The Evolution of Tyramides in Male Fungus-Growing Ants (Formicidae: Myrmicinae: Attini: Attina)

Ants use a variety of semiochemicals for essential activities and have been a source for many novel natural products. While ant taxa produce a wide variety of chemicals, the chemistry and ecology of male ants have remained understudied. Tyramides are a class of compounds that have been found only in males of the Myrmicinae ant subfamily. Tyramides found in the fire ant Solenopsis invicta are transferred to gynes during mating where they are converted to tyramine, leading to rapid reproductive development. To further understand the evolution of tyramide production in male ants, we determined the tyramide composition in males of 15 fungus-growing ant species (Formicidae: Myrmicinae: Attini: Attina) and a Megalomyrmex species (Formicidae: Myrmicinae: Solenopsidini). Thirteen tyramides were identified, four for the first time in natural sources, and their percent composition was mapped to the fungus-growing ant phylogeny.


Introduction
Ants (Formicidae Latreille, 1809) occupy virtually every ecological niche in the world. Thus, the over 16,500 described species have evolved a variety of mechanisms to solve similar problems (AntWeb 2022). Ant colonies depend on an array of semiochemicals for crucial functions, including food procurement, defense, and pheromonal communication (Vander Meer et al. 1998). Taxonomic and chemical divergence is evident within the two largest ant sub-families, Myrmicinae Lepeletier de Saint-Fargeau, 1835 (147 genera, 7,071 species) (Bolton 2022) and Formicinae Latreille, 1809 (52 genera, 3,220 species) (Bolton 2022). For example, Myrmicinae species have a well-developed sting apparatus (Kugler 1979) and produce a wide variety of bioactive alkaloid structural types that are injected or wiped onto adversaries (Fox and Adams 2022). In contrast, Formicidae species have no sting and spray formic acid for defense (Touchard et al. 2016).
The biology and chemistry of male ants are poorly understood compared to that of female alates (i.e., winged reproductive females or queens) and workers (Vargo and Laurel 1994;Bernadou and Heinze 2013). But males, like female alates, contribute to their natal colony's reproductive output and play an essential role in fitness. Males usually die soon after mating, often through predation in the air or desiccation on the ground (Hölldobler and Wilson 1990;Boomsma et al. 2005;Helms 2018). Male aggregation behavior can occur hundreds of meters above the ground to attract females during brief mating flights (Helms et al. 2016;Helms 2018), while species with "female calling" often remain on or close to the ground (Helms 2018). In species with female calling, males can survive outside the colony for months, waiting for females (Shik et al. 2012). Despite variation in mating strategies, males' relatively short lifespan and the difficulty of studying mating flights (but see Helms 2018 review) has limited research on male ants. But there has recently been a positive trend in male-focused research, including taxonomy (Boudinot 2013;Boudinot and Fisher 2013;Macgown et al. 2014;Probst et al. 2015) and chemical ecology (Vander Meer et al. 2021).
Males in the sub-family Myrmicinae produce a class of compounds called tyramides Adams et al. 2010;Chen and Grodowitz 2017). A breakthrough in our understanding of how male produced tyramides are used by newly-mated fire ant queens was recently published ( Vander Meer et al. 2021). Males transfer tyramides to female alates during mating, and female alates produce a tyramidespecific hydrolase enzyme in their reproductive system that quickly converts tyramides to tyramine, a biogenic amine. Tyramine floods the hemolymph of newly mated queens and triggers rapid reproductive development. In addition, artificial injection of tyramine into unmated female alates resulted in rapid dealation, ovariole development, and queen pheromone production. Vander Meer et al. (2021) conclude that male-produced tyramides enable young queens to rapidly shift to a reproductive stage after leaving their natal nest where reproduction is suppressed by their mother queen's primer pheromone. They further suggest that variations of this mechanism are likely to occur in other ant species.
