Origin and elaboration of a major evolutionary transition in individuality

Abstract

Obligate endosymbiosis, in which distantly related species integrate to form a single replicating individual, represents a major evolutionary transition in individuality1,2,3. Although such transitions are thought to increase biological complexity1,2,4,5,6, the evolutionary and developmental steps that lead to integration remain poorly understood. Here we show that obligate endosymbiosis between the bacteria Blochmannia and the hyperdiverse ant tribe Camponotini7,8,9,10,11 originated and also elaborated through radical alterations in embryonic development, as compared to other insects. The Hox genes Abdominal A (abdA) and Ultrabithorax (Ubx)—which, in arthropods, normally function to differentiate abdominal and thoracic segments after they form—were rewired to also regulate germline genes early in development. Consequently, the mRNAs and proteins of these Hox genes are expressed maternally and colocalize at a subcellular level with those of germline genes in the germplasm and three novel locations in the freshly laid egg. Blochmannia bacteria then selectively regulate these mRNAs and proteins to make each of these four locations functionally distinct, creating a system of coordinates in the embryo in which each location performs a different function to integrate Blochmannia into the Camponotini. Finally, we show that the capacity to localize mRNAs and proteins to new locations in the embryo evolved before obligate endosymbiosis and was subsequently co-opted by Blochmannia and Camponotini. This pre-existing molecular capacity converged with a pre-existing ecological mutualism12,13 to facilitate both the horizontal transfer10 and developmental integration of Blochmannia into Camponotini. Therefore, the convergence of pre-existing molecular capacities and ecological interactions—as well as the rewiring of highly conserved gene networks—may be a general feature that facilitates the origin and elaboration of major transitions in individuality.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: The evolution of four subcellular localization zones of germline genes that radically alter embryogenesis in C. floridanus.
Fig. 2: The Hox genes abdA and Ubx are rewired to regulate germline genes in C. floridanus.
Fig. 3: Blochmannia bacteria maintain and selectively regulate mRNA and proteins of maternal Hox and germline genes.
Fig. 4: Origin and elaboration of developmental integration of Blochmannia into Camponotini.

Data availability

All relevant data are included in the Article, Extended Data and Supplementary Information. Raw sequence data that support the findings of this study have been deposited in GenBank with accession code MH801205, and in NCBI Sequence Read Archive with the accession code PRJNA625680. All raw image data that support the findings of this study are available in FigShare with the following identifiers: reference number 78072 (https://figshare.com/projects/The_origin_and_elaboration_of_a_major_evolutionary_transition_in_ants/78072); Fig. 1, https://doi.org/10.6084/m9.figshare.12133308; Fig. 2, https://doi.org/10.6084/m9.figshare.12133311; Fig. 3, https://doi.org/10.6084/m9.figshare.12133314; Fig. 4, https://doi.org/10.6084/m9.figshare.12133326; Extended Data Fig. 1, https://doi.org/10.6084/m9.figshare.12133296; Extended Data Fig. 2, https://doi.org/10.6084/m9.figshare.12133287; Extended Data Fig. 3, https://doi.org/10.6084/m9.figshare.12133110; Extended Data Fig. 4, https://doi.org/10.6084/m9.figshare.12133278; Extended Data Fig. 5, https://doi.org/10.6084/m9.figshare.12130902; Extended Data Fig. 6, https://doi.org/10.6084/m9.figshare.12131022; Extended Data Fig. 7, https://doi.org/10.6084/m9.figshare.12132993; Extended Data Fig. 8, https://doi.org/10.6084/m9.figshare.12131430. Source data are provided with this paper.

References

  1. 1.

    Maynard-Smith, J. & Szathmary, E. The Major Transitions in Evolution (Oxford Univ. Press, 1997).

  2. 2.

    West, S. A., Fisher, R. M., Gardner, A. & Kiers, E. T. Major evolutionary transitions in individuality. Proc. Natl Acad. Sci. USA 112, 10112–10119 (2015).

    ADS  CAS  PubMed  Google Scholar 

  3. 3.

    Sachs, J. L., Skophammer, R. G. & Regus, J. U. Evolutionary transitions in bacterial symbiosis. Proc. Natl Acad. Sci. USA 108 (Suppl 2), 10800–10807 (2011).

    ADS  CAS  PubMed  Google Scholar 

  4. 4.

    Boomsma, J. J. & Gawne, R. Superorganismality and caste differentiation as points of no return: how the major evolutionary transitions were lost in translation. Biol. Rev. Camb. Philos. Soc. 93, 28–54 (2018).

    PubMed  Google Scholar 

  5. 5.

    Moran, N. A. Symbiosis as an adaptive process and source of phenotypic complexity. Proc. Natl Acad. Sci. USA 104 (Suppl 1), 8627–8633 (2007).

