doi 10.1098/rspb.2000.1217

Multipleoriginsof XYfemalemice(genus ): phylogeneticandchromosomalevidence HopiE. Hoekstra * and Scott V.Edwards Department of Zoology and Burke Museum, University of Washington, Seattle,WA98195,USA Despite the diversityin sex determination across organisms,theory predicts that the evolutionof XY females is rare inmammals dueto ¢ tness consequencesassociated with infertility orthe loss ofYY zygotes.W einvestigatedthis hypothesisfrom a phylogeneticperspective byexamining the inter- and intraspeci¢c distributionof Ychromosomes inmales andfemales (XYfemales) inSouth American ¢ eld mice (Akodon).Wefoundthat XYfemales occurredat appreciable frequencies (10^66%)in at least eight Akodon species, raisingthe possibilitythat this system ofsexdetermination has arisen multiple times inde- pendently.Todetermine the number oforigins of XY females in Akodon,weconstructed amolecular phylogenyof 1 6species of Akodon basedon mitochondrialDNA controlregion sequences. Bothparsimony andmaximum-likelihood reconstruction ofancestral states suggestthat multiple steps (gainsor losses of XYfemales) best explainthe evolutionof XY females, butdo not clearly di¡ erentiate betweensingle and multiple origins.W ethen directlycompared functional and non-functional Y chromosomes insix species bySouthern blot analysis. W efoundthat maleand female Y chromosomerestriction fragmentlength poly- morphism patterns wereidentical within species, butalways di¡ ered betweenspecies, providingevidence thatXY females aroseat least sixtimes withinthe Akodon lineage.T oourknowledge, this pattern in Akodon is the ¢rst documentationof a novelsex-determining system arisingmultiple times withina tight cladeof . Inaddition, this system providesa cleartest ofthe accuracyof phylogenetic methods toreconstruct ancestralstates. Keywords: sexdetermination ;Ychromosome;sexchromosomes; XY females; ancestralstates; Akodon

sex-determining mechanisms donot often evolve in species 1.INTRODUCTION withheteromorphic sex chromosomes likemammals Unlikemany basic developmental processes, the mechan- because(i) amale-determining mutation(formation of isms employedfor sex determination are highly diverse XXmales) facesthe lackof necessary Y-chromosome across organisms(Ohno 1 967;Bull1985; Wibbels et al. genesfor male function, and (ii) afemale-determining 1994).Howsuch di¡erent sex-determining mechanisms mutation(formation of XY females) facesthe production aroseand diversi¢ ed has long puzzled biologists (Bull ofone-quarter YY o¡spring and the potentialadverse 1985).Clearanswers to account for this diversityhave e¡ects ofY-chromosome genes on the feminizingprocess eludedscientists forthree primaryreasons: ¢ rst, the deep (Marin &Baker1 998).Thishypothesis predicts anevo- divergencebetween taxa with di¡ erent sex-determining lutionarilyconserved mechanism ofmammalian sex mechanisms complicatescomparative studies; second, determinationand very infrequent origins of novel sex- observinga sex-determining system earlyin its evolution determiningsystems, speci¢cally XX males orXY isdi¤cult; and¢ nally,the mechanisms ofsex determina- females. tionfor many clades are incompletely described, for SouthAmerican ¢ eldmice ( Akodon)present aunique examplereptiles, wherefewer than 10% of all species opportunityto study the evolutionof sex determination havebeen characterized (Viets et al.1994).Mammals becausethe aforementioneddi¤ culties areovercome by providean excellent system inwhich to study sex determi- the presence ofseveral closely related species inwhich nationbecause mechanisms areso well described across a XYfemales occur(see, forexample, Bianchi et al. 1971). varietyof species (Vorontsov et al.1980;F redga1 983). Inspecies withXY females, the presence orabsence of However,the diversityof sex determination is greatly the Ychromosomeno longer plays the decisiverole in sex reducedwithin mammals, whichgenerally employ a determination.In Akodon XYfemales, the Ychromosome system ofheterogametic (XY) males andhomogametic failsto trigger the malepathway and is hencereferred to (XX) females (Ohno1 967;Bull 1 983).Exceptionsto this as Y* (Lizarralde et al.1982).Evidencethat the causeof pattern generallyappear to have evolved once within a sex-reversal lies onthe Y * chromosomeincludes cytoge- genus.F orexample,the mole( Talpa; Sanchez et al. 1996), netic, molecular,breedingand phylogenetic studies, and whichhas hermaphroditic females; the woodlemming noindividuals of XYY * karyotypehave been reported (Myopus; Fredga et al.1976),whichhas XY females caused withinthis cladeof Akodon (Bianchi& Contreras1967; bya mutationon the Xchromosome;and¢ nally,asingle Lizarralde et al.1982;Vitullo et al.1986;Bianchi et al. species ofmicrotine vole( Microtus; Burgos et al. 1988), 1993;Espinosa & Vitullo1 996;H. E.Hoekstra,unpub- whichalso has XY females (fora review,see Fredga1 983). lisheddata) . Toexplainthe lackof diversity of sex-determining Inseveral species of Akodon, XY* females persist at schemes inmammals, Bull( 1983)suggested that novel highfrequency along with normal XX females innatural populations.Because XY * females occuronly in some *Author forcorrespondence ([email protected]) . species, the Akodon cladeallows us toaddress an

