Ecological and Evolutionary Applications for Environmental Sex Reversal of Fish

Ecological and Evolutionary Applications for Environmental Sex Reversal of FishAuthor(s): Alistair McNairSenior, P. Mark Lokman, Gerard P. Closs, Shinichi NakagawaSource: The Quarterly Review of Biology, Vol. 90, No. 1 (March 2015), pp. 23-44Published by: The University of Chicago PressStable URL: http://www.jstor.org/stable/10.1086/679762 .

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ECOLOGICAL AND EVOLUTIONARY APPLICATIONS FORENVIRONMENTAL SEX REVERSAL OF FISH

Alistair McNair Senior*Department of Zoology, University of Otago, Dunedin 9054, New Zealand and Charles Perkins Centre and

School of Biological Sciences, University of Sydney, Sydney, New South Wales 2006, Australia

e-mail: [email protected]

P. Mark LokmanDepartment of Zoology, University of Otago, Dunedin 9054, New Zealand

e-mail: [email protected]

Gerard P. ClossDepartment of Zoology, University of Otago, Dunedin 9054, New Zealand

e-mail: [email protected]

Shinichi NakagawaDepartment of Zoology, University of Otago, Dunedin 9054, New Zealand

e-mail: [email protected]

keywordsendocrine disrupting chemicals, hormones, sex determination, sex reversal,

teleosts, biological control

abstractEnvironmental sex reversal (ESR), which results in a mismatch between genotypic and phenotypic sex,

is well documented in numerous fish species and may be induced by chemical exposure. Historically,research involving piscine ESR has been carried out with a view to improving profitability in aquacultureor to elucidate the processes governing sex determination and sexual differentiation. However, recent studiesin evolution and ecology suggest research on ESR now has much wider applications and ramifications. Webegin with an overview of ESR in fish and a brief review of the traditional applications thereof. We thendiscuss ESR and its potential demographic consequences in wild populations. Theory even suggestssex-reversed fish may be purposefully released to manipulate population dynamics. We suggest new researchdirections that may prove fruitful in understanding how ESR at the individual level translates to

*Present Address: Charles Perkins Centre, University of Sydney, Sydney, New South Wales 2006 Australia

The Quarterly Review of Biology, March 2015, Vol. 90, No. 1

Copyright © 2015 by The University of Chicago Press. All rights reserved.

0033-5770/2015/9001-0002$15.00

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population-level processes. In the latter portion of the review we focus on evolutionary applications of ESR.Sex-reversal studies from the aquaculture literature provide insight in to the evolvability of determinants ofsexual phenotype. Additionally, induced sex reversal can provide information about the evolution of sexchromosomes and sex-linked traits. Recently, naturally occurring ESR has been implicated as a mechanismcontributing to the evolution of sex chromosomes.

Introduction

PHENOTYPIC sex can heavily influencethe selective forces, life history, and re-

productive advantages that an individual ex-periences (Lande 1980; Arnqvist and Rowe2005; Cox and Calsbeek 2009, 2010; Mank2009). As one might expect for such an in-fluential trait, mechanisms that are responsi-ble for the determination of sex are relativelywell conserved and stable within birds andmammals (Schartl 2004b; Ellegren 2010). Inthese taxa, sex is determined genetically bythe presence of sex chromosomes (XY/ZW;genetic sex determination or GSD) at con-ception and phenotypic sex is fixed through-out life (Schartl 2004b; Wilhelm et al. 2007).Such fixed phenotypic sex throughout lifeis termed gonochorism. Paradoxically, andin sharp contrast to the systems of sex de-termination in mammals and birds, sex-determination systems among fish arevariable (Francis 1992; Desjardins and Fer-nald 2009; Mank and Avise 2009). Somespecies display sequential or simultaneoushermaphroditism, expressing both maleand female sexual phenotypes during life(Robertson 1972; Devlin and Nagahama2002). There are also many gonochoristicfish species that, like mammals and birds,exhibit a fixed sex throughout life (Devlinand Nagahama 2002).

Classically, gonochoristic fish have beensegregated into those with discrete sex chro-mosomes that utilize GSD, and those withenvironmental sex determination (ESD),where an environmental factor initiates sex-ual differentiation. However, in fish, it maybe more useful to think of GSD and ESD asopposite ends of the same spectrum, withsome species inhabiting the middle of thatspace (Grossen et al. 2011). In these “middleground” species, a particular pairing of sexchromosomes can consistently lead to thedevelopment of one phenotypic sex or theother under stable environmental condi-

tions. However, in the same species environ-mental extremes (e.g., naturally occurringhigh temperatures or exogenous chemicalsof anthropogenic origin) during develop-ment may become the dominant force indetermining sexual phenotype (Baroiller etal. 2009b). Such environmental extremes in-terrupt or “override” genetically initiated go-nad development. Ultimately, in fish speciesthat do bear distinct sex-determining chro-mosomes, this interrupted development maylead to an individual with a genetic sex that ismost commonly associated with the oppositephenotypic sex; such a process is known asenvironmental sex reversal (ESR; Stelkensand Wedekind 2010).

The result of ESR is a sex-reversed fishthat, if capable of breeding, may produceoffspring at skewed sex ratios (Bull 1983).Figure 1 (A:F) shows how in a species with amale heterogametic (XY) and female ho-mogametic (XX) sex-determination system,various mating combinations of wild-typeand sex-reversed individuals can result inmonosex or sex ratio-skewed progeny. AsFigure 1.B demonstrates, production of fe-male-only offspring is straightforward, andcan be achieved by a cross between a wild-type female (XX) with a masculinized fishthat has a female genotype (XX). Figure 1Cshows the results of a mating between a wild-type male (XY) and a sex-reversed genotypicmale (XY); the sex ratio of the resultingprogeny would be skewed in favor of males.However, this mating pattern also allows forthe production of genotypic “supermales”(YY). If “supermales” are both viable andfertile, these individuals may then breed withwild-type females to produce entirely maleprogeny (Figure 1D). YY individuals maythemselves be further sex reversed andbreed with genotypic males or other “super-males,” producing only male offspring (Fig-ure 1E:F).