    ADS  CAS  PubMed  Google Scholar 

  6. 6.

    Ruiz-Trillo, I. & Nedelcu, A. M. Evolutionary Transitions to Multicellular Life: Principles and Mechanisms Vol. 2 (Springer, 2015).

  7. 7.

    Blochmann, F. Über das Vorkommen bakterienähnlicher Gebilde in den Geweben und Eiern verschiedener Insekten. Zbl. Bakteriol. 11, 234–240 (1892).

    Google Scholar 

  8. 8.

    Buchner, P. Endosymbiosis of Animals with Plant Microorganisms (Interscience, 1965).

  9. 9.

    Tanquary, M. C. Biological and Embryological Studies on Formicidae. PhD thesis, Univ. of Illinois (1912).

  10. 10.

    Wernegreen, J. J., Kauppinen, S. N., Brady, S. G. & Ward, P. S. One nutritional symbiosis begat another: phylogenetic evidence that the ant tribe Camponotini acquired Blochmannia by tending sap-feeding insects. BMC Evol. Biol. 9, 292 (2009).

    PubMed  PubMed Central  Google Scholar 

  11. 11.

    Zientz, E., Beyaert, I., Gross, R. & Feldhaar, H. Relevance of the endosymbiosis of Blochmannia floridanus and carpenter ants at different stages of the life cycle of the host. Appl. Environ. Microbiol. 72, 6027–6033 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Aranda-Rickert, A., Fracchia, S., Yela, N. & Marazzi, B. Insights into a novel three-partner interaction between ants, coreids (Hemiptera: Coreidae) and extrafloral nectaries: implications for the study of protective mutualisms. Arthropod-Plant Interact. 11, 525–536 (2017).

    Google Scholar 

  13. 13.

    Clark, R. E., Farkas, T. E., Lichter-Marck, I., Johnson, E. R. & Singer, M. S. Multiple interaction types determine the impact of ant predation of caterpillars in a forest community. Ecology 97, 3379–3388 (2016).

    PubMed  Google Scholar 

  14. 14.

    Feldhaar, H. et al. Nutritional upgrading for omnivorous carpenter ants by the endosymbiont Blochmannia. BMC Biol. 5, 48 (2007).

    PubMed  PubMed Central  Google Scholar 

  15. 15.

    de Souza, D. J., Bézier, A., Depoix, D., Drezen, J. M. & Lenoir, A. Blochmannia endosymbionts improve colony growth and immune defence in the ant Camponotus fellah. BMC Microbiol. 9, 29 (2009).

    PubMed  PubMed Central  Google Scholar 

  16. 16.

    Gil, R. et al. The genome sequence of Blochmannia floridanus: comparative analysis of reduced genomes. Proc. Natl Acad. Sci. USA 100, 9388–9393 (2003).

    ADS  CAS  PubMed  Google Scholar 

  17. 17.

    Kupper, M., Stigloher, C., Feldhaar, H. & Gross, R. Distribution of the obligate endosymbiont Blochmannia floridanus and expression analysis of putative immune genes in ovaries of the carpenter ant Camponotus floridanus. Arthropod Struct. Dev. 45, 475–487 (2016).

    PubMed  Google Scholar 

  18. 18.

    Ramalho, M. O., Vieira, A. S., Pereira, M. C., Moreau, C. S. & Bueno, O. C. Transovarian transmission of Blochmannia and Wolbachia endosymbionts in the neotropical weaver ant Camponotus textor (Hymenoptera, Formicidae). Curr. Microbiol. 75, 866–873 (2018).

    CAS  PubMed  Google Scholar 

  19. 19.

    Sauer, C., Dudaczek, D., Hölldobler, B. & Gross, R. Tissue localization of the endosymbiotic bacterium “Candidatus Blochmannia floridanus” in adults and larvae of the carpenter ant Camponotus floridanus. Appl. Environ. Microbiol. 68, 4187–4193 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Sauer, C., Stackebrandt, E., Gadau, J., Hölldobler, B. & Gross, R. Systematic relationships and cospeciation of bacterial endosymbionts and their carpenter ant host species: proposal of the new taxon Candidatus Blochmannia gen. nov. Int. J. Syst. Evol. Microbiol. 50, 1877–1886 (2000).

    CAS  PubMed  Google Scholar 

  21. 21.

    Wolschin, F., Hölldobler, B., Gross, R. & Zientz, E. Replication of the endosymbiotic bacterium Blochmannia floridanus is correlated with the developmental and reproductive stages of its ant host. Appl. Environ. Microbiol. 70, 4096–4102 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Ward, P. S., Blaimer, B. B. & Fisher, B. L. A revised phylogenetic classification of the ant subfamily Formicinae (Hymenoptera: Formicidae), with resurrection of the genera Colobopsis and Dinomyrmex. Zootaxa 4072, 343–357 (2016).

