Most popular form of manioc has been consumed for 9,000 years

Molecular study suggests sweet manioc or cassava has a different dispersal history from bitter manioc. Domestication involved selection of varieties with lower levels of prussic acid, making consumption possible.

By Peter Moon  |  Agência FAPESP – Manioc or cassava in English; mandioca, mandioca-mansa, macaxeira, aipim and various other names in Brazil; mandioca, manioc, yuca or casava in Spanish. There are many ways of designating the species Manihot esculenta, which has edible starchy tuberous roots and was domesticated some 9,000 years ago. Genetic and archeological research shows that domestication occurred in the upper Madeira River region of today’s Rondônia State in northern Brazil.

How manioc cultivation developed and evolved throughout the Americas is poorly understood. Some speculate that from the original center of domestication in southwestern Amazonia, it spread to other indigenous communities along the region’s great rivers, still the main thoroughfares for inhabitants of Amazonia.

To test this hypothesis, a genetic diversity study was conducted by Alessandro Alves-Pereira, a former PhD student affiliated with the University of São Paulo’s Luiz de Queiroz College of Agriculture (ESALQ-USP) in Brazil and now a postdoctoral researcher in the Plant Biology Department at the University of Campinas’s Biology Institute (IB-UNICAMP). The study was supervised by Maria Imaculada Zucchi, a researcher at the Agronomy Institute (IAC), and supported by FAPESP. Results were published in the journal Annals of Botany.

“Integrated archeological and ethnobotanical research suggests the spread of manioc growing was associated with human movement along the Amazon’s rivers in prehistoric times,” Alves-Pereira said. “We therefore decided to use molecular biology techniques to look for genetic signs of this dispersal by analyzing variation in the plant’s genome.” 

The group, which included other researchers affiliated with ESALQ-USP, the National Institute for Research on Amazonia (INPA) and the Federal University of Amazonas (UFAM), studied the two genomes of Manihot esculenta: the nuclear genome, which consists of the DNA found within cell nuclei, and the chloroplast genome. Chloroplasts are the plant cell organelles that perform photosynthesis.

Each genome provides a particular kind of information on evolutionary history. Most chloroplast genomes of angiosperms like manioc are transmitted maternally between generations, so that a matrilineal phylogenetic tree can be constructed by comparing the genomes of several samples of manioc collected in different regions. 

The nuclear genome, in contrast, undergoes recombination with every reproductive event, mixing parts of the father plant’s and mother plant’s nuclear DNA during embryo fertilization. 

“The nuclear genome gives us a more recent ‘snapshot’ of manioc’s diversity and reveals greater variation than the chloroplast genome, but it can’t take us very far back in time to find out when diversifications occurred,” Alves-Pereira said.

The material analyzed came from communities of small farmers in 44 municipalities on five major Amazonian rivers: the Negro, Branco, Madeira, Solimões and Amazon. Samples were also collected in southern Rondônia and northeastern Pará. 

Leaves were collected between 2010 and 2015 from 596 individuals, of which 325 were bitter manioc, 226 sweet manioc, 28 wild manioc (Manihot esculenta ssp. flabellifolia), and 17 undesignated (found outside croplands or kitchen gardens, and thus without associated traditional knowledge). 

The wild subspecies Manihot esculenta ssp. flabellifolia was domesticated 9,000 years ago. “Wild manioc has roots that accumulate starch, but they aren’t as large as the roots of the domesticated forms,” Alves-Pereira said.

“Wild manioc is also found in different forms in nature. It grows as a tall bush in open environments and as a vine in closed forest conditions. Domesticated manioc is a one- or two-meter shrub with less volume and ramification than in the wild.” 

The main difference between the different varieties is the degree of toxicity. Bitter manioc is highly poisonous. Its roots contain significant amounts of prussic acid (hydrogen cyanide) precursors and can be lethal if eaten raw or prepared incorrectly. 

Domestication involved selection of varieties with lower levels of poisonous substances, arriving at a plant with very little toxicity that can be consumed with practically no processing.

The product sold by supermarkets, street markets and corner stores is sweet manioc, which also contains a certain amount of toxic substances and therefore cannot be consumed immediately. The roots have to be chopped up, peeled and cooked to eliminate the poison.

The indigenous inhabitants of the region where wild manioc was domesticated developed techniques to remove the large amount of cyanide precursors, such as peeling and grinding the roots, pressing the resulting pulp to remove some of the toxins, and boiling to evaporate the acid or fermenting to produce cauim, a traditional alcoholic beverage enjoyed by indigenous people since pre-Columbian times.

