Evolutionary Anthropology 25:253–266 (2016)

ARTICLE

Digestive of Human and Nonhuman Primates

MAREIKE CORA JANIAK

All living organisms need to consume nutrients to grow, survive, and that were previously difficult or reproduce, making the successful acquisition of food resources a powerful impossible to digest. These changes selective pressure. However, acquiring food is only part of the challenge. can occur quite rapidly12 and thus While all animals spend much of their daily activity budget hunting, search- could be an important adaptive ing for, or otherwise procuring food, a large part of what is involved in response that allows animals to overall nutrition occurs once the meal has been swallowed. Most nutrition- carve out separate dietary niches in al components are too complex for immediate use and must be broken environments where several species down into simpler compounds, which can then be absorbed by the body. are competing for food resources. This process, , is catalyzed by enzymes that are either endoge- Both South America and Madagas- nous or produced by the host’s microbial population.1 Research shows car were populated by a small num- that the nutritional value of food is partially constrained by the digestive ber of primates rafting from the abilities of the microbial community present in the host’s gut and that African mainland.13 Upon arrival, these microbes rapidly adapt to changes in diet and other environmental these primates rapidly diversified 2 pressures. An accumulating body of evidence suggests that endogenously and filled the available dietary produced digestive enzymes also have been, and still are, common targets niches,14 evolving a suite of physio- of natural selection, further cementing their crucial role in an organism’s logical, morphological, and behavior- 3–5 digestive system. al characteristics to process a range of different diets.15–18 Since digestive adaptations are not just In this paper, I focus on the enzymes in it, has evolved in important for the ability to tolerate endogenous digestive enzymes that response to a multitude of pressures. new food resources, but also to max- are known to be important to pri- Recently, many research efforts have imize the energy obtained from mates. Primates exhibit a particular- focused on the gut microbiome, pro- them, changes in digestive enzymes ly diverse array of dietary ecologies. viding new insights into the interplay were likely part of this adaptive From exclusively insectivorous spe- between diet and gut adaptation for suite. Especially in human evolution, cies to grass-eating monkeys, the pri- a variety of animals, including maximizing the energy extracted mate digestive system, including the human and nonhuman primates.6–11 from foods may have been a crucial These are exciting new findings, but factor in fueling the growth of our to achieve a full picture of an ani- large brains.19 In nonhuman pri- mal’s digestive adaptations, the gut mates, many species depend on rela- Mareike Janiak is a doctoral candidate in microbiome and endogenously pro- tively low-quality foods, such as the Department of Anthropology at Rutgers University. She is interested in the diets and duced digestive enzymes should be leaves, which can only be digested dietary adaptations of primates. Her doc- viewed as complementary parts of efficiently with specific gut adapta- toral dissertation research seeks to identify the system. While the genes coding whether the digestive enzymes of New tions, such as foregut fermentation World monkeys exhibit adaptations for for digestive enzymes do not change and/or special digestive enzymes. insectivorous diets. as quickly as those of the micro- Recent work on primate nutrition- Email: [email protected] biome, the variety of endogenous al ecology has highlighted the many digestive enzymes within primates challenges primates face to meet not nevertheless constitutes a major just overall energy requirements, but Key words: dietary adaptations; ; ; adaptive strategy and warrants spe- also to balance micronutrients and chitinase; cial attention in this paper. intake,20–23 all while dealing Changes in the expression of diges- with fiber, tannins, and toxins con- 24,25 VC 2016 Wiley Periodicals, Inc. tive enzymes are important dietary tained in foods. The ability to DOI: 10.1002/evan.21498 adaptations that may allow an Published online in Wiley Online Library meet nutritional goals depends in (wileyonlinelibrary.com). organism to exploit food sources part on foraging decisions and the 254 Janiak ARTICLE

