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Parallel Evolution in the Integration of a Co-obligate Aphid Symbiosis

2020; Elsevier BV; Volume: 30; Issue: 10 Linguagem: Inglês

10.1016/j.cub.2020.03.011

1879-0445

David Monnin, Raphaella Jackson, E. Toby Kiers, Marie E. Bunker, Jacintha Ellers, Lee M. Henry,

Insect and Arachnid Ecology and Behavior

•Aphids have independently evolved dependence on Serratia symbiotica at least 4 times•The integration of the new co-obligate symbiont proceeds in a predictable manner•Loss of the riboflavin and peptidoglycan pathways in Buchnera leads to co-dependence•Amino acid synthesis is taken over by Serratia in a second phase of complementarity Insects evolve dependence—often extreme—on microbes for nutrition. This includes cases in which insects harbor multiple endosymbionts that function collectively as a metabolic unit [1McCutcheon J.P. McDonald B.R. Moran N.A. Convergent evolution of metabolic roles in bacterial co-symbionts of insects.Proc. Natl. Acad. Sci. USA. 2009; 106: 15394-15399Crossref PubMed Scopus (232) Google Scholar, 2McCutcheon J.P. von Dohlen C.D. An interdependent metabolic patchwork in the nested symbiosis of mealybugs.Curr. Biol. 2011; 21: 1366-1372Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 3Takiya D.M. Tran P.L. Dietrich C.H. Moran N.A. Co-cladogenesis spanning three phyla: leafhoppers (Insecta: Hemiptera: Cicadellidae) and their dual bacterial symbionts.Mol. Ecol. 2006; 15: 4175-4191Crossref PubMed Scopus (130) Google Scholar, 4Wu D. Daugherty S.C. Van Aken S.E. Pai G.H. Watkins K.L. Khouri H. Tallon L.J. Zaborsky J.M. Dunbar H.E. Tran P.L. et al.Metabolic complementarity and genomics of the dual bacterial symbiosis of sharpshooters.PLoS Biol. 2006; 4: e188Crossref PubMed Scopus (309) Google Scholar, 5Rao Q. Rollat-Farnier P.A. Zhu D.T. Santos-Garcia D. Silva F.J. Moya A. Latorre A. Klein C.C. Vavre F. Sagot M.F. et al.Genome reduction and potential metabolic complementation of the dual endosymbionts in the whitefly Bemisia tabaci.BMC Genomics. 2015; 16: 226Crossref PubMed Scopus (57) Google Scholar]. How do these dependences originate [6Douglas A.E. How multi-partner endosymbioses function.Nat. Rev. Microbiol. 2016; 14: 731-743Crossref PubMed Scopus (66) Google Scholar], and is there a predictable sequence of events leading to the integration of new symbionts? While co-obligate symbioses, in which hosts rely on multiple nutrient-provisioning symbionts, have evolved numerous times across sap-feeding insects, there is only one known case in aphids, involving Buchnera aphidicola and Serratia symbiotica in the Lachninae subfamily [7Pérez-Brocal V. Gil R. Ramos S. Lamelas A. Postigo M. Michelena J.M. Silva F.J. Moya A. Latorre A. A small microbial genome: the end of a long symbiotic relationship?.Science. 2006; 314: 312-313Crossref PubMed Scopus (257) Google Scholar, 8Lamelas A. Pérez-Brocal V. Gómez-Valero L. Gosalbes M.J. Moya A. Latorre A. Evolution of the secondary symbiont "Candidatus serratia symbiotica" in aphid species of the subfamily lachninae.Appl. Environ. Microbiol. 2008; 74: 4236-4240Crossref PubMed Scopus (60) Google Scholar, 9Manzano-Marín A. Simon J.C. Latorre A. 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.Genome Biol. Evol. 2016; 8: 1440-1458Crossref PubMed Scopus (40) Google Scholar]. Here, we identify three additional independent transitions to the same co-obligate symbiosis in different aphids. Comparing recent and ancient associations allow us to investigate intermediate stages of metabolic and anatomical integration of Serratia. We find that these uniquely replicated evolutionary events support the idea that co-obligate associations initiate in a predictable manner—through parallel evolutionary processes. Specifically, we show how the repeated losses of the riboflavin and peptidoglycan pathways in Buchnera lead to dependence on Serratia. We then provide evidence of a stepwise process of symbiont integration, whereby dependence evolves first. Then, essential amino acid pathways are lost (at ∼30–60 mya), which coincides with the increased anatomical integration of the companion symbiont. Finally, we demonstrate that dependence can evolve ahead of specialized structures (e.g., bacteriocytes), and in one case with no direct nutritional basis. More generally, our results suggest the energetic costs of synthesizing nutrients may provide a unified explanation for the sequence of gene losses that occur during the evolution of co-obligate symbiosis. Insects evolve dependence—often extreme—on microbes for nutrition. This includes cases in which insects harbor multiple endosymbionts that function collectively as a metabolic unit [1McCutcheon J.P. McDonald B.R. Moran N.A. Convergent evolution of metabolic roles in bacterial co-symbionts of insects.Proc. Natl. Acad. Sci. USA. 2009; 106: 15394-15399Crossref PubMed Scopus (232) Google Scholar, 2McCutcheon J.P. von Dohlen C.D. An interdependent metabolic patchwork in the nested symbiosis of mealybugs.Curr. Biol. 2011; 21: 1366-1372Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 3Takiya D.M. Tran P.L. Dietrich C.H. Moran N.A. Co-cladogenesis spanning three phyla: leafhoppers (Insecta: Hemiptera: Cicadellidae) and their dual bacterial symbionts.Mol. Ecol. 2006; 15: 4175-4191Crossref PubMed Scopus (130) Google Scholar, 4Wu D. Daugherty S.C. Van Aken S.E. Pai G.H. Watkins K.L. Khouri H. Tallon L.J. Zaborsky J.M. Dunbar H.E. Tran P.L. et al.Metabolic complementarity and genomics of the dual bacterial symbiosis of sharpshooters.PLoS Biol. 2006; 4: e188Crossref PubMed Scopus (309) Google Scholar, 5Rao Q. Rollat-Farnier P.A. Zhu D.T. Santos-Garcia D. Silva F.J. Moya A. Latorre A. Klein C.C. Vavre F. Sagot M.F. et al.Genome reduction and potential metabolic complementation of the dual endosymbionts in the whitefly Bemisia tabaci.BMC Genomics. 2015; 16: 226Crossref PubMed Scopus (57) Google Scholar]. How do these dependences originate [6Douglas A.E. How multi-partner endosymbioses function.Nat. Rev. Microbiol. 2016; 14: 731-743Crossref PubMed Scopus (66) Google Scholar], and is there a predictable sequence of events leading to the integration of new symbionts? While co-obligate symbioses, in which hosts rely on multiple nutrient-provisioning symbionts, have evolved numerous times across sap-feeding insects, there is only one known case in aphids, involving Buchnera aphidicola and Serratia symbiotica in the Lachninae subfamily [7Pérez-Brocal V. Gil R. Ramos S. Lamelas A. Postigo M. Michelena J.M. Silva F.J. Moya A. Latorre A. A small microbial genome: the end of a long symbiotic relationship?.Science. 2006; 314: 312-313Crossref PubMed Scopus (257) Google Scholar, 8Lamelas A. Pérez-Brocal V. Gómez-Valero L. Gosalbes M.J. Moya A. Latorre A. Evolution of the secondary symbiont "Candidatus serratia symbiotica" in aphid species of the subfamily lachninae.Appl. Environ. Microbiol. 2008; 74: 4236-4240Crossref PubMed Scopus (60) Google Scholar, 9Manzano-Marín A. Simon J.C. Latorre A. 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.Genome Biol. Evol. 2016; 8: 1440-1458Crossref PubMed Scopus (40) Google Scholar]. Here, we identify three additional independent transitions to the same co-obligate symbiosis in different aphids. Comparing recent and ancient associations allow us to investigate intermediate stages of metabolic and anatomical integration of Serratia. We find that these uniquely replicated evolutionary events support the idea that co-obligate associations initiate in a predictable manner—through parallel evolutionary processes. Specifically, we show how the repeated losses of the riboflavin and peptidoglycan pathways in Buchnera lead to dependence on Serratia. We then provide evidence of a stepwise process of symbiont integration, whereby dependence evolves first. Then, essential amino acid pathways are lost (at ∼30–60 mya), which coincides with the increased anatomical integration of the companion symbiont. Finally, we demonstrate that dependence can evolve ahead of specialized structures (e.g., bacteriocytes), and in one case with no direct nutritional basis. More generally, our results suggest the energetic costs of synthesizing nutrients may provide a unified explanation for the sequence of gene losses that occur during the evolution of co-obligate symbiosis. Sap-feeding insects have provided elegant case studies of the evolution of co-obligate symbioses, whereby organisms harbor multiple endosymbionts that function