Saturday, February 13, 2016

[tt] PNAS: Sticky bacteriophage protect animal cells
June 25, 2013

Proceedings of the National Academy of Sciences of the United States of

Sticky bacteriophage protect animal cells

Justin R. Meyer¹
Systems Biology Department, Harvard Medical School, Boston, MA 02115

For decades, biologists have discussed and experimented with bacterial
viruses, called phage, as a means of treating bacterial infections (1,
2). Remarkably, our own evolution may have beaten us to it. In PNAS,
Barr et al. present a compelling and unique hypothesis that animal
cells use phage as weapons against bacterial pathogens (3). Animal
cells ranging from cnidarian (coral) to human excrete mucus to protect
themselves from their external environment. Barr et al. propose a
unique function for mucus, to trap phage to intersect and destroy
invading bacteria before they reach the mucus-encapsulated tissue. This
hypothesis (termed BAM for bacteriophage adhering to mucus) suggests a
unique component of the animal immune system governed by a
cross-kingdom animal-phage mutualism.

The BAM model proposed by Barr et al. (3) is as follows: (i) Animal
epithelial cells secrete mucus that is rich with mucin glycoproteins,
which work like Velcro. These proteins have a unique structure that
incorporates hundreds of negatively charged glycan chains, which extend
nanometers into the surrounding environment (4). (ii) The other side of
the Velcro is the bacteriophage capsid, which has an Ig-like protein
domain that interacts with the glycoproteins. The capsid is the DNA
reservoir or "head" of the phage, from which the "tail" or other
structures that trigger infection extend. (iii) With the phage embedded
headfirst into the mucus matrix, their protruding tails may provide
protection by infecting and killing invading bacteria (Fig. 1).

| Fig. 1.
| Bacteriophage adherence to mucus. Ig-like properties of phage capsids
| (hexagon shape) adhere to glycan residues on mucin glycoproteins
| (branched structure) of epithelial cell's mucus layer. Once embedded
| within the mucus, phage defend animal cells against invading pathogenic
| bacteria.

In this interaction, animal cells benefit by gaining protection against
bacterial pathogens, but the phage associating with the mucus layer may
also benefit. Bacteria tend to congregate in and near mucus layers
because the mucus provides a high resource habitat (5). Therefore,
phage embedded in the mucus have an increased probability of
encountering a host. In this model, chemical properties of mucus and
the Ig-like domains of capsid proteins have coevolved to interact and
reinforce this mutualistic relationship.

In support of this complex model, Barr et al. (3) identify two natural
patterns consistent with BAM. First, and most importantly, mucus is
enriched with phage. This finding is true for all mucus layers sampled,
which ranged from invertebrates to humans. Second, phage with Ig-like
domains were overrepresented in samples taken on or near mucus.

To provide direct causal evidence for BAM, Barr et al. (3) conducted
experiments that incorporated techniques from many biological
disciplines. The first set used a laboratory-based model for the
three-species interaction, with human tissue cultures, the bacterial
pathogen Escherichia coli, and the bacteriophage T4. Barr et al. began
by showing that T4 adheres to mucus and that these phage particles
remain viable and able to reproduce despite being tangled in a mucus
web. Next, T4 were shown to efficiently kill invading E. coli. Most
importantly, phage bound to mucus protected tissue culture cells from
mortality caused by E. coli. Barr et al. also ran experiments to
diagnose the molecular mechanisms that facilitate the phage-mucus
interaction. As predicted, adherence of the phage to the mucus layer is
a result of the interaction between glycans and Ig-like domains on the

Taken together, the experiments show that the physical mechanics
proposed by BAM work under controlled laboratory settings. Combined
with the natural patterns, the work strongly suggests that phage
enrichment in mucus provides defense from bacterial pathogens.

The next big challenge will be finding evidence that BAM adds an
additional layer to the animal immune system under natural conditions.
In a more natural environment, animal cells encounter tens or hundreds
of potentially pathogenic strains of bacteria. On average, phage are
extremely strain specific (6, 7); thus, how a mucus layer creates a
reservoir of phage extensive enough to protect against a myriad of
bacterial diseases remains to be seen.

An additional limitation of BAM for providing immunity is that many
phage are not lethal and can donate beneficial genes to their hosts
(8). One example that challenges the effectiveness of BAM are
pathogenic enteric bacteria from the genera Escherichia and Shigella.
Many benign E. coli strains are made pathogenic after receiving toxin
genes from phage of the λ family (9, 10), which also possess Ig-like
domains on their capsids (11). Therefore, under some rare conditions,
BAM may increase the incidence of disease by improving the probability
of benign E. coli encountering a λ phage. Animal cells could avoid this
consequence by selectively binding with other phage. It will be
fascinating to know whether mucus is selective and if epithelial cells
can reap the benefits of BAM, but avoid this negative consequence.

Another promising line of research to explore will be the role of BAM
in the context of the animal microbiome. Recent work has revealed that
a healthy microbiome helps maintain host fitness (12, 13). One way
animal cells maintain a healthy microbiome is by producing compounds
that favor the proliferation of commensal bacteria in mucus (14, 15).
Perhaps BAM has a similar effect; phage may inhibit the growth of
certain microbes, clearing space and resources for commensals to
flourish. Unraveling the direct benefits of phage's ability to destroy
invading pathogens, and other indirect benefits moderated through the
microbiome will likely reveal new therapeutic possibilities for phage.

Overall, I believe BAM is a compelling hypothesis that has likely
revealed a hidden, but ubiquitous and beneficial interaction between
metazoans and phage. I look forward to seeing the important work this
report will undoubtedly instigate.


¹E-mail: justin.raymond.meyer{at}

Author contributions: J.R.M. wrote the paper.

The author declares no conflict of interest.


