The hologenome concept of evolution after 10 years

Quantitative considerations

The human gut contains approximately 4 × 10¹³ bacteria, similar to the total number of human cells in the body [9]. Because of the large diversity of bacterial species, the gut microbiome contains ca. 9 million unique protein-coding genes or 400 times more bacterial genes than human genes [10]. It is important to realize that the bacterial count of the different bacterial species can differ by several orders of magnitude. In some cases, as few as 40 abundant bacterial species accounts for ca. 75% of the human gut microbiome [11]. However, bacterial species that are present at less than 4 × 10⁷ copies (0.00001% of the total) cannot be detected by current methods. Such relatively rare bacterial species should not be ignored since they have the potential to amplify under different conditions and play a role in adaptation and evolution of the holobiont. Clearly, analyses of the taxonomic diversity associated with the human microbiome will continue to be an area of great importance.

The gut microbiome also contains abundant and diverse viruses [12] and fungi [13], but they have not been studied extensively.

Individual variation: core and conserved function

High-throughput sequencing has demonstrated that although the bacterial species composition within the human gut is unique to each person, microbiomes of different individuals are closer to each other than to microbiomes of other primates [14]. These data suggest that there is something common (a core) to the human microbiome. Part of the Human Microbiome Project (HMP) has provided an opportunity to examine and better define what constitutes the taxonomic core within and across body habitats and individuals [15]. Shapira [16] has emphasized the differences between conserved core microbiota and flexible, environmentally driven microbiota with regard to their maintenance and contributions to host adaptation. However, attempts to identify “core” bacterial species in the gut microbiome have yielded only a few common species. Nonetheless, as discussed above, the presence or absence of a bacterial species depends on technical limits of detection. Methods developed to detect rare species may reveal that there are many more common species than currently considered and that individual variation may be the result of quantitative rather than qualitative differences that are caused by a different diet or some other environmental factor.

An alternative explanation is possible. Although human gut microbiomes vary between individuals in taxonomic composition, the biological functions they perform are surprisingly invariable between different people. Humans harbor phylogenetically distinct gut communities that can carry out the same functions [17, 18]. This means that there is considerable metabolic redundancy, genes, or isogenes for the same function being distributed across many species, so that a healthy gut microbiome can be assembled in many ways [19]. This idea was expressed in 2009 by Turnbaugh et al. [20] and lately in an article by Doolittle and Booth [21], titled: “It’s the song, not the singer….”. This is consistent with the hologenome concept, which considers all of the genes of a holobiont, not necessarily specific species.

Variation with time and environmental conditions

The composition of human gut microbiomes and their corresponding hologenomes change with age, diet, medication, and many other factors. Gut microbiomes of newborns are dominated by facultative anaerobes such as the Proteobacteria, after which the diversity of strict anaerobes within the Firmicutes and Bacteroidetes phyla increases towards an adult-like profile by approximately 1 year of age [22]. Throughout this early developmental stage, microbial composition is shaped by mode of delivery [23], infant diet [24], antibiotic treatment [25], and exposure to environmental factors, such as furry pets [26]. During most of adult years, the microbiome seems to be more or less stable [27]. The gut microbiome in older people (> 65 years), however, is extremely variable between individuals and differs from the microbiome of younger adults [28]. Microbiomes of people in long-stay care centers are less diverse than that of community dwellers and are correlated with low fiber diets and increased frailty [29].

Food, food additives, and essentially any material that is put in the mouth affects the gut microbiota at all ages. Both long-term [30] and short-term [31] diet influences the human gut microbiota. An “animal-based diet,” rich in meats, eggs, and cheeses increases the abundance of bile-tolerant microorganisms (Alistipes, Bilophila, and Bacteroides), whereas a “plant-based diet,” composed of grains, legumes, fruits, and vegetables, increases the levels of Firmicutes that metabolize dietary plant polysaccharides. Not only macronutrients, but also other components that are consumed affect the microbiome, such as red wine [32], tea and coffee [33], chocolate [34], food emulsifiers [35], artificial sweeteners [36], and, of course, antibiotics [37]. Also, the microbiome is affected by other factors such as, physical activity [38] and illnesses, e.g., cancer [39] and diabetes [40].