Fungus-growing ants (Formicidae: Myrmicinae: Attini: Attina) are obligate symbionts that cultivate a fungal garden as their only food source. Attines are a diverse group composed of over 250 species, that vary in natural history traits, including mating frequencies and colony size (Branstetter et al. 2017;Murakami et al. 2000;Solomon et al. 2019). Before this paper, only two fungus-growing ant species Cyphomyrmex faunulus and Trachymyrmex septentrionalis, were shown to produce tyramides (N-[2-(4-hydroxyphenyl) ethyl]-propanamide and N-[2-(4-hydroxyphenyl)ethyl]-2-oxobutanamide, respectively) (Adams et al. 2010 (Branstetter et al. 2017;Solomon et al. 2019). Megalomyrmex milenae (Formicidae: Myrmicinae: Solenopsidini) was included as an outgroup. The aim of this study is to characterize the diversity of tyramide structural types produced by males across the fungus-growing ant subtribe in a phylogenetic context that will become the foundation for tyramide functional investigations within the Attina in the future.

Methods and Materials
Colony Collections Live queenright colonies and queenless sub-colonies were collected from the Panama Canal region from 2015-2019 during May, June, and July. Atta sexdens males were collected immediately preceding a mating flight in May of 2019, then placed directly into vials of methanol. Chemical Extraction Live male ants (whole) were placed in vials with 40-150 µL of HPLC grade methanol (Table 1). Other males were trisected in a glass dish (head, thorax, and abdomen). The trisected parts were each placed in their own vial with methanol and passively extracted for at least two weeks. The dish and tools used for trisection were rinsed in ethanol, methanol, and then pentane between trisections. See Table 1 for the extraction procedure used for each male collection.
Gas Chromatography-Mass Spectrometry GC-MS was carried out in the electron impact (EI) mode using a Shimadzu QP-2010 GC-MS or a Shimadzu QP-2020 GC-MS equipped with an RTX-5, 30 m × 0.25 mm i.d. column. The instrument was programmed from 60° C to 250° C at 10° C/ min. High-resolution mass spectrometry (HRMS) measurements were obtained by the Mass Spectrometry Laboratory of the School of Chemical Sciences at the University of Illinois Urbana-Champaign using a Waters Q-TOF Ultima ESI mass spectrometer or a Waters Micromass VG 70-VSE mass spectrometer. Previously identified tyramide compounds (1, 2, 3, 4, 6, 10, 11, 12, and 13) were determined through comparison to authentic samples (see Jones 2010; Adams 2010). Unidentified tyramides 5, 7, 8, and 9 were synthesized by the methods described below and had identical gas chromatographic retention times and mass spectra as the natural compounds. Retention indices were calculated by direct comparison with a Restek System Performance Test Standard Mixture of n-Alkanes (16 components) (Restek Pure Chromatography). Comparison of standard 0.6 µg/L and 60 µg/ µL solutions of N-[2-(4-Hydroxyphenyl)ethyl]-propanamide, 2, in methanol to the methanol extracts of Mycetomollerius zeteki, Cyphomyrmex costatus, and C. muelleri showed that they had approximately 1.1 µg/ant, 0.3 µg/ant, and 0.5 µg/ ant, respectively, of their most abundant tyramides.

Chemical Results
Chemical analyses identified thirteen unique tyramide structures, nine of which were reported previously Adams et al. 2010) (Table 2).
Four tyramides-5, 7, 8, and 9-are newly described compounds, identified by comparison to synthetic samples. Since the mass spectra of the tyramides found in various ant species are characterized by an intense base peak at m/z = 120 ( Fig. 1) Adams et al. 2010), they are easily visualized by a fragment ion search, even though they may co-elute with other compounds (e.g., fatty acids or their methyl esters) (Fig. 2). In addition, these compounds have discernable parent ions. Thus, the structures of novel tyramides 5, 7, 8, and 9 were identified from their mass spectra, microchemistry, and synthetic procedures. The four novel compounds include the first reported α-hydroxytyramide, 8, α,β-unsaturated tyramide, 9, and the second reported α-ketotyramide, 5, from natural sources.
