Please choose the correct phrase for each underlined spot. Thanks. 1) The hybrid v/g microbiome is dominated by Staphylococcus/ Providencia/ Propionibacterium/ Corynebacterium/ Proteus/ Enterobacteria

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Acknowledgments:We acknowledge J. T. Lis, I. Golding, R. Phillips, O. Bensaude, and E. Bertrand for valuable discussions. We thank members of the Darzacq, Dahan, and Howard Hughes Medical Institute–Janelia Farm Transcription Imaging Consortium groups for suggestions.

I.I.C. was supported by fellowships from the European Molecular Biology Organization and the Foundation PierreGilles de Gennes (FPGG). I.I. acknowledges the Netherlands Organization for Scientific Research and FPGG for financial support. A.S. was supported by La Ligue nationale contre le cancer. L.M. acknowledges support from Centre de Génétique Moléculaire UPR 3404. This work was supported by grants Agence Nationale de la Recherche Pol2Kinetics to X.D. and DYNAFT to X.D. and M.D.; X.D. and M.D. acknowledge the support of Nikon France. Supplementary Materialswww.sciencemag.org/cgi/content/full/science.1239053/DC1 Materials and Methods Supplementary Text Figs. S1 to S8 Table S1 References 11 April 2013; accepted 17 June 2013 Published online 4 July 2013; 10.1126/science.1239053 The Hologenomic Basis of Speciation:

Gut Bacteria Cause Hybrid Lethality in the GenusNasonia Robert M. Brucker 1*and Seth R. Bordenstein 1,2* Although the gut microbiome influences numerous aspects of organismal fitness, its role in animal evolution and the origin of new species is largely unknown. Here we present evidence that beneficial bacterial communities in the guts of closely related species of the genusNasonia form species-specific phylosymbiotic assemblages that cause lethality in interspecific hybrids.

Bacterial constituents and abundance are irregular in hybrids relative to parental controls, and antibiotic curing of the gut bacteria significantly rescues hybrid survival. Moreover, feeding bacteria to germ-free hybrids reinstates lethality and recapitulates the expression of innate immune genes observed in conventionally reared hybrids. We conclude that in this animal complex, the gut microbiome and host genome represent a coadapted“hologenome”that breaks down during hybridization, promoting hybrid lethality and assisting speciation. A lthough the gut microbiome influences numerous fitness traits in animals, little at- tention has been given to how the micro- biome is structured between closely related species and how the microbiome contributes to the origin of new species. By incorporating the microbiome into the biological species concept (1)andthe Bateson-Dobzhansky-Muller model of hybrid in- compatibilities (2), theoretical evidence specifies that negative epistasis between host genes and the host microbiome can accelerate the evolution of hybrid lethality and sterility (3).

We recently established that when environ- mental factors such as diet are controlled for, species of theNasoniawasp complex harbor phylosymbiotic gut microbiotas—atermintro- duced here to denote microbial community rela- tionships that recapitulate the phylogeny of their host (4). Similar to phylogenomics, phylosymbio- sis asserts that the relationships of microbiomesacross host species maintain an ancestral signal of the host's evolution. The phylosymbiotic signal could be a consequence of host immune genes that rapidly evolve in a continual arms race with components of the microbiome.

We hypothesize that the phylosymbiotic gut microbiome within species breaks down in hybrids via epistatic interactions between the microbiome and nuclear and/or cytoplasmic genomes, leading to hybrid lethality. Using the parasitoid wasp genus Nasonia, we set out to experimentally determine the influence of the microbiome on hybrid lethality.

TheNasoniagenus consists of several species of haplodiploid parasitoid wasps that are readily hybridized.Nasonia vitripennisdiverged approx- imately 1 million years ago from the ancestor of Nasonia giraultiandNasonia longicornis,which themselves diverged less than 400,000 years ago (5). In the laboratory, all three species are reared on the fly hostSarcophaga bullataunder iden- tical conditions.Nasoniaoffspring are oviposited by the mother inside the puparium of the fly, where the eggs develop to adulthood before emerging from the fly host in about 14 days. In the absence ofWo l b a c h i ainfections, reciprocal crosses be- tween species produce fertile, diploid, F 1hybridfemales (6). However, hybrid lethality is observed in F 2male offspring, because they are haploid recombinants of their grandparents. Interspecies crosses ofN. vitripennisandN. giraultiorN.

longicornisresult in F 2hybrid males exhibiting up to ~90% lethality during larval development, whereasN. giraultiandN. longicornishybrids only exhibit ~8% hybrid lethality (6–10).