Host genetics affects the microbiome

In addition to environmental factors, host genetics plays a role in the acquisition, maintenance and stability of gut microbiota [41–43]. The three components, environment, host genetics, and microbiome, interact to maintain homeostasis in the gut. The disruption of this stability by modifying one or more of these three interacting components may trigger the development of diseases. It has been shown that a single host gene can have a large effect on the diversity and population structure of the gut microbiota. Most of the genes shown to have an impact on the composition of the gut microbiome are components of the immune system.

Endosymbionts and exosymbionts

Although exosymbionts are present in all animals and plants, it is well documented that non-pathogenic endosymbiosis is common only among plants and invertebrates (e.g., insects and corals). To the best of our knowledge, endosymbionts are not present in vertebrates. Invertebrates and plants have developed homeostatic interactions with the endosymbionts to benefit both. Moreover, they have developed specific immunological systems that participate in maintaining and controlling this important relationship [44, 45]. The close proximity of endosymbionts to host nuclei may enhance exchange of genetic material, as discussed in the section on “Genetic variation and evolution of holobionts.” In vertebrates, the more complex immune system appears to limit penetration of microorganisms into organs and cells [46, 47], while enabling a homeostatic and often beneficial relationship to develop with the exosymbionts.

Protection against pathogens

In general, germfree (GF) animals are more sensitive to infection by pathogens than conventional (CV) animals [51]. Following oral infection, the numbers of Listeria monocytogenes, a pathogenic bacteria, were 10,000-fold higher in the small intestine of GF mice compared to CV mice [52]. Staphylococcus aureus infection is prevented by resident Corynebacterium species [53]. Recently acquired symbiotic bacteria protect corals against the bleaching pathogen Vibrio shiloi [54]. Production of antibiotics is a common mechanism by which resident bacteria protect the holobiont against pathogens [55]. One of the strongest arguments for the role of bacteria in combatting infectious disease is the successful treatment of patients, suffering from severe diarrhea caused by Clostridium difficile infection, with fecal transplants from healthy donors [56]. Bacteria have also been shown to protect plants against infectious diseases by inhibiting the phytopathogen and by inducing systemic resistance [57, 58].

Provision of nutrients

An important general fitness contribution of microbiomes to their hosts is carrying out metabolic processes that the animal or plant cannot carry out by themselves [59]. There are many examples: nitrogen fixation in legumes [60], cellulose degradation in ruminants [61], termites [62] and cockroaches [63], essential amino acid synthesis in insects [64], photosynthesis by microalgae in corals, mollusks and sponges [65], and oxidation of inorganic compounds [66] and hydrocarbons in deep-sea invertebrates [67].

In humans, gut bacteria have been shown to perform several beneficial biochemical reactions that cannot be carried out by the host. For example: (i) production of metabolites from dietary components, such as the conversion of dietary fiber to the short-chain fatty acids, acetate, propionate. and butyrate [68]; (ii) modification of metabolites that are produced by the host, such as primary bile acids that are converted to secondary bile acids, thus assisting in bile acid recycling [69]; (iii) de novo synthesis of compounds, such as the important microbial immune modulator polysaccharide A, produced by the common gut bacterium Bacteroides fragilis [70]; (iv) synthesis of vitamins. Certain gut bacteria can produce vitamin K as well as most of the water-soluble B vitamins [71].