collectively as a metabolic unit. These include species of mealybugs that depend on endosymbionts, which in turn harbor their own endosymbionts, and cicadas, in which one symbiont has fragmented into distinct but interdependent lineages [1McCutcheon J.P. McDonald B.R. Moran N.A. Convergent evolution of metabolic roles in bacterial co-symbionts of insects.Proc. Natl. Acad. Sci. USA. 2009; 106: 15394-15399Crossref PubMed Scopus (232) Google Scholar, 2McCutcheon J.P. von Dohlen C.D. An interdependent metabolic patchwork in the nested symbiosis of mealybugs.Curr. Biol. 2011; 21: 1366-1372Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 3Takiya D.M. Tran P.L. Dietrich C.H. Moran N.A. Co-cladogenesis spanning three phyla: leafhoppers (Insecta: Hemiptera: Cicadellidae) and their dual bacterial symbionts.Mol. Ecol. 2006; 15: 4175-4191Crossref PubMed Scopus (130) Google Scholar, 4Wu D. Daugherty S.C. Van Aken S.E. Pai G.H. Watkins K.L. Khouri H. Tallon L.J. Zaborsky J.M. Dunbar H.E. Tran P.L. et al.Metabolic complementarity and genomics of the dual bacterial symbiosis of sharpshooters.PLoS Biol. 2006; 4: e188Crossref PubMed Scopus (309) Google Scholar, 5Rao Q. Rollat-Farnier P.A. Zhu D.T. Santos-Garcia D. Silva F.J. Moya A. Latorre A. Klein C.C. Vavre F. Sagot M.F. et al.Genome reduction and potential metabolic complementation of the dual endosymbionts in the whitefly Bemisia tabaci.BMC Genomics. 2015; 16: 226Crossref PubMed Scopus (57) Google Scholar]. What processes drive multiple microbial species to join into co-obligate symbioses [6Douglas A.E. How multi-partner endosymbioses function.Nat. Rev. Microbiol. 2016; 14: 731-743Crossref PubMed Scopus (66) Google Scholar], and, more generally, is there a predictable, deterministic sequence of events leading to the genomic and anatomical integration of new symbionts? The aphids are an ideal lineage to study early-stage co-obligate symbioses. The majority of aphid species harbor a single obligate symbiont, Buchnera aphidicola, and a second non-obligate symbiont Serratia symbiotica (hereafter referred to as Buchnera and Serratia, respectively). While Serratia is found at intermediate frequencies in numerous aphid species, the symbiont has transitioned to a co-obligate relationship with Buchnera in the Lachninae subfamily [7Pérez-Brocal V. Gil R. Ramos S. Lamelas A. Postigo M. Michelena J.M. Silva F.J. Moya A. Latorre A. A small microbial genome: the end of a long symbiotic relationship?.Science. 2006; 314: 312-313Crossref PubMed Scopus (257) Google Scholar, 8Lamelas A. Pérez-Brocal V. Gómez-Valero L. Gosalbes M.J. Moya A. Latorre A. Evolution of the secondary symbiont "Candidatus serratia symbiotica" in aphid species of the subfamily lachninae.Appl. Environ. Microbiol. 2008; 74: 4236-4240Crossref PubMed Scopus (60) Google Scholar, 9Manzano-Marín A. Simon J.C. Latorre A. 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.Genome Biol. Evol. 2016; 8: 1440-1458Crossref PubMed Scopus (40) Google Scholar]. Such co-obligate functioning is marked by Buchnera's losing metabolic capabilities, namely the ability to synthesize the essential nutrients riboflavin and, in some species, tryptophan [10Manzano-Marín A. Latorre A. Settling down: the genome of Serratia symbiotica from the aphid Cinara tujafilina zooms in on the process of accommodation to a cooperative intracellular life.Genome Biol. Evol. 2014; 6: 1683-1698Crossref PubMed Scopus (47) Google Scholar]. Our aim was to determine whether (1) other cases of obligate co-dependences have arisxen across the aphids, and (2) to ask whether these transitions followed predictable genomic, metabolic, and anatomical trajectories. Such patterns can provide insight into the evolutionary processes that have led to the genome structure of more ancient multi-partner symbioses [6Douglas A.E. How multi-partner endosymbioses function.Nat. Rev. Microbiol. 