bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 Nitrogen conservation, conserved: 46 million years of N-recycling by the core symbionts of

2 turtle

3

4 Yi Hu1*, Jon G. Sanders2*, Piotr Łukasik1, Catherine L. D'Amelio 1, John S. Millar3, David R.

5 Vann4, Yemin Lan5, Justin A. Newton1, Mark Schotanus6, John T. Wertz6, Daniel J. C.

6 Kronauer7, Naomi E. Pierce2, Corrie S. Moreau8, Philipp Engel9, Jacob A. Russell1

7

8 1 Department of Biology, Drexel University, Philadelphia, PA 19104, USA

9 2 Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA

10 02138, USA

11 3 Department of Medicine, Institute of Diabetes, Obesity and Metabolism, University of

12 Pennsylvania, Philadelphia, PA 19104, USA

13 4 Department of Earth and Environmental Science, University of Pennsylvania, Philadelphia, PA

14 19104, USA

15 5 School of Biomedical Engineering, Science and Health systems, Drexel University,

16 Philadelphia, PA 19104, USA

17 6 Department of Biology, Calvin College, Grand Rapids, MI 49546, USA

18 7 Laboratory of Social Evolution and Behavior, The Rockefeller University, 1230 York Avenue,

19 New York, NY 10065, USA

20 8 Department of Science and Education, Field Museum of Natural History, Chicago, IL 60605,

21 USA

22 9 Department of Fundamental Microbiology, University of Lausanne, 1015 Lausanne,

23 Switzerland bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

24 * These authors contributed equally to this manuscript.

25 Category: Biological Sciences-Evolution

26 Key Words

27 Nutritional symbiosis, nitrogen metabolism, , metagenomics

28 Short title: A nitrogen-recycling symbiosis in turtle ants

29 bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

30 Abstract

31 Nitrogen acquisition is a major challenge for herbivorous , and the repeated origins of

32 herbivory across the ants have raised expectations that nutritional symbionts have shaped their

33 diversification. Direct evidence for N-provisioning by internally housed symbionts is rare in

34 animals; among the ants, it has been documented for just one lineage. In this study we dissect

35 functional contributions by bacteria from a conserved, multi-partite gut symbiosis in herbivorous

36 ants through in vivo experiments, (meta)genomics, and in vitro assays. Gut bacteria

37 recycle urea, and likely uric acid, using recycled N to synthesize essential amino acids that are

38 acquired by hosts in substantial quantities. Specialized core symbionts of 17 studied Cephalotes

39 species encode the pathways directing these activities, and several recycle N in vitro. These

40 findings point to a highly efficient N-economy, and a nutritional mutualism preserved for

41 millions of years through the derived behaviors and gut anatomy of Cephalotes ants.

42 43 Introduction

44 Nitrogen (N) is a key component of living cells and a major constituent of the nucleic acids and

45 proteins directing their structure and function. Like primary producers1, herbivorous animals face

46 the challenge of obtaining sufficient N in a world with limited accessible N, suffering

47 specifically due to the low N content of their preferred foods2. The prevalence of herbivory is,

48 hence, a testament to the many adaptations for sufficient N-acquisition. Occasionally featured

49 within these adaptive repertoires are internally housed, symbiotic microbes. Insects provide

50 several examples of such symbioses, with disparate herbivore taxa co-opting symbiont N-

51 metabolism for their own benefit3, 4, 5, 6. Such tactics are not employed by all herbivores7,

52 however, and few studies have quantified symbiont contributions to host N-budgets8, 9, 10, 11, 12.

53 bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

54 Ants comprise a diverse insect group with a broad suite of diets. Typically viewed as predators

55 or omnivores, several ants are functional herbivores, with isotopic N-ratios overlapping those of

56 known herbivorous insects13, 14. While occasionally obtaining N from tended sap-feeding insects,

57 most are considered plant canopy foragers, scavenging for foods such as extrafloral nectar,

58 pollen, fungi, vertebrate waste, and plant wound secretions14. Quantities of usable and essential

59 N in such foods are limiting15, 16. Hence, the repeated origins of functional herbivory provide a

60 useful natural experiment, enabling tests for symbiotic correlates of N-limited diets. The

61 concentration of specialized bacteria within herbivorous taxa suggests such a correlation17, 18.

62 But N-provisioning by internally housed symbionts has only been documented for carpenter ants,

63 whose intracellular Blochmannia provide them with amino acids made from recycled N19.

64

65 Herbivorous cephalotines (i.e. Cephalotes and Procryptocerus) and ants from other herbivore

66 genera (i.e. Tetraponera and Dolichoderus) exhibit hallmarks of a symbiotic syndrome distinct

67 from that in the Camponotini. Large, modified guts with prodigious quantities of extracellular

68 gut bacteria make up one defining feature20, 21, 22, 23, 24. Also characteristic are the oral-anal

69 trophallaxis events transmitting symbionts between siblings21, 25, 26, 27 and the domination of gut

70 communities by host-specific bacteria17, 28, 29. Such symbiotic “hotspots” stand out in relation to

71 several ant taxa, which show comparatively low investment in symbiosis18, 23.

72

73 N-provisioning by bacterial symbionts in these ants has been hypothesized as a mechanism for

74 their success in a seemingly marginal dietary niche14, 17. To investigate this, we focus on the

75 turtle ants of the Cephalotes (Fig. 1). With ~115 described species30, stable isotopes place

76 these arboreal ants low on the food chain14, 31. Workers are canopy foragers, consuming bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

77 extrafloral nectar and insect honeydew, fungi, pollen and leaf exudates32, 33, 34. Cephalotes also

78 consume mammalian urine and bird feces, excreta with large quantities of waste N accessible

79 only through the aid of microbes. Given this, the remarkably conserved gut microbiomes of

80 cephalotines28, 35 are proposed as an adaptation for their N-poor and N-inaccessible diets. Here

81 we measure symbiont N-provisioning in Cephalotes varians and gene content within the gut

82 microbiomes of 17 Cephalotes species (Table S1), describing symbiont N-metabolism across 46

83 million years of evolutionary history.

84

85 Results

86 Gut bacteria of turtle ants do not fix N2

36, 37, 38, 39, 40 87 Atmospheric N2-fixation is executed by bacterial symbionts of some invertebrates ,

88 and prior detection of nitrogenase genes in ants17, 29 has led to the proposal that symbiotic

89 bacteria fix nitrogen for their hosts. To test this, three Cephalotes varians colonies were

90 subjected to acetylene reduction assays within hours of field capture. In three separate

91 experiments no ethylene was produced within test tubes containing acetylene-exposed ants

92 (Table S2), arguing against active N-fixation.

93

94 Symbiont-upgrading of dietary amino acids has minimal impact on workers’ N-budgets

95 Based on precedents from intracellular symbionts of insects12, 19, 41, we then tested whether gut

96 bacteria could upgrade dietary nitrogen compounds, transforming non-essential or inaccessible N

97 compounds into essential amino acids that are acquired by hosts. Our efforts focused on

98 glutamate, an important pre-cursor in the synthesis of many amino acids. Cephalotes varians

99 workers from three colonies were reared on artificial diets42 varying in the presence/absence of bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

100 antibiotics and the presence/absence of heavy isotope labeled glutamate. 13C or 15N were used to

101 label glutamate across our two separate experiments. Heavy isotope enrichment in the free amino

102 acid pools from worker hemolymph, assessed via GC-MS (Table S3), allowed us to quantify

103 symbiont glutamate upgrading and provisioning back to hosts.

