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Convergent and complementary selection shaped gains and losses of eusociality in sweat bees

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Abstract

Sweat bees have repeatedly gained and lost eusociality, a transition from individual to group reproduction. Here we generate chromosome-length genome assemblies for 17 species and identify genomic signatures of evolutionary trade-offs associated with transitions between social and solitary living. Both young genes and regulatory regions show enrichment for these molecular patterns. We also identify loci that show evidence of complementary signals of positive and relaxed selection linked specifically to the convergent gains and losses of eusociality in sweat bees. This includes two pleiotropic proteins that bind and transport juvenile hormone (JH)—a key regulator of insect development and reproduction. We find that one of these proteins is primarily expressed in subperineurial glial cells that form the insect blood–brain barrier and that brain levels of JH vary by sociality. Our findings are consistent with a role of JH in modulating social behaviour and suggest that eusocial evolution was facilitated by alteration of the proteins that bind and transport JH, revealing how an ancestral developmental hormone may have been co-opted during one of life’s major transitions. More broadly, our results highlight how evolutionary trade-offs have structured the molecular basis of eusociality in these bees and demonstrate how both directional selection and release from constraint can shape trait evolution.

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Fig. 1: Comparative genomic resources for halictid bees.
Fig. 2: The maintenance of eusociality is associated with young genes and gene regulation.
Fig. 3: Signatures of selection associated with the gains and losses of eusociality in halictids.
Fig. 4: apolpp and associated lipid transport genes are expressed in glial cells.
Fig. 5: JH is higher in brains of eusocial foundresses and crosses the BBB.

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Data availability

Raw sequencing data are available at NCBI under the following accession numbers: PRJNA613468, PRJNA629833, PRJNA718331 and PRJNA512907 (Hi-C). Hi-C data and genome assemblies are also available at www.dnazoo.org. Genomes and browsers can be accessed at beenomes.princeton.edu and on NCBI: PRJNA613468. Please address inquiries or material requests to skocher@princeton.edu.

Code availability

Repository with code is on GitHub at https://github.com/kocherlab/HalictidCompGen.

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Acknowledgements

We thank our many colleagues who contributed samples and field support to this dataset, including: J. Gibbs, S. Droege, R. Jeanson, J. Milam, J. Straka, M. Podolak, J. Cane and M. Hagadorn; M. Richards, C. Plateaux-Quenu, J. Gibbs and L. Packer for discussion and insights on halictid life history and behaviour; T. Sackton, R. Corbett-Detig, N. Clark, A. Siepel and X. Xue for providing discussion and advice on data analysis; W. Tong for the bee drawings and M. Sheehan for assistance with RNA library preparation; and J. Rabinowitz and lab for support and access to LC–MS equipment. This work was supported by NSF-DEB1754476 awarded to S.D.K. and B.G.H., NIH 1DP2GM137424-01 to S.D.K., USDA NIFA postdoctoral fellowship 2018-67012-28085 to B.E.R.R., DFG PA632/9 to R.J.P., a Smithsonian Global Genome Initiative award GGI-Peer-2016-100 to W.T.W. and C.J.K., a Smithsonian Institution Competitive Grants Program for Biogenomics (W.T.W., K.M.K., B.M.J.), a Smithsonian Tropical Research Institute fellowship to C.J.K., and a gift from Jennifer and Greg Johnson to W.T.W. M.F.O. was supported by Vicerrectoría de Investigación, UCR, project B7287. E.L.A. was supported by an NSF Physics Frontiers Center Award (PHY1427654), the Welch Foundation (Q-1866), a USDA Agriculture and Food Research Initiative Grant (2017-05741), and an NIH Encyclopedia of DNA Elements Mapping Center Award (UM1HG009375). Sampling permit details: S.D.K., E.S.W. and M.F.O. (R-055-2017-OT-CONAGEBIO), S.D.K. (P526P-15-04026) and R.J.P. (Belfast City Council, Parks and Leisure Dept).

