From the organismal to biosphere levels: environmental impacts on the amphibian microbiota

Abstract

This review summarizes the role of environmental factors on amphibian microbiotas at the organismal, population, community, ecosystem, and biosphere levels. At the organismal-level, tissue source, disease status, and experimental manipulations were the strongest predictors of variation in amphibian microbiotas. At the population-level, habitat quality, disease status, and ancestry were commonly documented as drivers of microbiota turnover. At the community-level, studies focused on how species’ niche influence microbiota structure and function. At the ecosystem-level, abiotic and biotic reservoirs were important contributors to microbiota structure. At the biosphere-level, databases, sample banks, and seminatural experiments were commonly used to describe microbiota assembly mechanisms among temperate and tropical amphibians. Collectively, our review demonstrates that environmental factors can influence microbiotas through diverse mechanisms at all biological scales. Importantly, while environmental mechanisms occurring at each of the different scales can interact to shape microbiotas, the past 10 years of research have mostly been characterized by targeted approaches at individual scales. Looking forward, efforts considering how environmental factors at multiple organizational levels interact to shape microbiota diversity and function are paramount. Generating opportunities for meaningful cross-disciplinary interactions and supporting infrastructure for research that spans biological scales are imperative to addressing this gap.

frogs, salamanders, toads, microbial ecology, global change, reintegrating biology

Introduction

Host-associated microbiomes play an essential role in the health of organisms, including immune system activation, metabolism, and energy uptake (Baquero and Nombela 2012). Changes in microbiome diversity or composition can influence the contributions these communities bestow to host physiological processes (Petersen and Round 2014). It has been well-established that wildlife microbial communities may differ depending on multiple host physiological and behavioral factors (i.e. life stage, natural history, habitat use, and disease status; Wang et al. 2011, Kohl et al. 2013, Kueneman et al. 2014, Walke et al. 2014, Avena et al. 2016, Jiménez and Sommer 2017, Jani and Briggs 2018). As our understanding of microbiomes continues to grow, the role the environment plays in structuring host-associated microbial communities continues to be documented (i.e. temperature, available diet, and pollutants; Sanzo and Hecnar 2006, Walke et al. 2014, Antwis et al. 2014a, Avena et al. 2016, Heiman and Greenway 2016, Kohl and Yahn 2016, Zhang et al. 2016, Fontaine et al. 2018, Jani and Briggs 2018, Li et al. 2020, Yang et al. 2020a, Hernández-Gómez et al. 2020b, Huang and Liao 2021, Huang et al. 2021). While the effects of environmental variation on host-associated microbiomes have been studied extensively in humans, ruminants, and model organisms, this line of study has not been as deeply investigated in wildlife.

In the coming decades, wildlife populations are expected to face a multitude of threats associated with environmental change that can alter natural associations between hosts and microbiotas. For example, by altering natural sources of microbial symbionts in the environment, habitat degradation (i.e. fragmentation or deforestation) has been documented to alter microbiotas in amphibians, reptiles, birds, and mammals (Amato et al. 2013, Jiménez et al. 2020, Murray et al. 2020, Beale et al. 2022). Furthermore, in many cases, growing evidence suggests that shifts in microbial communities induced by environmental changes can result in cascading negative effects to host health (Redford et al. 2012, Bahrndorff et al. 2016, Jiménez and Sommer 2017). Given the association between overall health and host-associated microbiomes, the ability to make a priori predictions about how environmental changes will alter host-associated microbiomes in wildlife, and importantly when changes to microbiomes are expected to modify health, is both of fundamental biological interest and has critical conservation implications.

To understand when and how environmental changes influence microbiota structure and function, reintegrating our understanding across the silos of biological disciplines and scales is a fundamental contemporary challenge. This review will (1) summarize current understanding of how environmental factors influence amphibian microbiota at the organismal, population, community, ecosystem, and biosphere levels, (2) highlight gaps in our understanding of how environmental factors influence amphibian microbiota, and (3) identify avenues for addressing these gaps.

Amphibians as a model for microbiota community ecology

Amphibians are a useful model for understanding the role of environmental factors in shaping microbial communities. First, amphibian species vary in life-history traits from fully aquatic species to species that undergo metamorphosis where larval stages are fully aquatic and adult stages are terrestrial. As such amphibians utilize a diversity of environments that influence both diversity and composition of host-associated microbial communities (Kueneman et al. 2019).

Second, amphibians are globally distributed and live in diverse habitats from vernal pools in the arctic to permanent temperate ponds to tropical ephemeral bromeliad microecosystems (Ficetola et al. 2015). Additionally, populations of the same species can live in habitats that vary in environmental conditions. For example, North American wood frogs (Rana sylvaticus) live as far south as Alabama and as far north as Alaska; thus, wood frogs across their range can encounter drastically different abiotic and biotic environmental conditions (Larson et al. 2014). Similarly, populations of wood frogs can be found in ponds that vary in quality from populations highly impacted by human activities such as agriculture to relatively pristine ponds found in protected state lands (Hua et al. 2013). Therefore, the wide range of habitats utilized by amphibians make these taxa a useful model for understanding the effects of the environment on microbiotas.

Third, amphibians have long been established as useful model systems with research across biological scales and disciplines. For instance, the ability for some amphibian species to produce large numbers of offspring and the relative ease of caring for amphibians in the lab makes these taxa ideal for experimental work. Indeed, amphibians such as African clawed frogs (Xenopus laevis), leopard frogs (Rana sp.), and common frogs (Rana temporaria), are well-established across disciplines as models for asking genetic, physiological, toxicological, developmental, infectious disease, and numerous other research questions. In addition, amphibians are also useful models in the field of ecology and evolution as amphibian communities are well-described and the genomes of both model and nonmodel species have been sequenced. Collectively, this past interdiciplinary research provides a strong foundation for evaluating how environmental effects on microbiotas across biological scales.