104

105 Antibiotic treatment successfully suppressed gut microbial load in this and all below experiments

106 (Fig. S1), and workers survived treatments at rates sufficient for subsequent data generation (Fig.

107 S2). In addition, ants clearly absorbed nutrients from the administered diets, as hemolymph

108 glutamate pools showed 4-7% enrichment for heavy isotopes on the heavy vs. light isotope diets

109 in the absence of antibiotics (p=0.0033 15N vs. 14N diet; p=0.0018 13C vs. 12C diet). Yet, ant

110 acquisition of symbiont-processed C and N from dietary glutamate was minimal. For instance,

111 on the 13C-glutamate diet, antibiotic treatment reduced the fraction of heavy isotope-bearing

112 isoleucine (p=0.0147), leucine (p=0.0004), threonine (p=0.0029), and tyrosine (p=0.0169) in

113 worker hemolymph (Fig. S3), relative to estimates on this same diet without antibiotics. But

114 effect sizes for each amino acid were small, with changes of just 1.3-2.6% in ants with

115 suppressed microbiota. On the 15N-glutamate diet (Fig. S4), only phenylalanine (p=0.045)

116 showed heavy isotope enrichment in untreated vs. antibiotic treated workers, again, with small

117 effect size (2.9%).

118

119 Symbionts recycle, then upgrade dietary N-waste; hosts acquire this N

120 Cephalotes ants consume bird droppings and are attracted to mammalian urine. In addition, most

121 of their symbionts colonize the ileum25, the gut compartment where Malpighian tubules deliver

122 nitrogenous wastes (Fig. 1). Insects lack the capacity to convert their dominant nitrogenous bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

123 wastes—uric acid or urea—into usable forms of N. It has, thus, been posited that ant-associated

124 gut symbionts recycle N waste, further converting this N into essential amino acids that are

125 acquired by hosts21. To test this, we implemented an experiment similar to our dietary N-

126 upgrading assessments (above), using 15N isotope labeled urea instead of glutamate. After

127 consuming diets with heavy urea, 15 of the 16 detectable amino acids in C. varians hemolymph

128 were enriched for the heavy isotope signal when compared to diets with light (14N) urea (i.e. all

129 but asparagine; significant p-value range: 1.92E-14 - 0.0210) (Fig. 2; Table S3). On the 15N diet,

130 antibiotic treatment strongly reduced the heavy isotope signal in these same 15 amino acids

131 (significant p-values ranged from 1.92E-14 – 0.0410), directly implicating bacteria in the use of

132 diet-derived N-waste. The impact of bacterial metabolism on worker N-budgets was substantial,

133 with 15-36% enrichment of heavy essential amino acids in hemolymph of symbiotic, versus

134 aposymbiotic, ants within five weeks on the experimental diet.

135

136 Metagenomic analyses: strong taxonomic conservation

137 To address the mechanisms behind symbiont N-recycling and upgrading, we used shotgun

138 Illumina HiSeq sequencing to characterize microbiomes. Eighteen sequence libraries were

139 generated across seventeen Cephalotes species collected from four locales (Fig. 1; Table S1).

140 Two of these came from our experimental model C. varians. Shotgun sequencing efforts yielded

141 median values of 32,706,498 reads and 143.625 Mbp of assembled scaffolds per library (Table

142 S4). The median N50 for scaffold length was 1106.5 bp.

143

144 A prior study suggested that >95% of the Cephalotes gut community is comprised of core

145 symbionts from host-specific clades43. To assess whether the dominant bacteria sampled here bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

146 came from such specialized groups we extracted 16S rRNA fragments >200bp from each

147 metagenome library. Top BLASTn hits were downloaded for each sequence, and jointly used in

148 a maximum likelihood phylogenetic analysis. In the resulting tree (Fig. S5), 94.4% of our 335

149 Cephalotes symbiont sequences grouped within 10 cephalotine clades that included sequences

150 from prior in vivo studies. Inferences on metagenome content have, hence, been made using

151 partial genomes from the dominant, specialized core taxa.

152

153 Classification of assembled scaffolds took place using USEARCH comparisons against public

154 reference genomes in IMG and the KEGG database (see Materials & Methods, and

155 Supplementary Methods for more detail). Results from these analyses paralleled our 16S rRNA-

156 based discoveries of a highly conserved core microbiome (Fig. 3). In all metagenomes,

157 Cephaloticoccus symbionts44 from the Opitutales were the most dominant, with scaffolds from

158 these bacteria typically forming a single “cloud” differentiated from others by depth of coverage

159 and %GC content. Xanthomonadales scaffolds were ubiquitous and typically abundant, with

160 multiple scaffold clouds often evidencing co-existence of distinct strains, with up to ~10%

161 average %GC divergence. Less abundant, though still ubiquitous across metagenomes were

162 clouds of scaffolds from the Pseudomonadales, Burkholderiales, and Rhizobiales. Multiple

163 scaffold clouds at differing depths of coverage were consistent with multiple strain co-existence,

164 for each taxon, within most microbiomes. Unlike these core groups, Flavobacteriales,

165 Sphingobacteriales, and Campylobacterales were common but not ubiquitous. For instance,

166 presence/absence calls for N-metabolism genes (Table S5) suggested the absence or extreme

167 rarity of these symbionts in several metagenomes.

168 bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

169 Metagenomic analyses: urease is ubiquitous, N2 fixation is absent

170 Consistent with our acetylene reduction experiments using C. varians, IMG/ER based annotation

171 recovered no N-fixation genes in any of the 18 metagenome libraries. This absence encompassed

172 genes encoding the molybdenum-containing nitrogenase system (i.e. nifD, nifH, nifK), and those

173 from the iron-only (anfD, anfG, anfH, anfK) and vanadium-containing (vnfD, vnfG, vnfH, vnfK)

174 systems (Table S5). Together, our experiments and metagenomics suggest that prior

175 observations of nifH genes in Cephalotes workers arose from detection of rare or contaminant

176 bacteria17 or from a portion of the gut not included in the present study (exclusively the midgut,

177 ileum, and rectum).

178

179 Matching our discovery of symbiotic N-recycling in C. varians were findings of ureA, ureB, and

180 ureC genes in both C. varians metagenomes and in those of the 16 additional Cephalotes species

181 (Figs. 4, S6, S7; Table S5). The presence of complete gene sets for the core protein subunits of

182 the urease enzyme in all sampled microbiomes suggests that symbionts from most Cephalotes

183 species can make ammonia from N-waste urea. Taxonomic classification for urease gene-

184 encoding scaffolds suggested that abundant Cephaloticoccus symbionts (order: Opitutales)

185 encoded all three core urease genes. Complete copies of each gene were found on a single

186 Opitutales-assigned scaffold within 15 of 18 metagenomes. Genes encoding the urease accessory

187 proteins (ureF, ureG, and ureH) were often found on these same scaffolds, with a strong trend of

188 conserved architecture for this gene cluster (Fig. S6).