In this study, we scored the average number of hybrid and nonhybrid eggs, larvae, pupae, and adults produced byNasoniafemales (n=48 females, each parasitizing oneS. bullatahost per developmental period) and determined that 78% of hybrid lethality occurs between the first- and the fourth-instar (L4) larval stages (Fig.

1A). There was a slight asymmetry in the num- ber of survivingN. vitripennis/N. giraultiand N. giraulti/N. vitripennishybrids (the F 2hybrid genotype denotes grandfather or grandmother), but the difference was not statistically significant (P= 0.36, Mann-Whitney U test). In contrast, hy- brids of the younger sister speciesN. giraultiand N. longicornisexhibited little to no F 2hybrid le- thality, as previously reported (6)(fig.S1A).The hybrid lethality betweenN. vitripennisandN. giraulti is often diagnosed by larval melanization—aprom- inent immune response to pathogens in arthropods (Fig. 1B). We postulated that because parental Nasoniaspecies assemble phylosymbiotic gut mi- crobiomes (4) (Fig. 2), the melanization and lethality in larval hybrids result in part from al- tered gut microbiomes.

First, we tested the hypothesis that bacterial community differences occur between larval hy- brids and nonhybrids during the L2 larval stage, just before the point of F 2hybrid male lethality.

We f o c u s e d o n t h eN. vitripennis/N. giraultihy- brid microbiota, because this genotype elicits elevated lethality as compared to the reciprocal cross; however, this asymmetry was insignificant in our experiments. As hypothesized, the micro- biota ofN. vitripennis/N. giraultihybrids was unlike that of either parental species in both bacterial abundance (Fig. 2B) and diversity (Fig.

2C), whereas the negative controlN. longicornis/ N. giraultihybrids that survived (6)(fig.S1A)had a parental-like microbiota. Both hybridizations 1Department of Biological Sciences, Vanderbilt University, Nash- ville, TN 37232, USA. 2Department of Pathology, Microbiology, and Immunology, Vanderbilt University, Nashville, TN 37232, USA.

*Corresponding author. E-mail: [email protected] (R.M.B.); [email protected] (S.R.B.) www.sciencemag.orgSCIENCEVOL 341 9 AUGUST 2013 667 REPORTS had a substantial number of novel and rare op- erational taxonomic units (OTUs,P=0.06,chi square test with Tates correction, fig. S3). The single major difference in theN. vitripennis/ N. giraultihybrid microbiota was a shift in the dominant bacterial OTU fromProvidenciasp.

IICDBZ10 in pure species controls (81 and 96% of the reads inN. vitripennisandN. giraulti,re- spectively) toProteus mirabilisstrain SNBS in theN. vitripennis/N. giraultihybrid (86% of reads). These two species are natural residents of theNasoniaparental species.P. mirabilisSNBS is the dominant species inN. longicornis, and Providenciasp. IICDBZ10 is the dominant spe- cies inN. vitripennisandN. giraulti.

Second, to test whether theN. vitripennis/N.

giraultiF 2lethality in hybrids is conditional on the microbiome, we reared conventional (on a normalS. bullatahost), germ-free (without bacte- ria), and bacteria-inoculated (without bacteria first and subsequently inoculated with specific bacteria) hybrids and nonhybrids. For germ-free rearing ofNasonia,weusedNasoniarearing me- dium (NRM), a liquid medium that we recent- ly developed for culturingNasoniawithout its S. bullatafly host (11). If hybrid lethality in larvae is intrinsically based on negative epistasis between host incompatibility genes, then the null hypoth- esis is thatN. vitripennis/N. giraultiandN. giraulti/ N. vitripennishybrid lethality occurs regardless of germ-free or conventional rearing conditions. How- ever, if the lethality is conditional on the micro- biome, then we expect that germ-free rearing of hybrids will rescue hybrid lethality.

A comparison of results indicates a near- complete rescue of hybrid lethality in germ-free hybrids relative to pure species controls under germ-free conditions (Fig. 1C,F 1,46 = 1.207,P= 0.277 forN. vitripennistoN. giraulti/N. vitripennis, andF 1,46 = 0.824,P= 0.369 forN. giraultito N. vitripennis/N. giraulti). Therefore, hybrids that would typically show severe lethality under con- ventional rearing conditions exhibit a striking increase in survival. In a subsequent experiment, when F 1female hybrids oviposited their F 2male hybrid offspring into germ-free (GF)S. bullata fly hosts, there was also a marked increase in survival relative to hybrids that were reared on conventional (Cv)S. bullatafly hosts (fig. S1B).

In contrast, inN. longicornis/N. giraultiand N. giraulti/N. longicornishybrids, survival val- ues of germ-free hybrids and nonhybrid controls were expectedly high and similar to each other (fig. S1A, Mann-Whitney U test,P>0.5).

