# Genomic evidence of a complex supergene system linking dispersal to social polymorphism

Stefano Mona, Elise Gay, Antoine Taupenot, Joshua Ducancel, Romuald Laso-Jadart, Quentin Helleu, Pascaline Chifflet-Belle, Emilie Teodori, Jean-Marc Aury, Zijun Xiong, et al.

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## To cite this version:

Stefano Mona, Elise Gay, Antoine Taupenot, Joshua Ducancel, Romuald Laso-Jadart, et al. Genomic evidence of a complex supergene system linking dispersal to social polymorphism. Current Biology, 2025, 35 (24), pp.6155-6162.e5. 10.1016/j.cub.2025.10.065. hal-05456284

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1 **Genomic evidence of a complex supergene system**

2 **linking dispersal to social polymorphism**

3

4 Stefano Mona<sup>1,2,9*</sup>, Elise J. Gay<sup>1,2,9</sup>, Antoine Taupenot<sup>3</sup>, Joshua Ducancel<sup>1,2</sup>, Romuald Laso-

5 Jadart<sup>1,2</sup>, Quentin Helleu<sup>4</sup>, Pascaline Chifflet-Belle<sup>1,2</sup>, Emilie Teodori<sup>5</sup>, Jean-Marc Aury<sup>5</sup>, Zijun

6 Xiong<sup>6</sup>, Lukas Schrader<sup>7</sup>, Joel Vizueta<sup>8</sup>, Mathieu Molet<sup>3</sup>, Claudie Doums<sup>1,2</sup>

7 **Author Contact :**

8 Stefano MONA : stefano.mona@mnhn.fr

9 **Lead Contact :**

10 Stefano MONA : stefano.mona@mnhn.fr

11 **Author Affiliation :**

12 <sup>1</sup> Institut de Systématique, Évolution, Biodiversité (ISYEB), EPHE-PSL, Muséum national

13 d'Histoire naturelle, CNRS, Sorbonne Université, Université des Antilles, 75005 Paris, France

14 <sup>2</sup> EPHE, PSL Research University, 75014 Paris, France

15 <sup>3</sup> Institut d'Ecologie et des Sciences de l'Environnement de Paris (iEES-Paris), UMR 7618,

16 Sorbonne Université, Université Paris Est Créteil, Université Paris Diderot, CNRS, INRA, IRD,

17 75005 Paris, France

18 <sup>4</sup> Laboratoire Structure et Instabilité des Génomes - INSERM U1154 - CNRS 7196

19 Muséum National d'Histoire Naturelle, Sorbonne Université, 75005 Paris, France

20 <sup>5</sup> Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry,

21 Université Paris-Saclay, Evry, 91057, France

22 <sup>6</sup> School of Basic Medical Sciences, Jiangxi Medical College, Nanchang University, Nanchang

23 330031, China.
24 ⁷ Institute for Evolution & Biodiversity, University of Münster, 48149 Münster, Germany

25 ⁸ Section for Ecology and Evolution, Department of Biology, University of Copenhagen,
26 Universitetsparken 15, 2100, Copenhagen, Denmark

27 ⁹ These authors contributed equally

28 **Summary**

29 Supergenes play an important role in the evolution of complex phenotypes, locking specific
30 combinations of alleles¹⁻³. One such complex phenotypes is social polymorphism in ants, i.e.,
31 the presence of single (monogyne) or multiple (polygyne) reproductive queens within
32 colonies, known to be determined by social supergenes in at least five lineages⁴,⁵. This
33 polymorphism is associated with divergent individual and colonial traits, some linked to
34 dispersal⁶. We explored the idea that antagonistic selection between social forms favours the
35 emergence of regions of suppressed recombination. To this end, we studied the ant
36 *Myrmecina graminicola*, in which a social polymorphism is coupled with the presence/absence
37 of wings in queens. We discovered a new "social supergene" of ~20 Mb with three haplotypes.
38 Supergene genotypes determine the three queen phenotypes observed in nature: monogyne
39 winged, monogyne apterous and polygyne apterous. The two haplotypes associated with
40 aptery carry an additional copy of a gene probably involved in wing development as part of a
41 ~116 kb insertion predating the origin of the social supergene (~20Mya vs. ~1Mya). Syntenic
42 analyses showcased an independent evolution of the social supergene. The screening of
43 workers' genotypes suggests that assortative mating and segregation distortion may play a
44 role in preserving supergene polymorphism. This unique supergene system illustrates the
45 theoretically expected genetic link between social polymorphism and dispersal in ants. Its
46 modular evolution mirrors the role of sexually antagonistic selection in the origin of sex
47 chromosomes and makes ants a very promising model for studying supergene evolution.

48

49

50 **Keywords:** recombination, social insects, social polymorphism, wing polymorphism, dispersal, supergene, antagonistic evolution, *Myrmecina graminicola*

52
## 53 Results and discussion

The social supergenes in ants show striking similarities with sex chromosomes. Both represent remarkable cases of convergent evolution⁴,⁵,⁷ and antagonistic selection is thought to have played a major role in their origin⁷,⁸. In ants, the two main social environments (monogyne colonies headed by a single reproductive queen and polygyne colonies by multiple queens) generate antagonistic selective pressures, especially on traits linked to dispersal and colony foundation⁶. When founding a new colony alone, the young queen relies on its energetic reserves to raise the first workers⁹. Conversely, the queens need less energetic reserves if they are adopted by an existing colony or if they found new colonies with the help of workers or other queens⁹. In these cases, costly traits associated with dispersal (e.g., flight or increased energy reserves) will be selected against. Producing queens with low energetic reserves for independent colony foundation would be very risky while producing large, winged queens would be wasteful if queens are readopted or found new colonies with the help of workers. Thus, in species harbouring both social and wing polymorphisms, antagonistic selection should pave the way to the evolution of a genetic architecture linking the two traits.