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ESR can be intentionally induced by ex-posing fish to high levels of exogenouschemicals, a phenomenon that is very welldocumented and utilized by aquaculture re-searchers and fish physiologists (Johnstoneet al. 1978, 1979a,b; Pandian and Sheela1995; Pandian and Kirankumar 2002; Cnaaniand Levavi-Sivan 2009). Recently, ecologistsand evolutionary biologists have also be-come interested in ESR (Stelkens andWedekind 2010). Studies have shown thatnumerous wild fish populations may be ex-posed to ESR-inducing pollutants (e.g., sew-age and industrial outflows; Purdom et al.1994; Rempel and Schlenk 2008). Giventhat sex-reversed fish can produce progenyat skewed sex ratios, models have focusedon the effects of ESR on dynamics of wildpopulations (Hurley et al. 2004). It has evenbeen suggested that sex-reversed individualscan be purposefully released when it is de-sirable to manipulate the dynamics of wildpopulations (Cotton and Wedekind 2007a).Biologists working on the evolution of sex-determination systems have also shown in-terest in ESR. It has been suggested that sexreversal can play roles in transitions between

sex-determining systems, and in the mainte-nance of sex chromosomes (Wedekind2010; Wedekind and Stelkens 2010).

This article aims to bridge recent ESR-related evolutionary and ecological thinkingwith ESR, and the literature published onESR-inducing methods from aquacultureand physiology. In doing so, we hope toachieve two objectives: first, to familiarizeevolutionary biologists and ecologists withexisting applications of ESR techniques inaquaculture/physiology, thus providing po-tentially new model systems and, second, toincrease awareness of emerging ESR-relatedtopics among researchers working in aqua-culture and physiology. We begin by giving abrief overview of traditional applications ofchemically induced ESR. The rest of this re-view has then been split into two broad top-ics. The first deals with the implications ofESR for short- to medium-term populationdynamics. The second part deals with ESR asa mechanism in evolutionary processes andas a tool to study those processes. In eachsection, we begin by reviewing the publishedliterature before moving on to discuss out-

Figure 1. Predicted Results of Various Crosses Between Wild-Type (WT) and Sex-Reversed (SR)Individuals

Letters (X or Y) represent the sex chromosomes of individuals, while the phenotypic sex of individuals isrepresented by shape (circles and squares being phenotypic males and females, respectively). The sex ratio ofthe progeny of each cross is presented at the bottom of each panel.

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standing questions and future study direc-tions.

ESR in Aquaculture andPhysiological Research

For aquaculture, creating monosex popu-lations through sex reversal (or via thebreeding of sex-reversed fish; Figure 1) canbe advantageous for three main reasons.First, in food species, such as the Nile tilapia(Oreochromis niloticus), sexual dimorphisms inlife history such as growth rate or age atmaturity make one sex more profitable thanthe other (Cnaani and Levavi-Sivan 2009).Second, in monosex populations where fe-males and males are separated, reproductioncan be more easily managed. Finally, inmany ornamental species such as the fight-ing fish (e.g., Betta splendens), only one sexexpresses the colors and ornamental finspreferred by fish enthusiasts (Cnaani andLevavi-Sivan 2009).

Chemically induced ESR has proved avaluable tool in the study of fish physiologyand developmental biology (Yamamoto 1958,1962; Orban et al. 2009). By crossing wild-type individuals with individuals exposed tosex-reversing chemicals in a manner similarto that described above, and by analyzing thesex ratio of the progeny produced (Bull1983; Gomelsky et al. 2002; Hamaguchi et al.2004; Williamson and May 2005), it is possi-ble to determine: whether environment orgenotype is the “dominant force” in sex de-termination; and whether males or femalesare the heterogametic sex when GSD hasevolved (Figure 1; Bull 1983). Further, it ispossible to determine the mechanisms thatcontrol sexual differentiation by administer-ing various exogenous hormones, endocrinedisrupting chemicals (EDCs), or steroid syn-thesis inhibitors at different developmentalstages, and assessing their effect on gene ex-pression and sexual differentiation (e.g.,Chang et al. 1995; Bhandari et al. 2005).

As a result of aquaculture and physiologyresearch, to date the sex-reversing effects of avariety of chemicals have been tested onhundreds of fish species from a range offamilies (Pandian and Sheela 1995; Devlinand Nagahama 2002; Senior and Nakagawa2013). Important aquaculture taxa, such as

tilapia and salmonids, are without doubt themost intensively studied (Fitzpatrick et al.2005; Atar et al. 2009; Makino et al. 2009;Neumann et al. 2009). However, numeroussex reversal studies also focus on zebrafish(Danio rerio) and medaka (Oryzias latipes),as these species have become model organ-isms in developmental biology and genetics(Yamamoto 1958; Kobayashi and Iwamatsu2005; Hashimoto et al. 2009; Orban et al.2009). In fact, the latter of these speciesperhaps has the best understood sex-deter-mining system, and evolution thereof, of allnonmammalian vertebrates (Kondo et al.2004; Schartl 2004a; Herpin and Schartl2009).

ESR and Effects on PopulationDemography

reviewCentered on the idea that sex-reversed in-

dividuals may give rise to offspring at skewedsex ratios (Figure 1), a number of modelshave focused on the potential effects of ESRon the dynamics of wild populations (Ka-naiwa and Harada 2002, 2008). We now givea brief overview of the contexts in which ESRin wild populations has been modeled, andreview the findings of these models.

Ecologists have been considering the pos-sible effects of ESR on wild populations inthe context of anthropogenic pollutants,specifically, the effects of EDCs on fish (Pur-dom et al. 1994; Hotchkiss et al. 2008; Rem-pel and Schlenk 2008; Cotton and Wedekind2009; Allner et al. 2010). In the early 1990s,scientists in the U.K. began reporting unusu-ally high numbers of intersex rainbow trout(Oncorhynchus mykiss) in lakes receiving efflu-ent from sewage treatment plants. Furtherstudy using caged trout confirmed the efflu-ent was responsible for the intersex fish(Purdom et al. 1994). Subsequent reportsdemonstrated that the incidence of intersexfish reported in the U.K. is not a local orspecies-specific phenomenon (Jobling et al.1998), nor are sewage treatment plants thesole source of EDCs. For example, numerousstudies have demonstrated the effects of milleffluents in North American rivers on sexualdevelopment of mosquitofish (Gambusia sp.;

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Howell et al. 1980; Bortone et al. 1989; Bor-tone and Davis 1994; Toft et al. 2003; Toftand Guillette 2005; Orlando et al. 2007).Gomelsky et al. (1994) demonstrated thatthe water used to house fish that had beenfed on a hormone-treated diet was also po-tent enough to induce sex reversal, even afterbeing passed through a biofilter. Similarly, uri-nary or fecal waste from livestock farms (ei-ther runoff or having been applied asfertilizer) may contain substantial amountsof free steroids (Shore and Shemesh 2003).All in all, it seems that there is abundantevidence that chemicals of anthropogenicorigin can affect sexual differentiation inwild populations.