Lastly, amphibian microbiotas from captive and wild populations have received considerable attention in the literature over the past decade. The microbes that reside on (i.e. skin microbiota) or within (i.e. gut microbiota) amphibians are not only highly sensitive to environmental changes (Hughey et al. 2016, 2017b, Hernández-Gómez et al. 2020a, Wuerthner et al. 2022), but studies indicate that changes to amphibian microbiota have important implications for amphibian susceptibility to disease (e.g. Batrachochytrium dendrobatidis; Bd; Bates et al. 2018, Greenspan et al. 2020). Indeed, a number of bacterial isolates from the skin of amphibians can produce metabolites that inhibit amphibian fungal pathogens associated with global amphibian declines (Woodhams et al. 2015, 2018). The link between changes to the skin microbiota and amphibian health, as well as the potential role of bacterial symbionts in disease dynamics, contribute to the useful nature of amphibians as a model.

Amphibian microbiome literature collection

We identified amphibian microbiota manuscripts published between 2011 and 2022 by searching the phrases “amphibian,” “tadpole,” “salamander,” “caecilian,” “toad,” “frog,” “microbes,” “microbial communities,” “16S rRNA,” “18S rRNA,” “bacteria,” “DNA sequencing,” “microbiome,” “microbiota,” and “amplicon sequencing” on webofscience.com. The last search was performed on 19 December 2022 and returned 4337 hits. We performed an initial review of manuscript titles and abstracts to reduce the number of studies to those that implemented shotgun metagenomics or 16S rRNA amplicon sequencing to characterize the microbial communities. For the remaining 234 manuscripts, we evaluated each study’s methodology and assigned them to different biological categories based on their sampling strategies (Table 1). Studies may have been assigned to more than one category if the authors’ sampling strategy overlapped across multiple biological levels of organization (Table 1). Following characterization, we evaluated each study independently to determine the effect of environmental/individual factors on microbiota community diversity (i.e. alpha diversity), composition (i.e. beta diversity), and compositional heterogeneity (i.e. beta diversity dispersion).

Table 1.

The boundaries between organizational levels are not always distinct. For clarity, we define below how we interpreted each of the categories used in this review paper. While many papers could be categorized into a single level, there were a handful that spanned multiple scales. Therefore, we included discussions of these papers at each level they incorporated.

Biological organization levels	Description
	Organisms are individual living beings with an organized structure, can react to stimuli, reproduce, grow, adapt, and maintain homeostasis. In this review, organismal studies are those that focus on comparing microbiotas within the bodies of individuals (i.e. gut vs. skin) and among individuals living in different environmental conditions
	Populations are individuals of the same species living in a discrete area. In this review, population studies are those that sample the microbiotas of a particular species across more than one population of the same species
	Communities are an aggregate of groups of species in ecological systems. In this review, community studies are those that compare microbiota of multiple cohabiting host species
	Ecosystems are one or more communities of living organisms interacting with their nonliving physical and chemical environments. In this review, ecosystem studies are those that evaluate microbiota of hosts and nonliving, abiotic components
	Biospheres are all ecosystems on Earth. In this review, biosphere studies are those that include samples originating from more than one ecosystem

Organismal level

The vast majority of work evaluating the effects of environmental factors on amphibian microbiotas falls into the organismal level (Table S1). We categorize organismal-level studies as those that focus on measuring how environmental factors influence the microbiotas of individuals of a particular species. The environmental factors considered in the literature are diverse including studies that evaluate the effects of diet (Kohl et al. 2014, Chang et al. 2016, Edwards et al. 2017, Knutie et al. 2017b, Wang et al. 2020, 2021, Zhang et al. 2020, Huang and Liao 2021, Antwis et al. 2014a, c), temperature (Kohl and Yahn 2016, Longo and Zamudio 2017a,b, Fontaine et al. 2018, Fontaine and Kohl 2020, Muletz-Wolz et al. 2019, Li et al. 2020, Carter et al. 2021, Zhu et al. 2021), housing conditions (Loudon et al. 2014, 2016, Kueneman et al. 2016a, Kearns et al. 2017, Wuerthner et al. 2019, Tong et al. 2019a, 2020b,, 2020c, Michaels and Preziosi 2020, Jones et al. 2021, Piccinni et al. 2021, Walke et al. 2021, Zhu et al. 2022c), habitat choice/life-stage (i.e. aquatic–terrestrial habitat use correlates with developmental stages; Kueneman et al. 2014, 2016b, Vences et al. 2016, Davis et al. 2017, Sabino-Pinto et al. 2017, Sanchez et al. 2017, Bletz et al. 2017d, Bataille et al. 2018, Chai et al. 2018, 2022b, Prest et al. 2018, Flechas et al. 2019, Zhang et al. 2019, Long et al. 2020, Yang et al. 2020b, 2022, Xu et al. 2020b, Alexiev et al. 2021, Fontaine et al. 2021, Hou et al. 2022, Martinez-Ugalde et al. 2022, Michaels and Preziosi 2020, Wuerthner et al. 2022), social interactions (Hughey et al. 2017a, McGrath-Blaser et al. 2021, Kruger and Roth 2022), pathogen exposure/infection status (Jani and Briggs 2014, 2018, Becker et al. 2015,2019, 2021, Federici et al. 2015, Walker et al. 2015, Longo and Zamudio 2017a, b, Lopez et al. 2017, Harrison et al. 2019, Shu et al. 2019a, Hernández-Gómez et al. 2020b, Kruger 2020, Weeks et al. 2020, Jani et al. 2021, Jones et al. 2021, Medina et al. 2021, Hu et al. 2022, Jiang et al. 2022, Schmeller et al. 2022), pollutants (Kohl et al. 2015, Hughey et al. 2016, Knutie et al. 2018, Mu et al. 2018, Wiebler et al. 2018, Wang et al. 2019, Ya et al. 2019, 2020, Yao et al. 2019, Bie et al. 2020, Xie et al. 2020, Zheng et al. 2020, Yang et al. 2020a, Evariste et al. 2021, Gust et al. 2021, Gutierrez-Villagomez et al. 2021, Huang et al. 2021, 2022a, b, Jiménez et al. 2021, Zhao et al. 2021, Chapman et al. 2022, Li et al. 2022, Lin et al. 2022, Lv et al. 2022, Shen et al. 2022, Wan et al. 2022, Zhu et al. 2022a, b, Chai et al. 2022a, b, Liu et al. 2022a, b, c), seasonal variation (Longo et al. 2015, Tong et al. 2019b, 2020a, b, Walke et al. 2021, Longo 2022, Xu et al. 2022), and supplementation or removal of microorganisms (Rebollar et al. 2016b, Pereira et al. 2017, Weng et al. 2017 Madison et al. 2019, Kenison et al. 2020, Woodhams et al. 2020, Christian et al. 2021, Tong et al. 2021).