1. Levin BR, Bull JJ
(2004) Population and evolutionary dynamics of phage therapy. Nat
Rev Microbiol 2(2):166–173.
2. Pirnay J-P, et al.
(2012) Introducing yesterday's phage therapy to today's medicine.
Future Virol 7(4):379–390.
3. Barr JJ, et al.
(2013) Bacteriophage adhering to mucus provide a non–host-derived
immunity. Proc Natl Acad Sci USA 110:10771–10776.
4. Cone RA
(2009) Barrier properties of mucus. Adv Drug Deliv Rev 61(2):75–85.
5. Martens EC, Chiang HC, Gordon JI
(2008) Mucosal glycan foraging enhances fitness and transmission of
a saccharolytic human gut bacterial symbiont. Cell Host Microbe
6. Flores CO, Meyer JR, Valverde S, Farr L, Weitz JS
(2011) Statistical structure of host-phage interactions. Proc Natl
Acad Sci USA 108(28):E288–E297.
7. Flores CO, Valverde S, Weitz JS
(2013) Multi-scale structure and geographic drivers of
cross-infection within marine bacteria and phage. ISME J
8. Daubin V, Lerat E, Perrière G
(2003) The source of laterally transferred genes in bacterial
genomes. Genome Biol 4(9):R57.
9. Barondess JJ, Beckwith J
(1990) A bacterial virulence determinant encoded by lysogenic
coliphage lambda. Nature 346(6287):871–874.
10. Cheetham BF, Katz ME
(1995) A role for bacteriophages in the evolution and transfer of
bacterial virulence determinants. Molec Microbiol 18(2):201–208.
11. Veesler D, Cambillau C
(2011) A common evolutionary origin for tailed-bacteriophage
functional modules and bacterial machineries. Microb and Molec Biol
Rev 75(3):423–433.
12. Blaser M, et al.
(2013) The microbiome explored: Recent insights and future
challenges. Nat Rev Microbiol 11(3):213–217.
13. Bäckhed F, Ley RE, Sonnenburg JL, Peterson DA, Gordon JI
(2005) Host-bacterial mutualism in the human intestine. Science
14. Vaishnava S, et al.
(2011) The antibacterial lectin RegIII γ promotes the spatial
segregation of microbiota and host in the Intestine. Science
15. Schluter J, Foster KR
(2012) The evolution of mutualism in gut microbiota via host
epithelial selection. PLoS Biol 10(11):e1001424.

Christian "naddy" Weisgerber
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[tt] [PLOS Bio] Host Biology in Light of the Microbiome: Ten Principles of Holobionts and Hologenomes

Host Biology in Light of the Microbiome: Ten Principles of Holobionts and

Seth R. Bordenstein, Kevin R. Theis

Published: August 18, 2015
DOI: 10.1371/journal.pbio.1002226


Groundbreaking research on the universality and diversity of
microorganisms is now challenging the life sciences to upgrade
fundamental theories that once seemed untouchable. To fully appreciate
the change that the field is now undergoing, one has to place the
epochs and foundational principles of Darwin, Mendel, and the modern
synthesis in light of the current advances that are enabling a new
vision for the central importance of microbiology. Animals and plants
are no longer heralded as autonomous entities but rather as
biomolecular networks composed of the host plus its associated
microbes, i.e., "holobionts." As such, their collective genomes forge a
"hologenome," and models of animal and plant biology that do not
account for these intergenomic associations are incomplete. Here, we
integrate these concepts into historical and contemporary visions of
biology and summarize a predictive and refutable framework for their
evaluation. Specifically, we present ten principles that clarify and
append what these concepts are and are not, explain how they both
support and extend existing theory in the life sciences, and discuss
their potential ramifications for the multifaceted approaches of
zoology and botany. We anticipate that the conceptual and
evidence-based foundation provided in this essay will serve as a
roadmap for hypothesis-driven, experimentally validated research on
holobionts and their hologenomes, thereby catalyzing the continued
fusion of biology's subdisciplines. At a time when symbiotic microbes
are recognized as fundamental to all aspects of animal and plant
biology, the holobiont and hologenome concepts afford a holistic view
of biological complexity that is consistent with the generally
reductionist approaches of biology.

Citation: Bordenstein SR, Theis KR (2015) Host Biology in Light of the
Microbiome: Ten Principles of Holobionts and Hologenomes. PLoS Biol
13(8): e1002226. doi:10.1371/journal.pbio.1002226

Academic Editor: Matthew K. Waldor, Harvard University, UNITED STATES

Published: August 18, 2015

Copyright: © 2015 Bordenstein, Theis. This is an open access article
distributed under the terms of the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction
in any medium, provided the original author and source are credited

Funding: This publication was made possible by National Science
Foundation ( grants DEB 1046149 and IOS 1456778 to
SRB, and IOS 0920505 to KRT. KRT was supported, in part, by the BEACON
Center for the Study of Evolution in Action (National Science
Foundation Cooperative Agreement DBI 0939454). The funders had no role
in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.

Competing interests: The authors have declared that no competing
interests exist.


"The time has come to replace the purely reductionist 'eyes-down'
molecular perspective with a new and genuinely holistic, eyes-up,
view of the living world, one whose primary focus is on evolution,
emergence, and biology's innate complexity."—Carl Woese (2004) [1]

At the end of the 19th century, the theory of evolution via natural
selection was birthed with the appreciation that individual animals and
plants vary in their phenotypes and that competition at the individual
level drives gradual change in the frequencies of these phenotypes [2].
From this early vantage point, fusing evolution with Mendelian genetics
in the early 20th century was a seamless transition in biology, namely
one based on the framework that phenotypes in the individual animal and
plant are encoded by the nuclear genome under the laws of Mendelian
inheritance [3–5]. In the mid-20th century, the modern synthesis
grounded the nucleocentric foundation of zoology and botany in three
areas: (1) the nuclear mutability and recombinogenic nature of
organisms, (2) the sorting of this genetic variation by natural
selection, and (3) the observations that macroevolutionary processes
such as the origin of species can be explained in a manner that aligns
with Mendelian genetics and microevolutionary mechanisms [6].

The foundation of the modern synthesis remains as scientifically sound
today as when it was conceived. However, it is critical to recognize
that microbiology was largely divorced from these early epochs in the
life sciences. The modern synthesis commenced at a time when the germ
theory of disease dictated the prevailing wisdom on microbes, and the
molecular tools used to understand the microbial world and its
influence were inferior to those available now [7–11]. The theories of
gradual evolution and the modern synthesis were thus forged during
periods of eukaryocentricism and nucleocentrism that did not appreciate
the centrality of microbiology in zoology and botany because of
limitations in perspective and technology.

Today, there is an unmistakable transformation happening in the way
that life is comprehended [12–16], and it is as significant for many
biologists as the modern synthesis. Animals and plants are no longer
viewed as autonomous entities, but rather as "holobionts" [17–21],
composed of the host plus all of its symbiotic microbes (definitions in
Box 1). The term "holobiont" traces back to Lynn Margulis and refers to
symbiotic associations throughout a significant portion of an
organism's lifetime, with the prefix holo- derived from the Greek word
holos, meaning whole or entire. Amid the flourishing of host microbiome
studies, holobiont is now generally used to mean every macrobe and its
numerous microbial associates [19,22], and the term importantly fills
the gap in what to call such assemblages. Symbiotic microbes are
fundamental to nearly every aspect of host form, function, and fitness,
including in traits that once seemed intangible to microbiology:
behavior [23–26], sociality [27–30], and the origin of species [31].
The conviction for a central role of microbiology in the life sciences
has been growing exponentially, and microbial symbiosis is advancing
from a subdiscipline to a central branch of knowledge in the life
sciences [14,32–35].

