Undead food-webs: Integrating microbes into the food-chain

Accepted Manuscript Undead food-webs: Integrating microbes into the food-chain

Shawn A. Steffan, Prarthana S. Dharampal PII: DOI: Article Number: Reference:

S2352-2496(18)30047-8 https://doi.org/10.1016/j.fooweb.2018.e00111 e00111 FOOWEB 111

To appear in:

Food Webs

Received date: Revised date: Accepted date:

1 August 2018 25 October 2018 14 November 2018

Please cite this article as: Shawn A. Steffan, Prarthana S. Dharampal , Undead foodwebs: Integrating microbes into the food-chain. Fooweb (2018), https://doi.org/10.1016/ j.fooweb.2018.e00111

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ACCEPTED MANUSCRIPT Undead food-webs: Integrating microbes into the food-chain

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Shawn A. Steffan1,2, and Prarthana S. Dharampal2

US Dept. of Agriculture, Agricultural Research Service

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Dept. of Entomology, University of Wisconsin, Madison, WI

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Corresponding author:

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Shawn Steffan

Dept. of Entomology

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1630 Linden Dr.,

Madison, WI 53706

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University of Wisconsin-Madison

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[email protected]

Food webs: Review/Perspective Running head: Undead food-webs

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Abstract Detritivory represents the dominant trophic mode on Earth, and within this vast consumer group, microbes tend to predominate. Virtually all detritus is replete with microbes, many of which consume the detrital substrate, rendering it a complex of living and non-living

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components. A detrital mass, therefore, can be considered an ‘undead’ food-web in which

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the resident microbes busily displace non-living biomass with their own, while carnivorous

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microbes prey upon other organisms. In this food-chain, bacteria and fungi have been shown to register just as animals do, demonstrating that microbes are analogous to animals within

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trophic hierarchies. Given that microbial consumers are often suffused throughout detrital

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substrates, the detritus becomes a complex of multiple trophic groups. It has long been suggested that microbial consumers within soil systems represent distinct trophic groups,

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and in recent studies, these hypotheses have been tested empirically. The detrital complex,

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then, can be considered a dynamic microcosm of the broader food-web, and the trophic identities within the complex (as well as the metazoans that consume the complex), can be

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measured and interpreted. This multi-trophic aspect of ‘undead’ detritus tends to elevate the

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trophic positions of animals (i.e., trophic inflation), which derives from the assimilation of heterotrophic (microbial) and autotrophic proteins. Importantly, inclusion of microbes in

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trophic hierarchies provides a more comprehensive framework within which to interpret organismal-, population-, and community-scale trophic identities. However, in recalibrating our trophic ‘lens’ to include microbial consumers, there remain some long-standing concepts, questions, and definitions that will need to be revisited.

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“The first food-webs were composed of bacteria, Protozoa and fungi, which even nowadays form the basis for all food-webs.” – Price, 1988 (Price, 1988)

1. Background

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In terrestrial ecosystems, the vast majority of primary productivity (> 90%) tends to be

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consumed as non-living detritus (Hagen et al., 2012; Moore et al., 2004), which means that

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most food-webs in these systems rest upon a foundation of dead autotrophic biomass. In contrast to ‘green’ food-webs, which are driven by energy derived from the consumption of

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living plants, the ‘brown’ webs derive from non-living, decaying biomass (Kaspari and

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Yanoviak, 2009). Brown webs are widely known to be replete with decomposers (Schoener, 1989; Zou et al., 2016), which drive system productivity among all major ecosystem types

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(van der Heijden et al., 2008), while a relatively small proportion of primary productivity

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finds its way into ‘green’ food-webs (Hagen et al., 2012). Although its significance varies by ecosystem type and/or disturbance regime (Bardgett and Cook, 1998; Cebrian and

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Lartigue, 2004), the importance of detritivory as a dominant feeding mode, as well as a

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driver of system productivity and diversity, is both conspicuous and worthy of greater attention (Brose and Scheu, 2014; Evans-White and Halvorson, 2017; Ferreira et al., 2015;

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García-Palacios et al., 2016; Gessner et al., 2010; Moore et al., 2004). Characterizing food-webs as ‘green’ or ‘brown’ has provided a useful framework

with which to visualize these two trophic pathways, and establishes a basis for the concept of the ‘detrital shunt’ (Polis and Strong, 1996). The detrital shunt is so named because it describes the diversion of uneaten biomass from green to brown food-chains, and this phenomenon has been well-supported for decades (Hagen et al., 2012; Haraguchi et al.,

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2013; Moore et al., 1988; van der Heijden et al., 2008). Given the primacy of detrital foodwebs, especially in terrestrial systems, it appears however, that this shunt represents the dominant pathway for the ‘upward’ flow of energy and nutrient flow (Coleman, 1996; Hyodo et al., 2008; Moore and de Ruiter, 2012). Detritus represents the bulk of the biomass