Aside from the wide range in environmental conditions evaluated in the literature, many of these studies utilize a similar approach: characterizing and comparing the diversity (alpha and beta) of the skin or gut microbes in amphibians experiencing different environmental conditions. For example, Huang et al. (2021) exposed Pelophylax nigromaculatus tadpoles to atrazine and found that 500 ug/l of atrazine altered the composition and diversity of intestinal microbiota after 20 days. Using a similar study design, Walke et al. (2021) found that altering habitat structure by adding substrate into a pond shifted the composition of the microbiota community on the tadpole skin. Collectively, across these studies, there is an overwhelmingly consistent conclusion that environmental factors influence microbiota diversity and structure among individuals.

Despite consensus that shifts in environmental factors can induce shifts in microbiota diversity, how, when, and the mechanisms for how environmental change influences microbiota diversity and structure is not well-understood (Table 2). This is complicated by the challenge that a shift in the same environmental factor can induce different directional changes in microbiota diversity. For example, Fontaine and Kohl (2020), Li et al. (2020), and Kohl and Yahn (2016) found that shifts in temperature had no effect on microbiota alpha diversity. In contrast, Fontaine et al. (2018), Muletz-Wolz et al (2019), Fontaine et al. (2022), and Longo and Zamudio (2017a) found that increasing temperatures caused a decrease in the microbiota alpha diversity. In addition, Carter et al. (2021) found richer bacteriome communities at intermediate temperatures (e.g. 14°C) compared to low (6°C) and high temperatures (22°C) in experimental manipulations. The equivocal nature of these studies highlight a significant challenge in these types of investigations, while studies may manipulate the same environmental factor (temperature), variation in other environmental factors associated with experimental methods (field vs. lab), housing conditions (experimental unit size, water source), and host source (i.e. host-associated factors-species used, population) make developing generalizations about how specific environmental factors influence microbiota challenging. Collectively, this work underscores the importance of integrated approaches that work to standardize experimental work across disciplines, regions, and so on.

Table 2.

Amphibian microbiota research challenges at the organismal level.

Challenge #1: interactions between environmental and host-associated factors

In addition to environmental factors, host factors also influence microbiotas. For instance, in constant environmental conditions, host factors such as antimicrobial peptides (AMPs; Krynak et al. 2016, Woodhams et al. 2020), ontogeny (Griffiths et al. 2018), host body condition (Estrada et al. 2022), and immunogenetics (Hernández-Gómez et al. 2018, Belasen et al. 2021) can be associated with both alpha and beta microbiota diversity. While associations between host factors and microbiomes or environmental factors and microbiomes have been assessed independently, differentiating the contribution of the environment and host remains a challenge and should continue to be an area for continued research.

Challenge #2: standardize approaches to develop generalities

Though studies at the organismal level are most common, variation in methodology (i.e. exposure techniques to environmental factors, husbandry methodology, length of studies, and use of different species) currently limit our ability to develop generalizations regarding when and how environmental conditions influence microbiotas. Growing evidence demonstrates that housing conditions influence both microbiota community (Piccinni et al. 2021) and function (Wuerthner et al. 2017). Looking ahead, efforts at standardizing experimental, husbandry, and data collection efforts are critical. For instance, identifying appropriate developmental stages for experimentation, developing consistent husbandry practices (water source, housing containers), or identifying critical supplementary response variables to collect (i.e. developmental stage, mass, and so on) would be useful. Toward this goal, one important challenge to overcome will be to facilitate interactions between disciplines to identify commonalities and to develop best practices across questions asked at different scales. Symposiums that facilitate communication between researchers that focus on different biological scales may greatly improve our ability to break down the barriers across our research silos

Challenge #3: expand upon the type and geographic range of amphibian species considered

Most of the studies in our review fell into the organismal level. Though the amphibians studied represent diverse life-history strategies that encounter different habitats (i.e. direct developers vs. amphibians with larval stages; short vs. long larval stages, permanent vs. ephemeral habitats), there are limited replications across the strategies. Future work evaluating the generalities that can be drawn across species with different life histories will require substantial efforts toward systematically and sufficiently sampling across these categories as well as across geographic ranges.

Challenge #4: understand how environmentally induced changes to microbiota influence host fitness

While many studies show that environmental factors alter microbiota, few consider what are the cascading fitness consequences of these changes. Of those that do, the vast majority of studies focus on how shifts in microbiotas influence susceptibility to parasites (commonly just fungal pathogens such as Bd or Bsal—see Table 3). Given the diversity of health functions that microbiotas influence, we need to expand beyond just disease susceptibility to better understand the suite of effects it has (i.e. physiology, behavior, and social interactions) and importantly how these affect influences overall fitness. For example, shifts in microbiota may reduce susceptibility to disease but may have positive effects on growth and development. Understanding the net effects of these changes as opposed to just one metric is imperative to evaluating and making predictions about how environmentally induced changes are expected to alter microbiotas.

