Bacterial leaf symbiosis in angiosperms: host specificity without co-speciation

Bacterial leaf symbiosis is a unique and intimate interaction between bacteria and flowering plants, in which endosymbionts are organized in specialized leaf structures. Previously, bacterial leaf symbiosis has been described as a cyclic and obligate interaction in which the endosymbionts are vertically transmitted between plant generations and lack autonomous growth. Theoretically this allows for co-speciation between leaf nodulated plants and their endosymbionts. We sequenced the nodulated Burkholderia endosymbionts of 54 plant species from known leaf nodulated angiosperm genera, i.e. Ardisia, Pavetta, Psychotria and Sericanthe. Phylogenetic reconstruction of bacterial leaf symbionts and closely related free-living bacteria indicates the occurrence of multiple horizontal transfers of bacteria from the environment to leaf nodulated plant species. This rejects the hypothesis of a long co-speciation process between the bacterial endosymbionts and their host plants. Our results indicate a recent evolutionary process towards a stable and host specific interaction confirming the proposed maternal transmission mode of the endosymbionts through the seeds. Divergence estimates provide evidence for a relatively recent origin of bacterial leaf symbiosis, dating back to the Miocene (5-23 Mya). This geological epoch was characterized by cool and arid conditions, which may have triggered the origin of bacterial leaf symbiosis.     

Insect life history and the evolution of bacterial mutualism

Bacterial symbiosis has played a fundamental role in the evolution of eukaryotes. However, we still know little about how cooperative relationships with bacteria originate, and why they form in some host species but not others. Facultative symbionts that are beneficial, but not essential, provide unique insights into these processes. We use data from over a hundred aphid species to test if host life history is associated with the presence of facultative symbionts. We find that aphid species that have mutualistic associations with ants that protect them from natural enemies are less likely to carry symbionts that provide similar benefits. We also find one symbiont species occurs more frequently in unrelated aphid species that specialise on certain plant genera. In addition, aphid species that attack multiple plants often carry different symbiont complements. Our findings provide evidence of the ecological conditions that facilitate stable, mutually beneficial relationships between microbes and eukaryotic hosts.     

Screening for leaf-associated endophytes in the genus Psychotria (Rubiaceae)

Burkholderia endophytes were identified within the leaves of non-nodulated members of the genus Psychotria. In contrast to leaf-nodulated Psychotria species, which are known to accommodate their endosymbionts into specialized endosymbiont-housing structures, non-nodulated species lack bacterial leaf nodules and harbor endosymbionts intercellularly between mesophyll cells. Based on molecular data (rps16, trnG, and trnLF), the phylogenetic reconstruction of the host plants revealed a separate origin of leaf-nodulated and non-nodulated Psychotria species. Despite a distinct phylogenetic position of the two host clades, the endophytes of the non-nodulated plants were not placed into a single monophyletic group but were found to be closely related to the leaf-nodulated endosymbionts. The observation of genetically similar endophytes in both nodulated and non-nodulated Psychotria lineages suggests that the host plant is playing a crucial role in the induction of leaf nodule formation. Moreover, the concentration of endosymbionts into specialized leaf nodules may be considered as a more derived evolutionary adaptation of the host plant, serving as an interface structure to facilitate metabolic exchange between plant and endosymbiont.     

Burkholderia species are ancient symbionts of legumes

Burkholderia has only recently been recognized as a potential nitrogen-fixing symbiont of legumes, but we find that the origins of symbiosis in Burkholderia are much deeper than previously suspected. We sampled 143 symbionts from 47 native species of Mimosa across 1800 km in central Brazil and found that 98% were Burkholderia . Gene sequences defined seven distinct and divergent species complexes within the genus Burkholderia . The symbiosis-related genes formed deep Burkholderia -specific clades, each specific to a species complex, implying that these genes diverged over a long period within Burkholderia without substantial horizontal gene transfer between species complexes.     