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on which most terrestrial food-chains are supported, (Birkhofer et al., 2008; Hagen et al.,

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2012; Moore and de Ruiter, 2012; Peterson and Luxton, 1982), and detritivorous prey often

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represent the bulk of the ‘meat’ available to omnivores and carnivores (Chahartaghi et al., 2005; Digel et al., 2014; Haraguchi et al., 2013; Hyodo et al., 2008; Larsen et al., 2013;

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Moore et al., 1988; Steffan et al., 2017). Although green and brown webs are helpful to

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visualize trophic origins, the rampant co-mingling of these food streams within natural systems begs for conceptual models that desegregate green and brown webs, while

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accommodating the full diversity of heterotrophic macro- and microbiota.

2. The problem with ‘sanitization’ of food-webs

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For decades, food-web theory has recognized that within trophic hierarchies,

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heterotrophic microbes represent the primary decomposer group (Azam et al., 1983; Fenchel, 2008; Scheu and Setala, 2002). However, given the complexities associated with

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ascribing decomposer organisms to specific trophic levels, empirical evidence of the trophic function of these microbial decomposers has been difficult to obtain. Absent such measurable data, food-web conceptualizations have traditionally excluded detritivores/decomposers from the trophic hierarchies or topologies used to model food-web structure — this ‘sanitization’ of food-webs (Schoener, 1989), however, can be problematic because it effectively purges the bulk of heterotrophs from any given community.

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Microbes within brown webs often represent the single largest group of consumers within their respective communities (Coleman, 1996; Hagen et al., 2012; Moore and de Ruiter, 2012; van der Heijden et al., 2008). In light of recent evidence showing that microbes are trophic analogues of animals, (using contemporary molecular methods (Digel

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et al., 2014; Steffan et al., 2017, 2015)), the justification for sanitizing food-chains becomes

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quite tenuous. Of course, soil food-webs are often a ‘black box’ since the fauna therein are

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difficult to observe, (Brose and Scheu, 2014), as are their trophic pathways (Digel et al., 2014; Larsen et al., 2013; Ohkouchi et al., 2017; Pollierer et al., 2012). Moreover, the

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prevalence of generalist feeders, abundance of reticulate trophic links, and ubiquity of

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trophic omnivory make trophic interactions within soil communities exceptionally complex (Shurin et al., 2006; Strong, 1992). However, the exclusion of ‘decomposer groups’

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becomes particularly difficult to justify within terrestrial food-webs that tend to have higher

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levels of ‘refractory detritus’ (less palatable autotrophic biomass) than aquatic or marine webs (Cebrian, 1999). As a result, on land there tends to be a much larger standing stock of

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biomass that gets commandeered by detritivores (Cyr and Face, 1993; Hagen et al., 2012),

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and most of these consumers tend to be microbes (Coleman, 1996; Moore et al., 2004; van der Heijden et al., 2008). Thus, the primary issues associated with the exclusion of

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heterotrophic microbes from food-web models may be summarized as the following: A) species-rich subsets of the heterotroph community are excluded, based on qualitative, nontrophic criteria; B) on land, such exclusions represent the most significant consumer and prey groups (Digel et al., 2014; Larsen et al., 2013; Moore et al., 2004, 1988) disregarding the trophic equivalence (interchangeability) of bacteria, fungi, and animals within a foodchain effectively clouds interpretation of carnivore or omnivore trophic identity (Fig. 1).

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Collectively, these issues preclude the unification of the macro- and microbiomes within terrestrial food-web science, preventing the accurate assessment of species-level trophic identity and community-scale functional diversity. Given that green and brown trophic pathways may converge at lower, intermediate,

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and higher-order trophic positions (Coleman, 1996; Polis and Strong, 1996; Wolkovich et

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al., 2014; Zou et al., 2016), the respective consumer communities necessarily co-mingle,

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underscoring the importance that measurement of organismal trophic identity remain independent of the idiosyncrasies of any particular web. Indeed, a complex food-web can

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be seen as analogous to a ‘Jenga’ tower (de Ruiter et al., 2005), maintaining overall

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structural integrity even while its constituent ‘green’ or ‘brown pieces’ cycle in and out. The contemporary food-web models we employ as descriptors of nature will need to be as

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inclusive as possible, fully integrating the macro- and microbiomes, and will likely need to

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be constructed independent of consumer phylogeny, size, or trophic origin (Fig. 1). The overarching goal of this review, therefore, is to articulate the need to re-calibrate how we

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view detritus, detritivory, omnivory, and carnivory. We will marshal evidence to support

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three primary theses: 1) microbes and animals are trophically interchangeable within trophic hierarchies; 2) living biomass is no different, trophically, from nonliving biomass; 3) the

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mixing of living and non-living biomass within detritus creates ‘undead’ complexes, which not only requires the interdigitation of microbes and animals within food-chains, but also necessitates a trophic framework that can operate independently of green or brown web origins. Recent advances in trophic-biomarker-based assays have allowed the empirical measurement of microbial trophic identity. Examples of heterotrophic microbes as predators

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or prey span the major phyla of the Fungi and Bacteria (Ohkouchi et al., 2017; Steffan et al., 2015). Across both terrestrial and aquatic ecosystems, diverse communities of microbial decomposers reveal a large potential for facilitative interactions and resource partitioning between fungal and bacterial species (De Boer et al., 2005; Osono, 2007; Tiunov and Scheu,

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2005), which can increase decomposition rates and system productivity (Demi et al., 2018).

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Indeed, heterotrophic microbes can occupy virtually any trophic position along the

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continuum of strict herbivory to apex carnivory (Moore et al., 1988; Polis and Strong, 1996; Steffan et al., 2015; Wolkovich et al., 2014), toggling between living and non-living diets.

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Broadly viewed, microbial decomposer communities are functionally similar across

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terrestrial and aquatic systems (De Deyn and Van Der Putten, 2005; García-Palacios et al., 2016; Wagener et al., 1998). However, there are major systemic differences between the

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food-web architecture, pathways of energy flow, and magnitude of biomass partitioning of

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terrestrial and aquatic webs. Most notably, the higher concentrations of cellulose, lignin, and other defensive, recalcitrant compounds within terrestrial plants means that the majority of

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the primary productivity remains uneaten, resulting in far greater flow of dead biomass into

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the detrital pathway (Evans-White and Halvorson, 2017; Shurin et al., 2006). Often, terrestrial detritivorous animals lack the enzymes needed to exploit these recalcitrant

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compounds, but many microorganisms are able to exploit the unused resources. Using their superior enzymatic potential to digest and assimilate recalcitrant compounds, microbes extract and consolidate the resources, making them available to secondary decomposers (Scheu and Falca, 2000; Zieger et al., 2017). This ‘external rumen’ strategy of relying on external microbes as nutritional mutualists is rampant across many soil-dwelling animal taxa, (see the example below of

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fungus-growing leafcutter ants), and likely explains why most detritivores still have not evolved the capacity to digest cellulose and lignin (Lavelle et al., 2005; Scheu and Setälä, 2002; Swift et al., 1979). Tracer studies indicate that acquisition of such microbially-derived nutrients plays a critical role in the nutrition of detritivores (Chung and Suberkropp, 2009;

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Halvorson et al., 2016; Pollierer et al., 2012; Riggs et al., 2015). In fact, detritivorous

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animals show a strong dietary preference for microbe-digested substrates (and exhibit higher

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survival and increased growth rates) likely due to accumulation of microbial prey of higher nutritional value, increased palatability, and other changes in substrate chemistry (Cummins,

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1974; Danger et al., 2012; Frainer et al., 2016; Nalepa et al., 2001; Scheu and Falca, 2000).

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For the many detritivorous animals that rely on the external rumen, there is little difference between their habitat and food (Goncharov and Tiunov, 2014)—‘a situation reminiscent of

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paradise’ (Scheu and Setälä, 2002). Microbes or metazoa embedded within detritus become

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food for countless higher-order consumers, fueling niche diversity and heterotrophic

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productivity (Fitter et al., 2005; van der Heijden et al., 2008).

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3. Trophic equivalence among microbes and animals Microbes are pervasive, important components of virtually all food-webs. As with metazoa,

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heterotrophic microbes consume both dead and living nutritional resources and register within trophic hierarchies, whether detrital or not (Digel et al., 2014; Moore et al., 1988; Steffan et al., 2017). It has been shown empirically that microbes are analogous to animals within a food-chain—for example, a fungus feeding on plant biomass is not different, trophically, from a mouse feeding on plant biomass (Steffan et al., 2015). Both the fungus and the mouse in this example would be strict herbivores, and it has been demonstrated that

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when consumers such as fish, mammals, insects, crustaceans, or fungal predators feed exclusively on herbivorous microbes or animals, the consumer registers as a carnivore (Steffan et al., 2017, 2015). Functionally, then, microbes can be predators or prey, and consumption of heterotrophic microbial biomass represents carnivory—in other words,

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mold is meat (Fig. 1).

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To illustrate these trophic roles, the leafcutter ant ‘fungus-gardens’ serve as useful

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model communities, because they represent a tripartite symbiosis (Currie et al., 2003).

