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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.

References 1. Chakravarti LJ, Beltran VH, van Oppen MJH. Rapid thermal adaptation in photosymbionts of reef-build-

ing corals. Glob Chang Biol. 2017; 23:4675–4688. https://doi.org/10.1111/gcb.13702 PMID: 28447372

2. van Oppen MJH, Bongaerts P, Frade P, Peplow LM, Boyd SE, Nim HT, et al. Adaptation to reef habitats

through selection on the coral animal and its associated microbiome. Mol Ecol. 2018; 27:2956–2971.

https://doi.org/10.1111/mec.14763 PMID: 29900626

3. Rosado PM, Leite DCA, Duarte GAS, Chaloub RM, Jospin G, Nunes da Rocha U, et al. Marine probiot-

ics: increasing coral resistance to bleaching through microbiome manipulation. ISME J. 2019; 13:921–

936. https://doi.org/10.1038/s41396-018-0323-6 PMID: 30518818

4. Voolstra CR, Ziegler M. Adapting with microbial help: microbiome flexibility facilitates rapid responses

to environmental change. BioEssays. 2020; 42:2000004. https://doi.org/10.1002/bies.202000004

PMID: 32548850

5. Cohen ML, Mashanova EV, Rosen NM, Soto W. Adaptation to temperature stress by Vibrio fischeri

facilitates this microbe’s symbiosis with the Hawaiian bobtail squid (Euprymna scolopes). Evolution.

2019; 73:1885–1897. https://doi.org/10.1111/evo.13819 PMID: 31397886

6. Cornejo-Granados F, Gallardo-Becerra L, Leonardo-Reza M, Ochoa-Romo JP, Ochoa-Leyva A. A

meta-analysis reveals the environmental and host factors shaping the structure and function of the

shrimp microbiota. PeerJ. 2018; 6:e5382. https://doi.org/10.7717/peerj.5382 PMID: 30128187

7. Huggett MJ, Apprill A. Coral microbiome database: integration of sequences reveals high diversity and

relatedness of coral-associated microbes. Environ Microbiol Rep. 2019; 11:372–385. https://doi.org/10.

1111/1758-2229.12686 PMID: 30094953

PLOS BIOLOGY

PLOS Biology | https://doi.org/10.1371/journal.pbio.3001322 August 19, 2021 13 / 18

8. Sullam KE, Essinger SD, Lozupone CA, O’Connor MP, Rosen GL, Knight R, et al. Environmental and

ecological factors that shape the gut bacterial communities of fish: a meta-analysis. Mol Ecol. 2012;

21:3363–3378. https://doi.org/10.1111/j.1365-294X.2012.05552.x PMID: 22486918

9. Thomas T, Moitinho-Silva L, Lurgi M, Björk JR, Easson C, Astudillo-Garcı́a C, et al. Diversity, structure

and convergent evolution of the global sponge microbiome. Nat Commun. 2016; 7:11870. https://doi.

org/10.1038/ncomms11870 PMID: 27306690

10. Lozupone CA, Stombaugh J, Gonzalez A, Ackermann G, Wendel D, Vázquez-Baeza Y, et al. Meta-

analyses of studies of the human microbiota. Genome Res. 2013; 23:1704–1714. https://doi.org/10.

1101/gr.151803.112 PMID: 23861384

11. Antwis RE, Griffiths SM, Harrison XA, Aranega-Bou P, Arce A, Bettridge AS, et al. Fifty important

research questions in microbial ecology. FEMS Microbiol Ecol. 2017; 93:fix044. https://doi.org/10.1093/

femsec/fix044 PMID: 28379446

12. Cullen CM, Aneja KK, Beyhan S, Cho CE, Woloszynek S, Convertino M, et al. Emerging priorities for

microbiome research. Front Microbiol. 2020; 11:136be. https://doi.org/10.3389/fmicb.2020.00136

