The Hologenome Theory

The growing understanding of the central role of microorganisms in biology has sparked a shift in how biological individuality and the units of natural selection are viewed. The Hologenome Theory suggests that the evolution of organisms should be considered not only at the level of its genome but rather in the context of the genome and its microbiome. While the host genome changes slowly, the microbiome genome is highly adaptable and can respond rapidly to environmental changes by altering microbial populations, horizontal gene transfer, and mutation. These interactions between the host and microbiome are crucial to evolution, as alterations in either can lead to selective pressures. Furthermore, rapid changes in the microbiome genome can provide a significant advantage for holobionts in adapting and surviving under changing environmental conditions. According to the Anna Karenina principle, exposure of a holobiont, an animal or plant with all its associated microorganisms, to adverse conditions may compromise host mechanisms, resulting in an increased likelihood of stochastic processes. This high stochasticity can lead to a succession of microbial communities with varying effects on the host, including fitness, speciation, and genetic variation. Therefore, this paper will present evidence supporting the notion that hosts and microorganisms together make up a single genome, and any change can drastically affect the microbiome's integrity, ultimately impacting the evolution, diversification, and speciation of organisms and their future offspring.

Microbes are ubiquitous in the lives of multicellular organisms, with evidence supporting almost all forms of life existing in beneficial, detrimental, or neutral symbiosis. In the 19th century, after the germ theory of disease by Koch and Pasteur was publicized, the public primarily saw microbes as sources of illness and decay (Inglis, 2007). However, this once widely held view that microbes are inherently harmful to their host has been replaced with the understanding that they play an essential role in the survival and development of many organisms. 

From the earliest forms of life to the most complex organisms, microbes have profoundly impacted their hosts in various ways, including respiration and ATP production, protection against pathogens, and provision of nutrients (Rosenberg et al., 2016). For example, the mitochondria, which evolved endosymbiotic events with alphaproteobacteria, play a crucial role in energy production and are considered “extreme symbionts” in most eukaryotic organisms (Emelyanov, 2003). The chloroplast, which is responsible for photosynthesis, fatty acid and amino acid synthesis, and plant immune response in plant cells, also happens to evolve from bacteria, specifically cyanobacteria (Gould et al., 2008). Additionally, studies have demonstrated that legumes rely on nitrogen fixation by symbiotic bacteria to obtain nitrogen for growth, and ruminants, termites, and cockroaches rely on cellulose degradation by their microbiota to digest plant material (Oldroyd et al., 2011; Watanabe et al., 2010). Furthermore, as scientists continue to explore the complexities of the microbial world, it has become increasingly evident that the relationship between host and microbe is far more complex than previously imagined.

As more evidence arises supporting the significant impact of microbes on host development, many microbiologists have wondered how the presence of microbiomes affects the evolution of both the host and microbial residents. While theoretical models suggest that competition within a microbiome can promote the evolution of dependencies between different species,  models like this only demonstrate the stability of mutualisms in microbial symbioses and tend to represent only a small portion of the diverse and dynamic microbiome (Morris, 2018). As a result, the role of other microbial taxa in host ecology and evolution is poorly understood. One way of approaching the impact of microbiomes on host evolution and ecology is taking a holistic approach and viewing the host-microbiome system as a holobiont. The term ‘holobiont’ was first coined by Lynn Margulis et al. (1991) in the late 20th century in her book “Symbiosis as a Source of Evolutionary Innovation: Speciation and Morphogenesis.” In this book, she proposed that macroscopic hosts and their symbiotic microbes form a unified organism and that the physiology of the holobiont arises from the joint activities of its genome and the genomes of its microbiomes (Bordenstein et al., 2015). The hologenome theory was introduced shortly by another scientist but lost most of its traction outside of microbiology until recently, with the advancements of high-throughput sequencing revitalizing the concept and its study (Hurst, 2017). 