To understand how manioc cultivation spread, the researchers had to find out how and where sweet and bitter manioc differentiated from their wild ancestor. Alves-Pereira’s laboratory used conventional genetic techniques to extract DNA from manioc leaf cells. The next step was to look for molecular markers that could serve as reference points in comparing the genomes of the various lineages.

More specifically, the team looked for microsatellites, short segments of DNA with repetitive sequences that are inherited from both parents and are useful for kinship analysis. “By identifying microsatellites, we were able to study the genetic relationships between individuals. We used 14 nuclear microsatellites and four chloroplast microsatellites,” he said.

Diversification and domestication

When the researchers compared the genomes of the 596 samples, they were surprised to find that genetic variation among them did not point to a biogeographical bias. In other words, analysis of the nuclear genomes did not reveal the existence of regional varieties. “We thought genetic analysis of manioc varieties would give us clues to how cultivation spread via Amazonia’s rivers. That’s not what happened,” Alves-Pereira said.

According to Zucchi, they expected to find genetic evidence to explain manioc’s geographical dispersal. “We failed to detect the expected significant variation between individuals collected near different rivers,” she said. “What we did detect was substantial diversity between the sweet and bitter varieties.”

If nuclear genome analysis proved inconclusive, by contrast, chloroplast genome analysis revealed something unknown. Because manioc domestication is a process that has been under way for thousands of years, the researchers assumed it must have been necessary to derive thousands of generations from a wild ancestor in order to arrive at sweet manioc. By the same token, they thought the emergence of bitter manioc must have marked an intermediate stage in the domestication process that ended with sweet manioc.

“However, the data pointed to an unexpected result. Sweet manioc displays a higher level of heterozygosity and significant divergence compared to bitter manioc,” Alves-Pereira said.

In the case of sweet manioc, the larger amount of heterozygotes (different genotypes for the same allele) suggests a longer diversification period after domestication than for bitter manioc. 

According to Alves-Pereira, the lower degree of heterozygosity observed in the case of bitter manioc may indicate that less time has elapsed since its domestication.

The evidence of less endogamy for sweet manioc reinforces this theory. The larger or older a species population or group of individuals undergoing domestication is, the less likely that there will be crossbreeding between siblings or cousins “if different varieties are selected for distinct preferences by different growers,” Alves-Pereira said.

The higher levels of heterozygosity and lower endogamy found for sweet manioc can be seen as the signature of a longer period of diversification following its domestication. In the case of bitter manioc, less heterozygosity and more endogamy may indicate a shorter diversification period.

“We concluded that a possible interpretation for the genetic variation data and how this variation is spatially distributed was that sweet manioc was domesticated first, some 9,000 years ago, as suggested in the genetic and archeological literature. Bitter manioc was domesticated much later. So, the dispersal process appears to have been very different for each variety in both time and space,” Alves-Pereira said.

Selection of wild manioc varieties with low levels of toxicity by pre-Columbian indigenous populations until they arrived at sweet manioc must have been an older process, he added, because the Amazon is believed to have been sparsely inhabited at that time, and these communities were nomadic, with a smaller demand for food that could be met by sweet manioc growing close to wherever they were living. 

What about bitter manioc? Once they had domesticated sweet manioc, ancient groups of hunter-gatherers began to abandon nomadism for a more sedentary life in villages, growing their own manioc. The archeological record indicates the onset of population growth among pre-Columbian peoples as occurring between 4,000 and 3,000 years ago. More manioc had to be cultivated to feed more mouths.

“Today, most inhabitants of the Amazon grow sweet manioc in their backyards, while bitter manioc is cultivated in much larger areas of cleared forest,” Alves-Pereira said.

Is this how it was 4,000 years ago? The theory that bitter manioc was domesticated at a time of population growth in sedentary communities raises an as yet unanswered question. Was it the need to feed more mouths that forced them to look for new food sources and ultimately to develop detoxification techniques in order to be able to consume bitter manioc or was population growth made possible by the increase in food supply due to domestication of bitter manioc?

This is not a question geneticists can answer but a hypothesis to guide future archeological prospecting in the Amazon. According to Zucchi, research on the manioc genome continues. Alves-Pereira is currently processing more than 5,000 markers called single nucleotide polymorphisms (SNPs), which are being used for the construction of a more refined genetic analysis.

The article “Patterns of nuclear and chloroplast genetic diversity and structure of manioc along major Brazilian Amazonian rivers” (doi:10.1093/aob/mcx190) by Alessandro Alves-Pereira, Charles R. Clement, Doriane Picanço-Rodrigues, Elizabeth A. Veasey, Gabriel Dequigiovanni, Santiago L.F. Ramos, José B. Pinheiro and Maria I. Zucchi is published at: academic.oup.com/aob/article-abstract/121/4/625/4791086.