    PubMed  Google Scholar 

  23. 23.

    Sinotte, V. M., Freedman, S. N., Ugelvig, L. V. & Seid, M. A. Camponotus floridanus ants incur a trade-off between phenotypic development and pathogen susceptibility from their mutualistic endosymbiont Blochmannia. Insects 9, 58 (2018).

    PubMed Central  Google Scholar 

  24. 24.

    Stoll, S., Feldhaar, H., Fraunholz, M. J. & Gross, R. Bacteriocyte dynamics during development of a holometabolous insect, the carpenter ant Camponotus floridanus. BMC Microbiol. 10, 308 (2010).

    PubMed  PubMed Central  Google Scholar 

  25. 25.

    Sameshima, S., Hasegawa, E., Kitade, O., Minaka, N. & Matsumoto, T. Phylogenetic comparison of endosymbionts with their host ants based on molecular evidence. Zool. Sci. 16, 993–1000 (1999).

    CAS  Google Scholar 

  26. 26.

    Degnan, P. H., Lazarus, A. B., Brock, C. D. & Wernegreen, J. J. Host–symbiont stability and fast evolutionary rates in an ant-bacterium association: cospeciation of Camponotus species and their endosymbionts, Candidatus Blochmannia. Syst. Biol. 53, 95–110 (2004).

    PubMed  Google Scholar 

  27. 27.

    Extavour, C. G. & Akam, M. Mechanisms of germ cell specification across the metazoans: epigenesis and preformation. Development 130, 5869–5884 (2003).

    CAS  PubMed  Google Scholar 

  28. 28.

    Lehmann, R. & Nüsslein-Volhard, C. The maternal gene nanos has a central role in posterior pattern formation of the Drosophila embryo. Development 112, 679–691 (1991).

    CAS  PubMed  Google Scholar 

  29. 29.

    Lehmann, R. Germ plasm biogenesis—an oskar-centric perspective. Curr. Top. Dev. Biol. 116, 679–707 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Khila, A. & Abouheif, E. Reproductive constraint is a developmental mechanism that maintains social harmony in advanced ant societies. Proc. Natl Acad. Sci. USA 105, 17884–17889 (2008).

    ADS  CAS  PubMed  Google Scholar 

  31. 31.

    Lynch, J. A. et al. The phylogenetic origin of oskar coincided with the origin of maternally provisioned germ plasm and pole cells at the base of the Holometabola. PLoS Genet. 7, e1002029 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Lynch, J. A. & Roth, S. The evolution of dorsal–ventral patterning mechanisms in insects. Genes Dev. 25, 107–118 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Akam, M. Hox genes, homeosis and the evolution of segment identity: no need for hopeless monsters. Int. J. Dev. Biol. 42, 445–451 (1998).

    CAS  PubMed  Google Scholar 

  34. 34.

    Hughes, C. L. & Kaufman, T. C. Hox genes and the evolution of the arthropod body plan. Evol. Dev. 4, 459–499 (2002).

    CAS  PubMed  Google Scholar 

  35. 35.

    Braendle, C. et al. Developmental origin and evolution of bacteriocytes in the aphid–Buchnera symbiosis. PLoS Biol. 1, e21 (2003).

    PubMed  PubMed Central  Google Scholar 

  36. 36.

    Matsuura, Y., Kikuchi, Y., Miura, T. & Fukatsu, T. Ultrabithorax is essential for bacteriocyte development. Proc. Natl Acad. Sci. USA 112, 9376–9381 (2015).

    ADS  CAS  PubMed  Google Scholar 

  37. 37.

    Höhna, S. et al. RevBayes: Bayesian phylogenetic inference using graphical models and an interactive model-specification language. Syst. Biol. 65, 726–736 (2016).

    PubMed  PubMed Central  Google Scholar 

  38. 38.

    Wernegreen, J. J., Degnan, P. H., Lazarus, A. B., Palacios, C. & Bordenstein, S. R. Genome evolution in an insect cell: distinct features of an ant–bacterial partnership. Biol. Bull. 204, 221–231 (2003).

    CAS  PubMed  Google Scholar 

  39. 39.

    Bhatkar, A. & Whitcomb, W. Artificial diet for rearing various species of ants. Fla. Entomol. 53, 229–232 (1970).

    Google Scholar 

  40. 40.

    Khila, A. & Abouheif, E. In situ hybridization on ant ovaries and embryos. Cold Spring Harb. Protoc. 2009, pdb.prot5250 (2009).

  41. 41.

    Rafiqi, A. M., Lemke, S. & Schmidt-Ott, U. Megaselia abdita: fixing and devitellinizing embryos. Cold Spring Harb. Protoc. 2011, pdb.prot5602 (2011).

