critical review
reshma ghimire
ESSAY
Natural experiments and long-term
monitoring are critical to understand and
predict marine host–microbe ecology and
evolution
Matthieu LerayID 1‡*, Laetitia G. E. WilkinsID
2¤‡ , Amy ApprillID
3 , Holly M. BikID
4 ,
Friederike Clever 1,5
, Sean R. ConnollyID 1 , Marina E. De León
1,2 , J. Emmett DuffyID
6 ,
Leïla Ezzat7, Sarah Gignoux-WolfsohnID8, Edward Allen Herre1, Jonathan Z. KayeID9, David I. KlineID
1 , Jordan G. KuenemanID
1 , Melissa K. McCormickID
8 , W. Owen McMillan
1 ,
Aaron O’DeaID 1,10*, Tiago J. PereiraID
4 , Jillian M. PetersenID
11 , Daniel F. PetticordID
1 ,
Mark E. Torchin 1 , Rebecca Vega ThurberID
12 , Elin VidevallID
13,14 , William T. WcisloID
1 ,
Benedict YuenID 11
, Jonathan A. EisenID 2,15,16
1 Smithsonian Tropical Research Institute, Balboa, Ancon, Republic of Panama, 2 UC Davis Genome
Center, University of California, Davis, Davis, California, United States of America, 3 Marine Chemistry and
Geochemistry Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts, United
States of America, 4 Department of Marine Sciences and Institute of Bioinformatics, University of Georgia,
Athens, Georgia, United States of America, 5 Department of Natural Sciences, Manchester Metropolitan
University, Manchester, United Kingdom, 6 Tennenbaum Marine Observatories Network, Smithsonian
Environmental Research Center, Edgewater, Maryland, United States of America, 7 Department of Ecology,
Evolution and Marine Biology, University of California Santa Barbara, Santa Barbara, California, United
States of America, 8 Smithsonian Environmental Research Center, Edgewater, Maryland, United States of
America, 9 Gordon and Betty Moore Foundation, Palo Alto, California, United States of America,
10 Department of Biological, Geological and Environmental Sciences, University of Bologna, Bologna, Italy,
11 Centre for Microbiology and Environmental Systems Science, University of Vienna, Vienna, Austria,
12 Department of Microbiology, Oregon State University, Corvallis, Oregon, United States of America,
13 Center for Conservation Genomics, Smithsonian Conservation Biology Institute, Washington, DC, United
States of America, 14 Department of Ecology and Evolutionary Biology, Brown University, Providence,
Rhode Island, United States of America, 15 Department of Evolution and Ecology, University of California,
Davis, Davis, California, United States of America, 16 Department of Medical Microbiology and Immunology,
University of California, Davis, Davis, California, United States of America
¤ Current address: Max Planck Institute for Marine Microbiology, Department of Symbiosis, Bremen, Germany
‡ These authors share first authorship on this work.
* [email protected] (ML); [email protected] (AO)
AbstractAU : Pleaseconfirmthatallheadinglevelsarerepresentedcorrectly: Marine multicellular organisms host a diverse collection of bacteria, archaea, microbial
eukaryotes, and viruses that form their microbiome. Such host-associated microbes can sig-
nificantly influence the host’s physiological capacities; however, the identity and functional
role(s) of key members of the microbiome (“core microbiome”) in most marine hosts coexist-
ing in natural settings remain obscure. Also unclear is how dynamic interactions between
hosts and the immense standing pool of microbial genetic variation will affect marine eco-
systems’ capacity to adjust to environmental changes. Here, we argue that significantly
advancing our understanding of how host-associated microbes shape marine hosts’ plastic
and adaptive responses to environmental change requires (i) recognizing that individual
host–microbe systems do not exist in an ecological or evolutionary vacuum and (ii)
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OPEN ACCESS
Citation: Leray M, Wilkins LGE, Apprill A, Bik HM,
Clever F, Connolly SR, et al. (2021) Natural
experiments and long-term monitoring are critical
to understand and predict marine host–microbe
ecology and evolution. PLoS Biol 19(8): e3001322.
https://doi.org/10.1371/journal.pbio.3001322
Published: August 19, 2021
Copyright: This is an open access article, free of all
copyright, and may be freely reproduced,
distributed, transmitted, modified, built upon, or
otherwise used by anyone for any lawful purpose.
The work is made available under the Creative
Commons CC0 public domain dedication.
Funding: Financial support for the workshop was
provided by grant GBMF5603 (https://doi.org/10.
37807/GBMF5603) from the Gordon and Betty
Moore Foundation (W.T. Wcislo, J.A. Eisen, co-
PIs), and additional funding from the Smithsonian
Tropical Research Institute and the Office of the
Provost of the Smithsonian Institution (W.T.