2016; 14: 731-743Crossref PubMed Scopus (66) Google Scholar]. Using data on the symbiont prevalence in 131 aphid species from [11Henry L.M. Maiden M.C.J. Ferrari J. Godfray H.C.J. Insect life history and the evolution of bacterial mutualism.Ecol. Lett. 2015; 18: 516-525Crossref PubMed Scopus (90) Google Scholar], we identified species that carry Serratia at a high frequency, and then tested aphid populations in both the United Kingdom and the Netherlands for obligate dependence on the symbiont. We defined species as having evolved obligate reliance on Serratia if (1) all individuals within populations carry the symbiont and (2) they experience a significant fitness reduction when the symbiont is removed. Symbionts that do not meet these criteria are referred to as "facultative," as they are not essential for host survival. We screened for the presence of Serratia using PCR and measured dependence by "curing" individual aphids with antibiotics that selectively removed Serratia without affecting Buchnera, and then counted their total offspring to determine the lifetime fecundity of the aphids in the presence and absence of the symbiont. We identified ubiquitous Serratia symbioses in seven aphid species (Table S1), representing three independent co-obligate transitions in Microlophium carnosum, Aphis urticata, and in the Periphyllus genus. In the Periphyllus genus, Serratia was consistently present in the five species we surveyed, and we confirmed obligate dependence via curing Serratia in both P. hirticornis and P. lyropictus. These data suggest a single transition into an obligate relationship with Serratia at the origins of the Periphyllus genus (see below). Curing had the most dramatic effect in species of the Periphyllus genus, potentially reflecting a longer-term evolutionary association with Serratia (Figure 1A). We confirmed that the antibiotic treatments had no significant effect on the fecundity of our control aphid species Acyrthosiphon pisum, which harbors Serratia as a facultative symbiont, and the uninfected Macrosiphoniella artemisiae (Figures 1A and S1; Data S1A). Likewise, we confirmed with qPCR that the antibiotic treatment reduced Serratia density (Figure 1B; Data S1B), but did not reduce Buchnera density (Figure 1C; Data S1B). We next estimated the origins of obligate dependence on Serratia using deep coverage 16S amplicon sequencing from our field-collected populations and previous data on Serratia associations in aphids [11Henry L.M. Maiden M.C.J. Ferrari J. Godfray H.C.J. Insect life history and the evolution of bacterial mutualism.Ecol. Lett. 2015; 18: 516-525Crossref PubMed Scopus (90) Google Scholar]. First, we found evidence of a more ancient relationship between Serratia and aphids in the Periphyllus genus: amplicon sequencing confirmed that Serratia was absent from Chaitophorus aphids, a sister lineage to the Periphyllus clade (Data S1C). This suggests that dependence on Serratia originated at the divergence of these 2 genera an estimated 63–79 mya (Figures 2 and S2). Second, we found evidence of more recent origins of Serratia obligate dependence (<30 mya) in A. urticata and M. carnosum. Specifically, Serratia was either absent or present only as a facultative infection in some individuals in the species of A. idaei and A. fabae related to A. urticata. Lack of obligate dependence was likewise confirmed in Macrosiphum euphorbiae and A. pisum, related to M. carnosum. We then asked whether evolving obligate dependence on Serratia was associated with a consistent genomic signature in aphids, and more specifically whether Buchnera-Serratia metabolic complementarity originates in a predictable manner across host lineages. We obtained whole-genome sequencing data for M. carnosum, A. urticatasymbiont; aphid; co-obligate;Serratia symbiotica;Buchnera aphidicola; evolution of dependence; metabolic complementation, and three Periphyllus species. We then used previously published data from the Lachninae subfamily (Cinara cedri, C. tujafilina and Tuberolachnus salignus) to compare the gene losses in Buchnera from the four independent transitions into an obligate relationship with Serratia. This included the three new cases of co-obligate dependence identified here, and the previously identified cases in the Lachninae subfamily. Our analysis centered on the pathways and genes involved in essential nutrient provisioning to the host (Data S1D). Specifically, we focused on pathways that have experimental evidence for being essential for the aphid: riboflavin [14Nakabachi A. Ishikawa H. Provision of riboflavin to the host aphid, Acyrthosiphon pisum, by endosymbiotic bacteria, Buchnera.J. Insect Physiol. 