Lastly, while a number of studies evaluate how environmental factors modify microbiota diversity and composition changes, only a handful consider the functional consequences of these shifts in microbial communities. Of these studies, the most common response variable assessed is the effect of shifting microbial communities’ on amphibian disease outcomes, specifically, amphibian susceptibility to the amphibian pathogenic fungal disease, Chytridiomycosis (Becker et al. 2015, Federici et al. 2015, Longo et al. 2015, Walke et al. 2015, Kueneman et al. 2016b, Edwards et al. 2017, Bletz et al. 2018, Madison et al. 2019, Woodhams et al. 2020, Tong et al. 2020b, Jani et al. 2021, Jiménez et al. 2021, Estrada et al. 2022, Chen et al. 2022a). A total of five other studies evaluated disease susceptibility to other amphibian pathogens: one using ranavirus (Harrison et al. 2019), three using nematodes (Knutie et al. 2017a, b, Shu et al. 2019a), and one using trematodes (Hernández-Gómez et al. 2020b). We need more studies that focus on how shifts in microbiota diversity influence other relevant functional metrics such as intestinal histology or morphology (as in Ya et al. 2019, 2020, Huang et al. 2021, and Liu et al. 2022c), reproductive success, growth/development (as in Hernández-Gómez et al. 2020b), metabolism (Chapman et al. 2022), and survival (as in Knutie et al. 2018, Harrison et al. 2019, and Chai et al. 2022a; Table 2).

Population level

Studies that incorporate population components into their amphibian design sampled microbiotas of a particular variety across more than one population of the same species. Much of this work used natural observations to compare microbiotas across geographically distant populations within the range of a particular taxon. Across the range of multiple aquatic and terrestrial amphibian species, numerous studies document a distance decay relationship in the similarity of microbiotas among populations (Griffiths et al. 2018, Loudon et al. 2020, Walker et al. 2020, Song et al. 2021). The negative relationship between distance and microbiota similarity across broad host ranges suggests that there are limits to the dispersal of bacteria among populations, that likely result from dispersal barriers or environmental change. Microbial turnover among populations has been associated with differences in disease histories (Jani and Briggs 2014, 2018, Rebollar et al. 2016a, Jani et al. 2017, 2021, Kueneman et al. 2017, Hernández-Gómez et al. 2017b, Bletz et al. 2018, Campbell et al. 2019, Ellison et al. 2019b, Wilber et al. 2020, Jervis et al. 2021, Bates et al. 2022, Basanta et al. 2022a), habitat type (Medina et al. 2017, Sanchez et al. 2017, Hernández-Gómez et al. 2020a, Belasen et al. 2021, Basanta et al. 2022a, b), and habitat quality (Becker et al. 2017, Hughey et al. 2017b, Muletz-Wolz et al. 2018, Assis et al. 2020, Goff et al. 2020, Jiménez et al. 2020, Preuss et al. 2020, Su et al. 2022, Wuerthner et al. 2022). In addition to characterizing the microbial communities, a number of these works have also investigated how environmental differences among populations influence bacteria associated with important physiological factors. For example, the presence of heavy metals in the environment influenced the relative abundance of bacteria associated with digestive functions in gut microbiotas of Mongolian toads (Strauchbufo raddei; Zhang et al. 2016). In addition, several studies have observed a negative effect on the relative abundance of Bd-inhibitory bacteria on the skin of amphibian populations experiencing disturbance from roads (Wuerthner et al. 2022), agriculture (Jiménez et al. 2020, Preuss et al. 2020), invasive vegetation (Hernández-Gómez et al. 2020a), human land-use (Assis et al. 2020, Buttimer et al. 2021), captivity (Bates et al. 2019), and pollutants (Costa et al. 2016). The body of work evaluating natural variation in microbiotas within a species’ range and among populations experiencing different abiotic/biotic pressures highlight environmental filters as a strong driver shaping the distribution of bacterial symbionts across geographical space.

Included in the population category are a few studies that were also assigned to organismal, ecosystem, and biosphere groups because they sampled individuals from distinct populations of the same species. In these cases, expanding the number of populations included in the experimental work have allowed authors to make conclusions as to the generality of amphibian microbiota responses to host/environmental filters. For example, Belasen et al. (2021) observed less diverse skin microbial communities on river frogs that were homozygous for the MHC IIB gene compared to heterozygotes. While river frogs collected in island habitats also exhibited less diverse microbiotas, these differences in alpha diversity between MHC IIB heterozygote and homozygote hosts were maintained between the two habitats (Belasen et al. 2021). Other host characteristics such as Bd infection status (Jani and Briggs 2014), microhabitat use (Wuerthner et al. 2022), sex (Krynak et al. 2016, Hernández-Gómez et al. 2018), and life-history status (Kueneman et al. 2014, Song et al. 2021) have also been documented as consistent predictors of amphibian microbiota compositional/diversity regardless of population source. In captivity, populations can also have a lingering effect on the composition and stability of microbiotas. Several seminatural and artificial experiments were included in the population category because they included individuals from more than one population in their study design. In these examples, a degree of population legacy has been observed in the response of individual microbiotas to influence from probiotic therapies (Davis et al. 2017), captivity (Passos et al. 2018, Xiang et al. 2018, Hernández-Gómez et al. 2019, Vaissi et al. 2019), and infection with Bd (Jani and Briggs 2014, 2018, Bletz et al. 2018, Wilber et al. 2020). Both natural and artificial experiments that consider population variation have been helpful in identifying key host and environmental factors important to the microbial ecology of amphibian-associated microbiotas.

Genomic approaches that look at variation in host and microbiome genetics are necessary to identify key host factors influencing microbiotas and responding to environmental change (Table 3). Endemic vs. epidemic studies have shown a microbiota response to Bd infection history; however, most of these techniques do not quantify whether these altered communities differ in their ability to inhibit Bd in vitro or in vivo. As DNA sequencing becomes more accessible, utilizing technologies such as metagenomics and genomics (see Rebollar et al. 2018) may allow researchers to identify (1) how host adaptive genes respond to pathogen invasion/environmental change? (2) Does genetic adaptation to disease/environmental change affect how amphibian hosts interact with microorganisms in their environment or bodies? (3) Is there a parallel between microbial symbiont turnover and the function of the microbial communities? An additional consideration in factors that shape microbiota assemblage is the contribution of evolutionary history, which is a more cryptic contribution of environmental change. More specifically, different environmental backgrounds may lead to evolutionary responses that may also lead to modifications to host-associated immune factors related to microbial community assemblage. For example, costs associated with salt tolerance made amphibians release more cortisones, which might modify the environment where microbes establish (Shidemantle et al. 2021). Collectively, we need to expand our environmental factors to also consider the interaction between population legacy and environmental change.

