nowadays Ecuador), found evidence of small plantations of an apparently native cocoa tree , . It is very likely that the ’Nacional’ Ecuadorian cocoa population existed for several centuries prior to the arrival of the Europeans , but its origin in the Ecuadorian coastal region has never been clarified. Two hypotheses have been put forward: according to Allen and Lass , the Nacional cocoa variety could have originated from a local wild population, that has nowadays completely disappeared along with the original forest cover of the region, or Nacional could have been introduced in the coastal region from the Amazonian area of Ecuador where wild cocoa is common –. There is no evidence that T. cacao and its products played any part in the lives of Ecuadorian coastal inhabitants during the Chorrera phase of the pre-Columbian Valdivia culture (2000 BC), contrary to the extensive amount of information available on this phenomenon with respect to Mesoamerican people, most notably the Mayas and Aztecs. In Ecuador, available information related to the history of Nacional cocoa cultivation is linked with the Spanish colonization history in this country.
Since the time when the first Spanish colonists began deforestation of the Ecuadorian coastal region, a large number of native cocoa trees have been reported –, principally along the Guayas basin. Apparently, these colonists began to sow seeds of these native trees approximately 100 years after the discovery of America, and when the native Mesoamerican populations started to decline. The cocoa cultivation areas expanded, and the native Nacional variety rapidly became known worldwide due to its strong floral so-called “Arriba” aroma, exclusively generated by Nacional cocoa beans. The fine flavour quality of chocolate products obtained from Nacional cocoa beans has always been highly appreciated by chocolate manufacturers.
The native Nacional variety was the only one planted in Ecuador until the early 1890s, when foreign germplasm was first introduced in this country. In 1890, due to the quality traits of Nacional cocoa beans, Ecuador had a privileged position in the markets of Hamburg and London . From 1910, foreign germplasm introductions progressively increased due to the appearance of two fungal diseases known as witches’ broom (Moniliophthora perniciosa) and frosty pod (Moniliophthora roreri), which together devastated the native plantations.
A large genetic admixture between the native variety and foreign germplasm is currently found in modern Ecuadorian cocoa plantations . The fine flavour cocoa aroma has decreased in this hybrid complex, and 25% of the Ecuadorian cocoa production was recently classified as ‘bulk’ cocoa by the International Cocoa Organization (ICCO). There is nowadays increasing demand for fine flavour cocoa, which presently represents 6.8% of the world cocoa production and for which Ecuador remains the main supplier .
In a previous work, a few non-introgressed Nacional types were identified within this hybrid population as potential representatives of the native Nacional variety . Here we analyze their relationships with a wide range of wild cocoa genotypes covering a broad geographical range from upper and lower Amazonian regions , , , , , in order: a) to identify the putative centre of origin of Nacional, and b) to trace the domestication history of the Nacional cocoa variety.
Materials and Methods
A total of 176 individuals from different geographical origins were used for simple sequence repeat (SSR) analyses in this study (Table 1 and Table S1):
Origin of the 14 groups of accessions used in this study.
- Seven putative ancient Nacional variety individuals cultivated along the Ecuadorian coastal region .
- Sixty-five wild genotypes collected in the northern region A (LCT-EENa), central region B (LCT-EENb) and southern region C (LCT-EENc) of the upper Amazonian region of Ecuador (Fig. 1b) .Geographic distribution and locations of the 14 groups of cocoa accessions used in this study.
Eighty SSR markers were chosen for these analyses (Table 2). Seventy four SSRs were isolated from expressed sequence tags (EST) and six from genomic sequences, and mapped –. They are distributed on all of the 10 cocoa chromosomes.
List of SSR loci (mTcCIRx) and number of alleles per locus identified in this study.
DNA Isolation and PCR Amplification
Amplified SSR fragments were scored as alleles. The following genetic parameters were calculated for each population using GENETIX software : unbiaised expected heterozygosity  (HE), observed heterozygosity (HO), proportion of polymorphic loci at which the frequency of the most common allele does not exceed 95% (P0.95) and 99% (P0.99), and mean number of alleles per locus.
We also estimated allelic richness (Rs) for each population, using a rarefaction approach to correct for unequal sample sizes  with the program FSTAT V. 2.9.3 .
Wright’s F-statistics were estimated according to Weir and Cockerham  using the GENETIX software: FIS, an estimate of heterozygote deficiency or excess, calculated for the whole population sample and for each population, and FST, the proportion of variance due to population differenciation, calculated for the whole population sample and between pairs of populations; their significance were assessed using a 1000-fold permutation test.
As most mutations in SSR involve the addition or subtraction of a small number of repeat units, according to the stepwise mutation model population, genetic differentiation was also estimated by RST . RhoST, an unbiased version of RST in which allele sizes are transformed to standardize variances, was estimated for pairs of populations using GENEPOP software, WEB version 4.0.10 –.
Analyses of genetic distance among pairs of populations  were carried out to determine the genetic similarity between the Nacional pool and the wild and cultivated genotypes using GENETIX.
In addition, a dendrogram was constructed using a dissimilarity index (simple matching) and the neighbour-joining (NJ) method with 500 bootstrap replicates. Neighbour-joining cluster analyses were carried out using DARWIN software 5.0 .
Finally, a paternity analysis was performed using the software CERVUS, version 2.0 , to identify the most likely ancestral population of the Nacional variety. An 80% confidence threshold was used for paternity assignment.
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