This revelation brings forth several newly appreciated facets of the
life sciences, including the testable derivation that the nuclear
genome, organelles, and microbiome of holobionts comprise a hologenome
[35–37]. The hologenome concept is a holistic view of genetics in which
animals and plants are polygenomic entities. Thus, variation in the
hologenome can lead to variation in phenotypes upon which natural
selection or genetic drift can operate. While there is a rich
literature on coevolutionary genomics of binary host–microbe
interactions, there have been few systematic attempts to align the true
complexity of the total microbiome with the modern synthesis in a way
that integrates these disparate fields [38–40].

The object of this essay is to make the holobiont and hologenome
concepts widely known. We clarify and append what they are and are not,
explain how they are both consistent with and extend existing theory in
ecology and evolutionary biology, and provide a predictive framework
for evaluating them. Our goal is to provide the main conceptual
foundation for future hypothesis-driven research that unifies perceived
divisions among subdisciplines of biology (e.g., zoology, botany, and
microbiology) and advances the postmodern synthesis that we are now
experiencing [41,42]. We distill this topic with evidence-based
reasoning to present the ten principles of holobionts and hologenomes
(summarized in Box 1).

| Box 1. The Ten Principles of Holobionts and Their Hologenomes
| 1. I. Holobionts and hologenomes are units of biological organization
| + Complex multicellular eukaryotes are not and have never been
| autonomous organisms, but rather are biological units
| organized from numerous microbial symbionts and their genomes.
| + Biomolecular associations between host and microbiota are more
| conceptually similar to an intergenomic, genotype x genotype
| interaction than a genotype x environment interaction.
| 2. II. Holobionts and hologenomes are not organ systems,
| superorganisms, or metagenomes
| + As holobionts are complex assemblages of organisms consisting
| of diverse microbial genomes, biology should draw a clear
| distinction between holobionts/hologenomes and other terms
| that were not intended to describe host–symbiont associations.
| + Organ systems and superorganisms are biological entities
| comprised of one organism's genome; metagenome means "after"
| or "beyond" the genome, does not intrinsically imply
| organismality, and obviates the fundamentals of symbiosis in
| the holobiont.
| 3. III. The hologenome is a comprehensive gene system
| + The hologenome consists of the nuclear genome, organelles, and
| microbiome.
| + Beneficial, deleterious, and neutral mutations in any of these
| genomic subunits underlie hologenomic variation.
| 4. IV. The hologenome concept reboots elements of Lamarckian evolution
| + Although Lamarck never imagined microbes in his theory,
| applying the tenets to holobionts rebirths some major aspects
| of Lamarckism.
| + The nuclear genome is inherited mainly within a Mendelian
| framework, but the microbiome is originally acquired from the
| environment and may become inherited.
| + Host–microbe associations can forge disequilibria via parental
| transfer or stable environmental transmission.
| 5. V. Hologenomic variation integrates all mechanisms of mutation
| + Every hologenome is a multiple mutant, meaning that there is
| variation across many individual genomes spanning the nucleus,
| organelles, and microbiome.
| + Base pair mutation, horizontal gene transfer, recombination,
| gene loss and duplication, and microbial loss and
| amplification are all sources of variation.
| 6. VI. Hologenomic evolution is most easily understood by equating a
| gene in the nuclear genome to a microbe in the microbiome
| + Evolution for both genes and symbionts is fundamentally a
| change in population frequency over successive generations,
| i.e., the fraction of holobionts carrying that particular
| nuclear allele or microbe.
| + Covariance of hosts and microbes in a holobiont population
| (i.e., community genetics) follows a theoretical continuum
| directly to coinheritance of gene combinations within a genome
| (i.e., population genetics).
| + A grand unified theory of evolutionary and ecological genetics
| deserves priority attention.
| 7. VII. The hologenome concept fits squarely into genetics and
| accommodates multilevel selection theory
| + Multilevel selection theory asserts that selection operates
| across multiple levels of genetic variation with phenotypic
| effects, from genes to hologenomes and beyond.
| + Holobionts are exclusive to hosts and their associated
| microbiota; different holobionts, such as a pollinator and a
| flower, interact with each other under standard ecological
| principles.
| 8. VIII. The hologenome is shaped by selection and neutrality
| + Natural selection can work to remove deleterious nuclear
| mutations or microbes while spreading advantageous nuclear
| mutations or microbes; in the absence of selection, the
| neutral spread of hologenomic variation through populations is
| an inherently stochastic process.
| + Mixed ecological models of stochastic and deterministic
| community assembly likely reflect natural systems, and
| partitioning the microbiota into stochastic versus
| deterministic subunits will be an important future goal of the
| field.
| 9. IX. Hologenomic speciation blends genetics and symbiosis
| + The Biological Species Concept was never intended to be
| exclusive of symbiosis, though history largely divorced the
| two and created unnecessary controversy.
| + Antibiotic or axenic experiments in speciation studies must be
| a routine, if not obligatory, set of experiments in genetic
| analyses of speciation for an all-inclusive understanding of
| the origin of species.
| 10. X. Holobionts and their hologenomes do not change the rules of
| evolutionary biology
| + Although the concepts redefine that which constitutes an
| individual animal or plant, they are not a fundamental
| rewriting of Darwin's and Wallace's theory of evolutionary
| biology.
| + Simply put, if the microbiome is a major, if not dominant,
| component of the DNA of a holobiont, then microbiome variation
| can quite naturally lead to new adaptations and speciation,
| just like variation in nuclear genes.

I. Holobionts and Hologenomes Are Units of Biological Organization

Host–microbial symbioses are familiar to most biologists [14,32], yet
detailed examples are often limited to very defined, often pairwise,
associations [14,35,43]. The holobiont and hologenome concepts upgrade
this conventional vision to encompass the vast ecological and genomic
complexity of a host and its total microbiota (see Box 2). These
concepts assert that macrobes are not and have never been autonomous
individuals, but rather are organized biological units, i.e.,
holobionts, composed of hundreds to thousands of individual organisms
[32,33,35,44]. Host-associated microbes have an overwhelmingly evident
influence on the physiology, anatomy, behavior, reproduction, and
fitness of holobionts [14,23–25,45–49]. The holobiont and hologenome
concepts therefore raise the discussion of individuality [33] and
organismality [50] beyond its historical perspective to a level that
challenges and extends current thinking. Although there has been
widespread discussion and applied success of the ecological theories
underlying host–microbial interactions [51–55], the specific
evolutionary principles governing these multifarious interactions
remain fundamentally unexplored.