  42. 42.

    Rothwell, W. F. & Sullivan, W. in Drosophila Protocols (eds Sullivan, W. et al.) 141–157 (Cold Spring Harbor Laboratory Press, 2000).

  43. 43.

    Bownes, M. A photographic study of development in the living embryo of Drosophila melanogaster. J. Embryol. Exp. Morpol. 33, 789–801 (1975).

    CAS  Google Scholar 

  44. 44.

    Kearse, M. et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28, 1647–1649 (2012).

    PubMed  PubMed Central  Google Scholar 

  45. 45.

    Bonasio, R. et al. Genomic comparison of the ants Camponotus floridanus and Harpegnathos saltator. Science 329, 1068–1071 (2010).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Kosman, D. et al. Multiplex detection of RNA expression in Drosophila embryos. Science 305, 846 (2004).

    CAS  PubMed  Google Scholar 

  47. 47.

    Sambrook, J., Fritsch, E. F. & Maniatis, T. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989).

  48. 48.

    Rafiqi, A. M., Lemke, S. & Schmidt-Ott, U. Megaselia abdita: preparing embryos for injection. Cold Spring Harb. Protoc. 2011, pdb.prot5601 (2011).

  49. 49.

    Holland, P. M., Abramson, R. D., Watson, R. & Gelfand, D. H. Detection of specific polymerase chain reaction product by utilizing the 5′–3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl Acad. Sci. USA 88, 7276–7280 (1991).

    ADS  CAS  PubMed  Google Scholar 

  50. 50.

    Xie, F., Xiao, P., Chen, D., Xu, L. & Zhang, B. miRDeepFinder: a miRNA analysis tool for deep sequencing of plant small RNAs. Plant Mol. Biol. (2012).

  51. 51.

    Vandesompele, J. et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, research0034.0031 (2002).

  52. 52.

    Silver, N., Best, S., Jiang, J. & Thein, S. L. Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Mol. Biol. 7, 33 (2006).

    PubMed  PubMed Central  Google Scholar 

  53. 53.

    Pfaffl, M. W., Tichopad, A., Prgomet, C. & Neuvians, T. P. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper—Excel-based tool using pair-wise correlations. Biotechnol. Lett. 26, 509–515 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Andersen, C. L., Jensen, J. L. & Ørntoft, T. F. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 64, 5245–5250 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Borowiec, M. L. et al. Compositional heterogeneity and outgroup choice influence the internal phylogeny of the ants. Mol. Phylogenet. Evol. 134, 111–121 (2019).

    PubMed  Google Scholar 

  56. 56.

    Blaimer, B. B. et al. Phylogenomic methods outperform traditional multi-locus approaches in resolving deep evolutionary history: a case study of formicine ants. BMC Evol. Biol. 15, 271 (2015).

    PubMed  PubMed Central  Google Scholar 

  57. 57.

    Mezger, D. & Moreau, C. S. Out of South-East Asia: phylogeny and biogeography of the spiny ant genus Polyrhachis Smith (Hymenoptera: Formicidae). Syst. Entomol. 41, 369–378 (2016).

    Google Scholar 

  58. 58.

    Lilienstern, M. Beiträge zur Bakteriensymbiose der Ameisen. Zeitschrift für Morphologie und Ökologie der Tiere 26, 110–134 (1932).

    Google Scholar 

  59. 59.

    Jungen, H. Endosymbionten bei Ameisen. Insectes Soc. 15, 227–232 (1968).

    Google Scholar 

  60. 60.

    Pagel, M., Meade, A. & Barker, D. Bayesian estimation of ancestral character states on phylogenies. Syst. Biol. 53, 673–684 (2004).

    PubMed  Google Scholar 

  61. 61.

    Rambaut, A., Drummond, A. J., Xie, D., Baele, G. & Suchard, M. A. Posterior summarization in Bayesian phylogenetics using Tracer 1.7. Syst. Biol. 67, 901–904 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. 62.

    Fan, Y., Wu, R., Chen, M.-H., Kuo, L. & Lewis, P. O. Choosing among partition models in Bayesian phylogenetics. Mol. Biol. Evol. 28, 523–532 (2011).

    CAS  PubMed  Google Scholar 

  63. 63.

    Rueden, C. T. et al. ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics 18, 529 (2017).