Wcislo, J.P. Meganigal, and R.C. Fleischer, co-PIs).
JP was supported by a WWTF VRG Grant and the
ERC Starting Grant ’EvoLucin’. LGEW has received
funding from the European Union’s Framework
Programme for Research and Innovation Horizon
2020 (2014-2020) under the Marie Sklodowska-
Curie Grant Agreement No. 101025649. AO was
supported by the Sistema Nacional de
Investigadores (SENACYT, Panamá). A. Apprill was
supported by NSF award OCE-1938147. D.I. Kline,
M. Leray, S.R. Connolly, and M.E. Torchin were
expanding the field toward long-term, multidisciplinary research on entire communities of
hosts and microbes. Natural experiments, such as time-calibrated geological events associ-
ated with well-characterized environmental gradients, provide unique ecological and evolu-
tionary contexts to address this challenge. We focus here particularly on mutualistic
interactions between hosts and microbes, but note that many of the same lessons and
AU : Anabbreviationlisthasbeencompiledforthoseusedinthemaintext:Pleaseverifythatallentriesarecorrect:approaches would apply to other types of interactions.
Main
It is widely recognized that host-associated microbes play profound roles in the health of their
marine hosts and the ecosystems they inhabit. Although some such interactions with microbes
are transient, many are more persistent and can be generally described as symbioses. Symbio-
ses come in many flavors including parasitism, commensalism, and mutualism (see Box 1),
and, in this paper, we focus in particular on the mutually beneficial (i.e., mutualistic) subset of
such interactions involving marine hosts. Despite the wide recognition of the importance of
such mutualisms, it remains less clear how these associations scale up to drive broader ecologi-
cal and evolutionary patterns and processes. For example, the contribution of microbes to host
acclimatization and adaptation (see Box 1 for definitions) is an active new field of experimental
research with much potential. Studies, mostly conducted in controlled laboratory settings,
have evaluated the ecological costs/benefits for hosts to associate temporarily with different
microbes (e.g., corals [1–4]) or to engage in obligate intimate relationships (e.g., bobtail squid
with the bioluminescent bacteria Aliivibrio fischeri [5]). Experimental studies are, however, intrinsically limited in several ways. They limit them-
selves to a small number of experimentally tractable hosts and microbes, and, in doing so,
fail to account for the enormous complexity of interactions and variation that exist in nature
between multiple hosts and their multitudes of associated microbes. Short-lived experiments
(e.g., days to weeks) cannot replicate the scales of time and space involved in the potential
coevolution of hosts and microbes (Box 1). Attempts to merge long-term datasets to reveal
overarching patterns (e.g., [6–9]) have provided valuable insights but are shadowed by the lim-
its and biases introduced by mixing information from different contexts or methodologies
[10]. These limitations obscure general principles on the roles (mutualistic or otherwise) of
host-associated microbes across host individuals, species, and communities [11–13].
Here, we demonstrate the value of moving beyond taxon-centric approaches to studying
host–microbe associations in their natural evolutionary and ecological context. We suggest
intensifying long-term research in well-documented “natural experiments”. Such natural
experiments, including well-calibrated geological events (e.g., vicariance and creation of novel
habitats accurately dated using fossil and geological data) and environmental gradients where
multiple hosts and associated microbes are subjected to the same range of environmental con-
ditions, can be particularly useful (Fig 1). These phenomena provide a unique framework for
comparative studies where the processes of interest occur over spatial and evolutionary time
scales that are nearly impossible to capture in laboratory experiments. The value of combining
experimental and long-term field studies at natural experiments has been recognized by ecolo-
gists [14–16]. We argue that similar approaches should be applied to the study of host–microbe
interactions. We highlight several natural experiments that can advance our understanding of
the ecological and evolutionary mechanisms shaping host–microbe interactions (with a focus
on mutualistic ones) in marine communities and ecosystems.
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supported by a Rohr Family Foundation grant for
the Rohr Reef Resilience Project, for which this is
contribution #2. This is contribution #85 from the
Smithsonian’s MarineGEO and Tennenbaum
Marine Observatories Network. 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.
Abbreviations: LTER, Long-Term Ecological
Research; MarineGEO, Marine Global Earth
Observatory; MBON, Marine Biodiversity
Observation Network; TEP, Tropical Eastern Pacific.
Identifying important players
Marine organisms have evolved complex structural, behavioral, and chemical mechanisms to
regulate the presence, abundance, and activity of their microbial associates. Hosts can limit
colonization by transient opportunistic microbes that would use space and resources without
providing any benefits, and some hosts can even block pathogens entirely [17–19]. Host-spe-
cific and obligate microbial associates, often called the “core microbiome” of a host population
Box 1. Definitions of key terms
Acclimatization: The process by which an organism becomes accustomed to new envi-
ronmental conditions during its lifetime.