189

190 Urease genes were occasionally assigned to other bacteria (Fig. 4; Table S5), suggesting that

191 more than one symbiont can participate in this recycling function. Notable were cases from C. bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

192 rohweri (Xanthomonadales), C. grandinosus (Burkholderiales), and C. eduarduli (unclassified

193 Bacteria), which hosted additional bacteria encoding complete sets of urease core and accessory

194 proteins. In these cases, urease genes mapped to single scaffolds with identical gene order to that

195 seen for Cephaloticoccus (Fig. S6). Urease function was also inferred for Rhizobiales bacteria in

196 several Cephalotes species. Rhizobiales-assigned scaffolds encoding urease genes differed from

197 those of Cephaloticoccus with respect to gene order, the presence of the ureJ accessory gene,

198 and the existence of a gene fusion between ureA and ureB (Fig. S6).

199

200 A maximum likelihood phylogenetic analysis of UreC proteins encoded by the sampled

201 microbiomes identified two distinct Cephalotes-specific lineages (Fig. S8). The first (bootstrap

202 support = 99%) consisted of Rhizobiales-assigned UreC proteins, with relatedness to homologs

203 from various families in the Rhizobiales. The second (bootstrap support = 93%) was comprised

204 of proteins assigned to Opitutales, Burkholderiales, Xanthomonadales, and unclassified Bacteria,

205 showing relatedness to homologs from bacteria in the Rhodocyclales (Betaproteobacteria).

206

207 Metagenomic analyses: ammonia assimilation and amino acid synthesis genes found across

208 numerous core taxa

209 The above results provide genetic mechanisms to explain symbiont-mediated urea recycling in

210 C. varians, suggesting a broad distribution for this function across the Cepalotes genus. Also

211 necessary to explain our experiments are: 1. symbiont genes to assimilate the ammonia made

212 from urea degradation, and 2. symbiont genes using this assimilated N to synthesize amino acids.

213 Assessment of our metagenomes met these expectations in C. varians and all 16 other host bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

214 species. But in contrast to our findings for a small number of urea recyclers, genes involved in

215 these processes assigned to all core symbiont taxa, suggesting extensiove metabolic overlap.

216

217 Within all metagenomes, numerous taxa encoded complete gene sets for ammonia assimilation

218 (e.g. Figs. 4, S7, S9, S10; Table S5). Similarly, gene sets for the synthesis of each essential and

219 non-essential amino acid were complete in all metagenomes. With the exception of histidine

220 synthesis, complete only for Campylobacterales, each amino acid biosynthesis pathway was

221 complete within multiple bacterial taxa (Figs. 4, S6, S7, S9, S10; Table S5). Xanthomonadales

222 and Burkholderiales bins (outside of C. angustus) encoded all genes to synthesize the remaining

223 eight essential amino acids. This paralleled findings for Opitutales, Rhizobiales, and

224 Pseudomonadales: the former typically showed an incomplete pathway for lysine, while the

225 latter two often seemingly lacked a single gene for methionine biosynthesis. Sphingobacteriales

226 and Flavobacteriales lacked required genes in the lysine and methionine pathways. And

227 pathways for threonine, valine, leucine, isoleucine, tryptophan, and phenylalanine were

228 occasionally missing all or most genes within the Flavobacteriales. Many of the core taxa also

229 possessed complete gene sets for the synthesis of non-essential amino acids.

230

231 Metagenomic analyses: uric acid synthesis and degradation, and other means of urea production

232 Uric acid is a major waste product of many insects. This compound is also found in bird excreta,

233 a common Cephalotes food. To analyze capacities to recycle N from uric acid we examined gene

234 content in the pathway converting this compound into urea. Scaffolds assigning to ,

235 thus likely originating from Cephalotes genomes, often contained a subset of genes involved in bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

236 uric acid degradation, including one encoding the canonical uricase enzyme (uaZ) and one

237 encoding breakdown of 5-HIU (uraH; Table S5; Fig. S11).

238

239 Beyond those scaffolds, Burkholderiales bacteria were implicated in uricase function in all but

240 two metagenomes (Figs. 3, 4, S6, S7; Table S5). First, the puuD uricase homolog was detected

241 in 14 metagenomes. Encoding a membrane-associated form of this enzyme, with a C-terminal

242 cytochrome c domain45, this gene was found on Burkholderiales-assigned scaffolds in all cases

243 where detected. Genes encoding the remaining steps in the uric acid degradation pathway (i.e. 5-

244 HIUOHCUallantoinallantoateurea) also classified to Burkholderiales (Fig. S6). In total,

245 our analyses suggested complete gene sets for this pathway and taxon in 13 out of 18

246 metagenomes (Table S5; Figs. 4, S7). Maximum likelihood phylogenies of the bacterially

247 encoded PuuD and UraH proteins revealed monophyly of homologs from Cephalotes-associated

248 Burkholderiales (bootstrap support = 98% for PuuD and 76% for UraH; Fig. S12). Lineages in

249 both trees showed relatedness to homologous proteins from free-living Burkholderiales and other

250 Proteobacteria.

251

252 Genes synthesizing urea from uric acid mapped to numerous scaffolds across several

253 metagenomes. However, in seven libraries, they mapped to just one or two Burkholderiales-

254 assigned scaffolds. Synteny was conserved in these cases, and such scaffolds also possessed

255 additional genes encoding the subunits of xanthine dehydrogenase (Fig. S6), an enzyme

256 converting xanthine into uric acid. Core symbionts appear to produce xanthine via purine

257 recycling, as genes for guanine deaminase enzymes (Fig. S13) classified to Burkholderiales in 16 bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

258 metagenomes (Table S5). Adenine deaminase enzymes were similarly encoded by bacteria,

259 namely Rhizobiales, across 10 metagenomes.

260

261 Further analyses of our metagenomes revealed that bacteria aside from Burkholderiales can

262 produce urea through other mechanisms (Fig. 4; Table S5). For instance, across most hosts,

263 microbes from the Burkholderiales, Rhizobiales, Xanthomonadales, and/or Pseudomonadales

264 possessed arginase genes, catalyzing a reaction that converts arginine to urea and ornithine. In

265 several metagenomes, arginase genes also binned to Hymenoptera, suggesting their presence in

266 Cephalotes genomes. Genes for a separate, two-step pathway converting arginine to urea (Fig.

267 S14) were present in most metagenomes. But only Burkholderiales encoded both steps.

268

269 Refining symbiont role assignments: fine scale metagenome binning, cultured core symbiont

270 genomes, and in vitro N-recycling assays

271 Multiple strains for many of the aforementioned core taxa often co-exist within a single gut

272 community43. So despite pathway “completeness” assessed at the level of host order, it remains

273 unclear whether individual symbiont strains encode complete pathways for key aspects of N-

274 metabolism. We addressed this through genome sequencing of cultured symbiont strains from

275 five of the eight core bacterial taxa across two host species (i.e. C. varians and C. rohweri). The

276 14 strains prioritized for sequencing were chosen based on 16S rRNA gene identity (or near

277 identity) in comparison to core symbionts previously sampled through culture-independent

278 techniques (Fig. S5). Alignments of C. varians isolates to scaffolds from conspecific

279 metagenomes (Fig. S15) indicates that these strains or very close relatives are present in vivo,

280 supporting the relevance of in vitro findings from these strains to the natural gut community.. bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

281

282 Highlights of this work (Fig. 5; Supplementary Results; Table S7) included the discovery of a

283 Burkholderiales strain (Cv33a) with a capacity to convert uric acid into urea. This strain lacked

284 urease genes, but three cultured symbionts encoded all genes necessary for urease function,

285 including Cephaloticoccus isolates from C. varians (Cv41) and C. rohweri (Cag34) and a

286 Xanthomonadales symbiont from C. rohweri (Cag60). Thirteen out of fourteen isolates encoded

287 the glutamate dehydrogenase gene (gdhA) converting ammonia into glutamate, and most

288 encoded complete pathways for synthesizing most amino acids. As expected from metagenomic

289 analyses, all genomes lacked nitrogenase genes.