Proc. R.Soc.Lond. B (2000) 267, 1825^1831 1825 © 2000The RoyalSociety Received 8 March 2000 Accepted 2 June 2000 1826H. E.Hoekstraand S. V.Edwards Multiple origins of XYfemale mice importantquestion regarding the evolutionof novel sex- primersequences are 1 2S1:5 ’-CGTTCATTGCTTAATTTTTAT determiningsystems that cannotbe addressed inother CACTGC-3’ and C2: 5’-TGGTCTTGTAAATCAGTAATG-3 ’. mammalianclades, namely ,howmany times didXY * PCR productswere cleaned using a puri¢cation kit ( Quiagen, females evolvein Akodon?Bull’s(1983)hypothesis predicts Valencia,CA, USA).Cyclesequencing using £ uorescently asingleancient origin of XY * females becausethe mode labelleddye terminators of both strands was carried out on an ofsex determination is aconservedtrait, whereasif sex ABI 373Aautomated sequencer. Sequences were aligned manu- determinationin Akodon ismore labile,XY * females may allyusing the program Sequencher ( GeneCodes)and adjusted haveevolved independently in several species. Inorder to byeye as necessary .Sequenceswere deposited in GenBank determine the number oforigins of the Y * chromosome, (accessionnumbers AF29 6258^AF296278). wetested aseries ofpredictions specifying single and Topologieswere generated using neighbour-joining (NJ) and multiple originsof the Y * chromosomeat both the species maximum-likelihood( ML)methods. T reeswere rooted by a andchromosomal levels (¢ gure 1 ).Weaddress these designatedoutgroup, Auliscomysp ictus ,anotherakodontine predictionsthrough a combinationof phylogeneticanalysis (Smith & Patton1 993).Todevelopan empirical model of of Akodon species lineagesusing mitochondrial DN A sequenceevolution, ML constructionwas based on an initialNJ (mtDNA) controlregion sequences, aswell as an exami- treefrom which MLestimatesof ¬ (0.377;among-site rate nationof the ancestryof the Y * chromosomeitself. variation), µ (3.901;transitionbias) and the proportion of invar- iantsites (0.507) were generated. These values were then used in combinationwith anHKY85 model of sequence evolution, 2. METHODS which waschosen on the basis of hierarchical hypothesis testing (a) Tissue samples (Huelsenbeck& Rannala1997) ,togenerate an ML tree Samples of Akodonazarae and Akodonmolinae werecollected in (Voelker& Edwards1 998).Treetopologies and bootstrap Argentinaduring March ^April 1998.Remaining tissue samples supportwere generated using P AUP4.0(Swo¡ ord 1999) . fordetection of XY * femalesand phylogenetic reconstruction from1 4specieswere obtained from the Museum of V ertebrate (d) Reconstruction ofXY * evolution Zoology,Berkeley,CA;FieldMuseum of N aturalHistory , The presence(1 )orabsence (0) of XY * femaleswithin a Chicago,IL; National Museum of Natural History ,Washington, specieswas coded as a binarycharacter (Wiens 1 999)and DC;andMuseum of Southwestern Biology ,Albuquerque,NM. mappedon tothe ML topologyusing MacClade ( Maddison& Althoughthere are ca.25species of Akodon currentlydescribed Maddison1 992).Maximum-parsimony(MP) algorithmswere fromacross South America (Smith & Patton1 991),themajority usedto determine ancestral states. Ambiguous lineages were ofspecimens are inaccessible and not represented in museum resolvedusing the A CCTRAN (acceleratedtransitions) and collections.However, we havetissues from a geographically DELTRAN(delayed transitions) options of MacClade.ML esti- diversesample of well-described species from all of the major mationsof ancestral states were calculated using DISCRETE clades of Akodon (seeAppendix A) . 1.01b(Pagel1994, 1997) .Ratesof characterchange and ML esti- matesof ancestral character states were calculated following (b) Screening for XY * females Mooers& Schulters(1 999)using branch lengths estimated from Todetectthe presence of XY * females in Akodon species,poly- theNJ tree. Because DISCRETE does not handle unknown merasechain reaction ( PCR)genotyping was performed on 1 79 states,the unknown tips were either assigned state by MP ,or phenotypicfemales from 16 species of Akodon.PCR primers alternativelyall possible permutations were averaged. designedto amplify an intronof the Smcy geneon the Y chromo- someof mammals (Agulnik et al.1994)were used to determine (f) Southern blotanalysis thepresence or absence of theY * chromosomein phenotypically Highmolecular weight DNA was isolated from an XX female Akodon.MaleDNA of the same species was used as a female, XY* femaleand XY malefor each of six Akodon species positivecontrol in everycase. A secondPCR reaction,using the forwhich highquality tissues were available. XX femaleDNA singlecopy autosomal gene tubulin (Palumbi1 996),wasused as a servedas a controlto ensure that bands were Y -chromosome controlto ensure that the absence of abandin aPCR reaction speci¢c. