DOI: 10.1126/science.1206360, 216 (2011);333 Science

, et al.Levi T. MorranBiparental SexRunning with the Red Queen: Host-Parasite Coevolution Selects for

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Running with the Red Queen:Host-Parasite Coevolution Selectsfor Biparental SexLevi T. Morran,* Olivia G. Schmidt, Ian A. Gelarden, Raymond C. Parrish II, Curtis M. Lively

Most organisms reproduce through outcrossing, even though it comes with substantial costs. TheRed Queen hypothesis proposes that selection from coevolving pathogens facilitates the persistenceof outcrossing despite these costs. We used experimental coevolution to test the Red Queenhypothesis and found that coevolution with a bacterial pathogen (Serratia marcescens) resulted insignificantly more outcrossing in mixed mating experimental populations of the nematodeCaenorhabditis elegans. Furthermore, we found that coevolution with the pathogen rapidly droveobligately selfing populations to extinction, whereas outcrossing populations persisted throughreciprocal coevolution. Thus, consistent with the Red Queen hypothesis, coevolving pathogens canselect for biparental sex.

Outcrossing (mating between different in-dividuals) is the most prevalent mode ofreproduction among plants and animals.The maintenance of outcrossing on such a largescale strongly suggests that there is a selective ad-vantage for outcrossing relative to self-fertilizationor asexual reproduction. Nonetheless, the preva-lence of outcrossing is puzzling, because it oftenincurs costs that are not associated with uni-parental modes of reproduction (13). For exam-ple, many outcrossing species produce malesthat facilitate outcrossing but are incapable ofbearing offspring themselves, resulting in thecost of males. Every male takes the place of anoffspring-bearing progeny (female or hermaph-rodite) that could have been produced (2). Thesystematic loss of offspring-bearing progeny canreduce the numerical contribution of a lineageby as much 50% (2). Therefore, the selective ben-efits of outcrossing must more than compensatefor this fitness deficit to achieve a high frequencyin nature.

One selective benefit of outcrossing, relativeto self-fertilization, is the capability to produceoffspring with greater fitness under novel envi-ronmental conditions (4, 5). Outcrossing can in-crease fitness and accelerate a populations rateof adaptation to novel conditions by permittinggenetic exchange between diverse lineages, pro-moting genetic variation among offspring, andallowing beneficial alleles to be quickly assembledinto the same genome (6, 7). In contrast, obligateselfing can impede adaptation by preventing ge-netic exchange, which results in the loss of within-lineage genetic variation and ultimately confinesbeneficial alleles to a single lineage (8, 9). Undernovel environmental conditions, the benefits ofoutcrossing can compensate for the cost of maleproduction, but these benefits may be short-lived(5). Outcrossing is less likely to be favored after

populations adapt to a novel environment, as ge-netic exchange becomes less imperative or per-haps even deleterious (8, 9). Hence, the long-termmaintenance of outcrossing would seem to requirethat populations are constantly exposed to novelenvironmental conditions.

The Red Queen hypothesis provides a pos-sible explanation for the long-term maintenanceof outcrossing. Specifically, under the RedQueenhypothesis, coevolutionary interactions betweenhosts and pathogensmight generate ever-changingenvironmental conditions and thus favor the long-term maintenance of outcrossing relative to self-fertilization (10) or asexual reproduction (11, 12).The reason is that hosts are under selection toevade infection by the pathogen, whereas thepathogen is selected to infect the hosts. Assumingthat some form of genetic matching between hostand pathogen determines the outcome of inter-actions, pathogen genotypes that infect the mostcommon host genotypes will be favored by natu-ral selection (11, 13). This may produce substan-tial and frequent change in pathogen populations,thus rapidly changing the environment for thehost population. Under these conditions, outcross-ing can facilitate rapid adaptation by generating

offspring with rare or novel genotypes, which aremore likely to escape infection by coevolving path-ogens (1013). Conversely, selfing and asexualreproduction generate offspring with little or nogenetic diversity, thus impeding the adaptive pro-cess and leaving them highly susceptible to infec-tion by coevolving pathogens (1013).

The Red Queen hypothesis has been empir-ically supported in studies of natural snail popu-lations, which show that sexual reproduction ismore common where parasites are common andadapted to infect the local host population (14, 15).Outcrossing also seems to reduce the degree ofinfection relative to biparental inbreeding andasexual reproduction in fish (16). Finally, thecapability of antagonistic interactions to drive rap-id evolutionary change has also been determinedfor several different systems (1720). Nonetheless,direct controlled tests for the effect of coevolutionon the maintenance of sex have proven difficult,because they require biological systems in whichhost and pathogen populations can coevolve formultiple generations in a manner that selects forincreased infectivity by a pathogen as well as in-creased resistance (or enhanced avoidance) bythe host. Further, the host species should exhibitgenetic variation in its degree of outcrossing. Thus,we chose to examine the nematodeCaenorhabditiselegans and its pathogenic bacteria Serratiamarcescens, which exhibit these desired properties.

Populations of the host species, C. elegans,are composed of males and hermaphrodites. Thehermaphrodites can reproduce through eitherself-fertilization or by outcrossingwithmales (21).Although usually low (

in response to S. marcescens exposure (5), andS. marcescens can evolve greater infectivity whensuccessful infection ofC. elegans is its onlymeansof proliferation. Selection for increased infectiv-ity can be imposed by propagating only thosebacterial cells that have been harvested from thecarcasses of hosts, which were killed by the bacte-ria within 24 hours of exposure. Therefore, theC. elegans/S. marcescens system can be used togenerate antagonistic coevolution when a host pop-ulation and a pathogen population are repeatedlypassaged under selection together, thus permittinga direct test of the Red Queen hypothesis.

