Convergent evolution of a nutritional symbiosis in ants

16 Ants are among the most successful organisms on earth. It has been suggested that forming symbioses 17 with nutrient-supplementing microbes may have contributed to their success, by allowing ants to 18 invade otherwise inaccessible niches. However, it is unclear whether ants have repeatedly evolved 19 symbioses to overcome the same nutrient limitations. Here, we address this question by comparing the 20 independently evolved symbioses in Camponotus , Cardiocondyla , Formica and Plagiolepis ants. Our 21 analysis reveals the only metabolic function consistently retained in all of the symbiont genomes is 22 the capacity to synthesise tyrosine, which is essential for insect cuticles. We also reveal that in certain 23 multi-queen lineages, only a fraction of queens carry the symbiont, suggesting ants differ in their 24 colony-level reliance on symbiont-derived nutrients. Our results suggest symbioses can arise to solve 25 common problems, but hosts may differ in their dependence on symbionts, highlighting the 26 evolutionary forces influencing the persistence of long-term endosymbiotic mutualisms.


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Ants are among the most ecologically dominant organisms in terrestrial ecosystems, and part of their 31 success lies in their ability to occupy a wide range of habitats. It has been suggested that acquiring   with essential amino acids that can improve brood production, especially when proteins are scarce 1 .

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Blochmannia is also thought to aid hosts in nitrogen recycling and synthesises the aromatic amino 46 acid tyrosine, which is an important component of insect cuticles 5 . The symbiont of Cardiocondyla 47 obscurior, Candidatus Westeberhardia cardiocondylae, hereafter Westeberhardia, despite having a 48 highly reduced genome, has also retained the capacity to synthesise tyrosine through a shared 49 metabolic pathway with its ant host 6 . Two additional ant genera, Formica and Plagiolepis, are also 50 known to harbour symbionts within bacteriocytes surrounding the midgut, suggesting they also play a 51 role in provisioning nutrients for their hosts 7-9 . However, the functional role of the symbionts in 52 Formica and Plagiolepis is currently unknown.

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While the acquisition of nutrient provisioning symbionts has repeatedly allowed insects to 54 invade nutrient imbalanced niches, such as plant sap and blood feeding, it is less clear why these 55 3 relationships evolve in predominantly omnivorous insects such as ants. In particular, it is unclear 56 whether ants have repeatedly evolved symbioses to overcome the same vital nutrient limitations. This 57 has limited our understanding of the metabolic challenges facing omnivorous insects, and how 58 nutritional symbioses evolve to overcome them.

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The aim of this study is to determine whether the four bacteriocyte-associated symbioses in 60 ants represent ancient nutritional mutualisms that have evolved to serve similar functions for their 61 hosts. We first characterise the genomes of the symbionts in Formica and Plagiolepis, and several 62 new strains of Westeberhardia from phylogenetically divergent Cardiocondyla lineages. Using a 63 comparative approach, we then ask whether the symbionts from all four ant lineages have retained 64 metabolic pathways in their highly reduced genomes that suggest they serve similar nutrient-65 provisioning roles for their hosts. We then investigate the phylogenetic and intracolony distributions 66 of symbionts in diverse Formica and Cardiocondyla species to determine the origins of each 67 symbiosis and its prevalence across species and castes. This survey reveals that in many ant lineages 68 that maintain multi-queen (polygynous) colonies, only a fraction of queens carry the symbiont,

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suggesting species differ in their dependence on symbiont-derived nutrients at the colony level. We

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We first tested the hypothesis that each of the four ant lineages hosts its own ancient strictly vertically 77 transmitted symbionts that have co-speciated with its host. To address this aim, we compared the 78 genomes of symbionts from 13 species of ants, representing four independently evolved symbioses.

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This includes symbionts from three Formica, two Plagiolepis, and an additional three Cardiocondyla 80 species that we sequenced, in addition to four previously published genomes from Blochmannia, the 81 obligate symbiont of Camponotus ants, and the one pre-existing Westeberhardia genome from 82 Cardiocondyla obscurior 6,10-13 .

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We found the gene order of single copy orthologs in symbionts is perfectly conserved in ant 84 species belonging to the same genus (Fig. 1). This type of extreme structural stability of genomes only 85 occurs in symbionts that have been strictly vertically transmitted within a matriline 14 and has been 86 documented in the obligate symbionts of whiteflies, psyllids, cockroaches and aphids 15-18 . In contrast, 87 genome structure differed substantially between symbionts from different ant genera (Fig. 1, Fig S1).