Table 3.

Amphibian microbiota research challenges at the population and community levels.

Challenge 1: what is/are the mechanism(s) behind the effect of environmental change on microbial communities?

For the most part, studies that have found compositional differences between disturbed and pristine populations of amphibians assumed that environmental change bestows a direct effect on the microbial communities of amphibian hosts. Host and environmental factors likely interact, and we still do not understand the mechanisms behind patterns of variation among populations of amphibians. For example, Wuerthner et al. (2022), observed richer skin microbiotas in salamanders inhabiting disturbed habitats by sampling individuals living in habitats located near/far from roads. In this case, the study design did not allow the authors to identify whether the change in alpha diversity results from differences in environmental reservoirs caused by roads or because of a host physiological response to environmental change. The effect of environmental change on prey communities, habitat availability, and population density can result in physiological stress among hosts. Physiological stress responses can in turn lead to changes in host-associated microbiotas and their functions. Controlled experiments in the field or mesocosms are needed to track indirect and direct effects of environmental change on amphibian microbiotas. Incorporating individuals from multiple populations in experimental studies is important to evaluate natural variation in the response of amphibian microbiotas to environmental change.

Challenge 2: expanding beyond microbiota–host–parasite interactions

While the contribution of the microbiota to individual-level processes (i.e. physiology, mass, development, and so on) are relatively well-studied, studies evaluating cascading ecological consequences of host-associated microbiome change are not yet common (see Prado-Irwin et al. 2017). The growing exception being studies that evaluate how shifts in microbiota caused by environmental changes influence amphibian–pathogenic fungal (Bd/BSal) interactions. While our knowledge is growing in the area of fungal pathogens, amphibians are vulnerable to a diversity of other types of micro- (ranavirus) and macroparasites (trematodes). Future work evaluating the generalizability of microbiota induced changes to amphibian host–parasite interactions has broad ecological and conservation implications. Understanding how shifts in microbiota influence disease outcomes has important conservation implications for the amphibian taxa that are declining globally; however, there are many other ecological interactions to consider (i.e. predator–prey or inter/intraspecific competition). For example, in amphibian communities with gape-limited predators that preferentially consume smaller tadpole prey (i.e. newts), shifts in tadpole microbiota that result in reduced tadpole mass may indirectly modify newt–tadpole interactions by making tadpole prey more vulnerable to predation. Future studies might evaluate how shifts in microbiota induce changes in community level cascades (bottom-up vs. top-down control) or whether they modify the strength or outcome of species interaction via changes in abundance or behavior (i.e. mutualism, competition, facilitation, predation, parasitism, and so on).

Challenge 3: understanding how shifts in microbiotas associated with human-induced environmental changes alter ecological interactions

As human populations continue modifying natural ecosystems, human-induced environmental changes that modify amphibian microbiotas may play a substantial role in shaping ecological interactions. For example, global climate change is expected to lead to warmer springs, which can compress the traditional phenological timing of amphibian breeding leading to increased temporal niches overlaps across species that typically do not interact (Carey and Alexander 2003, Rivers-Moore and Karssing 2014). A number of studies have documented that amphibian species have unique specific microbial communities due to host-associated factors but as temporal niches shrink and amphibian species begin to interact, will this influence transmission of microbes between amphibian species? By altering the potential environmental pool of microbes, will this lead to shifts in amphibian microbiotas? Changes in temperature are only one of many ways that human activities can influence natural ecosystems. Future studies are needed that both characterize how human-induced environmental change influences amphibian microbiota and evaluate the ecological consequences of these changes. Importantly, given the diversity of environmental factors that may influence amphibian microbiota, designing studies in a way that allows for developing generalizations will be imperative (i.e. standardized approaches, communication between research groups, and so on).

Challenge 4: manipulative studies for understanding microbiota assemblage and maintenance

To date, most community-level studies evaluate field-collected samples or compare between field and lab environments (Sabino-Pinto et al. 2016, Bates et al. 2019). The outcome of these studies is often equivocal where some studies find that amphibian microbiota are closely associated with environmental microbes (Xu et al. 2020a) while others find that microbiota are distinct from environmental microbes (McKenzie et al. 2012). The complexity of these environments makes isolating factors important to microbiota maintenance and assemblage challenging. While there are only a handful of manipulative studies, these studies suggest that community-level ecological interactions can both influence microbiota communities and have cascading ecological effects. For instance, Hughey et al. (2022) demostrate that parasite exposure selects for defensive microbes making the host-associated microbiota less likely to be disturbed during future parasite exposures. Overall, to disentangle the relative contribution of host vs. environment vs. host-by-environmental factors it is imperative to integrate the complexity of real-world ecology with the ability to conduct manipulative and controlled studies. Toward this end, future work would be greatly benefitted by the ability to design mechanistic studies that directly manipulate microbial communities of hosts that vary in genetic and environmental backgrounds (similar to what can be achieved in other model systems—fruit flies). However, to date, an important challenge for amphibian research is the current limitations in axenic amphibian models. While model amphibians like Xenopus can be useful, the ability to create axenic amphibians of different species living in different communities is crucial to asking community level questions about how environmental factors influence microbiota assemblage and also how this might have cascading ecological consequences.

