Jonathan Romiguier
email: jonathan.romiguier@umontpellier.fr

Marek L Borowiec

Arthur Weyna

Quentin Helleu

Etienne Loire

Christine La Mendola

Christian Rabeling

Brian L Fisher

Philip S Ward

Laurent Keller

Ant phylogenomics reveals a natural selection hotspot preceding the origin of complex eusociality

The evolution of eusociality has allowed ants to become one of the most conspicuous and ecologically dominant groups of organisms in the world. A large majority of the current ~14,000 ant species belong to the formicoids 1 , a clade of nine subfamilies which exhibit the most extreme forms of reproductive division of labour, large colony size 2 , worker polymorphism 3 and extended queen longevity 4 . The eight remaining non-formicoid subfamilies are less well studied, with few genomes having been sequenced so far and unclear phylogenetic relationships 5 . By sequencing 65 genomes, we provide a robust phylogeny of the 17 ant subfamilies, retrieving high support to the controversial leptanillomorph clade (Leptanillinae and Martialinae) as the sister-group to all other extant ants. Moreover, our genomic analyses revealed that the emergence of the formicoids was accompanied by an elevated number of positive selection events. Importantly, the top three gene functions under selection are linked to key features of complex eusociality with histone acetylation being implicated in caste differentiation, gene silencing by RNA in worker sterility and autophagy in longevity. These results show that key pathways associated with eusociality have been under strong selection during the Cretaceous, suggesting that the molecular foundations of complex eusociality may have evolved rapidly in less than 20 Ma.

Results and discussion

A reference tree of ant subfamilies

To build a comprehensive phylogenetic tree including representatives of all extant ant subfamilies, we conducted two main types of analyses on the 4,300,911 amino acids from 4,151 single-copy protein-coding genes that we generated from 83 species (including 9 hymenopteran outgroups, and 65 newly sequenced genomes; see Material and Methods). First, we performed supermatrix approaches, where all genes were concatenated for estimating a single species tree. Second, we performed supertree approaches, where the species trees were estimated from all gene trees. The two best resulting trees from each approach are summarized in Figure 1. Each node of the tree is supported with maximal support by the supermatrix analyses, and nearly all nodes (78/82) are congruently supported by both the supermatrix and supertree approaches. Importantly, the deepest and most important nodes of the ant phylogeny, including every relationship among the 17 ant subfamilies are maximally supported by both the supermatrix and supertree approaches.

Our results confirm that the so-called poneroid subfamilies (Ponerinae, Paraponerinae, Agroecomyrmecinae, Proceratinae, Apomyrminae, Amblyoponinae) are monophyletic [START_REF] Moreau | Phylogeny of the ants: diversification in the age of angiosperms[END_REF][START_REF] Ward | Tales of dracula ants: the evolutionary history of the ant subfamily Amblyoponinae (Hymenoptera: Formicidae)[END_REF][START_REF] Branstetter | Enriching the ant tree of life: enhanced UCE bait set for genome-scale phylogenetics of ants and other Hymenoptera[END_REF][START_REF] Borowiec | Compositional heterogeneity and outgroup choice influence the internal phylogeny of the ants[END_REF] , rather than paraphyletic [START_REF] Rabeling | Newly discovered sister lineage sheds light on early ant evolution[END_REF][START_REF] Kück | Improved phylogenetic analyses corroborate a plausible position of Martialis heureka in the ant tree of life[END_REF][START_REF] Brady | Evaluating alternative hypotheses for the early evolution and diversification of ants[END_REF] . Both the supermatrix and supertree provide maximum support for the poneroid monophyly. The relationships among poneroid subfamilies are also all congruently supported by both approaches (Figure 1), including for the Paraponerinae (one living species) and Agroecomyrmecinae (two living species) which are inherently difficult to relate with other subfamilies as their deep phylogenetic divergence resulting in long branches. An analysis of ultraconserved elements (UCE) markers for an increased data set of 166 taxa (see STAR Methods) retrieved the same subfamily relationships (Figure S1), further supporting the view that our phylogeny (Figure 1) is robust.

These analyses are important because the rooting of the ant phylogeny has been a controversial issue since the discovery of Martialis heureka, an extremely rare and morphologically divergent ant species that was initially inferred as the sister-group of all other ants [START_REF] Rabeling | Newly discovered sister lineage sheds light on early ant evolution[END_REF] . Some studies suggested that Leptanillinae is the subfamily sister to all other ants [START_REF] Kück | Improved phylogenetic analyses corroborate a plausible position of Martialis heureka in the ant tree of life[END_REF][START_REF] Moreau | Testing the museum versus cradle tropical biological diversity hypothesis: phylogeny, diversification, and ancestral biogeographic range evolution of the ants[END_REF] , while a recent study suggested that Leptanillinae and Martialinae may form a monophyletic group [START_REF] Borowiec | Compositional heterogeneity and outgroup choice influence the internal phylogeny of the ants[END_REF] . Our analyses support this last hypothesis, with Leptanillinae+Martialinae forming a clade (hereafter referred to as the leptanillomorph clade) that is the sister-group to all other ants (see Figure 1). This conclusion was also supported by two further analyses controlling for outgroup composition, which has been suggested to affect the rooting of the ant tree [START_REF] Borowiec | Compositional heterogeneity and outgroup choice influence the internal phylogeny of the ants[END_REF] . First, we built an alternative supermatrix of 2,343 genes (983,951 amino acids) containing many outgroups, with 115 non-ant aculeate species borrowed from published transcriptomes [START_REF] Peters | Evolutionary history of the Hymenoptera[END_REF] . These analyses revealed that the rooting of the ant phylogeny and subfamily relationships are not affected by the inclusion of all these outgroups (see Figure S1B). Second, using random combinations of outgroups (see STAR Methods for details), we always found the same rooting, with strong support for the leptanillomorph clade being sister to all other ants (bootstrap values: 100 for 109 trees and 99 for the remaining 6 trees). Because all species of the leptanillomorph clade are pale, blind and have a similar hypogeic ecology, this suggests that some early ants may have escaped extinction by retreating to these stable, subterranean habitats before other lineages diversified by developing novel morphological and behavioral adaptations [START_REF] Rabeling | Newly discovered sister lineage sheds light on early ant evolution[END_REF] . According to our divergence date estimates, the common ancestor of the leptanillomorphs lived around the Jurassic-Cretaceous boundary (~145 Ma) shortly after the common ancestor of all extant ants (~150 Ma). This suggests the possibility that subterranean lifestyles existed in the ancestors of extant ants or, more likely, that a hypogeic lifestyle originated at an early stage in the history of leptanillomorphs. This result contrasts with the fossil evidence, because the earliest-known fossilized crown ants were not specialized to subterranean habitats and come from Burmese amber deposits that are approximately 99 million years old [START_REF] Barden | Adaptive radiation in socially advanced stem-group ants from the Cretaceous[END_REF][START_REF] Boudinot | Evolution and systematics of the Aculeata and kin (Hymenoptera), with emphasis on the ants (Formicoidea: †@@@idae fam. nov., Formicidae)[END_REF] . Set against our divergence date estimates, this indicates a gap in the ant fossil record of ~50 million years, further emphasizing an existing discrepancy between fossils and molecular data when it comes to the question of ant origins [START_REF] Borowiec | Ants: Phylogeny and classification[END_REF] . This is reminiscent of the debate on the origin of placental mammals, estimated be in the middle Cretaceous by molecular data, whereas there is no fossil record before the K-T crisis [START_REF] Dos Reis | Phylogenomic datasets provide both precision and accuracy in estimating the timescale of placental mammal phylogeny[END_REF] . It has been suggested that the lack of fossils may stem from the occurrence of only a few lineages of placental mammals and perhaps small populations sizes during the Cretaceous [START_REF] Cooper | Evolutionary explosions and the phylogenetic fuse[END_REF] . Similarly, it is possible that the abundance of crown ants was low at first, and only sufficiently increased with the rise of angiosperm [START_REF] Moreau | Phylogeny of the ants: diversification in the age of angiosperms[END_REF] to be represented in the fossil record. Alternatively, there may be methodological biases leading to overestimation of divergence dates [START_REF] Springer | Evolutionary models for the diversification of placental mammals across the KPg boundary[END_REF][START_REF] Bromham | Bayesian molecular dating: opening up the black box[END_REF] or incorrect phylogenetic placement of early ant fossils [START_REF] Boudinot | Evolution and systematics of the Aculeata and kin (Hymenoptera), with emphasis on the ants (Formicoidea: †@@@idae fam. nov., Formicidae)[END_REF] .

