Phase formation in the N-B-Ti system

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Special Issue: From One to Many

[23_TD$IF]Review

Symbiont Acquisition and Replacement as a Source of Ecological Innovation Sailendharan Sudakaran,1,5,* Christian Kost,2,3,6 and Martin Kaltenpoth1,4,* Nutritional symbionts play a major role in the ecology and evolution of insects. The recent accumulation of knowledge on the identity, function, genomics, and phylogenetic relationships of insect–bacteria symbioses provides [236_TD$IF]the opportunity to assess the effects of symbiont acquisitions and replacements on the shift into novel ecological niches and subsequent lineage diversification. The megadiverse insect order Hemiptera presents a particularly large diversity of symbiotic associations that has frequently undergone shifts in symbiont localization and identity, which have contributed to the exploitation of nutritionally imbalanced diets such as plant saps or vertebrate blood. Here we review the known ecological and evolutionary implications of symbiont gains, switches, and replacements, and identify future research directions that can contribute to a more comprehensive understanding of symbiosis as a major driving force of ecological adaptation.

Trends Microbial symbionts can confer beneficial traits on their insect hosts and contribute to the hosts’ adaptation to novel ecological niches. In the megadiverse insect order Hemiptera, obligate symbionts have been acquired and replaced many times independently. Symbiont acquisitions and replacements can mitigate the perilous effects of genomic decay in long-term symbioses and/or facilitate the adaptation to new host plants, thus providing the opportunity for niche expansion and diversification.

Symbiosis as a Source of Evolutionary Innovation in Plant–Insect Interactions

Our current understanding of the ecological implications of symbiont acquisitions and replacements is hampered by a lack of detailed knowledge [237_TD$IF]about the nutritional ecology of closely related insect taxa with different symbionts.

Symbiotic associations with microbes are ubiquitous in nature and represent major driving forces of evolutionary innovation by conferring novel phenotypic traits on the host, thereby allowing for the expansion into previously inaccessible ecological niches and subsequent lineage diversification [1–3]. A prime example is the acquisition of aerobic heterotrophic bacteria by a proto-eukaryote that later evolved into mitochondria, which enabled the diversification of eukaryotes from strictly anaerobic protists into the three major multicellular kingdoms [4]. In terrestrial ecosystems, plants and insects constitute not only the most diverse groups of macroorganisms, but they also interact with each other in a variety of ways. In particular, the evolutionary innovation that allowed insects to feed on angiosperms during the Cretaceous was one of the milestones that contributed significantly to the rich diversity of insect species we see today [5,6]. However, plant tissues are composed of complex plant polymers, such as cellulose, hemicellulose, pectin, and lignin, that are difficult for herbivores to digest [7]. Additionally, low nitrogen levels and imbalanced amino acid profiles in some plant tissues can constitute significant nutritional challenges to herbivorous insects and create the need for complementing the diet [8,9]. In addition, plants produce a wide range of toxic chemicals to protect themselves against herbivore attacks. To overcome these plant defenses and nutritional challenges, insects have evolved a diverse array of morphological, physiological, and behavioral adaptations [10].

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Insect Symbiosis Research Group, Max Planck Institute for Chemical Ecology, Jena, Germany 2 Experimental Ecology and Evolution Research Group, Max Planck Institute for Chemical Ecology, Jena, Germany 3 Institute of Microbiology, Friedrich Schiller University, Jena, Germany 4 Department for Evolutionary Ecology, Institute [23_TD$IF]of Organismic and Molecular Evolution, Johannes Gutenberg University, Mainz, Germany 5 Present address: Systems Biology, Wisconsin Institute for Discovery, University of Wisconsin-Madison,

http://dx.doi.org/10.1016/j.tim.2017.02.014 © 2017 Elsevier Ltd. All rights reserved.

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In addition to adaptive changes of the insects themselves, their association with microbial symbionts can profoundly influence their interaction with plants [11–13]. Symbiotic microbes possess the ability to supplement limiting nutrients, break down plant polymers, or detoxify plant defense compounds, thereby directly impacting the insects’ ability to exploit certain host plants as nutritional resources [2,11,14–16]. While a plethora of studies have elucidated the nutritional contributions of microbial symbionts to herbivorous insects, we are only beginning to appreciate the importance of microbial symbionts for enhancing the insects’ ability to adapt to new ecological niches [11,12].

Diversity of Symbiotic [238_TD$IF]Associations in Insects A diverse range of symbioses with nutritional benefits occurs across insect taxa [12,17]. First, many obligate (or primary) symbionts (see Glossary) are localized intracellularly in specialized organs, so-called bacteriomes, within the insect body (Figure 1). These symbionts are vertically transmitted between host generations and usually supplement nutrients to their insect host that are deficient in the host’s plant diet [18]. The second category comprises facultative (or secondary) symbionts that infect their host sporadically, can be located

(B) (A) (C)

(D)

Figure 1. [24_TD$IF]Schematic Overview of Different Symbiotic Associations That Occur in Hemipteran Insects. (A) Intracellular localization of symbionts in a bacteriome (symbiont cells in green). (B) Bacteriome with dual intracellular symbionts housed in independent bacteriocytes (symbionts are shown in orange and green). (C) Extracellular symbionts (indicated in purple) localized in the midgut. (D) Extracellular symbionts (indicated in red) harbored in specialized gastric caeca or crypts. Host cell nuclei are indicated in blue.

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[234_TD$IF]Madison, WI, USA 6 Present address: Department of Ecology, School of Biology/Chemistry, Osnabrück University, Osnabrück, Germany

*Correspondence: [email protected] (S. Sudakaran) and [email protected] (M. Kaltenpoth).