TABLE 1. Endogenously Produced Digestive Enzymes of Primates Enzyme Gene symbol Substrate Alpha-Amylase AMY1 Starch MGAM Disaccharide maltose (product of starch digestion by a-amylase) Chitinase CHIA (present in walls of fungi and exoskeletons) Pepsin A PGA Protein CYM Protein Ribonuclease RNASE1 In ruminants and colobines: foregut bacteria LYZ, LZM In ruminants and colobines: foregut bacteria PRSS1 Protein Gastricsin PGC Protein Lipases PNLIP, CEL Lipids Lactase LCT Lactose (main carbohydrate in milk) TREH Trehalose (disaccharide found in insects, fungi, and plants) SI Sucrose, maltose nutritional composition of food disaccharide maltose, which can shows that the ability to express a- items.25–27 However, it is also con- then be hydrolyzed into glucose and amylase in saliva evolved after sever- strained by the gut’s capability to absorbed into the bloodstream.32 All al duplications of the pancreatic extract these nutrients, which is vertebrates express this digestive amylase gene AMY2 within the pri- where digestive enzymes and varia- enzyme in their , but only mate lineage.34 Two insertions occur tion in them undoubtedly play a key some mammals have evolved to addi- in the promoter region of AMY1, role.1,3,4,28 tionally express a-amylase in their resulting in the expression of a- The enzymes discussed here mouths, where it is secreted by amylase in saliva. Comparing the include amylase, lactase, pepsin A, parotid and/or submaxillary glands.33 gene structures between different chymosin, chitinase, ribonuclease, Species that express a-amylase in primates shows that the first inser- and lysozyme. While this is not an their saliva include some primates, tion, a g-actin pseudogene, arose exhaustive list of the enzymes at rodents, lagomorphs, and .34 after the divergence of the New work in primate digestive tracts, they Strikingly, the secretion of a-amylase World monkey lineage.34 The second are the more important ones because by the salivary glands has evolved insertion, an endogenous retrovirus, they usually represent the first step independently several times, sugges- occurred after the split from the Old in the digestion of their respective ting that this phenotype provides a World monkeys and is found only in 1 substrates, and research in primates selective advantage for certain spe- hominoids (Fig. 1).34 Studies with has largely been limited to them. cies.32 Furthermore, there is consid- transgenic mice indicate the retrovi- Table 1 details both the enzymes dis- erable variation, both between and ral insertion is required to change cussed here and ones that have not within species in which salivary amy- the expression site of amylase from been studied in depth in primates. I lase has evolved, in the amount of the pancreas to the parotid gland.36 describe the specific role of each enzyme that is expressed.3,31,35 Evi- This is consistent with the lack of , summarize what is dence that this may also be the case salivary amylase activity in New known about its inter- and intra- for pancreatic amylase comes from a World monkeys. Old World monkeys, species variability, and discuss the study comparing copy number varia- however, express salivary amylase adaptive implications of such varia- tion of pancreatic amylase genes in despite lacking the retroviral inser- tion. I include information on diges- wolves and domestic dogs.4 tion. More work is needed to tease tive enzymes in nonprimate Within primates, only Old World apart whether the insertion of the g- mammals to build a comparative monkeys, apes, and humans express actin pseudogene plays a role in sali- evolutionary framework. a-amylase in their saliva; New World vary amylase expression or if anoth- monkeys do not (Fig. 1).3,34 No er mechanism is responsible for this AMYLASE investigation of salivary amylase phenotype in Old World monkeys. Starches are a staple in the diets activity in strepsirrhines has been The latter would suggest an addition- of many contemporary human popu- published, suggesting a potential al independent evolution event with- lations.29 They are also present in avenue for future research. However, in the primate order.34 the diets of some nonhuman pri- given current understanding of the Rodents also express salivary amy- mates and other mammals in the evolutionary pattern of this trait in lase, but must have evolved the abili- form of underground storage organs, primates, it is unlikely that strepsir- ty independently from primates.36 unripe fruits, and seeds.30,31 Alpha- rhines express a-amylase in their However, similarities between pan- amylase is the enzyme that catalyzes saliva. creatic and salivary amylase genes in the breakdown of starch into sugar A comparison of the amylase gene mice indicate that the latter resulted by cleaving the glycosidic bonds of structures in New World and Old from duplication of the pancreatic the to produce the World monkeys, apes, and humans amylase gene, as it did in the primate ARTICLE Digestive enzymes of human and nonhuman primates 255

Figure 1. Salivary amylase expression in primates relative to that in humans and the proposed timing of the two insertions thought to cause amylase expression in saliva.11 Cercopithecines express salivary amylase despite lacking the retroviral insertion, suggesting the possibility of an alternate mechanism. lineage.37 Within rodents, the salivary Independent evolution of the would have had an advantage over amylase gene appears to have under- expression of a-amylase by salivary dogs that could not.4 gone duplication early in the muroid glands in several taxa strongly sug- Copy numbers also vary widely for lineage (including hamsters, gerbils, gests that this digestive enzyme pro- the AMY1 gene within humans and this true mice, and rats), but only arvico- vides a selective advantage.34,36 genetic variation is correlated with the line rodents (voles, lemmings, and Several studies have provided evi- level of salivary amylase expressed.3 muskrat) and hamsters express two dence of the possible selective bene- Individuals from populations whose gene copies. It is unclear whether or fits for salivary amylase.3,42,43 The diets have traditionally included large not this pattern is actually homolo- primary role of a-amylase, in both amounts of starch, such as Europeans, gous, as the two groups are only dis- the pancreas and in saliva, is starch Japanese, and the Hadza, tend to have tantly related.37 digestion. There is strong evidence more copies of the salivary amylase Lagomorphs, pigs, and some bats that a diet rich in starch acts as a gene than do members of populations also produce a-amylase in their sali- selective pressure on the evolution of that eat little starch, such as the Mbuti, vary glands,33 but no studies investi- amylase genes.3,4 Canines express Datoga, Yakut, and Biaka.3 Individuals gating the genetic bases of this only pancreatic, not salivary amy- with higher salivary amylase levels phenotype have been conducted in lase, but an interesting variation was have been shown to perceive starch as these groups. Evidence for salivary discovered in a recent study that less viscous and, as compared to indi- amylase has been found in several compared pancreatic amylase gene viduals with low salivary amylase levels, species of bats, including Eidolon hel- copy numbers in wolves and domes- report that starch viscosity decreases vum,38 Epomophorus labiatus,39 and ticated dog breeds.4 While wolves more quickly during mastication.42 Myotis grisescens,40 but a systematic consistently had only two copies of Changes in viscosity, such as viscosity survey comparing variation in diges- the AMY2B gene, diploid copy num- thinning, are considered desirable and tive enzymes, including salivary amy- bers in dogs ranged from 4 to 30. an important factor in determining lik- lase, across the order Chiroptera has This increase in copy number corre- ing of foods.42 These differences may not been done. Given that bats exhib- lated with a significant increase in a- lead to, in individuals that express high it great dietary diversity, feeding on amylase activity in dogs, leading the levels of amylase in their saliva, a stron- , insects, small vertebrates, nec- authors to conclude that efficient ger preference for starchy foods and tar, fruit, and pollen,41 knowledge of starch digestion represented a selec- result in the adoption of a high-starch the digestive enzymes found in differ- tive benefit in the process of dog diet in populations with a high fre- ent species could provide a better domestication, presumably because quency of such a phenotype.42 Thus, it understanding of the types of selec- dogs that were able to digest the is possible that the variation in AMY1 tive pressures under which dietary potentially high-starch food dis- copy number and salivary amylase lev- physiology evolves. carded or provided by humans els predates the observed dietary 256 Janiak ARTICLE