Rosenberg et al. (2007) reintroduced the hologenome theory with their study on corals after observing that the invasion of specific pathogens did not cause certain diseases in these organisms but rather the development of pathogenic microbiome communities, which human-caused environmental changes may have triggered. Furthermore, the elaborate relationship between corals and their symbiotic Zooxanthellae and the complex network of mutualisms in the coral reef ecosystem made the hologenome theory a natural fit for studying co-evolutionary feedback across multiple levels (Rosenberg et al., 2007). Ultimately, this led to more research on coral systems, and unique theoretical implications of the hologenome concept began to emerge. These implications supported the notion that the concept was relevant throughout the Tree of Life, potentially impacting the study of the ecology and evolution of every macroscopic organism on the planet (Rosenberg et al., 2009). 

Various external stressors, such as predators, parasites, social disruption, habitat modification, and degradation, can affect the composition of a microbiome (Lavrinienko et al., 2020). Stress can produce deterministic changes to the microbiome or increase the variance of microbiota, leading to higher inter-individual differences due to microbiome instability (Arnault et al., 2022). Many researchers have proposed the Anna Karenina principle (AKP) to characterize the differences between healthy and disease-associated microbiota (Arnault et al., 2022). The Anna Karenina principle was derived from Leo Tolstoy’s novel “Anna Karenina,” where he famously writes, “Happy families are all alike; every unhappy family is unhappy in its own way.” (Bornmann et al., 2011). Tolstoy’s novel inspired scientist Diamond J. to expand on his principle as a way to explain why plants and animals might demonstrate undomestic behavioral traits (Diamond, 1994). In the context of host-associated microbiomes, the AKP suggests that certain stressors have stochastic and chaotic effects on microbial community composition, insinuating that all healthy microbiomes are alike, but each dysbiotic microbiome is dysbiotic in its own way (Zaneveld et al., 2017). The AKP concept is essential for diagnosing microbiome dysbiosis in biomedical research with humans and animals and has recently been implemented in dysbiosis in wild animals and plants (Lavrinienko et al., 2020).

As the hologenome theory has grown in popularity, many aspects of the concept have received criticism on whether the holobiont should be considered the primary unit of natural selection or whether hosts and microorganisms comprise a single genome. Nonetheless, even with their speculations, the growing recognition of the critical role played by microorganisms in biology has undoubtedly brought a shift in the perspective on biological individuality and units of natural selection (Stencel et al., 2018). In addition to the fitness, developmental, and functional benefits of microbes, they can also be inherited vertically, such as from the host's parents, or horizontally, including the environment, other host species, or other members of the same species, inevitably modifying the host's evolution and ecology (Morris, 2018). Therefore, this paper will provide evidence to support the notion that the interaction between hosts and their microbiome is mutually dependent, and any external stressors can profoundly affect the microbiome's composition. Furthermore, these microbiomes can be transferred to offspring, influencing diversification, speciation, genetic variation, and fitness, ultimately impacting the host's and future generations' evolution and development. 

Findings and observations based on the hologenome theory of evolution have created a solid set of principles that set the foundation of the concept. The first principle states that all animals and plants harbor diverse and abundant microbiota, which means they can be regarded as holobionts (Rosenberg et al., 2016; 2018). Analyses have shown that the holobiont can include various other organisms, such as archaea, protists, and viruses (Hurst, 2017). In a quantitative consideration, multicellular organisms have large populations of microbes residing on their surfaces and within their fluids, referred to as exosymbionts, and found inside some cells of animals and plants, referred to as endosymbionts (Rosenberg et al., 2016). For example, the human gut contains a vast number of bacteria, estimated to be approximately 4 x 10^13, and contains around 9 million unique protein-coding genes, 400 times more bacterial than human genes (Yang et al., 2009). However, these reported values are minimum numbers, and minor species may not be detected using current detection methods. Despite seemingly having little effect on the holobiont, these rare bacteria have the potential to become more abundant under different conditions, hence contributing to the holobiont's fitness, adaptation, and evolution (Rosenberg et al., 2018). Lastly, examinations of the human microbiota suggest that there is a ‘core’ microbiota consisting of bacterial species present the majority of the time in relatively large numbers and are common to most individuals (Rosenberg et al., 2018; Aries et al., 2015). The ‘noncore’ microbiota encompasses readily exchangeable species that vary due to factors like environmental conditions, diet, and diseases (Zilber-Rosenberg et al., 2021). In some cases, like with the dual symbiosis in the deep-sea hydrothermal vent snail Gigantopelta aegis, these ‘noncore’ microbiota can become stable holobiont members and form part of the core microbiota (Aries et al., 2015; Lan et al., 2021).