Pervasive positive selection associated with the origin of the socially diverse formicoid clade

To investigate the molecular changes associated with the evolution of complex eusociality, we conducted positive selection analyses on the 4,151 ortholog genes of the 75 ant genomes (see STAR Methods). The percentage of genes under positive selection varied greatly among the 38 branches ranging from 0 to 2.6% in a single branch (Figure 2). Strikingly, the branch leading to the formicoid clade stands out as a clear outlier with a 30fold higher rate of positive selection compared to the average of other tree branches. There were 110 positively selected genes on the branch subtending the formicoid clade while the average number of genes with positive selections was only 3.1 in other branches (maximum value = 20 genes). This finding is particularly remarkable given that the genes considered in our analysis are highly conserved universal orthologs across Hymenoptera [START_REF] Seppey | BUSCO: assessing genome assembly and annotation completeness[END_REF] . This indicates that extensive molecular changes in well-conserved core genes occurred along the branch giving rise to the formicoids. By contrast, there was no evidence of a further burst of positive selection later in the evolutionary history of the formicoids, including in the multiple branches leading to the most complex eusocial species (Figure 2). This suggests that most of the genetic innovations specific to complex eusociality in formicoids occurred in less than ~20 Ma during the early Cretaceous (see Figures 1 and2).

Functional enrichment analyses for the formicoid branch revealed that histone acetylation was the most significantly over-represented function among the 110 positively selected genes. Histone acetylation is well-known for controlling transcriptional activity [START_REF] Struhl | Histone acetylation and transcriptional regulatory mechanisms[END_REF] , reprograming foraging behaviour of the major worker caste into the minor worker caste [START_REF] Simola | Epigenetic (re)programming of castespecific behavior in the ant Camponotus floridanus[END_REF] , colony activity rhythms [START_REF] Libbrecht | A role of histone acetylation in the regulation of circadian rhythm in ants[END_REF] , and the longevity/fecundity trade-off in workers [START_REF] Choppin | Histone acetylation regulates the expression of genes involved in worker reproduction and lifespan in the ant Temnothorax rugatulus[END_REF] . Histone acetylation is also involved in caste determination of honeybees through the effect of royal jelly [START_REF] Spannhoff | Histone deacetylase inhibitor activity in royal jelly might facilitate caste switching in bees[END_REF] and has been identified as a key caste-specific enhancer of transcription regulating the differential larval development of queens and workers [START_REF] Wojciechowski | Phenotypically distinct female castes in honey bees are defined by alternative chromatin states during larval development[END_REF] . Interestingly, our analyses revealed positive selection on histone acetyltransferase (Data S1), a gene previously linked to functions potentially relevant to eusociality, such as the regulation of worker polymorphism [START_REF] Alvarado | Epigenetic variation in the Egfr gene generates quantitative variation in a complex trait in ants[END_REF] . The second most significant function was autophagy. Autophagy has repeatedly been shown to be essential for queen lifespan extension [START_REF] Madeo | Essential role for autophagy in life span extension[END_REF][START_REF] Nakamura | Autophagy and Longevity[END_REF] and the castespecific programmed cell death responsible for the divergent ovary development in queen and worker honeybees [START_REF] Barchuk | Molecular determinants of caste differentiation in the highly eusocial honeybee Apis mellifera[END_REF] . Finally, the third most significant function was gene silencing by RNA (Figure 2). From our results, we retrieved the gene Tudor-SN, which is a candidate for controlling worker sterility in honeybees [START_REF] Hartfelder | The ovary and its genes-developmental processes underlying the establishment and function of a highly divergent reproductive system in the female castes of the honey bee, Apis mellifera[END_REF] . Altogether,these results reveal that the common ancestor of the formicoid ants underwent important genomic changes relative to the regulation of gene expression (e.g., histone acetylation and gene silencing RNA) and soma maintenance (e.g., autophagy). These changes may have been important in allowing the evolution of extreme division of labour in formicoids, the ant clade comprising the vast majority of species exhibiting extreme forms of complex eusociality (e.g. maximum colony size of 3 million polymorphic workers in formicoid Dorylus species, compared to a maximum of 50,000 monomorphic workers in some poneroid species of the genus Leptogenys 34 ). However, given that the nine formicoid subfamilies also display some species with less complex levels of eusociality, this implies that while the genomic changes that occurred during the early Cretaceous may have favoured the emergence of extreme division of labour and more overtly complex forms of eusociality, they did not necessarily lead to such changes in social organisation. Knowing which selective pressures triggered these dramatic molecular changes remains an open and intriguing question.

Conclusions

By providing genome-wide data for all ant subfamilies, this study infers the leptanillomorph clade as the sister-clade to all other extant ants and clarifies controversial subfamily relationships which will be important for further comparative studies in ants. The comparative genome analysis also reveals important changes in key molecular pathways implicated in the differential gene expression of queens and workers. This burst of molecular innovations, which occurred over ~20 million years in the early Cretaceous, possibly played an important role in facilitating the evolution of complex eusociality including the large colony sizes, extensive caste polymorphism and extreme fecundity/longevity of queens which characterize multiple lineages of formicoids.

Figures

Figure 1: Phylogeny and timeline of ant evolution based on whole-genome data. The topology is inferred according to the main supermatrix analysis (1,692,052 amino acid sites after cleaning, maximum likelihood, IQ-TREE PMSF C20 profile mixture model, Figure S1D), ultrafast bootstrap support is displayed first when node support is not maximal. Node support of the main supertree analyses (from gene trees of the 1,552 alignments of more than 500 amino acid sites, gene trees inferred with model search in IQ-TREE, species tree with ASTRAL, Figure S1E) are displayed second when not maximal (dash when node shows incongruence with the supermatrix analysis). Time divergence has been estimated using chronos with 12 calibration nodes (Table S2). Ant images from Alex Wild, used with permission. See also Figure S1, Table S1 and Table S2. [START_REF] Blanchard | Defensive traits exhibit an evolutionary trade-off and drive diversification in ants[END_REF] . Significantly enriched functional categories (Biological Process) under positive selection are represented as a word cloud for the formicoid branch. The size of the font is proportional to the p-value (Fisher's exact test, larger font indicating the most significant ones, from 0.011 to 0.047). Darker colours indicate the three functions with the highest numbers of significant annotated genes under positive selection. See also Data S1.

STAR Methods

Resource availability

Lead contact

Further information and requests for resources be directed to and will be fulfilled by the lead contact, Jonathan Romiguier (jonathan.romiguier@umontpellier.fr).

Material availability

This study did not generate new unique reagents.

Data and code availability

Experimental model and subject details

We sampled 65 species (64 ants, 1 jewel wasp Ampulex compressa) across all ant subfamilies. We collected specimen from various sources and collectors (details and full overview of samples available in Table S1).

Method details

Sequencing and genome assembly DNA extractions have been performed based on a high salt method [START_REF] Aljanabi | Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques[END_REF] on samples conserved in ethanol or in -80 °C freezers. We sequenced the genomes of 65 samples (64 ants and the jewel wasp Ampulex compressa) using Illumina Hiseq technology (paired-end, 150 bp reads). Reads were cleaned using Trimmomatic [START_REF] Bolger | Trimmomatic: a flexible trimmer for Illumina sequence data[END_REF] and first assembled using AbySS 2.0.2 [START_REF] Jackman | ABySS 2.0: resource-efficient assembly of large genomes using a Bloom filter[END_REF] with kmer size set on 61 or the optimal value as estimated by KmerGenie v1.7016 [START_REF] Chikhi | Informed and automated k-mer size selection for genome assembly[END_REF] . We removed potential contaminations using the blobtools pipeline [START_REF] Laetsch | BlobTools: Interrogation of genome assemblies[END_REF] , with the exclusion of contigs that blasted on any non-arthropod phylum against the Genebank database (nt). Reads mapping on these contaminant contigs were filtered and remaining reads were used for a second genome assembly using SPAdes 3.9.0 [START_REF] Bankevich | SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing[END_REF] . For each species, we selected the best assembly between SPAdes and AbySS based on the number of complete and single copy ortholog genes using BUSCO [START_REF] Seppey | BUSCO: assessing genome assembly and annotation completeness[END_REF] with a 4,415 Hymenoptera ortholog dataset from OrthoDB v9 [START_REF] Zdobnov | OrthoDB v9. 1: cataloging evolutionary and functional annotations for animal, fungal, plant, archaeal, bacterial and viral orthologs[END_REF] (see Table S1 for assembler, BUSCO scores and N50 of each species).

Phylogeny

We built our phylogenomic dataset by complementing the 4,415 ortholog genes of our 65 species with 17 (9 ants and 8 hymenopteran outgroups) supplemental reference genomes from OrthoDB v9 [START_REF] Zdobnov | OrthoDB v9. 1: cataloging evolutionary and functional annotations for animal, fungal, plant, archaeal, bacterial and viral orthologs[END_REF] . We aligned the amino-acid sequences using mafft [START_REF] Katoh | MAFFT version 5: improvement in accuracy of multiple sequence alignment[END_REF] and cleaned the resulting alignments with Spruceup 43 and trimal [START_REF] Capella-Gutiérrez | trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses[END_REF] with the "automated1" option.

We concatenated the alignments in a supermatrix that we analysed with two different substitution models with IQ-TREE v 2.0.5 [START_REF] Nguyen | IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies[END_REF] and 1,000 ultrafast bootstraps [START_REF] Hoang | UFBoot2: improving the ultrafast bootstrap approximation[END_REF] . First, we performed a gene partitioned analysis (all partitions share the same set of branch lengths but have their own evolution rate) with LG+F+G4 models after having removed partitions that failed at symmetry tests testing stationarity and homogeneity assumptions [START_REF] Naser-Khdour | The prevalence and impact of model violations in phylogenetic analysis[END_REF] (resulting tree is presented in Figure S1C and has been used as the guide tree for the next analysis).

Second, because the most controversial ant subfamily relationships are expected to be affected by long-branch-attraction artefacts (Martialinae and Paraponerinae are monotypic subfamilies with long branches), we used the posterior mean site frequency model (PMSF model, LG+C20+F+G) which has been designed to correct for such artefacts by modeling site heterogeneity [START_REF] Wang | Modeling site heterogeneity with posterior mean site frequency profiles accelerates accurate phylogenomic estimation[END_REF][START_REF] Tihelka | The evolution of insect biodiversity[END_REF] . The resulting tree is presented in Figure 1 + Figure S1D and is referred in the results as the main supermatrix analysis. We also used this tree for estimating divergence times via penalised maximum likelihood approaches [START_REF] Paradis | ape 5.0: an environment for modern phylogenetics and evolutionary analyses in R[END_REF] and a set of 12 node calibrations (details of calibrations and references in Table S2).

A coalescent-based species tree analysis was performed by first producing gene trees using IQ-TREE [START_REF] Nguyen | IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies[END_REF] with the substitution model selected for each alignment by the built-in ModelFinder option MFP+MERGE. Because coalescent-based species tree approaches are sensitive to inaccurate gene trees [START_REF] Gatesy | Phylogenetic analysis at deep timescales: unreliable gene trees, bypassed hidden support, and the coalescence/concatalescence conundrum[END_REF] , we only kept gene trees from long alignments of more than 500 amino-acids (n=1,366) and used ASTRAL 5.7.4 [START_REF] Zhang | ASTRAL-III: polynomial time species tree reconstruction from partially resolved gene trees[END_REF] for producing the supertree (Figure S1E).

In case outgroup composition affected our results [START_REF] Borowiec | Compositional heterogeneity and outgroup choice influence the internal phylogeny of the ants[END_REF] , we built an alternative dataset containing 115 outgroups of Aculeate species by matching our OrthoDB IDs with the IDs provided by the alignments of an Hymenoptera phylogenomic dataset [START_REF] Peters | Evolutionary history of the Hymenoptera[END_REF] , resulting in a 2,343 gene supermatrix (983,951 sites) after an automated trimal cleaning. We first performed the same PMSF analysis as described above (see tree in Figure S1B). Second, we produced a reduced supermatrix by keeping only alignments with at least 90% of species and sites with 90% of non-ambiguous characters, resulting in a supermatrix with 271,959 sites. We then removed from 0 to 115 random outgroups from this supermatrix, producing 116 new supermatrices for testing the effect of random outgroup removal. The same PMSF tree inference as described above was then performed, resulting in 116 phylogenetic trees (available in the Zenodo repository).

We built an alternative dataset of ultra-conserved elements loci (UCE) from our genomes and merged the data with the phylogenetic dataset of Branstetter et al. [START_REF] Branstetter | Enriching the ant tree of life: enhanced UCE bait set for genome-scale phylogenetics of ants and other Hymenoptera[END_REF] . We used phyluce [START_REF] Faircloth | PHYLUCE is a software package for the analysis of conserved genomic loci[END_REF] to extract UCE loci from our genome assemblies. 2,510 loci were retrieved but we only kept the 1,855 that were common with the Branstetter et al. [START_REF] Branstetter | Enriching the ant tree of life: enhanced UCE bait set for genome-scale phylogenetics of ants and other Hymenoptera[END_REF] dataset. We aligned the data using mafft [START_REF] Katoh | MAFFT version 5: improvement in accuracy of multiple sequence alignment[END_REF] , cleaned the alignment using trimal [START_REF] Capella-Gutiérrez | trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses[END_REF] with the -automated1 option and removed alignments that contained fawer than 75% of the total number of taxa (n = 166). We concatenated the 1,230 remaining alignments in a supermatrix with 426,015 sites and inferred a tree using IQ-TREE with a locus partitioned analysis (all partitions share the same set of branch lengths but have their own evolution rate) with GTR+I+G4 models and 1000 ultrafast bootstraps (Figure S1A).

Divergence dating analyses

First, we used a simple approach exploiting the largest supermatrix by using the topology and branch lengths of Figure S1D (supermatrix of 1,692,052 amino acids, PMSF model LG+C20+F+G) to estimate divergence times via penalised maximum likelihood approaches [START_REF] Paradis | ape 5.0: an environment for modern phylogenetics and evolutionary analyses in R[END_REF] and a set of 12 node calibrations (details of calibrations and references in Table S2). To confirm the retrieved estimations (presented in Figure 1), we analysed a reduced dataset using a Bayesian approach, as implemented in MCMCTree, a part of the PAML package, v4.10 72 . MCMCTree utilizes rapid approximate likelihood computation [START_REF] Dos Reis | Approximate likelihood calculation on a phylogeny for Bayesian estimation of divergence times[END_REF] , which makes it suitable for divergence dating of genome-scale data sets [START_REF] Dos Reis | Phylogenomic datasets provide both precision and accuracy in estimating the timescale of placental mammal phylogeny[END_REF] . Due to computational constraints, we used an alignment with loci containing a minimum of 95% of our 83 taxa, totalling 182,809 amino acid sites. We fixed the topology to be the same as our analysis of the full alignment. We constrained our root node with a soft bound maximum age of 236 Ma, corresponding to the lower bound of the 95% highest posterior density (HPD) interval for that split in Hymenoptera tree estimations [START_REF] Peters | Evolutionary history of the Hymenoptera[END_REF] . We also set soft bounds on the root of the Formicidae to be 103 Ma and 169 Ma, corresponding to the upper 95% bound of HPD in Borowiec et al. [START_REF] Borowiec | Compositional heterogeneity and outgroup choice influence the internal phylogeny of the ants[END_REF] and lower bound in Economo et al. [START_REF] Economo | Macroecology and macroevolution of the latitudinal diversity gradient in ants[END_REF] , the most divergent of recent estimates for the crown age of the family [START_REF] Borowiec | Ants: Phylogeny and classification[END_REF] . We also used minimum node age constraints based on fossils presented in Table S2. We ran each analysis unpartitioned, under the LG model for 5 million generations. We examined each run's statistics in Tracer [START_REF] Rambaut | Posterior summarization in Bayesian phylogenetics using Tracer 1.7[END_REF] and confirmed convergence and sufficient effective sample sizes (>>200) for all parameters. Retrieved estimations were close to those retrieved with penalised likelihood on the whole dataset (Figure 1) and are available with all output files in the zenodo repository (https://doi.org/10.5281/zenodo.5705739).

Positive selection analysis

We performed a positive selection detection analysis by using 4,415 nucleotide alignments. Nucleotide alignments have been produced and refined from amino-acid alignments using the command reportGapsAA2NT and refineAlignment from macse v1.2 [START_REF] Ranwez | MACSE: Multiple alignment of coding sequences accounting for frameshifts and stop codons[END_REF] .