PMID: 32140140

13. Wilkins LGE, Leray M, O’Dea A, Yuen B, Peixoto RS, Pereira TJ, et al. Host-associated microbiomes

drive structure and function of marine ecosystems. PLoS Biology. 2019; 17:e3000533. https://doi.org/

10.1371/journal.pbio.3000533 PMID: 31710600

14. Sagarin R, Pauchard A. Observational approaches in ecology open new ground in a changing world.

Front Ecol Environ. 2010; 8:379–386. https://doi.org/10.1890/090001

15. Barley SC, Meeuwig JJ. The power and the pitfalls of large-scale, unreplicated natural experiments.

Ecosystems. 2017; 20:331–339. https://doi.org/10.1007/s10021-016-0028-5

16. Hewitt JE, Thrush SF, Dayton PK, Bonsdorff E. The effect of spatial and temporal heterogeneity on the

design and analysis of empirical studies of scale-dependent systems. Am Nat. 2007; 169:398–408.

https://doi.org/10.1086/510925 PMID: 17243075

17. Douglas AE. Housing microbial symbionts: evolutionary origins and diversification of symbiotic organs

in animals. Philos Trans R Soc Lond B Biol Sci. 2020; 375:20190603. https://doi.org/10.1098/rstb.2019.

0603 PMID: 32772661

18. Foster KR, Schluter J, Coyte KZ, Rakoff-Nahoum S. The evolution of the host microbiome as an eco-

system on a leash. Nature. 2017; 548:43–51. https://doi.org/10.1038/nature23292 PMID: 28770836

19. McLaren MR, Callahan BJ. Pathogen resistance may be the principal evolutionary advantage provided

by the microbiome. Philos Trans R Soc Lond B Biol Sci. 2020; 375:20190592. https://doi.org/10.1098/

rstb.2019.0592 PMID: 32772671

20. Shade A, Handelsman J. Beyond the Venn diagram: the hunt for a core microbiome. Environ Microbiol.

2012; 14:4–12. https://doi.org/10.1111/j.1462-2920.2011.02585.x PMID: 22004523

21. Bright M, Bulgheresi S. A complex journey: transmission of microbial symbionts. Nat Rev Microbiol.

2010; 8:218–230. https://doi.org/10.1038/nrmicro2262 PMID: 20157340

22. Moran NA, McCutcheon JP, Nakabachi A. Genomics and evolution of heritable bacterial symbionts.

Annu Rev Genet. 2008; 42:165–190. https://doi.org/10.1146/annurev.genet.41.110306.130119 PMID:

18983256

23. Chomicki G, Werner GDA, West SA, Kiers ET. Compartmentalization drives the evolution of symbiotic

cooperation. Philos Trans R Soc Lond B Biol Sci. 2020; 375:20190602. https://doi.org/10.1098/rstb.

2019.0602 PMID: 32772665

24. van Oppen MJH, Medina M. Coral evolutionary responses to microbial symbioses. Philos Trans R Soc

Lond B Biol Sci. 2020; 375:20190591. https://doi.org/10.1098/rstb.2019.0591 PMID: 32772672

25. Clavijo JM, Donath A, Serôdio J, Christa G. Polymorphic adaptations in metazoans to establish and

maintain photosymbioses. Biol Rev Camb Philos Soc. 2018; 93:2006–2020. https://doi.org/10.1111/

brv.12430 PMID: 29808579

26. Dubilier N, Bergin C, Lott C. Symbiotic diversity in marine animals: the art of harnessing chemosynthe-

sis. Nat Rev Microbiol. 2008; 6:725–740. https://doi.org/10.1038/nrmicro1992 PMID: 18794911

27. Risely A. Applying the core microbiome to understand host–microbe systems. J Anim Ecol. 2020;

89:1549–1558. https://doi.org/10.1111/1365-2656.13229 PMID: 32248522

28. Astudillo-Garcı́a C, Bell JJ, Webster NS, Glasl B, Jompa J, Montoya JM, et al. Evaluating the core

microbiota in complex communities: a systematic investigation. Environ Microbiol. 2017; 19:1450–