Using mathematical models, Hurley et al.(2004) showed that under certain circum-stances, EDC-induced masculinization couldresult in the population becoming depend-ant on sex reversal for reproduction. Theirmodel showed that in a theoretical male het-erogametic population, after a number ofgenerations of EDC-induced sex reversal, Ychromosomes could become extinct (Hurleyet al. 2004). At this point the theoretical pop-ulation became reliant on sex-reversed geno-typic females to provide male gametes forfertilization. Consequently, once the mascu-linizing agent is removed from the environ-ment, the EDC-exposed populations maycrash.

On the other hand, the model from Hur-ley et al. (2004) suggested that environmen-tal feminization does not appear to be sodamaging. Direct effects on sex ratio wereobserved, with phenotypic females becom-ing more prevalent in the population. How-ever, once the pollutant was removed fromthe environment, the population returnedto stability within a few generations (Hurleyet al. 2004). That being said, a later publica-tion suggested that if sex-reversed individualswere sufficiently prevalent within the popu-lation (i.e., a very strong sex-reversing chem-ical was used), feminization may lead to anoverrepresentation of Y chromosomes, andultimately the loss of X chromosomes (Cot-ton and Wedekind 2009). Assuming the re-moval of the feminizing agent, the extinctionof X chromosomes will lead to populationcollapse (Cotton and Wedekind 2009). It is

worth noting that conclusions from modelssuch as those of Hurley et al. (2004) shouldbe inverted in a female heterogametic sex-determination system (ZW); i.e., environ-mental feminization would bring aboutpopulation collapse, whereas the popula-tion could recover from masculinization.

Consequently, models have also focusedon the potential for ESR as a tool to purpose-fully manipulate wild populations, namedTrojan sex chromosome (TSC) theory (Gu-tierrez and Teem 2006; Cotton and Wede-kind 2007a,b; Stelkens and Wedekind 2010;Gutierrez et al. 2012; Senior et al. 2013).Under TSC theory, repeated introductionsof sex-reversed or “Trojan” individuals wouldtheoretically tip sex ratios in a desired direc-tion, resulting in population growth or crash(Stelkens and Wedekind 2010). In decliningpopulations, it may be desirable to increasepopulation size by tipping the sex ratio infavor of females. Such a sex-ratio skew couldbe achieved through repeated introductionsof Trojan fish that only produce femalegametes (Cotton and Wedekind 2007b). Forexample, in a male-heterogametic species(XY), a genotypic female (XX) could be mas-culinized. This “Trojan male” could then bereleased and would only sire female off-spring (Figure 2A; as evidenced by routinepractices in aquaculture). A similar processcan also be carried out in species with afemale heterogametic system of sex determi-nation, but it requires an extra sex-reversalstep (Figure 2B).

Populations of introduced species may re-quire control. In such situations, TSC may beused to skew the sex ratio in favor of malesand bring about population decline (Gutier-rez and Teem 2006; Cotton and Wedekind2007a; Figure 2C and 2D). In theory, as abiocontrol measure, TSC has three majoradvantages over alternative techniques forcontrol of invasive fish. First, invasive fishtend to be particularly difficult to control viaspecific removal of individuals owing to thehigh fecundity of females; i.e., if a relativelylow number of females are left in the en-vironment, the invasive population islikely to recover. Second, unlike othersimilar biological controls, such as thedaughterless gene theory (Thresher and

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Bax 2003; Thresher et al. 2007, 2008),TSC does not require the release of ge-netically modified organisms into theenvironment. Finally, unlike controltechniques such as the release of preda-tory species or the use of toxins, TSC ishighly species specific and can be readilyhalted.

Although the aforementioned modelsdemonstrate the theoretical effects of ESRon population sex ratio, their applicability toreal-world populations remains unproven.Alho et al. (2010) explored the effects ofESR on the dynamics of a wild population ofcommon frogs (Rana temporaria) in whichapproximately 9% of the population was sub-ject to sex reversal. Such sex-reversed individ-uals did indeed breed within the population

and, as theory predicts, produced offspringat a skewed sex ratio (Alho et al. 2010). How-ever, when Alho et al. (2010) applied amodeling approach similar to the modelsdescribed above (Hurley et al. 2004), theyfound that empirical data on the populationsex ratio did not match model predictions,perhaps due to fitness differences between sex-reversed and wild-type individuals (Wedekind2010). If ESR at the level of individual ani-mals does not translate to predicted fluctu-ations in the population sex ratio, previousmodels may be overstating the influences ofsex reversal on population viability. Addi-tionally, population control tools such asTSC may prove unviable. In the followingsection, we explore outstanding issues thatsurround ESR at the level of the individual,

Figure 2. The Production of Trojan Fish in Both Male (XY) and Female Heterogametic (ZW) Systemsof Sex Determination

Circles denote phenotypic females and squares denote phenotypic males. Genotypic sex is given by the lettersinside the circles/squares. Solid lines indicate breeding, which is possible between any phenotypic male(square) and female (circle). Solid arrows denote the production of offspring from a given breeding. Dashedlines indicate sex reversal, either masculinization (M), or feminization (F) by chemicals such as testosteroneand estradiol. Individuals in bold shapes represent “Trojan” fish released from captivity, which then breed toproduce offspring at a skewed sex ratio. A. The production of a Trojan male to boost population size in a maleheterogametic system of sex determination. B. The production of a Trojan male to boost a female heteroga-metic population. C. The production of Trojan females to decrease a population with a male heterogameticsex-determination system. D. The production of a Trojan female to decrease population size in a populationwith female heterogametic sex-determination systems.

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and how that ESR may translate to population-level processes.

outstanding issues and futuredirections

ESR and Reproductive PerformanceFor ESR to profoundly affect population

dynamics (either intentionally for TSC orunintentionally as a result of EDCs), sex-reversed individuals must reproduce success-fully. When models of the population-leveleffects of ESR have considered reduced re-productive ability of sex-reversed individuals,sex reversal had a less profound effect on thepopulation (Cotton and Wedekind 2009; Se-nior et al. 2013).

In instances where exogenous chemicalsinduce ESR (e.g., in the models of Hurley etal. 2004), it may be important to distinguishbetween two types of effects: the toxic effectsof exposure to EDCs on reproduction (i.e.,endocrine disruption); and the effects ofESR on reproduction (i.e., the mismatch be-tween genotypic and phenotypic sex). Todate, a number of studies have linked expo-sure to EDCs/ESR-inducing chemicals toimpaired gonadal function and reducedexpression of sexual characteristics in wildand captive animals (Toft and Baatrup 2001;Toft et al. 2003, 2006, 2007; Mills and Chich-ester 2005; Toft and Guillette 2005; Morleyet al. 2010; Senior et al. 2014). A recentmeta-analysis combined studies of chemicallyinduced ESR to gain insight into the ques-tion of sex reversal and reproductive capacity(Senior et al. 2012). That analysis found thatexposure to sex-reversing chemicals reducedgonadosomatic index (GSI), but that this re-duction was more likely the result of chemi-cal exposure, rather than downstream ESR(Senior et al. 2012). Therefore, exposure toEDCs may compromise gonadal develop-ment among sex-reversed and wild-type fishequally. It should be noted, however, thatthis meta-analysis reported mean effectsacross a range of species, and that somespecies-specific effects of ESR and EDCswere likely to be present. To truly teaseapart the effects of EDC exposure andESR, future studies will need to comparethe effects of chemical exposure on the

reproduction of those individuals identi-fied as sex reversed and those individualsidentified as wild-type.