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Neotropical leafcutter ants (e.g., Acromyrmex) are prodigious harvesters of plant biomass, which is used to sustain their fungus-gardens (i.e., their fungal symbiont, Leucoagaricus)

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(Currie et al., 1999). When the harvested plant biomass is included in the community

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module, these fungus-gardens have been shown to host four discrete trophic levels, three of which are occupied by microbes (Steffan et al., 2015). Specifically, it was revealed that the

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fungus is the sole herbivore within the leafcutter community (feeding at trophic position

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‘2’), with the ants registering as carnivores (trophic position ‘3’). A bacterium, Pseudonocardia, which exists within microscopic crypts in the ant exoskeleton (Currie et

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al., 2006), was shown to be the apex carnivore of the community (trophic position ‘4’).

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While it was interesting to find four trophic levels within a single fungus-garden, it was perhaps equally novel to suggest that the fungus-garden, itself, was quite literally an herbivore. As such, the fungus represented ‘meat’ to the developing ant larvae. Not surprisingly, the fungivorous ants were measured at distinctly carnivorous trophic positions, which would seem incongruous with their traditional characterization as the dominant herbivores of the Neotropics. However, given the acknowledgement that their fungal symbiont was, trophically, no different from the flesh of an animal herbivore, the 9

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carnivorous trophic position measured in the ants was predictable, as was the trophic position of the bacteria feeding upon the ants. Here, the integration of microbes into the food-chain revealed remarkable trophic diversity within a single fungus-garden community, and shed light on the dominant herbivores of the Neotropics—fungi.

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This represents evidence that animals, fungi, and bacteria are interchangeable in a food-chain. Why does it matter to interdigitate microbes and animals within food-chains?

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The trophic attributes of any species represent important dimensions of its niche (Chase and

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Leibold, 2003). There is a measurable ‘microbe effect’ within food-chains, which tends to inflate the trophic positions of detritivorous fauna and create non-integer trophic positions

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(Steffan et al., 2017). The preponderance of trophic omnivory (non-integer trophic

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positions) among higher-order consumers in real food-webs has been well-documented (Chikaraishi et al., 2011; Steffan et al., 2017; Thompson et al., 2007). Altogether, these

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observations underscore the importance of the inclusion of microbes in food-chains, because

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it appears that the microbe effect in food-chains is integral to the assessment of omnivore

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and carnivore trophic identity.

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4. Living vs. non-living biomass—a needless dichotomy Biomass capture is a fundamental aspect of heterotrophy and a major global ecosystem service, hence the phase state of biomass (living or dead) represents a key, unresolved issue in trophic ecology. Biomass is apportioned along trophic hierarchies (Elton, 1946; Lindeman, 1942), and this apportionment is shared, (albeit to varying degrees in terrestrial and aquatic ecosystems) among the overlapping trophic spectra of populations and communities (Darnell, 1961; Shurin et al., 2006; Strong, 1992). As biomass gathers along 10

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this hierarchy, the consumed diets may toggle between living and non-living states, but these phase changes do not transform the trophic identity of the food, itself. In other words, these non-living resources appear to be retain their trophic identity (i.e., their trophic position) and are no less useful for heterotroph dietary needs as when they were alive—

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many organisms, such as fungi, arthropods, and vertebrates, develop on diets of living

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and/or nonliving biomass (Moore et al., 1988; Polis and Strong, 1996) (Fig. 2). Indeed,

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given the necessary digestive physiology, it is conceivable that non-living food represents a more accessible diet, considering that it is less likely to be well-defended by the host

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organism.

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Since most primary productivity does not pass into green webs, the uneaten majority of plant biomass senesces, dies, and becomes food for vast populations of organisms within

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the detrital pathway. In time, detritus is changed markedly by microbial colonization and

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consumption (Fig. 2), but until then, a dead leaf is essentially just a leaf, and an animal carcass is still just carrion—its trophic identity has not changed. Importantly, at an

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elemental level, a unit mass of detritus is indistinguishable from living biomass, and

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2013).

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trophically, plant detritus is indistinguishable from living plant biomass (Steffan et al., 2017,

5. Integrating microbes into the food-chain Virtually any given detrital mass has been colonized by microbes. In the process of consuming the mass, microbial populations displace the non-living components with their own biomass. These microorganisms actively transform the non-living substrate into one that is rather exuberant with life, giving the detritus what may be referred to, in 11

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anthropomorphic terms, as an ‘afterlife.’ This microbially-mediated process of decomposition has generally been viewed as simple mineralization of compounds for plants, rather than the trophic transformation of proteins once locked within dead biomass. Trophically however, microbial consumption of a detrital mass creates new trophic positions

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within the detritus, rendering the detritus a food-web microcosm exhibiting a hierarchy of

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consumer groups (Ohkouchi et al., 2017; Steffan et al., 2017). The mixing of trophic groups

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within detritus creates a multi-trophic complex, and the identities of the trophic groups (positions in the trophic hierarchy) do not change as a function of their green or brown

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‘lineage’ (Scheu and Setälä, 2002; Steffan et al., 2017). The metazoan fauna consuming

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microbe-colonized detritus or animal carrion necessarily feed as omnivores. As these animal consumers (e.g., earthworms, springtails, mollusks, mustelids) feed upon detrital complexes,

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they purposely or inadvertently consume microbes. For faunal consumers, then, detritivory

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represents trophic omnivory because such fauna are consuming multiple trophic groups, including their own (Coll and Guershon, 2002; Digel et al., 2014).

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Indeed, microbes can be viewed as ‘meat’ in the food-chain and, by this same token,

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may also function as carnivores at virtually any trophic position (Steffan et al., 2015). The ‘upward’ flow of detritus-derived biomass being channeled into higher-order consumers

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commonly passes through the cells of microbes at some point (Larsen et al., 2009; Ohkouchi et al., 2017; Steffan et al., 2017), and likely will find its way back into microbial cells during the consumption/decomposition of carrion. As the typical first line of consumers, fungi and bacteria become prey for the larger meso- and macro-fauna (Moore et al., 1988; Polis and Strong, 1996). Detritivorous prey are known to bolster predator populations, which may then shift to preying upon herbivorous prey, suppressing herbivore populations and

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indirectly protecting plant productivity (Moore et al., 2004; Scheu, 2001). In this way, microbes not only mediate both the bottom-up (nutrient mineralization) but also top-down factors (detritivorous prey for higher-order consumers) that shape community structure (Moore and de Ruiter, 2012; van der Heijden et al., 2008). Microbial populations’ roles in

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the mineralization of detritus for plants and as food for meso-fauna are well-documented

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(Bardgett and Cook, 1998; Moore et al., 1988; Shurin et al., 2006; van der Heijden et al.,

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2008), but less studied are their roles as higher-order consumers. In fact, microbes can function at any position in a food-chain (Digel et al., 2014; Polis and Strong, 1996; Steffan

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1996; Hagen et al., 2012; Moore et al., 2004).

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et al., 2015), and tend to do so in greater numbers and biomass than animals (Coleman,

Consumption of microbial meat appears to be not only unavoidable but also

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potentially beneficial, so much so that animal-microbe symbioses are remarkably common

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(Crotty, 2011; Crotty et al., 2011; Ferlian et al., 2015; López-Mondéjar et al., 2018; Nalepa et al., 2001; Pollierer et al., 2010; Tsunoda et al., 2017). It is often suggested that meso- and

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macro-fauna do not indulge in significant fungivory or bacterivory, but this is quite unlikely,

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and recent evidence is bearing this out (Epps and Penick, 2018; Hyodo et al., 2014; Keebaugh et al., 2018). Such microbivory is likely exceedingly common given that plant

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detritus and/or carrion is rife with microbes when consumed by animals. Recent measurements of earthworm, bark beetle, springtail, and bee trophic positions provide evidence of the rampant consumption of ‘microbial meat’ within detritus (Steffan et al., 2017). Additionally, springtails and various micro-fauna are extraordinarily common within terrestrial soils, and it has been shown that many of these invertebrate fauna are largely ‘grazers’ upon fungi, protozoa, bacteria, and nematodes within the soil profile (Brose and

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Scheu, 2014; Chahartaghi et al., 2005; Maraun et al., 2011). The strong reliance on brown food-web resources has been demonstrated empirically using non-stable carbon (C) isotopes (i.e., 14C concentrations tightly linked to specific time signatures within the global, atmospheric CO2 pool), demonstrating that detritivorous prey are major food sources for

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certain terrestrial predators (Haraguchi et al., 2013). Broad surveys of the carbon ‘age’ of

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omnivores and carnivores have shown that across all major terrestrial ecosystem types,

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carnivores tend to be composed of ‘old carbon’ (= carbon that is significantly older than the consumer’s food source) (Hyodo et al., 2014; Tayasu and Hyodo, 2010). For example,

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carnivores that live 2 years or less (most global fauna) are often composed of carbon that is

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3-8 years old, suggesting that both the carnivores and their prey were ‘assembled’ mostly from resources deriving from relatively old primary productivity (i.e., brown webs).