Community level

Studies that evaluate the effects of environmental factors on microbiotas at the community-level address questions including: (1) how do different amphibian species within a community differ in their microbiota? (2) How does living in communities with different environmental conditions influence amphibian microbiota communities? (3) How do other members of the community (e.g. other amphibians and other taxa that function as predators, coinhabitants, and microbes) influence a focal amphibian’s microbiota? Across the community level studies, the majority focus on characterizing and comparing variation in microbiotas of different amphibian species living in the same community to understand the relative contribution of host vs. environmental factors (Table 3). For example, both McKenzie et al. (2012) and Bletz et al. (2017b) compared the diversity and composition of the bacterial communities on the skin of cohabitating amphibians across different localities. They found that cohabiting amphibian species harbor unique skin bacterial communities indicating that in this system host-specific factors appear to more strongly regulate symbiont communities compared to environmental factors (McKenzie et al. 2012, Bletz et al. 2017b). In addition, a central objective in studies comparing microbiotas of cohabiting amphibians is to explore whether community interactions among amphibian hosts influences microbiota communities in a way that alters patterns of susceptibility or prevalence of pathogenic fungi (Bd or Bsal). For example, Jiménez et al. (2019), Rebollar et al. (2019), Hughey et al. (2022), Martins et al. 2022, and McKnight et al. (2022) compared microbiotas of sympatric tropical/subtropical species and found variation in total/fungal-inhibitory bacteria richness and relative abundance was related to Bd infection probability. Alternatively, Neely et al. (2022) and Kruger (2020) observed marginal or no differences in skin microbiome structure between Bd-infected and uninfected frogs in temperate environments. Overall, these studies are consistent with the notion that both host and environmental factors independently and interactively influence amphibian microbiotas and ultimately host health.

Of the community-level studies, several evaluated how living in communities with different environmental conditions influence microbiotas. For example, Buttimer et al. (2021) compared the microbiota of several terrestrial salamander species across different communities in the San Francisco Bay Area, CA and found that variation in community environmental variables (% forest cover and annual precipitation) influenced the skin microbial communities of some salamander genera more strongly than others. Similarly, Bletz et al. (2017c), Garcia-Recinos et al. (2019), Ellison et al. (2019a), García-Sánchez et al. (2022), Hill et al. (2022), and Muletz-Wolz et al. (2018) characterized the skin bacterial microbiome of several species of closely related salamander/frogs living in communities that differ in habitat quality or ecotype. Overall, studies that have included taxonomy and habitat characteristics have found that phylogenetic history strongly influences the diversity and community structure of the total bacterial microbiome at higher taxonomic levels (e.g. between orders), but on lower scales (e.g. within genera and species), the effect of habitat predominates. Collectively, these studies underscore the importance of evaluating environmental by host factor interactions and support the growing consensus that environmental factors exert a strong effect on microbiome structure and composition, especially within a species.

Only three studies consider how other members of the community [other amphibians or other taxa (predators, coinhabitants, and microbes)] influence amphibian microbiota. Greenspan et al. (2020) used replicated bromeliad microecosystems to test how arthropod communities influence amphibian host microbiome assembly and pathogen burden. They found that arthropods influenced amphibian microbiome assembly by altering the pool of environmental bacteria, reducing host colonization by bacteria and promoting antimicrobial components of aquatic bacterial communities. Lyra et al. (2018) describe differences in both bacterial microbiota alpha diversity and composition between invertebrates and tadpoles inhabiting the same habitat. Although, organisms with similar diets (e.g. snails and tadpoles) shared the highest proportion of OTUs. Finally, Weitzman et al. (2018) describe strong variation in microbiome composition between sympatric amphibians and reptiles. In nature, amphibians face a diversity of inter and intraspecific interactions that may influence microbiota communities yet our understanding of when these ecological interactions influence microbiotas and the functions they perform remain limited.

Ecosystem level

Studies that look at amphibian microbiomes from the ecosystem level incorporated multiple biotic and abiotic microbial reservoirs in their microbiome surveys to understand the distribution of amphibian bacterial symbionts across hosts and the environment. At the ecosystem level, we included studies that looked at communities of amphibians, or one species, and an environmental source of microorganisms in their sampling design. Most investigations in the ecosystem category used natural observations to study the distribution of microorganisms between amphibians and abiotic environmental components (Table S1). Across all these studies, amphibians shared up to half of their microbial symbionts with the tested environmental source. In aquatic settings, substrates (e.g. water or soil) and amphibians possessed divergent communities, with the former typically possessing a low relative abundance of amphibian-associated bacteria/microeukaryotes (Kueneman et al. 2014, 2017, Walke et al. 2014, Hernández-Gómez et al. 2017a, Bletz et al. 2017c, Bates et al. 2018, Hughey et al. 2019, Alexiev et al. 2021, Douglas et al. 2021, Martinez-Ugalde et al. 2022). However, studies that looked at host/environment microbiota overlap in terrestrial systems found correlations in the composition of soil and amphibian microbiotas (Fitzpatrick and Allison 2014, Prado-Irwin et al. 2017, Bird et al. 2018). These observations are evidence that there is some degree of microbial dispersal between the environment and amphibian bodies, and the level of transmission might depend on characteristics of the microhabitat amphibians use across the ecosystem.

Natural observations have also been used to assess the effect of environmental change on environmental reservoirs within amphibian habitats. For example, variation in habitat temperature and precipitation because of altitudinal or latitudinal differences among habitats have been correlated with microbiota turnover in amphibians and environmental surfaces (Albecker et al. 2019, Ruthsatz et al. 2020, Xu et al. 2020b). Seminatural experiments have also reported amphibian and sometimes environmental microbial community differences when comparing sites across ecosystems that differ in vegetation (Hernández-Gómez et al. 2020a), pollution (Su et al. 2022, Zhu et al. 2022b), and human land use intensity (Hernández-Gómez et al. 2017a, Barnes et al. 2021). One study found richer skin microbial communities on California slender salamanders (Batrachoseps attenuatus) inhabiting forest patches dominated by native oak trees compared to invasive eucalyptus trees in central California (Hernández-Gómez et al. 2020a). In addition, Hernández-Gómez et al. (2020a) documented lower body condition indices in salamanders collected from the invasive eucalyptus forest patches indicating a negative impact of dysbiosis, environmental change, or both on host health. Despite these observations, the authors found similar numbers/composition of bacteria among the soil collected from underneath the log where the salamanders were collected from (Hernández-Gómez et al. 2020a). Thus, environmental change might directly or indirectly influence amphibian microbiotas through alterations of environmental reservoirs, degradation of host health, and/or changes in other a/biotic sources that have not yet been measured.