As well as potentially different effects ofESR and of EDCs on reproductive fitness,models (particularly models of TSC) need toconsider the viability and reproductive ca-pacity of “YY”-carrying individuals, or “su-permales.” YY fish are of value to the aquacul-ture industry and, thus, their production iswell documented (Yamamoto 1964, 1975;Scott et al. 1989; Kavumpurath and Pandian1992; Cnaani and Levavi-Sivan 2009). Ka-maruzzaman et al. (2009) found no differ-ences in growth rate between YY and wild-type Nile tilapia. In the guppy (Poeciliareticulata), YY animals are known to becapable of reproduction, and may even befurther feminized and still reproduce (Ka-vumpurath and Pandian 1992, 1993). Stud-ies have gone so far as testing for differencesbetween the sperm quality of YY and XYmales, and found little difference (Kowalskiet al. 2011; Gennotte et al. 2012). Thus, itseems that YY individuals can be both viableand reproductively capable. However, wenote that this may not be universally true forall fish species (George et al. 1994), and thatthe viability of YY animals is likely predictedby the extent to which sex chromosomes areheteromorphic.

Do Sex-Reversed and Wild-Type FishBehave Alike?

As well as morphological differences,quantifying the behavioral differences be-tween sex-reversed and wild-type fish is likelyto be important in evaluating the effects thatESR can have on populations (althoughmodels suggest fecundity parameters may bemore influential; Senior et al. 2013). Behav-ioral differences have been identified be-tween individuals from populations exposedto EDCs (which can cause ESR) and thosenot exposed. For example, Toft et al. (2004)found differences in behaviors directly re-lated to reproduction between mosquitofishexposed to mill effluents and those frompopulations inhabiting unpolluted habitat.The next key step is to identify those individ-uals that have undergone ESR as a result ofchemical exposure, and examine their be-

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havior relative to wild-type fish both exposedand not exposed to chemicals. Such behav-ioral studies are currently few and far be-tween, although insights from a number ofimportant studies can be highlighted.

First, Kobayashi and Nakanishi (1999)demonstrated that male spawning behaviorscould be induced in the gynogenetic cruciancarp (Carassius auratus langsdorfii) by testos-terone implant. Kirankumar and Pandian(2002) demonstrated that hormonally mas-culinized fighting fish display behaviors anal-ogous to their induced male phenotype, butthat they may be less competitive than wild-type male fish. Larsen and Baatrup (2010)compared the behaviors of zebrafish thatwere/were not exposed to masculinizingchemicals, but did not find any statisticallysignificant differences between exposed andunexposed males (Larsen and Baatrup 2010).That said, the effect sizes they describe sug-gest that, if anything, the competitive abili-ties of sex-reversed males may be slightlycompromised. Kuramochi et al. (2011) ex-amined nest-building behavior (a male-specific trait) in masculinized Mozambiquetilapia (Oreochromis mossambicus). Their re-sults showed that the majority of, but not all,masculinized females displayed male-specificbehaviors. The above studies highlight thatsex-reversed fish can adopt behaviors thatare representative, but not necessarily iden-tical, to those of wild-type fish of the samesexual phenotype. Studying the behavioraldifferences between sex-reversed and wild-type fish, perhaps that have both been ex-posed to EDCs, is of importance if we are tounderstand how ESR affects populations.However, as we have already mentioned, veryfew studies have achieved this aim, and this isclearly a question that requires further ex-perimentation.

Species-Specific Factors that MayExacerbate/Relieve the Population-Level

Effects of ESRTo date, the literature on both Trojan sex

chromosomes and EDCs has recognized fac-tors associated with the reproductive fitnessof sex-reversed fish. Somewhat less discussedare the additional interactions that sex-ratioimbalance (which is predicted to result from

ESR) can have with species-specific biology.In many species, sex-ratio imbalances canhave unpredictable consequences. For ex-ample, some species are able to adjust thesex of their progeny in response to skewedpopulation sex ratios (West 2009; Barbosaand Magurran 2010). If populations targetedfor management by TSC possess an ability toallocate sex accordingly, then these popula-tions may respond unpredictably to thosebiological control attempts. Alternatively,sex-ratio skews can exacerbate existing sex-ual conflicts (Le Galliard et al. 2005; Kokkoand Rankin 2006). For example, an excess ofmales could begin to overexploit dwindlingfemale numbers leading to increased extinc-tion risk (Rankin et al. 2011). Thus, sexualconflicts may be exploited to make methodslike TSC a more effective biological controltool. Finally, some fish species are thought tobe inherently sperm-limited (Levitan and Pe-tersen 1995), although we acknowledge thatexperimental evidence for this phenomenonis currently mixed (Yund 2000). Neverthe-less, if a species is sperm-limited, then skew-ing a collapsing fish population in favor offemales (via TSC) may do little to boost pop-ulation size. Alternatively, skewing a sperm-limited invasive population in favor of malesmay have the undesirable effect of actuallyincreasing the population in subsequentgenerations.

Failing to account for inherent aspects of aspecies’ biology/ecology, such as those out-lined above, when attempting to managewild populations has, in the past, led to un-predicted consequences. For example, afailure to account for sex allocation in thehighly endangered parrot, the kakapo (Strigopshabroptilus), meant that a supplementaryfeeding program lead to an unintended in-crease in the proportion of males within thepopulation (Robertson et al. 2006). Modelsof sex-ratio skews that result from ESR in fishpopulations should perhaps be married withpublished models and with data fromspecies-specific evolutionary ecology studies(e.g., Le Galliard et al. 2005). In this way wemay gain a more precise insight into howESR can affect population viability in a givenspecies.

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Studying ESR in Wild Fish PopulationsStudies that make use of sex-specific ge-

netic markers, such as that by Alho et al.(2010), to date provide the best insights as tohow ESR may affect population dynamics(Wedekind 2010). On the surface, fish pop-ulations seem ideal for such studies. As men-tioned earlier, many fish populations areexposed to EDCs and the sexual differentia-tion processes of many species are known tobe plastic and, thus, sex reversals may becommonplace. However, distinguishing be-tween wild-type and sex-reversed individualsis difficult, as only a handful of sex-specificgenetic markers have been successfully de-veloped in fish (Devlin et al. 1994; Matsudaet al. 1997; Griffiths et al. 2000; Kovacs et al.2000; Khoo et al. 2003; Watanabe et al. 2004;Felip et al. 2005; Shikano et al. 2011a), per-haps due to the relatively high turnover ofgenetic sex-determination systems in teleosts(Kikuchi and Hamaguchi 2013).