We tested this premise in the West Palearctic ant species *Myrmecina graminicola*, in which crossing experiments suggest a simple Mendelian determinism of the wing polymorphism¹⁰. Three types of colonies are known in this species: polygyne colonies, always headed by more than one apterous reproductive queen and monogyne colonies headed either by a single winged (later named monogyne winged) reproductive queen (dealated after her mating flight) or by an apterous queen¹¹. Note that a monogyne apterous colony type could simply result from the loss of queens from a polygyne colony, either following queen death or incomplete collection. In our study population (the Fontainebleau Forest, Ile-de-France), we found 29 queenless colonies out of the 254 sampled (11.4%), suggesting queen death to occur frequently enough to misclassify a polygyne colony as monogyne. The remaining 225 colonies displayed a high frequency of the monogyne winged type (88.9%) and a lower frequency of the polygyne (8.4%) and monogyne apterous types (2.7%) (see experimental procedures for more details). Note that 22% of monogyne colonies contained unmated non reproductive dealated queens (see more details in the supplementary materials). The three colony types were similar in size (see experimental procedures for more details). The queen phenotypes did not visually differ in size and had similar egg-laying rate¹².
84

85

We built a new chromosome-scale genome assembly (see experimental procedures, Figure S1, 14 chromosomes known in *M. graminicola*¹³) from a single male from a polygyne apterous colony and further obtained whole genome sequences of 68 unrelated queens (40 monogyne winged, six monogyne apterous and 22 polygyne apterous queens, see experimental procedures) for population genomic analyses. We first compared the monogyne winged and polygyne apterous queens, the two phenotypes not subject to identification errors, to detect regions harbouring supergene characteristics. We discovered a large genomic region spanning over 20.6Mb on chromosome 1 showing: i) high level of differentiation between the two queen morphs (*Fst* values larger than the 95th percentile of the empirical distribution, Figure 1A); ii) high level of linkage-disequilibrium (LD) compared to genome-wide average (Figure 1B); iii) a strict separation of the two queen morphs along the principle component 1 of a PCA. When adding the six monogyne apterous queens on the PCA, one queen clustered with the polygyne group (being likely the result of a misclassification) and the remaining five clustered with the monogyne group (Figure 1C). The new social supergene we identified on Chr1 showed similarities with other social supergenes, with the ancestral phenotype (monogyne colonies) associated with the homozygous state⁴,⁵ (Figure 1D). Finally, we found no evidence of population structure between the sampled queens when investigating the whole genome after removing the social supergene. Both PCA and ancestry-based clustering supported the occurrence of a single panmictic population with similar levels of heterozygosity among the three morphs (Figure S1), with average pairwise *Fst* never exceeding ~0.005.

To disentangle the correlation between the social and wing polymorphisms and uncover the genetic determinant of the latter, we took advantage of the presence of the five genetically monogyne but apterous queens. We performed an *Fst* genomic scan between them and the winged monogyne queens and identified several adjacent windows within the social supergene containing higher than average *Fst* values (Figure 2A). Closer inspection revealed that this region harbours two *Fst* peaks: the first at position 22.859 Mb reaching ~0.29 and the second at position 22.975 Mb reaching ~0.54 (Figure S2). The two peaks flank a ~116 kb stretch deprived of SNPs as compared to the genomic average. To further investigate this region, we plotted the mean sequencing depth by queen morphs and observed a ~50% reduction
compared to the genomic average in all apterous queens (monogyne or polygyne) and a depth
being virtually zero in monogyne winged queens (Figure 2B). This suggested the presence of
an insertion in the social supergene found only in apterous queens. By analysing the
sequencing depth per individual, we found that all five monogyne apterous queens and 22 out
of 23 polygyne queens were hemizygote in the inserted region (see below).

Combining the two *Fst* genomic scans and the sequencing depth analysis (Figure 1A, Figure 2A,B, Figure S2), we were able to identify three haplotypes at the social supergene. We called the three haplotypes: Mo, the ancestral haplotype associated with monogyne winged queens; Ma, the haplotype associated with monogyne apterous queens which is similar to Mo but contains the insertion that is associated with the absence of wings; Pa, the derived haplotype associated with polygyne apterous queens, which also contains the insertion. Note that we will refer to M* either when the Mo and Ma cannot be distinguished (for example using RFLP digestion, see below) or when they were pooled for specific analyses. We found four out of the six possible genotypes in queens: MoMo (monogyne winged), MoMa (monogyne apterous), MoPa (polygyne apterous) and one MaPa (polygyne apterous). The Pa haplotype is therefore only found in heterozygous queens, at least in our population. This pattern mirrored the observation in the social supergene of *S. invicta*, where the derived haplotype is recessively lethal<sup>14</sup>. However, the evolutionary mechanism determining the absence of PaPa queens has yet to be investigated. It is likely that the derived Pa may behave more similarly to the Y chromosome in mammals and is more prone to degeneration than the derived P haplotypes in *F. selysi*, where the presence of PP queens should allow recombination to better purge deleterious mutations<sup>15</sup>. Conversely, the insertion determining apterism is dominant and, though it is present at low frequency in our sample, it is also viable in homozygosity. The physical linkage between aptery and polygyny is consistent with theoretical predictions suggesting that the coevolution of social organization and dispersal should lead to linked polymorphisms in both traits<sup>16</sup> but the presence of monogyne apterous queens bearing the Ma haplotype is puzzling.