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Author notes

  1. These authors contributed equally: Beryl M. Jones, Benjamin E. R. Rubin, Olga Dudchenko.

  2. These authors contributed equally: Erez Lieberman Aiden, Sarah D. Kocher.

Authors and Affiliations

  1. Department of Ecology and Evolutionary Biology, Princeton University, Princeton, NJ, USA

    Beryl M. Jones, Benjamin E. R. Rubin, Callum J. Kingwell, Ian M. Traniello, Z. Yan Wang, Eli S. Wyman, Weijie Liu, Andrew E. Webb, Kennedy S. Omufwoko, Nikki Van Dorp & Sarah D. Kocher

  2. Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA

    Beryl M. Jones, Benjamin E. R. Rubin, Callum J. Kingwell, Ian M. Traniello, Z. Yan Wang, Eli S. Wyman, Weijie Liu, Lance R. Parsons, S. RaElle Jackson, Katharine Goodwin, Shawn M. Davidson, Matthew J. McBride, Andrew E. Webb, Kennedy S. Omufwoko, Nikki Van Dorp & Sarah D. Kocher

  3. The Center for Genome Architecture, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA

    Olga Dudchenko, Per A. Adastra, Melanie Pham, Arina D. Omer, David Weisz & Erez Lieberman Aiden

  4. Center for Theoretical Biological Physics, Rice University, Houston, TX, USA

    Olga Dudchenko & Erez Lieberman Aiden

  5. Smithsonian Tropical Research Institute, Panama City, Republic of Panama

    Callum J. Kingwell, Karen M. Kapheim & William T. Wcislo

  6. Department of Biology, Utah State University, Logan, UT, USA

    Karen M. Kapheim

  7. Department of Chemistry, Princeton University, Princeton, NJ, USA

    Matthew J. McBride

  8. Biodiversity and Tropical Ecology Research Center (CIBET) and School of Biology, University of Costa Rica, San José, Costa Rica

    Mauricio Fernández Otárola

  9. Department of Biology, Temple University, Philadelphia, PA, USA

    Joshua Schraiber & Fernando Villanea

  10. Illumina Artificial Intelligence Laboratory, Illumina Inc, San Diego, CA, USA

    Joshua Schraiber

  11. Department of Anthropology, University of Colorado Boulder, Boulder, CO, USA

    Fernando Villanea

  12. Institute of Biology, Martin-Luther University Halle-Wittenberg, Halle, Germany

    Robert J. Paxton

  13. German Centre for Integrative Biodiversity Research (iDiv), Halle-Jena-Leipzig, Germany

    Robert J. Paxton

  14. Department of Entomology, University of Georgia, Athens, GA, USA

    Brendan G. Hunt

Authors
  1. Beryl M. Jones
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  2. Benjamin E. R. Rubin
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  29. Sarah D. Kocher
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Contributions

B.M.J., B.E.R.R. and S.D.K. designed the study. B.M.J., B.E.R.R. and S.D.K. wrote the initial draft of the manuscript. All authors revised and commented on the manuscript. B.M.J., B.E.R.R., E.S.W., B.M.J., S.D.K., M.F.O., K.M.K., R.J.P., W.T.W., C.J.K. and K.S.O. collected samples. B.E.R.R., C.J.K., Z.Y.W. and N.V.D. generated genomic libraries. O.D., P.A.A., M.P., A.D.O. and D.W. performed Hi-C sequencing and genome assembly. B.E.R.R., L.R.P. and A.E.W. conducted genome annotation/alignment. B.M.J., B.E.R.R., S.D.K., O.D., K.M.K., B.G.H., J.S., F.V. and I.M.T. conducted genomic analyses. A.E.W. curated the database. W.L., E.S.W., S.R.J., K.G., S.M.D., C.J.K., Z.Y.W., M.J.M., K.S.O. and N.V.D. performed laboratory experiments. E.L.A. supervised Hi-C sequencing and genome assembly. S.D.K. provided overall project supervision.

Corresponding author

Correspondence to Sarah D. Kocher.

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Nature Ecology & Evolution thanks Guojie Zhang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Typical life cycle for solitary and eusocial sweat bees.

In a typical solitary species, reproductive females produce a single brood that is a mix of males and females. However, some solitary species are multivoltine and can produce multiple reproductive broods in a year. In typical eusocial halictid species, females produce two broods: first workers, then reproductives. At the end of the season, females mate and overwinter as adults.

Extended Data Fig. 2 Genome assembly statistics.

Nineteen genomes are included in this comparative dataset. 15 de novo assemblies were generated using a combination of 10x genomics and/or Hi-C, and 2 previously-published genomes (N. melanderi11, L. albipes3) were improved by scaffolding with Hi-C data. M. genalis13 and D. novaeangliae12 were used as-is. (a) Genome assembly lengths ranged from ~300 to 420 Mb. (b) Scaffold N50s for each species following Hi-C scaffolding, and (c) GC content was consistent across species. (d) RepeatModeler was used to characterize different types of repeats in the halictid assemblies. (e) Numbers of genes (in thousands) for each species following individual annotation. (f) Conserved non-exonic elements (CNEEs) were called using phastCons on a progressive Cactus alignment. (g) microRNAs were also characterized using brain tissue from available species and from34. For some species, fresh tissue was not available (N/A).