1462. https://doi.org/10.1111/1462-2920.13647 PMID: 28078754

29. Shade A, Stopnisek N. Abundance-occupancy distributions to prioritize plant core microbiome

membership. Curr Opin Microbiol. 2019; 49:50–58. https://doi.org/10.1016/j.mib.2019.09.008 PMID:

31715441

PLOS BIOLOGY

PLOS Biology | https://doi.org/10.1371/journal.pbio.3001322 August 19, 2021 14 / 18

30. Clever F, Sourisse JM, Preziosi RF, Eisen JA, Rodriguez Guerra EC, Scott JJ, et al. The gut micro-

biome stability of a butterflyfish is disrupted on severely degraded Caribbean coral reefs. bioRxiv. 2020;

https://doi.org/10.1101/2020.09.21.306712

31. Zhang C, Derrien M, Levenez F, Brazeilles R, Ballal SA, Kim J, et al. Ecological robustness of the gut

microbiota in response to ingestion of transient food-borne microbes. ISME J. 2016; 10:2235–2245.

https://doi.org/10.1038/ismej.2016.13 PMID: 26953599

32. Sharp KH, Pratte ZA, Kerwin AH, Rotjan RD, Stewart FJ. Season, but not symbiont state, drives micro-

biome structure in the temperate coral Astrangia poculata. Microbiome. 2017; 5:120. https://doi.org/10.

1186/s40168-017-0329-8 PMID: 28915923

33. Brandl SJ, Tornabene L, Goatley CHR, Casey JM, Morais RA, Côté IM, et al. Demographic dynamics of

the smallest marine vertebrates fuel coral reef ecosystem functioning. Science. 2019; 364:1189–1192.

https://doi.org/10.1126/science.aav3384 PMID: 31123105

34. Kuempel CD, Altieri AH. The emergent role of small-bodied herbivores in pre-empting phase shifts on

degraded coral reefs. Sci Rep. 2017; 7:39670. https://doi.org/10.1038/srep39670 PMID: 28054550

35. Cleary DFR, Swierts T, Coelho FJRC, Polónia ARM, Huang YM, Ferreira MRS, et al. The sponge

microbiome within the greater coral reef microbial metacommunity. Nat Commun. 2019; 10:1–12.

36. Brothers CJ, Van Der Pol WJ, Morrow CD, Hakim JA, Koo H, McClintock JB. Ocean warming alters pre-

dicted microbiome functionality in a common sea urchin. Proc R Soc B. 2018; 285:20180340. https://

doi.org/10.1098/rspb.2018.0340 PMID: 29925614

37. Cavalcanti GS, Shukla P, Morris M, Ribeiro B, Foley M, Doane MP, et al. Rhodoliths holobionts in a

changing ocean: host-microbes interactions mediate coralline algae resilience under ocean acidifica-

tion. BMC Genomics. 2018; 19:701. https://doi.org/10.1186/s12864-018-5064-4 PMID: 30249182

38. Alverdy JC, Luo JN. The influence of host stress on the mechanism of infection: lost microbiomes,

emergent pathobiomes, and the role of interkingdom signaling. Front Microbiol. 2017; 8:322. https://doi.

org/10.3389/fmicb.2017.00322 PMID: 28303126

39. Brooks AN, Turkarslan S, Beer KD, Lo FY, Baliga NS. Adaptation of cells to new environments. Wiley

Interdiscip Rev Syst Biol Med. 2011; 3:544–561. https://doi.org/10.1002/wsbm.136 PMID: 21197660

40. Pita L, Rix L, Slaby BM, Franke A, Hentschel U. The sponge holobiont in a changing ocean: from

microbes to ecosystems. Microbiome. 2018; 6:46. https://doi.org/10.1186/s40168-018-0428-1 PMID:

29523192

41. Apprill A. The role of symbioses in the adaptation and stress responses of marine organisms. Ann Rev

Mar Sci. 2020; 12:291–314. https://doi.org/10.1146/annurev-marine-010419-010641 PMID: 31283425

42. Kikuchi Y, Tada A, Musolin DL, Hari N, Hosokawa T, Fujisaki K, et al. Collapse of insect gut symbiosis

under simulated climate change. mBio. 2016; 7:e01578–16. https://doi.org/10.1128/mBio.01578-16

PMID: 27703075

43. Zhang B, Leonard SP, Li Y, Moran NA. Obligate bacterial endosymbionts limit thermal tolerance of

insect host species. Proc Natl Acad Sci USA. 2019; 116:24712–24718. https://doi.org/10.1073/pnas.