We cleaned the alignments of potential errors further by using hmmcleaner v1.8, a tool that has been reported as especially effective for reducing false positives for detection of positive selection [START_REF] Franco | Evaluating the usefulness of alignment filtering methods to reduce the impact of errors on evolutionary inferences[END_REF] . We used the value of 5 for the threshold parameter then removed every species with fewer than 20% nucleotides remaining after the cleaning. To ensure that gap-rich regions did not bias our analyses, we applied two different supplemental cleaning treatments by keeping only codons shared with more than 50 and 75% of the species of the alignment. All of the following analyses were performed with the three cleaning strategies (hmmcleaner only; hmmcleaner+50% complete codons; hmmcleaner+75% complete codons) and retrieved consistent results regarding the relatively high percentage of genes under positive selection in the Formicoid branch compared to other branches (38.13, 36.26 and 30.58-fold increases, respectively, see Data S1). Only the results of the "hmmcleaner+more than 75% complete codons" treatment are presented in the main text. We used the aBSREL method (adaptive Branch-Site Random Effects Likelihood) from the HyPhy package [START_REF] Smith | Less is more: an adaptive branch-site random effects model for efficient detection of episodic diversifying selection[END_REF] , an improved implementation of the branch-site model typically used to test whether positive selection has occurred on some branches via the estimation of dN/dS (non-synonymous substitution rate over synonymous substitution rate) [START_REF] Yang | Statistical properties of the branch-site test of positive selection[END_REF] . All branches were tested for positive selection for each gene, with p-values corrected for multiple testing on multiple branches (using the built-in correction in aBSREL.An additional correction was conducted for multiple testing on multiple genes [START_REF] Benjamini | Controlling the false discovery rate: a practical and powerful approach to multiple testing[END_REF] . For each internal branch in Figure 2, we only considered alignments containing at least one species for each of its three connected clades, ensuring that the positive selection test reflects this exact part of the evolutionary history of the gene. For each gene, we reported the gene ontology function of the Apis mellifera ortholog gene as available in OrthoDB [START_REF] Zdobnov | OrthoDB v9. 1: cataloging evolutionary and functional annotations for animal, fungal, plant, archaeal, bacterial and viral orthologs[END_REF] . Gene ontology enrichment analyses have been performed using topGO [START_REF] Alexa | Gene set enrichment analysis with topGO[END_REF] with Fisher exact tests and the default weight01 algorithm.

GC-content variations are known to potentially bias detection of positive selection methods via the process of biased gene conversion [START_REF] Galtier | Adaptation or biased gene conversion? Extending the null hypothesis of molecular evolution[END_REF][START_REF] Romiguier | Analytical biases associated with GC-content in molecular evolution[END_REF] . Particularly for our results, strong GCcontent variations among Formicoid and Poneroid species could lead to an overestimation of positive selection in the branch leading to Formicoids. To ensure that it is not the case, we measured the average GC-content of all species and retrieved no significant difference between Formicoids and Poneroids when considering all genes (45.43% vs 46.14% in Formicoids and Poneroids, p-value = 0.14 from Welch two sample test) or only considering the 110 genes retrieved as positively selected from the main analysis (48.10% vs 48.92% in Formicoids and Poneroids, p-value = 0.15 from Welch two sample test).

Gene predictions and gene family analyses

We performed gene predictions for our 65 genomes by using MAKER2 v 2.31.8 [START_REF] Holt | MAKER2: an annotation pipeline and genome-database management tool for second-generation genome projects[END_REF] , a pipeline for genome annotation using ncbi-blast v 2..2.28 [START_REF] Boratyn | BLAST: a more efficient report with usability improvements[END_REF] , RepeatMasker v 4.0.5 [START_REF] Chen | Using RepeatMasker to Identify Repetitive Elements in Genomic Sequences[END_REF] , exonerate v 2.2.0 54 , snap v 2013.11.29 [START_REF] Korf | Gene finding in novel genomes[END_REF] , augustus v 3.2.2 [START_REF] Stanke | AUGUSTUS: a web server for gene prediction in eukaryotes that allows user-defined constraints[END_REF] , tRNAscan-SE v1.3.1 and snoscan 0.9 [START_REF] Schattner | The tRNAscan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs[END_REF] . We filtered genes with fewer than 1000 nucleotides and individual protein sets were blasted against each other as well as against 28 additional Hymenoptera protein sets (detailed list available in output files available on 1https://doi.org/10.5281/zenodo.5705739) using orthofinder v 2.2.1 [START_REF] Emms | OrthoFinder: phylogenetic orthology inference for comparative genomics[END_REF] . The resulting gene count data file was then used for a gene family evolution analysis with CAFE v5 [START_REF] Mendes | CAFE 5 models variation in evolutionary rates among gene families[END_REF] with base model default setting values. After trying several filtering methods, we removed gene families with difference in gene number larger than 50 to prevent "-inf" likelihood scores.

We assigned GO terms for our 65 protein sets using eggNOG v5 [START_REF] Huerta-Cepas | eggNOG 5.0: a hierarchical, functionally and phylogenetically annotated orthology resource based on 5090 organisms and 2502 viruses[END_REF] and used it to assign GO terms to gene families analysed with CAFE. To identify gene functions overrepresented in gene families that underwent significant expansion/contraction, GO term enrichment analyses were performed using topGO [START_REF] Alexa | Gene set enrichment analysis with topGO[END_REF] , with Fisher exact tests and the default weight01 algorithm. Gene functions significantly over-represented and potentially related to eusociality include autophagy, determination of adult lifespan, oogenesis, detection of chemical stimulus involved in sensory perception of smell, maintenance of chromatin silencing, olfactory receptor activity, histone deacetylase binding, histone kinase activity (see the topGOresults tables in the Zenodo repository for the whole list). However, these results should be taken with caution because our analyses showed that the genomes available in public databases tended to exhibit greater increases/decreases of gene families than our 65 genomes. This is probably due to the fact that we had to use short-read sequencing technologies to be able to analyse low amounts of DNA and/or degraded DNA for some species that are rare and very difficult to collect. We therefore chose not to present these results in the main text but instead made them available in a Zenodo repository (https://doi.org/10.5281/zenodo.5705739) where we provide MAKER2 control file and options, resulting protein fasta files, orthofinder main output files, CAFE5 input and output files, eggNOG annotations and topGO analysis input/output.