If the NRM generally increased survival, it should also affect nonhybrid survival as it would that of hybrids. However, average survival rates of controls decreased insignificantly on NRM.

Further, germ-free hybrids that were reared on NRM inoculated with bacterial strains once again yielded higher lethality as compared to parental controls (Fig. 1C). The commonNasoniabacteria Providencia rettgeristrain IITRP2 andProteus mirabilisstrain SNBS were isolated from parental Nasoniaand added to germ-free NRM. Upon con-suming a 1:1 inoculum of these bacteria, germ-free hybrids exhibited severe lethality in comparison to nonhybrids (Fig. 1C,F 1,46 = 23.863,P< 0.001) and at levels similar to those of conven- tionally reared hybrids (Fig. 1C, Mann-Whitney Utest,P> 0.5). Furthermore, mono-inoculants of antibiotic-resistant (AR)P. rettgeriandEntero- coccus faecalisstrain XJALT-127-2YG1 isolated fromNasonia, as well as green fluorescent protein (GFP)–expressingE. coli, also recapitulated sig- nificant hybrid lethality (fig. S1B,F 1,46 = 36.372, P< 0.001, fig. S2A,F 1,46 =7.281,P< 0.01; see the supplementary text).

The requirement of gut bacteria for interspe- cific hybrid lethality inNasoniais remarkable given that several studies previously mapped quantita- tive trait loci with hybrid lethality to the waspchromosomes and mitochondria (5,7,8,10).

Indeed, marker transmission ratio distortion (MTRD) analyses in survivingNasoniaF 2hybrids dem- onstrate an allelic bias at several loci in the genome toward one parental genome, indicating that an incompatible allele from the other parental species contributes to hybrid lethality (5,9–11). Based on our observation that germ- free hybridNasoniaexhibit a significant increase in survival, we predicted that MTRD would re- vert to near-Mendelian inheritance ratios in the germ-free hybrids. We selected four markers— three within genomic regions associated with hybrid lethality and one control locus that is ex- pected to have 50:50 inheritance ratios—and geno- typedN. vitripennis/N. giraultiandN. giraulti/ N. vitripennishybridNasoniaL4 larvae. Larvae 0 20 40 60 80 100 v/v g/v v/g g/g v/v g/v v/g g/g v/v g/v v/g g/g Conventional Germ-free C Inoculated Percent survival (+/-SE) ** ** **** A D 0.2 0.4 0.60.81 1.2 1.4 1.6 Genome OXPHOS Immune Germ-free InoculatedRelative RPKM to conventionally reared 0.2mm B Viable Inviable 0 10 20 ** ** ** 30 40 50 Eggs L1 L4 Yellow Pupae Adult Average # of individuals (+/-SE) v/g g/v v/v g/g ** Fig. 1. The symbiotic and genetic basis of hybrid lethality.(A)AveragenumberofF 2males (TSEM) within theS. bullatahost during development of the egg, L1 larvae, L4 larvae, yellow red-eye pupae, and eclosed adults conventionally reared onS. bullatahosts. The F 2hybrid genotype is indicated as paternal/ maternal wherev=N. vitripennisandg=N.giraulti. Mann-Whitney U test, **P< 0.001.n=48 replicates per developmental stage. (B)(Top)N. vitripennisL3 larva that is healthy and alive; (bottom) hybridv/gL3 larva that is melanized and dead. (C)Percentofsurvival(TSEM) from egg to pupae of conventionally reared (red), germ-free (blue), and inoculated (purple; germ-free individuals inoculated withProvidencia andProteusbacteria in the NRM)N. vitripennis(v)andN. giraulti(g) parental species and hybrids. Mann- Whitney U test, **P< 0.001,F 1,46 = 23.863 and 12.962, ***P< 0.001, an average of triplicate experiments withn=48 hosts for egg and pupae counts per conventional cross, andn=24 wells containing 12 to 42 larvae per germ-free and inoculated cross. (D) Average hybridv/ggene expression relative to con- ventionally reared hybridsfor the total genome, OXPHOS genes, and immunity genes (ttest, **P< 0.001).

RPKM denotes reads per kilobase per million mapped reads. Conventionaln=14, germ-freen=20, and inoculatedn=20 individuals were sequenced and averaged.

9 AUGUST 2013 VOL 341SCIENCEwww.sciencemag.org 668 REPORTS reared conventionally yielded the expected dis- torted frequencies for each of the MTRD markers (5), whereas germ-free hybrids exhibited typ- ical Mendelian inheritance at all markers ex- cept for MM5.03 (table S1). A large bias against N. vitripennisalleles remained (a frequency of 0.08), although the frequency was significantly higher from the MTRD under conventional rearing (N. vitripennisfrequency of 0.20,Ztest of pro- portions,P< 0.001).