One exception to this rule is in Chinooksalmon (Oncorhynchus tshawytscha). In thisspecies, the sex-specific genotypic markerOtY1 has been identified, initially with a viewto increasing the detectability of sex-reversedfish for aquaculture brood stocks (Devlin etal. 1994). The OtY1 marker has been appliedto several wild salmon populations in NorthAmerica. The results have, however, beensomewhat misleading. Initial studies re-ported relatively high rates of ESR (rangingfrom 16-60% feminization) in wild popula-tions, perhaps suggesting that feminizingEDCs were affecting sexual development(Nagler et al. 2001; Williamson and May2002; Chowen and Nagler 2004). Later stud-ies of captive fish, however, found that OtY1may not effectively detect ESR in these wildpopulations (Williamson and May 2005; Wil-liamson et al. 2008). Reasons for the failureof OtY1 in this instance are speculative, butinclude an X-Y recombination event (whichmay itself have been due to ESR; see thesection Could ESR be a “Fountain of Youth”for Sex Chromosomes? below), or the loss ofa male-determining factor on a Y chromo-some, meaning the individual is phenotypi-cally female, but carries a Y-chromosomethat still amplifies with OtY1 (Williamson and

May 2005; Williamson et al. 2008). Thosestudies making use of the OtY1 marker inwild populations are a clear example of thedifficulties of identifying mismatches be-tween genotypic and phenotypic sex in fish,due to the dynamic nature of teleost sexchromosomes (cf. Shikano et al. 2011a).

The three-spined stickleback (Gasterosteusaculeatus) may make a good model organismfor studying ESR in wild fish populations(Katsiadaki et al. 2007; Scholz and Mayer2008). Sex-specific markers have been devel-oped in this species (Griffiths et al. 2000;Peichel et al. 2004), and in some cases ap-plied to wild populations (e.g., Kitano et al.2007). We note that ESR has not yet beendetected in wild stickleback populations (Ar-nold et al. 2003; Hahlbeck 2004). Neverthe-less, the sexual-differentiation process of thespecies is known to be sensitive to endocrinedisruption, and laboratory studies demon-strate that chemically induced ESR can oc-cur (Hahlbeck et al. 2004a). In addition,sticklebacks are relatively widespread (Mun-zing 1963), and known to inhabit pollutedareas (Pettersson et al. 2007; Sanchez et al.2007). Therefore, a wider application of thestickleback sex-specific marker may identifypopulations containing sex-reversed individ-uals. As a model organism for ESR, stickle-backs may also be particularly valuable as theeffects of EDCs on stickleback physiologyare relatively well studied (Hahlbeck et al.2004b). Finally, the stickleback has been amodel organism in behavioral as well asevolutionary ecology, thus processes such assexual selection are well studied. Such behav-ioral studies lay the groundwork for furtherwork on the interactive effects of sex reversal,sex ratio, reproductive behaviors, and endo-crine disruption (e.g., Wibe et al. 2002; Brianet al. 2006).

ESR and the Evolution of SexDetermination and Sex Chromosomes

reviewRecently, work on the evolution of sex

determination has adopted a threshold-dichotomy model of sex determination (Mit-twoch 2006; Grossen et al. 2011; Quinn et al.2011; Schwanz et al. 2013). Under such mod-

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els, sex is determined by interacting genotypicsex (e.g., ZZ/ZW) and environmental factors(e.g., temperature). These combined fac-tors drive expression of physiological traits(e.g., testosterone production) toward aninternal threshold. If this threshold is sur-passed, individuals develop one of two phe-notypic sexes. Individuals who fall short ofthe “sex-determining threshold” developthe opposing phenotypic sex. In a consistentenvironment, one genotype will usually sur-pass the threshold (e.g., ZZ�male), whereasthe other does not (ZW�female). However,under environmental extremes one geno-typic sex may fail to meet an ordinarily sur-passed threshold, or potentially overshoot athreshold that is not ordinarily met. Suchinstances result in ESR.

Threshold-dichotomy models of sex de-termination have lead to the identificationof novel mechanisms that may underlietransitions between sex-determining systems(Schwanz et al. 2013). For example, Quinnet al. (2011) demonstrate that, where ESRcan occur as a result of unusually met/un-met thresholds, an evolutionary shift in sex-determining thresholds resulted in transitionsbetween sex-determination systems. As anaside, we note that those models also as-sume that individuals homogametic for thesex chromosome usually restricted to heter-ogametic sex (e.g. YY “supermales”) are via-ble (see the section ESR and ReproductivePerformance above).

The threshold-dichotomy model of sex de-termination is in accordance with abundantobserved data from reptiles, where temper-ature-sex chromosome interactions deter-mine phenotypic sex (Mittwoch 2006), aswell as from fish where ESR is ubiquitous.However, to truly understand how selectionacts to alter sex-determining thresholds, re-searchers studying the evolution of sexdetermination require a mechanistic under-standing of sexual differentiation (Uller andHelanterä 2011). As previously described(see the section ESR in Aquaculture andPhysiological Research above), induced ESRand the application of exogenous steroidhormones/EDCs are often used to exploreprocesses of sex differentiation and, ulti-mately sex determination (e.g., Kitano et al.

2000; Gomelsky et al. 2002; Hamaguchi etal. 2004). Thus, these methods can be usedto aid biologists in understanding the evo-lution of sex-determination systems.

As well as in sex-determination systems,ESR may play an important role in the evo-lution of sex chromosomes themselves. Whatis more, ESR-inducing treatments can be atool to explore sex-chromosome evolution.Notably, sex-chromosome evolution (i.e., thestructure of sex chromosomes and genes lo-cated thereon) has traditionally been treatedas a topic separate from the evolution ofsex-determining mechanisms because: theselective mechanisms that drive transitionsbetween sex-determining systems can bestudied without invoking knowledge aboutthe structure of sex chromosomes; and thestructure of sex chromosomes can evolvewithout altering sex determination (Bull1983).

With regards to investigating sex-chromo-some evolution, ESR may be used to elucidatethe extent to which certain traits are sex-linked.Sex-reversed individuals theoretically expressall of the sex-specific genes carried; i.e., genesspecific to their phenotypic sex (Mag-Muresanet al. 2004). Therefore, in species with XY/ZWsex determination, a sex-reversed individualof the homogametic sex (i.e., XX/ZZ) can-not express traits associated with the sexchromosome specific to the heterogameticsex (i.e., Y or W; Figure 3). Perhaps the mostinteresting recent example of the use ofESR to demonstrate sex linkage was by Gor-don et al. (2012).