Adaptation: A heritable trait of an organism that increases its fitness in its surrounding
environment. In comparison to acclimatization, adaptations will be passed on to the
next generation.
Convergent evolution: Independent origins of similar features in different organisms in
response to separately experiencing similar selective pressures. Importantly, conver-
gently originated features, also known as analogous features, were not present in the
common ancestor of the taxa in question.
Genetic drift: Change in the relative frequency of genotypes due to random variation in
reproduction. Such drift is more common in small populations and leads to changes in
genotype frequencies independent of adaptive forces.
Host–microbe coevolution: During host–microbe coevolution, multicellular hosts and
their associated microbes show a concerted and heritable response to an environmental
change.
Homologous recombination: The process by which two pieces or stretches of DNA that
are very similar in their sequence physically align and exchange nucleotides.
Horizontal gene transfer: The unidirectional movement of DNA, usually only small frac-
tions of a genome, from one organism to another. Though this generally occurs more
frequently within species than between, it can also occur across vast evolutionary
distances.
Metagenomics: Studies of the genetic material of communities of organisms.
Phenotypic plasticity: Phenotypic plasticity is the ability of a specific genotype to pro-
duce more than one phenotype in response to a changing environment during an indi-
vidual’s lifetime. These phenotypic changes may include an organism’s behavior,
morphology, physiology, or other features. Phenotypic plasticity is adaptive if it increases
an individual’s survival and if the ability is passed on to the next generation.
Symbioses: Symbioses are broadly defined as intimate interactions between at least two
organisms where at least one of them benefits. We focus here specifically on mutually
beneficial interactions (aka mutualisms) between multicellular eukaryotes and their
associated microbes. These interactions may include disease resistance, predator avoid-
ance, and nutrition. These interactions will ultimately increase host survival and fitness.
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Fig 1. Examples of marine natural experiments as observatories of host–microbe interactions. Regionally focused, long-term,
and taxonomically broad research programs will help fill key knowledge gaps about the nature of microbe functions and the
dynamics of host–microbe interactions in changing oceans. We highlight areas of the world’s oceans where environmental
gradients are well characterized, where the taxonomy and evolutionary history of the local host fauna and flora is already well
established, where paleoecological studies can provide important historical context, where a long-term monitoring program is
ongoing, and where there is significant research infrastructure. Long-term monitoring sites (white dots) include sites of the NSF’s
LTER Network, the Smithsonian Institution’s MarineGEO network of partners, the MBON, the AIMS, and the ASSEMBLE. (1)
NASA MODIS data; (2) Adapted from [93]; (3) Adapted from [73]; (4) Adapted from [74]; (5) Adapted from [94]; (6) Adapted
from [95]. AIMSAU : AbbreviationlistshavebeencompiledforthoseusedinFigs1and4:Pleaseverifythatallentriesarecorrect:, Australian Institute of Marine Science; ASSEMBLE, Association of European Marine Biological Laboratories; LTER, Long-Term Ecological Research; MarineGEO, Marine Global Earth Observatory; MBON, Marine Biodiversity
Observation Network.
https://doi.org/10.1371/journal.pbio.3001322.g001
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or species, are generally assumed to play more important functional roles than opportunistic
and transient taxa [20]. This core microbiome is exemplified by an obligate nutritional micro-
bial symbiosis, in which the host relies extensively on microbial partners for survival by syn-
thesis of food, often in a nutrient-limited habitat. The host may acquire these partners
horizontally (from the surrounding environment), vertically (from the parent to the offspring),
or in both ways (mixed mode) [21]. Many evolved symbioses result in codependency; for
example, the genomes of host-associated microbes have lost genes encoding pathways that
were previously essential, such as those for motility or environmental stress responses, but that
became obsolete in obligate symbiotic lifestyles [22]. In return, hosts have evolved mechanisms
to maintain their associated microbes in stable intracellular environments and to support their
nutritional needs [23]. Some of these nutritional associations are clearly identifiable because
symbionts form massive and dense populations, sometimes only consisting of a single micro-
bial species, in or on the bodies of their hosts. Examples include photosynthetic symbioses in
cnidarians [24] and chemosynthetic symbioses in invertebrate animals such as bathymodiolin
mussels, lucinid clams, Riftia tubeworms, and Astomonema nematodes [25,26]. Although widespread, host reliance on a single or few microbes for nutrition are the exception rather
than the rule. The vast majority of animals and plants are instead associated with a diverse
assemblage of microbes where it is challenging to differentiate between members of the core
microbiome and the myriad of transient microbes and even more challenging to determine
what, if any, key functional roles such microbes play.