We used experimental coevolution in theC. elegans/S. marcescens system to test the pre-diction that antagonistic coevolution betweenhost and pathogen populations can maintain highlevels of outcrossing despite the inherent cost ofmales. We used obligately selfing, wild-type, andobligately outcrossing populations of C. eleganswith a CB4856 genetic background (5). Where-as the reproductive modes of the obligately self-ing and obligately outcrossing populations aregenetically fixed, the wild-type populations can

reproduce by either selfing or outcrossing [thebaseline outcrossing rate is ~20 to 30% (5)], andthe rate of outcrossing can respond to selection(5). Before the experiment, we mutagenized fiveindependent replicate populations of each matingtype (obligate selfing, wild-type, and obligate out-crossing) by exposing them to ethyl methane-sulfonate (EMS) to infuse novel genetic variationin each population. The five replicate populationswere then passaged under three different para-site treatments (table S1): (i) control (no exposureto S. marcescens), (ii) evolution (repeated expo-sure to a fixed, nonevolving strain ofS.marcescens),and (iii) coevolution. The coevolution treatment in-volved repeated exposure (30 host generations) toa potentially coevolving population of S.marcescens,which was under selection for increased infectiv-ity. S. marcescens Sm2170 served as the ancestralstrain in the coevolution treatment, as well as thefixed strain in the evolution treatment.

The results were consistent with the RedQueen hypothesis. In the coevolution treatment,all of the obligately selfing populations becameextinct within 20 generations (fig. S1). However,

none of the obligately selfing populations wentextinct in either the evolution treatment or in thecontrol treatment. In addition, all of the obligatelyoutcrossing and wild-type populations persistedthroughout the experiment in all three treatmenttypes (fig. S1). Thus, extinction was only ob-served in obligately selfing hosts when confrontedwith coevolving pathogens.

We also found that the presence of coevolvingS. marcescens selected for and maintained highlevels of outcrossing in wild-type C. elegans pop-ulations (Fig. 1). Over the first eight generationsof the experiment, outcrossing rates increasedfrom 20% to more than 70% in both the evo-lution and coevolution treatments (Fig. 1) (F2,11 =8.26; P = 0.006). However, the wild-type popu-lations consistently exposed to a fixed populationof S. marcescens (evolution treatment) exhibiteda steady decline in outcrossing rates after this ini-tial increase, eventually returning to control levelsof outcrossing (Fig. 1), as previously observed (5).In contrast, populations in the coevolution treat-ment consistently maintained high levels of out-crossing throughout the experiment, relative tothe control treatment (Fig. 1) (F1,12 = 209.5; P a: F1,71 = 21.2; P < 0.0001) or to the nonco-evolving control bacteria (Fig. 2A) (c > b: F1,71 =31.9; P < 0.0001). Therefore, the bacteria in thecoevolution treatment evolved greater infectivityin response to selection. Further, the obligatelyselfing hosts did not adapt to the evolution ofincreased infectivity in the bacteria, becausethe bacteria from the coevolution treatment in-duced greater levels of mortality against the wormsafter 10 generations of coevolution than againstthe ancestral hosts (Fig. 2A) ( f > c: F1,71 = 69.2;P < 0.0001). Finally, an increase in mortalityby more than a factor of 3 was observed in theobligately selfing hosts in only 10 generations(Fig. 2A) ( f > a: F1,71 = 173.7; P < 0.0001),which could explain why these hosts were drivento extinction.

The pathogens that coevolved with the wild-type and obligate outcrossing populations alsoevolved greater infectivity (Fig. 2, B andC) ( i > h:F1,104 = 69.5;P < 0.0001; i > g:F1,104 = 32.9;P n: F1,60 = 141.1; P < 0.0001; o > m:F1,60 = 50.9;P < 0.0001). However, thewild-typeand obligately outcrossing populations adaptedto the changes in their respective coevolving path-ogen populations. Specifically, both the wild-typeand obligately outcrossing populations exhibitedlower mortality rates against the pathogens withwhich they were currently evolving than did their

Fig. 2. Coevolutionary dynamics ofhosts and pathogens. We exposedhosts evolved under the coevolutiontreatment and their ancestral popu-lations (before coevolution) to threepathogen populations: (i) an ancestorstrain (ancestral to all S. marcescensused in this study), (ii) a noncoevolv-ing strain (evolved without selection),and (iii) their respective coevolvingstrain (coevolving with the host pop-ulation). We evaluated host mortal-ity after 24 hours of exposure to thepathogens and present the meansacross the replicate host populations.(A) Three obligately selfing C. eleganspopulations persisted beyond 10 hostgenerations in the coevolution treat-ment. These populations were assayedbefore extinction. (B) All five wild-type C. elegans populations in thecoevolution treatment and their an-cestors were assayed at the endpointof the experiment (30 host gener-ations). (C) All five obligately out-crossing C. elegans populations in thecoevolution treatment and their an-cestors were assayed at the endpointof the experiment. Error bars, 2 SEM.

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ancestors (Fig. 2, B and C) (i > l:F1,104 = 27.9;P r: F1,60 = 166.2; P < 0.0001), thusindicating reciprocal coevolution in the outcross-ing host populations. Whereas the obligate selfingpopulations in the coevolution treatment becamemore infected over time (Fig. 2A), the wild-typepopulations maintained the same level of infec-tivity over the course of the experiment (Fig. 2B)(g = l:F1,104 = 0.35;P= 0.554), while the obligateoutcrossing populations were significantly lessinfected at the end of the experiment relative tothe beginning (Fig. 2C) (m > r: F1,60 = 33.1; P