Figure 2. Frequency of the lactase persistence phenotype and frequencies of known associated alleles of the LCT gene. Phenotype fre- quencies predicted by surface interpolation are based on measurements at 235 locations.57 Allele frequencies are based on data from the Global Lactase Persistence Association Database57 and Jones and colleagues.67

ecologies and, in turn, drove the die- amount of food resources available to of the coding sequence suggests that tary choices of these populations rath- them, resulting in rising levels of obe- these copies may all be nonfunctional3 er than vice versa. However, sity and type 2 diabetes. Therefore, (but see Behringer and coworkers35). geographic location is not a good pre- increased availability of glucose fol- For gorillas, only relative (not abso- dictor of copy-number variation,3 lowing starch consumption due to lute) copy numbers of AMY1 have providing evidence against this higher salivary amylase levels may been reported; these numbers are rela- hypothesis and suggesting instead that now actually be maladaptive. A recent tively higher than those of chimpan- diet acts as a selective pressure on study found a link between lower zees and relatively lower than those of digestive enzymes.3 AMY1 copy number and increased humans.50 Although no information on One study showed that blood obesity risk,46 but these results could copy numbers in orangutans has been glucose levels are higher when not be replicated.47,48 published, a recent article providing high-starch foods are chewed before Variation in salivary amylase measurements of salivary amylase lev- swallowing than they are when high- expression and salivary amylase gene els expressed in all hominoids suggests starch foods are swallowed whole.44 copy numbers is also observed across that they may be similar to those of This suggests that the absorption of other hominids. A recent study of gorillas.35 Gorillas and orangutans glucose, and thus the amount of ener- ancient DNA showed that both Nean- have very similar levels of salivary a- gy made available from starch, is derthals and Denisovans had only a amylase, which are significantly higher increased by contact with saliva, likely single copy of AMY1 per chromosome, than those of both chimpanzees and due to salivary amylase. An improved suggesting that the copy number vari- bonobos (Fig. 1). Alpha-amylase levels ability to digest starch, increasing the ation observed in modern humans are somewhat higher in bonobos than amount of easily-absorbed glucose, originated comparatively recently.49 in chimpanzees, which not only is in may have conferred an important fit- As opposed to humans, individual accordance with the higher AMY1 copy ness advantage on individuals living chimpanzees (Pan troglodytes verus) number reported for this species, but in an environment where resources do not differ in AMY1 copy number also indicates that the copies are not were limited.45 Many modern human and, instead, uniformly have two cop- actually nonfunctional, as had been populations, however, especially those ies of the gene.3 Bonobos (Pan panis- suggested.3,35 living in urban and industrialized set- cus) consistently have four copies of The observed salivary amylase levels tings, certainly are not limited by the the AMY1 gene, although, a disruption further correspond to the diets ARTICLE Digestive enzymes of human and nonhuman primates 257

Box 1. Cheek Pouches

The cercopithecines are a sub- (hamadryas baboons) and Theropi- are important sites for the initial group of Old World monkeys that thecus gelada (gelada baboons) digestion of .120 are characterized by the evolution have salivary amylase levels that When a potato is inserted into the of cheek pouches, which they often are twice as high as those of cheek pouch of a restrained bonnet use to store food.3,119 Lambert119 humans and about eight times macaque (Macaca radiata), more has suggested that cheek pouches higher than those Pan troglodytes than 50% of its starch is converted may facilitate the digestion of (common chimpanzees). This is into simpler sugars within five unripe fruit, seeds, underground consistent with the diets of these minutes, indicating that the high storage organs, or foods containing species, which, in addition to being amylase levels in the primate cheek high levels of starch.31,119 The high high in starch, are also likely to be pouches are extremely effective at levels of salivary amylase expressed high in tannins.31 One study has starch digestion.120 in these species31 support this provided evidence that cheek hypothesis. Papio hamadryas pouches in cercopithecine primates

Box 1. Long-tailed macaques (Macaca fascicularis) on Sumatra with filled cheek pouches. Photo by Joram Berlowitz. [Color figure can be viewed at wileyonlinelibrary.com]