141 Evolution of the social supergene and origin of the insertion

We aligned the nucleotide sequence of the social supergene against the three ant lineages
where a similar genetic architecture has been previously described (Solenopsis invicta<sup>14</sup>,
144 *Formica selysi*¹⁵, *Pogonomyrmex californicus*¹⁷). We found no large synteny blocks between
145 *M. graminicola* and these other three social supergenes suggesting another instance of
146 independent evolution of genetically determined sociality in ants (Figure 3).

147 We then focused on the inserted region, present exclusively in Ma and Pa. A portion of this
148 insertion, spanning 60 Kb, aligned with a 26 Kb region on chromosome 4 (Figure 2C and Figure
149 S3A,B), which in turn displayed similarities with *Pristomyrmex punctatus* and *Acanthomyrmex*
150 *ferox*, two closely related species¹⁸ (Figure S3C,D). The duplicated region between CHR1 and
151 CHR4 harbours a single gene that we have named Awing. This gene is highly conserved across
152 Hymenoptera, and is also found in Diptera (excluding the family Drosophilidae), Lepidoptera
153 and Coleoptera. It is mostly present as a single copy, but its function is unknown. This implies
154 that all apterous queens (both MoMa and M*Pa) harbour an additional (duplicated) copy of
155 *Awing* in the inserted region of CHR1 (*AwingDpl*) as compared to winged queens. While we
156 could not yet establish the functional link between the insertion and the wing polymorphism,
157 the most parsimonious explanation is that the latter is determined by the presence of the
158 additional copy of *Awing*. However, it remains to be determined whether the absence of
159 developed wings depends on the number of copies of *Awing* or on the specific protein motif
160 of the duplicated version. Copy number variation of specific genes in non-recombining regions
161 are known to affect phenotypic traits. In seed beetles (*Callosobruchus maculatus*), male body
162 size is determined by the number of copies of the TOR gene on the Y chromosome, with larger
163 males having a single copy¹⁹. Similarly, wingless males of the pea aphid (*Acyrthosiphon pisum*)
164 carry an X chromosome with an insertion harboring an additional expressed copy of the
165 *follistatin* gene²⁰.

166 We tentatively dated the origin of social and wing polymorphisms by estimating the number
167 of fixed SNPs between M* and Pa and the net divergence between the *Awing* and *AwingDpl*.
168 The social polymorphism originated at ~1 Mya (95% CI estimated from the distribution of
169 annotate genes: 0-3.7Mya) while the duplication at ~10 (95% HPD: 3.2-19.3) to ~23 Mya after
170 Bayesian inference or net divergence calculation, respectively (Figure S3F). While this dating
171 should be taken cautiously, the difference in the molecular divergence estimated in the two
172 comparisons is large enough to robustly conclude that wing polymorphism predates the social
173 one. This is consistent with the presence of wing polymorphism in the related but
174 predominantly monogyne species *M. nipponica*²¹ and *M. americana*²². A genomic comparison
within the genus would help to clarify the evolutionary history of the duplication. If its origin is confirmed to be older than the social supergene, this would be an interesting case illustrating the modularity of supergene evolution with the co-option of a pre-existing duplication, probably favored by socially antagonistic selection. Indeed, the duplication in the Pa haplotype agrees with general theories on dispersal that predict cooperation (here polygyny) in philopatric phenotypes (here apterous queens)¹⁶,²³,²⁴. This finding reinforces the association between dispersal and social organisation⁶,²⁵ and the importance of social antagonistic selection put forward by Scarparo et al.⁸ in the ant *Formica cinerea*. On the contrary, the presence of the Ma haplotype, linking apterism to monogyny, even if found at low frequency in our population, might simply reflect the survival of the ancestral wing polymorphism. We predict that ants that evolved convergent social polymorphism will offer exceptional model systems, paralleling the sex chromosomes⁷,²⁶ to inspire research for understanding the origin and expansion of autosomal supergenes³,²⁷ and for functional assessments of key functional genes²⁸.