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intra- or extracellularly, and are transmitted via various routes of vertical and horizontal transmission. Some of these symbionts are known to manipulate the reproductive capacity of their hosts, but others provide benefits such as defense or nutrition [17,19]. The third group encompasses extracellular microbes that infect insect guts and are localized in the gut lumen or in gut-associated sacs (gastric caeca) (Figure 1). These symbionts are often transmitted vertically through egg-surface contamination, coprophagy, or special symbiont-containing capsules or mucus, but can also be horizontally acquired from the environment or from conspecifics by social transmission [20–23]. Members of this third category can contribute to their host’s fitness through nutrient provisioning, breaking down of plant polymers, nitrogen recycling, or detoxification of plant defenses (Figure 1) [24]. Finally, some microbial symbionts live outside of the host’s body on the food source, assisting in its breakdown to simple substrates suitable for consumption or enriching the diet with assimilated nitrogen [12]. Such interactions are common in the gardens or galleries of fungus-farming insects [25,26]. Each symbiotic system has its unique functional capabilities constrained by the symbionts’ identity and localization as well as the evolutionary stability of their association with the host insect [12]. For example, intracellular symbionts that form long-term mutualistic associations with their host insects [239_TD$IF]can contribute important nutritional benefits, but are limited in their ability to secrete products into the gut lumen to break down complex compounds or detoxify plant toxins, as they are spatially confined to the cytoplasm of host cells [27]. By contrast, gut symbionts or environmental, food-associated microbes are well suited for such functions [24]. Furthermore, intracellular symbionts undergo massive gene loss over extended evolutionary timescales, and horizontal acquisition of new genes is severely restricted, thereby limiting the symbionts’ metabolic flexibility [27–32]. If a phytophagous insect shifts from its current host plant to a different food source, its intracellular symbionts are unlikely to compensate for the nutrient deficiencies and other ecological challenges. Concordantly, such shifts require the insect host to either replace the current symbiont with a better-suited one, or alternatively acquire an additional symbiont to overcome the new challenges. By contrast, extracellular gut symbionts or food-associated microbes often possess dynamic genomes that can rapidly gain and lose functional genes, as is typical for most free-living bacterial taxa [33]. However, their transmission between generations can be less reliable, thereby compromising the evolutionary stability of their contributions towards host fitness [12]. Overall, shifts in symbiotic associations are expected to occur in insect lineages that switch to a new food source, because bacterial symbionts may compensate for the ecological challenges or provide the opportunity to switch in the first place. Below, we review symbiotic associations in the four hemipteran suborders and discuss possible implications of symbiont acquisitions and replacements for ecological adaptation of their hosts. While many symbiont replacements may enhance host fitness simply by escaping the ‘evolutionary rabbit hole' of increasingly degenerate symbiont genomes rather than providing novel ecological traits to the host [29], some affect the host’s ability to adapt to different nutritional resources [34]. Although we focus primarily on obligate symbionts, we also briefly summarize the knowledge on the involvement of facultative symbionts to the exploitation of novel host plants.

Diversity of Obligate Symbionts in the Insect Order Hemiptera The Hemiptera represent the most diverse hemimetabolous insect order and comprise taxa that occupy very different ecological niches. The order consists of around 82 000 described species in four suborders, that is, Sternorrhyncha, Auchenorrhyncha, Coleorrhyncha, and Heteroptera [35]. [240_TD$IF]Many hemipteran taxa are associated with obligate symbionts (Figure 2) that complement the deficiencies of the food source [30,36]. However, our knowledge on the role of

Glossary Bacteriocyte: specialized cell in a bacteriome containing intracellular bacterial symbionts. Bacteriome: a specialized organ (mainly found in insects) that hosts endosymbiotic bacteria. Codiversification: simultaneous speciation of, for example, host and symbiont. Co-obligate symbiont: second essential symbiont co-occurring with the original obligate symbiont. Coprophagy: consumption of feces. Crypt: invagination of an insect’s organ (e.g., cuticula, gut) and potential living space for symbiotic bacteria. Dual symbiosis: interaction between a host and two cooccurring endosymbionts. Endosymbiont: organism (e.g., a bacterium) that lives within the body or cells of another organism. Facultative symbiont: symbiont that is not essential for host survival, development, and reproduction, but can confer fitness benefits that are often context-dependent. Gastric caeca: projections of the digestive tract found in many insects. Hemimetabolous insects: insects with incomplete metamorphosis that gradually change from larval stages (nymphs) to adults and lack a pupal stage. Horizontal transmission: symbiosis that is newly established in every host generation. Symbionts are acquired from environmental sources, interacting organisms, or unrelated conspecifics. Monophyletic clade: a group of phylogenetically related organisms that comprises a common ancestral taxon and all its descendants. Mono-symbiosis: interaction between a host and a single species of bacterial endosymbiont. Obligate symbiont: symbiont that is essential for successful development and reproduction of its host. Phylogenomics: phylogenetic analysis of evolutionary relationships between taxa based on full genome sequences. Primary symbiont: see Obligate symbiont. Secondary symbiont: see Facultative symbiont. Syncytium: a multinucleated cell that results from the fusion of multiple uninuclear cells.

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Aphalaridae Psylloidea

Psyllidae: Heteropsylla Liviidae: Diaphorina Aleyrodoidea

Sternorrhyncha Aphidinae Aphidoidea

Lachninae Hormaphidinae Adelges

Adelgidae Phylloxeroidea

Pineus Phylloroxidae Pseudococcinae

Pseudococcinae Phenacoccinae Coccoidea Lecanodiaspididae, Coccidae, Eriococcidae, Ortheziidae, Coelostomidiidae, Monophlebidae

Diaspididae Achilidae Acanaloniidae Cixiidae Fulgoridea

Dictyopharidae Delphacidae Fulgoridae

Auchenorrhyncha Cicadoidea

Sulcia BetaSymb

Cercopoidea Evacanthinae Membracidae

Cicadomorpha

Cicadellinae Membracoidea

Typhlocybinae Deltocephalinae Ledrinae Coleorrhyncha Reduviidae Mirididae

Cimicomorpha

Cimicidae

Carsonella GammaSymb Carsonella GammaSymb Carsonella Profftella Porera Hamiltonella Buchnera Wolbachia Buchnera Serraa Buchnera Yeast-like Proffa & Valloa Gilleellia/Ecksreinia & Steffania Annandia Serraa Annandia Hargia Tremblaya Moranella, Mikella, Doolilea, Gullanella, Hoaglandella Brownia/Flavobacteriales Enterobacteriaceae SymB Walczuchella Enterobacteriaceae SymB Burkholderia Uzinura Sulcia Vidania Sulcia Vidania Purcelliella Sulcia Vidania Yeast-like Sulcia Vidania Sulcia Hodgkinia Sulcia Zinderia Sodalis-like Sulcia BetaSymb Sulcia BetaSymb Baumannia Sulcia Nasuia Yeast-like Sulcia Ophiocordyceps Evansia Rhodococcus Rickesia Wolbachia Wolbachia

