16S rRNA gene sequences of gut symbiotic bacteria of pentatomid stinkbugs
From the midgut symbiotic organs of all 143 stinkbug individuals, representing 28 genera and 35 species, bacterial 16S rRNA gene was subjected to PCR amplification, cloning and sequencing. For each sample, five clones were sequenced and all yielded the same sequence, indicating monosymbiotic status of pentatomid stinkbugs in general. Within the same species, the sequences were either completely identical (most of the species) or nearly identical (ex. Alcimocoris japonensis > 99.86%; Dybowskyia reticulate > 99.86%; Glaucias subpunctatus > 99.86%; Gonopsis affinis > 99.93%; Graphosoma rubrolineatum > 99.93%) (Additional file 1). Within the same genus, sequences from different species tended to show high similarities to each other (ex. Homalogonia spp. 98.64%; Menida spp. 93.89–98.10%; Pentatoma spp. 98.29%; Scotinophara spp. 97.69–98.23%).
Molecular phylogenetic analysis of gut symbiotic bacteria of pentatomid stinkbugs
In addition to the 16S rRNA gene sequences of the gut symbiotic bacteria of the pentatomid stinkbugs determined in this study, we retrieved already-published 16S rRNA gene sequences of gut symbiotic bacteria of pentatomid stinkbugs [13–15, 20–22, 26, 28] (Additional file 2), those of gut symbiotic bacteria of other stinkbugs representing the families Scutelleridae, Cydnidae, Parastrachiidae, Acanthosomatidae, Plataspidae and Urostylididae [15, 18, 25, 37–41] (Additional file 2), and those of closely-related free-living bacteria [42–52] (Additional file 2). Molecular phylogenetic analysis revealed that the gut symbionts of the pentatomid stinkbugs were all placed within the Enterobacteriaceae of the γ-Proteobacteria (Fig. 1). Of these, the gut symbionts of Scotinophara spp. constituted a distinct basal clade, clustering with the gut symbionts of Edessa spp. and the genome-reduced gut symbiont, Candidatus Benitsuchiphilus tojoi, of the stinkbug family Parastrachiidae. The other stinkbug gut symbionts formed a large and coherent cluster together with such free-living γ-proteobacteria as Pantoea, Enterobacter, Erwinia, etc. Among them, the gut symbionts of Nezara spp. clustered with the genome-reduced gut symbionts of other stinkbug families, Candidatus Ishikawaella capsulata of the Plataspidae, Candidatus Rosenkranzia clausaccus of the Acanthosomatidae, and Candidatus Tachikawaea gelatinosa of the Urostylididae. Outside of this cluster were placed the gut symbiont of Palomena angulosa and the gut symbiont of a cydnid stinkbug, and further outside of them were placed Escherichia coli and allied free-living γ-proteobacteria (Fig. 1).
Intraspecific uniformity and exceptional diversity of gut symbiotic bacteria among pentatomid stinkbugs
As described above, bacterial 16S rRNA gene sequences obtained from different individuals of the same species were completely or nearly identical across different populations for most of the pentatomid species examined (Fig. 1). It should be noted, however, that several exceptional cases were observed in Plautia stali, Axiagastus rosmarus and Carbula crassiventris, as reported in a previous study [22], wherein different individuals and populations of the same pentatomid species may be associated with distinct bacterial symbionts (Fig. 1).
Intrageneric coherence and diversity of gut symbiotic bacteria among pentatomid stinkbugs
In the phylogeny, the gut symbiotic bacteria of different pentatomid species belonging to the same genus tended to be closely related to each other (ex. Homalogonia spp., Menida spp., Nezara spp., Pentatoma spp., Scotinophara spp., etc.) (Fig. 1), as had been recognized in previous studies (ex. Chlorochroa spp., Eurydema spp., Edessa spp., etc.) [13, 15, 20]. On the other hand, some pentatomid species belonging to the same genus were associated with phylogenetically distinct gut symbiotic bacteria (ex. Carbula abbreviata vs. C. crassiventris, Euschistus heros vs. E. sp., Plautia stali vs. P. splendens, etc.) [13, 20–22] (Fig. 1).
Multiple evolutionary origins of gut symbiotic bacteria among pentatomid stinkbugs
These phylogenetic patterns indicate that the gut symbiotic bacteria are polyphyletic in the stinkbug family Pentatomidae, as has been suggested in previous studies [13, 20, 22, 23]. Among diverse pentatomid species, presumably, their gut symbiotic bacteria have evolved in a dynamic manner, repeatedly acquired from environmental bacteria, horizontally transferred from different stinkbug species, and/or replacing pre-existing symbiotic bacteria. For further understanding of the dynamic evolutionary processes in detail, molecular phylogenetic analysis of the host stinkbugs is also needed, which should be pursued in future studies.