Biodiversity of Mimosa pudica rhizobial symbionts (Cupriavidus taiwanensis, Rhizobium mesoamericanum) in New Caledonia and their adaptation to heavy metal-rich soils

Rhizobia are soil bacteria able to develop a nitrogen-fixing symbiosis with legumes. They are taxonomically spread among the alpha and beta subclasses of the Proteobacteria. Mimosa pudica, a tropical invasive weed, has been found to have an affinity for beta-rhizobia, including species within the Burkholderia and Cupriavidus genera. In this study, we describe the diversity of M.?pudica symbionts in the island of New Caledonia, which is characterized by soils with high heavy metal content, especially of Ni. By using a plant-trapping approach on four soils, we isolated 96 strains, the great majority of which belonged to the species Cupriavidus taiwanensis (16S rRNA and recA gene phylogenies). A few Rhizobium strains in the newly described species Rhizobium mesoamericanum were also isolated. The housekeeping and nod gene phylogenies supported the hypothesis of the arrival of the C.?taiwanensis and R.?mesoamericanum strains together with their host at the time of the introduction of M.?pudica in New Caledonia (NC) for its use as a fodder. The C.?taiwanensis strains exhibited various tolerances to Ni, Zn and Cr, suggesting their adaptation to the specific environments in NC. Specific metal tolerance marker genes were found in the genomes of these symbionts, and their origin was investigated by phylogenetic analyses.     

Legume-nodulating betaproteobacteria: diversity, host range, and future prospects

Rhizobia form specialized nodules on the roots of legumes (family Fabaceae) and fix nitrogen in exchange for carbon from the host plant. Although the majority of legumes form symbioses with members of genus Rhizobium and its relatives in class Alphaproteobacteria, some legumes, such as those in the large genus Mimosa, are nodulated predominantly by betaproteobacteria in the genera Burkholderia and Cupriavidus. The principal centers of diversity of these bacteria are in central Brazil and South Africa. Molecular phylogenetic studies have shown that betaproteobacteria have existed as legume symbionts for approximately 50 million years, and that, although they have a common origin, the symbiosis genes in both subclasses have evolved separately since then. Additionally, some species of genus Burkholderia, such as B. phymatum, are highly promiscuous, effectively nodulating several important legumes, including common bean (Phaseolus vulgaris). In contrast to genus Burkholderia, only one species of genus Cupriavidus (C. taiwanensis) has so far been shown to nodulate legumes. The recent availability of the genome sequences of C. taiwanensis, B. phymatum, and B. tuberum has paved the way for a more detailed analysis of the evolutionary and mechanistic differences between nodulating strains of alpha- and betaproteobacteria. Initial analyses of genome sequences have suggested that plant-associated Burkholderia spp. have lower G+C contents than Burkholderia spp. that are opportunistic human pathogens, thus supporting previous suggestions that the plant- and human-associated groups of Burkholderia actually belong in separate genera.     

Symbionts in excess? No correlation between symbiont density and the ability of mealybug hosts to exploit plant species or tolerate insecticide stress

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Limited geographic distribution of certain strains of the bioluminescent symbiont Photobacterium leiognathi

Photobacterium leiognathi is a facultative bioluminescent symbiont of marine animals. Strains of P.?leiognathi that are merodiploid for the luminescence genes (lux-rib operon) have been previously obtained only from Japan. In contrast, strains bearing a single lux-rib operon have been obtained from all the areas sampled in Japan and the western Pacific. In this study, we tested whether distribution of merodiploid P.?leiognathi is limited by physical barriers in the environment, or because fish in the western Pacific preferentially form symbiosis with bacteria bearing a single lux-rib operon. We collected light organ symbionts from Secutor indicius, a fish species that is typically found in the western Pacific and has only recently expanded its geographic range to Japan. We found that all S.?indicius specimens collected from Japan formed symbiosis only with single lux-rib operon-bearing strains, although fish from other species collected from the same geographic area frequently contained merodiploid strains. This result shows that S.?indicius were preferentially colonized by bacteria bearing a single lux-rib operon and suggests that the limited geographic distribution of merodiploid P.?leiognathi can be attributed to preferential colonization of fish species found in the western Pacific by strains bearing only a single lux-rib operon.     