1999; 45: 1-6Crossref PubMed Scopus (91) Google Scholar] and essential amino acids [15Douglas A.E. Prosser W.A. Synthesis of the essential amino acid tryptophan in the pea aphid (Acyrthosiphon pisum) symbiosis.J. Insect Physiol. 1992; 38: 565-568Crossref Scopus (172) Google Scholar, 16Douglas A.E. Sulphate utilization in an aphid symbiosis.Insect Biochem. 1988; 18: 599-605Crossref Scopus (61) Google Scholar, 17Febvay G. Liadouze I. Guillaud J. Bonnot G. Analysis of energetic amino acid metabolism in Acyrthosiphon pisum: a multidimensional approach to amino acid metabolism in aphids.Arch. Insect Biochem. Physiol. 1995; 29: 45-69Crossref Scopus (78) Google Scholar, 18Liadouze I. Febvay G. Guillaud J. Bonnot G. Metabolic fate of energetic amino acids in the aposymbiotic pea aphid Acyrthosiphon pisum (Harris) (Homoptera: Aphididae).Symbiosis. 1996; 21: 115-127Google Scholar, 19Brinza L. Viñuelas J. Cottret L. Calevro F. Rahbé Y. Febvay G. Duport G. Colella S. Rabatel A. Gautier C. et al.Systemic analysis of the symbiotic function of Buchnera aphidicola, the primary endosymbiont of the pea aphid Acyrthosiphon pisum.C. R. Biol. 2009; 332: 1034-1049Crossref PubMed Scopus (33) Google Scholar, 20Wilson A.C.C. Ashton P.D. Calevro F. Charles H. Colella S. Febvay G. Jander G. Kushlan P.F. Macdonald S.J. Schwartz J.F. et al.Genomic insight into the amino acid relations of the pea aphid, Acyrthosiphon pisum, with its symbiotic bacterium Buchnera aphidicola.Insect Mol. Biol. 2010; 19: 249-258Crossref PubMed Scopus (147) Google Scholar, 21Shigenobu S. Wilson A.C.C. Genomic revelations of a mutualism: the pea aphid and its obligate bacterial symbiont.Cell. Mol. Life Sci. 2011; 68: 1297-1309Crossref PubMed Scopus (92) Google Scholar]. Of particular interest was the riboflavin pathway in Buchnera, as the loss of this pathway has been hypothesized to trigger the dependence on Serratia in the Lachninae aphids [10Manzano-Marín A. Latorre A. Settling down: the genome of Serratia symbiotica from the aphid Cinara tujafilina zooms in on the process of accommodation to a cooperative intracellular life.Genome Biol. Evol. 2014; 6: 1683-1698Crossref PubMed Scopus (47) Google Scholar]. We found a consistent signature for the loss of the riboflavin pathway of Buchnera in both M. carnosum and aphids in the Periphyllus genus (Figure 3). In M. carnosum, Buchnera is missing one gene, part of the ribD complex, which is essential to the riboflavin pathway. In the Periphyllus genus, by contrast, the full pathway is missing, as it is in the Lachninae subfamily. Previous work in the Lachninae aphids suggests that Buchnera has also lost the capacity to synthesize the amino acid tryptophan in certain species (e.g., C. cedri and T. salignus). We find similar losses in the Periphyllus lineage. Here, the majority of genes in the tryptophan pathway have either been lost or pseudogenized, and orthologous gene copies, which are predicted to serve the same function, have been retained in the Serratia genome (Figure 3). Conversely, the tryptophan pathway has been retained in the Buchnera genomes of both M. carnosum and A. urticata, the more recent co-obligate relationships. This result suggests an advanced stage of functional losses in Buchnera of Periphyllus aphids, further supported by losses in several additional amino acid pathways that also appear to have been taken over by Serratia. In contrast, Buchnera has retained the complete pathways to synthesize all of these essential nutrients in A. urticata. This is surprising, given the consistency of gene losses in Buchnera of M. carnosum, aphids in the Periphyllus genus, and the Lachninae aphids, all which are co-obligately dependent on Serratia. Compared to aphid lineages in which Buchnera is the sole obligate symbiont (A. pisum, Myzus persicae, and Aphis glycines), the co-obligate association of Serratia and Buchnera in A. urticata has only six genes missing in Buchnera, in which there are orthologous gene copies in Serratia. None of the six genes has direct links to essential nutrient pathways (see Table S2 for more detail). This suggests that co-obligate dependence can arise in this system through alternative starting