Several approaches have been proposed to identify key microbes or functions within com-
plex host microbiomes (reviewed in [27]). The most common practice is to identify microbial
taxa that are consistently associated with a host population or species using marker gene
sequencing, usually above some arbitrary prevalence threshold ([28]; but see [29,30] for alter-
native methods). The prevalence of a host–microbe association is typically measured without
explicit attention to co-occurring and closely related host taxa, the surrounding environment,
or adequacy of spatial and temporal sampling. This limited sampling and lack of context, often
resulting from funding constraints, leads to several major limitations. First, a microbial taxon
can be prevalent in a host population for reasons unrelated to its functional role. For example,
it may originate from the host’s food or habitat, including seawater or sediment [31]. Second,
even the core microbiome can change over time [32]. Functionally important microbes may
fluctuate in abundance throughout host ontogeny and may also vary seasonally. Essential host-
associated microbes may be overlooked if the sampling method cannot detect low abundance
reliably, resulting in false negatives, or if sampling is sporadic, missing the life stage or season
when particular microbes are essential. Third, many studies rely upon sequencing of rRNA
genes to characterize communities, yet rRNA genes are generally too conserved to distinguish
closely related taxa and reveal little directly about genomic functional potential. Clearly, under-
standing the functional roles of host-associated microbes requires analyses that go far beyond
individual marker gene profiles and instead encompass other types of information such as
whole genomes or metagenomes, transcriptomes, metabolomes, localization, biochemistry,
and more. Fourth, taxon-focused studies may miss valuable information about interactions
that could be gleaned from broader comparative analyses. Microbes that are specific to particu-
lar host genotypes, host species, or closely related groups of hosts, indicating a shared evolu-
tionary history, are likely candidates for core microbes with specialized functions (e.g., gut
fermenters associated with herbivores). These existing limitations could be robustly circum-
vented via whole-ecosystem studies where long-term collection of comprehensive genomic-
level datasets (e.g., ‘omic scale information) would transform our understanding of host–
microbe interactions at all levels.
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To instigate this new approach, we recommend strategically intensifying research within a
few ocean regions. This entails collecting large scale data on host-associated microbes across
phylogenetically diverse sets of co-occurring host organisms, together with data on surround-
ing free-living microbes (i.e., in seawater and sediments) through time in areas where the sur-
rounding abiotic environment and community dynamics have been well characterized. A
regionally focused and coordinated approach will allow identifying environmental sources
and hosts that serve as reservoirs of key host-associated microbial taxa and genes. Long-term
investments in research on particular communities of hosts and microbes will also help estab-
lish links between changes in core microbiome composition, environmental factors, ecosystem
function, and resilience. Public archival of genomic data and samples (available for comple-
mentary analysis using emerging technologies) collected from a few intensively studied ocean
regions will foster transformative discoveries on dynamic host–microbe relationships. Habi-
tat-forming corals, sponges, seagrasses, and mangrove trees are important focal groups, since
breakdowns in the associations between these species and their microbiomes likely dispropor-
tionately influence other taxa and ecosystem functions. However, this should not come at the
expense of research on more inconspicuous and overlooked, yet functionally important taxa
that comprise the majority of the oceans’ biological diversity (e.g., small fish that fuel marine
food webs [33] and urchins and crustaceans that feed on algae that can displace corals [34]).
Systematic biases toward studying certain taxa (vertebrates, species with large body sizes,
charismatic fauna), partly caused by the lack of coordination, have clearly affected our under-
standing of the distribution and roles of host-associated microbes. For example, a recent
microbiome comparison of several Indo-Pacific invertebrate species demonstrated that
sponges have a less specific microbiome than had been assumed for many years [35]. Expand-
ing the taxonomic breadth of host–microbe studies will be most fruitful in areas where taxo-
nomically rigorous field guides, ecological survey data, and functional trait databases are
available. Substantial progress will also occur where phylogenetic relationships are known and
local expert taxonomists can be engaged. One of the numerous potential outcomes includes
building community-wide association matrices to unveil the extent of reliance between hosts
and microbial partners (specificity versus ubiquity, obligate versus facultative) and the interac-
tions that promote the stability of core microbiomes.