Because ecosystems may exhibit a nonuniform distribution of microhabitats, sympatric species of amphibians, or even individuals of the same species, may exhibit different communities with different functionalities based on what habitat they utilize. Across more than one study, terrestrial amphibians possessed higher alpha diversities and different microbial communities compared to aquatic or arboreal forms (Kueneman et al. 2014, Rebollar et al. 2016a, Bletz et al. 2017c, Albecker et al. 2019, Wuerthner et al. 2022), and this pattern was mirrored when comparing the communities of biotic/abiotic reservoirs. In a reciprocal transfer experiment between pond and stream habitats, Bletz et al. (2016) tracked and found changes in the predicted functions of the gut and skin microbiota of salamander larvae associated with habitat shift. Interestingly, there are numerous examples where there is a correlation between microhabitat use and susceptibility to Bd, with aquatic amphibians being more resistant to the fungus than terrestrial ones (Hossack et al. 2013, Mesquita et al. 2017). Bd zoospores spread via the aquatic environment; thus, it is possible that continuous selective pressure from the pathogen on aquatic amphibians have led to increased immunocompetence. In fact, greater susceptibility to chytridiomycosis has been observed in terrestrial species that interact with bodies of water less frequently (Bancroft et al. 2011, Mesquita et al. 2017). As a result, different exposure histories among sympatric amphibians generate an interesting phenomenon where Bd can exist both as an endemic and epidemic state within the same geographic area.

Amphibian microbiotas are not closed systems; thus, experimental approaches that consider how disturbance factors such as climate change, invasive species, pollution, captivity, and habitat change influence microbial reservoirs continue to be important. Small-scale ecosystem experiments or even artificial experiments performed in mesocosms that incorporate environmental reservoirs are imperative to measure the response of host-associated and environmental microbiotas to environmental change (Table 4). Measuring the contribution of biotic and abiotic microbial reservoirs to natural amphibian microbiotas is necessary to identify sources of beneficial and pathogenic microorganisms in the environment. Identifying key environmental reservoirs might benefit the management of captive amphibian microbiotas, as substrates such as water, soil, or plants can be simply incorporated into enclosures under controlled conditions (Loudon et al. 2014, 2016, Kenison et al. 2020, Michaels and Preziosi 2020). Incorporating abiotic and biotic sources of microorganisms in captive settings might better prepare future translocates by incorporating new and functionally important bacteria into the microbiota (Loudon et al. 2014, Kenison et al. 2020) and/or pre-exposing them to potential pathogens in a controlled setting (Waddle et al. 2021). Thus, identifying key sources of microbial symbionts in the environment might not only allow amphibian microbiome researchers to encapsulate the complexity of host-associated microbiota, but may also have important repercussions for the way captive microbiomes are managed.

Table 4.

Amphibian microbiota research challenges at the ecosystem and biosphere levels.

Ecosystem challenge: capturing the complexity of an ecosystem in the laboratory

Experimental studies at the ecosystem level remain a challenge in some amphibian systems due to the complexity and sampling scale necessary to capture microbial reservoirs in the environment. A few studies have found a solution to this problem by using miniature ecosystems, such as bromeliad tanks, that make it more efficient to track the amphibian host, nonamphibious hosts, and abiotic substrates in a natural environment. When full of water, the core of bromeliad tanks can host diverse communities of aquatic arthropods, microorganisms, and algae, in addition to being used by some tropical frogs for egg and tadpole development. Greenspan et al. (2019) used these systems to quantify the negative effect that detrivore arthropods may have on substrate bacterial communities and Batrachochytrium zoospores in the environment. In a different study, Greenspan et al. (2020) observed changes in arthropod and environmental reservoir microbial communities in bromeliad microecosystems that experienced artificial warming under field conditions, and these changes negatively affected tadpole growth rates and the stability of their microbial communities. By monitoring associations between amphibian microbiotas, arthropod reservoirs, environmental reservoirs, and amphibian health, more experimental ecosystem studies are necessary to quantify the cascade effects that environmental and biodiversity change can have across multiple ecological scales.

Biosphere challenge #1: assessing the effect of climate change on the health of amphibian populations and their microbiotas

Climate change is disrupting temperature and moisture norms across the world that are likely to result in amphibian host range shifts toward higher latitudes and altitudes (Botts et al. 2015, Duan et al. 2016). At the landscape scale, climatic shifts and their consequences (e.g. wildfires) are altering the availability and distribution of microhabitats across the landscape (Cole et al. 2020, Loehman et al. 2020). As a result of environmental change and range expansions, we can expect fluctuations in the current patterns of microbial diversity and composition on the skin of future amphibian taxa across the globe (Xie et al. 2016). However, how climate-related changes in host–microbe associations will affect the susceptibility of future amphibian populations to infectious diseases such as B. dendrobatidis or B. salamandrivorans remains largely unexplored.

Biosphere challenge #2: evaluating whether pathogens are a source of selective pressure on host-associated microbiota functionalities

The global distribution of Batrachochytrium variants across the world poses an ideal natural experiment to assess the coevolution of microbiotas and pathogens. For example, population genomics work identifies east Asia as the geographical origin of Bd, with a more recent lineage Global Panzootic Lineage (GPL) being found across most parts of the world (O’Hanlon et al. 2018). Therefore, our current understanding indicates that across the world amphibians and their microbiotas have interacted with Bd for different lengths of time. To our knowledge, the influence of time since Bd introduction on the structure and functionality of amphibian microbiotas remains mostly unexplored.