points, including non-nutritional pathways. General genomic features of the different Buchnera strains likewise support the hypothesis that the co-obligate Serratia symbioses found in Lachninae and Periphyllus aphids are more ancient compared to M. carnosum and A. urticata (Figure S3). Both the genome size and GC content of Buchnera are highly reduced in the Lachninae and Periphyllus clades, which is suggestive of a more advanced degree of degradation. Gene redundancies are also indicative of the age of the co-obligate associations. In M. carnosum and A. urticata, the genomes of Serratia and Buchnera still contain a significant number of the same genes involved in synthesizing nutrients that are essential for the host aphid (72.5% and 39.2%, respectively). Conversely, in the Periphyllus lineages, both P. acericola and P. aceris have only 11.5% gene redundancy between the two symbionts. In P. lyropictus, there is a 47.1% overlap. The higher redundancy in P. lyropictus is likely due to Serratia being recently replaced by another Serratia strain within this aphid lineage. As Buchnera-Serratia complementarity could also arise through pathways that are not essential for host nutrition, we investigated genes involved in additional pathways involved in translation, as it underlies essential functions in all bacteria [22Gil R. Silva F.J. Peretó J. Moya A. Determination of the core of a minimal bacterial gene set.Microbiol. Mol. Biol. Rev. 2004; 68: 518-537Crossref PubMed Scopus (402) Google Scholar] and peptidoglycan synthesis, which has been shown to be important in other symbiotic systems [23Bublitz D.C. Chadwick G.L. Magyar J.S. Sandoz K.M. Brooks D.M. Mesnage S. Ladinsky M.S. Garber A.I. Bjorkman P.J. Orphan V.J. McCutcheon J.P. Peptidoglycan Production by an Insect-Bacterial Mosaic.Cell. 2019; 179: 703-712.e7Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar]. We also investigate the pathways to synthesize the precursors chorismate, homoserine, and vitamins B5 and B9, and lipoic acid. The additional B vitamins showed similar patterns of metabolic patchwork to riboflavin. However, genes to complete the pathways are either absent in both symbiont genomes (B5 in A. urticata and the Lachninae) or are missing where Buchnera is the sole symbionts (B9 in A. pisum), so it is unclear whether these vitamins are essential for the symbiosis. Genes involved in translation, chorismate, homoserine, and lipoic acid were for the most part conserved in all of the Buchnera genomes (Data S1E). The pathway for peptidoglycan, however, was entirely lost in the ancient co-obligate Buchnera (Lachninae and Periphyllus aphids). In contrast, only one or two genes were missing in the most recent lineages of M. carnosum and A. urticata. In all cases, the missing genes to synthesize peptidoglycan in Buchnera were retained on the Serratia genomes. This suggests that Serratia may be co-obligate because of its contributing role to the peptidoglycan synthesis of Buchnera, in addition to providing nutrients to the host. The only gene consistently missing in the peptidoglycan pathway from all co-obligate Buchnera, including M. carnosum and A. urticata, was murF. However, it is unclear whether this gene is essential for Buchnera as it is pseudogenized in A. glycines, which is a species not known to host any obligate symbiont other than Buchnera. Lastly, we studied the abundance and localization of symbionts within their host to look for anatomical signatures of co-obligate symbiosis. Likewise, we expected that a greater degree of metabolic reliance on Serratia in the Periphyllus aphids would correspond with greater anatomical integration—for example, through the formation of a specialized organ (bacteriome) to house Serratia. To test this idea, we performed fluorescent in situ hybridization (FISH) using probes specifically targeting Buchnera and Serratia. As predicted, we found increased anatomical integration of Serratia in host lineages corresponding to a greater reliance on Serratia (Figure 4). In the most extreme case, we found that the Periphyllus aphids evolved a large organ (bacteriome) containing numerous bacteriocytes to house Serratia in their abdomens (Figure 4). In contrast to high anatomical integration in the