Reinventing the Wheel and Making It Round Again: Evolutionary Convergence in Buchnera-Serratia Symbiotic Consortia between the Distantly Related Lachninae Aphids Tuberolachnus salignus and Cinara cedri

Abstract

Virtually all aphids (Aphididae) harbor Buchnera aphidicola as an obligate endosymbiont to compensate nutritional deficiencies arising from their phloem diet. Many species within the Lachninae subfamily seem to be consistently associated also with Serratia symbiotica We have previously shown that both Cinara (Cinara) cedri and Cinara (Cupressobium) tujafilina (Lachninae: Eulachnini tribe) have indeed established co-obligate associations with both Buchnera and S. symbiotica However, while Buchnera genomes of both Cinara species are similar, genome degradation differs greatly between the two S. symbiotica strains. To gain insight into the essentiality and degree of integration of S. symbiotica within the Lachninae, we sequenced the genome of both Buchnera and S. symbiotica endosymbionts from the distantly related aphid Tuberolachnus salignus (Lachninae: Tuberolachnini tribe). We found a striking level of similarity between the endosymbiotic system of this aphid and that of C. cedri In both aphid hosts, S. symbiotica possesses a highly reduced genome and is found exclusively intracellularly inside bacteriocytes. Interestingly, T. salignus' endosymbionts present the same tryptophan biosynthetic metabolic complementation as C. cedri's, which is not present in C. tujafilina's. Moreover, we corroborate the riboflavin-biosynthetic-role take-over/rescue by S. symbiotica in T. salignus, and therefore, provide further evidence for the previously proposed establishment of a secondary co-obligate endosymbiont in the common ancestor of the Lachninae aphids. Finally, we propose that the putative convergent split of the tryptophan biosynthetic role between Buchnera and S. symbiotica could be behind the establishment of S. symbiotica as an obligate intracellular symbiont and the triggering of further genome degradation.

Keywords: Buchnera aphidicola; Lachninae; Serratia symbiotica; aphid endosymbiont; co-obligate; symbiont settlement.

Fig. 1.—

Buchnera phylogenomic reconstruction, genetic-repertoire reduction in the Lachninae and convergent loss of tryptophan biosynthetic genes. (A) Bayesian phylogenomic reconstruction and divergence time estimates between Tuberolachnus salignus–Cinara cedri, and C. tujafilina–C. cedri. Subfamily names are displayed in bold lettering. Mean divergence time estimates are shown in gray under the subfamily or genus clade name. Erwinia spp. and Pantoea ananatis are the outgroups. As all posterior probabilities were equal to 1, they have been excluded from the tree (B) Venn-like diagram displaying the results of the clustering of orthologous proteins. The Lachninae and Aphidinae are represented as pangenomes reconstructed from currently available sequences. (C) Genetic maps of the chromosomal trp genes of different Buchnera strains displaying the convergent loss of the trpD, trpC, trpB, and trpA genes in T. salignus and C. cedri. Nonconserved genes among all strains are displayed in white, while the conserved ones are displayed in different coloring. The Aphidinae plot was done using Buchnera strain APS from the aphid Acyrthosiphon pisum. It was chosen as it would represent the ancestral gene order and content for the Aphidini–Macrosiphini common ancestor (shared by all Aphidinae but Buchnera from Aphis glycines, which has lost both the sohB and topA genes).

Fig. 2.—

Serratia symbiotica localization in Tuberolachnus salignus bacteriomes of early embryos. Whole-mount fluorescence in situ hybridization of early T. salignus embryos using 16S rRNA-directed probes. (Left) Eubacterial staining using EUB338 (Amann et al. 1990) 6-FAM labeled probe. (Center) S. symbiotica staining using STs (see Materials and Methods) DY-405 labeled probe. (Right) Merged image of both eubacterial and S. symbiotica staining, showing double-labeling of S. symbiotica and single-labeling of Buchnera with eubacterial probe in bacteriocytes of T. salignus in (Bottom) early and (Top) later embryos.