290

291 The fastidious nature of some symbionts limited our ability to infer strain functions for common

292 core taxa. Insights for these groups were gained through draft genome assembly from our best

293 sampled metagenome (i.e. C. varians colony PL010) using the Anvi’o platform (version 1.2.3)46

294 in conjunction with the CONCOCT differential coverage-based binning program47. The 11 near

295 complete draft genomes, where >87% of universal single copy genes were detected, spanned

296 seven of our eight core symbiont taxa (Fig. 5; Tables S8, S9, S10). Gene content analyses

297 supported findings from metagenomics and cultured isolate genomes (Supplementary Results).

298 In short, the dominant symbiont strains individually encoded up to 17 complete amino acid

299 biosynthesis pathways. Incomplete pathways were often missing just one to two genes. Nearly

300 all draft strain genomes showed capacities to assimilate ammonia into glutamate. N-recycling

301 pathways appeared incomplete within predicted N-recycling Burkholderiales and

302 Cephaloticoccus strains. This suggests the occasional absence of this function from these taxa bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

303 (but see Fig. 6) or, possibly, incomplete genome assembly due to challenges of scaffold binning

304 (Supplementary Results).

305

306 To test whether genetic signatures reflect actual N-recycling capacities, and to study the

307 conservation of this role within key taxa, we performed a series of in vitro assays, expanding the

308 number of targeted cephalotine species. Urease activity was qualitatively assessed by the

309 generation of ammonia in the presence of urea, and we obtained positive results for seven of

310 fifteen tested isolates (Fig. 6). All Cephaloticoccus (Opitutales) were positive, as was one of six

311 Xanthomonadales isolates. Results for four isolates with sequenced genomes accurately reflected

312 predictions from the presence/absence of urease genes.

313

314 Production of urea from allantoin served as a proxy for activity of the xanthine/uric acid pathway

315 (Fig. 6). Urea was produced from allantoin for 11 of the 17 assayed Burkholderiales isolates

316 (Table S11), suggesting function for at least part of this pathway. Coding capacity from the five

317 isolates with sequenced genomes accurately predicted the results of these assays.

318

319 In summary, genomic inferences on N-recycling seem to accurately reflect the metabolism of

320 core symbionts. And importantly, the phylogenetic placement of strains with in vitro assay data

321 reveal sporadic distributions of N-recycling, with notable enrichment in two clades

322 (Cephaloticoccus and a specific, unnamed lineage of Burkholderiales) suggesting long-standing

323 roles in the efficient use of N by the Cephalotes holobiont.

324

325 Discussion bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

326 Our findings show that ancient, specialized gut bacteria of Cephalotes ants recycle waste N

327 acquired through the diet (urea) and, likely, through ant waste metabolism (urea and uric acid).

328 Workers acquire large amounts of symbiont-recycled N in the form of essential and non-essential

329 amino acids. Symbionts encode genes to derive their own uric acid and urea, suggesting a third

330 potential origin for the influx of waste N into this system. Across a broad range of Cephalotes

331 species, gene content for N-metabolism varies little within core taxa and N-recycling roles

332 appear conserved within specific symbiont lineages. These findings depict an efficient N-

333 economy retained across 46 million years of Cephalotes evolution. They also support the

334 hypothesis that this multi-partite gut microbiome plays an adaptive role within an N-poor dietary

335 niche.

336

337 The magnitude of symbiont contributions to host N-budgets has rarely been calculated. But,

338 findings from wood-feeding termites implicate N-fixing bacteria in providing up to 60% of the N

339 in termite colonies10. Measures from the leaf-cutter ant system suggest that N-fixing bacterial

340 symbionts provide 45-61% of the fungus garden’s N-supply48. Our estimate in C. varians that

341 15-36% of the free amino acid pool was derived from symbiont-recycled N, within five weeks of

342 feeding, was notable, though not directly comparable to either of these estimates. Reduced

343 survival of antibiotic-treated workers, on diets where urea was the only source of N (Fig. S2), do

344 however suggest the importance of symbiont N-metabolism in adults. A similar importance of

345 bacteria was previously suggested for Cephalotes atratus49. In carpenter ants Blochmannia have

346 noticeable impacts on worker performance, larval and pupal development, and colony growth;

347 and the detriments of Blochmannia removal can be partially alleviated by the addition of

348 essential amino acids to the diets of aposymbiotic ants19. While the impacts of Cephalotes bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

349 worker microbiomes on larvae have not been measured, adult N-stores are implicated in larval

350 nourishment for several ants50. These results suggest a large potential for symbionts of adults to

351 shape performance at all stages within the colony.

352

353 The ubiquity of N-recycling Blochmannia across the Camponotini19, 51 combine with our

354 findings to support the hypothesized importance of nutritional symbionts in canopy-dwelling,

355 herbivorous ants14. A trend of “convergent associations”52 has, thus, emerged: canopy foraging

356 for N-poor or N-inaccessible foods has evolved separately in association with unrelated, yet

357 functionally similar symbionts. Future work on other ant herbivores and their conserved

358 symbionts23, 29 will assess the generality of such functional convergence. Also of interest will be

359 studies of symbiont-independent strategies for navigating N-poor diets7, 53.

360

361 The conserved nature of symbiont community composition across cephalotines is remarkable

362 compared to patterns for many arthropods54, 55, 56, adding to a trend across eusocial insects.

363 Within the termites, for instance, many core symbionts hail from host-specific lineages,

364 revealing ancient, specialized relationships3. Among the corbiculate bees, some relationships

365 with gut symbionts date back to 80 million years57. But even for these hosts, occasional symbiont

366 turnover takes place—in association with dietary shifts, for termites58, and among major

367 phylogenetic divisions in bees57.

368

369 Evolved behaviors have likely preserved partner fidelity in these groups. Among eusocial bees,

370 symbiont transfer takes place within the hive, through a combination of trophallaxis,

371 coprophagy, or contact with nest materials59, 60, 61. Termite siblings transmit symbionts through bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

372 oral-anal trophallaxis62, 63. A similar mode of passage has been noted for Cephalotes and

373 Procryptocerus ants and for other ant herbivores as well21, 25, 26, 27. Of likely further importance

374 for cephalotines is a fine-mesh filter, enveloping the proventriculus, which can bar the passage of

375 particles as small as 0.2 μM beyond the crop. This filter develops shortly after young adults

376 solicit trophallactic symbiont transfers25. Symbionts acquired during early adulthood will, thus,

377 be sealed off within the midgut, ileum, and rectum, with minimal opportunities for subsequent

378 colonization by additional, ingested microbes. These dual drivers of partner fidelity64 may

379 collectively explain the preservation of an ancient nutritional mutualism and sustained

380 exploitation of N-poor foods by successful canopy herbivores.