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank L. Davis, R. Johnson, A. Suarez, J. Gibson, J. Rand, A. Wild and E. LeBrun for help with collecting ants; A. Vasquez-Correa, S. Joly, P. Ward and T. Oakley for help with ancestral-state reconstruction; M. Zayd and T. Chen for help with qPCR analysis; C. Metzl for translations; P. Lasko, S. F. Gilbert, J. Liebig, D. W. Wheeler, R. Rajakumar, C. Extavour, Y. Idaghdour, D. Schoen, A. Khila and members of the laboratory of E.A. for discussions or comments on the manuscript; and McGill University’s Integrated Quantitative Biology Initiative (IQBI) and Advanced BioImaging Facility (ABIF) for imaging support. This work was supported by a doctoral fellowship from FQRNT (Quebec) to A.R., a Bezmialem Vakif University Grant (Turkey) to A.M.R., and an NSERC Discovery Grant and Steacie Fellowship (Canada), John Simon Guggenheim Fellowship (USA) and KLI Fellowship (Austria) to E.A.

Author information

Affiliations

Authors

Contributions

E.A., A.M.R. and A.R. conceived the project, designed experiments and collected ants. A.R. and A.M.R. performed all experiments. E.A. performed phylogenetic analyses. E.A., A.M.R. and A.R. wrote the manuscript.

Corresponding author

Correspondence to Ehab Abouheif.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Cameron R. Currie, Yannick Wurm and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Distribution of Blochmannia during oogenesis and the subcellular localization and expression of germline genes in Cfloridanus oocytes and embryos.

af, f′, Ovaries showing nuclear-stain DAPI in blue and Blochmannia in white: germline stem-cell niche without Blochmannia (a), germarium in which Blochmannia colonization occurs (b), Blochmannia initially fill the entirety of the cytoplasm of young oocytes (c) and progressively localize to the posterior pole of older oocytes (df), where Blochmannia surrounds the germplasm (f′). f′, gi, Mature oocytes showing maternal expression of germline genes in oocytes, showing osk mRNA in magenta (f′), Vas protein in yellow (g), nos mRNA in blue (h), Aub protein in green (i) and nuclear-stain DAPI in blue. jo, Subcellular localization zones in stage (st)-1 freshly laid eggs showing Aub protein in green (j), Gcl protein in orange (k), Tud protein in white (l), Hsp90 protein in red (m), smg mRNA in blue (n) and stau mRNA in blue (o). pu, Expression in stage-6 cellular blastoderm embryos showing Aub protein in green (p), Gcl protein in orange (q), Tud protein in white (r), Hsp90 protein in red (s), smg mRNA in blue (t) and stau mRNA in blue (u). Arrowheads indicate subcellular localization or expression zones of germline genes: zone 1, zone 1a, zone 1b, zone 2, zone 3 and zone 4. Anterior is to the left, dorsal is to the top. In situ hybridization and immunohistochemistry experiments were repeated at least 8 times independently on n ≥ 30 oocytes or embryos per developmental stage.

Extended Data Fig. 2 Blochmannia segregates between bacteriocytes and germline capsule, and makes up 97.2% of DNA content in freshly laid eggs of C. floridanus.

af, Blochmannia: at the posterior pole in freshly laid stage-1 eggs (a); inside bacteriocytes in stage-8 embryos (b); in bacteriocytes that line the midgut of stage-17 embryos (c); together with germline-precursor nuclei (yellow arrowheads) along the crest of the future germline capsule (d); surrounding the novel germline within the germline capsule (e); and as a small seed population for vertical transmission in the germline capsule (f). g, Freshly laid egg with DAPI in white, showing few zygotic nuclei in the anterior and Blochmannia at the posterior pole. hk, Pie charts representing the number of Illumina Hi-Seq reads that match each of the indicated genera from DNA of freshly laid eggs. h, High abundance of Blochmannia DNA (blue) compared to that of host DNA (orange) and of other associated microorganisms (slim black slice) (shown in more detail in ik) of decreasing abundance. We used a sequence similarity (e-value) of e−3 as a cut-off value for including any genus in our analysis. Numbers in j and k represent the following species: 8, Serratia; 9, Leuconostoc; 10, Cupriavidus; 11, Cutibacterium; 12, Corynebacterium; 13, Mycobacterium;14, Candida; 15, Cyberlindnera; 16, Lactobacillus; 17, Brevibacterium; 18, Methylobacterium; 19, Pan; 20, Staphylococcus; 21, Sphingomonas; 22, Bradyrhizobium; 23, Plasmopara; 24, Bacillus; 25, Streptococcus; 26, Sphingopyxis; 27, Hyphomicrobium, 28, Acinetobacter; 29, uncultured; 30, see k; 31, Burkholderia; 32, Achromobacter; 33, Pichia; 34, Hyphopichia; 35, Penicillium; 36, Cyprinus; 37, Paenibacillus; 38, Brachybacterium; 39, Stenotrophomona; 40, Variovorax; 41, Streptomyces; 42, Sphingobium; 43, Nocardiopsis; 44, Dermabacter; 45, Sphingobacteriu; 46, Klebsiella; 47, Morganella; 48, Acidovorax; 49, Malassezia; 50, Lysobacter; 51, Rothia; 52, Pongo; 53, Rhodoplanes; 54, Microbacterium; 55, Rhodopseudomona; 56, Acheta; 57, Exiguobacterium; 58, Paraburkholderi; 59, Enterococcus; 60, Ramlibacter; 61, Actinomyces; 62, Bordetella; 63, Xanthomonas; 64, Brevundimonas; 65, Citrobacter; 66, Drosophila; 67, Lactococcus; 68, Mesorhizobium; 69, Candidatus; 70, Gluconobacter; 71, Rhodococcus; 72, Rubrivivax; 73, Saccharomyces; 74, Chelatococcus; 75, Hydrogenophaga; 76, Micrococcus; 77, Rhizobium; 78, Thauera; 79, Azospirillum; 80, Bosea; 81, Micromonospora; 82, Caulobacter; 83, Triticum; 84, Tsukamurella. DAPI staining was repeated at least 4 times on n ≥ 30 embryos per developmental stage.