Extended Data Fig. 3 Human-mouse chromosomal dotplot.

Dotplots comparing the chromosomes of two mammalian species, human and mouse, separated by comparable evolutionary distance to the bees examined in this study (~80MY to common ancestor). The vertical and horizontal lines outline the boundaries of chromosomes in respective species, and the numbers on the axes mark the relevant chromosome name and orientation, with ‘-’ in front of the chromosome name representing a reverse complement to the chromosome sequence as reported in the assembly. See Extended Data Fig. 4 for more details. The human-mouse one-to-one alignments file was downloaded from https://github.com/mcfrith/last-genome-alignments.

Extended Data Fig. 4 Pairwise chromosomal dotplots for halictid species.

Dotplots showing alignment of chromosome-length scaffolds from 16 bee assemblies against the N. melanderi (NMEL) chromosome-length genome assembly. The NMEL reference (generated as part of this study) is shown on the y-axis. The x-axis shows the chromosome-length scaffolds in the respective bee assemblies that have been ordered and oriented to best match the NMEL chromosomes in order to facilitate comparison. The vertical and horizontal lines outline the boundaries of chromosomes in respective species, and the numbers on the axes mark the relevant chromosome name and orientation, with ‘-’ in front of the chromosome name representing a reverse complement to the chromosome sequence as reported in the assembly. Each dot represents the position of a 1000 bp syntenic stretch between the two genomes identified by Progressive Cactus alignments. The colour of the dots reflects the orientation of individual alignments with respect to NMEL (red indicates collinearity, whereas blue indicates inverted orientation). The dotplots illustrate that, with the exception of a few species, highly conserved regions belonging to the same chromosome in one species tend to lie on the same chromosome in other bee species, even though their relative position within a chromosome may change dramatically. This rearrangement pattern accounts for the characteristic appearance of the dotplots with large diffuse blocks of scrambled chromosome-to-chromosome alignments appearing along the diagonal. The pattern is visibly different from those characteristic of mammalian chromosome evolution (for example, see Extended Data Fig. 3). The few exceptions are species with multiple fissions (L. marginatum, L. albipes) and fusions (Augochlora pura, L. vierecki, L. pauxillum, L. malachurum) events. In the fission species, the syntenic regions that belonged to two different chromosomes in one bee species tend to belong to different chromosomes after the fission. The analysis of the fusion species suggests that the regions corresponding to separate chromosomes in the closely related species (and likely the ancestral species) remain separate in the new genome, possibly corresponding to the two chromosome arms. The alignments have been extracted from the hal file using the cactus hal2maf utility with the following parameters: –maxRefGap 500 –maxBlockLen 1000 –refGenome NMEL.

Extended Data Fig. 5 Relationship between selection and gene age associated with eusocial origins, maintenance, and reversions to solitary life histories.

Gene age ranges from the oldest Bilaterian group (Age=1) to the youngest, halictid-specific taxonomically restricted genes (Age=9). The relaxation panel demonstrates that there is a greater proportion of young genes experiencing relaxed selection when eusociality is lost (HyPhy RELAX, FDR < 0.1; Pearson’s r = 0.869, p = 0.002); we find no significant association with relaxation on extant, eusocial branches (Pearson correlation, r = −0.044, p = 0.91). Next, we looked at the sets of genes that show intensification of selection pressures (HyPhy RELAX, FDR < 0.1), but neither of these sets showed any significant association with gene age (Eusocial: Pearson correlation, r = 0.244, p = 0.492; Solitary: Pearson correlation, r = 0.627, p = 0.071). Finally, we looked at genes that showed evidence of positive selection (HyPhy aBSREL, FDR < 0.05). We found no relationship between gene age and the proportion of genes with evidence of positive selection on at least 1 branch representing the origins of eusociality (Positive, gains: Pearson correlation, r = 0.156, p = 0.688). Likewise, there was no relationship between gene age and the proportion of genes with evidence for positive selection (HyPhy aBSREL, FDR < 0.05) on at least 1 loss branch in the Halictini and on 1 loss branch in the Augochlorini (Positive, losses: Pearson correlation, r = 0.403, p = 0.283). Shading represents the 95% confidence intervals.

Extended Data Fig. 6 Evidence of positive selection in domains of Hex110 and ApoLpp.