Figure 2 :

 2 Figure 2: Branch subtending socially diverse formicoid ants shows increased rates of positive selection. Colours indicate the percentage of genes significantly under positive selection (aBSREL analysis) on each branch. Colony size and polymorphism data represent the maximum observed value in the genus and have been extracted from the literature[START_REF] Blanchard | Defensive traits exhibit an evolutionary trade-off and drive diversification in ants[END_REF] . Significantly enriched functional categories (Biological Process) under positive selection are represented as a word cloud for the formicoid branch. The size of the font is proportional to the p-value (Fisher's exact test, larger font indicating the most significant ones, from 0.011 to 0.047). Darker colours indicate the three functions with the highest numbers of significant annotated genes under positive selection. See also Data S1.

-

  -Raw reads of sequenced genomes data have been deposited at ENA (European Nucleotide Archive). Accession numbers are listed in the key resources table. Genome assemblies, data, raw results and command lines for reproducibility are available in the following Zenodo repository and are publicly available as of the date of publication https://doi.org/10.5281/zenodo.5705739. DOIs are listed in the key resource table. This paper does not report original code.-Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgements

We thank the FEBS (Federation of European Biochemical Societies) for the long-term fellowship granted to Jonathan Romiguier and funding from the French ANR (t-ERC grant RoyalMess) and ERC (RoyalMess, grant 948688), ERC and Swiss NSF to Laurent Keller, a grant from US National Science Foundation (CAREER DEB-1943626) to Christian Rabeling, and three reviewers for very useful comments. We thank the following for helping us to collect samples: C.S. Moreau, F.A. Esteves, S. Van Noort, N. Gunawardene, C. Poteaux, T. Bruscht, J.M. Gómez Durán, M. Molet, E. Vargo, Bui Tuan Viet, M.D. Goodisman, T. Delsinne and J. Orivel.

Author contributions

JR and LK conceived the study, JR, MB, CR, BLF, PSW and LK coordinated sample collection efforts, CLM performed DNA extractions, JR, MB, QH, AW and EL performed analyses, JR, MB, CR, BLF, PSW and LK wrote the manuscript.

Declaration of interests

The authors declare no competing interests. ). E) Significant GO-terms for the formicoid branch (Biological Process). F) Significant GO-terms for the formicoid branch (Molecular Function). G) Significant GO-terms for the formicoid branch (Cellular componment).

Supplemental information titles and legends


Ugo Jacovella
email: ugo.jacovella@universite-paris-saclay.fr

Corentin Rossi

Claire Romanzin

Christian Alcaraz

Roland Thissen

C Ro- Manzin

Structural elucidation of C 6 H +• 4 using chemical reaction monitoring: Charge transfer versus bond forming reactions

Keywords: Ion-molecule reaction, Charge transfer, Distonic radical cations, Structure elucidation

Mass spectrometry is a powerful tool but when used on its own, without specific activation of ions, the ion mass is the single observable and the structural information is absent. One way of retrieving this information is by using ion-molecule reactions. We propose a general method to disentangle isomeric structures by combining mass spectrometry, tunable synchrotron light source, and quantum-chemistry calculations. We use reactive chemical monitoring technique, which consists in tracking reactivity changes as a function of photoionization energy i.e. the ionic structure. We illustrate the power of this technique with charge transfer reactions of C 6 H +• 4

isomers with allene and propyne and discuss its universal applicability. Furthermore, we emphasize the special reactivity characteristics of distonic ions, where strong charge transfer reactivity but very limited reactivity involving bond formation and following cleavages were observed and attributed to the unconventional ortho-benzyne distonic cation. isomers is done by monitoring different structurespecific ion-molecule reaction channels with propyne and allene, highlighting an abnormally large charge transfer cross section for the distonic ortho-benzyne cation together with a surprisingly very weak chemical reactivity.

Table of contents

The group of C 6 H 4 hydrocarbons comprises a rich number of closed-shell stable isomers and diradical isomers. [1] The cyclic benzyne species own an exotic Kekule-type diradical structures where the two radical sites can be in ortho, meta or para arrangement as shown in Scheme 1. This diradical nature provides them hetarynes, and other related molecules have been isolated and investigated in cryogenic matrices. [1] Because the matrix isolation and spectroscopic characterization of the didehydrobenzenes 1-3 has been reviewed extensively, [1] we will focus here on rather new developments and especially on the tridehydrobenzenes 4-6 (Scheme 4) that have recently become amenable to experimental investigations. An interesting class of dehydroaromatic systems are the tetradehydrobenzenes (or benzdiynes), which have been thoroughly studied using matrix isolation spectroscopy by Sato et al. [9] Since this topic is covered by a separate Review in this Research Front, [10] it will not be discussed in detail here.

RESEARCH FRONT

The s determin defining

The (positive when th hetarynes, and other related molecules have been isolated and investigated in cryogenic matrices. [1] Because the matrix isolation and spectroscopic characterization of the didehydrobenzenes 1-3 has been reviewed extensively, [1] we will focus here on rather new developments and especially on the tridehydrobenzenes 4-6 (Scheme 4) that have recently become amenable to experimental investigations. An interesting class of dehydroaromatic systems are the tetradehydrobenzenes (or benzdiynes), which have been thoroughly studied using matrix isolation spectroscopy by Sato et al. [9] Since this topic is covered by a separate Review in this Research Front, [10] it will not be discussed in detail here. with an extremely rich chemistry, [2] making them important intermediates in a great number of classical reactions in organic chemistry (nucleophilic aromatic substitution, hexadehydro-Diels-Alder, ...). [3][START_REF] Hoffmann | Dehydrobenzene and cycloalkynes[END_REF][START_REF] Dubrovskiy | [END_REF][6] Additionally, derivatives of p-benzyne were discovered to be the biologically active intermediates of the potent enediyne anti-tumor antibiotics [7,8] through a Masamune-Bergman cyclization. [9] The short lifetime and high reactivity of benzynes complicate investigations in solution. An historical review on benzynes has been compiled by Wentrup, [10] whose group specialized in studying highly reactive species in gas phase such as benzyne by combining pyrolysis techniques with spectroscopic tools. Another elegant approach extensively employed by the group of Kenttämaa to overcome experimental difficulties faced in solution was to study diradical species in the gas phase utilizing charged tagged derivatives in mass spectrometric experiments. [11] Evidence of the longer lifetime of benzyne in the gas phase was recently demonstrated by the detection of the ortho-benzyne in the Taurus molecular cloud using radio astronomy. [12] The removal of an electron from closed-shell C 6 H 4 species generates highly reactive "classical" radical cations, whereas the removal of an electron from biradical benzyne will create a species where the positive charge and the radical sites are spatially separated. These surprising ions have been named distonic ions in 1984 by Yates, Bouma, and Radom [13] and often possess a much greater stability with respect to unimolecular decomposition compared to their neutral counterparts. Reactivity characteristics of distonic ions have been proven to differ greatly from more conventional ions. General characterization of their reactivity has not been reached yet. This is partly due to experimental challenges in creating these ions and disentangling them from isomeric conventional ions. [14][15][16][17][18] Information about distonic radical benzyne cations are extremely scarce. The experimental work of Liu and coworkers [19] that generated o-, m-, and p-benzyne radical cations from the photolysis of Mg +difluorobenzene complexes has laid the foundation for the isolation, characterization, and investigations of the reactivity of distonic radical benzyne cations. Nevertheless, to the best of our knowledge there are no experimental data reported on the reactivity of isolated distonic benzyne cation. Most of the studies, theoretical and experimental, have focused on the determination of the adiabatic ionization energy of the o-benzyne