Fig. 3.—

Serratia phylogenomic reconstruction, gene-order rearrangements and shared genetic repertoire between STs and SCc. (A) Bayesian phylogenomic reconstruction of different Serratia strains using Yersinia pestis strain CO92 as outgroup. Serratia symbiotica forms a monophyletic clade sister to the clade mainly composed of S. marcescens. Strain names are shown after species name in gray. Asterisks at nodes stand for a posterior probability equal to 1 (B) On the left, circular plot displaying the chromosomes of STs and SCc. From outermost to innermost ring, the features on the direct strand, the reverse strand, and GC-skew plot. Lines going from one genome to another represent orthologous genes in direct (orange) or reverse (black) orientation. On the right, Venn-like diagram displaying the shared (core) and unshared protein-coding genes between STs and SCc. (C) Minimum number of rearrangement phylogeny for Serratia strain as calculated by MGR. Numbers on top of branches indicate the number of inferred rearrangements undergone in each branch. NR, number of rearrangements. (D) Genetic maps of the chromosomal trp genes of different Serratia strains displaying the convergent loss of the trpE and trpG genes in Tuberolachnus salignus and Cinara cedri. Dendogram on the left displays phylogenetic relationships of the aphid hosts associated to each S. symbiotica strain. Nonconserved genes among all strains are displayed in white, while the conserved ones are displayed in different coloring. The trp locus of STs is marked with a “(-)” to indicate it has been reverse-complemented (from its original orientation in the genome) to facilitate comparison.

Fig. 4.—

Metabolic reconstruction of Tuberolachnus Salignus’s endosymbiotic consortium. Metabolic reconstruction of (A) Buchnera and (B) Serratia symbiotica of T. salignus. Intact pathways are represented with solid black lines, and unclear ones (missing a specific gene or having it pseudogenized by a frameshift) with solid gray lines. Importers are displayed using green ovals, while exporters and exporters/importers are displayed using blue ovals. The name inside each oval states the family/superfamily they belong to (following TCDB’s classification [Saier et al. 2014]), otherwise the protein name is used. Essential and nonessential amino acids are shown in red and purple lettering, respectively, while cofactors and vitamins are shown in blue. Blurred compounds represent those for which biosynthesis or import cannot be accounted for based on genomic data. Blurred transporters represent those for which a part of the transporter is missing, therefore recently pseudogenized.

Fig. 5.—

Proposed metabolic complementations found in the obligate endosymbiotic systems of different aphids and riboflavin biosynthesis take-over/rescue by Serratia symbiotica in the Lachninae. Diagram representing the proposed metabolic complementations in the biosynthesis of tryptophan and biotin in the currently available endosymbiotic systems of aphids as well as the riboflavin biosynthesis take-over/rescue by S. symbiotica in the endosymbiotic systems of the Lachninae aphids. The gene names are used as column names, while the abbreviations for the different endosymbiotic bacteria are used as row names, except for Aphididae last common ancestor (ALCA). Asterisks after gene names indicate a putative alternative enzyme could be performing the enzymatic function.

Fig. 6.—

Proposed evolutionary scenarios for the establishment of Serratia symbiotica as a co-obligate endosymbiont in currently sequenced Lachninae aphids. Dendogram representing the evolutionary history of Buchnera within aphids and the proposed (A) convergent and (B) nonconvergent scenarios for the establishment of S. symbiotica as a co-obligate endosymbiont in the Lachninae. Blue and red lines represent Buchnera and S. symbiotica, respectively. The blue diagonal lines represent losses in Buchnera, while the red diagonal lines represent those in S. symbiotica. Names on top and below of branches indicate the aphid familiar name (bold) and tribal name. Scale bar at the bottom represents timing in Ma for the branching of the different aphids. IN, infection events; Replace, symbiont replacement events; GRed, Genome reduction events; trpEGloss, trpEG gene-loss events.