Extended Data Fig. 3 Tracking the four functionally distinct zones through C. floridanus embryogenesis.

ai, Embryos showing Vas protein staining in yellow and DAPI in blue: freshly laid stage-1 and -2 eggs (a, b), cellular blastoderm stage-4 and -6 (c, d), gastrulation stage-7 (e), germband extension stage-8 to -10 (fh) and segmentation stage-12 (i) embryos. j, j′, j′′, k, l, Embryos showing higher-magnification confocal images of zone 1–4: freshly laid stage-1 egg, showing small germplasm foci budding off of the ancestral germplasm (j, j′, j′′), stage-8 embryo showing novel germline (k), stage-6 embryo showing germband (zone 3) and yolk sac (zone 4) expression (l). ns, onset of Vas expression throughout the nervous system, brain and central nervous system in embryos from stage 9 onwards. mu, osk mRNA in blue in stage-1 freshly laid egg (m, n), cellular blastoderm stage-3 and -4 embryo (or), gastrulation stage-7 embryo (s), and germband extension stage-8 and -9 embryo (t, u). n, o, Dorsal view, showing localization of small germplasm foci within the centre of bacteriocytes (zone 1b). qu, Formation of the novel germline (zone 2). u, Embryo, showing loss of zone 1a and zone 1b. v, v′, v′′, Small foci budding off the ancestral germplasm (zone 1). w, x, Higher-magnification confocal images of embryos, showing osk mRNA in magenta and DAPI in white. w, Stage-8 embryo, showing osk mRNA in magenta in the centre of bacteriocytes (zone 1b) surrounded by bacteria. x, Stage-8 embryo, showing expression of osk mRNA in the novel germline (zone 2). Zones are indicated with arrowheads. Anterior is to the left, dorsal is to the top. In situ hybridization and immunohistochemistry experiments were repeated at least 8 times independently on n ≥ 30 embryos per developmental stage.

Extended Data Fig. 4 abdA and Ubx are upstream of the germline genes.

a, b, Mature oocytes stained for abdA mRNA (a) or Ubx mRNA (b) in blue. ce, Colocalization (yellow and orange) of Ubx and AbdA (UbdA) protein in red, Vas protein in green and DAPI in blue in freshly laid stage-1 eggs (c), and stage-6 (d) and stage-12 (e) wild-type embryos. fp, Expression of the germline genes in YFP RNAi (n = 81) (fh) and high-concentration abdA RNAi embryos (n = 61 out 69) with DAPI in blue (jp), stained for Tud in white (f, i, l), Aub in green (g, j, m) or Vas in yellow (h, k, n, p), and DIC of stage-6 embryo with severe phenotype (o). ik, abdA RNAi embryos that are split along the midline (n = 21 out 61). lp, Severe abdA RNAi phenotypes with an undifferentiated stub (n = 34 out of 61) (ln) or in which the embryo is not detectable (o, p) (n = 6 out 61). Dotted outlines show changes in germband morphology and zone-3 expression after abdA RNAi. Zones are indicated with arrowheads. Asterisks indicate loss of germline gene expression within a specific zone. bc, bacteriocytes; cap, giant capsule; ys, yolk sac. Anterior is to the left, dorsal is to the top. q, r, Tissue-specific qPCR of nine germline genes (x axis; ago3, cad, gcl, nos, osk, stau, tud, vasa and wun2) from zone 1 (bacteriocytes), zone 2 (germline capsules), and zone 3 + zone 4 (embryonic germband + yolk sac) following YFP RNAi, low-concentration abdA RNAi and Ubx RNAi. Open bars represent mean relative quantification (RQ) values (y axis) and error bars represent standard error of the mean of: abdA RNAi (q) or Ubx RNAi (r). Black bars represent mean relative quantification values (y axis) and error bars represent standard error of the mean of YFP RNAi controls. Each individual data point (red squares) represents relative quantification value of a technical replicate from abdA or Ubx RNAi treatment relative to the average of all replicates of YFP RNAi control treatments (black diamonds) in that tissue. Two-tailed two-way ANOVA with replication for abdA RNAi versus YFP RNAi in zone 1 (F = 129.311, degrees of freedom (d.f.) = 1, n = 54, P = 5.95504 × 10−16 for abdA RNAi); zone 2 (F = 20.733, d.f. = 1, n = 54, P = 3.04542 × 10−5 for abdA RNAi); zone 3 + zone 4 (F = 38.932, d.f. = 1, n = 54, P = 7.02605 × 10−8 for abdA RNAi). Two-tailed two-way ANOVA with replication for Ubx RNAi versus YFP RNAi in zone 1 (F = 66.278, d.f. = 1, n = 54, P = 5.84252 × 10−11 for Ubx RNAi); zone 2 (F = 12.628, d.f. = 1, n = 54, P = 0.000798519 for Ubx RNAi); zone 3 + zone 4 (F = 40.841, d.f. = 1, n = 54, P = 4.00577 × 10−8 for Ubx RNAi). Raw data are in Source Data. In situ hybridization and immunohistochemistry experiments (ae) were repeated at least 8 times independently on n ≥ 30 embryos per developmental stage. Source data

Extended Data Fig. 5 Antibiotic treatment does not show unspecific effects.

ac, Stage-6 cellular blastoderm C. floridanus embryos with DAPI in white from wild-type colonies (n ≥ 30 embryos) (a), colonies treated with ampicillin (n ≥ 15 embryos) (b) or colonies treated with rifampicin (n ≥ 15 embryos) (c). do, C. floridanus embryos stained for nos mRNA (df, jo) and osk mRNA (gi) in blue collected from wild-type colonies (n ≥ 30 embryos each) (d, g, j, m), colonies treated with ampicillin (n ≥ 15 embryos each) (e, h, k, n) or colonies treated with rifampicin (n ≥ 15 embryos each) (f, i, l, o). p, q, Stage-12 mild-phenotype embryos collected from rifampicin-treated C.-floridanus colonies, showing expression of the segment polarity gene en in blue (n ≥ 15 embryos) (p) or abdA mRNA in blue (n ≥ 15 embryos) (q). r, s, Lasius niger embryos collected from rifampicin-treated colonies showing nos mRNA in blue in stage-6 embryos with normal primordial germ cells (pgc) (n ≥ 5 embryos) (r) and stage-12 embryos with normal germ cells (gc) (n ≥ 5 embryos) (s). Segments marked are as following: maxillary (mx), thoracic segments 1–3 (t1–t3) and abdominal segments 1–10 (a1–a10). White arrowheads indicate presence of Blochmannia (bl). White and black asterisks in embryos from rifampicin-treated colonies indicate loss of Blochmannia or loss of germline gene expression. di, Black arrowheads indicate zones. jl, Black arrowheads indicate germline capsule(s) (cp). mo, Black arrows indicate normal bacteriocyte (bc) and gonads (gc), development. Anterior is to the left, dorsal is to the top (af, p-r); dorsal is towards the reader in gi, mo, s; and ventral is towards the reader in jl. In situ hybridization experiments were repeated at least 8 times (C. floridanus) or 4 times (L. niger) independently.

Extended Data Fig. 6 Blochmannia maintains and selectively regulates mRNAs and proteins of maternal Hox and germline genes.

ar, Embryos from rifampicin-treated colonies stained for Ubx mRNA in blue (ac), Tud protein in white (df), Aub protein in green (gi), osk mRNA in blue (jl), nos mRNA in blue (mo) or stau mRNA in blue (pr). a, d, g, j, m, p, Freshly laid stage-1 eggs showing no effect on the number of zones relative to wild type, except for in d Tud in white showing loss of zone 1 relative to wild type. b, e, h, k, n, q, Stage-6 mild-phenotype embryos with no observable morphological defects: asterisks indicate loss of specific mRNAs and proteins of Ubx and germline gene expression. c, f, i, l, o, r, Stage-6 severe-phenotype embryos showing morphological defects and loss or misexpression of germline and Hox genes. di, Fluorescent images with DAPI in blue. Zones of germline and Hox gene expression are indicated with arrowheads. Question marks indicate presumptive zones. Anterior is to the left, dorsal is to the top. In situ hybridization and immunohistochemistry experiments were repeated at least 4 times independently on n ≥ 15 embryos per developmental stage.