RESEARCH FRONT

Thermochemistry and

to gain information about singlet-triplet splittings in neutral o-benzyne and heats of formation. [20][21][22][23][24][25][26] However, these thermodynamic quantities were most precisely obtained from benzyne anions using negative ion photoelectron spectroscopy. [27] Indeed, negative ion photoelectron spectroscopy has proven to be a powerful tool to retrieve the structures of (di)radicals including distonic species from their corresponding anionic counterparts. [28][29][30] Finally, computational and laser spectroscopic explorations of the C 6 H +• 4 potential energy surfaces including cyclic and acyclic isomers [31,32] have shown that the most stable structure on the potential energy surface of C 6 H +• 4 is a cyclic meta-benzyne, while for the neutral the most stable structure is ortho-benzyne. [1] When ions are not activated in specific means, mass spectrometric experiments usually suffer from their inherent blindness to isomeric distribution of ions. Ion mobility spectrometry, often combined with laser spectroscopy, is nowadays becoming the most prevalent technique to characterize an isomeric mixture of ions. [33,34] A powerful, though unconventional way is to use chemical reactivity to probe ion structures, which in addition to providing information on the shape of the ions also retrieves information on their chemical behavior. We used the chemical monitoring experimental technique, consisting in following the evolution of products from ion-neutral reaction as a function of the photoionization energy. This technique has been established and first applied with success to experimentally evaluate the barrier height of the unimolecular keto/enol tautomerization of the acetamide radical cation. [35] In the present study, we probe the isomeric distribution of C 6 H +• 4 species generated by dissociative photoionization using the the DESIRS undulator-based vacuum ultraviolet (VUV) beamline at the SOLEIL synchrotron facility in Saint-Aubin (France). [36]

+ C 2 H 2 + e - (1)
C 10 H 8 hνVUV ----→ C 6 H +• 4 + 2C 2 H 2 + e - (2)
Reactive ions were produced by photoionization in the 9 to 20 eV photon energy range. Higher order light was removed from the beam by using a gas filter either filled with Kr or Ne depending on the energy range (8-14 and 13.5-20 eV respectively). The reaction products are monitored using a tandem mass spectrometer with photoionization source known as CERISES, [37] enabling to retrieve reaction cross sections at well controlled and defined collision energies. In the present study, the collision energy was set to 0.4 eV. The structure elucidation is done by searching for reactivity changes as a function of the photon energy. The increase in photon energy will produce ions with higher internal energy and eventually drive unimolecular isomerization reactions, where the newly formed isomer might exhibit different reactivity with the neutral target. Reactivity changes among different ionic isomers might also be dependent on the chemical nature of the neutral reactants. We used the two C 3) are summarized in Table 1. The practically flat behavior of the CT above the transition indicates that a constant ratio of the two isomeric populations is produced. The change of the neutral reactant partner from allene to propyne leads to Reaction (4).

C 6 H +• 4 + H 2 C C CH 2 -→ Products (Table 1) (3)
C 6 H +• 4 + H 3 C C CH -→ Products (Table 2) ( 4
)
Similarly to Figure 1b), the bottom part (panel c) of Figure 1 displays the reaction cross sections for the Reaction (4), which are cataloged in with the propyne and allene in the high energy region is quasi -identical for all reactions passing through an adduct (reaction complex). The decrease by a factor 20 in the σ of the CT from allene to propyne is indicative of the ionization energy (IE) of neutral counterpart(s) of the C 6 H +• 4 isomer(s) responsible for the CT reaction. Typically, for a CT reaction to occur, the IE of the neutral partner should be smaller than the IE of the neutral counterpart of the ionic partner. Furthermore, the cross section is usually governed by Franck-Condon factors and energy resonance, with a prevalence for the latter, being in a simple approach the difference in IE of the two reactive species. Therefore, from the results of Reactions ( 3) and ( 4), it can be retrieved that the neutral counterpart(s) of the C 6 H +• 4 isomer(s) formed upon VUV dissociative ionization of phenylacetylene should have an IE at least greater than the one of allene (9.69 eV) [38] and closer to the latter than to the IE of propyne (10.37 eV). [39] The fourteen most stable isomers are depicted in the upper part of Figure 2 with their relative energies with respect to A + -the global minimum of the potential energy surface of C 6 H +•

4

-the energies are given in eV (light blue) in the bottom of each box. The IEs of the corresponding neutral counterparts are indicated on top of each box (red) and are also given in eV. All calculations were obtained using the CBS-QB3 composite method [40] implemented in Gaussian 16 package. [START_REF] Frisch | Gaussian˜16 Revision C.01[END_REF] The use of the B3LYP functional for the geometry optimization in the CBS-QB3 method might not capture correctly the geometries of all included isomers, but the choice of CBS-QB3 was guided by the robustness of the method with established accuracy for IEs of ≈ 0.05 eV, [START_REF] Montgomery | [END_REF] and its ease of use so that it can be utilized even by non-specialists.

Furthermore, the spread in the collision and internal energy of the reacting partners would contribute for more than the 0.05 eV of accuracy, making the presence of a high density of isomers with similar IEs the major limitation of the approach. The calculated IEs are schematically represented in the bottom part of species. The experimental values of the IEs of propyne [39] and allene [38] are represented by dash lines with the shaded area corresponding of the 0.4 eV of energy deposited in the system by kinetic energy (collision energy).