Role of microbes in host acclimatization and adaptation
Host-associated microbes can rapidly respond to extrinsic factors such as extreme or anoma-
lous environmental conditions (e.g., heatwaves, hypoxia), pathogens, anthropogenic distur-
bances (e.g., pollution, overfishing, aquaculture, invasive species), and acute and chronic
stressors [36,37]. They can also quickly change in response to factors intrinsic to the host (e.g.,
changes in host physiology [38]). The dynamic nature of microbes may provide a source of
ecological and evolutionary novelty to support potential host response mechanisms that aug-
ment the host’s own evolutionary potential. Host-associated microbial communities can shift
rapidly through the loss, gain, or replacement of individual members. Individual microbial
cells can make rapid physiological adjustments during their lifetime (plasticity) or within a few
generations (adaptation) [39] (Fig 2). In many microbes, relatively high rates of mutation and
exchange of genetic material among divergent lineages (through homologous recombination
and horizontal gene transfer) generate a high frequency of new genetic variants, some of
which may be better suited to novel conditions (Fig 2). These mechanisms contribute to fuel-
ing an immense standing pool of genetic variation that hosts can potentially draw upon. The
outcomes of the collective ecological and evolutionary response of hosts and their associated
microbes to environmental change may comprise 1 of 4 nonmutually exclusive scenarios
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[40,41]: (1) Imbalance: a temporary or permanent change of host fitness and microbial func-
tions leading to increased disease susceptibility; (2) Resistance: the microbiome continues per-
forming its functions and the host does not lose or gain fitness; (3) Acclimatization: the newly
formed microbial community in conjunction with host phenotypic plasticity enable the indi-
vidual host to adjust and maintain performance under changing environmental conditions
(Fig 2); and (4) Adaptation: in the long term, newly formed interactions between host geno-
types and associated microbes increase the fitness of the symbiosis and they become heritable
(Fig 2).
The role that host-associated microbes play in their host’s response to environmental
change is also influenced by their mode of transmission (Fig 3). While vertical transmission
may help ensure the intergenerational stability of mutualistic symbioses, the dependence on
symbionts with highly simplified and inflexible genomes is a risky strategy under variable or
unpredictable stressful conditions [42,43]. Vertically transmitted symbionts have fewer oppor-
tunities to exchange genes with the vast pool of genetic diversity available in the external
Fig 2. Conceptual representation of the role of microbes in host acclimatization and adaptation. Microbes can frequently adapt to environmental
changes more rapidly than their host because of shorter generation times and higher standing genetic variation. Changes that occur at the levels of
individual microbes and microbiomes can rapidly generate phenotypic plasticity in a broad range of host traits (i.e., one host genotype expresses multiple
phenotypes induced by microbes). Microbially induced phenotypes may promote host adaptation if they become heritable traits. Within microbiomes,
transient microbes (thin dashed circles) have limited effects on host phenotype. On the other hand, core microbes (thick dashed circles) that engage in
prolonged relationships with hosts and potentially coevolve with hosts likely alter host phenotypes and promote host adaptation. Note that the time scale at
which evolutionary changes occur varies widely between organisms, but adaptation is generally slower than acclimatization. Plain line: nonaltered
interaction; dashed line: altered interaction; colors of microbes represent different microbial taxa.
https://doi.org/10.1371/journal.pbio.3001322.g002
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environment, which could constrain the adjustment of these associations to rapidly changing
conditions. In the marine environment, the vast majority of mutualistic symbionts are
acquired horizontally from the surrounding environment or from other hosts [44]; this
includes associations where a host is entirely dependent upon a single or a few symbionts for
nutrition (e.g., tubeworms [45]; mussels [46]). Horizontal transmission has important implica-
tions for the adaptive potential of hosts [47]. The ability to acquire microbes and genes from
the surrounding environment allows hosts to access the huge evolutionary potential contained
within the larger microbial communities. Hosts with horizontally acquired microbes could
thus be better positioned to adjust and become resilient to changing environmental condi-
tions. Selection that maintains and fine-tunes the relationship could subsequently lead to adap-
tive genetic change.
Several key bottlenecks currently impede our understanding of how host-associated
microbes drive the initial response as well as long-term, evolutionary adaptation to climate
change–related disturbances in hosts with diverse microbial communities. First, changes in
microbiomes that confer adverse or beneficial outcomes for the host cannot be distinguished
from natural variability without adequate measures of host phenotypes that covary with fitness.
Unlike photosymbiotic organisms that exhibit quantifiable phenotypic responses to stress
Fig 3. The role of microbes in the host’s response to environmental changes is contingent upon their predominant mode of transmission.
Microbes that are present in the marine environment represent a vast pool of standing genetic variation. The majority of marine species with horizontal
(e.g., lucinid clams and snapping shrimps) or mixed mode of symbiont acquisition (e.g., sponges) interact with a large number of microbes that they
acquire during their lifetime. The ability to draw on this large evolutionary potential by switching microbes or gaining new genes potentially allows
hosts to respond rapidly to environmental changes. At the other end of the spectrum, the few marine hosts with strictly vertically transmitted symbionts
(e.g., flatworms) have less opportunity to exchange genes to rapidly adjust the symbiosis to changing conditions.