## *Signs of assortative mating and segregation distortion*

To further confirm the genotype/phenotype association highlighted by the genomic scan and to investigate the supergene genotypic distribution of workers, we devised three PCR-RFLP assays to identify the supergene haplotypes (Table S1, Figure S4). We confirmed the genotype-phenotype association and the absence of PaPa queens on 67 additional monogyne winged queens and on 49 additional reproducing polygyne apterous queens (from the same 20 polygyne colonies used for the whole-genome sequencing). We investigated the genotypic composition of 539 workers from a subset of 42 monogyne winged colonies (two workers per colony), and the five monogyne apterous colonies and 20 polygyne colonies (an average of 18.2 workers per colony). The monogyne winged queens mated with Mo males whereas a minimum of 20% of polygyne queens mated with a Pa male (under the restrictive assumption that a single queen of each polygyne colonies mated with a Pa male) (Figure 4, Table S2). Our results strongly suggest the occurrence of assortative mating, as observed in *F. selysi*²⁹,³⁰. The presence of polygyne colonies with MoMo workers also supports the idea that gene flow between monogyne and polygyne social forms in this population is mainly mediated by Mo males produced by monogyne colonies. The extent of this gene flow is sufficient to hamper
differentiation between social forms (i.e., the lack of population structure in the Fontainebleau Forest). Focusing on polygyne colonies, our data support a bias in favour of the M*Pa (MoPa or MaPa) workers. Regardless of the fathers' genotypes, we would expect to observe 50% of M*Pa workers in the absence of segregation distortion. Instead, we found 65% of M*Pa workers (Figure 4), a ratio significantly higher than the expected 50% (Chi² test = 33.54, p < 0.0001). At the colony level, the pattern was significant in six colonies (Table S2). It remains to be investigated whether it results from selfish effects and/or differential lethality during developmental stages and whether this bias occurs likewise in sexuals. Previous experimental work¹¹ strongly suggests that this should be the case as isolated polygyne queens, mated with Mo males, reared only 20% of winged young queens, whereas a ratio of 50% would be expected. The genotyping of sexuals associated to behavioural experiments will be necessary to determine whether the M*Pa genotype had larger than expected frequency due to selfish behaviour or other evolutionary mechanisms such as in *F. selysi*³¹ and *S. invicta*³². In the presence of selfish drive, assortative mating is theoretically required for the maintenance of supergene polymorphism³³ whereas disassortative mating is considered as a major force when homozygotes at the derived haplotypes are less fit³⁴. The absence of PaPa queens is striking when considering the frequency of PaPa workers (Fisher exact test, p < 0.001). This pattern is consistent with the deleterious effects, especially in the queen caste, of the non-recombining haplotype previously observed in other species, including ants⁵,³⁵. A more thorough investigation of the mating dynamics and segregation bias in our species is still required to fully demonstrate the occurrence of both assortative mating and selfish behaviours, two major selective forces at play for maintaining social polymorphisms⁵,²⁸.

227

## Conclusion

Our study reveals the occurrence of a complex social supergene system of ~20 Mb characterized by three haplotypes, whose genotypic combinations determine both a social trait (monogyne/polygyne) and the presence/absence of wings in queens. Indeed, two of the three haplotypes carry an insertion, always hemizygous in queens, harbouring an additional copy of the *Awing* gene and associated with apterism. The social supergene is not syntenic to the other known social supergenes, suggesting a novel instance of convergent evolution. Wing
235 polymorphism appears to predate social polymorphism in *Myrmecina*, consistently with its
236 occurrence in the two congeneric species *M. americana*²² and *M. nipponica*²¹, which are
237 supposedly predominantly monogyne. This finding suggests that the social polymorphism
238 emerged more recently, linking polygyny and apterism through the insertion of the duplicated
239 *Awing* gene in the Pa haplotype. The absence of polygyne winged queens supports the recent
240 suggestion that socially antagonistic selection could be a major driver of recombination
241 suppression and thereby of supergene evolution⁸. *M. graminicola* provides new insights into
242 the causal relationship between restricted dispersal and polygyny, suggesting the former as
243 an evolutionary promoter of the latter. Furthermore, it would be interesting to investigate
244 how the monogyne apterous colony persisted, either through neutral mechanisms or via some
245 as yet unknown selective advantages and assess the extent of differential female dispersal
246 between winged and apterous queens. Future investigations into distant populations and
247 sister species will shed light on the evolution of supergenes and on the selective pressures
248 operating spatially and temporally for the maintenance of their polymorphism.

249 RESOURCE AVAILABILITY

250 Lead contact

251 Further information and requests for resources and reagents should be directed to, and will
252 be fulfilled by, the lead contact, Stefano Mona (stefano.mona@mnhn.fr).

253

254 Materials availability

255 This study did not generate new unique materials or reagents.

256

257 Data and code availability

258 • The raw Illumina sequence obtained on the 68 queens used in this study are available
259 at EBI (PRJEB96731).

260 • The raw PacBio and Hi-C data used to generate the reference genome assembly are
261 available at EBI (PRJEB96881). This study did not generate any unique datasets or code
262

263

* The raw data on the PCR-RFLP and colony information and the list of genes contained in the insertion is available from Zenodo (DOI:10.5281/zenodo.17055977).

264

265

* Additional Information: Any additional information required to reanalyse the data reported in this paper is available from the lead contact upon request.

266

## 267 Acknowledgements

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We wish to thank B. Finand and T. Jean-François (master student) for their help in field sampling and A. Belly (master student) for his help in PCR-RFLP experiments. We are grateful to the Genotoul bioinformatics platform Toulouse Occitanie (Bioinfo Genotoul, https://doi.org/10.15454/1.5572369328961167E12) for providing computing resources. This work was granted access to the HPC resources of MesoPSL financed by the Region Ile de France and the project Equip@Meso (reference ANR-10-EQPX-29-01) of the Programme Investissements d'Avenir supervised by the Agence Nationale pour la Recherche (ANR). This work was financially supported by the MNHN (ATM DEGE-AILE; 2023) and the ANR (Grant N° ANR-23-CE02-0024-01; 2024-2028).

