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.
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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.
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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.
The authors declare no competing interests.
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 C. floridanus oocytes and embryos.
a–f, 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 (d–f), where Blochmannia surrounds the germplasm (f′). f′, g–i, 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. j–o, 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). p–u, 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.
a–f, 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. h–k, 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 i–k) 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.
a–i, 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 (f–h) 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. m–u, osk mRNA in blue in stage-1 freshly laid egg (m, n), cellular blastoderm stage-3 and -4 embryo (o–r), 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). q–u, 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.
a, b, Mature oocytes stained for abdA mRNA (a) or Ubx mRNA (b) in blue. c–e, 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. f–p, Expression of the germline genes in YFP RNAi (n = 81) (f–h) and high-concentration abdA RNAi embryos (n = 61 out 69) with DAPI in blue (j–p), 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). i–k, abdA RNAi embryos that are split along the midline (n = 21 out 61). l–p, Severe abdA RNAi phenotypes with an undifferentiated stub (n = 34 out of 61) (l–n) 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 (a–e) were repeated at least 8 times independently on n ≥ 30 embryos per developmental stage.
a–c, 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). d–o, C. floridanus embryos stained for nos mRNA (d–f, j–o) and osk mRNA (g–i) 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. d–i, Black arrowheads indicate zones. j–l, Black arrowheads indicate germline capsule(s) (cp). m–o, Black arrows indicate normal bacteriocyte (bc) and gonads (gc), development. Anterior is to the left, dorsal is to the top (a–f, p-r); dorsal is towards the reader in g–i, m–o, s; and ventral is towards the reader in j–l. 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.
a–r, Embryos from rifampicin-treated colonies stained for Ubx mRNA in blue (a–c), Tud protein in white (d–f), Aub protein in green (g–i), osk mRNA in blue (j–l), nos mRNA in blue (m–o) or stau mRNA in blue (p–r). 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. d–i, 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.
a–z, za, zb, zc, zd, ze, Cellular blastoderm stage embryos from Formicinae (a–z) and two sister subfamilies (za, zb, zc, zd) Myrmicinae and Dolichoderinae (ze), stained for Vas protein in yellow with DAPI in blue. a–n, 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. t–y, 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.
a–j, l–x, Freshly laid stage-1 eggs from the Formicinae (a–u) 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. a–k, 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. q–t, 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.
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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
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