1915307116 PMID: 31740601

44. Russell SL. Transmission mode is associated with environment type and taxa across bacteria-eukary-

ote symbioses: a systematic review and meta-analysis. FEMS Microbiol Lett. 2019; 366:fnz013. https://

doi.org/10.1093/femsle/fnz013 PMID: 30649338

45. Nussbaumer AD, Fisher CR, Bright M. Horizontal endosymbiont transmission in hydrothermal vent

tubeworms. Nature. 2006; 441:345–348. https://doi.org/10.1038/nature04793 PMID: 16710420

46. Salerno JL, Macko SA, Hallam SJ, Bright M, Won Y-J, McKiness Z, et al. Characterization of symbiont

populations in life-history stages of mussels from chemosynthetic environments. Biol Bull. 2005;

208:145–155. https://doi.org/10.2307/3593123 PMID: 15837964

47. Eberhard WG. Evolution in bacterial plasmids and levels of selection. Q Rev Biol. 1990; 65:3–22.

https://doi.org/10.1086/416582 PMID: 2186429

48. Hooks KB, O’Malley MA. Dysbiosis and its discontents. mBio. 2017; 8:e01492–17str. https://doi.org/10.

1128/mBio.01492-17 PMID: 29018121

49. Relman DA. Thinking about the microbiome as a causal factor in human health and disease: philosophi-

cal and experimental considerations. Curr Opin Microbiol. 2020; 54:119–126. https://doi.org/10.1016/j.

mib.2020.01.018 PMID: 32114367

50. Bénard A, Vavre F, Kremer N. Stress & symbiosis: heads or tails? Front Ecol Evol. 2020; 8:167. https://

doi.org/10.3389/fevo.2020.00167

51. Maher RL, Rice MM, McMinds R, Burkepile DE, Vega Thurber R. Multiple stressors interact primarily

through antagonism to drive changes in the coral microbiome. Sci Rep. 2019; 9:6834. https://doi.org/

10.1038/s41598-019-43274-8 PMID: 31048787

PLOS BIOLOGY

PLOS Biology | https://doi.org/10.1371/journal.pbio.3001322 August 19, 2021 15 / 18

52. Baker AC. Reef corals bleach to survive change. Nature. 2001; 411:765–766. https://doi.org/10.1038/

35081151 PMID: 11459046

53. Roach TNF, Dilworth J, H CM, Jones AD, Quinn RA, Drury C. Metabolomic signatures of coral bleach-

ing history. Nat Ecol Evol. 2021;1–9.

54. Awany D, Allali I, Dalvie S, Hemmings S, Mwaikono KS, Thomford NE, et al. Host and microbiome

genome-wide association studies: current state and challenges. Front Genet. 2019; 9:637. https://doi.

org/10.3389/fgene.2018.00637 PMID: 30723493

55. Geier B, Sogin EM, Michellod D, Janda M, Kompauer M, Spengler B, et al. Spatial metabolomics of in

situ host–microbe interactions at the micrometre scale. Nat Microbiol. 2020; 5:498–510. https://doi.org/

10.1038/s41564-019-0664-6 PMID: 32015496

56. Lozupone CA. Unraveling interactions between the microbiome and the host immune system to deci-

pher mechanisms of disease. mSystems. 2018; 3:e00183–17. https://doi.org/10.1128/mSystems.