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## 279 Author contributions

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**Original:** Conceptualization, S.M., C.D, M.M.; Methodology, S.M., C.D., E.J.G., A.T; Investigation, A.T., P.C.B; Formal analysis, A.T., E.J.G., R.J.L., J.V., Z.X., L.S.; Data curation, E.J.G., J.V., Z.X., L.S.; Visualization, A.T., E.J.G., R.J.L., C.D.; Writing – Original Draft, A.T., S.M., C.D., E.J.G., J.V., Z.X., L.S.; Supervision, S.M., C.D., M.M; Project Administration, S.M., C.D., M.M; Funding Acquisition, C.D., S.M., M.M. **Revised:** Conceptualization, S.M., C.D, M.M.; Methodology, S.M., C.D., E.J.G., A.T; Investigation, A.T., P.C.B., J.D.; Formal analysis, E.J.G., A.T., J.D., Q.H., R.J.L., J.V., Z.X., L.S.; Data curation, E.J.G., J.V., Z.X., L.S., JM.A., E.T., Q.H.; Visualization, E.J.G., J.D., R.J.L., Q.H., C.D.; Writing – Original Draft, S.M., C.D., E.J.G., J.V., Z.X., L.S.; Writing – Review & Editing, S.M., E.J.G., C.D.; Supervision, S.M., C.D., M.M; Project Administration, S.M., C.D., M.M; Funding Acquisition, C.D., S.M., M.M.

290

## 291 Declaration of interests
292 The authors declare no competing interests.

293 **Figure titles and legends**

294 **Figure 1. Characteristics of the social supergene of *M. graminicola*.** (A) *Fst* sliding window analysis computed between the 40 monogyne winged and the 22 polygyne apterous queens in the 14 chromosomes. The 95th quantile threshold of the *Fst* distribution is indicated by the dashed red line. (B) Heatmap of the linkage disequilibrium (*r*²) computed between the 62 queens. (C) PCA computed on the 68 queens on the social supergene with the monogyne winged in red, the polygyne apterous in blue and the additional six monogyne apterous in grey (the arrow indicates the only apterous queen identified as monogyne in the field which resulted as polygyne). (D) Proportion of heterozygous genotypes computed in the social supergene for the three morphs (see also Figure S1C).

303 **Figure 2. Genomic evidence of the wing polymorphism.** (A) *Fst* sliding window analysis computed between the 40 monogyne winged and the five monogyne apterous queens in the 14 chromosomes. Red peak indicates the inserted region. (B) Genomic scan of the sequencing depth for the 68 queens grouped by morph in *CHR1* (red: monogyne winged, grey: monogyne apterous, blue: polygyne apterous). Depth is averaged over morphs in windows of 500 bp with a slide of 200 bp. Genes were annotated in the inserted region (hemizygous in almost all apterous queens, see Main Text) from position ~22.859 MB to ~22.975 MB, with exons represented by arrows and introns by lines. The asterisk indicates the position of the gene *AwingDpl*, the duplicated copy of *Awing* from *CHR4*. The dashed line indicates the region homologous to *CHR4* and amplified by PCR in individuals harbouring at least one copy of *Ma* or *Pa*. No other annotated element within the insertion region has a clear relationship with wing development (results are provided in Zenodo: DOI:10.5281/zenodo.17055977). (C) Dot plot alignment of the inserted sequence detected in *CHR1* and its match found in *CHR4* (coordinates: ~18.998 MB to ~19.024 MB). The sequence identity is represented by the colour scale. The identity values are computed by the algorithm used by MiniMap2 implemented in the D-Genies tool where *I* = *M* / *N*, with *M* is the number of matching bases and *N* is the number of bases, including gaps. See also Figure S2, S3 and S4.

320 **Figure 3. Social supergene syteny.** Alignments between the social supergene of *M. graminicola* and the reference genomes of *S. invicta*¹⁴ (in purple), *F. selysi*¹⁵ (in green), and *P*.
californicus¹⁷ (in orange). The social supergene of *F. selysi* and *S. invicta* is located in the chromosomes 3 and 16 respectively. The social supergene of *P. californicus* spans seven scaffolds (6, 14, 22, 23, 38, 42, 48). Scaffolds harbouring the social supergenes are represented in darker colours. Alignments between social supergenes are coloured according to the target species, the alignment between the nucleotide sequence of *Awing* in CHR4 and *AwingDpl* in CHR1 is displayed in black, all other alignments are displayed in grey. Only alignments longer than 200 bp are shown.

**Figure 4. Frequency of the three colony types and their genotypic composition.** (A) Frequency of the three colony types at the two sampling dates (up pie chart: October 2021, down chart: April 2022). The number of collected colonies of each type is given in the pie chart. (B) Genotype composition at the social supergene for the three colony types. The genotypes of the queens, of the inferred male partner and of the workers are provided based on the PCR-RFLP (see Table S1 and Figure S4). All available monogyne apterous queens were whole-genome sequenced, implying that Mo and Ma could be distinguished. The number of workers of each genotype is provided within the pie charts (see also Table S2). A photograph of each queen type is provided, along with a schematic representation of the thoracic structure (upper left corner), modified from Figure 3 of Buschinger and Schreiber¹¹.

339
341 EXPERIMENTAL MODEL AND SUBJECT DETAILS

342 We sampled 254 colonies in the Fontainebleau Forest (48.493 N 2.652 W, Ile de France, 343 France) in an area of ~0.166 hectares in two time periods. We collected 102 colonies in 344 October 2021 and 152 colonies end of March/beginning of April 2022, just after the 345 hibernation period. Nests of *M. graminicola* consist in a single chamber and were found under 346 mosses. Particular attention was paid to collect the entire colony. Over the two samplings, 347 twenty-nine colonies were queenless (11.4%) and were not considered in this study. 348 Reproductive queens were identified through dissection by the presence of filled spermatheca 349 and developed ovaries. The winged (dealated) queens were identified by their well-developed 350 thorax¹¹ containing wing muscles whereas apterous queens showed a more simplified thorax 351 (Figure 4B). We found three colony types (excluding the queenless ones): monogyne winged 352 colonies (200 colonies, 88.9%) with a single winged reproductive queen (note that some of 353 these colonies also contained unmated non reproductive dealated queens, see the section 354 quantification and statistical analysis), polygyne apterous colonies (19 colonies, 8.4%) with 355 multiple apterous reproductive queens and six monogyne apterous colonies (2.7%). The 356 monogyne apterous phenotype is potentially subject to misidentification, as failure to collect 357 all queens or the recent death of all but one would result in a functionally polygyne colony 358 being classified as phenotypically monogyne. Five of the six monogyne apterous colonies were 359 genetically monogyne whereas one (C8s from the 2021 sampling) was genetically polygyne 360 (see results). Unless otherwise stated, our final data is therefore based on 20 polygyne and 361 five monogyne apterous colony types. 362