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(e.g., using a bleaching index or symbiont density), the early signs of physiological stress are
difficult to observe and measure in the vast majority of marine host–microbe associations. Sec-
ond, studies are rarely designed to disentangle causes from effects. Before–after studies corre-
late seemingly altered microbial communities with perturbations or diseases, often without
establishing causality in the relationship [48,49]. Third, most research in this field has been
conducted over temporal scales that are not suited for understanding processes of acclimatiza-
tion and adaptation that may occur over months to decades [50]. Single or multistressor
laboratory experiments conducted over days to weeks are powerful means to identify environ-
mental thresholds beyond which the host–microbiome interactions become disrupted [51].
However, how experimental results can be extrapolated to understand the response of natural
systems exposed to ambient microbes and heterogeneous stressors in their natural environ-
ment remains unclear. Fourth, the response of host–microbe mutualistic symbioses to stress-
ors is partly shaped by the environmental conditions experienced during the lifetime of the
host and by previous generations, although that information is rarely considered or available.
For example, the susceptibility of corals to future environmental changes is partly contingent
upon changes in algal symbiont composition that occurred as a result of previous exposures
to temperature anomalies (i.e., symbiont shuffling in the controversial adaptive bleaching
hypothesis [52]). Therefore, the tolerance of hosts and their host-associated microbes to envi-
ronmental change is rarely interpretable without ecological context [53]. Finally, there is a
dearth of paired host and microbial genomes in public databases. The lack of population-wide
data relating traits of interest to host and microbial genomic variation at the individual level
(i.e., genome-wide association studies) limits our understanding of how genomic innovations
contribute to host acclimatization and adaptation [54].
Bolstering our understanding of the mechanisms of host–microbe evolution requires
investing resources into long-term multidisciplinary research on diverse communities of hosts
and microbes distributed across well-characterized environmental gradients. Rigorously
designed comparative population genomic studies and field experiments (e.g., reciprocal
transplants) combined with measures of host phenotypes using methods such as in situ imag-
ing [55], immunological assays [56], gene expression [57], metabolomic profiling [58], and
behavioral assays [59] will illuminate adaptive genetic variants, how they are transferred
among microbial strains across host communities, and their impacts upon host fitness.
Repeated through time, these measures will provide unique insights into how microbiome-
mediated phenotypic plasticity may allow hosts to rapidly accommodate to novel environ-
ments or resources (e.g., microbes allow some host individuals to obtain nutrients from novel
foods) through periodic (e.g., seasonal fluctuations) and transient environmental changes (e.g.,
heat waves). For foundational, long-lived, and large colonial host species, noninvasive methods
exist for repetitive sampling of tagged individuals (e.g., for corals [60]). The focus should also
expand beyond foundation species to include small, ecologically important host organisms
and those with life history strategies that make them particularly tractable for transgenera-
tional studies. This approach will only be fruitful if integrated measures of hosts and micro-
biomes are collected over multiple generations (i.e., beyond the time scale of a typical scientific
project), where physiochemical parameters are being monitored, and where the evolutionary
history of the local host fauna and flora is already well established. Targeted comparative
research can similarly leverage natural experiments that have played out over longer time
scales. Sudden discontinuities in the distribution of many closely related populations and
species have been linked to geological vicariant effects, sharp environmental gradients, or a
combination of both [61]. Organisms on opposing sides of dispersal barriers (sometimes
impassable) follow different evolutionary trajectories under the influence of local environmen-
tal conditions [62]. These systems provide unique historical contexts in which researchers can
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generate testable hypotheses about the role that host-associated microbes played in the evolu-
tion of host traits. Signatures of convergent evolution, evident at the ecosystem-wide level (i.e.,
similar patterns observed across many hosts and symbionts that have been exposed to similar
selective pressures), likely reflect fundamental principles of adaptation [63].
Examples of natural experiments
Natural experiments are past events or gradients that allow researchers to explore biological
patterns and processes on spatial and temporal scales that far exceed those possible in the labo-
ratory. Natural experiments may or may not be created or altered by humans and have been
the bread and butter of natural historians, biogeographers, and evolutionary biologists for
decades. Building on this substantial body of conceptual work, we propose that natural experi-
ments can also enlighten our understanding of the evolution and ecology of host-associated
microbes and their hosts. We present examples of natural experiments where the outcomes of
complex interactions can be observed with replication to provide insights into the processes
underlying host–microbe evolution. Our examples focus on well-characterized systems where
host evolution has already been well explored, thereby allowing “tests” that approach the rigor
of laboratory experiments. We expect that studying natural experiments like these will allow
general principles of host–microbe evolution to emerge when repeated patterns are observed
within a system or across different systems.
Biogeography
The formation of the Isthmus of Panama presents an unparalleled opportunity for exploring
the roles of biogeographic isolation and environmental change in structuring host-associated
microbes (Fig 4). In the Miocene, populations of marine organisms and their microbial symbi-
onts moved freely between the Tropical Eastern Pacific (TEP) and Caribbean in a large, unified
tropical faunal province dominated by high primary productivity and seasonal upwelling [64].
Gradually, over millions of years, this shared faunal province became severed by uplift of the
Isthmus of Panama, which finally closed approximately 2.8 Ma (million years ago) [65]. The
Caribbean became nutrient poor, causing widespread extinction and a concurrent prolifera-
tion of coral reefs and immigration of new biotas [66]. In contrast, the TEP continued to expe-
rience strong seasonal upwelling and nutrient-rich conditions. In many cases, closely related
animal hosts diverged and followed separate evolutionary trajectories, adapting to the strongly
contrasting environments on opposite sides of the Isthmus. Presumably, their associated
microbiomes did so too. Today’s Caribbean and TEP marine ecosystems of Panama and
Central America are home to hundreds of sister species that emerged through transisthmian
vicariance, representing all major taxonomic groups. Decades of research have identified phy-
logenetic relationships between hosts, as well as the behavioral, physiological, and genetic
mechanisms involved in host divergence and reproductive isolation [65]. These data place
host-associated microbes into an unrivaled ecological and evolutionary framework.
Ocean gateways that remain open today also present unique attributes suitable for natural
experiments. The narrow Strait of Bab al Mandab connects the warm and saline semi-enclosed
Red Sea with the open and more variable Arabian Sea. The Red Sea is host to many endemic
species (5% to 13% endemic across a range of taxa [67]), while the pronounced seasonal varia-
tions in the Arabian Sea have driven fine-scale local adaptations [68]. Although the Mediterra-
nean has been connected to the Atlantic through the Strait of Gibraltar since the end of the
Messinian Salinity Crisis 5.3 Ma [69], the modern Mediterranean fauna bears the more recent
imprint of Pleistocene glacial and interglacial cycles. Temperature shifts in the basin over the
last 2 to 3 Million years dictated whether subtropical or higher latitude taxa could successfully
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colonize the basin from the Atlantic and subsequent basin wide extinctions [70]. The historical
context of these ocean gateways and their impacts on gene flow have been explored in a myriad
of organisms ranging from plants to invertebrates, fish, and mammals.
Other important biogeographic regions characterized by unique environmental conditions,
long-term data collection, and good scientific infrastructure include the Great Barrier Reef
[71], the Baltic Sea [72], the Larsen B ice shelf [73], Ischia Island [74], and the French Polyne-
sian island of Moorea [75] (Fig 1). Extensive research networks such as the National Science
Foundation’s Long-Term Ecological Research (LTER) Network, the Smithsonian Institution’s
Marine Global Earth Observatory (MarineGEO) network of partners, and the Marine
Fig 4. Methodological approach to leveraging a natural experiment, the Isthmus of Panama, for the long-term study of host–microbe ecology and
evolution. Present-day organisms physically separated by the Isthmus of Panama are adapted to the distinct environmental conditions of the
productive TEP and the oligotrophic Caribbean. In the Gulf of Panama of the TEP, organisms experience some of the most drastic annual fluctuations
in temperature, pH, oxygen, salinity, and nutrients, due to intense seasonal upwelling. Conversely, the nearby Gulf of Chiriquı́ of the TEP experiences
weak to no upwelling due to trade winds being largely blocked by the Cordillera Central mountain range. Multidisciplinary and long-term research on
hosts and associated microbes across these environmental spatiotemporal gradients, where decades of taxonomic, ecological, and evolutionary research
can be leveraged, will help capture the dynamics of host–microbe interactions. TEP, Tropical Eastern Pacific.
https://doi.org/10.1371/journal.pbio.3001322.g004
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Biodiversity Observation Network (MBON; Fig 1) are set to play a fundamental role in provid-
ing researchers with logistical access (field labs and sites) to these marine ecosystems and rig-
orously collected physicochemical and biological contextual data [via, for example, long-term
deployment of sondes (CTDs) and data loggers, standardized visual surveys, and other meth-
ods] at a global scale (Fig 1). The many examples of crucial long-term support networks typi-
cally overlook host-associated microbes. They can serve as a good model going forward or
they could be leveraged to facilitate comparative studies that map microbial variation across
communities of hosts from unique marine ecosystems to help us elucidate how host–microbe
associations adjust to changes in their environment at multiple temporal (from seasonal to
geological) and spatial scales (from local to biogeographical; Fig 4).