Geocoridae Rhyparochromidae

Artheneidae Berydae

Lygaeoidea

Blissidae Lygaeidae Pachygronthidae

Burkholderia Rohrkolberia cinguli Burkholderia Ischnodemia utricula Schneideria nysicola Kleidoceria schneideri Arocaa carayoni Burkholderia

Alydidae, Coreidae, Stenocephalidae

Heteroptera

Coreoidea

Burkholderia

Rhopalidae Pyrrhocoridae

Pyrrhocoroidea

Largidae

Coriobacterium, Gordonibacter Clostridium & kledsiella Burkholderia

Pentatomidae

Pantoea/Enterobacter spp.

Scutelleridae Plataspidae Ishikawaella capsulata

Parastrachiidae Pentatomomorpha

Benitsuchiphilus tojoi

Cydnidae

Pentatomoidea

Acanthosomadae Rosenkranzia clausaccus

Urostylidae Tachikawaea gelanosa

Aradoidea

(See figure legend on the bottom of the next page.)

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Vertical transmission: symbionts are transmitted from parents to offspring and hence remain associated with the host’s offspring when a new generation is formed.

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shifts in the bugs’ symbiotic associations [241_TD$IF]in allowing them to exploit novel food sources is limited to a few examples.

Symbiont-Assisted Phloem Feeding in Sternorrhyncha In the suborder Sternorrhyncha, most taxa feed on phloem sap that is rich in sugars, but poor in amino acids and cofactors [9]. The genomic and experimental analysis of symbionts associated with sternorrhynchan insects revealed the microbial provisioning of essential amino acids, vitamins, and other beneficial compounds to their respective hosts [37–41]. These symbiontprovided benefits enabled the exploitation of phloem sap as a primary food source and allowed Sternorrhyncha to exploit a diverse range of host plants [12,30]. Interestingly, sternorrhynchan symbionts have experienced numerous replacements and transitions from mono- to dual symbioses in the course of their evolutionary history (Figure 2). Psyllids (Psylloidea) and whiteflies (Aleyrodoidea) harbor closely related intracellular symbionts, ‘Candidatus Carsonella ruddii’ and ‘Candidatus Portiera aleyrodidarum’ (both Gammaproteobacteria), respectively (Figure 2) [18]. It is possible that the symbiont colonization occurred in their shared ancestor (i.e., the ancestor of all Sternorrhyncha), and the symbionts subsequently codiversified with their insect hosts. Carsonella is harbored in specialized bacteriomes that are located in the abdomen of the host. It has one of the smallest genomes (160 kb) identified so far [37] that has retained the pathways responsible for the biosynthesis of most, but not all, amino acids required by the host [42]. In several species of psyllids, this is compensated by a co-obligate intracellular symbiont that inhabits the syncytial bacteriocyte cells between the uninucleate bacteriocytes within the bacteriome [43] and provides the nutritional supplements [24_TD$IF]for which the biosynthetic capabilities were lost in Carsonella [42]. However, some other taxa, such as Pachypsylla spp. and Heteropsylla texana, are not associated with additional symbionts to compensate for the essential amino acid deficiency [42]. One plausible explanation for their metabolic independence from the symbiont-provided amino acids is that psyllids may modify the physiology of their host plant in such a way that it provides sufficient amounts of several essential amino acids. As a consequence, the corresponding biosynthetic capabilities may have been lost from the genome of Carsonella [42]. This interpretation is corroborated by the finding that hackberry psyllids of the genus Pachypsylla elicit dramatic changes in the host plant during the formation of galls [44], and H. texana although not a gall former also causes extensive morphological and physiological alterations to the leaf and floral shoot structures of its Prosopis spp. host plants [45]. A second possible explanation is that the host could have acquired genes for the biosynthesis of some essential amino acids horizontally from its symbionts or other bacterial associates [42]. Even though several events of symbiont-to-host gene transfer have been reported in insects [46–48], the functional relevance of these horizontally acquired genes remains elusive in most cases (but see e.g. [49,50]). Analogous to the situation in psyllids, the obligate Portiera symbionts in the whitefly Bemisia tabaci lack entire pathways for cofactor biosynthesis as well as several genes necessary for the production of certain essential amino acids [51]. Recent findings indicate that Portiera’s metabolic capabilities are complemented by a putative co-obligate symbiont, Hamiltonella

Figure 2. E [27_TD$IF] volutionary Shifts in Symbiotic Associations across Hemiptera. A schematic host insect phylogeny is indicated in grey. Symbionts are color-coded based on their taxonomic identity at phylum or class level: Gammaproteobacteria green (light and dark green were used for dual gammaproteobacterial symbioses), Betaproteobacteria blue, Alphaproteobacteria purple, Bacteriodetes red, Firmicutes yellow, Actinobacteria brown, yeast-like fungal symbionts orange. Black dashed lines represent putative losses of symbionts, and colored dashed lines indicate possible relatedness between symbionts that requires further experimental support. Transition events are based on previous studies revealing the identity of microbial symbionts and/or reconstructing the hosts’ molecular phylogenies [12,35,76,114,119,120,140]. Data on Auchenorrhyncha have been adapted from [76], with kind permission from Oxford University Press.