362 METHOD DETAILS

363 *M. graminicola* genome assembly

364 We performed the sequencing on one single male haploid individual collected from a polygyne 365 colony. We extracted high-quality DNA from the whole body excluding the abdomen using the 366 MagAttract HMW (High Molecular Weight) DNA Kit protocol. Given the low number of long 367 fragments extracted, the library was prepared following the PacBio ultra-low input protocol 368 and sequenced in one SMRT Cell on the Sequel II System. The dataset included 2,551,751 reads 369 with an average length of 9,925.8 bp (N50: 10,031 bp).
We first assembled the HiFi reads using hifiasm v0.19.8-r603³⁶ with default parameters, generating an initial assembly of 488.254 Mb spread across 3,294 contigs. We improved the scaffolding using the Hi-C data, obtained by sequencing the library prepared on a pool of six haploid Pa males (the social supergene haplotype was established using the PCR-RFLP assay, see below). We mapped the Hi-C reads against the assembly with Chronomap v0.2.6-r490³⁷ and scaffolded the genome using YaHs v1.2⁵⁵ with the --no-contig-ec option. We manually curated misarrangements and defined chromosome boundaries using Pretext-suite (PretextMap v0.1.9 and PretextView v0.2.5). The final genome assembly has an N/L50 of 12/14.767 Mb and contains 2,658 scaffolds. In particular, the 14 largest scaffolds are likely to be representative of the 14 known chromosomes and account for ~55.9% of the total assembly (Figure S1).

## 381 Genome annotation

RepeatModeler v2.0.452³⁸ was used to build de novo *M. graminicola* repeat library, which was subsequently used to annotate repeats using RepeatMasker v4.0.7³⁹ with default parameters. This resulted in ~50% of the assembled genome being annotated and soft-masked as repeat sequence. For the genome annotation, RNA-seq data generated by the GAGA project⁵⁶ from *M. graminicola* brood were aligned to the reference repeat soft-masked genome assembly using STAR v2.7.2b default options⁴⁰. In addition, we retrieved the publicly available gene annotations from the fruit fly *Drosophila melanogaster*, the red flour beetle *Tribolium castaneum*, the parasitoid wasp *Nasonia vitripennis*, the honeybee *Apis mellifera*, the clonal raider ant *Ooceraea biroi* and the Florida carpenter ant *Camponotus floridanus*⁵⁷⁻⁶². The annotations from these insect species were used to conduct homology-based gene predictions using GeMoMa v1.9, which also incorporates the RNA-seq evidence for splice site prediction⁴¹. This general gene annotation was then combined with the specific gene models for Odorant receptors annotated using ABCENTH, generating the final GFF3 annotation file. We also generated a GFF3 file including only one representative isoform per gene (i.e., the longest isoform is kept as the representative). Transposon-related proteins were identified and filtered using: i) a homology-based search against the SwissProt database; ii) the transposable element protein database from RepeatMasker; iii) the repeat-masked regions in the genome assembly.

## 399 Whole genome resequencing and data processing
We retrieved whole-genome sequence data for a subset of 40 monogyne winged reproductive queens, all the six queens of the putative monogyne apterous colonies and one queen of each of the 19 polygyne colonies. We further selected one additional queen from three of the 19 colonies, as a parallel ongoing project showed them to be unrelated (Ducancel, unpublished data). After the misidentification of one monogyne apterous queen (which resulted to be genetically polygyne, see below), the final dataset is composed of 68 unrelated queens (see below): 40 monogyne winged, five monogyne apterous, 23 polygyne apterous.

DNA was isolated from the head and the thorax of 68 queens using DNeasy Blood and Tissues Kit (QIAGEN). DNA libraries were paired-end sequenced with an Illumina NovaSeq 6000. Two runs were performed, pooling the 22 and 46 individuals from 2021 and 2022 sampling respectively. All the 68 resequenced queens were confirmed to be unrelated, after estimating the pairwise relatedness under the maximum-likelihood algorithm of Queller and Goodnight implemented in the R library *related*.

We trimmed raw reads using trimmomatic-0.39⁴² and aligned them against the *de novo* reference genome with the BWA-MEM algorithm⁴³. We tagged PCR duplicates using the 'MarkDuplicates' function of the Picard toolkit v2.25.6 (https://broadinstitute.github.io/picard/). We performed variant calling following GATK v4.0 best practices⁴⁴. First, we fed BAM files to HaplotypeCaller with -ERC BP_RESOLUTION to produce gVCFs containing all positions. Then, we used GenomicsDBImport and GenotypeGVCFs on each autosome separately, setting --include-non-variant-sites to true. We applied a filtering step using VariantFiltration to retain only positions with MQ > 30. Based on empirical distributions, we removed genotypes with a sequencing depth below 10 or above 50 and discarded positions within repeat regions. Finally, we retained only biallelic markers for further analyses.