Molecular evolutionary rate and nucleotide composition among gut symbiotic bacteria of pentatomid stinkbugs
In the phylogeny, the gut symbiotic bacteria conserved in their host genera (ex. those of Nezara spp., Menida spp., Homalogonia spp., Pentatoma spp., Scotinophara spp., etc.) tended to exhibit elongated branches, whereas the gut symbiotic bacteria promiscuously intermingled with free-living bacteria tended to exhibit very short branches (Fig. 1). Relative rate tests of the 16S rRNA gene sequences confirmed these observations: molecular evolutionary rates were remarkably accelerated (K1/K2 values 1.5 to 2.4, P values < 10−5) in the gut symbiotic bacteria of Menida spp., Homalogonia spp., and Pentatoma spp., and other long-branched gut symbiont lineages associated with a variety of pentatomid stinkbugs including Gonopsis affinis, Piezodorus hybneri, Niphe elongata, Erthesina fullo, Hermolaus amurensis, Halyomorpha halys, and Lelia decapunctata; molecular evolutionary rates were moderately accelerated (K1/K2 values 1.1 to 1.6, P values 0.001–0.04) in the gut symbiotic bacteria of Scotinophara spp., Nezara spp., Palomena angulosa, Rhynchocoris humeralis, Glaucias subpunctatus and Carpocoris purpureipennis; and no accelerated molecular evolution was detected (K1/K2 values around 1, P values > 0.05) in the gut symbiotic bacteria of promiscuous type from Chalazonotum ishiharai, Bathycoelia indica, Vitellus orientalis, Carbula abbreviata, Alcimocoris japonensis, Laprius gastricus, Aelia fieberi, Graphosoma rubrolineatum, Dybowskyia reticulata, Agonoscelis femoralis, Rubiconia intermedia, and Paraholcostethus breviceps (Additional file 3; Fig. 1). Notably, AT contents of the 16S rRNA gene sequences exhibited overall correlation with the categories: 46.3 to 49.5% for the highly accelerated sequences; 44.3 to 45.7% for the moderately accelerated sequences; and 43.9 to 44.9% for the sequences exhibiting no acceleration (Additional file 1).
Molecular evolutionary rate and nucleotide composition among gut symbiotic bacteria of other stinkbug groups and allied free-living γ-proteobacteria
Furthermore, 16S rRNA gene sequences of gut symbiotic bacteria of stinkbugs representing the Pentatomidae and other families published in previous studies, and also those of allied free-living γ-proteobacteria, all of which are members of the Enterobacteriaceae (Fig. 1), were similarly subjected to molecular evolutionary analyses (Additional file 4). Relative rate tests revealed the following patterns: in the genome-reduced and co-speciating gut symbiotic bacteria from the stinkbug families Plataspidae, Acanthosomatidae and Urostylididae [11, 12, 18], molecular evolutionary rates were extremely accelerated (K1/K2 values 3.4–5.0, P values < 10−7) with extremely high AT contents (>50.3%); in the genome-reduced gut symbiotic bacterium from the stinkbug family Parastrachiidae [37], a highly accelerated molecular evolutionary rate was observed (K1/K2 value 1.5, P value < 10−4) with extremely high AT content (51.0%); in the uncultivable gut symbiotic bacteria of pentatomid stinkbugs, some species (Eurydema spp. and Halyomorpha halys) [15, 28] exhibited remarkably high molecular evolutionary rates (K1/K2 values 1.8–2.0, P values < 10−7) with relatively high AT contents (48.1–48.6%), other species (Nezara viridula and Plautia stali type A) [14, 22] showed moderately high molecular evolutionary rates (K1/K2 values 1.1–1.6, P values around 0.02) with low AT contents (44.7–45.7%), and other species (Plautia splendens and Plautia stali B type) [21, 22] entailed no accelerated molecular evolution (K1/K2 values around 1.1, P values > 0.05) with low AT contents (43.9–44.3%); in the cultivable gut symbiotic bacteria of pentatomid stinkbugs (Axiagastus rosmarus C-E types and Plautia stali C-F types) [22], no accelerated molecular evolution was detected (K1/K2 values around 1.0, P values 0.3–1.0) with low AT contents (44.4–44.9%).
Relationships between molecular evolutionary rate, nucleotide composition, genome size, and cultivability of gut symbiotic bacteria of pentatomid stinkbugs
Previous studies have revealed that, through intimate and long-lasting host-symbiont co-evolution, obligate endocellular symbiotic bacteria of diverse insects tend to exhibit a characteristic genomic syndrome, including accelerated molecular evolution, AT-biased nucleotide composition, massive gene loss, and reduced genome size. This has been ascribed to stable and nutrition-rich endocellular environment and to strong population bottlenecks associated with the lifestyles of vertically-transmitted symbiotic bacteria [53–56]. Recently, remarkable genome reduction has also been identified among extracellular gut symbiotic bacteria of various stinkbugs [11, 12, 17, 18, 22, 28, 37, 41], elucidating that endocellularity cannot be the main driver of the symbiosis-associated reductive genome evolution [7, 11, 57]. In this study, a wide variety of gut symbiotic bacteria of pentatomid stinkbugs at different evolutionary stages, ranging from cultivable through uncultivable to genome-reduced, were found together with free-living bacteria and diverse gut symbiotic bacteria of other stinkbug families (Fig. 1), which provided an ideal opportunity to comparatively analyze the evolutionary processes underpinning the gut symbiotic associations. For that purpose, we focused on “the stinkbug gut symbiont clade subjected to comparative evolutionary analyses” (see Fig. 1 on the right side), defined an outgroup of the clade (Yersinia pestis KIM10+), and calculated the value K (genetic distance from the outgroup) for each member of the clade (Additional file 5), which enabled comparison of molecular evolutionary rates across all members of the clade. Figure 2a shows the relationship between K values and AT contents within the clade, indicating a fairly strong positive correlation (R = 0.955). On the plot, the gut symbionts of diverse pentatomid stinkbugs (black, red and blue) were scattered from the lower left (= low evolutionary rate and low AT content) to the upper right (= high evolutionary rate and high AT content). When uncultivable and genome-reduced gut symbionts of other stinkbug families (green) and free-living γ-proteobacteria (grey) were analyzed on the same plot, the former tended to be located on the upper right extreme (= highest evolutionary rate and highest AT content), whereas the latter were concentrated on the lower left extreme (= lowest evolutionary rate and lowest AT content). For some of the gut symbionts of pentatomid stinkbugs, we examined whether they are cultivable or not [14, 15, 21, 22, 28] (see Additional file 2). Interestingly, the uncultivable gut symbionts of pentatomid stinkbugs (red) tended to be distributed from the middle to the lower left, whereas the cultivable gut symbionts of pentatomid stinkbugs (blue) were concentrated on the lower left extreme as the free-living γ-proteobacteria. Figure 2b shows the relationship between K values and genome sizes within the clade, exhibiting a strong negative correlation (R = −0.847). In the plot, the uncultivable and genome-reduced gut symbionts of other stinkbug families (green) were located to the lower right extreme, the cultivable gut symbionts of pentatomid stinkbugs (blue) and the free-living γ-proteobacteria (grey) were concentrated to the upper left extreme, and the uncultivable gut symbionts of pentatomid stinkbugs (red) exhibited an intermediate distribution. Figure 2c shows the relationship between AT contents and K values within the clade, exhibiting a similar pattern to Fig. 2b with a strong negative correlation (R = −0.778).
Multiple evolutionary origins and different evolutionary stages of gut symbiotic bacteria in the Pentatomidae
These results collectively highlight the following dynamic evolutionary aspects of the gut symbiotic bacteria in the stinkbug family Pentatomidae: (i) each of the pentatomid host species examined in this study is monosymbiotically associated with a specific γ-proteobacterium within the midgut symbiotic organ; (ii) gut symbiotic bacteria are of multiple evolutionary origins in the Pentatomidae, presumably originating from free-living γ-proteobacteria belonging to the Enterobacteriaceae repeatedly; (iii) some are of relatively old origin and stably maintained across closely related host species, some are of relatively recent origin and stably associated with a specific host species, and others are of promiscuous nature whose infections may be polymorphic within and/or between host species; (iv) stable gut symbiotic bacteria tend to exhibit uncultivability, accelerated molecular evolution, AT-biased nucleotide composition and reduced genome; (v) by contrast, promiscuous gut symbiotic bacteria tend to exhibit cultivability, non-accelerated molecular evolution, unbiased nucleotide composition and non-reduced genome, which are similar to free-living environmental bacteria; and (vi) a number of gut symbiont lineages at different evolutionary stages of different symbiotic intimacy coexist within the stinkbug family Pentatomidae.