Gordon et al. (2012) showed that, in mul-tiple wild populations of guppies, as individ-uals colonize upstream into low-predationenvironments, Y-linkage of color genes be-comes broken. Within a number of river sys-tems, Gordon et al. (2012) sampled femalesfrom downstream high-predation sourcepopulations, and respective upstream low-predation “daughter” populations. Sampledfemales were then exposed to testosteronetreatments in order to force expression ofany “male-related” genes that they may carry.Their results showed that females from high-predation sites were less likely to expresscolor after testosterone treatment than fe-males from respective low-predation environ-

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ments (Gordon et al. 2012); i.e., as guppiescolonize upstream into low-predation environ-ments, a transition from Figure 3, scenario B toFigure 3, scenario A, occurs. The mechanismsthat underlie the aforementioned changes insex linkage with predation are currently un-known. However, further studies incorporatingESR may be used to elucidate these causes, atopic we will return to below (see the sectionElucidating Sex Linkage and Predation inGuppies).

Finally, ESR has been proposed as a mech-anism that may maintain the homomorphicityof sex chromosomes. The sex chromosomes ofsome groups (e.g., mammals and birds) arerelatively heteromorphic (Ellegren 2010). Thisheteromorphism is thought to occur becausethe chromosome specific to the heterogameticsex (Y or W) is prevented from recombining

and, thus, over time, accumulates deleteriousmutations (Figure 4, Population A). Such het-eromorphism is, however, not universal. Forexample, many sex chromosomes in fish arerelatively homomorphic (Schartl 2004b). Ex-ceptions such as the western mosquitofish(Gambusia affinis) and nine-spined stickleback(Pungitius pungitius) with heteromorphic sexchromosomes can be noted (Black and Howell1979; Shikano et al. 2011b), but such casesseem infrequent. Perrin (2009) suggested thatnaturally occurring ESR may act as a mecha-nism to maintain sex chromosome homomor-phicity in some taxa, such as fish.

Perrin (2009) begins by arguing that thepresence/absence of recombination betweensex chromosomes is specific to phenotypic sex.That is to say, whether sex chromosomes re-combine depends on phenotype, not geno-

Figure 3. Environmental Sex Reversal (ESR) Can Be Used to Ascertain Whether Sexually DimorphicTraits Are Sex-Linked

Two hypothetical scenarios in species with male heterogametic genetic sex determination (XY/XX) areshown. Genotype (XY or XX) is given and shapes represent phenotypic sex: squares for males and circles forfemales. Males and females are sexually dimorphic whereby males express a grey phenotype. In scenario A,genes controlling grey phenotype (depicted by �) are x-linked, and thus carried by both genotypic males andfemales. Hence, masculinized (via ESR) genotypic females express the grey phenotype. We note that a similaroutcome would be observed if genes controlling grey phenotype were not sex-linked at all; i.e., autosomal. Inscenario B, genes for the male-specific grey phenotype are Y-linked, and thus only carried by genotypic males.Because genotypic females (XX) do not carry genes for grey phenotype, masculinization does not result in agrey phenotypic male. In an alternative female heterogametic species (ZW/ZZ), feminization of genotypicmales (ZZ) could be used to ascertain whether female-specific traits are W-linked.

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type. If sex chromosome recombination isphenotype specific, it follows that ESRmay offer sex chromosomes specific to theheterogametic sex the chance to recombine.This recombination would allow sex chromo-somes to purge themselves of the deleteriousmutations accumulated in the heterogameticsex. ESR may therefore act as a “fountain ofyouth” for sex chromosomes, ultimately in-creasing sex-chromosome homomorphicity(see Figure 4 for a visualization and furtherdescription of this process). We now go on to

explore ESR-based future directions andmodel systems for the “fountain of youth” hy-pothesis and the other evolutionary processesdescribed above.

outstanding issues and futuredirections

Can Sex Reversal Respond to Selection?A number of evolutionary models of sex

determination, such as that by Quinn et al.(2011) described above, assume that sex-

Figure 4. A Visualization of the “Fountain of Youth” Hypothesis (Perrin 2009)Two hypothetical populations with a male heterogametic system of sex determination are depicted. In

population A, sexual differentiation is a nonplastic process, and thus environmental sex reversal (ESR) is notpossible. In population B, sexual differentiation is plastic and ESR can occur. X and Y represent X and Ychromosomes, and circles and squares represent female and male sexual phenotype, respectively. Recombi-nation between chromosomes is represented by interchanging arrows. The size of the chromosome representsthe buildup of deleterious mutations over time; a smaller chromosome equates to more degeneration. Overtime, in population A, the Y chromosome decreases in size because it is not able to recombine and builds updeleterious mutations. X chromosomes, on the other hand, are regularly offered a chance to recombine inphenotype females and thus show little degeneration. Perrin (2009) hypothesizes that recombination betweensex chromosomes is governed by sexual phenotype rather than genotype and that, in line with this idea, ESRfacilitates recombination between sex chromosomes ordinarily prevented from recombining. In population B,the Y chromosome begins to degenerate, but is then offered a chance to recombine with X owing to afeminizing sex-reversal event (ESR). Ultimately, in population B, sex chromosomes appear more homomor-phic than in population A.

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determining thresholds below/above whichESR may occur, can evolve/shift in responseto selection. To ascertain whether mecha-nisms of sexual differentiation can respondto selection in this way, we can ask the fol-lowing three questions: “Is there variationbetween individuals in their susceptibility toESR within populations?” “Can variability inESR be heritable?” “Ultimately, does suscep-tibility to ESR respond to selection?” Evi-dence from fish species and aquaculturestudies suggests that the answer to all threequestions is “yes.”

With regards to the first question, abun-dant literature from aquaculture researchprovides us with strong evidence that thereare between-individual differences in strengthof environmental stimuli required to induceESR. Individuals within experimental treat-ments do not uniformly display the sameresponses to ESR-inducing environmentalfactors. For example, almost all fish studiesof ESR-inducing chemicals on group sex ra-tio show a dose-dependent response (Seniorand Nakagawa 2013). Such a response toESR-inducing factors suggests that some in-dividuals will undergo ESR readily, whileothers require more intense treatments.

With regards the second question, sex re-versal can occur as a result of presence/ab-sence of specific alleles at a single autosomallocus (Kato et al. 2011). Whether polymor-phisms in such alleles interact with envi-ronmental factors and actually alter thethreshold of ESR remains to be seen.Nevertheless, evidence for sex reversal asa result of a single locus suggests thatheritable factors within populations cancontribute to sex reversal.