Both Hex110 (a) and ApoLpp (b) show evidence for domain-specific positive selection associated with the origins of eusociality. Predicted binding pockets by PHYRE are shown with pink rectangles, glycosylation sites in orange squares. Pink hexagons denote MEME-identified sites that also had a mutational effect score > =1 for Hex110 and >1 for ApoLpp. In both JHBPs, MEME identifies sites in functional regions of the protein, including the receptor binding domain and predicted binding pocket for ApoLpp as well as in all three Hemocyanin domains (associated with storage functions in these proteins) and in the predicted binding pocket of Hex110. Moreover, for both proteins, we also find region-specific, faster rates of evolution on eusocial branches compared to non-eusocial outgroups in this phylogeny: the predicted binding pocket for ApoLpp and all Hemocyanin domains for Hex110. Taken together, these results suggest that positive selection shaped protein function as eusociality emerged in this group of bees.

Extended Data Fig. 7 Apolipoprotein has experienced pervasive positive selection throughout the insects.

HyPhy aBSREL was used to identify branches with evidence of positive selection (highlighted in blue). Arrows indicate the direction of significant rate shifts detected on relevant branches. The only branch tested that did now show a significant rate shift is indicated by an ‘o’.

Extended Data Fig. 8 JH III is present in multiple bumblebee tissue types, including the brain.

LC-MS was used to measure JH III levels in dissected brain tissue and hemolymph from worker bumblebees (Koppert). Tissues were dissected in PBS. Negative control=fresh PBS, PBS control=3uL of buffer collected following brain dissections from each sample. RCC = retrocerebral complex. The RCC synthesizes JH and immediately releases it, it is not known to store JH78. Hemolymph (3uL) was collected by centrifuging thorax tissue from each sample. Ovaries and brains were dissected in PBS and the entire organ was used for JH III quantification. In all samples, we find detectable levels of JH III in the hemolymph, brain, and ovaries. N = 5 for all sample/tissue types except negative control, where n = 1. Note that because all samples were generated by extraction in a constant volume of buffer from the total input tissue, estimated amounts are not directly comparable across different tissue types. Boxes show median, 25th and 75th percentiles. Whiskers show minimum and maximum values without outliers.

Extended Data Fig. 9 Stable Isotope Tracing of Juvenile Hormone (JH).

Absolute quantification of JH III in the brain (a) and bodies (d) of bumblebees treated with acetone or JH III-D3. Absolute quantification of labelled JH III in the brains (b) and bodies (e) of bees treated with acetone or JH III-D3. Labelled JH III-D3 levels are high after 24 hours, but decay significantly by 72 hours. (c) Labelled JH III-D3 applied to abdomens of bumblebees is detectable in brains 24 h later (acetone is control). n = 3 tissues/condition. (f) JH III-D3 accounts for nearly all the total JH III in bee bodies after 24 hours, indicating that the labelled compound is well-absorbed by the bee. n = 3 bumblebee workers for each experimental condition. Boxes show median, 25th and 75th percentiles. Whiskers show minimum and maximum values.

Extended Data Fig. 10 JH III and JH III-D3 quantification.

(a) Chemical structure of JH III in positive ionization. JH III can exist in two forms of equal m/z and produces one fragment that retains the D3 label. (b) Parent and fragment m/z values for unlabelled and labelled JH III. (c) Chromatograph of unlabelled JH III. (d) Chromatograph of labelled JH III showing similar retention time to unlabelled JH III. (e) Mass spectra of unlabelled JH III showing the expected masses based on B. (f) Mass spectra of a 50/50 mix of labelled and unlabelled JH III showing the expected masses based on B. (g) Standard curve demonstrating a range of detection of JH III. (h) Chromatograph of unlabelled JH III in the brains of bees painted with acetone (grey dotted line) or with JH III-D3 (purple solid line). (i) Chromatograph of labelled JH III in the brains of bees painted with acetone (grey dotted line) or with JH III-D3 (purple solid line). (j) Mass spectra of JH III in bee brains painted with acetone showing the expected masses based on B. (k) Mass spectra of JH III in bee brains painted with JH III-D3 showing the expected masses based on B. (l) Absolute quantification of JH III in the brains of bees painted with acetone or JH III-D3. (m) Absolute quantification of JH III-D3 in the brains of bees painted with acetone or JH III-D3. n = 3 replicates for each panel. Boxes show median and 25th and 75th percentiles. Whiskers show minimum and maximum values.

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Jones, B.M., Rubin, B.E.R., Dudchenko, O. et al. Convergent and complementary selection shaped gains and losses of eusociality in sweat bees. Nat Ecol Evol 7, 557–569 (2023). https://doi.org/10.1038/s41559-023-02001-3

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  • DOI: https://doi.org/10.1038/s41559-023-02001-3

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