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defensa, that resides in the same bacteriome cells, as well as enzymes encoded in the insect’s own genome that were acquired from bacteria by horizontal gene transfer [52]. In addition to the symbiont-mediated production of amino acids and cofactors, the Portiera symbionts provision their hosts with carotenoids, which are limiting in phloem sap [38,51]. Interestingly, another group of phloem-feeding insects, the aphids, have independently evolved a different solution for acquiring their carotenoids: aphids have horizontally acquired a fungal gene and integrated it into their genome [50]. The symbiont of most aphids (Aphidoidea), ‘Candidatus Buchnera aphidicola’ (Gammaproteobacteria), provides essential amino acids and riboflavin to the host and also assists in nitrogen recycling [39,53]. Even though Buchnera is associated with the majority of aphid species, it was replaced by yeast-like extracellular symbionts in the clade comprising the aphid genera Cerataphis, Tuberaphis, Hamiltonaphis, and Glyphinaphis [54]. Moreover, a recent study on the banana black aphid Pentalonia nigronervosa provided evidence for Wolbachia being a co-obligate symbiont complementing the metabolic capacities of Buchnera, in particular with regard to functional lysine and riboflavin pathways [55]. If substantiated, this finding would not only provide another case of the reproductive manipulator Wolbachia adopting a mutualistic role in an insect [56], but also support the hypothesis that riboflavin supplementation may be a widespread benefit provided by Wolbachia in insects that facilitated its invasion and spread in host populations [57]. In cedar aphids (Cinara cedri), Buchnera coexists with the additional intracellular symbiont ‘Candidatus Serratia symbiotica’ [58]. Interestingly, the genome of Buchnera in C. cedri (425 kb) is one of the smallest of all Buchnera genomes described so far, having lost several functional genes important to meet the nutritional requirements of the aphid, with the respective functions being provided by the co-obligate Serratia symbiont [58,59]. Serratia symbiotica has been previously identified in other aphid species (Acyrthosiphon pisum), where it occurs as a facultative symbiont that is involved in protecting the host from heat stress [60] and defending it against parasitoid wasps [61]. A comparative analysis of Serratia genomes indicated that these symbionts are in the initial stages of genome reduction and currently transition from a facultative to an obligate endosymbiotic lifestyle in cedar aphids [58,59]. In addition to Serratia, several other facultative symbionts have been described in aphids, and some of these have been reported to affect host plant utilization (Box 1).

Box 1. Effect of Facultative Symbionts on Host Plant Utilization in Aphids In addition to their obligate symbionts, aphids are associated with facultative symbionts that can confer important ecological traits to their hosts [30,129]. In the pea aphid, Acyrthosiphon pisum, the facultative symbiont Regiella insecticola was found to significantly improve host performance on white clover (Trifolium pretense), but not on vetch (Medicago sativa) [127]. Interestingly, experimental transfer of this symbiont strain to naturally Regiella-free vetch aphids (Megoura crassicauda) improved survival on white clover, suggesting that interspecific transfer of facultative symbionts can affect performance of herbivorous insects [130]. By contrast, several other studies on A. pisum failed to find consistent plant-specific effects of facultative symbionts on aphid performance, indicating that symbiont-incurred costs and benefits are dependent on symbiont and host genotype [128,131–134]. In another aphid species, Aphis craccivora, natural infection or experimental transfection with the facultative symbiont Arsenophonus improved performance on locust (Robinia pseudoacacia), but was associated with fitness costs on vetch [135]. Comparative analyses on the distribution of the facultative symbionts Hamiltonella defensa, Serratia symbiotica, and R. insecticola within and across aphid species consistently yielded significant effects of the host plant [131,136–139] and provide evidence that colonization of particular host plants is associated with the acquisition of particular symbiont strains [139]. In addition, a recent study reported that S. symbiotica occurs more frequently in increasingly specialized aphids, suggesting that it may provide a nutritional benefit mediating host plant adaptation and specialization [138]. Thus, even though it is clear that facultative symbionts can have significant implications for host plant utilization and dietary breadth in aphids, the fitness effects of symbiont infection are governed by complex interactions between host and symbiont genotype and the environment.

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The Phylloxeroidea superfamily, which consists of the two families [243_TD$IF]Adelgidae and Phylloxeridae, is the sister clade to the aphids (Aphidoidea). The Adelgids (woolly conifer aphids) feed on parenchyma cells or phloem sap of various conifers [62]. [24_TD$IF]Multiple events of symbiont acquisition and replacement have been reported in this group [63,64]. Adelgids contain two major lineages: Adelges and Pineus. Pineus strobi harbors the two gammaproteobacterial symbionts ‘Candidatus Hartigia pinicola’ and ‘Candidatus Annandia pinicola’ within bacteriocytes [64]. Interestingly, ‘Ca. A. pinicola’ forms a monophyletic clade with ‘Candidatus Annandia adelgesuga’, the intracellular symbiont of Adelges tsugae [64,65], indicating that Annandia may have occurred ancestrally in the Adelgidae. One of the closest relatives to Annandia is Buchnera, suggesting that these symbionts may originate from a common ancestor. However, this hypothesis is currently based on phylogenetic analyses of 16S and 23S rRNA gene sequences only and needs to be substantiated by phylogenomic evidence [64]. ‘Ca. H. pinicola’, the gammaproteobacterial co-obligate symbiont of Pineus strobi, is only distantly related to Annandia and the symbionts of other sternorrhynchan taxa, indicating that this symbiont is likely a recent acquisition [64]. Within the Adelges lineage, further events of symbiont replacement have been reported (Figure 2). First, the gammaproteobacterial symbiont ‘Candidatus Gillettellia colleyia’/‘Candidatus Ecksteinia adelgidicola’ of A. cooleyi/A. voweni and A. nordmannianae/A. piceae, respectively, was replaced by the gammaprotebacterial symbiont lineage comprising ‘Candidatus Profftia tarda’ and ‘Candidatus Profftia virida’ in the ancestor of Adelges laricis/A. tardus and Adelges abietis/A. viridis, respectively [63]. Second, the betaproteobacterial symbiont ‘Candidatus Vallotia’ was replaced in the Adelges nordmannianae/A. piceae complex by the Gammaproteobacteria ‘Candidatus Steffania’. Based on the genomic data for ‘Ca. Steffania adelgidicola’, all genes responsible for the biosynthesis of key metabolites (i.e., essential amino acids, vitamins, and cofactors) are still intact [40]. The observed transitions in the symbiotic associations of adelgids may be related to the changed nutritional demands of the insect host due to a shift to a novel food source (i.e., parenchyma cell content or phloem sap of conifers). Once acquired, the new set of symbionts may have allowed adelgids to also use other host plant species within this novel ecological niche [63]. In the adelgids’ sister family Phylloxeridae, no obligate symbiont has been identified so far [66]. Further genomic and experimental data are required to gain a better understanding of the symbionts’ functional role and the implications of shifts in the symbiotic associations of aphids belonging to the superfamily Phylloxeroidea. In the last sternorrhynchan superfamily, Coccoidea, several shifts in symbiotic associations have been observed [67]. Mealybugs (Pseudococcidae) are associated with a unique nested symbiosis composed of ‘Candidatus Tremblaya princeps’ (Betaproteobacteria) and an intracellular gamma-proteobacterial symbiont, named ‘Candidatus Moranella endobia’ in Planococcus citri [41,67]. The two symbionts complement each other metabolically and provide essential amino acids to their host [41]. Surprisingly, recent findings indicate that the gammaproteobacterial symbionts living in Tremblaya cells have been replaced repeatedly in the Pseudococcidae and show differential degrees of genome erosion [68]. Additionally, one particular species of Pseudococcidae, Phenacoccus avenae, harbors only ‘Candidatus Tremblaya phenacola’ and lacks the Moranella symbiont. However, this Tremblaya symbiont retains a larger repertoire of genes encoding the biosynthetic pathways for a full set of essential amino acids required by the host, likely representing a more ancestral state of Tremblaya’s metabolic capabilities [47]. In some Phenacoccinae, both Tremblaya and Moranella have been replaced by a symbiotic assemblage consisting of a Flavobacteriales and a Gammaproteobacterium [69–71]. The metabolic functions conferred by these symbionts to their hosts, however, remain unknown. Armored scale insects (Diaspididae) are associated with the obligate endosymbiont ‘Candidatus Uzinura diaspidicola’ [72]. The Uzinura symbiont belongs to the Flavobacteria