Biosphere level

Studies that incorporate biosphere components into their amphibian microbiota survey design sampled across more than one ecosystem. While amphibian microbiota studies in this category are not numerous, and almost all exclusively are based on natural or seminatural experiments (Table S1), these studies give a glimpse into the distribution of bacterial symbionts across regional, intercontinental, or sometimes global scales. At the largest scales, biosphere studies use available data from previously published work (Kueneman et al. 2019, 2022) or global swab sample libraries (Medina et al. 2019) to evaluate the distribution of bacteria across ecosystems and different taxa. Other studies took a more regional approach and compared amphibian microbial communities between sites in two or more distinct ecosystems (Belden et al. 2015, Vences et al. 2016, Abarca et al. 2018b, Wagener et al. 2021).

Most studies observed the presence of richer communities in the skin of temperate amphibians compared to tropical ones (Belden et al. 2015, Kueneman et al. 2019). Differences in amphibian skin microbiota richness among habitats (e.g. aquatic, terrestrial, and arboreal) varied between temperate and tropical areas as well. For example, aquatic amphibians possessed richer skin microbial communities compared to terrestrial ones in tropical systems, but the reverse has been documented in temperate systems (Kueneman et al. 2019, Medina et al. 2019). In contrast, gut microbiotas between tadpoles sampled in Brazil and Madagascar overlapped, suggesting conserved relationships that might be due to diet or physiology (Vences et al. 2016). Microbial turnover among the skin of amphibians inhabiting different ecosystems occurs mostly at lower taxonomic levels; however, at the phylum level most amphibian skin microbiotas were dominated first by Proteobacteria, followed by Actinobacteria and Firmicutes. Temperate and tropical communities of amphibians also differed in the relative abundance of these phyla. Two transcontinental studies observed lower representation of Proteobacteria in the microbial communities of temperate amphibians compared to tropical ones (Belden et al. 2015, Kueneman et al. 2019). Documented variation between temperate and tropical systems indicate that temperature and moisture are also important filters that influence the microbial ecology of amphibian skin microbiota at global scales.

Experimental studies at global scales are quite difficult to perform (Table 4). However, we encourage future researchers to continue to use the growing list of amphibian microbiota datasets and sample libraries to ask questions related to the distribution of microorganisms across global scales. One caveat that meta-analyses using datasets should take into consideration is how different laboratories, library preparation materials, and DNA-sequencing instruments/runs can influence low-taxonomic turnover across samples. Ensuring that the data included in these meta-analyses originate from studies that utilize similar preparation techniques might be one way to limit the influence of variation in library preparation on sequencing bias. On the other hand, establishing standard sample collection, sequencing library preparation, and sequence handling/reporting practices across different laboratories might also facilitate comparisons across studies.

Taking advantage of natural/artificial large-scale events such as species range expansions, drought, or floods might also allow researchers to draw generalities on how these events influence amphibians and their microbiotas. For example, amphibians with broad latitudinal ranges present an opportunity to study how microbiota and host immunity will respond at both the northern and southern extremes of species ranges, as these are likely the areas to exhibit host species expansions/reductions in response to climate change. The effects of local filters on the assembly of amphibian microbiotas is even more evident in studies that compared invasive amphibians inside and outside of their home range. In these investigations, microbiota composition differed between invasive and native populations of Asian common toads (Duttaphrynus melanostictus; Santos et al. 2021), cane toads (Rhinella marina; Abarca et al. 2018b), guttural toads (Amietophrynus gutturalis; Wagener et al. 2021), and American bullfrogs (Lithobates catesbeianus; Kueneman et al. 2019), with the invasive species’ microbiota often resembling that of resident amphibians of the invaded ecosystem (Kueneman et al. 2019). In this case, studying the response of broadly distributed amphibians to environmental change might provide valuable information to predict how vulnerable and endemic species will respond.

Conclusion

Host-associated microbiomes play an essential role in the health of organisms. Understanding how environmental factors interact with host-associated factors to shape microbiotas has critical biological, health, and conservation implications. Collectively, our review demonstrates that environmental factors can influence microbiotas through diverse mechanisms at all biological scales. Importantly, while environmental mechanisms occurring at each of the different scales can interact to shape microbiotas, the past 10 years of research have mostly been characterized by targeted approaches at individual scales (65.11%). Several studies examine two biological organizational levels (27.23%) but few examine three or more levels (8.94%). Looking forward, efforts considering how environmental factors at multiple organizational levels interact to shape microbiota diversity and function are paramount. Generating opportunities for meaningful cross-disciplinary interactions and supporting infrastructure for research that spans biological scales are imperative to addressing this gap. Additionally, we note that while this review focuses on amphibian microbiota research, the insights, approaches, and areas for future directions are broadly relevant across diverse study systems. Indeed, interest in the role of environmental factors in shaping host-associated microbiomes has grown substantially and span diverse fields within biological sciences from microbiology to ecology and evolution to applied disciplines from medicine, veterinary science, and conservation biology. Despite important advances in our understanding, an important challenge that remains is the need to integrate these efforts across these siloed disciplines. Toward this end, we call for generating opportunities for meaningful cross-disciplinary interactions and supporting infrastructure for research that spans biological scales through:

• Continued efforts that facilitate interactions across disciplines such as joint conferences or targeted symposiums that bring researchers from different disciplines together.

• Efforts at developing standardized approaches for diverse study organisms (including nonmodel organisms) across disciplines and across biological scales.

• Continued hierarchical approaches that simultaneously and systematically evaluate the effect of environmental factors and their interaction with host-factor to shape microbiotas including:

• ○ studies that include more than one population/community/ecosystem, and

• ○ studies that evaluate environmental conditions and systematically assess microbiota responses from organismal to biosphere levels.

• Improved data transparency and accessibility to facilitate collaborations and application of novel statistical tools (e.g. machine learning approaches that facilitate efforts to utilize data to generate predictive models for applied purposes in health or conservation).

Funding

This work was supported by a USA National Science Foundation grant (#2042970).

Conflict of interest

None declared.