Starch consumption stands out as a significant characteristic of agricultural societies and hunter-gatherers thriving in arid environments. Conversely, those in rainforests, circum-arctic regions, and certain pastoralist communities consume considerably less starch. This variation in dietary habits suggests that different selective pressures may have influenced amylase, the enzyme responsible for starch hydrolysis.
Amylase: The Starch-Digesting Enzyme
Amylase is a biological compound produced by humans and other animals to break down starch, a complex carbohydrate that serves as a vital source of nutrition for many mammals. Humans cultivate it in the form of rice, wheat, corn, potatoes, and oats, while rats scavenge for scraps of pizza and bread, and wild boars root for tubers.
The Correlation Between AMY1 Copy Number and Diet
Research has revealed a positive correlation between the copy number of the salivary amylase gene (AMY1) and salivary amylase protein levels. Individuals hailing from populations with high-starch diets tend to possess, on average, more AMY1 copies compared to those with traditionally low-starch diets. Comparisons with other loci in a subset of these populations suggest that the extent of AMY1 copy number differentiation is highly unusual. This example of positive selection on a copy number-variable gene is among the first discovered in the human genome.
The Gene-Diet Connection
Mammals with starchy diets appear to have adapted, genetically, to stomach more carbs. Of the species studied, those with starch in their diets generally have more copies of the amylase gene, which carries instructions for making amylase, than animals like carnivores and herbivores whose strict diets tend to exclude starch. Carb-munching humans, house mice, brown rats, dogs, pigs, and boars have lots of copies, while mammals like mountain lions, which subsist on meat, and hedgehogs, which dine on foods such as insects and snails, have few. This is important because the gene is akin to a mold in a factory: the more units you have, the more amylase you can theoretically produce. As for how the extra copies of the amylase gene evolved, “It’s like the chicken and the egg - we cannot really tell what came first,” Ruhl says. “Starch in the diet may have led to more amylase, and the ability to digest starch may have led to increased starch intake, and so forth.”
Convergent Evolution: A Useful Adaptation
The genetic expansion of amylase likely occurred independently in multiple species. Based on genetic evidence, the study concluded that mice, rats, dogs, pigs, and humans likely acquired some of their extra copies of the amylase gene independently, at separate times in their evolution, rather than inheriting all the copies from a common ancestor. This phenomenon, called convergent evolution, can signal a particularly useful adaptation.
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Salivary Amylase: More Widespread Than Previously Thought
Amylase in saliva is more widespread than previously known (some pet dogs produce it, for example). Most amylase is produced in the pancreas, but some animals also secrete it in saliva. The new research finds that this capability is more common than previously known and proposes salivary amylase as another adaptation that may have arisen through convergent evolution in some species. When scientists tested for amylase in the drool of 22 mammalian species, they found it in 15 species, including six species that were not previously known to have amylase in saliva. Perhaps unsurprisingly, baboons and rhesus macaques that store food in cheek pouches for long periods of time were among the most prolific producers of salivary amylase among the mammals tested. Pet dogs were among the species that were newly identified as salivary amylase producers. While not all dogs have amylase, the research found it in several breeds, such as English cream golden retrievers, Labradors, and pitbulls.
The Evolutionary Advantage of Salivary Amylase
For animals who don’t store food in their cheeks, the evolutionary advantage of having amylase in saliva is unclear. But Ruhl, a leading salivary researcher, says one theory is that it helps animals and humans identify starchy foods as desirable to eat. Humans have a lot of salivary amylase, but why? Unlike the baboons who predigest food in their cheek pouches, we humans do not keep food in our mouths long enough for any substantial digestion to happen. One idea is that salivary amylase evolved to help our ancestors detect starch. They would not be able to taste it otherwise. Amylase liberates sugar in starch, and this may help animals develop a taste preference for starch-rich foods like potatoes or corn. Other hypothesized purposes for salivary amylase include cleaning sticky starch residues from teeth. Amylase in saliva might act as a kind of biochemical toothbrush nature has provided us with.
Dietary Shifts and Human Evolution
Hominin evolution is characterized by significant dietary shifts, facilitated in part by the development of stone tool technology, the control of fire, and most recently the domestication of plants and animals. Starch, for instance, has become an increasingly prominent component of the human diet, particularly among agricultural societies. It stands to reason, therefore, that studies of the evolution of amylase in humans and our close primate relatives may provide insight into our ecological history.
AMY1 Copy Number Variation and Protein Expression
Because the human salivary amylase gene (AMY1) shows extensive variation in copy number, it is important to assess whether a functional relationship exists between AMY1 copy number and the level of amylase protein expression in saliva. Studies estimated diploid AMY1 gene copy number for 50 European-Americans using an AMY1-specific real-time quantitative polymerase chain reaction (qPCR) assay. Extensive variation in AMY1 copy number in this population sample was observed, consistent with previous studies. Western blot experiments with saliva samples from the same individuals were performed in order to estimate salivary amylase protein levels. These experiments revealed a significant positive correlation between salivary amylase gene copy number and protein expression level.