generally consumed by these species. to bind to , inhibit salivary a- amylase in their saliva. Indeed, the lev- Chimpanzees and bonobos feed on amylase in both humans and nonhu- els are often even higher than those in large amounts of ripe fruit and appear mans.43,54 In response to being fed a humans (see Box).31 to have similarly low intake levels of diet high in tannins, mice exhibited a To summarize, the expression of sal- starch.51 Gorilla diets vary consider- significant increase in the expression ivary a-amylase is found only in some ably across habitats, with some popu- of salivary a-amylase, presumably to mammalian taxa and appears to have lations feeding on large amounts of counteract the inhibitory effects of the evolved multiple times. The precise fruit, while the diets of others are leaf- tannins. This suggests that having number and sequence of convergent dominated.52 However, compared to increased levels of this enzyme secreted evolutionary events is unclear at this chimpanzee and bonobo diets, those in saliva may be an adaptive feature for time. Additional research is needed to of gorillas tend to include more struc- species that consume large amounts of delineate the full genetic basis of sali- tural carbohydrates, potentially tannins.43 An interesting avenue for vary a-amylase expression in all spe- including starch from roots and, pre- future research would be to investigate cies. Further research should also include colobine monkeys and other sumably, higher tannin levels.51,52 It is salivary amylase expression in species nonprimate taxa with highly variable likely that high tannin levels are also with high-tannin diets. A good choice diets, such as the Chiroptera. included in the diet of orangutans, would be colobine monkeys, as the diet which, during times of fruit scarcity, of these primates consists mostly of may consume large amounts of seeds leaves and other herbaceous vegeta- LACTASE and cambium, both of which are rich tion,aswellasunripefruit,allofwhich All mammals lactate and nurse in starch and tannins.30,51,53 are presumed to contain high levels of their offspring with milk.32 To digest Studies have indicated that tannins, tannins.54 All cercopithecines that have their mothers’ milk, young mammals which are characterized by an affinity been tested express high levels of produce the digestive enzyme lactase 258 Janiak ARTICLE in the small intestine. Lactase pro- majority of people exhibit this pheno- gene flow that have occurred in duction is restricted to infancy; in type include Central and Northern these regions is mirrored in the dis- most mammals, its activity begins to Europeans, as well as various peoples tribution of the LP alleles (Fig. 2).5,67 decline after the offspring is weaned. in Africa and the Middle East. A com- The G-13915 variant likely originated Some humans, however, continue to mon background for these individuals on the Arabian peninsula and was produce lactase throughout their is that they descend from populations spread to Africa by nomadic Arabian lives and can successfully digest milk with a long history of pastoralism groups in the sixth or seventh centu- as adults.55 and fresh milk consumption.5 ry,66,68 while an East African origin The main components of milk are There are several problems with is most probable for the G-13907, G- water, lipids, carbohydrates, pro- accurately determining the number 14009 and C-14010 alleles (Table teins, and salt, but the exact propor- of people who are lactase persistent 2).5,61,67 tions of each varies from species to rather than nonpersistent. First, dif- Lactase persistence has evolved species.32 Lactose, the principal car- ficulties and costs involved in access- multiple times in humans, sugges- bohydrate in milk, is a disaccharide ing remote populations will lead to ting a strong selective pressure oper- that is cleaved into the monosacchar- an overrepresentation of people from ating on this phenotype. In fact, ides glucose and galactose by the industrialized and Western coun- several studies have shown that the enzyme lactase-phlorizin-, tries, which may have a different fre- LCT locus is under the strongest pos- also called lactase, found in the quency of the phenotype. Second, itive selection seen in humans so small intestine. While it is mainly the noninvasive measures used to far58,69 and it has become a textbook known for its ability to digest the determine lactase activity in most example of recent human adapta- sugar lactose, lactase can also hydro- studies may not be completely accu- tions. The evidence of both strong lyze b-galactosides, phlorizin (found rate. They involve administering a positive selection acting on the in the bark of some fruit trees), and dose of lactose to a person, followed alleles associated with lactase persis- several other b-glucosides.56 Produc- by measuring either the blood glu- tence and multiple convergent evolu- tion of this enzyme is essential for cose response or the presence of tion events show that there seems to young mammals because they need hydrogen in the breath. However, be adaptive significance in the ability to be able to digest the lactose in the amount of lactose used in these to digest fresh milk in adulthood. their mother’s milk, which they tests is equivalent to that in 1-2 liters Several authors have suggested depend on for nutrition during the of cow’s milk, which is significantly that lactase persistence is an exam- first part of their lives.55 If mammals more than most people consume in ple of gene-culture coevolution: The with small intestines that no longer one sitting and may lead to overdiag- cultural practice of dairying influ- produce lactase consume fresh milk, nosis of the lactase nonpersistent enced the evolution and spread of lactose passes to the colon undigest- phenotype.58 Furthermore, individu- the lactase persistence-causing allele ed; there it may be fermented by als who are genetically lactase persis- and vice versa.70 However, did dairy- bacteria, producing fatty acids and tent may lose the ability to produce ing evolve in response to the evolu- gases that cause flatulence and phys- the enzyme secondarily due to intes- tion of lactase persistence or did ical discomfort. Undigested lactose tinal diseases.58 These limitations lactase persistence evolve following that passes into the colon can also should be kept in mind when discus- the adoption of dairying practices? cause diarrhea, which may be a sing the relative proportions of each Most evidence suggests that pastoral- much more serious problem, espe- phenotype that have been measured ism and the consumption of milk cially in environments lacking an in various populations. predates the emergence of lactase adequate supply of safe drinking The production of lactase is persistence. A recent study of a large water.55 encoded by the gene LCT, which in sample of ancient Eurasian remains Like all other mammals, the humans is located on the long arm supports a relatively recent spread of majority of humans cease to produce of chromosome 2.58 To date, at least the allele.69 The earliest evidence of lactase after infancy. The actual pro- five independent single nucleotide LP in this sample comes from an portion of humans who are “lactase polymorphisms (SNPs) have been individual dated to 2450-2140 BCE.69 nonpersistent” is difficult to ascer- identified that are associated with Age estimates of the various lactase tain, but most studies suggest that it the LP trait: G-13907, T-13910, G- persistence alleles are also consistent is around 65% worldwide.57 The pro- 13915, G-14009, and C-14010 (Fig. with the hypothesis that the pheno- portion of people who are lactase 2).58–62 The T-13910 allele is linked type spread following the adoption nonpersistent varies widely between with LP in European, Indian, and of dairying practices.5,12,58,66 These different populations, from less than Central Asian populations.58,63,64 In age estimates and archeological evi- 10% in Northern Europe to more African and Arabic populations, it is dence for the advent of dairying are than 95% in Southern China (Fig. found only at very low frequencies, summarized in Table 2. 2).55 Some humans continue to pro- despite locally high prevalence of the As noted, all of this provides evi- duce lactase past infancy and LP phenotype.58,65,66 Several alleles dence that consuming milk and throughout their lives, a condition are associated with LP in Africa and being able to digest lactose has sig- referred to as “lactase persistence” the Arabian Peninsula. The complex nificant adaptive benefits. Several (LP). Populations in which the history of human migrations and hypotheses have been proposed to ARTICLE Digestive enzymes of human and nonhuman primates 259