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(phylum Bacteroidetes), making it distantly related to most other known sternorrhynchan symbionts [70]. Uzinura has an extremely reduced genome, yet retains all genes necessary for the biosynthesis of essential amino acids and the recycling of nitrogen [72]. Insect taxa belonging to this family are known to feed on photosynthetic cells (i.e., mesophyll), rather than phloem sap, as is the case for most other Sternorrhyncha. In addition to Diaspididae, the flavobacterial symbionts have been characterized in multiple families of scale insects such as Lecanodiaspididae, Coccidae, Eriococcidae, Pseudococcidae, Ortheziidae, Coelostomidiidae, and Monophlebidae [71]. In most cases, the flavobacterial symbionts are accompanied by an Enterobacteriaceae symbiont (Gammaproteobacteria), and genomic analyses indicate that both symbionts contribute to nitrogen recycling and provisioning of essential amino acids to their host [71,73]. Phylogenetic analyses based on the 16S rRNA genes reveal that all of the flavobacterial symbionts with the exception of ‘Candidatus Brownia rhizoecola’ (in Pseudococcidae: Phenacoccinae) form a single monophyletic clade, implying an ancient acquisition event and subsequent codiversification with their hosts [71]. Interestingly, a recent study reported on a completely different bacterial symbiont in the eriococcids Acanthococcus aceris and Gossyparia spuria: females of these scale insects harbor Burkholderia (Betaproteobacteria) in their fat-body cells and transmit them transovarially to the offspring [74]. Unfortunately, nothing is known yet about the functional contributions of these symbionts to their host, but the metabolic and ecological versatility of Burkholderia and its widespread occurrence in diverse insect taxa make this a particularly interesting genus for further studies.

Symbiont-Assisted Transition from Phloem- to Xylem-Feeding in Auchenorrhyncha Like most Sternorrhyncha, several groups of the Auchenorrhyncha such as planthoppers (Fulgoroidea: Cixiidae, Delphacidae, Flatidae, Fulgoridae), treehoppers (Membracoidea: Membracidae), and leafhoppers (Membracoidea: Cicadellidae) are phloem feeders [75]. Most Auchenorrhyncha engage in an obligate dual symbiosis: ‘Candidatus Sulcia muelleri’ (Bacteroidetes) synthesizes a set of seven or eight essential amino acids, and a co-obligate betaproteobacterial symbiont synthesizes the two or three remaining essential amino acids [76]. While Sulcia was retained in most host lineages since the origin of the symbiosis about 270 million years ago [77], the betaproteobacterial symbiont has been repeatedly lost or replaced [36,77–79]. The clade consisting of ‘Candidatus Nasuia deltocephalinicola’ in deltocephaline leafhoppers [80], ‘Candidatus Zinderia insecticola’ in spittlebugs [79], and ‘Candidatus Vidania fulgoroideae’ in fulgorid planthoppers [81] likely represents the ancestral betaproteobacterial coobligate symbiont partner of Sulcia [76]. This is supported by phylogenetic analyses revealing its codiversification with Sulcia muelleri and its hosts since the origin of the symbiosis [76–79]. Subsequent replacement events include the switch to ‘Candidatus Hodgkinia cicadicola’ (Alphaproteobacteria) in cicadas [79], to Sodalis-like symbionts (Gammaproteobacteria) in philaenine spittlebugs [78], and to ‘Candidatus Baumannia cicadellinicola’ (Gammaproteobacteria) in some leafhopper taxa [1]. These shifts in symbiotic associations may have significantly impacted the diet and ecology of the hosts. In particular, the acquisition of Hodgkinia and Baumannia symbionts, respectively, may have permitted cicadas and leafhoppers to transition from phloem feeding to utilizing xylem sap. Although both Baumannia and Hodgkinia originate from different bacterial groups, they have functionally converged to produce the same essential amino acids (i.e., methionine and histidine) that were produced by the ancestral symbiont they replaced [82]. Baumannia’s comparatively large genome (686 kb) additionally encodes for pathways to produce vitamins and cofactors, which may have enabled leafhoppers to shift to xylem sap [83], a resource that is likely even more

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nutritionally limiting than phloem sap [84,85]. In contrast, Hodgkinia has a severely reduced genome (144 kb) that lost most of its vitamin and cofactor biosynthesis pathways [82]. Unlike leafhoppers, the Hodgkinia-harboring cicadas feed exclusively on xylem of plant roots [86,87], where the metabolite concentration is different than in other parts of the plant [82,88][245_TD$IF]. Interestingly, the recent sequencing of the whole genome of Baumannia associated with the green sharpshooter revealed the loss of the entire methionine biosynthetic pathway, suggesting that the host acquires this amino acid from its diet [89]. Additionally, symbiont replacements or losses have also occurred in phloem-feeding Auchenorrhyncha. Both Sulcia and Nasuia have been lost from the leafhopper Scaphoideus titanus (Deltocephalinae). Instead, a transovarially transmitted Cardinium (Bacteroidetes) plus a yeastlike symbiont have been acquired [90]. Similarly, Nasuia has been replaced by a yeast-like symbiont of the genus Ophiocordyceps in the Ledrinae, and some species even lost Sulcia in addition [91]. Some leafhopper groups like the Typhlocybinae (Cicadellidae) switched their diet from phloem to the more nutritious parenchyma plant tissue and presumably as a consequence have lost the dual symbionts [36,77]. However, whether or not these and other shifts in symbiotic associations that have been reported for Auchenorrhyncha indeed reflect altered nutritional demands of the host has not yet been investigated and should be studied in the future.

Symbiont-Enabled Exploitation of Bryophyte Host Plants by Coleorrhyncha The moss bugs (suborder Coleorrhyncha) are associated with the intracellular symbiont ‘Candidatus Evansia muelleri’ (Gammaproteobacteria) that is closely related to Carsonella and Portiera, the obligate endosymbionts of psyllids (Hemiptera: Sternorrhyncha: Psyllidae) and whiteflies (Hemiptera: Sternorrhyncha: Aleyrodidae), respectively [92,93]. However, genomic structure and metabolic differences indicate that despite the monophyly of Evansia, Carsonella, and Portiera, the moss bug symbionts likely represent an independent infection event [93]. In addition to supplementing essential amino acids, Evansia synthesizes cofactors and is involved in sulphur metabolism, presumably to compensate for the low level of nitrogen- and sulfurcontaining compounds found in the moss bugs’ diet (i.e., plant sap of mosses and liverworts) [93–95].

Frequent Shifts in the Symbiotic Associations of Heteroptera The true bugs (suborder Heteroptera) consist of around 40 000 species and represent the largest hemipteran suborder. Five of the seven infraorders (Dipsocoromorpha, Enicocephalomorpha, Gerromorpha, Leptopodomorpha, and Nepomorpha) are almost exclusively predatory and lack any known nutritional symbionts [96]. By contrast, symbiotic associations have been identified in the two most diverse infraorders Cimicomorpha and Pentatomomorpha [36,97]. In the Cimicomorpha, blood-sucking species of the genus Rhodnius (kissing bugs) in the family Reduviidae (assassin bugs) harbor symbiotic Rhodococcus (Actinobacteria) in their gut cavity [98], and Cimicidae (bedbugs) are associated with Wolbachia (Alphaproteobacteria) in specialized bacteriomes [56]. Although belonging to different bacterial phyla and being localized in different host tissues, both symbionts complement the hosts’ diet with B vitamins that are deficient in vertebrate blood [56,98]. In the infraorder Pentatomomorpha, almost all members are phytophagous (mostly seed feeders), and most harbor symbiotic bacteria in the gastrointestinal tract or less commonly in bacteriomes. The specialized gastric caeca or crypts in the distal part of the midgut harboring extracellular proteobacterial symbionts appear to be the ancestral symbiont-bearing structures in Pentatomomorpha [97,99]. While little is known about symbiotic bacteria in the ancestral superfamily Aradoidea, most species in the superfamily Pentatomoidea have midgut crypts that are inhabited by one of several distinct lineages of vertically transmitted

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Gammaproteobacteria [100–105]. While the localization and molecular identification of symbionts has been reported for multiple lineages within the Pentatomoidea, less is known about their contributions to the hosts’ nutrition. Bugs from the family Plataspidae (Heteroptera; Pentatomoidea) harbor the crypt-associated symbiont ‘Candidatus Ishikawaella capsulata’ (Gammaproteobacteria) [106]. This symbiont features a reduced genome that encodes the biosynthesis of essential amino acids and vitamins and contains, in addition, a plasmid with an oxalate decarboxylase gene, which may provide protection to the host against the widespread plant toxin oxalic acid [106,107]. Importantly, reciprocal symbiont exchange between the two plataspid species Megacopta punctatissima and M. cribaria resulted in the complete reversal of the bugs’ performances on their respective host plants, indicating that the symbiont strain, not the host genotype, governs host plant specialization [34]. As M. punctatissima is an important pest on legumes, the pest status is thus conferred by the bacterial symbionts and can be transferred experimentally to M. cribaria. Interestingly, a population genomic analysis of symbionts associated with invasive M. cribaria in the USA that infest legume plants revealed a high degree of similarity to the symbiont genome of native Japanese M. punctatissima, suggesting that a horizontal symbiont transfer from M. punctatissima to M. cribaria occurred naturally and conferred the pest status to the invasive bug [108]. Similar to the Plataspidae, the Urostylididae, another pentatomoid family, harbor a gammaproteobacterial symbiont (‘Candidatus Tachikawaea gelatinosa’) that possesses the biosynthetic capabilities for provisioning essential amino acids and vitamins to the host [109]. In addition, experimental removal of symbionts in several other closely related pentatomoid families resulted in high mortality and reduced growth [102,110–113]. Overall, this indicates that these bug-associated symbionts play an important role for the fitness of their insect host by providing essential amino acids and/or vitamins. Recent studies on the symbionts of the superfamilies Coreoidea, Lygaeoidea, and Pyrrhocoroidea suggest that this large monophyletic clade of bugs ancestrally harbored Burkholderia symbionts in midgut crypts, which were secondarily lost or replaced by other symbiotic associations [20,114]. The symbiotic Burkholderia are phylogenetically diverse, and in some cases there is experimental evidence demonstrating horizontal acquisition from the environment in every generation, indicating a high degree of flexibility in the symbiosis [115,116]. Even though little is known about the original function of Burkholderia symbionts in bugs, one report implicates the bacteria in insecticide resistance of their host, the bean bug Riptortus pedestris [117]. When this insect was exposed to the organophosphate fenitrothion in the laboratory or in the field, environmental Burkholderia strains resistant to this insecticide were acquired by the bugs and conferred protection to the host [117]. As detoxification of the synthetic insecticide fenitrothion cannot be the ancestral benefit conferred by Burkholderia symbionts to their hosts, future studies addressing potential benefits through nutritional supplementation or detoxification of plant secondary metabolites are necessary to fully understand the evolutionary and ecological implications of the widespread association of bugs with these betaproteobacterial symbionts. Likewise, functional analyses of symbiont-provided benefits are urgently needed to understand the ecological factors underlying the repeated changes in symbiotic associations from crypt-associated Burkholderia to gammaproteobacterial symbionts localized in bacteriomes or midgut epithelial cells in several lygaeoid families (Figure 2) [118–120]. An evolutionary transition in symbiotic associations with probable ecological implications has recently been reported in the superfamily Pyrrhocoroidea [114]. While members of the family Largidae are associated with the ancestral crypt-associated Burkholderia symbionts [121,122], bugs in the sister family Pyrrhocoridae lost their crypts. Instead, they harbor a stable bacterial consortium made up of two Actinobacteria (Coriobacterium glomerans and Gordonibacter sp.), one Firmicute (Clostridium sp.), and one Gammaproteobacterium (Klebsiella sp.) in the lumen of