Periphyllus aphids, we found that both M. carnosum and A. urticata exhibit minimal integration of Serratia. In A. urticata, Serratia is localized in a small cluster of relatively large cells (∼4), forming a small bacteriome surrounded by Buchnera-containing bacteriocytes. In M. carnosum, Serratia is the least integrated, with the symbiont being localized in sheath cells surrounding the Buchnera-containing bacteriocytes. This pattern is similar to the one found in A. pisum where Serratia maintains a consistently facultative relationship with its host. We likewise expected Serratia abundance within the aphid to increase as the symbiont takes on a more metabolically demanding role. Here, we used qPCR to quantify the copies of Serratia genomes compared to the host aphid. In line with our predictions, we found a substantial increase in the abundance of Serratia that coincided with its greater metabolic role of synthesizing amino acids. Specifically, we found that the ratio of Serratia to host genome copies increased dramatically to an abundance ratio of 512:1 in P. hirticornis. This is compared to a 3:1 ratio found in the less integrated co-obligate of M. carnosum (Figure 1B). Dependence on multiple co-obligate symbionts has originated numerous times in the evolution of eukaryotes. However, the vast majority of co-obligate symbioses are ancient. Data on recent associations are needed to reveal the evolutionary processes that initiate dependence and provide insight into the intermediate steps leading to the extreme genomic and anatomical integration observed in ancient associations. By comparing ancient and recent associations in aphids, we find strong evidence that the mechanisms initially binding symbiotic partners in obligate relationships occur in a deterministic, predictable manner. Specifically, we find that dependence on Serratia originates through parallel evolutionary trajectories marked by repeated losses of the same nutrient pathways in Buchnera across multiple host lineages. Our genomic and FISH data show stepwise processes of symbiont integration, with the losses of essential amino acid pathways occurring between 30 and 60 million years after the co-obligate symbiosis evolves. This is followed by a second phase of dependence characterized by greater anatomical integration of Serratia in the more ancient obligate partnerships of the Periphyllus genus compared to the more recently adopted co-obligate associations of A. urticata and M. carnosum. Our results provide the first evidence that Buchnera has repeatedly lost the capacity to produce the essential nutrient riboflavin in multiple aphid lineages. In each case in which the pathway to synthesize riboflavin has been lost, Serratia has retained genes to compensate for these metabolic changes in Buchnera. Studies have shown that an aphid's demand for riboflavin is relatively low compared to other nutrients, such as amino acids [14Nakabachi A. Ishikawa H. Provision of riboflavin to the host aphid, Acyrthosiphon pisum, by endosymbiotic bacteria, Buchnera.J. Insect Physiol. 1999; 45: 1-6Crossref PubMed Scopus (91) Google Scholar, 24Sasaki T. Hayashi H. Ishikawa H. Growth and reproduction of the symbiotic and aposymbiotic pea aphids, Acyrthosiphon pisum maintained on artificial diets.J. Insect Physiol. 1991; 37: 749-756Crossref Scopus (78) Google Scholar, 25Russell C.W. Poliakov A. Haribal M. Jander G. van Wijk K.J. Douglas A.E. Matching the supply of bacterial nutrients to the nutritional demand of the animal host.Proc. Biol. Sci. 2014; 281: 20141163Crossref PubMed Scopus (33) Google Scholar]. This may explain why riboflavin is lost first as the modest host demand for this vitamin may be easily met by a new symbiont even at a relatively low abundance. In several species within the Lachninae sub-family, the tryptophan pathway is also missing. This suggests that once the co-obligate symbiosis with Serratia is established, the loss of amino acid pathways in Buchnera can follow [10Manzano-Marín A. Latorre A. Settling down: the genome of Serratia symbiotica from the aphid Cinara tujafilina zooms in on the process of accommodation to a cooperative intracellular life.Genome Biol. Evol. 2014; 6: 1683-1698Crossref PubMed Scopus (47) Google Scholar]