The next principle states that the holobiont functions as a distinct biological entity during development, anatomically, metabolically, immunologically, and in evolution (Rosenberg et al., 2016; 2018). As previously stated, the fitness of holobionts greatly depends on positive interactions between the host and its symbionts and between the microbiota themselves, resulting in a more well-adapted holobiont. In addition, many studies have shown that microbes protect against pathogens. For instance, Corynebacterium species resident in human bodies can protect against pathogenic Staphylococcus aureus infection (Ramsey et al., 2016), and corals are protected from bleaching caused by the symbiotic bacteria Vibrio shiloi (Rosenberg et al., 2007). Furthermore, microbes also contribute to the development of certain behavioral features. For example, studies have shown that germ-free mice exhibited significantly different behavior than control mice, such as spending more time in the light compartment of a box, displaying the influence of gut microbiota on the development of the mammalian brain (Heijtz et al., 2011). Lastly, the role of microbiomes in providing heat is often overlooked yet vital (Rosenberg et al., 2018). Microbiomes generate heat as a byproduct of enzymatic catabolism and cell material synthesis, and previous studies have shown that bacteria can produce approximately 168 milliwatts of heat per gram (Russel, 1986). Moreover, evidence has shown that antibiotics, which can disrupt microbiomes, decrease the body temperature of mammals like rats and rabbits (Kluger et al., 1990). 

The following principle states that a significant proportion of the microbiome genome, in combination with the host genome, is transmitted from one generation to the next, thereby allowing unique characteristics of the holobiont to propagate (Rosenberg et al., 2016; 2018). For holobionts to be recognized as units of selection in evolutionary processes, both the host and microbiome genomes, referred to as the hologenome, must be passed down to future generations (Bordenstein et al., 2015; Rosenberg et al., 2009). While the mechanisms involved in host DNA transmission are well documented, microbiome transmission is less precise and occurs through various mechanisms (Stencel et al., 2018). Vertical transmission (VT) of symbionts, where symbiotic microbes are directly transmitted from parent to offspring, is the most known mode of transmission in organisms (Hurst, 2017). Maternal transmission is commonly observed in species with separate sexes, where transmission occurs exclusively from mother to progeny (Hurst, 2017). For example, a large portion of the colonization of the newborn gut is established through various factors such as inoculation with maternal vaginal and fecal microbes during birth and breastfeeding, as shown by a complex bacterial community dominated by Lactobacilli and enteric bacteria in an infant's first postpartum bowel movement (Moles et al., 2013; Fernández et al., 2013). Indirect or horizontal transmission is another mode of symbiotic transmission. A well-known example of horizontal transmission is the squid light organ-Vibrio fischeri symbiosis (Rosenberg et al., 2018). The female squid lays hundreds of fertilized eggs, and subsequently, adult squids release large amounts of V. fischeri into the water, which colonizes the immature light organ of the developing squid (Nyholm et al., 2009). Nevertheless, most transmission occurs via a mixed mode, where vertical and horizontal transmission both occur subsequently or simultaneously, and accurate evidence supporting microbiomes transferred over multiple generations is difficult to pinpoint (Hurst, 2017; Bordenstein et al., 2015). For example, Moeller et al. used gyrB gene sequencing in fecal samples from humans, wild chimpanzees, and wild bonobos to study the strain diversity within their gut microbiomes (Moeller et al., 2016). They found that strains of Bacteroidaceae and Bifidobacteriaceae have been maintained exclusively within host lineages across thousands of host generations, indicating co-speciation between gut bacteria and Hominidae. 