CT reaction observed. As aforementioned, the ∆E IE is expected to be smaller with the IE of allene than with propyne. The ortho-benzyne distonic cation (F + ) is the only C 6 H +• 4 isomer fulfilling all the enunciated requirements to be responsible for the CT reaction with allene. Mechanistically, the acetylene unit eliminated from the phenylacetylene should favour the formation of ortho-benzyne distonic cation. Indeed, only a H migration from the ortho position to the acetylenic group is needed to form the acetylene leaving group. The non-zero σ for the CT reaction with propyne indicates that a small fraction of the ortho-benzyne is formed with at least few hundreds meV of internal energy.

We have now identified the isomeric population of the low energy region to be simply constituted of the ortho-benzyne distonic cation. The current study does not allow to discriminate the isomers present in the high energy region. We tentatively proposed that the isomers are those of more conventional nature i.e radical cations -typically more chemically reactive -so the 3-and 4-membered rings and the acyclic isomers are privileged. These isomers require 4 eV of internal energy to be formed, indicating that the isomerization most likely occurs on the C 8 H + 6 potential energy surface rather than on the C 6 H +• structures within a certain IE range. The only prerequisite is then the knowledge of the IE of the different isomeric structures, which for molecules with less than 20 atoms can be obtained ab initio with sufficient precision at relatively low computational cost using basic composite methods. The proxy reaction chosen to illustrate this concept reveal once more how the chemistry of distonic ions can differ significantly from their isomeric radical cations structures.

  Chemical monitoring technique is used to disentangle the structural diversity of a C 6 H +• 4 isomeric mixture. The structural elucidation of C 6 H +•

4

 4

  Scheme 3.

  Scheme 4.

  Scheme 3.

3 H 4 4 using

 344 photon energy (hν).

  The data clearly indicate two distinct reactivity behaviors in the [9.8 ; 14 eV] and [14 ; 20 eV] energy regions. The low-energy region exhibits only charge transfer (CT) reaction products, whereas the high-energy region reveals a very rich chemistry with reactions involving new chemical bond formation and further bond cleavages. The reaction cross section (σ) of the CT significantly decreases from 200 Å2 in the low-energy region to 3 Å2 in the the high-energy region, indicating an evolution of the C 6 H +•

Figure 1 :

 1 Figure 1: a) Photoion yield of C 6 H +• 4 resulting from dissociative ionization from phenylacetylene. b) and c) are reaction cross sections for the products resulting from C 6 H +• 4 produced from phenyacetylene reacting with allene and propyne, respectively, as a function of photon energy. Light and dark data-set correspond to the two gas-filter conditions aforementioned.

Figure 2

 2 Figure 2 and are color-coded as a function of the chemical nature of the corresponding isomer. The dashed lines indicate the referenced values of the IEs of propyne and allene, with the shaded zone representing the 0.4 eV of the experimental collision energy. If we assume that the C 6 H +• 4 are formed with no internal energy

Figure 2 :

 2 Figure 2: Upper part: Geometries and relative energies to A + given in eV (light blue at the bottom of each box). The IEs of the corresponding neutral counterparts are shown on top of each box and are also given in eV (red). All calculations were done with the CBS-QB3 composite method. Bottom part: Distribution of the calculated IEs as a function of the isomeric nature of the C 6 H +• 4

+ + + o-benzyne m-benzyne p-benzyne Diradical neutrals Distonic cations

		Scheme 1
			The s
			determin
			defining
			The
			(positive
			when th
			taking t
	of Dehydrobenzenes 2.1. Didehydrobenzenes	Electronic Structure	available cals can of form 7 (80.5

  Three different precursors have been used in an attempt of stirring the isomeric distribution obtained. The phenylacetylene (C 8 H 6 ) that generates C 6 H +• 4 upon C 2 H 2 elimination (Reaction (1)) and from the two C 10 H 8 isomers (naphthalene and azulene), where two successive acetylene losses lead to C 6 H +•

	4	(Reaction (2)) .
		C 8 H 6	hνVUV ----→ C 6 H +• 4

Table 1 :

 1 Reaction products and corresponding cross sections for C 6 H +•

	4	from phenylacetylene reacting with

* Converged value above 17 eV.

Table 2 .

 2 The reactivity behavior of C 6 H +• 4

Table 2 :

 2 Reaction products and corresponding cross section for C 6 H +•

	4	from phenylacetylene reacting with

structures produced above 14 eV. From this observation, it can be inferred that at least two isomeric populations are present, one below and one above 14 eV. The fact that the σ of the CT plunges drastically but do not reach zero indicates that most of the low-energy population is transferred to the high-energy population (≈ 98.5%) but

one where all isomers

Acknowledgments U.J. is grateful to the SOLEIL facility for providing beamtime under project 20201123. C.A and C.R. acknowledge the synchrotron SOLEIL for the support to the associated CERISES setup of ICP since 2008 and subsistence expenses during beamtime periods. The ICP group acknowledges support from the LabEx PALM (ANR-10-LABX-0039-PALM, Project ERACOP).

The CT reaction can thus be used as a proxy for the formation of ortho-benzyne distonic cation. We have tested this approach with the prototypical polycyclic aromatic hydrocarbons (PAHs), naphthalene and azulene. The reaction cross sections of the C 6 H +• 4 obtained from Reaction (2) with allene are presented in Figure 3b) and d) for azulene and naphthalene neutral precursors, respectively. No CT reaction is observed with either precursors, revealing that ortho-benzyne distonic cation is not formed or, with an extremely low yield, upon dissociative ionization, which in turn informs about the C 8 H + 6 intermediate generated. It appears that the acetylene elimination from naphthalene and azulene cations will preferentially form bicyclic structure such as pentalene or bicyclobutane, confirming previous experimental findings [43,44] and calculations. [45] Indeed, difficulties of forming ortho-benzyne upon acetylene loss from C 8 H + 6 bicyclic structures compared to the phenylacetylene structure can be anticipated and may explain the present observations.

In this paper, we demonstrate how chemical reaction monitoring combined with a tunable photoionization light source can be used to disentangle the composition of an isomeric mixture. We used the CT reaction of C 6 H +• 4 with allene as a proxy for the formation the ortho-benzyne distonic cation. This technique could be extended by using neutral precursors with IEs equally spaced -a chemical ruler -to enclose isomeric