| Box 2. Terminology
| Coevolution: reciprocal evolution of interacting species
| Commensalism: a relationship benefiting one party while the other is
| unaffected
| Mutualism: a relationship benefiting both parties
| Parasitism: a relationship benefiting one party to the other's
| detriment
| Symbiosis: two or more species living closely together in a long-term
| relationship
| Macrobe: a eukaryotic host, most being visible by eye
| Microbiota: the microbes in or on a host, including bacteria, archaea,
| viruses, protists, and fungi
| Microbiome: the complete genetic content of the microbiota
| Holobiont: a unit of biological organization composed of a host and its
| microbiota
| Hologenome: the complete genetic content of the host genome, its
| organelles' genomes, and its microbiome
| Microbe flow: the exchange of microbes between holobionts
| Phylosymbiosis: microbial community relationships changing in parallel
| with the host nuclear phylogeny
| Hologenome Concept of Evolution
| The hologenome concept of evolution was first explicitly introduced in
| 1994 during a symposium lecture by Richard Jefferson [56], and it was
| independently derived in 2007 by Eugene Rosenberg and Ilana
| Zilber-Rosenberg [57]. It posits that hosts and their microbiota are
| emergent individuals, or holobionts, that exhibit synergistic
| phenotypes that are subject to evolutionary forces [35–37]. Via
| fidelity of transmission from parents to offspring or stable
| acquisition of the microbiome from the environment, covariance between
| the host and microbiota can be established and maintained.
| Consequently, as with phenotypes encoded by nuclear genomes, phenotypes
| encoded by beneficial, deleterious, and neutral microbes in the
| microbiome are subject to selection and drift within holobiont
| populations. Genetic variation among hologenomes can arise through
| changes to host genomes as well as through changes to the genomes of
| constituent symbiotic microbes [35–37,58]. The microbiomes, and thus
| their encoded phenotypes, can change through differences in the
| relative abundances of specific symbiotic microbes, the modification of
| the genomes of existing resident microbes, or the incorporation of new
| microbial symbionts into holobionts, which can occur even within the
| reproductive lifetime of hosts [58]. Importantly, genetic variation in
| the microbiome vastly exceeds that in the host genome and accumulates
| much more rapidly than variation in host genomes. Therefore, given that
| genetic variation is the raw material upon which evolution ultimately
| acts, microbial sources of hologenomic variation are potential targets
| of evolution, and, despite its inherent complexity, biologists must
| consider the incorporation of the microbiome in the overall study of
| evolution.

A default position in modeling host and symbiont associations would be
to define them as genotype-by-environment (G host x E microbiota)
interactions. Another simplistic vision is that the microbiota is a
phenotype encoded by the host genome [44,59–63]. These tenets are
useful to a degree but merit a reexamination. Ample evidence shows that
members of the microbiota are not subjected unilaterally to the host's
intent but instead colonize specific hosts over other biotic or abiotic
habitats [64–66]. Thus, microbes are not solely an E that succumbs to
the control of a G host. They are an evolving G themselves, with their
own genomes, transcriptomes, metabolomes, etc. If we took this
host-centric view to its extreme opposite, then we end with the equally
wrong conclusion that hosts are just an environment for microbes.
Moreover, a framework for this biological organization already exists
in which the genome and microbiome forge networks of G x G interactions
that can in turn interact with E to potentially forge multispecific
geographic mosaics of coevolution [67,68]. That is, these symbioses are
best viewed as neither G × E nor G × G, but rather G × G × E. The key
point here is that the biomolecular associations between host and
microbiota are more conceptually similar to an intergenomic G x G
network or epistasis than any alternative vision that is incapable of
dealing with the nonlinear intricacies of symbioses.

Intergenomic epistasis is when genes of one species interact with
specific genes in another. The interactions, and sometimes
intertwining, of genomes and gene products between the host and
microbiota can carry out many functions of a hologenome, such as the
synthesis of essential amino acids [69], chemosynthesis [70], or
metabolite production [71]. These symbiotic combinations can be
transmitted across holobiont generations and are critical for the
maintenance of mutualisms, homeostasis, and potential coevolutionary
outcomes, such as those exemplified between the nuclear genome and
mitochondria [72]. An important and appealing aspect of intergenomic
epistasis is that it unifies, rather than separates, the genetics of
populations and communities [73]. For instance, there is a conceptual
continuum between intragenomic (or cytonuclear) interactions and
intergenomic interactions between the host genome and the microbiome.
The novelty and future challenge is identifying the number and types of
intergenomic interactions that are ecologically and evolutionarily
relevant (Box 3). This will likely require new theoretical and
statistical models, e.g., from complex systems science [74–76], that
may ultimately have as much bearing on contemporary and future
evolutionary theory as the models underlying the modern synthesis

| Box 3. Long-Term Inquiries of the Hologenome
| Hologenomic homeostasis
| Although microbiota are host specific [77–84], they are often highly
| diverse. The same can be said of nuclear genetic variation across the
| genome. Thus, an important area of scholarship will be to determine the
| homeostatic mechanisms within hologenomes that maintain such diverse
| but specific host–microbe assemblies. On the surface, the challenge for
| selection on holobiont traits seems extraordinary given the multitude
| of microbes that can potentially colonize hosts. It is presumably
| accomplished through the host's immune system and through competitive
| exclusion and antimicrobial production by members of the microbiota
| itself [14,35,80,85–87]. This area of inquiry, which is approachable
| from many disciplines, is among the primary frontiers for biologists to
| tackle.
| Hologenomic breadth
| It is important that we increase the comparative breadth and depth of
| study systems in host–microbial evolution. Much of the novelty of the
| hologenome concept lies in its emphasis on the integrative roles of
| hosts and their diverse microbiota in holobiont fitness. Well-defined
| host–microbial systems, in which one or two microbial partners exhibit
| great effect on their hosts, are tremendously valuable in elucidating
| the proximate aspects of symbiosis given their general tractability and
| ease of manipulation. However, if the hologenome concept, or any other
| allied theory, is robust, it must be evaluated using systems in which
| hosts are populated by complex microbial communities as well. While
| continuing to capitalize on well-defined systems, we should
| additionally encourage studies assessing the routes and fidelity of
| transgenerational host–microbial association, the strength of
| functional integration, and the fitness consequences of comprehensive
| microbiome variation in complex host–microbial systems. This will
| require concomitant advances in multi-omics analytical techniques and
| complex systems modeling, thereby catalyzing transdisciplinary
| discoveries in the process.
| Population and community genetics
| To determine if evolutionary changes at the hologenomic level are
| indeed concordant with evolutionary changes at the nuclear level, there
| are a handful of critical questions that must be answered across a
| broad swath of animal and plant clades. How stable is the interspecific
| covariance, or correlation, between a host and its microbiota and their
| interacting genes? How consistent is microbial transmission from one
| holobiont generation to the next? Is genetic disequilibria between host
| and microbial genes strong enough for evolution to drive covariance and
| changes in their frequencies over multiple holobiont generations? How
| much intergenomic epistasis occurs in the hologenome such that one
| nuclear allele's effect on a trait depends on the state of another
| microbial allele? Although these inquiries are formidable, they are
| unquestionably within the realm of population and community genetics
| approaches.

The debatable and testable issue of the hologenome is whether nuclear
genes and microbes are coinherited to a degree that evolution can
operate on their interaction. Coinheritance of hologenomic interactions
can occur either by vertical transmission via internal (e.g.,
transovarial) or external (e.g., breast milk) transfer mechanisms or
through stable symbioses acquired faithfully from the environment. We
discuss these crucial transmission mechanisms further in principle IV.

II. Holobionts and Hologenomes Are Not Organ Systems, Superorganisms, or

There appears to be a considerable number of misplaced
characterizations and colloquialisms used to refer to host-microbiota
symbioses, and these misnomers can potentially act as impasses to new
advances. In this section, we adapt and append the lucid clarifications
previously noted in The Hologenome Concept [35]. First, neither the
holobiont nor the microbiota should be labeled as an organ system or
organ, despite frequent uses in the popular media and scientific
literature [88–90]. An organ or organ system is strictly composed of
cells from the same genome that perform one or more specific functions.
In contrast, the microbiota is a multispecies consortia of cells with
many genomes that can contribute to multiple functions throughout the
body. Second, the holobiont is not a superorganism. This term is
exclusively used in the context of an assembly of multiple individuals
from the same species, such as in colony-forming ants, wasps, bees, and
termites [91,92]. The holobiont is instead composed of multiple domains
of life, as well as viruses. Finally, the term metagenome is not
equivalent to hologenome. Metagenome refers to the sum of genetic
information from an environmental sample and was first used in this
context to describe the collective genomes of soil microbes [93]. Meta
means "after" or "beyond" in Greek. Equating an environmental
metagenome to a host's hologenome obviates the fundamentals of
symbiosis in the holobiont. Consider the thought exercise of removing
the bacterial metagenome from soil and hosts. In nature, soil would
persist, but the host would not. While we understand that metagenomics
will not be restrained by any one definition, we and others also
recognize the salience of clear definitions in this nascent field,
particularly ones that distinguish the metagenome "beyond" the soil
from the hologenome that encompasses the "whole" collection of genomes
in a holobiont. To summarize, biology can and should draw a clear
distinction between the hologenome and other terms that were never
intended to describe host-symbiont associations, including organ,
superorganism, and metagenome.

III. The Hologenome Is a Comprehensive Gene System

The geneticist Sewall Wright stated that "selection, whether in
mortality, mating or fecundity, applies to the organism as a whole and
thus to the effects of the entire gene system rather than to single
genes" [94]. In other words, selection operates on phenotypes encoded
by the organism's underlying gene system. In this light, the hologenome
is the entire gene system of the holobiont, including elements of the
nuclear genome, organelles, and microbiome that increase fitness,
decrease fitness, or do not affect fitness at all. Within these genomic
subunits, mutations are constantly arising at their own finite rates.
In the nuclear genome, selection fixes favorable variants and purges
the deleterious ones, or "selfish" genes can spread to enhance their
own fitness. In the microbiome, selection favors the spread of
beneficial microbes involved in nutrition, defense, or reproduction
[20], while pathogenic microbes are either purged by holobiont
selection or the pathogens deploy adaptations such as reproductive
distortions to enhance their selfish transmission to the next
generation [95,96]. Moreover, neutral mutations in the nuclear genome
can drift to fixation or extinction across generations, as do microbes
without any fitness consequences. Thus, nuclear genes with adaptive,
deleterious, and neutral mutations that change their frequencies in a
holobiont population are generally analogous to beneficial, parasitic,
or neutral microbes that also change their frequencies in a holobiont
population. How these entities change their frequencies can of course
vary with transmission mode, and we address similarities and
differences below. Also, classifying microbes at just one end of the
symbiotic spectrum pigeonholes the reality that microbial symbioses can
be pleiotropic or context-dependent. These varied evolutionary forces
can sufficiently explain why animal and plant holobionts harbor
species-specific microbial communities that are segregated into their
own limited supply of hologenomic variability [31,35].

If hologenomic variation underscores fitness differences, then
manipulating the total microbiota will alter host fitness, and
therefore germ-free, gnotobiotic, and transbiotic (i.e., populated by
an atypical microbiota) hosts will exhibit reduced fitness compared to
wild-type and conventionalized hosts. Such predictions need assessment
among a broad phylogenetic range of hosts, but ample evidence already
exists. For example, in hemipteran insects, germ-free and interspecific
gut microbiota cause a decrease in survivorship and delayed development
in comparison to control or conventionalized species [97,98], and mice
with human gut microbiota have a global immunodeficiency including less
T cell proliferation and increased susceptibility to enteric infection
[99]. Moreover, interspecific hybridizations can lead to a breakdown in
hologenomic interactions within species [100,101].

IV. The Hologenome Concept Reboots Elements of Lamarckian Evolution

The nuclear genome is inherited mainly within a Mendelian framework,
and the microbiome is presumed to be mostly acquired from the
environment or inherited uniparentally [102–106]. Whether these
different transmission modes can be unified into a coherent
evolutionary theory depends in part on whether dynamics between host
and symbiont genes in the hologenome (e.g., intergenomic epistasis and
coinheritance) are similar to dynamics between genes in the same
nuclear genome [107]. In considering how genome-microbiome
disequilibria, i.e., statistical associations of covariance, among
hologenomes could arise, let's begin with the simplistic assumption
that hologenomic change commences with Lamarck's fundamental
evolutionary theory [58], generally defined as inheritance of acquired
characteristics. Although evolution has had a long and tenuous history
with Lamarckism [108,109], it is time to integrate it to a degree
alongside Darwinism in light of modern advancements. Consider the cases
of mitochondria and insect endosymbionts as textbook examples of
bacteria that were once acquired from the environment during an
organism's lifetime but now are vertically inherited over generations.
It follows that the principal tenets of Lamarckism are operational in
the origins of intimate symbioses: holobionts can gain symbiotic traits
through environmental acquisition of microbes, and holobionts can
potentially pass these traits on to the next generation via vertical
transmission. Although Lamarck never imagined microbes in his theory,
applying the tenets to holobionts rebirths Lamarckism, as some have
duly noted [35,58,110].

Once new host-microbe associations are established, they can be
maintained in disequilibria via vertical or stable environmental
transmission [35,111]. Persuasive evidence is thoroughly reviewed
elsewhere [35,105,106]. The more generations for which hologenomic
disequilibrium is maintained, and the more significant the variants'
fitness effects, the more likely it is that selection will operate on
them to drive changes in their frequencies. While some microbes are
vertically transmitted and thus fit seamlessly into current population
genetic theory, other microbes are generally not assumed to be
vertically transmitted sensu strictu from one generation to the next,
though we need to delve much deeper into these areas. Some fraction of
the microbiota may also be acquired in a stable manner from the
environment each generation, while the other fraction may be more
permissive across holobiont generations. It is also important to note
that vertically transmitted microbes do not have to remain present
through a holobiont's lifetime nor comprise a major fraction of the
microbiota to play out their evolutionary role. For instance, they may
come and go across a lifetime or body site to be vertically transmitted
and may also shape critical microbial successions that occur during
development. Lastly, although the relationships between hosts and
microbial symbionts could be fragile when there is deviation from
vertical transmission [112], such concerns are no more or less valid
than those for gene-gene interactions within a nuclear genome that can
be broken up by recombination [107]. A critical point is that
covariance of genes within and between genomes is fundamentally
similar, and associations can be reinforced by population structuring
and symbiont-host epistasis.