381

382

383 Materials & Methods

384 Collections and experimental assays

385 Details on ant collections and the uses of ants from particular locales are presented in Fig. 1 and

386 Table S1. For many of these protocols, additional details can be found in the Supplementary

387 Methods.

388

389 Experiments on live ants were performed on Cephalotes varians colony fragments collected

390 from the Florida Keys. Acetylene reduction assays were used to assess the capacity for N-

391 fixation. To achieve this, we incubated adult workers (and also, in some instances, queens,

392 larvae, and pupae) in air-tight syringe chambers loaded with acetylene very shortly after

393 collection in the field. After incubation, samples were analyzed with a gas chromatography-

394 flame ionization detector to quantify levels of acetylene and ethylene. bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

395

396 Controlled lab experiments were performed to quantify microbial contributions toward N-

397 upgrading of non-essential dietary amino acids and, separately, N-recycling, and subsequent

398 upgrading, of dietary N-waste. Adult workers from each experimental colony (n=3 colonies per

399 each of three experiments) were divided into three groups with equal number. In the first

400 treatment, workers were fed antibiotics to suppress or eliminate their gut bacteria for three

401 weeks. After this time, workers transitioned to the trial period where they were continuously fed

402 antibiotics in addition to a diet of glutamate (with 15N or 13C) or a diet with urea containing 15N.

403 Feeding for this trial period lasted four to five additional weeks. For the second and third

404 treatment groups, bacterial communities were not disrupted. Diets for workers in these groups

405 were identical to those of treatment group one, save for antibiotics, during the three week pre-

406 trial period. For the four to five week trial period, workers from the second treatment group were

407 fed on the aforementioned heavy-isotope diets; those from the third group were fed otherwise

408 identical diets in which glutamate or urea consisted of standard isotope ratios (i.e. biased toward

409 lighter isotopes).

410

411 During the trial period we recorded worker mortality, noting an elevation in the 15N urea feeding

412 group treated with antibiotics, but not in antibiotic-treated workers from the one examined

413 glutamate experiment (Fig. S2). Efficacies of antibiotic treatments were quantified via qPCR on

414 bacterial 16S rRNA genes and, for a subset of specimens, amplicon sequencing of 16S rRNA

415 (Fig. S1; Table S14). Worker hemolymph was harvested at the end of the four to five week trial,

416 from three to ten surviving workers per replicate. Hemolymph was then pooled, used for amino bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

417 acid derivitization, and subjected to GC-MS to quantify proportions of free amino acids

418 containing the heavy isotopes (see details in Table S12).

419

420 Metagenomics

421 Adult workers were dissected under a light microscope using fine forceps, with removal of gut

422 tissues from each dissected Cephalotes worker. DNA extractions were performed on ten single

423 guts for ten workers from each of two colonies for C. varians or on pools of guts from ten

424 workers in a single colony for each of the remaining Cephalotes species. Separate extractions

425 from C. varians siblings were then pooled within colonies. The resulting two DNA samples and

426 the 16 samples from other Cephalotes species were then used for metagenomic library

427 preparation. After size selection, adapter ligation, amplification, and clustering, samples were

428 sequenced (2x100bp or 2x150 paired end reads) on an Illumina HiSeq2500 machine. Sequences

429 were trimmed for quality, with removal of adapter sequences after de-multiplexing. Assembly of

430 reads from individual libraries then proceeded using a variety of k-values with the IDBA-UD

431 metagenomic assembler. Scaffolds were uploaded to the Integrated Microbial Genomes with

432 Microbiome Samples Expert Review (IMG/M-ER) website65. Classification in IMG/MER

433 proceeded based on USEARCH similarity against all public reference genomes in IMG and the

434 KEGG database. Due to some incorrect scaffold assignments (to errant bacterial taxa not known

435 from Cephalotes, such as Rhodocyclales), seven genomes from cultured bacterial isolates

436 belonging to core Cephalotes-associated taxa were used to obtain more accurate information of

437 phylogenetic binning. IMG/M-ER was used to annotate gene content from our scaffolds and

438 taxonomic bins. Based on these annotations, we focused on N-metabolism, using KEGG66 and

439 Metacyc67 as guides to manually construct degradation pathways for N-waste products and bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

440 biosynthetic pathways for amino acids. We examined the completeness of the N-waste

441 degradation pathways based on Fig. 6A and the completeness of the amino acid biosynthetic

442 pathways based on Figs. S7 and S9 across 18 metagenomes, 14 isolate genomes and 11 draft

443 genomes (as described below).

444

445 Homologs from N-recycling pathways and 16S rRNA genes were extracted from each dataset

446 and used in phylogenetic analyses with closely related homologs from the NCBI database. To

447 further aid in understanding taxonomic composition and to illustrate depth of coverage for the

448 taxa in our libraries, we generated “blob plots” based on read mapping to classified scaffolds

449 using BWA68 and modified scripts from a prior publication69. These graphs showed the depth of

450 coverage for each scaffold in relation to our classifications, along with the %GC content, a

451 taxonomically conserved genomic signature that further aided us in our efforts to visualize the

452 diversity of symbionts within microbiomes (Fig. 3)

453

454 Fine-scale metagenomic binning to generate draft symbiont genomes

455 To improve the assignment of metabolic capabilities to individual symbiont strains we used the

456 Anvi’o metagenome visualization and annotation pipeline (version 1.2.3)46 in conjunction with

457 the CONCOCT differential coverage-based binning program47. In doing so, we binned

458 assembled scaffolds from the metagenomic datasets of C. varians colony PL010—the library

459 with best symbiont coverage—into draft genomes of symbiont strains. Reconstruction of N-

460 metabolic pathways was then performed to comprehend the range of metabolic capabilities of

461 individual symbionts.

462 bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

463 Genomics and in vitro assays on cultured bacterial isolates

464 Gut tissues from Cephalotes and Procryptocerus worker ants were dissected and macerated.

465 Contents were then plated on tryptic soy agar plates, and plates were incubated at 25°C under an

466 atmosphere of normal air supplemented with 1% carbon dioxide in a CO2-controlled water-

467 jacketed incubator. After colony sub-cloning, pure isolate cultures were maintained under these

468 same conditions on the aforementioned plates or in tryptic soy broth. DNA extracted from these

469 cultures was subsequently used for 16S rRNA PCRs to compare isolates to bacteria previously

470 sampled through culture-independent studies. Isolates from C. varians and C. rohweri (both

471 previously well-studied through culture-independent means) were prioritized for genome

472 sequencing when their 16S rRNA sequences were identical or nearly identical to those of from

473 prior in vivo studies. Extracted bacterial DNA was used for library preparation and Illumina or

474 PacBio SMRT sequencing. Assembled genomes were uploaded to IMG/ER for annotation, with

475 N-metabolism pathway reconstruction and extraction of genes for phylogenetics occurring as

476 described above. Alignments of isolate genomes to metagenomes were done with Icarus70 as

477 implemented in MetaQuast71 and visualized in Circos72

478

479 A subset of cultured isolates was subjected to assays to detect ammonia production from urea.

480 Several were also tested to determine whether allantoin, a derivative of uric acid breakdown,

481 could be used to synthesize urea. Methodological details on these assays are described in the

482 Supplementary Methods. As described above for genome sequencing prioritization, strains

483 prioritized for assays were those deemed highly related to specialized core Cephalotes

484 symbionts.