In several species, we observed a tyramide, M + = 221, whose mass spectrum had a diagnostic fragment at m/z = 121 (40%), along with the base peak at m/z = 120, similar to the mass spectrum of an α-ketotyramide found in males from some Monomorium species ). 2-Oxobutanoyltyramide, 5, was prepared from 2-oxobutanoic acid using carbodiimide coupling, and direct comparison of synthetic 5 with the ant extracts confirmed this structure.
In those extracts containing 5, a later eluting tyramide, M + = 223, whose mass spectrum had diagnostic ions at m/z = 194 and m/z = 59, was present. These fragments would be expected from fragmentation on either side of the alcohol group of the α − hydroxytyramide corresponding to 5. An authentic sample of 2-hydroxybutanoyltyramide, 8, was prepared by NaBH 4 reduction of a few milligrams of 5. Direct comparison of synthetic 8 with the ant extracts confirmed this structure.
In several species we observed a tyramide, M + = 235, corresponding to the molecular weight of the saturated n-hexanoyltyramide, but with a notably shorter retention time that  For tyramide structures and retention indices, see Table 2. The asterisk indicates where the peak representing compound 4 would be, if present in this sample matched that of 2-methylpentanoyltyramide, 7. Samples of 3-methyl-and 4-methylpentanoyltyramidqe were also prepared from the corresponding 3 and 4-methylpentanoic acids, and their retention times did not match that of 7. In those species containing 7, we also observed a longer retention time compound, M + = 233, with intense ions at m/z = 97 and m/z = 114 that could be converted to tyramide 7 by microhydrogenation over PtO 2 . Direct comparison with a synthetic sample confirmed the structure of 2-methyl-(E2)-pentenoyltyramide, 9.
Taxonomic Comparisons Tyramides were observed in all ant samples examined in this study, except for Apterostigma dentigerum, where only one of four colonies sampled showed trace amounts of 1 and 2. Apterostigma dentigerum is unusual among the sampled Attina in that it appears to lack or have minimal amounts of tyramides.
Tyramide 2 was the most prevalent tyramide observed in this study and the largest profile component in the four sampled leaf-cutter species (two Atta spp. and two Acromyrmex spp.).
It was present to some extent in all attine species sampled, but not in previously sampled Cyphomyrmex faunulus and Trachymyrmex septentionalis (Adams et al. 2010). Tyramide 1 is found in smaller proportions within all species sampled in this study but absent in C. faunulus (Adams et al. 2010) (Fig. 3).
Generally, we found that closely related species had similar tyramide compositions. Based on our samples, the four leaf-cutter species are largely consistent; their tyramide profiles are comprised mainly of tyramide 2, relatively small proportions of tyramide 3 in Acromyrmex species, and trace or small amounts of tyramide 1 in Atta and Acromyrmex, respectively. Mycetomollerius zeteki, My. opulentus, and a single (out of five) My. mikromelanos colony (RMMA190506-03) have similar profiles to the leaf-cutter species, despite being more closely related to Sericomyrmex. The other four My. mikromelanos colonies were consistent with one another, but not with any other sampled taxa. Paratrachyrmex cornetzi and P. bugnioni are qualitatively similar, with only three minor non-overlapping tyramides. The three Cyphomyrmex species (C. muelleri, C. longiscapus, Fig. 3 Tyramide composition-shown as a percentage of total peak area in the profile-across the phylogeny of the subtribe Attini (Branstetter et al. 2017). Data for C. faunulus and T. septentrionalis (indicated by *) are from Adams et al. (2010). Megalomyrmex milenae (Formicidae: Myrmicinae: Solenopsidini) is an outgroup. Each row represents the tyramide composition of the species, with darker shades representing larger proportions within the species; for species with multiple sampled colonies, the tyramide compositions are averaged across colonies. Absence of a compound is represented by dashes (-), trace amounts are indicated by ( +) and C. costatus), all members of the C. wheeleri species group, are qualitatively consistent (tyramides: 1, 2, 5, 6, 7, and 8), but vary in quantities (Fig. 3).
The two Sericomyrmex species, S. amabilis and S. opacus, showed the most variation between congeneric species, despite both being members of the S. amabilis species group. The tyramide profile of S. amabilis contains seven structures and is qualitatively similar to the C. wheeleri species group. In contrast, S. opacus contains 12 of the 13 tyramides found in this study, making its tyramide composition the most diverse (Fig. 3).