The last principle states that genetic variation in the hologenome can occur due to changes in the host and microbiome genome (Rosenberg et al., 2018). While the host genome changes slowly, the microbiome genome is highly adaptable and can respond rapidly to environmental dynamics more quickly than the host genome (Morris, 2018). Along with the well-known mechanisms of genetic variation, such as mutation, sexual recombination, and chromosome rearrangement, three modes of genetic variation specific to microbiomes in holobionts are often overlooked (Rosenberg et al., 2018). The first one is the increase or decrease in the abundance of a particular microbiota species, which can occur in response to changing external conditions. Various environmental factors, such as nutrient availability, artificial sweeteners, diseases, pH, temperature, and antibiotics, have been reported to lead to changes in symbiont populations and variations in hologenomes (Zilber-Rosenberg et al., 2021; Rosenberg et al., 2009). Prebiotics, which are food ingredients that stimulate the growth or activity of beneficial microorganisms, is a prime example of the amplification concept (Rosenberg et al., 2013). Since, in proportion, the diverse microbial population in holobionts encodes more genetic information than the host genome, amplification and reduction of microbes can be a powerful mechanism contributing to the evolution of holobionts (Zilber-Rosenberg et al., 2021; Yang et al., 2009). The second mechanism is the acquisition of new microbes. For several centuries, scientists have documented the acquisition of microorganisms through various means, such as air, water, and direct contact with organic or inorganic surfaces, and sometimes some of these microbes can establish themselves in the host under suitable conditions (Casadevall et al., 2000). An example of a significant evolutionary event driven by the acquisition of bacteria is the ability of many insects to use plant material as a nutrient source (Rosenberg et al., 2016). While animal genomes, such as termites, cockroaches, and ruminants, lack the genes for synthesizing enzymes needed to break down cellulose, they depend on cellulolytic microorganisms in their digestive tract to convert cellulose into fatty acids (Watanabe et al., 2010). The evolution of these hindgut microbiotas in these organisms likely occurred through the gradual process of internalizing microorganisms that digest plant litter from the environment or dinosaur feces (Dietrich et al., 2014). The last mechanism is horizontal gene transfer (HGT), which involves transferring groups of genes between bacteria of different taxa. An intriguing example of human evolution by HGT is demonstrated in the ability of Japanese individuals to break down agar, an abundant ingredient in their diet, because they harbor a gut bacterium containing genes that degrade the agarose polysaccharide of agar (Hehemann et al., 2010). They acquired this bacterium from a marine bacterium present on raw seaweed to a resident gut bacterium via HGT. Although HGT usually occurs between bacteria in the same ecological niche, the marine bacterium was present in the gut long enough to transfer some of its genes to a resident gut bacterium, ultimately spreading the bacteria with transferred genes throughout the Japanese population by vertical and horizontal transmission (Hehemann et al., 2010; Rosenberg et al., 2018).

In closing, there is an extreme disservice in investigating the evolution and development of life on Earth without earnestly considering the implications of microbes and microbiomes. Studying the impacts of microbes has invoked various theories and hypotheses to explore the evolutionary significance of the interdependent relationship between host and microbiota. In particular, the hologenome theory and the Anna Karenina principle have revolutionized our understanding of evolution and microbes' role in shaping their hosts' phenotypes. The hologenome concept acknowledges microbial communities' vital role in their host species' evolution and challenges the traditional view of evolution as a linear process governed solely by genetic mutations. Rather, it suggests that evolution is a complex and dynamic interplay between the host genome and its associated microbiome. Correspondingly, the Anna Karenina principle provides a powerful framework for understanding the role of microbial symbiosis in evolution, emphasizing the importance of a stable and balanced microbial community in promoting the health and adaptation of the host. Therefore, the transfer of microbiomes to offspring through vertical and horizontal transmission can influence diversification, speciation, genetic variation, and fitness, ultimately shaping the host's and future generations' evolution and development. While the hologenome concept and the Anna Karenina principle are still subject to debate and further research, they offer a promising avenue for exploring the complex relationships between host organisms and their associated microbial communities, with potential implications for medicine, agriculture, and ecology.