485 bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

486 Fluorescence In Situ Hybridization

487 The fixed, dissected gut of an adult worker from Cephalotes sp. JGS2370 was hybridized with a

488 set of eubacterial probes labeled with AlexaFluor488 and AlexaFluor555 and imaged following

489 the protocol in SI Appendix, Supplementary Methods on a Leica M165FC microscope.

490 Fluorescent microphotographs taken using blue and green excitation filters were then merged with

491 a photograph taken under white light.

492

493

494 Acknowledgments

495 We thank Ryuichi Koga for assistance with Fluorescence In Situ Hybridization and Amit Basu

496 for help with amino acid derivitization. YH’s dissertation committee members Sue Kilham,

497 Shivanthi Anandan, Mike O’Connor and Sean O’Donnell provided useful suggestions on

498 statistics and experimental design. Dr Pamela Plantinga provided advice on statistical analyses

499 for in vitro symbiont assays. This study was funded by NSF grant #s 1050360 and 1442144 to

500 JAR, NSF grant #1442316 to CSM, and NSF grant #1442156 to JTW. Funding was also

501 provided by SNFS grant IZK0Z3_164213 to YH and PE, and by SNFS grant 31003A_160345 to

502 PE.

503

504

505

506

507

508 bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

509 References

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646 39. Lechene CP, Luyten Y, McMahon G, Distel DL. Quantitative imaging of nitrogen 647 fixation by individual bacteria within animal cells. Science 317, 1563-1566 (2007). 648 649 40. Breznak JA, Brill WJ, Mertins JW, Coppel HC. Nitrogen fixation in termites. Nature 650 244, 577-580 (1973). 651 652 41. Shigenobu S, Wilson ACC. Genomic revelations of a mutualism: the pea aphid and its 653 obligate bacterial symbiont. Cellular and Molecular Life Sciences 68, 1297-1309 654 (2011). 655 656 42. Straka J, Feldhaar H. Development of a chemically defined diet for ants. Insectes 657 Sociaux 54, 100-104 (2007). 658 659 43. Hu Y, Lukasik P, Moreau CS, Russell JA. Correlates of gut community composition 660 across an ant species (Cephalotes varians) elucidate causes and consequences of 661 symbiotic variability. Molecular Ecology 23, 1284-1300 (2014). 662 663 44. Lin JY, Russell JA, Sanders JG, Wertz JT. Cephaloticoccus gen. nov., a new genus of 664 ‘Verrucomicrobia’ containing two novel species isolated from Cephalotes ant guts. 665 International Journal of Systematic and Evolutionary Microbiology 66, 3034-3040 666 (2016). 667 668 45. Doniselli N, Monzeglio E, Dal Palù A, Merli A, Percudani R. The identification of an 669 integral membrane, cytochrome c urate oxidase completes the catalytic repertoire 670 of a therapeutic enzyme. Scientific Reports 5, 13798 (2015). 671 672 46. Eren AM, et al. Anvi'o: an advanced analysis and visualization platformfor 'omics 673 data. Peerj 3, (2015). 674 675 47. Alneberg J, et al. Binning metagenomic contigs by coverage and composition. Nat 676 Methods 11, 1144-1146 (2014). 677 678 48. Pinto-Tomas AA, et al. Symbiotic nitrogen fixation in the fungus gardens of leaf- 679 cutter ants. Science 326, 1120-1123 (2009). 680 681 49. Jaffe K, et al. Sensitivity of ant (Cephalotes) colonies and individuals to antibiotics 682 implies feeding symbiosis with gut microorganisms. Canadian Journal of Zoology 79, 683 1120-1124 (2001). 684 685 50. Wheeler DE, Martinez T. Storage proteins in ants (Hymenoptera:Formicidae). 686 Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 687 112, 15-19 (1995). 688 689 51. Williams LE, Wernegreen JJ. Genome evolution in an ancient bacteria-ant symbiosis: 690 parallel gene loss among Blochmannia spanning the origin of the ant tribe 691 Camponotini. Peerj 3, (2015). bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

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736 64. Kaltenpoth M, et al. Partner choice and fidelity stabilize coevolution in a Cretaceous- 737 age defensive symbiosis. Proceedings of the National Academy of Sciences of the 738 United States of America 111, 6359-6364 (2014). 739 740 65. Chen IMA, et al. IMG/M: integrated genome and metagenome comparative data 741 analysis system. Nucleic Acids Res 45, D507-D516 (2017). 742 743 66. Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids 744 Res 28, 27-30 (2000). 745 746 67. Caspi R, et al. The MetaCyc database of metabolic pathways and enzymes and the 747 BioCyc collection of Pathway/Genome Databases. Nucleic Acids Research 42, D459- 748 D471 (2014). 749 750 68. Li H, Durbin R. Fast and accurate long-read alignment with Burrows-Wheeler 751 transform. Bioinformatics 26, 589-595 (2010). 752 753 69. Kumar S, Jones M, Koutsovoulos G, Clarke M, Blaxter M. Blobology: exploring raw 754 genome data for contaminants, symbionts and parasites using taxon-annotated GC- 755 coverage plots. Frontiers in Genetics 4, (2013). 756 757 70. Mikheenko A, Valin G, Prjibelski A, Saveliev V, Gurevich A. Icarus: visualizer for de 758 novo assembly evaluation. Bioinformatics 32, 3321-3323 (2016). 759 760 71. Mikheenko A, Saveliev V, Gurevich A. MetaQUAST: evaluation of metagenome 761 assemblies. Bioinformatics 32, 1088-1090 (2016). 762 763 72. Krzywinski M, et al. Circos: an information aesthetic for comparative genomics. 764 Genome research 19, 1639-1645 (2009). 765 bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was A not certified by peer review) is the author/funder. All rightsB reserved. No reuse allowed without permission. 1 EBCC, Peru 2 Herradura, n=1 n=1 7.5 12 n=1 n=1 n=1 n=1 5.0 8 n=1 n=1 2.5 4 Trophic level (delta 15N) n=2 3 Ocampo, Argentina 4 Otamendi, Argentina 10.0 15

7.5 10 5.0 5 2.5 1 Trophic level (delta 15N)

Plants Plants

Predators Predators Cephalotes Cephalotes Sap-feeders n=5 Camponotus Sap-feeders Camponotus 2 Leave-chewers Leave-chewers 3 Other- n=10 Other-Myrmicinae 4 C

Ileum Locale Experimental and genomic studies Midgut Malpighian Tubules Florida, USA Arizona, USA Metagenomics Genomics Texas, USA In vivo colony Madre de Dios, Peru Cultured isolate Minas Gerais, experiments fragment experiments French Guiana Hindgut

766 Figure 1: Ecology of Cephalotes ants and origins of specimens used in our study.

767 A) Map showing sampling locales for cephalotines (Cephalotes and Procryptocerus) used in this

768 study (stars), the activities they were used for, along with sample size (i.e. # of species).