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For soil-dwelling heterotrophs, another major source of energy is derived from the

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rhizosphere. Plants can transport and release up to 25% of their net fixed carbon into the soil as root exudates (Kawasaki et al., 2016). This belowground-derived high quality substrate

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includes a nutritious blend of various amino acids, organic acids, sugars, and secondary

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metabolites (Baudoin et al., 2003). Findings from multiple trophic studies reveal substantial contributions of such ‘root carbon’(Goncharov and Tiunov, 2014) within the biomass of

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root-associated microbiota, and their consumers (Albers et al., 2006; Hu et al., 2018; Jones et al., 2009; Ostle et al., 2007; Pollierer et al., 2012, 2007; Scheunemann et al., 2015; Zieger et al., 2017). As an organic resource, root exudates are made from newly assimilated carbon (Hyodo et al., 2006), which is typical of diets within grazer food-webs. Yet, because it is comprised of non-living pools of compounds, root exudate is trophically no different than any other forms of plant-based detritus. From the rhizosphere perspective, this presents a

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trophic cross-road, where consumer trophic identity is not exclusively aligned with either green or brown webs. However, trophically, it does not appear to matter. Whether feeding as an herbivore (direct rhizophagy of living root tissue and exudate) or detritivore (digestion and assimilation of root-derived non-living organic molecules) (Goncharov and Tiunov,

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2014), consumer trophic identity can be accurately assessed using compound-specific

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analyses of nitrogen isotopes.

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In reference to terrestrial food-webs, Strong (1992) describes a ‘trophic tangle’ of consumers (a nod to Darwin’s ‘tangled bank’ of intermingled species), referring to the

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reticulate links between consumers and the consumed, as well as the trophic gradients

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within and among populations. Omnivory appears to be exceedingly common within such trophic tangles and is particularly rampant among organisms capable of carnivory

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(Thompson et al., 2007). For omnivorous organisms, populations, and communities that

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derive partly from green and partly from brown lineages, it may be exquisitely difficult to track and disentangle the relative contributions of green/brown lineages across all positions

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in the trophic hierarchy. Given that green/brown food-webs are trophically

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indistinguishable, it is not necessary to track the relative contributions of a population’s green/brown trophic lineages in order to get a valid estimate of the species’ trophic

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tendency. Considerably more important is the measurement and interpretation of microbial contributions to the food-chain, primarily those within the detrital sphere. While microbes are viewed as the dominant agents of nutrient cycling in most ecosystems (Vanni, 2002), their portrayal as mere decomposers of dead biomass, devoid of a trophic identity, is a gross generalization, and mischaracterization of real world food-webs (Polis, 1999) (See Section 6 below). It is important to reflect on the relative importance of decomposers not only in the

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framework of green- versus brown- streams, but also within the context of whole food-webs where these channels frequently coalesce (Polis and Strong, 1996; Zou et al., 2016). With empirically-based estimates of microbial trophic roles in the community, the trophic identities of higher-order consumers will better reflect the broad interdigitation of

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heterotrophic species within the community. Thus, the trophic tangle need not be

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disentangled to characterize consumer trophic identity. At community scales, it may be

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more informative to disregard taxonomic identity altogether, in favor of trophic taxa, or consumer groups that each share a particular trophic tendency. Erecting and analyzing

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trophic taxa, independent of organismal phylogeny, should represent a powerful means of

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quantifying the functional diversity, omnivory, and redundancy in a food-web, especially

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since it would include microbiota alongside meso- and macrobiota (Fig. 2).

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6. Revisiting terminology to facilitate a conceptual pivot The hallmarks of brown pathways, broadly referred to as ‘decomposition, decay, or

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mineralization,’ merely refer to the outcome of microbial consumption, and tend to diminish

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the concept that microbes actually occupy a place in the food-chain and serve significant roles as predators and prey (Anderson et al., 2017; Hall Jr and Meyer, 1998; Halvorson et

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al., 2016; Haubert et al., 2009). This oversight, and general exclusion of microbes from food-chains, is a bit ironic because detritivorous microbes, on any given day or place on Earth, tend to be the dominant consumer groups (Coleman, 1996; Hedlund et al., 2004; Polis and Strong, 1996). Given that globally, most primary productivity gets channeled through ‘brown’ food-chains (Hagen et al., 2012) and that food-web productivity and biodiversity generally rest upon detrital foundations (Moore et al., 2004; van der Heijden et al., 2008),

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virtually all fauna, particularly terrestrial omnivores and carnivores, are ‘assembled’ with proteins and lipids that have microbial ‘fingerprints’ (Arthur et al., 2014; Dharampal and Findlay, 2017; Larsen et al., 2009; Ohkouchi et al., 2017; Steffan et al., 2017). Heterotrophic microbes within the food-chain represent ‘meat’ to their consumers, and thus,

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the trophic diversity within the microbiome will tend to shape the trophic identities of most

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higher-order consumers. Currently, ‘trophic diversity within the microbiome’ is not a

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concept that has gained much traction, partly because it has been difficult to measure, but also because microbes simply are not viewed as the trophic analogs of animals.