00183-17 PMID: 29556546

57. Strader ME, Wong JM, Hofmann GE. Ocean acidification promotes broad transcriptomic responses in

marine metazoans: a literature survey. Front Zool. 2020; 17:7. https://doi.org/10.1186/s12983-020-

0350-9 PMID: 32095155

58. Galtier d’Auriac I, Quinn RA, Maughan H, Nothias L-F, Little M, Kapono CA, et al. Before platelets: the

production of platelet-activating factor during growth and stress in a basal marine organism. Proc R Soc

B. 2018; 285:20181307. https://doi.org/10.1098/rspb.2018.1307 PMID: 30111600

59. Vuong HE, Yano JM, Fung TC, Hsiao EY. The microbiome and host behavior. Annu Rev Neurosci.

2017; 40:21–49. https://doi.org/10.1146/annurev-neuro-072116-031347 PMID: 28301775

60. Greene A, Leggat W, Donahue MJ, Raymundo LJ, Caldwell JM, Moriarty T, et al. Complementary sam-

pling methods for coral histology, metabolomics and microbiome. Methods Ecol Evol. 2020; 11:1012–

1020. https://doi.org/10.1111/2041-210X.13431

61. Bowen BW, Gaither MR, DiBattista JD, Iacchei M, Andrews KR, Grant WS, et al. Comparative phylo-

geography of the ocean planet. Proc Natl Acad Sci USA. 2016; 113:7962–7969. https://doi.org/10.

1073/pnas.1602404113 PMID: 27432963

62. Lessios HA. The Great American schism: divergence of marine organisms after the rise of the Central

American Isthmus. Annu Rev Ecol Evol Syst. 2008; 39:63–91. https://doi.org/10.1146/annurev.ecolsys.

38.091206.095815

63. Sheppard SK, Guttman DS, Fitzgerald JR. Population genomics of bacterial host adaptation. Nat Rev

Genet. 2018; 19:549–565. https://doi.org/10.1038/s41576-018-0032-z PMID: 29973680

64. O’Dea A, Jackson JBC, Fortunato H, Smith JT, D’Croz L, Johnson KG, et al. Environmental change pre-

ceded Caribbean extinction by 2 million years. Proc Natl Acad Sci USA. 2007; 104:5501–5506. https://

doi.org/10.1073/pnas.0610947104 PMID: 17369359

65. O’Dea A, Lessios HA, Coates AG, Eytan RI, Restrepo-Moreno SA, Cione AL, et al. Formation of the

Isthmus of Panama. Sci Adv. 2016; 2:e1600883. https://doi.org/10.1126/sciadv.1600883 PMID:

27540590

66. O’Dea A, Jackson J. Environmental change drove macroevolution in cupuladriid bryozoans. Proc R

Soc B. 2009; 276:3629–3634. https://doi.org/10.1098/rspb.2009.0844 PMID: 19640882

67. DiBattista JD, Roberts MB, Bouwmeester J, Bowen BW, Coker DJ, Lozano-Cortés DF, et al. A review

of contemporary patterns of endemism for shallow water reef fauna in the Red Sea. J Biogeogr. 2016;

43:423–439. https://doi.org/10.1111/jbi.12649

68. DiBattista JD, Saenz-Agudelo P, Piatek MJ, Cagua EF, Bowen BW, Choat JH, et al. Population geno-

mic response to geographic gradients by widespread and endemic fishes of the Arabian Peninsula.

Ecol Evol. 2020; 10:4314–4330. https://doi.org/10.1002/ece3.6199 PMID: 32489599

69. Garcia-Castellanos D, Estrada F, Jiménez-Munt I, Gorini C, Fernàndez M, Vergés J, et al. Catastrophic flood of the Mediterranean after the Messinian salinity crisis. Nature. 2009; 462:778–781. https://doi.

org/10.1038/nature08555 PMID: 20010684

70. Patarnello T, Volckaert F a. MJ, Castilho R. Pillars of Hercules: is the Atlantic–Mediterranean transition

a phylogeographical break? Mol Ecol. 2007; 16:4426–4444. https://doi.org/10.1111/j.1365-294X.2007.