If portions of the microbiome are transmitted with fidelity across
holobiont generations or stably acquired from the environment, we
expect at least three types of evolutionary outcomes. First,
offsprings' microbiota and/or microbiomes should be more similar to
those of the respective organs of their parents at a similar age than
to those of other unrelated adults in the population. Second, for
inherited microbes, experimentally tagged (e.g., genetically labeled
[113,114]) microbes in adult organs should appear in the respective
organs of their offspring at a similar age more often than their
offsprings' peers. Third, host immune systems, morphological
structures, and/or behavioral repertoires should include mechanisms to
promote the effective transmission of beneficial microbes from parents
to offspring. Some illustrative model systems are already well
developed [115–117]. Broadly evaluating immunological and behavioral
mechanisms for transmission of microbial partners across holobiont
generations should be a future research priority [23,118].

V. Hologenomic Variation Integrates All Mechanisms of Mutation

Every hologenome is a multiple mutant, meaning that there is variation
across many individual genomes spanning the nucleus, organelles, and
microbiome. Without this variation, there can of course be no
evolutionary change in a population of holobionts. Random nucleotide
changes are the most obvious source of variation in the hologenome,
followed by recombination within and between chromosomes, horizontal
gene transfer within and between holobionts, and duplications/losses of
gene regions. These changes can occur in any portion of the hologenome,
so there is potential for immense genetic diversity across the entire
gene network.

Features of the microbiome such as fluctuations in microbial abundances
within holobionts are also sources of variation [35]. Indeed, they are
akin to gene duplication events driving changes in a nuclear gene's
abundance. For instance, the same microbial lineage that occurs at
different relative abundances in two otherwise genetically identical
holobionts could have different functional consequences that selection
can act upon. The most obvious illustration is when a microbe operates
as a commensal when rare but as a pathogen when relatively abundant
[119–122]. Here, the fitness of the holobiont can change dramatically.
Moreover, since no two holobionts develop in exactly duplicate
environments, there can be continuous establishment and evolution of
holobiont-specific microbes at different relative abundances that may
drive evolutionary change.

Any analysis of holobionts and their hologenomes must also account for
the multiple generations that microbes experience within the host's
single generation. These differences in generation time are not fatal
to the concepts, but they likely affect evolutionary outcomes of the
symbiosis. For example, the propensity for symbiosis to drive molecular
complexity is now a foundational premise [123], such as in obligate
symbionts (with their own generation times) supplementing the missing
nutrients in the inadequate diets of thousands of holobiont species
spanning cicadas, bedbugs, and aphids [124]. In cicadas, the case is so
extreme that genomic and cellular complexity has increased even in the
absence of new symbionts via symbiotic heteroplasmy [125]. Notably,
even nuclear genomes of mammalian species including humans, nonhuman
primates, rodents, and elephants increase in complexity via microbial
symbiosis and independent gene transfer events from virus-derived
elements [126]. Similarly in Drosophila melanogaster, viral sequences
are endogenized adjacent to retrotransposon DNA, and when transcribed,
the RNA is altered by the RNA interference (RNAi) machinery and used as
part of the immune system to combat lethal viral infections [127].

VI. Hologenomic Evolution Is Most Easily Understood by Equating a Gene in the
Nuclear Genome to a Microbe in the Microbiome

Is the hologenome concept refutable? We believe it is and suggest the
implementation of the following litmus test: are evolutionary changes
at the hologenomic level fundamentally in conflict with evolutionary
changes at the nuclear gene level? Or to put it more simply, how is the
evolution of a nuclear gene any different than the evolution of a
microbial symbiont in a holobiont population? While the strength of
selection, levels of genetic variation, and transmission strategies
differ between nuclear genes and microbes, they also vary among
different types of genes in the same nuclear genome. The important
point is that evolution for both genes and symbionts is fundamentally a
change in frequency over successive generations, i.e., the fraction of
holobionts carrying that particular nuclear allele or microbe.
Therefore, there is no intellectual disparity in contemplating the
spread of a nuclear gene as akin to the spread of a microbe through a
holobiont population. Hologenomic evolution occurs when one whole
animal or plant, i.e., holobiont, leaves a different number of
reproducing progeny than another, thereby changing the frequencies of
their associated genes in the next generation.

Covariance of hosts and microbes (i.e., community genetics) in a
holobiont population is important to this discussion as it follows a
theoretical continuum directly to coinheritance of gene combinations
within a genome (i.e., population genetics) [73]. The parameter Θ is
useful here as it is the degree of coinheritance of polygenic or
hologenomic combinations. When Θ is low because of recombination of
nuclear genes or random horizontal transmission between hosts and
microbes, there is little heritability and therefore selection will
have little effect on the combinations. When Θ is high because of
linkage disequilibria in the same genome or covariance of hosts and
microbes, then evolution will operate on the combinations in a manner
similar to as if they were single genes. Intermediate levels of Θ are
likely to reflect natural systems and the limits of inference.

Historically, models of evolution have not properly accounted for
genetically complex traits, even in the nuclear genome, because
multiple genetic signals underlying phenotypes are more diffuse [128].
Yet, high-throughput sequencing techniques have enabled genome-wide
association studies that map many small-effect alleles associated with
phenotypic variations. Similarly in the microbial sciences, it is
becoming increasingly appreciated that animal and plant holobionts are
multispecies modules in which polygenic and complex systems theories of
phenotypic variation are needed to identify signals of hologenomic
functions and evolutionary events. These questions and ideas are an
important priority for future research and emphasize the theoretical
and genetic continuum between polygenic traits in the nuclear genome
and host–microbe interactions in the hologenome [107]. Thus, holistic
theoretical and experimental models deserve priority attention in which
the genes and organisms underlying hologenomic traits vary in their
inheritance mode, heritability for the traits, and linkage
disequilibria. Moreover, a hologenomic framework may lead to resolving
part of the missing heritability problem for complex traits that are
attributable to both the nuclear genome and the microbiome.

VII. The Hologenome Concept Fits Squarely into Genetics and Accommodates
Multilevel Selection Theory

Multilevel selection theory asserts that selection operates across
multiple levels of genetic variation with phenotypic effects [129],
i.e., genes, chromosomes, genomes, cytonuclear interactions, groups,
symbionts, communities, species, etc. Evolution as a change in allelic
frequencies undoubtedly applies as all these entities have mutations
that could lead to phenotypic variation. Under a framework in which
evolutionary individuality includes hologenomic networks, fitness
differences can arise not only from nuclear or cytoplasmic mutations,
but also from host–microbe associations. Therefore, both evolutionary
and ecosystem adaptive change are relevant to the study of fitness.
Indeed, the proposal of an "eco-evolutionary" framework, the interplay
between evolutionary and ecological dynamics [130–134], is worthy of
serious attention as biology evolves to handle the combinatorial nature
of hologenomic units of evolution.

A limitation of scaling the so-called "individual" unit of evolution to
a holobiont is that biologists may ask: where does the multiorganismal
assembly of the holobiont end? Does it proceed ad infinitum?
Interactions between symbiotic microbes and their hosts make sense, but
should interacting holobionts themselves be considered part of the same
inclusive holobiont? For instance, do the genomes of an insect
pollinator and flower constitute a hologenome? The answer here is not
complex. Holobionts and their hologenomes are exclusive to the hosts
and their associated microbiota. Different holobionts, such as the
aforementioned pollinator and flower, clearly interact, but these
interactions are not new to biology, as they form the basis for all
past and present ecological investigations [135,136]. They are simply
holobionts themselves interacting with each other.

VIII. The Hologenome Is Shaped by Selection and Neutrality

Natural selection acts on holobiont phenotypes encoded by any potential
source of variation in the hologenome. As previously introduced in
principle III, selection can work to remove deleterious nuclear
mutations or microbes while spreading advantageous nuclear mutations or
microbes. In the absence of selection or when variants are selectively
equivalent, the neutral spread of hologenomic variation through
populations is an inherently stochastic process. For instance, many
microbes could replace each other over holobiont generations because of
redundant functions [137]. This may explain why animals generally have
an evident core microbiota at higher taxonomic levels, i.e., phylum,
but not at lower levels, i.e., species [85,138–140]. Neutral evolution
in the nuclear genome can also occur when nuclear allelic variants with
the same function replace each other. It is crucial to remember that
neutrality does not necessarily mean that variants are functionless.
Functional constraints and therefore negative selection are consistent
with the neutral theory. Thus, both natural selection and neutral
evolution can be seen as part of the spectrum of evolutionary
possibilities operating on the hologenome.

Beyond this evolutionary framework, various ecological theories of
community assembly are also relevant for determining whether the
microbiome is constructed stochastically or deterministically
[52,141–143]. Neutral theories of ecology emphasize the role of chance
in community assembly because ecological drift and random dispersal can
affect which microbial species inhabit a holobiont. If microbial
community structure and dynamics are primarily stochastic, then
community composition should not differ from expectations based on
random community assembly models [142,143]. In contrast, if the
host-associated microbiota is deterministically assembled, i.e., by
host-microbiota interactions, then its composition will consistently
deviate from neutral expectations. Mixed models of stochastic and
deterministic community assembly likely reflect natural systems, and
partitioning the microbiota into stochastic versus deterministic
subunits will be an important future goal of the field.

What experiments can detect non-neutral dynamics in the hologenome?
Consider the following scenario involving a genus of closely related
holobiont species reared in an unbiased manner. As horizontal
transmission is the presumed main mode of acquisition for the
microbiota [35,106], the microbial community is not a priori expected
to change in parallel with the host nuclear phylogeny unless
hologenomic interactions generate specificity and codivergence between
the genome and microbiome—a process that we previously termed
"phylosymbiosis" [100]. Phylosymbiosis does not assume that microbial
communities are stable or vertically transmitted from generation to
generation. Instead, phylosymbiosis predicts that for each generation,
intraspecific microbial communities are more similar to each other than
to interspecific microbial communities, and the levels of genetic
divergence between hosts will associate with the relative differences
between their microbial communities, yielding phylosymbiotic
concordance. Thus, given a genus of closely related animal or plant
species, the host and microbiota can either assemble (1) randomly by
stochastic processes without concordant relationships or (2)
phylosymbiotically by deterministic processes in which the
relationships of the microbiota are concordant with ancestry. At
present, evidence for phylosymbiosis under diet-controlled regimes
exists only in Nasonia [144] and Hydra [145], but the pattern also
occurs in wild populations of sponges [146], ants [147], and apes
[148,149]. Testing null models of population genetics and ecology for
the hologenome will require the application of current and new
statistical tests to distinguish selection from neutrality at both
evolutionary and ecological scales.

IX. Hologenomic Speciation Blends Genetics and Symbiosis

The Biological Species Concept [150] importantly offers a research
program to explain the origin of species—namely, the evolution of
barriers to interbreeding, i.e., reproductive isolation. In the absence
of reproductive isolation and unlimited interbreeding between
holobionts, complete gene flow and "microbe flow," a term we introduce
here to denote the exchange of microbes between holobionts, can act as
cohesive forces merging holobiont populations back into a cohesive
group. In contrast, isolating mechanisms such as ecological isolation,
mate discrimination, and hybrid incompatibilities may serve as traits
that drive holobiont populations into incipient species with unique
sets of hologenomic associations [151–154].

Despite the century-long paradigm of studying speciation genes in
nuclear genomes of model systems, the Biological Species Concept was
never intended to be exclusive of speciation symbionts [31,155].
Indeed, Theodosius Dobzhansky's graduate student Lee Ehrman pioneered
studies of symbiosis to explain Haldane's rule in Drosophila [156].
Today, there are numerous holobiont systems wherein speciation microbes
have been identified [31]. In fact, the number is similar in scope to
the quantity of known speciation genes, and we ponder how many
genetically mapped traits involved in intrinsic isolation could be
"cured" if the microbiome was removed. Antibiotic or axenic experiments
in speciation studies must be a routine, if not obligatory, set of
experiments in genetic analyses of speciation. The simple ability to
rear closely related animal species and their hybrids free of bacteria
and then to inoculate bacteria back into axenic animals permits a
gain-and-loss investigation of whether microorganisms underlie any
isolating barrier between holobiont species. The study of hologenomic
speciation is no longer optional—it is a necessary frontier that must
be traversed for an all-inclusive understanding of the origin of
species (Box 4).

| Box 4. Hologenomic Speciation
| Animal and plant species do not arise exclusively from divergence in
| their nuclear genomes [31]. Instead, symbiotic and nuclear genetic
| components can cause isolation barriers that influence the evolution of
| new animal and plant species. We argue that a combinatorial nature of
| hologenomic speciation is a far more accurate vision of speciation than
| has been traditionally recognized. Just as a speciation geneticist
| might inquire how many genes cause reproductive isolation and identify
| their functions [157,158], a speciation microbiologist would inquire
| how many host-associated microbes cause reproductive isolation and
| determine what kinds of microbes they are [31]. By simultaneously
| pursuing both sets of the questions rather than one or the other as is
| usually done, speciation biologists can achieve a unified theory of the
| Biological Species Concept that fuses symbiosis and Mendelian genetics.
| For instance, in the case of mushroom-feeding Drosophila flies or
| Nasonia parasitoid wasps, both symbiotic and nuclear genetic components
| combine to cause nearly complete reproductive isolation between young
| species [100,159,160]. All that matters is that the hologenomic
| components, the collection of host, organelle, and microbial DNA,
| function in isolation barriers.
| Pioneering work on symbiont-induced speciation traces back to Lee
| Ehrman and her studies of infectious hybrid sterility between
| subspecies of D. paulistorum [161]. The bacterial infections in the
| testes were later identified as beneficial Wolbachia within the
| subspecies that functionally breakdown in hybrids [162], similar to how
| adaptive nuclear genes within species can also breakdown in hybrids.
| Another salient example is the evolution of Wolbachia-induced F[1]
| hybrid inviability in the incipient stages of speciation between
| closely related Nasonia species [160,163]. Symbiont-induced, behavioral
| barriers to reproduction occur as well. For instance, variation in the
| gut microbiota, and consequently host odor profiles, causes premating
| isolation between strains of D. melanogaster [152].
| Speciation genetic experiments are classically designed to rule in
| nuclear genes by mapping traits to chromosomal regions, but they fail
| to assess microbes as causes of reproductive isolation. As a result,
| the significance of microbial-induced isolation has undoubtedly been
| underassessed. We propose that microbe-free experiments be universally
| implemented in speciation studies to upgrade this narrow approach. By
| way of illustration, one of the best-studied genes involved in
| Drosophila adaptive evolution and hybrid inviability, Nup96 [164,165],
| encodes a component of the nuclear pore complex that is hijacked by
| viruses to breach the nucleus [166]. Thus, mapping speciation genes to
| nuclear chromosomes is not evidence against hologenomic speciation
| sensu strictu, as some have previously noted. Rather, speciation genes
| in the nucleus may be half of the story as they often interact with the
| microbiota to cause reproductive isolation. This precedent is evident
| in Nasonia in which quantitative trait loci that associate with F[2]
| hybrid lethality are contingent on the presence of the Nasonia gut
| microbiota [100].
| The large and integral role of immune genes on reproductive isolation
| in both animals and plants has been previously termed the "Large Immune
| Effect" [31]. The immune system rapidly evolves to handle the resident
| microbiota of the holobiont, namely a finite subset of host-associated
| microbes spanning mutualists, pathogens, and commensals. For instance,
| molecular population genetic studies demonstrate in Drosophila, humans,
| and chimps that defense and immunity genes evolve more rapidly and are
| under more positive selection than the rest of the genome [167–169].
| Immunity genes can also be preferentially misexpressed (i.e., either an
| increase or decrease in levels of expression compared to the parental
| expression) in some hybrids, suggesting that the genes subject to high
| rates of positive selection within species are also the ones likely to
| be aberrantly expressed in hybrids. For example, in the hybridization
| of D. melanogaster and D. simulans, we previously calculated that 93%
| of the immune genes were differentially expressed relative to the
| nonhybrid controls, compared with 57% of the nonimmune genes [31,170].
| Hybrid autoimmunity is a frequent occurrence in plants as well
| [171,172]. Immune gene breakdowns in hybrids are likely windows into
| speciation by symbiosis and the hologenomic complexities maintaining
| host–microbe homeostasis. Indeed, in a recent study of the house mouse
| hybrid zone, hybrids displayed numerous differences in their
| microbiota, increased gut pathology, and altered immune gene expression
| [173]. Cases of accelerated rates of immune system evolution and
| positive selection within species coupled with aberrant immune gene
| function and gut microbiota in postmating reproductive isolation are
| verifications for hologenomic speciation. Moreover, the microbiota
| itself is now recognized as essential in the training and function of
| the holobiont immune system [47], including the remarkable possibility
| that mucus-associated bacteriophages operate as part of the adaptive
| and innate antimicrobial immune system [174]. Finally, the study of
| microorganisms associated with disease agents is poised to greatly
| impact our knowledge and therapeutic treatments of infectious diseases
| [175]. Collectively, these efforts and views should lead to deeper
| insights into host–microbial relationships and provide exciting new
| opportunities for the study of the origin of animal and plant species.

X. Holobionts and Their Hologenomes Do Not Change the Rules of Evolutionary

It is possible that preconceptions about how evolution works might
cause some to think that the hologenome concept changes the way they
understand evolutionary biology. However, there is no fundamental
rewriting of Darwin's and Wallace's theory of evolutionary biology
involved in this concept. Like single nucleotide mutations, acquisition
of new symbionts births raw genetic variation that evolution can
operate on. If one looks at the host-associated microbiome as a major,
if not dominant, component of the DNA of an animal or plant, then
transmissible changes in the microbiome can quite naturally lead to new
adaptations and speciation just like changes in nuclear genes. We
adhere to this general view and invite the community to consider an
expansive but not revolutionary extension of evolutionary genetics in
light of the heritable [60,78] and inherited microbiome [103,105]. In
the perennial debate about whether evolutionary biology needs a rethink
[42], it has already been noted that "this expansion of evolutionary
biology does not denigrate Darwin in the least but rather emphasizes
the fertility of his ideas" [41].


At a time when symbiotic microbes are recognized as fundamental to
nearly all aspects of animal and plant biology, the holobiont and
hologenome concepts afford holistic, eyes-up views of the multicellular
eukaryotic world that are consistent with the generally reductionist
approaches of evolutionary biology. Rather than transforming
evolutionary thought, the hologenome concept develops it in a
substantive and timely way. From a specific standpoint, the holobiont
and hologenome concepts redefine that which constitutes an individual
animal or plant by asserting that hosts and their symbiotic microbes
are complex units of biological organization upon which ecology and
evolution can act. From a general standpoint, the concepts assert that
macrobe evolution has been driven by both population and community
genetics and that symbiotic microbes and nuclear genes hold equivalent
significance in the origin of new holobiont species. Like all good
scientific theories, the concepts are subject to refutation, and in
this essay, we have explained how they can be empirically and
experimentally falsified. We anticipate that the conceptual foundation
provided in this essay will serve as a roadmap for hypothesis-driven,
experimentally validated research on holobionts and their hologenomes.


We thank colleagues Robert Brucker, Tony Capra, Tal Dagan, Nolwenn
Dheilly, Devin Drown, Jonathan Klassen, Georgiana May, Edward Van
Opstal, Nick Parrish, Maulik Patel, and Katrine Whiteson for their
critical reading and feedback on this manuscript. We also deeply
appreciate the feedback of the external reviewers, including three
transparent reviewers who exemplify progress in the peer review system.
Together, our colleagues made us think in diverse ways that reflect the
genuine breadth of this topic. Any opinions, findings, and conclusions
or recommendations expressed in this material are those of the
author(s) and do not necessarily reflect the views of the National
Science Foundation.


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