The answer to the last question providesperhaps the strongest evidence that thresh-olds of sex determination are evolvable traits.Nile tilapia is an important aquaculture spe-cies, and perhaps the most extensively stud-ied with regard to ESR. The species displaysgenetic sex determination (Cnaani 2013).However, temperature effects are also pres-ent, whereby genotypic females develop asmales in response to high temperatures dur-ing development (reviewed in Baroiller et al.2009a). The phenomenon of temperature-induced ESR has been extensively observed

in wild (e.g., Bezault et al. 2007) and captivetilapia populations (e.g., Rougeot et al.2008). Most interestingly, however, the ther-mal sensitivity of individuals within popula-tions appears to be both variable and heritableand responds to selection because it canbe artificially selected for (Wessels andHörstgen-Schwark 2007, 2011).

Elucidating Sex Linkage and Predation inGuppies

The selective mechanisms that drive abreakdown of Y-linked color with predationin guppies (as described by Gordon et al.2012) are not currently well understood. Fur-ther application of ESR may help to eluci-date these mechanisms. One hypothesis isthat genes for coloration act additively (Gor-don et al. 2012); i.e., the more genes carried,the more color expressed. If sex linkage lim-its the number of color genes per male,breaking sex linkage would allow males tocarry a higher number of color genes, ulti-mately increasing phenotypic color. In high-predation environments, the benefits ofbeing colorful (more attractive to mates)have to be offset against a very strong cost(being visible to predators; Endler 1980).However, in a low-predation environment,the costs of being colorful are not so great.Therefore, under low predation, geneticchanges that break sex linkage and allow forincreased coloration may be released fromnegative selection by predation.

Cross-breeding experiments may be ableto demonstrate whether genes for color actadditively in guppies. For example, if colorgenes act additively, a male from a high-predation population (Y-linked color) bredwith a female from the same populationshould theoretically produce, on average,less colorful sons than when bred with afemale from a low-predation population(nonsex-linked color). This test of additivecolor requires that the Y-linkage of color inexperimental females be confirmed. ESR-inducing techniques (Figure 3), such asthose outlined by guppy breeders in theaquaculture industry (Mag-Muresan et al.2004), will make useful screening methodsfor the presence of color genes in females.

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The genomic mechanism that breaksY-linkage of color in guppies also requireselucidation. One potential mechanism, al-ready discussed, may be sex-chromosomerecombination in sex-reversed individu-als (Perrin 2009). If portions of the Y chro-mosome recombine in naturally feminizedguppies, color genes may become locatedon X. If feminization (leading to XY recom-bination) is relatively common in wild pop-ulations, and genes for color act additively(as previously described), the patterns ob-served by Gordon et al. (2012) could beproduced.

Naturally occurring ESR (i.e., in the ab-sence of chemicals) is known to occur inguppies (Mag-Muresan et al. 2004). Thewidespread application of sex-specific ge-netic markers (Khoo et al. 2003; Tripathi etal. 2009) to wild guppy populations mayreveal the frequency of naturally occur-ring feminization, although we note thatESR studies in wild fish populations mustbe interpreted with caution (see the sec-tion Studying ESR in Wild Fish Popula-tions above). Laboratory studies may alsoprovide evidence for the hypothesis thatESR breaks Y-linkage of color genes inguppies; such a study is discussed belowin the section Could ESR be a “Fountainof Youth” for Sex Chromosomes?

ESR, Sex Chromosomes, andReproductive Isolation

Born out of the idea that ESR may be usedto elucidate sex linkage within species, wealso wonder whether ESR may be used tostudy the roles that sex chromosomes andsex linkage play in reproductive isolation.Indeed, fish are already known as model sys-tems for exploring sex linkage, sex-specificselection, and reproductive isolation (Lind-holm and Breden 2002; Kitano and Peichel2012). What is more, sex reversal has beenimplicated as a mechanism that may contrib-ute to speciation in fish (Lande et al. 2001).However, it seems to us that evolutionarystudies may not have gone as far as incorpo-rating induced ESR into hybridization exper-iments (although sex reversal of hybrids iscertainly possible; e.g., Gomelsky et al. 1999or Omoto et al. 2002). In some instances,

clear predictions for the results of ESR-hybridization experiments under hypothesesof sex linkage and reproductive isolation areidentifiable.

For example, Volff (2004) suggests an ex-pansion of an earlier “divergent-resolution”hypothesis (Lynch and Force 2000; Taylor etal. 2001) in which sex linkage of genes vitalto male viability/sterility may lead to repro-ductive isolation. Briefly, Volff (2004) as-sumes two genes are vital to male viability/sterility, and that in a population these genesare simultaneously present on both X and Ychromosomes. Following divergence fromthis ancestral population, if one “male-essential” gene were to become X-linked,and the other Y-linked, in one of the daugh-ter demes (due to inactivation/loss of cer-tain loci resulting in pseudogenes), butoppositely sex-linked in the other daughterpopulation (i.e., divergent resolution), maleinviability would be observed in hybridcrosses (Figure 5). Volff (2004) notes thatsuch a mechanism would also be in keepingwith, and may contribute to, the well-knownreproductive phenomenon Haldane’s rule,which states that, in hybrids, if one sex isinviable or sterile that sex is the heteroga-metic sex.

We suggest that under the aforemen-tioned mechanism, the following fourpredictions from an ESR-hybridizationstudy can be made. First, feminization ofXY individuals from the daughter popu-lations should yield viable females. Sec-ond, masculinization of XX individuals fromeach of the daughter populations wouldyield inviable/sterile males (depending onthe function of the X- and Y-linked genes).Third, although “wild-type” (XY) male hy-brids would be inviable/sterile, masculinizedXX individuals should develop as fully func-tional males, as they would carry copies ofboth “male-essential” genes. Finally, if onewere able to induce feminization at a veryearly stage (perhaps via some method ap-plied to Y gametes), XY hybrid individualsshould develop as functional females. Thesepredictions, and the mechanisms that giverise to those outcomes, are summarized inFigure 5.

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Could ESR be a “Fountain of Youth” forSex Chromosomes?