Lack of endosymbiont release by two Lucinidae (Bivalvia) of the genus Codakia: consequences for symbiotic relationships

Associations between marine invertebrates and chemoautotrophic bacteria constitute a wide field for the study of symbiotic associations. In these interactions, symbiont transmission must represent the cornerstone allowing the persistence of the association throughout generations. Within Bivalvia , in families such as Solemyidae or Vesicomyidae , symbiont transmission is undoubtedly vertical. However, in Lucinidae , symbiont transmission is described in the literature as 'environmental', symbionts being acquired from the environment by the new host generations. Hence, if there is transmission, symbionts should be transmitted from adults to juveniles via the environment. Consequently, we should observe a release of the symbiont by adults. We attempted to detect such a release within two Lucinidae species of the genus Codakia . We sampled 10 Codakia orbicularis and 20 Codakia orbiculata distributed in 10 crystallizing dishes containing filtered seawater. During 1 month of investigation, we analyzed water of the dishes in order to detect any release of a symbiont using catalyzed report deposition-FISH techniques. For 140 observations realized during this period, we did not observe any release of symbionts. This suggests that the idea of host-to-host passage in Lucinidae is inaccurate. We could therefore consider that the transmission mode from generation to generation does not occur within Lucinidae , symbiosis appearing to be advantageous in this case only for the host, and constitutes an evolutionary dead-end for the bacteria.     

Coexistence of Wolbachia with Buchnera aphidicola and a secondary symbiont in the aphid Cinara cedri

Intracellular symbiosis is very common in the insect world. For the aphid Cinara cedri, we have identified by electron microscopy three symbiotic bacteria that can be characterized by their different sizes, morphologies, and electrodensities. PCR amplification and sequencing of the 16S ribosomal DNA (rDNA) genes showed that, in addition to harboring Buchnera aphidicola, the primary endosymbiont of aphids, C. cedri harbors a secondary symbiont (S symbiont) that was previously found to be associated with aphids (PASS, or R type) and an alpha-proteobacterium that belongs to the Wolbachia genus. Using in situ hybridization with specific bacterial probes designed for symbiont 16S rDNA sequences, we have shown that Wolbachia was represented by only a few minute bacteria surrounding the S symbionts. Moreover, the observed B. aphidicola and the S symbionts had similar sizes and were housed in separate specific bacterial cells, the bacteriocytes. Interestingly, in contrast to the case for all aphids examined thus far, the S symbionts were shown to occupy a similarly sized or even larger bacteriocyte space than B. aphidicola. These findings, along with the facts that C. cedri harbors the B. aphidicola strain with the smallest bacterial genome and that the S symbionts infect all Cinara spp. analyzed so far, suggest the possibility of bacterial replacement in these species.     

Host hybridization as a potential mechanism of lateral symbiont transfer in deep‐sea vesicomyid clams

Deep‐sea vesicomyid clams live in mutualistic symbiosis with chemosynthetic bacteria that are inherited through the maternal germ line. On evolutionary timescales, strictly vertical transmission should lead to cospeciation of host mitochondrial and symbiont lineages; nonetheless, examples of incongruent phylogenies have been reported, suggesting that symbionts are occasionally horizontally transmitted between host species. The current paradigm for vesicomyid clams holds that direct transfers cause host shifts or mixtures of symbionts. An alternative hypothesis suggests that hybridization between host species might explain symbiont transfers. Two clam species, Archivesica gigas and Phreagena soyoae, frequently co‐occur at deep‐sea hydrocarbon seeps in the eastern Pacific Ocean. Although the two species typically host gammaproteobacterial symbiont lineages marked by divergent 16S rRNA phylotypes, we identified a number of clams with the A. gigas mitotype that hosted symbionts with the P. soyoae phylotype. Demographic inference models based on genome‐wide SNP data and three Sanger sequenced gene markers provided evidence that A. gigas and P. soyoae hybridized in the past, supporting the hypothesis that hybridization might be a viable mechanism of interspecific symbiont transfer. These findings provide new perspectives on the evolution of vertically transmitted symbionts and their hosts in deep‐sea chemosynthetic environments.     

Evolutionary changes in symbiont community structure in ticks

Ecological specialization to restricted diet niches is driven by obligate, and often maternally inherited, symbionts in many arthropod lineages. These heritable symbionts typically form evolutionarily stable associations with arthropods that can last for millions of years. Ticks were recently found to harbour such an obligate symbiont, Coxiella‐LE, that synthesizes B vitamins and cofactors not obtained in sufficient quantities from blood diet. In this study, the examination of 81 tick species shows that some Coxiella‐LE symbioses are evolutionarily stable with an ancient acquisition followed by codiversification as observed in ticks belonging to the Rhipicephalus genus. However, many other Coxiella‐LE symbioses are characterized by low evolutionary stability with frequent host shifts and extinction events. Further examination revealed the presence of nine other genera of maternally inherited bacteria in ticks. Although these nine symbionts were primarily thought to be facultative, their distribution among tick species rather suggests that at least four may have independently replaced Coxiella‐LE and likely represent alternative obligate symbionts. Phylogenetic evidence otherwise indicates that cocladogenesis is globally rare in these symbioses as most originate via horizontal transfer of an existing symbiont between unrelated tick species. As a result, the structure of these symbiont communities is not fixed and stable across the tick phylogeny. Most importantly, the symbiont communities commonly reach high levels of diversity with up to six unrelated maternally inherited bacteria coexisting within host species. We further conjecture that interactions among coexisting symbionts are pivotal drivers of community structure both among and within tick species.     

A specific mix of generalists: bacterial symbionts in Mediterranean Ircinia spp

Microbial symbionts form abundant and diverse components of marine sponge holobionts, yet the ecological and evolutionary factors that dictate their community structure are unresolved. Here, we characterized the bacterial symbiont communities of three sympatric host species in the genus Ircinia from the NW Mediterranean Sea, using electron microscopy and replicated 16S rRNA gene sequence clone libraries. All Ircinia host species harbored abundant and phylogenetically diverse symbiont consortia, comprised primarily of sequences related to other sponge-derived microorganisms. Community-level analyses of bacterial symbionts revealed host species-specific genetic differentiation and structuring of Ircinia-associated microbiota. Phylogenetic analyses of host sponges showed a close evolutionary relationship between Ircinia fasciculata and Ircinia variabilis, the two host species exhibiting more similar symbiont communities. In addition, several bacterial operational taxonomic units were shared between I.?variabilis and Ircinia oros, the two host species inhabiting semi-sciophilous communities in more cryptic benthic habitats, and absent in I.?fasciculata, which occurs in exposed, high-irradiance habitats. The generalist nature of individual symbionts and host-specific structure of entire communities suggest that: (1) a 'specific mix of generalists' framework applies to bacterial symbionts in Ircinia hosts and (2) factors specific to each host species contribute to the distinct symbiont mix observed in Ircinia hosts.     

Rhizobien in der Pflanzenmikrobiota

Rhizobia in the plant microbiota The plant microbiota is of critical importance for plant growth and survival in soil. To explore mechanisms underlying plant‐microbiota interactions, defined commensal communities can be composed from microbiota culture collections and co‐cultivated with germ‐free plants to determine their impact on plant growth and health. The order Rhizobiales belongs to the core microbiota and includes nitrogen‐fixing bacteria that are known to engage in symbiotic interactions with legumes. Compatible host‐symbiont pairs are needed for a functional symbiosis, which involves the activation of highly specialized and interdependent signaling pathways between the two partners. Comparative genome analysis of more than 1,300 legume symbionts and rhizobial root commensals from non‐leguminous plants revealed that the most recent common ancestor of rhizobia lacked the gene repertoire needed for symbiosis and was able to colonize roots of a wide variety of plants. During evolution, key symbiosis genes were acquired multiple independent times by commensals belonging to different families of the Rhizobiales order.     

Reduced genome of the thioautotrophic intracellular symbiont in a deep-sea clam, Calyptogena okutanii

Although dense animal communities at hydrothermal vents and cold seeps rely on symbioses with chemoautotrophic bacteria [1, 2], knowledge of the mechanisms underlying these chemosynthetic symbioses is still fragmentary because of the difficulty in culturing the symbionts and the hosts in the laboratory. Deep-sea Calyptogena clams harbor thioautotrophic bacterial symbionts in their gill epithelial cells [1, 2]. They have vestigial digestive tracts and nutritionally depend on their symbionts [3], which are vertically transmitted via eggs [4]. To clarify the symbionts' metabolic roles in the symbiosis and adaptations to intracellular conditions, we present the complete genome sequence of the symbiont of Calyptogena okutanii. The genome is a circular chromosome of 1,022,154 bp with 31.6% guanine + cytosine (G + C) content, and is the smallest reported genome in autotrophic bacteria. It encodes 939 protein-coding genes, including those for thioautotrophy and for the syntheses of almost all amino acids and various cofactors. However, transporters for these substances to the host cell are apparently absent. Genes that are unnecessary for an intracellular lifestyle, as well as some essential genes (e.g., ftsZ for cytokinesis), appear to have been lost from the symbiont genome. Reductive evolution of the genome might be ongoing in the vertically transmitted Calyptogena symbionts.     

Can maternally inherited endosymbionts adapt to a novel host? Direct costs of Spiroplasma infection,but not vertical transmission efficiency,evolve rapidly after horizontal transfer into D. melanogaster

Maternally inherited symbionts are common in arthropods and many have important roles in host adaptation. The observation that specific symbiont lineages infect distantly related host species implies new interactions are commonly established by lateral transfer events. However, studies have shown that symbionts often perform poorly in novel hosts. We hypothesized selection on the symbiont may be sufficiently rapid that poor performance in a novel host environment is rapidly ameliorated, permitting symbiont maintenance. Here, we test this prediction for a Spiroplasma strain transinfected into the novel host Drosophila melanogaster. In the generations immediately following transinfection, the symbiont had low transmission efficiency to offspring and imposed severe fitness costs on its host. We observed that effects on host fitness evolved rapidly, being undetectable after 17 generations in the novel host, whereas vertical transmission efficiency was poorly responsive over this period. Our results suggest that long-term symbiosis may more readily be established in cases where symbionts perform poorly in just one aspect of symbiosis.     

Candidatus Sodalis melophagi sp. nov.: phylogenetically independent comparative model to the tsetse fly symbiont Sodalis glossinidius

Bacteria of the genus Sodalis live in symbiosis with various groups of insects. The best known member of this group, a secondary symbiont of tsetse flies Sodalis glossinidius, has become one of the most important models in investigating establishment and evolution of insect-bacteria symbiosis. It represents a bacterium in the early/intermediate state of the transition towards symbiosis, which allows for exploring such interesting topics as: usage of secretory systems for entering the host cell, tempo of the genome modification, and metabolic interaction with a coexisting primary symbiont. In this study, we describe a new Sodalis species which could provide a useful comparative model to the tsetse symbiont. It lives in association with Melophagus ovinus, an insect related to tsetse flies, and resembles S. glossinidius in several important traits. Similar to S. glossinidius, it cohabits the host with another symbiotic bacterium, the bacteriome-harbored primary symbiont of the genus Arsenophonus. As a typical secondary symbiont, Candidatus Sodalis melophagi infects various host tissues, including bacteriome. We provide basic morphological and molecular characteristics of the symbiont and show that these traits also correspond to the early/intermediate state of the evolution towards symbiosis. Particularly, we demonstrate the ability of the bacterium to live in insect cell culture as well as in cell-free medium. We also provide basic characteristics of type three secretion system and using three reference sequences (16 S rDNA, groEL and spaPQR region) we show that the bacterium branched within the genus Sodalis, but originated independently of the two previously described symbionts of hippoboscoids. We propose the name Candidatus Sodalis melophagi for this new bacterium.     

The specificity of Burkholderia symbionts in the social amoeba farming symbiosis: Prevalence,species, genetic and phenotypic diversity

The establishment of symbioses between eukaryotic hosts and bacterial symbionts in nature is a dynamic process. The formation of such relationships depends on the life history of both partners. Bacterial symbionts of amoebae may have unique evolutionary trajectories to the symbiont lifestyle, because bacteria are typically ingested as prey. To persist after ingestion, bacteria must first survive phagocytosis. In the social amoeba Dictyostelium discoideum, certain strains of Burkholderia bacteria are able to resist amoebal digestion and maintain a persistent relationship that includes carriage throughout the amoeba's social cycle that culminates in spore formation. Some Burkholderia strains allow their host to carry other bacteria, as food. This carried food is released in new environments in a trait called farming. To better understand the diversity and prevalence of Burkholderia symbionts and the traits they impart to their amoebae hosts, we first screened 700 natural isolates of D. discoideum and found 25% infected with Burkholderia. We next used a multilocus phylogenetic analysis and identified two independent transitions by Burkholderia to the symbiotic lifestyle. Finally, we tested the ability of 38 strains of Burkholderia from D. discoideum, as well as strains isolated from other sources, for traits relevant to symbiosis in D. discoideum. Only D. discoideum native isolates belonging to the Burkholderia agricolaris, B. hayleyella, and B. bonniea species were able to form persistent symbiotic associations with D. discoideum. The Burkholderia–Dictyostelium relationship provides a promising arena for further studies of the pathway to symbiosis in a unique system.     

Genomic signatures of obligate host dependence in the luminous bacterial symbiont of a vertebrate

The majority of bacteria engaged in bioluminescent symbiosis are environmentally acquired and facultatively symbiotic. A few enigmatic bioluminescent symbionts have not been successfully cultured, which has led to speculation that they may be obligately dependent on their hosts. Here, we report the draft genome of the uncultured luminous symbiont of an anomalopid flashlight fish, ‘Candidatus Photodesmus katoptron’. The genome of the anomalopid symbiont is reduced by 80% compared with close relatives and lacks almost all genes necessary for amino acid synthesis and for metabolism of energy sources other than glucose, supporting obligate dependence on the host for growth. ‘Candidatus Photodesmus katoptron’ is the first described obligate mutualistic symbiont of a vertebrate. Unlike most other obligate mutualists, the anomalopid symbiont genome has retained complete pathways for chemotaxis and motility as well as most genes involved in cell wall production, consistent with the hypothesis that these bacteria may be transmitted environmentally during an extra‐host phase.     

Buchnera has changed flatmate but the repeated replacement of co‐obligate symbionts is not associated with the ecological expansions of their aphid hosts

Symbiotic associations with bacteria have facilitated important evolutionary transitions in insects and resulted in long‐term obligate interactions. Recent evidence suggests that these associations are not always evolutionarily stable and that symbiont replacement, and/or supplementation of an obligate symbiosis by an additional bacterium, has occurred during the history of many insect groups. Yet, the factors favouring one symbiont over another in this evolutionary dynamic are not well understood; progress has been hindered by our incomplete understanding of the distribution of symbionts across phylogenetic and ecological contexts. While many aphids are engaged into an obligate symbiosis with a single Gammaproteobacterium, Buchnera aphidicola, in species of the Lachninae subfamily, this relationship has evolved into a ‘ménage à trois’, in which Buchnera is complemented by a cosymbiont, usually Serratia symbiotica. Using deep sequencing of 16S rRNA bacterial genes from 128 species of Cinara (the most diverse Lachninae genus), we reveal a highly dynamic dual symbiotic system in this aphid lineage. Most species host both Serratia and Buchnera but, in several clades, endosymbionts related to Sodalis, Erwinia or an unnamed member of the Enterobacteriaceae have replaced Serratia. Endosymbiont genome sequences from four aphid species confirm that these coresident symbionts fulfil essential metabolic functions not ensured by Buchnera. We further demonstrate through comparative phylogenetic analyses that cosymbiont replacement is not associated with the adaptation of aphids to new ecological conditions. We propose that symbiont succession was driven by factors intrinsic to the phenomenon of endosymbiosis, such as rapid genome deterioration or competitive interactions between bacteria with similar metabolic capabilities.