TABLE 2. Ages and Origins of LP Alleles and Archeological Evidence of the Advent of Dairying Archeological Evidence for Estimates of Allele Likely Location/ Advent of Dairying in Region Origin (years BP) Population of Origin of Origin T-13910 8000-900058 Central Balkans12 Slaughtering age profiles of 5000-1200065 sheep, goats, and cattle sug- 6256-868312 gest milk use~11,000 years BP116 Organic residues on pottery provide evidence for milk use by 9000 years BP117 G-13915 40005,66 Arabian Peninsula5,66 Domestication of the camel occurred~6000 years BP66 G-13907 50005 Cushitic Speakers/Eastern Ethiopia5 Stable isotope analyses of pot- G-14009 Ethiopia/East Africa (?)62 sherds indicate dairying in C-14010 2700-680058 Cushitic Speakers/East Africa5 Saharan Africa began by 7000 years BP118 explain the adaptive significance of of lactose. The lactase persistence phe- capuchin monkey (Cebus capuci- digesting fresh milk. Flatz and Rot- notype was present in 50% of Hadza nus)76 and a potto (Perodicticus pot- thauwe71 proposed the calcium hunter-gatherers, even though this to).77 However, such experiments are assimilation hypothesis, arguing that population is not known to consume extremely invasive and do not allow being able to digest lactose is partic- milk.58 This may mean that the Hadza for differentiation between enzymes ularly important in high latitudes, descended from a pastoralist popula- that are endogenously produced or where levels of ultraviolet light are tion or that lactase aids in the diges- of dietary origin, since chitinases are 58 low. Exposure to UV light is neces- tion of another food resource. present in many plant resources.1 sary for mammals to synthesize vita- Lactase does catalyze the hydrolysis of Despite a lack of concrete evidence, min D, a necessary step for the phlorizin, a bitter glycoside found in it has been assumed that insectivo- absorption of calcium, which is the roots and bark of plants in the rous animals, including primates, essential for bone health and growth. Rosaceae family, which is native to are able to synthesize chitinolytic 5,58 Because milk provides both vitamin Tanzania, where the Hadza live. enzymes to digest the exoskeletons D and calcium, these authors suggest The of lac- of insects or, alternatively, have gut that the ability to digest lactose and tase persistence in multiple human microbes that are able to do so.74 consume fresh milk without adverse populations is a classic example of Genetic research has identified a effects was an important adaptation the strong selective pressure that diet family of chitinase and chitinase-like for humans living in northern of an organism can represent. proteins in mammals that are pre- Europe.71 Since the hypothesis was Worldwide correlations of the lactase sumed to have arisen by gene dupli- proposed, it has become clear that persistence phenotype and the cation and evolved to fulfill a variety the lactase persistence phenotype is known genotypes suggest that we of functions,78,79 including protec- widespread in additional populations still have not found all of the under- tion from pathogens and aiding in not living in high latitudes, so there lying genetic mechanisms of lactase the hydrolysis of polysaccharides.78 persistence and that more research are likely other adaptive benefits to 5 Discovered in 2000, acidic mamma- dairying in addition to calcium is needed (Fig. 2). lian chitinase (AMCase) is a chitinase assimilation.72 Milk and milk prod- found in mammals. It is structurally ucts are high-calorie foods and, as CHITINASE very similar to other chitinases, but opposed to plants, which are only Chitin is one of the most common functions optimally at a much lower 80 available seasonally, are often avail- structural carbohydrates in nature, pH and was named accordingly. able year-round. Fresh milk is also a making up 58%-85% of the exoskele- AMCase seems to play an important valuable source of clean and uncon- tons of and 8%-60% of role as a digestive enzyme in mam- taminated fluids, which may be par- the cell walls of fungi.1 All primates mals, in addition to being involved in 78 ticularly important in arid, semi- include some insects in their diet, pathogen defense. 12 desert environments. Camels, for whether through accidental con- An Italian study found varying lev- example, can survive in extremely sumption or active insectivory (Fig. els of AMCase activity in the gastric arid conditions by metabolizing the 3),73 and fungi are a dietary staple of juices of 20 out of 25 human partici- fat stored in their humps. Their milk some New World monkeys, as well pants.81 The AMCase purified from could be an important source of flu- as humans.74,75 It is unclear, howev- participants’ gastric juice was able to ids for the humans keeping them. er, whether any primates are actually hydrolyze fluorescein isothiocyanate- There is limited evidence that lac- able to digest chitin. chitin in an experimental setting, tase may have beneficial digestive Chitinolytic enzymes have been leading the authors to hypothesize properties in addition to the hydrolysis isolated from the gastric mucosa of a not only that it represents an 260 Janiak ARTICLE

macaques. The finding that MACase appears to be significantly more effi- cient at chitin digestion than is human AMCase raises the exciting possibility that MACase is an impor- tant digestive enzyme for nonhuman primates. Until recently, it was unclear whether primates or other mammals produced endogenous chi- tinases in their digestive systems, because an exogenous or microbial origin of any chitinolytic activity could not be excluded.1,83 Further- more, almost intact exoskeletons are reportedly found in the feces of pri- mates,86 which has led many to pre- sume that they are indigestible.87 The discovery of the MACase gene Figure 3. A cotton-top tamarin (Saguinus oedipus) feeds on an insect. Whether primates family has provided a new avenue produce enzymes that can digest insects’ chitinous exoskeletons has not yet been con- for the study of dietary adaptations clusively determined. Photo by Mickey Samuni-Blank via Wikimedia Commons. [Color fig- in insectivores. ure can be viewed at wileyonlinelibrary.com] Future research should investigate the mCHIA gene across the primate order to test whether mCHIA might adaptation for the digestion of While both bats and primates have exhibit genetic variation, such as arthropods, but that the varying lev- been shown to harbor microorgan- higher copy numbers, in more insec- 9,83 els, as well as absence in some par- isms capable of degrading chitin, tivorous primate species. Such varia- ticipants, were due to decreased producing an endogenous digestive tion would provide evidence that consumption of insects in the West- enzyme for the hydrolysis of chitin 81 MACase is important in the digestive ern diet. While this is an intriguing would allow faster and more efficient system of primates that rely on hypothesis, Muzzarelli and col- digestion of prey, provid- 61 chitin-containing food resources, leagues have described an unpub- ing an important adaptive benefit for such as arthropods and fungi. lished follow-up study, which found insectivorous species. that human gastric juice containing In addition to rodents, bats, and high levels of AMCase was unable to humans, only macaques have so far PEPSINOGENS OR digest the wings of the bluebottle fly been investigated for AMCase activi- Pepsins are enzymes that provide (Calliphora vomitoria).82 ty. Using the human AMCase gene, the first step in the digestion of pro- More conclusive evidence for a CHIA as a guide, researchers were teins, an essential component of the digestive function of AMCase comes able to identify homologous genes, diet of all animals. In order to avoid from studies of mice and bats. named mCHIA, in the rhesus any unwanted digestion of the host Murine AMCase is optimally active (Macaca mulatta) and long-tailed tissue, all proteolytic digestive 85 at a low pH level and even remains macaque (M. fascicularis) genomes. enzymes are secreted as inactive pre- functional in extremely acidic condi- A comparison of enzymatic activity cursors known as zymogens. The tions, such as those found in the of the proteins cloned from the zymogens are converted into their stomach, suggesting that it has been macaque and human CHIA genes active form in the gastric lumen. adapted for a digestive function.80 showed that both enzymes are most Although it was previously thought active at pH 5.0, but remain func- Pepsinogen or Pepsin A that any chitinolytic activity in the tional at pH 2.0. Unlike of mammals was AMCase, the enzyme cloned from Pepsinogen A is the zymogen of due to chitinases expressed by intes- the macaque sequence also remained pepsin A, the most abundant gastric tinal bacteria,83 both mice and bats active at pH 8.0, giving it a much in most adult mammals, have recently been shown to secrete broader pH range.85 The most strik- which appears to be highly polymor- AMCase from the chief cells at the ing difference was that the macaque phic in primates.88,89 This enzyme, base of the gastric glands, where oth- AMCase (MACase) was 50 times secreted by the chief cells of the gas- er digestive enzymes are also secret- more efficient than human AMCase tric mucosa, is maximally active at a ed.84 The diets of wild mice often at hydrolyzing a chitin substrate.85 pH of about 2, in accordance with include significant amounts of Like AMCase in humans, mice, its role in the acidic environment of insects and many species feed and bats, MACase is expressed at the stomach.90 Pepsin A hydrolyzes almost exclusively on insects.84 high levels in the stomachs of the bonds of proteins, ARTICLE Digestive enzymes of human and nonhuman primates 261 creating smaller chains of amino Platyrrhines differ from other pri- chymosin is often better known as ren- acids that can then be further mates in that multiple forms of pep- nin. Chymosin was first discovered in digested by the enzymes trypsin, chy- sinogen A do not appear to be neonate cattle and, as noted, its ability motrypsin, and elastase.1 typical in this group. Of the four spe- to clot milk has long been used by Pepsin A has been shown to be cies of New World monkeys tested, humans in the production of highly polymorphic at both the pro- only capuchin monkeys (Cebus cheese.1,98 Indeed, its importance to tein level and genetic level in various apella) had two isozymogens of pep- the dairy industry has led to the publi- primates. Humans have a cluster of sinogen A; the common marmoset cation of many articles regarding the three genes that are known to be (Callithrix jacchus), squirrel monkey clotting activity of chymosin. Its physi- present in variable copy numbers.91 (Saimiri sciureus), and cotton-top ological role in animal digestive sys- 99 The other great apes exhibit even tamarin (Saguinus oedipus) all had a tems is less well understood. 97 greater variation in pepsinogen A single form of pepsinogen A. As suggested by its function in the isozymogens than do humans. Narita Whether this pattern is related to the clotting of milk, chymosin is com- and colleagues92 purified numerous dietary ecologies of the platyrrhines mon to a variety of mammalian spe- forms of pepsinogen A from the gas- is unclear. More research is needed cies, including, but most likely not tric mucosa of a gibbon (Hylobates to determine if these results are also limited to cows, zebras, horses, seals, lar), an orangutan (Pongo pygmaeus), found in the rest of the New World cats, pigs, kangaroos, opossums, por- a gorilla (Gorilla gorilla), and a chim- monkey species. As a group, the pla- cupines, and rats. In almost all of tyrrhines tend to rely more heavily these species it is found only in the panzee (Pan troglodytes). The num- 99 ber of pepsinogen A isozymogens on insects than foliage for protein, stomachs of fetuses and neonates. while the opposite is generally seen With one exception, chymosin is found ranged from seven in the 30 gorilla to eight in the gibbon, thir- in the catarrhines. More insectivo- absent from adult mammals. Experi- teen in the chimpanzee, and fourteen rous species may not require multi- ments with rats and pigs have shown in the orangutan.92 A follow-up study ple pepsinogens to digest protein, that there appears to be a switch in but rather express chitinolytic around the time of wean- by the same group predicted that ing.96 While pepsinogen A, in the five and three genes respectively pig, and progastricsin, in the rat, encode pepsinogen A1 and A2 in the were almost undetectable in fetuses orangutan.89 These genes are also and neonates, chymosin was present in the chimpanzee, while the It is unclear, however, expressed at high levels in the first human pepsinogen genes are most whether any primates phase of development. When chymo- like those for pepsinogen A1. This sin production began to cease in rats led the authors to conclude that pep- are actually able to seven days after birth, progastricsin sinogen A diverged into types A1 and digest chitin. expression increased rapidly and A2 in the hominoid lineage, but that 89 continued to be expressed strongly the latter was lost in humans. It 96 throughout adulthood. Similarly, has been suggested that the extreme porcine stomachs reduced produc- multiplicity of pepsinogen A in great tion of chymosin 5-10 days after enzymes for the digestion of insect apes is related to a dietary reliance birth, while pepsinogen A production 92 exoskeletons. Future research on on herbaceous material. However, began to increase one week after both pepsinogens and chitinases in while gorillas and orangutans may birth and showed a rapid increase at New World monkeys may provide consume relatively high amounts of three weeks of age.95 additional insight into the relation- foliage, chimpanzees and gibbons Newborn Japanese macaques are considered to be more ship between these digestive (Macaca fuscata) were not shown to frugivorous. enzymes and dietary ecology. produce chymosin or any other Macaques express four closely Many primate species, including neonate-specific digestive enzymes related forms of pepsinogen A that most of the cercopithecines and all although, as noted, relative expres- are apparently encoded by four sepa- of the strepsirrhines, have not been sion of different pepsinogens 88,93 rate genes. A study of Japanese tested for genetic variation relating changes with age.88 Human infants macaques (Macaca fuscata) found to pepsinogen A. However, as dis- also lack chymosin and the human that relative expression of the four cussed, some interesting patterns gene (hPC) for this enzyme appears pepsinogen A forms varied with have already emerged in the great to be nonfunctional.99,100 It is plausi- 88 88,89,93 age ; this was similar to what had apes and the macaques. ble that the hPC pseudogene is a previously been found in rabbits.94 shared trait of the catarrhines. New This increase in pepsinogen A pro- PROCHYMOSIN OR CHYMOSIN World primates, on the other hand, duction commonly occurs among differ from both other primates and mammals and is likely related to the Prochymosin, the zymogen of chy- other mammals in their expression changing digestive demands associ- mosin, is secreted by the gastric muco- of chymosin. Platyrrhines express ated with weaning and the adoption sa of neonate mammals. Because of its chymosin and furthermore, express of an adult diet.95,96 use in the cheesemaking industry, it in adulthood. Prochymosin was 262 Janiak ARTICLE purified from the gastric mucosa of are transferred via the placenta because they have evolved quite similar a common marmoset (Callithrix jac- before birth rather than through digestive systems. Both colobines and chus), a cotton-top tamarin (Sagui- their mothers’ milk. The vast litera- ruminants have sacculated forestom- nus oedipus), a squirrel monkey ture on immune markers in human achs wherein bacteria break down (Saimiri sciureus), and a capuchin breast milk (for a review, see Agar- foliage consumed. The animal, in turn, monkey (Cebus apella), suggesting wal and colleagues102) and studies receives important nutrients from that this trait is common to all pla- linking breastfeeding to improved digesting these bacteria once they tyrrhines. More research is needed to infant health103 cast doubts on the have entered the true stomach and confirm this and to explain why this validity of this hypothesis. small intestine.30 Two enzymes that enzyme persists into adulthood.97 Both humans and rabbits transfer are involved in digesting the forestom- Compared to pepsin, chymosin some immunoglobulins through the ach bacteria of colobines and rumi- tends to exhibit weaker general pro- placenta before birth, which may nant artiodactyls are pancreatic teolytic activity and approximately explain why the neonates of both ribonuclease and lysozyme. These similar milk clotting activity.99 The taxa lack chymosin. Rabbits, howev- enzymes perform other, nondigestive latter finding suggests that chymosin er, do express a different neonate- functions in most animals, but have is not essential for the digestion of specific protease, pepsinogen F.94 been adapted for a digestive function milk in neonate mammals and begs Kageyama and colleagues94 suggest in foregut-fermenters.104,105 the question of why chymosin is that the presence of a neonate- Lysozyme is usually expressed in expressed in neonates and only grad- specific pepsin might be due to the macrophages, where it digests the pep- ually replaced by pepsin A. high protein content of rabbit milk. tidoglycan cell walls of bacteria as part It has been suggested that chymo- Because primate milk is comparative- of the host animal’s immune defense sin is advantageous during the post- ly low in protein and high in lactose, system.106 In ruminant artiodactyls natal transfer of immunoglobulins, specific proteases may not be neces- and colobines, however, lysozyme is which are contained in the colos- expressed at high levels in the stomach trum.95,101 Experiments with porcine and has evolved to be better suited for pepsin have shown that this protease the task of digesting foregut bacteria. cleaves immunoglobulins into smaller The of rumi- The lysozymes of ruminants and colo- fragments. It is likely that pepsin bines are an excellent example of con- from other species has the same nants and colobines are vergent evolution: They have effect.95 Considering that the general an excellent example of undergone extremely similar changes proteolytic activity of chymosin is without sharing a common origin.107 weaker than that of other proteases, convergent evolution: As opposed to ruminant artiodactyls, it presumably limits damage to the They have undergone which have up to ten different genes immunoglobulins while retaining the extremely similar for lysozyme, colobines have retained ability to clot milk. The postnatal the single gene that occurs in most transfer of immunoglobulins through changes without sharing mammals.106,107 This gene has accu- the colostrum is critically important a common origin. mulated nine substitu- to many mammalian species in which tions,107 which appear to create immunoglobulins are not transferred functional differences that improve through the placenta before birth. the resulting protein’s performance as Placental transfer of immunity occurs sary to digest it efficiently.88 While a digestive enzyme. In order to func- in primates, rabbits, and possibly oth- this certainly is a reasonable explana- tion in the acidic environment of the er mammals,88 but species in which tion for the lack of chymosin in stomach, ruminant and colobine neonatal chymosin activity has been humans and macaques, it makes the digestive lysozyme has an optimum demonstrated tend to rely on postna- finding that adult New World mon- pH of 5.0, as opposed to nondigestive tal transfer through the colostrum.95 keys express chymosin even more lysozymes, which function best at a The presence of trypsin inhibitors in puzzling. More research is needed to neutral pH.108 In addition, digestive the colostrum provides further evi- elucidate the adaptive significance of lysozyme is more resistant to digestion dence not only that proteases are dis- this enzyme in platyrrhine digestion. by pepsin. Stomach lysozymes of the advantageous to the transfer of A more thorough survey of chymosin cow (Bos taurus) and the langur mon- immunoglobulins, but that chymosin expression throughout the platyr- key (Presbytis entellus) retained may have evolved as a neonate- rhines may reveal patterns of varia- around 75% of their activity after an specific digestive enzyme that effec- tion that could provide clues to a hour of exposure to pepsin, whereas tively clots milk while, at the same potential adaptive benefit of this the lysozymes of rats and humans time,havingalowenoughgeneral digestive enzyme. retained less than 7.5% of their proteolytic activity that it does not activity.108 damage immunoglobulins.95,99 Like lysozyme, ribonuclease has RIBONUCLEASE AND LYSOZYME Kageyama90 hypothesizes that been exapted for a digestive function most infant primates do not express The leaf-eating primates, the colo- in ruminants and colobines. However, chymosin because immunoglobulins bines, are often compared to ruminants the digestive function of ribonuclease ARTICLE Digestive enzymes of human and nonhuman primates 263