Indirect evidence that ESR may act as a“fountain of youth” for sex chromosomes(Figure 4) has been gained using compara-

tive molecular techniques. Stöck et al. (2011)carried out such a study in the tree froggenus Hyla. In this genus, males bear homo-morphic sex chromosomes (XY), which donot recombine in wild-type individuals. Stöck

Figure 5. Environmental Sex Reversal (ESR) May Be Used to Ascertain Whether DivergentResolution of Genes That Are Located on Sex Chromosomes, and Essential to MaleViability, Contributes to Reproductive Isolation Following Divergence

Modified from Volff (2004). Shown is a hypothetical set of species with male heterogametic (XY) sexdetermination. Dashed lines indicate Y chromosomes, and solid lines indicate X chromosomes. Two genes, Aand B, are depicted as boxes present on each chromosome. Black squares represent functioning genes and greysquares represent inactivated pseudogenes (ps). Phenotypic sex is denoted by male and female symbols. Aviable phenotypic male can only develop if functioning A and B are both present in an XY individual. In theancestral population, functional alleles for A and B genes are present on both X and Y chromosomes. Followingdivergence, in one deme B becomes inactivated on the Y chromosome and A becomes inactivated on X. Thereciprocal process also occurs in the sister deme, i.e., divergent resolution. In subsequent hybridization, malesare inviable; hybrid XY individuals do not carry functional copies of both A and B. XX female hybrids wouldbe functional as neither A nor B is necessary for female development, although these individuals do carrypotentially functional copies of both genes. Phenotypic males are thus not naturally present in hybrids, however,masculinization of XX hybrids would theoretically yield viable males, as these individuals carry functional A andB. Other predictions for ESR experiments are presented (the right side of the diagram). Wild-type XXindividuals (nonhybrids) should theoretically not yield viable males following masculinization, as these indi-viduals do not carry functional copies of both A and B. Hybridized XY individuals may theoretically be viableas females, although we note that feminizing environmental effects may have to act at a very early stage beforea missing A/B became lethal to male development (e.g., prezygotically on Y-gametes).

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et al. (2011) have shown that three species ofEuropean tree frog share ancestral sex chro-mosomes; i.e., autosome recruitment has notoccurred postspeciation. However, the levelof overlap in the allelic distribution of within-species X and Y chromosomes was higherthan the level of overlap of between-species Ychromosomes. That is to say, the Y chro-mosomes of sister species showed lesssimilarity to one another than they do totheir conspecific X chromosomes. Theseresults are suggestive of the fact that re-combination between conspecific X andY chromosomes has occurred postspecia-tion (Perrin 2009; Stöck et al. 2011).Stöck et al. (2011) argue that ESR maycause these recombination events.

Two direct tests of the “fountain of youth”hypothesis have also been proposed (Perrin2009; Stelkens and Wedekind 2010). First, evi-dence for recombination can be gained usingsex-linked allelic markers in sex-reversed indi-viduals. Using such methods, Matsuba et al.(2010) demonstrated that the rate of recom-bination of sex chromosomes in the com-mon frog (Rana temporaria) is dependent onphenotype. Their results indicated that in mas-culinized genotypic females (XX), recombi-nation of X chromosomes did not occur,although in wild-type females (also genotypi-cally female; XX), X chromosomes do recom-bine. Second, by cross-breeding sex-reversedgenotypic males (XY) with wild-type males (XYphenotypic males) and observing the variancein fitness of heterogametic offspring, onemight be able to detect the mutation-purgingeffects of ESR (Stelkens and Wedekind 2010).If ESR does allow sex chromosomes to recom-bine, then the variance in fitness of heteroga-metic offspring from sex-reversed/wild-typecrosses should be higher than the variance infitness of heterogametic offspring from wild-type/wild-type crosses (Stelkens and Wedekind2010).

To date, most evidence that ESR may re-sult in sex-chromosome recombination hasbeen garnered from amphibians. However,fish also seem to be good model systems fortesting the “fountain of youth” hypothesis(Perrin 2009). Protocols to induce ESR arewell established in many species (see Pand-ian and Sheela 1995; Cnaani and Levavi-

Sivan 2009). The wide diversity of sexchromosomes among fish species makes fur-ther comparative molecular studies, such asthat described in frogs above, possible. Fi-nally, the large number of progeny thatmany fish produce in a single generationmakes detecting recombination among off-spring far easier than it would be amongother vertebrate taxa; e.g., if exploring the“fountain of youth” hypothesis with the off-spring fitness variance study outlined above.

The guppy may lend itself to the study ofXY recombination in sex-reversed individu-als. Sex-linkage maps for guppies have beenproduced (Khoo et al. 2003; Tripathi et al.2009), making direct molecular tests possi-ble. What is more, as discussed above (seethe section Elucidating Sex Linkage and Pre-dation in Guppies), in many guppy popula-tions genes for coloration are Y-linked. If X-Yrecombination were to occur in feminizedgenotypic males, then Y-linkage of colorgenes could be broken by ESR; a processtestable with a two-step ESR experiment.First, feminized genotypic male fish (XY)need to be bred with wild-type males(Figure 1C). Note that these males needto be sourced from populations with strictY-linkage. Second, the daughters (XX) of thisbreeding can be exposed to masculinizingchemicals to ascertain whether they carrygenes for coloration on an X-chromosome(Mag-Muresan et al. 2004; Figure 3). Ifdaughters from such a breeding expresscolor more frequently than daughters fromwild-type crosses of fish from the same pop-ulation, one may infer the breakdown ofY-linkage following ESR.

ConclusionThe sex-determination processes that op-

erate in fish taxa are highly diverse relative tothose in other vertebrates. Even where ge-netic sex-determination systems analogous tothose in other vertebrates have evolved, themechanisms of differentiation that result insexual phenotype are plastic and may re-spond to environmental factors. Environ-mental sex reversal is a widely observedphenomenon in fish, and can be induced bychemical exposure. Chemically induced ESRhas long been exploited by the aquaculture

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industry to increase profitability in fish farm-ing, and by physiologists to understand sex-ual differentiation. However, such researchhas applicability to wild fish populations, par-ticularly where pollutants induce ESR, or ininvasive or struggling populations where sex-reversed individuals may be released to pur-posefully manipulate population dynamics.Better understanding of the effects of ESRon wild-populations may be gained by: differ-entiating between the toxic effects of chem-ical exposures and the effects of mismatchedgenetic and phenotypic sex; and studyinghow sex-ratio skews that result from ESR in-teract with species-specific factors, such assexual conflicts. ESR research may also beused to understand how sex-determination

systems and sex chromosomes evolve. Chem-ically induced ESR may be a research toolused to elucidate the extent to which traitsbecome sex-linked in response to selectivepressures, or ultimately how sex linkage con-tributes to reproductive isolation. Finally,naturally occurring ESR may even be an evo-lutionary mechanism that plays a significantrole in sex-chromosome evolution.

acknowledgments

We would like to thank Andy Hicks, Eduardo Santos,Malgorzata Lagisz, three anonymous reviewers, andthe editorial team at the QRB for their time in proof-reading and making comments on this review. Wewould also like to thank the Marsden Fund(UOO0812) and the Department of Zoology, Univer-sity of Otago, for financial support.

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