Megalomyrmex milenae, a non-attine species and our outgroup, has a distinctly different profile from the other species in this study. While tyramide 7 is found in some the attines as a minor compound, it is a major component in Me. milenae. Additionally, only Me. milenae produced greaterthan-trace amounts of compound 4 (Fig. 3). Megalomyrmex milenae also differs from the tyramide composition of S. invicta (composed almost entirely of tyramides 1 and 11), another Solenopsidini species (Vander Meer et al. 2021).

Discussion
In the current study, some genera (i.e., Atta, Acromyrmex, and Cyphomyrmex) show strong within-genus similarity in their tyramide profiles. Chemical profiles are, overall, most similar among closely related species; however, there was not a directional trend of structural diversity or profile complexity across our sampled taxa. Tyramides have been consistently observed in all Myrmicinae species examined thus far, though not without exception (i.e., Apterostigma dentigerum) Adams et al. 2010;Chen and Grodowitz 2017;Vander Meer et al. 2021).
In the fire ant, S. invicta, tyramides stored in the male endophallic bladder are transferred to winged females during mating, where they are converted to the biogenic amine tyramine. Tyramine activates rapid reproductive development in the newly mated queen. This male/female sexual co-evolved system rapidly overcomes the effects of the queen's primer pheromone that inhibits competitive reproductive development in her female sexual daughters ( Vander Meer et al. 2021). While the tyramide compositions of S. invicta and fungus-growing ants differ, their hydrolysis product is the same biogenic amine, tyramine. It is probable that tyramine plays a similar role in fungus growing ants-initiating rapid reproductive development in newly mated queens. Interestingly, S. invicta queens mate only once (Ross and Fletcher 1985), but some leaf-cutter queens mate with multiple males (Murakami et al. 2000). These polyandrous queens have sperm from multiple males (Den Boer et al. 2009); however, each male is anticipated to transfer the same blend of tyramides to the new queen. In theory, the newly mated queens will hydrolyze the tyramide mix to tyramine regardless of which male or combination of males produced them, resulting in the rapid onset of reproductive development.
Multiple reproductive isolation mechanisms have evolved to prevent cross-species mating, but movement of species to new habitats may compromise some behavioral, phenological, physiological, or geographic mating isolation mechanisms (Vander Meer et al. 1985;Ross et al. 1987;Vander Meer and Lofgren 1989). We posit that species-specific tyramide hydrolysis could be another mechanism for reproductive isolation. In the event of cross-species mating, tyramide hydrolysis (tyramine production) would not occur, reducing the rate of founding success of the newly mated queen. Alternatively, variation in tyramide composition may be a result of genetic drift, which would be consistent with the general similarity of tyramide compositions in congeneric species. If there is selection for the presence of tyramides, but little to no selection on specific tyramide structures or compositions, species may vary in this trait.
Enzymes often show a degree of substrate specificity (e.g., tyramide hydrolase found in the gynes of S. invicta) (Draganov et al. 2005;Horton et al. 2010;Kaur et al. 2014), but some attine males produce many tyramides with a diversity of structures, including a-ketoamides, simple alkyl amides, a-branched alkyl amides, and a, b-unsaturated amides. The surprising discovery of a diversity of tyramide structures will prompt future research to test the degree of tyramide hydrolysis specificity.
Within ants, evidence supports the specificity of tyramides to Myrmicinae males and its presence across the subfamily (Vander Meer unpublished). However, there may be exceptions. For example, it appears that Ap. dentigerum males produce little to no tyramides; however, we did not control for the age of males in this study. Sampling more Myrmicinae species will reveal broader phylogenetic tyramide patterns, and collecting males at light traps, outside the nest, will ensure sexual maturity. Such studies will provide further insight into the pervasiveness of male tyramides across ants and the functional link between tyramides and reproductive suppression of female alates inside natal nests. coordination with STRI. RMMA was supported by The Ohio State University. Thank you to our two reviewers and journal editor for their invaluable feedback on this manuscript.