One explanation for a microbial basis for hy- brid lethality is that negative epistasis (mismatched gene-gene interactions) occurs between chromo- somal genes and the microbiome. These inter- actions can accelerate the number of potential hybrid incompatibilities under the Bateson- Dobzhansky-Muller model of genetic incom- patibilities (3). To better understand the mechanisms behind host-microbe interactions that underscore hybrid lethality, we compared the transcriptomes of germ-free L2 hybrid larvae with those of con-ventionally reared and bacteria-inoculated L2 hybrid larvae (just before lethality). The genome- wide expression patterns and the oxidative phos- phorylation (OXPHOS) family of genes, a family thought to be causative in hybrid lethality, were similar across all three rearing conditions (Fig.

1D and supplementary text). However, the 489 innate immune genes inNasoniathat we pre- viously annotated (12)yielded,onaverage,asig- nificant decrease in transcript levels in germ-free individuals relative to conventional or inoculated hybrids (Fig. 1D,ttest,P< 0.001). Specifically, 39.7% of the immune genes were underexpressed by twofold or greater in germ-free hybrids rela- tive to conventional and inoculated hybrids, and 4.9% were overexpressed (fig. S4 and table S2).

Conventionally reared and inoculated hybrids, in turn, have similar immune gene expression (Fig.

1D,ttest,P= 0.104). However, it is important to note that immune genes may be only one of sev- eral possible functional categories that break downbetween the host and microbiome during hybrid lethality.

Causes for the postzygotic hybrid lethality in Nasoniahave traditionally been attributed to host cytonuclear interactions and host gene-by- environment interactions (7–10). However, this study shows that severe hybrid lethality in lar- vae can also be due to gene-microbe interactions with beneficial members of the phylosymbiotic gut microbiome. In this light, the phylosymbiotic microbiome can be understood as an addition to the coadapted genomes of a host organism rather than an arbitrary amalgam. Linking the mi- crobiome and host genome underscores the holo- genome as a unit of evolution and blurs the lines between what biologists typically demarcate as the environment (13) and the genotype of a spe- cies. Based on the mounting evidence for spe- ciation by symbiosis (3), it is becoming clearer that a unified theory of evolution that considers the nuclear genome, cytoplasmic organelles, and microbiome as interacting components in the origin of new species is an emerging frontier for biology. References and Notes1. E. Mayr,Animal Species and Evolution(Harvard Univ.

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Acknowledgments:Sequences are available at the Dryad Digital Repository, doi:10.5061/dryad.3c190. We thank S. Bordenstein and A. Williams for technical assistance during the development of the in vitro cultivation method; L. Funkhouser, D. Sutherland, and C. Wogsland for their assistance in genotypingNasoniahybrids; R. Pauly for bioinformatic assistance; and B. Jovanovic, L. Funkhouser, K. Jernigan, and N. Renner for providing feedback on an earlier version of the manuscript. We apologize in advance to colleagues whose papers we could not cite due to space restrictions. This research was made possible by NSF award DEB 1046149 to S.R.B.

Supplementary Materialswww.sciencemag.org/cgi/content/full/science.1240659/DC1 Materials and Methods Supplementary Text Figs. S1 to S4 Tables S1 to S6 References (14–26) 17 May 2013; accepted 8 July 2013 Published online 18 July 2013; 10.1126/science.1240659 Corynebacterium Propionibacterineae Propionibacterium Staphylococcus Bacillaceae Methylobacterium Enterobacteriaceae Morganella Proteus Providencia v/v g/g l/l S. bullata A B Most abundant OTUs C v/v g/g l/g l/lv/g 0.005S. bullata l/l g/g v/v v/g S. bullata l/g 0.04 Fig. 2. Phylosymbiosis and speciation inNasonia.(A) Simplified phylogeny ofNasonia(bold lines) andS. bullataflies.N. vitripennis(v),N. giraulti(g), andN. longicornis(l). (B)Aweighted,toabundanceof each OTU, UniFrac cluster analysis depicting the microbial relationships of the threeNasoniaspecies (bold lines) and two hybrids for the L2 larval microbiota, as well as the unparasitizedSarcophagahost pupa. The tip of each branch is a pie chart depicting the abundance of each genus of bacteria within the host insects.

Genera of the top 10 most abundant bacterial genera are listed in the key. (C) An unweighted UniFrac cluster analysis depicting the microbial relationships; pie charts at the tip of each branch represent the genera level, bacteria diversity sampled.

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