Fat storage and obesity

Not only is the composition of the gut microbiome of obese and lean individuals different [72], but more significantly, fecal bacteria transferred from obese humans to germ-free mice caused a greater increase in body weight than transplants from lean humans [73], suggesting that the microbiome in combination with diet and genetic factors causes obesity. Although bacteria that contribute to obesity could be considered harmful, under certain conditions they are beneficial. During the third trimester of pregnancy, these so-called “obese bacteria” become abundant and induce metabolic changes that promote energy storage in fat tissue that in turn encourages growth of the fetus [74] and milk production in the mother. Also, during our evolution, food insecurity was a frequent occurrence, and the ability to store energy in the form of fat was probably advantageous for survival.

Development and behavior

It is has been known for many years that certain microbial symbionts (once termed primary symbionts) interact with their hosts to benefit the holobionts: Rhizobia strains cooperate with legume plants to produce root nodules that perform nitrogen fixation [75]. Vibrio fisheri triggers the formation of the light organ in squid, where luminescence occurs to help the squid avoid predation [76]. Intracellular algae of the genus Symbiodinium carry out photosynthesis that provides nutrients to their host coral [77]. In recent years, it has been shown that co-development of the microbiome with animals and plants is not limited to primary symbionts. In plants, microbes associated with root tips acquire nutrients from plant secretions and in turn produce indole acetic acid that stimulates root elongation and lateral root formation [78]. In vertebrates, the gut microbiome promotes the development of the immune system and body organs [79]. Exposure to microorganisms educates the immune system, induces innate and adaptive immunity [80, 81], and initiates memory B and T cells that are essential to combat various pathogens. In addition, the gut microbiome encourages also the development of bone mass [82] and blood vessels in the intestinal wall [83].

Hydra is an evolutionarily ancient multicellular organism which has been used as a model system in developmental biology. Recently, it has been shown that Hydra microbiota plays an essential function in reproduction [84] and influences the spontaneous contractions, likely by modulating the pacemaker activity movement [85].

Bacteria in the mammalian gut also modulate brain development and behavior, including anxiety and mood disorders [86]. Data from experiments performed in rodents with altered intestinal microbiota, whether germ-free mice, or conventionally raised animals treated with probiotics and/or antibiotics, all indicate that rodent behavioral responses are impacted when the bacterial status of the gut is manipulated [87]. Microbial gut–brain signaling is bidirectional. The circuitry of neurons, hormones, and chemical neurotransmitters allows messages to be transmitted between the brain and the gut. For example, the rate at which food is being moved and how much mucus is lining the gut—both of which can be controlled by the brain—have a direct impact on the environmental conditions the microbiota experiences. On the other hand, the gut microbiota influences the body’s level of the potent neurotransmitter serotonin, which promotes in addition to gut functions also feelings of happiness and peacefulness [88].

Microbiomes warm their hosts

Recently, we suggested that provision of heat is an underappreciated general contribution of microbiomes to holobionts [89]. Microbiomes produce heat as a by-product of the enzymatic catabolism of substrates and synthesis of cell material. It was reported that bacteria have specific rates of heat production of ca. One hundred sixty-eight milliwatts per gram [90, 91]. Based on these findings, it can be calculated [92] that about 70% of human body heat production at rest is the result of bacterial metabolism in the gut.

Consistent with the concept that microbes warm their hosts are reports that treatment of rabbits [93] and rodents [94] with antibiotics lowered their body temperature. Heat output by gut microbiota may also help explain the observation that germ-free mice had 40% less total body fat than conventionally raised mice, even if their caloric intake was 29% higher [95]. The warming effect of microbiomes has also been reported in plants [96]. Heat produced by the sugar catabolism of yeast populations inhabiting floral nectar increased the temperature of the nectar and modified the within-flower thermal microenvironment.

Though the significance of heat production by microbiomes has scarcely been studied, its contribution may have far-reaching implications. It may help warm-blooded animals avoid hypothermia, and in cold-blooded animals, it can raise their body temperature.

Amplification or reduction

Amplification/reduction refers to the increase or decrease of one group (e.g., species) of symbionts relative to others, which can occur rapidly when conditions change. The holobiont is a dynamic entity with certain microorganisms multiplying and others decreasing in number as a function of local conditions within the holobiont. An increase in the number of a particular microbe is equivalent to amplification of a whole set of genes. Considering the large amount of genetic information encoded in the diverse microbial population of holobionts, often more than in the host genome [10], microbial amplification/reduction can be a powerful mechanism for contributing to adaptation, development, and evolution of holobionts. Reported examples of environmental factors that lead to changes in symbiont populations and thereby to variations in hologenomes include nutrient availability [130–134], artificial sweeteners [36, 135], food emulsifiers [136], disease states [137–140], pH [141], temperature [142], and of course antibiotics [143–145]. Prebiotics, food ingredients that induce the growth or activity of beneficial microorganisms [146], is an applied example of the amplification principle [147].

Since genetic variation by amplification is driven by the environment, it has a Lamarckian aspect to it, as discussed by us in a previous paper [148]. As will be reviewed in the next section, amplification is a crucial step in genetic variation and evolution by acquisition of novel microbes. For a pioneer microbe to become established in its host it must multiply.

Acquisition of novel microbes from the environment

Microbes were on this planet for 2.1 billion years before there were any animals or plants. During this time, they evolved enormous biochemical diversity and split into two domains, Bacteria and Archaea. The first eukaryote was probably formed by the acquisition of bacteria to eventually form mitochondria [149] and chloroplasts [150] and possibly by the uptake of an Archaea by Bacteria to form the nucleus [151]. Uptake of microbes into multicellular organisms continued to provide genetic variation for holobionts throughout evolution. Many of the beneficial interactive fitness traits of holobionts discussed above fit into this category.

Animals and plants come into random contact with billions of microorganisms during their lifetime, via air, water and interaction with organic and inorganic surfaces. Occasionally some of these microbes will find a niche and under appropriate conditions amplify in the host and affect the phenotype of the holobiont. Unlike mutation, which causes small changes in existing genomes, acquisition of a microbe introduces hundreds of new genes into the holobiont. Rather than reinvent the wheel, animals and plants can acquire pre-evolved genetic information in the form of microbes. It is likely that after the microbe is acquired, mutations and selection occur in the microbe and host to optimize the interaction.

An example of a major evolutionary event that was driven by the acquisition of bacteria is the ability of many animals to use plant material, in the form of cellulose and other complex polysaccharides, as nutrients. However, animal genomes do not contain the information for synthesizing enzymes for degrading cellulose. Instead, animals such as termites, cockroaches, and ruminants rely on cellulolytic microorganisms that are present in their digestive tract. These microbes anaerobically convert cellulose to fatty acids that are the major source of carbon and energy for their host animal [134]. How did they gain access to these specialized microbes? It is likely that the evolution of termite and cockroach hindgut microbiotas occurred by the gradual process of internalizing from the environment microorganisms that digest plant litter. Instead of plant cellulose being broken down in the soil prior to ingestion, it “rots” in the hindgut after consumption [152]. It has also been suggested that cockroaches acquired cellulolytic microbes by eating the dung of dinosaurs [153], which are known to have been hindgut fermenters [154].

There are many other examples of important evolutionary events that were driven by the acquisition of microbes by animals and plants. Such examples include acquisition of zooxanthellae by corals and other marine invertebrates, which formed photosynthetic animals and led to the construction of coral reefs [155], acquisition of diverse chemosynthetic bacteria by deep sea animals, which allowed for life in the absence of light [156], acquisition of anaerobic bacteria by the gut of ants, which supported herbivory [157], and acquisition of nitrogen-fixing bacteria by legumes, which permitted plant growth under limiting nitrogen conditions [158].

Horizontal gene transfer

Another important mode of genetic variation in holobionts, referred to as horizontal gene transfer (HGT) or lateral gene transfer, involves the transfer of groups of genes between bacteria of different taxa and from microbiomes to their hosts. HGT is generally associated with gene transfer between different bacteria, but can also take place from microorganisms to animals and plants and the other way around. The intimate contact between microbes and host genomes in holobionts would promote HGTs [159]. On average, bacteria in the human gut contain a minimum of 49 observed horizontally acquired genes [160]. It has been suggested that nutritional adaptation is one of the key selective pressures on the microbiome in the mammalian gut and that HGT processes contribute to that adaptation [161].

An interesting example of evolution of humans by HGT between bacteria is the ability of Japanese to break down agar (an abundant ingredient in their diet) since they have a bacterium in their gut that contains genes that code for the porphyranases that degrade the polysaccharide agarose of agar. Westerners lack this bacterium in their gut and therefore cannot digest agar. The group of genes coding for agarose digestion was driven into a resident gut bacterium by HGT from a marine bacterium that was present on raw seaweed [162]. Although HGT usually occurs between bacteria in the same ecological niche, apparently the marine bacterium was present in the gut long enough to have some of its genes transferred to a resident gut bacterium. Furthermore, the bacteria with the transferred genes spread throughout the Japanese population by vertical and horizontal transmission [163].

Until 2010, only a few examples of HGT from microbes to animals were recognized: Wolbachia genes to the chromosomes of their insect hosts [164], bacterial and fungal genes into the telomere region of rotifers [165], fungal genes to aphids [166], and cellulose genes from bacteria to nematodes [167]. However, an examination of the recent availability of a large number of high-quality genomes has led to the conclusion that HGT in animals and plants typically results in tens or even hundreds of active foreign genes [168]. Analysis of the 13.7-megabase genome of the extremophile red alga Galdieria sulphuraria revealed that 5% of its protein-coding genes were acquired by HGT from bacteria and archaea [169]. Examination of the genomes of 12 Drosophila species showed on average 40 foreign genes that had been horizontally transferred from bacteria and fungi [170]. When the Drosophila species were placed on a phylogenetic tree, there was a correspondence between the number of HGT events and the length of each branch, suggesting that HGT has occurred throughout Drosophila evolution and is likely to be ongoing. This paper also pointed out that HGT events were more frequent in invertebrates than in vertebrates, an observation that may be explained by the closer proximity between invertebrate endosymbiont (intracellular) and host genetic material.

In humans, 145 genes (not present in other primates) were attributed to HGT [170]. These genes are distributed throughout the genome and play a variety of roles, such as amino-acid metabolism (two genes), macromolecule modifications (15 genes), lipid metabolism (13 genes), antioxidant activities (5 genes), and innate immune response (7 genes). Most of the 145 genes identified in the study came from bacteria, but some originated from viruses and yeasts. Analysis of the moss Physcomitrella patens identified 128 genes found in land plants but absent from algae [171]. These genes were acquired by HGT from prokaryotes, fungi or viruses. Many of these genes are involved in some essential or plant-specific activities such as xylem formation, plant defense, nitrogen recycling, and the biosynthesis of starch, polyamines, hormones, and glutathione.

A key event in the evolution of placental mammals, including humans, was the acquisition by HGT, from a retrovirus, of the gene coding for the protein syncytin [172]. Initially, the function of syncytin was to allow retroviruses to fuse host cells so that viruses could move from one cell to another. Now, syncytin is necessary for the development of the placental syncytium, the essential part of the mother-fetus barrier. Knockout of syncytin genes in genetically modified mice provided evidence for their absolute requirement for placenta development and embryo survival [173]. Similarly, retroviral-derived molecules appear to have played a crucial function in the generation of the progesterone-sensitive uterine decidual cell, allowing nutrient provision to the developing embryo [174]. These data indicate that the integration of viral DNA into a host genome played a primary role in a major evolutionary leap by enabling growth and maturation of the fetus in placental mammals. In general, it is clear that introduction of genes by HGT into eukaryote genomes has been a major force propelling genetic variation and evolution.