Figure 4. The independently duplicated ribonucleases in Asian and African colobines and ruminants exhibit parallel functional changes, including similar optimal pH.105,109 was not only acquired in parallel by in the small intestine of colobines, duplications in carnivores.115 Never- ruminants and leaf-monkeys, but which has a pH between 6 and 7.109 theless, the parallel evolution of a actually evolved independently at least As opposed to the colobine-specific digestive system and the convergent twice within the colobine lineage, ribonucleases, ribonuclease 1 is adaptation of digestive enzymes in once in Asian and once in African found in other tissues besides the ruminants and colobines illustrate colobines (Fig. 4).105,109,110 Gene pancreas. There it degrades double- the power that common selective duplications were followed by parallel stranded RNA and is hypothesized to pressures can have, especially when functional changes in the daughter have an antiviral function, although related to diet. genes, but these changes, unlike those its role is not completely under- 112 in digestive lysozyme, are caused by stood. The enzymes RNASE1B, CONCLUSION AND DIRECTIONS different amino acid substitutions in RNASE1b and RNASE1g have lost FOR FUTURE RESEARCH the ruminants versus the colobines. the ability to degrade double- All primates share one gene for stranded RNA, suggesting that they Digestive enzymes are important pancreatic ribonuclease called have also lost the original physiologi- in the primate digestive system and RNASE1. In numerous Asian colo- cal role of the parent enzyme in provide significant adaptive benefits. bines, a second pancreatic ribonucle- favor of specialization for a function This is demonstrated by the fact that ase gene, RNASE1B, has been in the colobine digestive system.109 lactase persistence, salivary amylase, found.110,111 It is presumed to be Interestingly, RNASE1 duplications digestive ribonuclease, and lysozyme common to all Asian colobines, since have also been found in carnivores have all evolved independently in its origin around 3.5 million years and bats, animals that are not fore- response to convergent selective ago coincides with the timing of the gut fermenters.113,114 In the super- pressures and have done so not just radiation of this taxon.111 In the Afri- family Musteloidea (order twice, but often multiple times. can colobines, RNASE1 was duplicat- Carnivora), RNASE1 was indepen- While the research reviewed here ed twice, resulting in two additional dently duplicated in four families: has shown the powerful impact that pancreatic ribonuclease genes, the Procyonidae (raccoons), Ailuri- digestive enzyme variation can have, RNASE1b and RNASE1c.109,110 RNA- dae (red pandas), Mephitidae many gaps remain in our knowledge SE1B and the common ancestor of (skunks), and Mustelidae (wea- of primate and, more broadly, mam- RNASE1b and RNASE1c share three sels).115 In bats (order Chiroptera), malian digestive enzymes. Although amino acid substitutions that are not RNASE1 duplications are present in primates are naturally the focus of found in the parent gene and have five species of the Vespertilionidae biological anthropology, the evolu- been shown to lower the optimal pH (Myotis lucifugus, M. altarium, M. tionary and ecological significance of of the enzyme. The original ribonu- ricketti, Ia io, and Murina leu- a trait is best understood when it clease, RNASE1 is optimally active cogaster) and two species of the can be placed in a comparative con- in all primates at a pH of 7.4, while Molossidae (Tadarida brasiliensis and text. Digestive enzyme variation in the Asian colobine enzyme, RNA- T. insignis).114 All of these bat spe- other mammals presents the relevant SE1B, and the African colobine cies are insectivorous, suggesting the evolutionary context within which enzymes, RNASE1b and RNASE1g, possibility of a dietary adaptation. At primate variation can be evaluated. respectively have optimal pH levels this point, however, there is no con- Among primates, strepsirrhines of 6.3 and 6.7 (Fig. 4).109,111 The con- clusive evidence to support this (especially lemurs) and platyrrhines vergence of this functional change is hypothesis.114 Likewise, more are the two groups that have been strong evidence that it represents an research is needed to understand the the most neglected when it comes to adaptation for bacteriolytic activity functional significance of RNASE1 digestive enzyme research. This gap 264 Janiak ARTICLE needs to be corrected in future stud- implies that an alternative haplotype human gut microbiota. Microb Ecol Health Dis ies. Lemurs and New World monkeys is present that allows adults in this 26:26164. 3 Perry GH, Dominy NJ, Claw KG, et al. 2007. evolved following two dispersal population to digest milk. 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