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In Formica and Cardiocondyla ant species, we also find that the host and symbiont phylogenies are in 89 general concordance (Fig S2). This strongly suggests the symbioses in each of the four ant lineages

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Despite on-going genome reduction, obligate symbionts of insects typically retain gene networks 125 required for maintaining the symbiosis with their host, such as pathways for synthesising essential 126 nutrients. This has resulted in the symbionts of sap-and blood-feeding insects converging on genomes 127 that have retained the same sets of metabolic pathways -to synthesis essential nutrients missing in 128 their hosts' diets 23,24 . Here we test the hypothesis that the four bacteriocyte-associated symbionts of 129 ants have been acquired to perform similar functions. For this, we assess whether they have 130 consistently retained metabolic pathways to synthesis the same key nutrients. Two major patterns 131 stand out.

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First, we find that the four divergent ant symbionts have all retained the shikimate pathway, 133 which produces chorismate, along with most of the steps necessary to produce tyrosine from this 134 precursor (Table 1 and Table S2). Both the symbiont of Formica and Westeberhardia lack one of the 135 genes required to produce tyrosine. However in Westeberhardia it is believed the host provides the 136 gene to complete the final step of the pathway 6 , and we find this gene is also present in the Formica 137 ant genomes (Fig S4). In addition, all symbionts except Westeberhardia can produce phenylalanine 138 which is a precursor that can be converted to tyrosine by their hosts 5,25,26 . Tyrosine is important for 6 insect development as it is used to produce L-DOPA, which is a key component of insect cuticles 5 (Table S1). This suggests that provisioning of tyrosine, 154 or tyrosine precursors, is of general importance across all bacteriocyte-associated symbioses of ants.

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Second, our comparative analysis revealed clear differences in the pathways lost or retained 156 across symbionts (Table 1 and Table S2). This is most evident when comparing Blochmannia with 157 Westeberhardia, the latter of which has lost the capacity to synthesise most essential nutrients. The

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Previous work on the extracellular gut symbionts of several arboreal ant lineages identified 169 nitrogen recycling via the urease operon as a function that may be of key importance for ant 170 symbioses 1,2,31,32 . However, we do not find any evidence that the symbionts of Formica, Plagiolepis, 171 or Cardiocondyla play a role in nitrogen recycling via the urease operon (Table 1). This suggests 172 nitrogen recycling may play an important role for more strictly herbivorous ants, such as Cephalotes.

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Our results, however, indicate tyrosine may be universally required for cuticle synthesis across a

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Our investigation reveals the natural occurrence of uninfected queens is a widespread 205 phenomenon in many Formica and Cardiocondyla species (Figure 4). We confirmed the absence of 206 symbionts in queens, and that they have not been replaced with another bacterial or fungal symbiont, 207 using diagnostic PCR, whole genome and deep-coverage amplicon sequencing (Table S3, Table S4,

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The degradation and eventual loss of symbionts from bacteriocytes has been reported in 216 males, and in sterile castes of aphids and ants 37 , which do not transmit symbionts to offspring. In 217 reproductive females, bacteriocytes may degrade as a female ages; however, symbionts are typically 218 retained at high bacterial loads in the ovaries, as this is required to maintain the symbionts within the 219 germline 22 . It is of note that all of the symbiotic ant species we investigated maintain multi-queen 220 colonies, and the vast majority had at least one queen, often more, within a colony that carried the 221 symbiont (Table S5). We hypothesize that species that maintain colonies with uninfected queens may 9 be able to retain sufficient colony-level fitness with only a fraction of queens harbouring the symbiont 223 and receiving its nutritive benefits.

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Dependence on symbionts in a socioecological context

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The retention of symbionts in queens and workers of some species, but not others, suggests species 227 either differ in their dependence on symbiont-derived nutrients, or that symbionts have lost the 228 capacity to make nutrients in certain host lineages. Our analysis of symbiont genomes did not reveal 229 any structural differences, such as the disruption of metabolic pathways, which could explain 230 differences in symbiont retention between host species (Table S2). This suggests differences in the 231 retention of symbionts may reflect differences in host ecologies.

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In ants, which occupy a wide range of feeding niches, reliance on symbiont-derived nutrients 233 will largely depend on lineage-specific feeding ecologies. For example, Camponotus ants have been 234 shown to be predominantly herbivores. Blochmannia, in turn, has retained the capacity to synthesise 235 key nutrients missing in plant-based diets, such as essential amino acids. Blochmannia is also always 236 present in queens and workers 22 , which is a testament to the importance of these nutrients for the

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Our data suggests that symbiotic relationships can evolve to solve common problems but also 262 rapidly breakdown if the symbiosis is no longer required. We have identified tyrosine provisioning as 263 a unifying function across bacteriocyte-associated symbionts of ants. But we have also shown species 264 can vary in how much they depend on symbionts for nutrients. Our results demonstrate that ants have 265 a unique labile symbiotic system, allowing us to better understand the evolutionary forces that 266 influence the persistence and breakdown of long-term endosymbiotic mutualisms.             Table S2.

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*Tyrosine is considered a non-essential amino acid because it can be synthesised by most eukaryotic 533 hosts from phenylalanine.