Population-Based Differences in AMY1 Copy Number
While there is a considerable range of variation in dietary starch intake among human populations, a distinction can be made between “high-starch” populations for which starchy food resources comprise a substantial portion of the diet, and the small fraction of “low-starch” populations with traditional diets that incorporate relatively few starchy foods. Such diets instead emphasize proteinaceous resources (e.g., meats and blood) and simple saccharides (e.g., from fruit, honey, and milk). To determine if AMY1 copy number differs among populations with high- and low-starch diets, AMY1 copy number was estimated in three high-starch and four low-starch population samples. The high-starch sample included two agricultural populations, European-Americans and Japanese, and Hadza hunter-gatherers who rely extensively on starch-rich roots and tubers. Low-starch populations included Biaka and Mbuti rainforest hunter-gatherers, Datog pastoralists, and the Yakut, a pastoralist/fishing society. Mean diploid AMY1 copy number is greater in high-starch populations. Strikingly, the proportion of individuals from the combined high-starch sample with at least 6 AMY1 copies (70%) is nearly 2 times greater than that for low-starch populations (37%). To visualize the allele-specific number and orientation of AMY1 gene copies, high-resolution fluorescence in situ hybridization on stretched DNA fibers (fiber FISH) was performed; these results were consistent with diploid AMY1 copy number estimates from qPCR experiments.
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Natural Selection and AMY1 Copy Number
The among-population patterns of AMY1 copy number variation do not fit expectations under a simple regional-based model of genetic drift: high- and low-starch samples include both African and Asian populations, suggesting that diet more strongly predicts AMY1 copy number than geographic proximity. Based on this observation, it has been hypothesized that natural selection may have influenced AMY1 copy number in certain human populations.
Observations suggest that natural selection has shaped AMY1 copy number variation in either the Japanese or the Yakut, or in both populations. It is favored a model in which AMY1 copy number has been subject to positive or directional selection in at least some high-starch populations but has evolved neutrally (i.e., through genetic drift) in low-starch populations.
Several lines of evidence offer mechanisms by which higher salivary amylase protein levels may confer a fitness advantage for individuals with a high-starch diet. First, a significant amount of starch digestion occurs in the mouth during mastication. For example, blood glucose levels have been shown to be significantly higher when high-starch foods such as corn, rice, and potatoes (but not apples) are first chewed and then swallowed, rather than swallowed directly. In addition, it has been suggested that oral digestion of starch is critically important for energy absorption during episodes of diarrhea. Lastly, salivary amylase persists in the stomach and intestines after swallowing, thereby augmenting the enzymatic activity of pancreatic amylase in the small intestine.
AMY1 Copy Number Variation in Primates
To understand better the evolutionary context of human AMY1 copy number variation, patterns of AMY1 copy number variation in chimpanzees (Pan troglodytes) and bonobos (Pan paniscus) were analyzed. In contrast to the extensive copy number variation observed in humans, each of 15 wild-born western chimpanzees (P. t. verus) showed evidence of only 2 diploid AMY1 copies, which is consistent with previous findings. Although evidence of a gain in AMY1 copy number in bonobos relative to chimpanzees was observed, sequence-based analyses suggest that each of these AMY1 copies has a disrupted coding sequence and may be non-functional. Therefore, the average human has ∼3 times more AMY1 copies than chimpanzees, and bonobos may not have salivary amylase at all. Outgroup comparisons with other great apes suggest that AMY1 copy number was most likely gained in the human lineage, rather than lost in chimpanzees. Given that AMY1 copy number is positively correlated with salivary amylase protein level in humans, it stands to reason that the human-specific increase in copy number may explain, at least in part, why salivary amylase protein levels are ∼6-8 times higher in humans than in chimpanzees. These patterns are consistent with the general dietary characteristics of Pan and Homo; chimpanzees and bonobos are predominantly frugivorous and ingest little starch relative to most human populations.
Considering other primates, while New World monkeys do not produce salivary amylase and tend to consume little starch, cercopithecines (a subfamily of Old World monkeys including macaques and mangabeys) have relatively high salivary amylase expression, even compared to humans.
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Plant Underground Storage Organs and the Initial Increase in AMY1 Copy Number
The initial human-specific increase in AMY1 copy number may have been coincident with a dietary shift early in hominin evolutionary history. For example, it is hypothesized that starch-rich plant underground storage organs (USOs) were a critical food resource for early hominins. Changes in USO consumption may even have facilitated the initial emergence and spread of Homo erectus out of Africa.