## Genomic regions associated with sociality

Monogyne apterous queens are subject to misidentification in the field due to failure to collect all queens or the loss of all but one. To identify genomic regions associated to sociality we therefore focused only on the comparison between monogyne winged and polygyne queens. Candidate regions should fulfil three characteristics: i) *Fst* above the 95th percentile of the empirical distribution; ii) strong LD relative to genome-wide average; iii) clearly distinguishing
the social morph on the first axis of the PCA. Fst⁶³ was computed on sites with no missing data in windows of 10 kb with a 2.5 kb slide containing a minimum of 10 SNPs. Pairwise linkage disequilibrium (LD) was estimated by calculating r² values sampling one SNP every 100 using the *LDheatmap* R library⁴⁶ on a dataset with SNPs filtered as follows: < 20% of missing data; heterozygosity rate < 80% and a minor allele frequency < 0.05. Local PCA was computed in R with *pcadapt* v4.4⁴⁷. Using these criteria, we found a region of ~20 Mb located within the CHR1 (coordinates: ~5,940,000 to ~26,615,000), which we defined as the “social supergene”. We further compared the genotypic distribution of the biallelic SNPs with no missing data in the three morphs within the social supergene. We finally computed genome-wide both the PCA (on the 68 queens) and the genotypic distribution by morph, excluding the chromosome 1 (carrying the social supergene), and the chromosomes 4 and 7, as they present large structural variants with unknown phenotypic association.

442

443 Genetic basis of wing polymorphism

The PCA on the social supergene allowed us to define the five monogyne apterous colony as being genetically monogyne (see Results). To detect genomic regions associated with the wing polymorphism, we computed the *Fst* in sliding windows between monogyne winged and the five monogyne apterous queens (same parameters as before). We found a candidate region of ~116 kb within the social supergene (see Results, Figure S2) which we further inspected for sequencing depth variation. This region is absent in winged queens and hemizygotic in all but one (diploid) apterous queen (see Results). Finally, we mapped this region against the assembled genome using Minimap v2 implemented in the alignment tool D-Genies⁴⁸ and the Nucmer function of the MUMMER tool (v4.0.0beta2)⁴⁹. We found a significant match on CHR4 which was further cross-validated. The region duplicated between CHR1 and CHR4 contains a single conserved gene (named by us *Awing*), see Figure S3.

455 Evolution of the inserted region

We did a second variant calling using only apterous individuals hemizygotic for the insertion using the same pipeline as before but setting ploidy to 1. We retained SNPs with no missing data and with a depth of coverage between 5 and 50. We computed the divergence time between the copies of *Awing* present within CHR4 and CHR1 (hereafter, *AwingDPL*), a proxy
for the duplication event. First, we aligned the codons of *Awing* and *AwingDPL* from *M. graminicola* with the ortholog of *Awing* from seven other species (*Pristomyrmex punctatus* GCA_048543395.1, *Acanthomyrmex ferox* GCA_048543515.1, *Eutetramorium mocquerysi* GCA_048542615.1, *Temnothorax rugatulus* GCA_048541785.1, *Temnothorax nylanderi* GCA_048541665.1, *Temnothorax pilagens* GCA_048544075.1 Scaffold, *Vollenhovia emeryi* GCF_000949405.1), see Figure S3. We then ran MCMCTree⁵⁰ to estimate the age of the nodes of the phylogenetic tree by calibrating the clock rate using fossil records. We used three calibration points: 36 Mya divergence between *Vollenhovia emeryi* and *Eutetramorium mocquerysi*⁶⁴; 36 Mya divergence between *Myrmecina graminicola* and *Temnothorax rugatulus*⁶⁵, and 17 Mya divergence between *Temnothorax rugatulus* and *Temnothorax nylanderi*⁶⁶. Furthermore, we set the age of Crematogastrini at 52 Mya following⁵⁶. The second method was based on the equation $T_g = \text{Div}/2\mu$, where $T_g$ is the generation time, Div the estimated nucleotide divergence and $\mu$ the haploid mutation rate. We used a generation time of 3 years, a mutation rate of $3.5 \times 10^{-9}$ (the average between the $3.4 \times 10^{-9}$ for *Apis mellifera*⁶⁷ and $3.6 \times 10^{-9}$ for *Bombus terrestris*⁶⁸). The nucleotide divergence was estimated using the GTR+F+G4 substitution model on the codon alignment using IQ-TREE v2.4.0⁵¹ (Figure S3). Finally, we estimated the genetic divergence between Mo and Pa by computing the number of SNPs that are homozygote in all MoMo queens and heterozygotes in all MoPa queens, scaled by the total number of positions (variable and non-variable) called in the selected regions. We restricted the analysis to the annotated genes within the social supergene and converted the divergence in years with the above formula.

## Cross-species comparison

We aligned the ~20MB region of *CHR1* associated to the social polymorphism (including the insertion containing *AwingDPL*) and the region of *CHR4* containing *Awing* (see Results) against the whole-genome assemblies of *F. selysi* (GenBank assembly: GCA_009859135.1), *S. invicta* (GenBank assembly: GCA_016802725.1) and *Pogonomyrmex californicus* (GenBank assembly: GCA_024349325.1) using NUCMER function from the MUMMER tool (v4.0.0beta2)⁶⁹ with the default parameters. Alignments were represented by means of a circos plot (circos-0.69-9⁵²). To eliminate noise from the numerous small alignments and make the circos more readable, all alignments smaller than 200 bp were discarded except within the duplicated gene sequence, where the threshold was lowered to 100 bp.
To comprehensively identify genes in the two regions (on CHR1 and CHR4) associated with the wing polymorphism, we employed multiple complementary annotation strategies. First, we used the existing GAGA annotation⁵⁶. We then predicted genes using Braker 3 (v3.0.7.4)⁵³ in protein mode with a reference database composed of RefSeq ant proteomes (24 species; https://www.ncbi.nlm.nih.gov/datasets/genome/?taxon=36668; Accessed in 04 2024). Additionally, we applied the deep learning-based annotation tool Helixer (v0.3.4)⁵⁴ with the invertebrate model.