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the anoxic M3 region of the midgut [22,114,123]. The actinobacterial symbionts were shown to play an important role for supplementing limiting B vitamins to their insect host [21]. Furthermore, the pyrrhocorids’ microbiota may be involved in nitrogen metabolism, degradation of complex dietary components (specifically cellulose), and detoxification of noxious plant secondary metabolites, specifically cyclopropenoic fatty acids (CPFAs) [22,123]. The symbiontprovided benefits probably enabled their insect hosts to successfully exploit a nutritionally challenging food source (i.e., seeds of Malvales plants) that is inaccessible to many other insects, mainly due to the low concentrations of B vitamins as well as the presence of toxic CPFAs and other secondary metabolites. Even though seed feeding is widespread among pentatomomorphan bugs, the shift of the symbiotic association from crypt-associated to an anaerobic midgut microbiota appears to be confined to the Pyrrhocoridae [114]. The specialized nutritional challenges associated with the dietary switch to Malvales seeds may have driven this evolutionary change. This interpretation is supported by age estimations, which revealed that the origin of the pyrrhocorid symbiosis roughly coincided with the evolution of the Malvales plant order [114].

Ecological and Evolutionary Implications of Shifts in Symbiotic Associations With regard to the diversity of symbiotic associations, the Hemiptera represent the best studied insect order to date: numerous studies have characterized the microbial symbionts that are associated with a broad phylogenetic range of hemipteran hosts on a molecular level [12,20,22,37–39,41,63,71,76,77]. Taken together, these studies revealed an enormous diversity of symbiotic associations ranging from obligate intracellular [246_TD$IF]symbioses, such as in the plant-sap feeding suborders Sternorrhyncha, Auchenorrhyncha, and Coleorrhyncha [18,38,42,53,63,67,70,71,77,82,93], to extracellular symbioses within the suborder Heteroptera[247_TD$IF], where the symbionts are housed in specialized structures such as crypts [20,102,106,112] or in the gut lumen [22] [248_TD$IF](Figure 1). In several taxa, phylogenetic analyses revealed a long-term codiversification of hosts and symbionts, sometimes dating to the origin of the insect family or suborder itself, which in some cases goes back to the early stages of terrestrial herbivory on Permian vascular plants at least 270 million years ago [14,27]. Despite the evolutionarily ancient association of Hemiptera with microbial symbionts, their symbiotic associations have undergone frequent changes, including losses and replacements of obligate symbionts or the acquisitions of co-obligate and facultative symbionts [249_TD$IF](Figure 2). Moreover, also specialized symbiont-bearing structures have arisen multiple times independently, housing several distinct microbial taxa, including Actinobacteria, Alphaproteobacteria, Bacteroidetes, Betaproteobacteria, Gammaproteobacteria, and yeast-like fungal symbionts [58,63,67,71, 78,114,119,120,124]. Two mutually nonexclusive evolutionary scenarios may explain the numerous shifts in symbiotic associations that can be observed within the Hemiptera. First, due to their confinement to the insect host and strong population bottlenecks during transmission to the next host generation, obligate endosymbionts experience strong genetic drift, resulting in an accumulation of slightly deleterious mutations (Muller’s ratchet) [125]. As a result of this process, long-term symbiotic relationships should be faced with a quantitative or qualitative deterioration of the benefits that are provided by the symbionts [29]. To compensate for this effect, the host could acquire additional (facultative) symbionts. If sufficiently common, easily accessible, and/or stably [250_TD$IF]transferred between host generations, acquisition of a facultative symbiont should relax the selective pressure on the obligate symbiont to maintain nutritional pathways that were previously essential to the host [126]. Degradation of such capabilities by further genome erosion could rapidly increase the hosts’ dependence on the newly acquired symbiont and thus expedite the latter’s establishment as a co-obligate symbiont. Depending on the metabolic capabilities of both co-occurring symbionts, as well as the pace with which their genomes erode, this situation can result in either the complete loss of the original symbiont (i.e., symbiont

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Figure 3. Schematic Overview over Shifts in Symbiotic Associations and Their Potential Implications for Permitting Niche Expansion or Specialization. The schematic phylogeny represents the possible evolutionary trajectories of symbiotic associations. Coloring of symbiotic associations corresponds to their color-coding in Figure 1. Different plants represent different ecological niches. This schematic depicts the most frequently observed shifts in symbiotic associations in Hemiptera and their potential influence on the host’s niche expansion [230_TD$IF]or dietary specialization. In some cases, switches in symbiotic associations may not result in the [231_TD$IF]shift to a new ecological niche, but rather allow the host to replace or metabolically complement the existing symbiont.