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At its core, the characterization of detritivorous microbes as merely vehicles of

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‘decomposition’ or ‘decay’ is misleading (Schoener, 1989). However, these biochemical processes are, in essence, euphemisms for the effects of microbial populations eating,

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growing, and depleting a non-living organic resource. To better illustrate this idea, consider

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a mass of leaves and branches on a forest floor—this detritus will certainly decompose over time, but trophically, there is simply plant biomass being consumed by detritivorous

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organisms. Thus, terms such as microbial ‘decomposition’ or ‘decay’ are trophically

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imprecise because they ignore the fact that detritivorous microbes are, in fact, consumers that are ‘re-composing’ the detritus in their own form. In the same sense that it would be

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odd or incongruous to refer to wolf predation as the ‘decay’ of an elk herd, it is trophically imprecise to consider the consumption of non-living biomass as decay. This context is important to consider because the terminology used to characterize microbial consumption of detritus has often constrained our view of them, effectively relegating microorganisms to the spheres of decay, decomposition, and mineralization (all of which focus solely on servicing the needs of plants), while ignoring the fact that microbes eat detritus to sustain

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growth and reproduction. This inordinate focus on green food-webs, metazoan-centric perspective of trophic pathways, and relegation of microbes to the narrow ecological roles of decay/mineralization, undermines our understanding of microbial roles in food-chains,

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and perhaps further justifies the excessive sanitization of food-webs.

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7. Measuring trophic pathways and organismal trophic identity

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With the advent of novel techniques based on gas chromatography-mass spectrometry (GCMS), powerful new tools have emerged allowing investigators to trace individual molecules

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as they percolate through living and non-living pools of a trophic hierarchy (Evershed and

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Bull, 2007). Within the discipline of trophic ecology, two types of biomolecules have proven quite useful as proxies for measurement of consumer trophic position—glutamic

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acid and phenylalanine (Chikaraishi et al., 2009). Compound-specific isotopic analyses of

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glutamic acid and phenylalanine frequently are able to provide accurate and precise estimates of consumer trophic position (Chikaraishi et al., 2011; Steffan et al., 2013). In the

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area of microbial ecology, two primary categories of biomarkers—amino acids and fatty

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acids—have become established as useful biomarkers (Boecklen et al., 2011; Ohkouchi et al., 2017; Ruess et al., 2005). By quantifying the natural abundance of stable isotopes within

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amino acid pools (i.e., nitrogen and carbon) and fatty acids (carbon), recent studies have offered unprecedented insight into microbial reworking of living and dead organic matter (Calleja et al., 2013; Dharampal and Findlay, 2017; Kürten et al., 2013; Pollierer et al., 2010; Schmidt et al., 2006; Steffan et al., 2017; Yamaguchi et al., 2017). These techniques have unveiled cryptic biogeochemical pathways of nutrient transfer, and refocus the significance of the microbial ‘ooze’ within trophic hierarchies (Lindeman, 1942).

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8. Summary and future directions Under the banner of a unified macro- and microbiome, we have addressed the need for foodweb models to incorporate heterotrophic microbes into trophic hierarchies, as well as to

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desegregate green and brown webs within such models. Increasingly, the microbiome is recognized as a major driver of functional diversity and system productivity (Moore et al.,

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2004; van der Heijden et al., 2008). To better measure, interpret, and describe changes to

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organismal trophic identity, our conceptual models must accommodate the dominant yet largely invisible players in ecosystems—microbes. Here, we have examined trophic identity

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within the context of long-held theories that parse carnivores by size, and which

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compartmentalize consumers into simple integer-based trophic positions, or exclude detritus altogether from food-web analyses. Ultimately, we argue that detritus-based food-chains are

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trophically identical to the ‘grazing’ food-webs, and that the living and non-living pathways

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support consumers that can be interdigitated with one another, all of which are channeled into the biomass of higher-order consumers.

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Complete integration of microbes with their faunal analogs will facilitate accuracy in

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assessing the niches of species and the trophic diversity of communities. The rampant omnivory observed in ubiquitous detritivores, such as earthworms, flies, and beetles, is often attributable to microbivory associated with a diet of decaying plant matter. The integration of microbes within food-webs is especially important in terrestrial ecosystems given that the bulk of primary productivity on land tends to accrue as large standing stocks of undecomposed litter (more so than aquatic or marine systems) (Cebrian, 1999; Cebrian and Lartigue, 2004; Knops et al., 2001; Shurin et al., 2006). Here, decomposition is largely 19

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shared by fungi and bacteria. It is the fungi and bacteria in soils that simultaneously ‘fuel’ primary productivity and detritivore populations (Moore et al., 2004; Polis and Strong, 1996; van der Heijden et al., 2008). Future investigations of how climate change may be affecting animal and plant

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communities will need to be able to assess how the resident microbial communities mediate

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biomass flows into brown, green, and autotrophic pools (Bardgett and Cook, 1998;

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Coleman, 1996; Moore et al., 2004). As meso- and apex carnivores encounter forced niche shifts, observational data alone may be inadequate in distinguishing between the two groups

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(i.e., apex carnivores may not be at the apex of the food-chain) (Holt, 2006; Rooney et al.,

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2006; Terborgh and Estes, 2013). Since trophic description of higher-order consumption is predicated upon the accurate characterization of prey pools, it will be fundamental for

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dietary reconstruction to quantify the relative contribution of detrital subsidies to such prey.

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Natural food-webs often represent tangled networks, dominated by omnivores with non-integer trophic positions (Moore et al., 2004). These omnivorous, non-integer trophic

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positions are often attributable to the ‘microbe effect’ within trophic hierarchies. Indeed,

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food-webs are rendered more reticulate and circuitous by microbes at all trophic positions (Brose and Scheu, 2014; Pollierer et al., 2012; Scheu, 2001). Among intermediate and

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higher-order trophic positions, the blending of green and brown is ubiquitous, while strictly green consumption is rare. However, a vast body of early literature has focused on the ‘greenness’ of communities (Schoener, 1989), while subsidies from the brown channel, originating from prodigious microbial communities, remains largely underappreciated. The goal of this review, therefore, has been to better illuminate the ‘black box’ of brown foodwebs, and underscore the primacy of such webs as major drivers of heterotrophic

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productivity, diversity, and overall system stability. In moving towards an integrated framework, research endeavors should approach detritus as a microcosm of the broader food-web, replete with multiple trophic groups, and populated by herbivorous, omnivorous, and carnivorous microbes, all of which are knitted together within the ‘tangle’ of other

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consumers.

Acknowledgments

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The authors gratefully acknowledge the support of the U.S. Dept. of Agriculture,

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Agricultural Research Service, during the drafting of this review, and Dr. Stefan Scheu and

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another anonymous reviewer for their comments.

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Figure captions: Fig. 1. Schematic representation of macro- and microbiotic consumers, feeding within either green or brown food-webs, at simple integer-based trophic levels. For green food-webs, trophic level 1 is represented by living autotrophic producers; trophic level 2, by grazing

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herbivores that consume only the autotrophic producer biomass; trophic level 3, by strict carnivores that feed only on strict herbivores. For brown food-webs, trophic level 1 is

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represented by dead organic matter of autotrophic origin; trophic level 2, by detritivorous

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herbivores that feed only on the plant-based detritus; trophic level 3, by carnivores that feed only the nonliving biomass of herbivorous detritivores. Within their respective trophic

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hierarchies (green or brown food-webs), micro- and macrobiotic consumers occupying

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comparable trophic positions are trophically analogous.

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Ryan Hodnett; Olga K. Cotter.

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Photo credits: Don DeBold; flicker.com; Sheila Brown; Mike Pennington; Lyle Buss; National Park Service;

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Fig. 2. Simplified representations of omnivorous consumers within green and brown foodwebs (i.e., consumers that register at non-integer trophic positions). In green food-webs,

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omnivores registering near trophic position ~2.5, would obtain ~50% of their diet from living autotrophic producer biomass (trophic level 1), and the remainder from grazing herbivores (trophic level 2). At trophic position ~3.5, omnivores would likely rely on a mixed diet comprised of grazing herbivores (trophic level 2), other omnivores (trophic position ~2.5), and/or carnivores (trophic level 3) derived from living plants. In brown foodwebs, omnivores registering at trophic position ~2.5 might obtain ~50% of their diet from plant-based detritus (trophic level 1), and the remainder from herbivorous detritivores 30

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(trophic level 2). Omnivores registering near trophic position ~3.5 might rely on a mixed diet comprised of herbivorous detritivores (trophic position 2), lower level omnivores consuming such detritivores, and/or carnivores which exclusively consume herbivores (trophic level 3) within brown foodwebs. Both green and brown food-webs contain micro-

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and macrofaunal consumers representing lower (~2.5) and higher (~3.5) trophic omnivory,

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suggesting trophic equivalence among the micro- and macrobiota across food-webs.

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Photo credits: Robb Hannawacker, Katja Schulz, Ursula Skjonnemand, Guido Bohne, Hedwig Storch, Petr Kratochvil, James Gathany; Martin Cooper; Gilles San Martin; Andy Murray; Brocken Inaglory; Krzysztof

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Niewolny; Andreas Kay; Rachel Welfoot.

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Figure 1

Figure 2