769 Numbered circles show sites of ant sampling in two prior studies14,31, from which stable nitrogen

770 isotope data were extracted and plotted here. B) Nitrogen isotope ratios obtained from

771 Cephalotes ants, other ants in the Myrmicinae, and Camponotus ants, hosts of known N-recycling

772 bacteria. High ratios of 15N/14N in organismal tissues suggest a higher placement on the food

773 chain. Panels show results from different locales, with each illustrating N isotope ratios for plants,

774 known herbivores, and known predators. C) C. atratus workers tend honeydew-producing,

775 ant-mimicking membracids (upper left). C. eduarduli and C. maculatus (smaller worker) feeding

776 on bird droppings (lower left). Soldier caste of C. varians with an outlined digestive tract (upper

777 right). A FISH microscopy image of a digestive tract from a Cephalotes worker is shown at lower

778 right. Note the large bacterial mass in the ileum near the midgut-ileum junction, the site where

779 N-wastes are emptied via Malpighian tubules. A bioRxiv preprint doi:50 https://doi.org/10.1101/185314** ; this version***** posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

40

30 containing 15N

Percentage of amino acid pool 20

Ile Leu Lys Met Phe Thr Val B 50 ** *** ** *

40

30

20 containing 15N

10 Percentage of amino acid pool

0 Ala Arg Asn Asp Cys Glu Gly Pro Tyr

14N-labeled urea & bacteria + 15N-labeled urea & bacteria - 15N-labeled urea & bacteria +

780 Figure 2: Symbiont removal reduces proportions of free, 15N-labeled amino acids in

781 hemolymph of Cephalotes varians workers consuming 15N-labeled urea . (A) Essential, and

782 (B) non-essential amino acids in ant hemolymph measured through GC-MS. Asterisks indicate

783 that 15N in essential amino acids from ants consuming 15N-labeled urea (blue) was significantly

784 higher than that in antibiotic-treated ants on this same diet (green) and in those consuming diets

785 with unlabeled urea (red). bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

786 Figure 3: Taxon-annotated GC-coverage plots for 18 Cephalotes metagenomes, reveals

787 taxonomic conservation. Assembled scaffolds in each metagenome are plotted based on

788 their %GC content (x-axis) and their depth of sequencing coverage (y-axis, log scale). Bacterial

789 genomes vary in %GC genome content and core symbionts show variable abundance; these 790 bioRxivplots, preprint thus, doi: illustratehttps://doi.org/10.1101/185314 the existence ;of this numerous version posted dominant September symbiont7, 2017. The strainscopyright inholder Cephalotes for this preprint worker (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 791 guts. Phylogeny at lower right, based on 16S rRNA sequences from our metagenomes, identify

792 the Cephalotes-specific clades from which nearly all of our sequence data have been obtained.

793 Colors on the phylogeny match those in the blob plots, illustrating the taxa to which scaffolds

794 were assigned. Circle size reveals scaffold length. Not shown here are scaffolds binning to

795 Hymenoptera or to unclassified organisms. UAbioRxiv exported preprint doi: https://doi.org/10.1101/185314 UA from urea; this from version posted SeptemberTaxonomic 7, 2017. The assignments copyright holder for this preprint (which was from ant not certifiedbird dropping by peer review) ismammal the author/funder. urine All rights reserved. No reuse allowed without permission. malpighian tubules Cephalotes ant hosts Burkholderiales Pseudomonadales Xanthomonadales Rhizobiales Uric acid (UA) Opitutales 1 1 1 1 1 1 4 5 Xanthine 5-HIU Urea NH3 Glu 3 2 3 Arginine

Millions40 30 of 20 years10 ago 0 N-recycling & assimilation Essential amino acid biosynthesis Eocene Oligo. Miocene Plio. P. 1 2 3 4 5 Lys Thr Met Val Leu Ile His Trp Phe 1 C. atratus 0.5 0 1 C. rowheri 0.5 0 1 C. clypeatus 0.5 0 1 C. simillimus 0.5 0 1 C. minustus 0.5 0 1 C. spinosus 0.5

0 Pathway completeness 1 C. pusillus 0.5 0 1 C. umbraculatus 0.5 0 1 C. angustus 0.5 0 1 C. pellans 0.5 0 1 C. pallens 0.5 0 1 Cephalotes ant phylogeny C. varians 0.5 0 1 C. maculatus 0.5 0 1 C. grandinosus 0.5 0 1 C. persimilis 0.5 0 1 C. persimplex 0.5 0 1 C. eduarduli 0.5 0

796 Figure 4: Pathways for N-waste recycling and amino acid biosynthesis and their

797 distributions across core gut symbionts from 17 Cephalotes species. Various symbiotic gut

798 bacteria convert N-wastes into ammonia, incorporate ammonia into glutamate, and synthesize

799 essential amino acids. Upper panel shows sources of the N-wastes uric acid (bacterial

800 metabolism via xanthine degradation, bird droppings, host ant waste-metabolism via Malpighian

801 tubule delivery) and urea (mammalian urine, uric acid metabolism, and arginine metabolism). bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was 802 Arrows innot this certified panel by peer are review)colored is the to author/funder. reflect All rights reserved. of the coreNo reuse Cephalotes allowed without-specific permission. microbes

803 participating in these steps in multiple metagenomes. Numbers near arrows link particular

804 pathways to bar graphs (below), which in turn plot pathway completeness (i.e. proportion of all

805 genes present) for the dominant core taxa in each metagenome. At left on the lower panel below

806 is the phylogeny of Cephalotes species used for metagenomics including a chronogram dating

807 divergence events in these species’ history30.

Xanthine/UA degradation Urea degradation Non-essential AA biosynthesis Essential AA biosynthesis Glu Gln Tyr Pro Asp Asn Ala Ser Gly Cys Arg* Lys Thr Met Val Ile Leu Phe Trp His bioRxiv preprintSpingobacteriales_Bin11 doi: https://doi.org/10.1101/1853140; this0 version1 1 posted1 1 September 1 1 0 7, 12017.1 The 1 1copyright1 1 holder1 1for 1this 1preprint1 1 (which 1 was Flavobacteriales_Bin16not certified by peer review) is the0 author/funder.0 0 1 All1 rights0 1 reserved.0 0 No1 reuse1 0 allowed 0 1 without0 0 permission.0 0 0 1 0 0 Cephaloticoccus primus (Cag34) 0 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 Cephaloticoccus capnophilus (Cv41) 0 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 Opitutales_Bin7-1 0 0 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 Campylobacterales_Bin17 0 *0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Rhizobiales sp. (JR021-5) 0 0 1 1 0 0 1 0 0 0 1 1 0 1 1 1 0 0 0 0 0 0 Rhizobiales_Bin13 0 0 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Rhizobiales_Bin6-2 0 1 1 1 1 0 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Rhizobiales_Bin6-3 0 0 1 1 1 0 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Xanthomonadales_Bin14 0 0 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Xanthomonadales (Cag60) 0 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Ventosimonas gracilis (Cv58) 0 0 1 1 1 1 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Ventosimonas sp. (Cag320) 0 0 1 1 1 1 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Ventosimonas sp. (Cag27) 0 0 1 1 1 1 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Ventosimonas sp. (Cag26) 0 0 1 1 1 1 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Bacterial phylogeny Burkholderiales_Bin12 0 0 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Burkholderiales sp. (Cag20) 0 0 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Burkholderiales sp. (Cv44) 0 0 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Burkholderiales sp. (Cag25) 0 0 1 1 1 1 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Burkholderiales_Bin3-1 1 0 1 1 1 0 0 0 1 0 1 1 1 1 1 1 1 1 1 1 1 1 Burkholderiales sp. (Cv33a) 1 0 1 1 1 1 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Burkholderiales sp. (Cv36) 0 0 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Burkholderiales_Bin5-1 0 0 1 0 1 1 1 0 0 1 0 1 1 1 1 1 1 1 1 1 1 1 Burkholderiales (Cv52) 0 0 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 Color key: Pathway completeness Host 0 0 0 0 0 1 1 1 1 1 1 C. varians C. rohweri 0 0.10.20.3 0.4 0.5 0.6 0.7 0.80.9 1

808 Figure 5: Core symbiont strains possess complete or near complete pathways for N-

809 recycling and amino acid biosynthesis. Heatmap illustrates the proportion of genes present

810 from each N-metabolic pathway across distinct symbiont strains. Coding capacities for strains

811 were inferred from 14 fully sequenced cultured isolate genomes (symbionts from C. varians and

812 C. rohweri) and 11 draft genomes (assembled from C. varians colony PL010 metagenome;

813 identified by the term “Bin” within their names). The maximum likelihood phylogeny of

814 symbiotic bacteria on the left was inferred using an alignment of amino acids encoded by seven

815 phylogenetic marker genes obtained from symbiont genomes, and branch colors are used to

816 illustrate distinct bacterial orders. Red asterisk for urea recycling in the Cephaloticoccus-like

817 Opitutales bin (7-1) indicates that urease genes from the PL010 metagenome binned to

818 Opitutales, but not to the draft genome for the dominant strain. When combined with the likely

819 presence of just one Opitutales strain within the PL010 microbiome, it is likely that a completely

820 assembled genome would encode all urease genes. The black asterisk next to Arg denotes that

821 inferences on pathway completeness for arginine biosynthesis were based on the pathway

822 starting with glutamate (see Fig. S9), as opposed to other metabolic mechanisms. In vitro assayed N-recycling pathways: A bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Xanthine/Uric acid pathway Urea degradation

UreE UreG 1 kb UreF uraH allA xdhA xdhB xdhC UreA UreB UreC puuD TIGR03212PRHOXNBalc 1 kb UreH ureH ureA ureB ureC ureE ureF ureG Gene cluster in Burkholderiales bacteria Gene cluster in Optitutales Accessory Urease subunit xdhABC puuD uraH PRHOXNB TIGR03212 alc and Xanthomonadales bacteria proteins encoding proteins Xan UA 5-HIU OHCU Allantoin Allantoate urea urea ammonia urea production assay urea degradation assay

B Phylogenetic diversity of cultured core symbiont N-recyclers 0.01 JQ254782 Cultured Urea production CV33a Host Strain taxonomy JQ254474 symbiont strain from allantoin CSM3487-56 CV33a C. varians positive CSM3487-49 * CSM3490-51 CSM3487-56 Procryptocerus positive ¶ CSM3490-52 pictipes JDR108A-110-103 CSM3487-49 positive JDR108-110A-72 CSM3490-51 positive JQ254448 C. placidus SP57-223A CSM3490-52 positive ¶ SP57-225 JDR108-110A-103 positive ¶ JQ254421 C. texanus CV52 JDR108-110A-72 positive ¶ KF730291 SP57-223A positive ¶ C. rohweri CSM3487-15 SP57-225 positive Burkholderiales JR038B-95 ¶ FJ477560 CV52 C. varians negative KF730309 Procryptocerus * CSM3487-15 negative POW0550W-166 pictipes FJ477679 JR038B-95 C. texanus positive ¶ KF730294 FJ477612 POW0550W-166 positive POW0550W-2 POW0550W-2 negative ‡ POW0550W-160 POW0550W-160 C. varians negative FJ477592 ‡ CV44 CV44 negative CV32 CV32 * negative KF730310 ¶ Urea is produced without allantoin but* allantoin boosts urea production. ‡ Urea is produced and allantoin has no impact on urea produciton.

Cultured Urea degradation JDR108-110A-27 Host Strain taxonomy JDR108-110A-112 symbiont strain via urease JDR108-110A-105 JDR108-110A-27 positive JQ254444 JDR108-110A-112 C. texanus positive 0.01 MF187348 CAG34 JDR108-110A-105 positive Opitutales Cv41 CAG34 C. rohweri positive (Cephaloticoccus) FJ477619 Cv41 * positive POW0550W-89 C. varians JQ254448 POW0550W-89 * positive CV33 Cv33a C. varians negative Burkholderiales JQ254474 * Pseudomonadales KF730304 Cv58 C. varians negative CV58 * (Ventosimonas) FJ477623 POW0550W-4 negative POW0550-4 POW0550W-97 C. varians negative FJ477594 POW0550-97 POW0550W-103 negative KF730290 CAG62 C. rohweri positive Xanthomonadales KF730295 JDR108-110A-106 negative POW0550-103 FJ477554 JDR108-110A-108 C. texanus negative CAG62 JDR108-110A-57 negative JDR108A-110-106 JDR108A-110-108 JDR108A-110-57 ≥80% bootstrap support Core Cephalotini bacteria from culture independent efforts * Isolates with sequenced genomes: in all cases in vitro results match expectations from genome annotation. 823 Figure 6: A limited range of cultured core symbiont strains recycle N in vitro. Results

824 summarize findings from symbionts of five cephalotine ants, including four species from the 825 bioRxivgenus preprint Cephalotes doi: https://doi.org/10.1101/185314 and one from its; thissister version genus posted Procryptocerus. September 7, 2017. A) The Genes copyright and holder pathways for this preprint used (which by was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 826 specialized gut symbionts to recycle the N-wastes uric acid and urea. The architecture for

827 clusters of symbiont genes encoding the involved enzymes are illustrated. Red boxes within

828 these pathways represent the metabolic steps assayed for panel B. B) Shown at left are

829 phylogenies of cultured symbionts subject to metabolic assays in vitro and their closest relatives

830 in the NCBI database, which are enclosed within taxon-specific colored boxes. Most cultured

831 isolates had 16S rRNA sequences that were identical or most closely related to one obtained

832 from a Cephalotes ant through culture-independent means. Such isolates also showed high

833 identity to abundantly represented scaffolds from our metagenomes (Fig. S15). Nodes for

834 cultured symbionts are connected to relevant rows within data tables, where the results of assays

835 for urea production (from allantoin—part of the uric acid pathway) and urea degradation (to

836 ammonia) assays are illustrated. Asterisks highlight isolates with a sequenced genome; for each

837 of these, in vitro results matched expectations derived from gene content. Additional symbols

838 used in the urea production table indicate whether allantoin boosted urea production and whether

839 production was completely allantoin-dependent. Urea production without complete allanotin-

840 dependence is likely to stem from arginine metabolism.