03477.x PMID: 17908222

71. De’ath G, Fabricius KE, Sweatman H, Puotinen M. The 27-year decline of coral cover on the Great Bar-

rier Reef and its causes. Proc Natl Acad Sci USA. 2012; 109:17995–17999. https://doi.org/10.1073/

pnas.1208909109 PMID: 23027961

72. Herlemann DP, Labrenz M, Jürgens K, Bertilsson S, Waniek JJ, Andersson AF. Transitions in bacterial

communities along the 2000 km salinity gradient of the Baltic Sea. ISME J. 2011; 5:1571–1579. https://

doi.org/10.1038/ismej.2011.41 PMID: 21472016

PLOS BIOLOGY

PLOS Biology | https://doi.org/10.1371/journal.pbio.3001322 August 19, 2021 16 / 18

73. Convey P, Chown SL, Clarke A, Barnes DKA, Bokhorst S, Cummings V, et al. The spatial structure of

Antarctic biodiversity. Ecol Monogr. 2014; 84:203–244. https://doi.org/10.1890/12-2216.1

74. Hall-Spencer JM, Rodolfo-Metalpa R, Martin S, Ransome E, Fine M, Turner SM, et al. Volcanic carbon

dioxide vents show ecosystem effects of ocean acidification. Nature. 2008; 454:96–99. https://doi.org/

10.1038/nature07051 PMID: 18536730

75. McCliment EA, Nelson CE, Carlson CA, Alldredge AL, Witting J, Amaral-Zettler LA. An all-taxon micro-

bial inventory of the Moorea coral reef ecosystem. ISME J. 2012; 6:309–319. https://doi.org/10.1038/

ismej.2011.108 PMID: 21900967

76. Neall VE, Trewick SA. The age and origin of the Pacific islands: a geological overview. Philos Trans R

Soc Lond B Biol Sci. 2008; 363:3293–3308. https://doi.org/10.1098/rstb.2008.0119 PMID: 18768382

77. Randall JE. Reef and Shore Fishes of the Hawaiian Islands. Sea Grant College Program, University of

Hawai‘i; 2007.

78. Delrieu-Trottin E, Williams JT, Bacchet P, Kulbicki M, Mourier J, Galzin R, et al. Shore fishes of the Mar-

quesas Islands, an updated checklist with new records and new percentage of endemic species. Check

List. 2015; 11:1758. https://doi.org/10.15560/11.5.1758

79. McCosker JE, Rosenblatt RH. The fishes of the Galápagos Archipelago: an update. Proc Calif Acad

Sci. 2010; 61:167–195.

80. Shaw KL, Gillespie RG. Comparative phylogeography of oceanic archipelagos: hotspots for inferences

of evolutionary process. Proc Natl Acad Sci USA. 2016; 113:7986–7993. https://doi.org/10.1073/pnas.

1601078113 PMID: 27432948

81. Duarte CM, Agusti S, Barbier E, Britten GL, Castilla JC, Gattuso J-P, et al. Rebuilding marine life.

Nature. 2020; 580:39–51. https://doi.org/10.1038/s41586-020-2146-7 PMID: 32238939

82. Cavicchioli R, Ripple WJ, Timmis KN, Azam F, Bakken LR, Baylis M, et al. Scientists’ warning to

humanity: microorganisms and climate change. Nat Rev Microbiol. 2019; 17:569–586. https://doi.org/

10.1038/s41579-019-0222-5 PMID: 31213707

83. VanWormer E, Mazet J a. K, Hall A, Gill VA, Boveng PL, London JM, et al. Viral emergence in marine

mammals in the North Pacific may be linked to Arctic sea ice reduction. Sci Rep. 2019; 9:15569. https://

doi.org/10.1038/s41598-019-51699-4 PMID: 31700005

84. Salgado J, Vélez MI, González-Arango C, Rose NL, Yang H, Huguet C, et al. A century of limnologi-

cal evolution and interactive threats in the Panama Canal: long-term assessments from a shallow

basin. Sci Total Environ. 2020; 729:138444. https://doi.org/10.1016/j.scitotenv.2020.138444 PMID:

32380321

85. Albano PG, Steger J, Bošnjak M, Dunne B, Guifarro Z, Turapova E, et al. Native biodiversity collapse in

the eastern Mediterranean. Proc R Soc B. 2021; 288:20202469. https://doi.org/10.1098/rspb.2020.