Emergence of volcanic islands
Novel habitats such as remote island archipelagos that formed over relatively recent geological
history also offer exceptional opportunities to study evolutionary processes in marine and ter-
restrial host-associated mutualistic microbes. Initially barren, shallow coastal areas were colo-
nized by marine organisms from neighboring areas that subsequently evolved in conditions
that are often drastically different from their native environments. Three archipelagos in par-
ticular, Hawai’i and Marquesas, located at the periphery of the Indo-Pacific region, and the
Galapagos in the TEP, have provided tremendous opportunities to study evolution through
comparative phylogeography (Fig 1). All three are composed of young islands (25 to 0.75 Ma,
5.5 to 0.4 Ma, and 3.2 to 0.05 Ma, respectively; reviewed in [76]) with high proportions of
endemic species (25.0% [77], 13.7% [78], and 13.6% [79] for fishes, respectively). The shallow
coastal habitats of the islands within these archipelagos were colonized sequentially by marine
species as they formed, resulting in a “progression” pattern whereby evolutionarily older line-
ages consistently occur on older islands [80]. These regions provide a unique historical context
for understanding the evolution of host-associated microbes and their roles in driving host
ecological success when new ecological opportunities emerge.
Ongoing human-induced changes
Marine communities are changing rapidly in the face of climate change and other anthropo-
genic activities [81]. The physicochemical parameters associated with the catastrophic changes
occurring over contemporary timescales are now relatively well characterized, but the effects
on most host-associated microbes are still virtually unknown [82]. Coral bleaching is a notable
exception. As host species and their associated microbes shift in distribution, they often face
novel abiotic and biotic conditions. For example, melting of ice is opening new pathways for
the movement of animals, plants, and microbes through the Arctic, from the North Pacific to
the North Atlantic, leading to one of the largest species invasions ever observed [83]. The grad-
ual increase in salinity caused by the expansion of the Panama Canal, along with predicted
increased runoff and evaporation, will likely result in greater movement of marine species
between the tropical Western Atlantic and the TEP [84] (Fig 4). Construction of the Suez
Canal in 1869 caused an influx of saline water into the Mediterranean that was followed by the
intrusion of invasive species from the subtropical Red Sea [85]. Rats introduced to islands of
the Chagos Archipelago precipitated a decline in bird density, thereby reducing the nitrogen
input on land and in the sea with downstream effects on coral reef productivity [86]. Finally,
many tropical species are expanding their distributions with the warming climate [87]. For
example, mangrove trees take advantage of the lower frequency of freezes to colonize salt
marshes [88], which allows many invertebrate and fish species to simultaneously expand their
ranges. Additional anthropogenic pressures stem from episodic or localized disasters such as
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the 2010 Deepwater Horizon oil spill in the Gulf of Mexico [89], anoxic events (Bocas del Toro
[90]), sediment runoff events (Great Barrier Reef [91]), as well as water pollution and eutrophi-
cation around large urban centers such as Jakarta, Hong Kong, and Singapore [92] (Fig 1).
These anthropogenic changes provide multiple opportunities to understand how the rapid
evolutionary potential of host-associated microbes underpins adaptive evolution in hosts.
Conclusions
Understanding what changes in host-associated microbes mean for the maintenance of marine
communities and ecosystems requires measurements that go far beyond the typical life span of
a publicly funded scientific project. The integration of microbial sampling into long-term eco-
logical monitoring programs across key geographic locations will help us identify important
core and transient host-associated microbes and provide the fundamental basis for mechanis-
tic studies. Researchers should focus on the vast majority of marine animals and plants that
are able to interchange microbial partners, genes, and functions with surrounding microbial
communities. The future of marine ecosystems around the globe may in part depend upon
the ability of marine organisms to dip into the enormous pool of microbes and harness their
remarkable genetic potential.
Acknowledgments
We thank the staff of the Smithsonian Bocas del Toro Research Station, Rachel Collin, Jennifer
McMillan, and Patricia Leiro for helping with the logistics of the #istmobiome workshop
(December 9 to 13, 2019, Bocas del Toro) during which some of these ideas were discussed.
We thank Kendall D. Clements (ORCID: 0000-0001-8512-5977), A. Murat Eren (ORCID:
0000-0001-9013-4827), Niko Leisch (ORCID: 0000-0001-7375-3749), J. Patrick Megonigal
(ORCID: 0000-0002-2018-7883), Luis C. Mejı́a (ORCID: 0000-0003-2135-5241), Emilia M.
Sogin (ORCID: 0000-0001-7533-3705), and Blake Ushijima (ORCID: 0000-0002-1053-5207)
for participating in the discussions. Illustrations by Natalie Renier (http://nrenier.com/),
Woods Hole Oceanographic Institution.
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