replacement) or the coexistence of both microbes and their host in a tripartite dependency [29]. Examples for both cases have been described for several Hemipteran taxa (Figures 2 and 3). The second scenario relies on the capability of microbial symbionts to confer novel adaptive traits to their host. As a consequence, the symbiotic association may allow the insect to expand its ecological range or even invade an entirely new ecological niche [17]. Even though it is often difficult to establish the chain of causality that leads to these [251_TD$IF]changes (i.e., did the symbiont acquisition facilitate a host plant shift or did the host plant shift select for the acquisition of novel symbionts?), a temporal correlation between a dietary shift of the host and the origin of a new nutritional symbiosis strongly implicates the symbiont in the observed change of its host’s ecology (Figure 3). Several examples for such cases have been reported in Hemiptera, and the establishment of new symbiotic associations may even have facilitated the evolution of the major feeding habits that can be found in this insect order (Figure 2). For example, multiple independent acquisitions of obligate intracellular symbionts, which provide essential amino acids allowed the suborders Sternorrhyncha, Auchenorrhyncha, and Coleorrhyncha to exploit phloem sap as a food source [12,76,93]. In the case of leafhoppers, the transition from the ancestral symbiont to Baumannia, which possesses the genomic potential to produce vitamins, cofactors, and essential amino acids, likely facilitated the switch from phloem to xylem feeding [83]. Furthermore, the B-vitamin deficiency of vertebrate blood as a nutritional resource in blood-feeding Cimicomorphan bugs was compensated by two independent acquisition events of vitamin-supplementing symbionts, a gut-associated Actinobacterium in Reduviidae, and a bacteriome-localized Wolbachia in Cimicidae [56,98]. Finally, the association with an anoxic microbial gut community that provides vitamins and possibly aids in the detoxification of plant secondary metabolites probably enabled pyrrhocorid bugs to feed on seeds of Malvales plants [114]. In most other cases, however, the ecological implications of a shift in the host’s symbiotic association remain unknown. One of the major reasons for this lack of knowledge on the consequences of transitions in symbiotic associations is the difficulty of addressing this question directly through experimental manipulation. However, a convincing case for the host’s adaptation to a novel ecological niche by acquiring new symbionts can still be made by providing evidence for the following points: (i) a comparative molecular and/or histological characterization of the symbiotic associations across host taxa reveals a shift in symbiont identity and/or localization; (ii) mapping this

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transition onto the hosts’ phylogeny identifies the evolutionary origin of this association; (iii) functional assays (e.g., disrupting the host–symbiont association and quantifying the consequences for host fitness) and/or genomic analyses reveal nutritional benefits provided by the derived symbiont that (iv) complement novel nutritional challenges of the host after switching to a new diet. The advent of next-generation sequencing has greatly facilitated the reconstruction of molecular host phylogenies and significantly helped to identify symbiotic bacteria and characterize their genome sequences. However, functional assays are urgently required to elucidate the ecological relevance of these bacterial symbionts, particularly in light of nutritional challenges that are associated with dietary shifts of the insect hosts. Even if a herbivorous host switches to a different plant species yet still utilizes the same type of food source (e.g., phloem sap or seeds), a shift in the symbiotic association may have allowed for this change, for example by alleviating the challenges associated with imbalanced nutrient composition or detoxification of secondary plant metabolites [34,127]. Due to the rich knowledge on the diversity and genomics of symbiotic associations across Hemiptera, this insect order represents a promising model system for studying the role of symbiont switches in the host’s adaptation to a novel food source.

Concluding Remarks Symbiotic microbes play a major role in supplementing the nutrition of many herbivorous insect taxa, which is greatly facilitated by the rich metabolic repertoire that generally characterizes prokaryotic genomes. However, our understanding of the ecological factors underlying the transition in symbiotic associations and their evolutionary implications remains limited to a small number of examples. The high diversity of symbiotic associations associated with hemipteran insects, and their role in enabling their hosts to exploit particular nutritional resources, makes this group an ideal system for addressing this issue. So far, most studies have focused on molecular aspects of these symbioses (e.g., phylogenetics, genomics), which have tremendously advanced our knowledge of the symbionts’ identity, genome evolution, and metabolic capabilities across many hemipteran taxa. What is needed now are detailed comparative studies on the nutritional ecology of closely related insect taxa that are associated with different symbionts (see Outstanding Questions). In particular, fitness assays of symbiotic and aposymbiotic insects should be complemented by analyses of the nutritional composition of their herbivorous diet, linking symbiont-provided benefits to the hosts’ nutritional challenges. In addition, further studies are required to broaden our understanding of the importance of other symbiont-mediated traits, such as protection against pathogens, parasites, or environmental stresses, for the shift of its insect host to a novel ecological niche [128]. Overall, such studies are likely to significantly advance our understanding of symbiosis as a driving force of evolutionary innovation, ecological adaptation, and diversification in insects.

Outstanding Questions How commonly are acquisitions of novel symbionts associated with a dietary shift in the host? How do symbionts contribute to host fitness in cases where a host-plant shift occurs without a corresponding change in the exploited tissue type (e. g., phloem sap)? Are nutritional symbionts a major cause of adaptive radiations in herbivorous insects? What is the evolutionary potential of symbiont-mediated nutritional supplementation, digestion, and detoxification vs. the insect’s own metabolic capabilities for the adaptation to novel diets? How much flexibility do long-term obligate symbionts maintain for adjusting their metabolic contributions to their hosts’ needs when the plant nutritional composition is variable or changes over evolutionary time? Which factors hamper/facilitate the replacement of an existing symbiont by another one? What are the costs and benefits of carrying multiple interacting symbionts relative to harboring a single symbiont strain?

Competing Interests The authors declare that they have no competing interests. Acknowledgments We thank Laura Victoria Flórez and Tobias Engl for valuable comments on the manuscript, and we gratefully acknowledge financial support from the Max Planck Society (SS, CK, and MK), the German Science Foundation (MK, DFG KA2846/2-1), the Volkswagen Foundation (CK), and the Jena School of Microbial Communication (JSMC) (CK and MK).

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