2469 PMID: 33402072

86. Graham NAJ, Wilson SK, Carr P, Hoey AS, Jennings S, MacNeil MA. Seabirds enhance coral reef pro-

ductivity and functioning in the absence of invasive rats. Nature. 2018; 559:250–253. https://doi.org/10.

1038/s41586-018-0202-3 PMID: 29995864

87. Wernberg T, Bennett S, Babcock RC, de Bettignies T, Cure K, Depczynski M, et al. Climate-driven

regime shift of a temperate marine ecosystem. Science. 2016; 353:169–172. https://doi.org/10.1126/

science.aad8745 PMID: 27387951

88. Saintilan N, Wilson NC, Rogers K, Rajkaran A, Krauss KW. Mangrove expansion and salt marsh decline

at mangrove poleward limits. Glob Chang Biol. 2014; 20:147–157. https://doi.org/10.1111/gcb.12341

PMID: 23907934

89. Beyer J, Trannum HC, Bakke T, Hodson PV, Collier TK. Environmental effects of the Deepwater Hori-

zon oil spill: a review. Mar Pollut Bull. 2016; 110:28–51. https://doi.org/10.1016/j.marpolbul.2016.06.

027 PMID: 27301686

90. Altieri AH, Harrison SB, Seemann J, Collin R, Diaz RJ, Knowlton N. Tropical dead zones and mass mor-

talities on coral reefs. Proc Natl Acad Sci USA. 2017; 114:3660–3665. https://doi.org/10.1073/pnas.

1621517114 PMID: 28320966

91. MacNeil MA, Mellin C, Matthews S, Wolff NH, McClanahan TR, Devlin M, et al. Water quality mediates

resilience on the Great Barrier Reef. Nat Ecol Evol. 2019; 3:620–627. https://doi.org/10.1038/s41559-

019-0832-3 PMID: 30858590

92. Heery EC, Hoeksema BW, Browne NK, Reimer JD, Ang PO, Huang D, et al. Urban coral reefs: degra-

dation and resilience of hard coral assemblages in coastal cities of East and Southeast Asia. Mar Pollut

Bull. 2018; 135:654–681. https://doi.org/10.1016/j.marpolbul.2018.07.041 PMID: 30301085

93. Robertson DR, Christy JH, Collin R, Cooke RG, D’Croz L, Kaufmann KW, et al. The Smithsonian Tropi-

cal Research Institute: marine research, education, and conversation in Panama. Smithson Contrib

Mar Sci. 2009;73–93.

PLOS BIOLOGY

PLOS Biology | https://doi.org/10.1371/journal.pbio.3001322 August 19, 2021 17 / 18

94. Berumen ML, Voolstra CR, Daffonchio D, Agusti S, Aranda M, Irigoien X, et al. The Red Sea: environ-

mental gradients shape a natural laboratory in a nascent Ocean. In: Voolstra CR, Berumen ML, editors.

Coral Reefs of the Red Sea. Cham: Springer International Publishing; 2019. pp. 1–10.

95. Archana A, Thibodeau B, Geeraert N, Xu MN, Kao S-J, Baker DM. Nitrogen sources and cycling

revealed by dual isotopes of nitrate in a complex urbanized environment. Water Res. 2018; 142:459–

470. https://doi.org/10.1016/j.watres.2018.06.004 PMID: 29913387

PLOS BIOLOGY

PLOS Biology | https://doi.org/10.1371/journal.pbio.3001322 August 19, 2021 18 / 18

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