## RFLP screen of the supergene alleles

We designed a restriction fragment length polymorphism (PCR-RFLP) assay to rapidly assess the three haplotypes at the social supergene in a larger dataset. To differentiate M* from Pa, we set two pairs of primers to amplify two regions of the social supergene (outside the insertion). The two regions contained a restriction site for EcoRV, one cutting M* (M-CHR1) and the other Pa (P-CHR1) (Tables S1, S2). To differentiate the presence and absence of the insertion (Mo from Pa or Ma), we designed one pair of primers amplifying two regions: one on the insertion and the other on CHR4 (to which the insertion is orthologous). The region on CHR4 contains a restriction site for BsuRI enzyme in all queens (W-CHR4). This restriction site contains a mutation on CHR1, preventing the restriction (Table S2, Figure S4). Note that our test cannot distinguish whether the insertion is hemizygous or occurs in two copies (Figure S4). This implies that we cannot discriminate neither MoMa from MaMa nor MoPa from MaPa, for which we would need the depth of coverage. This is why we refer to the genotype assessed by the PCR-RFLP assay as M*Ma and M*Pa when lacking further information (i.e., resequencing data).

Each reaction mixture (total volume of 15 µl) contained 1 µl of genomic DNA, 0.21 µl of Taq polymerase, 0.375 µl of dNTPs, 1.5 µL of PCR 10X reaction buffer, and 0.375 µL of each primer. The thermal cycling profile was 5 min at 95°C, followed by 35 cycles of 60s at 95°C, 60s at the corresponding annealing temperature (58° for M-CHR1, 61° for P-CHR1, 59° for W-CHR4) and 90s at 72°C, ending with 10 min at 72°C. 10 µL of the PCR product was then digested with 2 µL of the corresponding restriction enzyme (Table S6) for 17 hours at 37°C in the appropriate buffer (total volume 31.5 µL). We visualized the DNA products with 2% agarose gel electrophoresis. The validity of the three assays was assessed by screening the 68 queens
retained for the whole genome resequencing. We retrieved the known genotypes from all of them, based on the three enzymes. We then successfully screened with the three PCR-RFLP tests: i) 67 monogyne winged queens (9 were non reproductive queens) from 58 additional colonies; ii) all the 54 additional polygyne apterous queens (from the same 20 polygyne colonies used for the whole-genome sequencing) but five could not be correctly genotyped after two trials leading to 49 polygyne apterous queens.

Finally, we successfully screened: i) two workers for 42 monogyne winged colonies (84 workers); ii) an average of 16.2 workers from the five monogyne apterous colonies (81 workers); iii) an average of 18.7 workers from the 20 polygyne apterous colonies (374 workers). All workers were screened with M-CHR1 and W-CHR4. Those genotyped as PaPa were double checked with the P-CHR1.

533

## 534 QUANTIFICATION AND STATISTICAL ANALYSIS

535

### 536 Frequencies of colony types

The frequency of the three colony types (confirmed by genetic screens) did not significantly differ between the two samplings (Fisher exact test, p = 0.64, Figure 1A). Note that 44 of the monogyne winged colonies contained multiple unmated non reproductive (dealated) winged queens, i.e., lacking filled spermatheca and developed ovaries. We found 16 colonies in 2021 sampling and 28 colonies in 2022 sampling, with an average of 5.4 non-reproductive queens (min = 2, max = 20). The number of unmated non-reproductive queens in these monogyne colonies was significantly higher in April 2022 (mean (min-max) = 6.7 (2-20)) than in October 2021 (3.1 (2-11)) (Wilcoxon nonparametric test, W = 133, p = 0.021). Determining the relatedness between these non-reproductive queens and the reproductive one will help to put forward evolutionary hypotheses to interpret this finding. These non-reproductive queens were not found in polygyne or monogyne apterous colonies. The number of reproductive queens in polygyne colonies were on average 4, with a minimum of 2 (not considering the misidentified colony with a single queen) and a maximum of 13, giving a total of 77 queens.
Shortly after collection, workers were counted for each colony type. Monogyne winged colonies (mean ± sd: 74.0 ± 45.8) did not significantly differ from polygyne apterous colonies (mean ± sd: 84.7 ± 51.1) (Type III SS, two-way Anova, F[1;216] = 0.006, p = 0.94) whatever the sampling date (interaction effect: F[1;216] = 0.006, p = 0.94). Not surprisingly, the colony size was significantly lower just after the hibernation period in April 2022 (mean ± sd : 63.8 ± 35.8) than in October 2021 (mean ± sd: 91.9 ± 54.8) (F[1;216] = 17.42, p < 0.0001). Note that we removed from the analysis the five monogyne apterous colonies (mean ± sd: 86.8 ± 60.0), their number being too low for conducting a proper statistical test.

All statistical analyses were conducted using the R software⁴⁵.

559

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777 **Figure 4**