Extended Data Fig. 7 Character states of germline localization zones, location of embryo, obligate endosymbiont and germline capsule.

az, za, zb, zc, zd, ze, Cellular blastoderm stage embryos from Formicinae (az) and two sister subfamilies (za, zb, zc, zd) Myrmicinae and Dolichoderinae (ze), stained for Vas protein in yellow with DAPI in blue. an, Camponotini tribe. a, Camponotus floridanus. b, Camponotus castaneous. c, Camponotus novaeboracensis. d, Camponotus pennsylvanicus. e, Camponotus americanus. f, Camponotus ocreatus. g, Camponotus sansabeanus. h, Camponotus festinatus. i, Polyrhachis illaudata. j, Polyrhachis schlueteri. k, Polyrhachis dives. l, Polyrhachis rastallata. m, Colobopsis leonardi. n, Colobopsis impressus. o, Gigantiopini tribe: Gigantiops destructor. p, Pleigiolepidini tribe: Anoplolepis gracilipes. q, Oecophyllini tribe: Oecophylla smaragdina. r, s, Formicini tribe. r, Formica subsericea. s, Formica occulta. ty, Lasiini tribe. t, Paratrechina longicornis. u, Nylanderia vividula. v, Nylanderia fulva. w, Lasius niger. x, Lasius emarginatus. y, Prenolepis imparis. z, Myrmelachistini tribe: Brachymyrmex patagonicus. za, zb, zc, zd, Myrmicinae. za, Aphaenogaster rudis. zb, Myrmica americana. zc, Veromessor pergandei. zd, Monomorium sp. ze, Dolichoderinae: Dolichoderus thoracicus. zf, zg, Freshly laid stage-1 eggs stained for Vas protein in yellow with DAPI in blue of F. occulta (zf) and A. gracilipes (zg). zf′, zg′, Endosymbiont at the posterior pole of F. occulta (zf′) and A. gracilipes (zg′). zi, zj, zk, zl, Cellular blastoderm stage embryos showing osk mRNA in blue, for L. niger (zi), F. occulta (zj), G. destructor (zk) and C. floridanus (zl). Zones of germline gene expression are indicated with white or black arrowheads. Magenta arrowheads indicate the location of the embryo within the egg. Experiments on all species were repeated 4 times independently on n ≥ 5 embryos, except for C. floridanus, which was repeated 8 times independently with n = 30. Anterior is to the left, dorsal is to the top.

Extended Data Fig. 8 Character states of maternal Hox localization zones.

aj, lx, Freshly laid stage-1 eggs from the Formicinae (au) and two sister subfamilies Myrmicinae (v, w) and Dolichoderinae (x) stained for UbdA (Ubx + abdA protein) in white or blue and (in k) abdA mRNA in blue. ak, Camponotini tribe. a, Camponotus floridanus. b, Camponotus novaeboracensis. c, Camponotus castaneous. d, Camponotus pennsylvanicus. e, Camponotus festinatus. f, Camponotus sansabeanus. g, Camponotus ocreatus. h, Polyrhachis rastallata. i, Polyrhachis dives. j, Colobopsis leonardi. k, Colobopsis impressus. l, m, Gigantiopini tribe: Gigantiops destructor. In m, UbdA protein in red co-stained with Vas protein in green and DAPI in blue to distinguish germ cells from zone 3. n, Pleigiolepidini tribe: Anoplolepis gracilipes. o, p, Formicini tribe. o, Formica occulta. p, Formica subsericea. qt, Lasiini tribe. q, Lasius niger. r, Lasius emargiatus. s, Nylanderia vividula. t, Paratrechina longicornis. u, Myrmelachistini tribe: Brachymyrmex patagonicus. v, w, Myrmicinae subfamily. v, Aphaenogaster rudis. w, Monomorium sp. x, Dolichoderinae subfamily: Dolichoderus thoracicus. Zones of maternal Hox localization are indicated with arrowheads: zone 1 (ancestral germline), zone 2 (novel germline), zone 3 (embryo) and zone 4 (anterior). Anterior is to the left, dorsal is to the top. Experiments on all species were repeated 4 times independently on n ≥ 5 embryos, except for C. floridanus, for which experiments were repeated 8 times independently with n = 30 embryos.

Extended Data Table 1 Combinatorial and dynamic localization or expression of germline and Hox genes across zones in stage-1 eggs and stage-6 embryos
Extended Data Table 2 Posterior probabilities under the unequal- and equal-rates model for the five developmental characters

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-16 and Supplementary Tables 1-3.

Reporting Summary

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Rafiqi, A.M., Rajakumar, A. & Abouheif, E. Origin and elaboration of a major evolutionary transition in individuality. Nature 585, 239–244 (2020). https://doi.org/10.1038/s41586-020-2653-6

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing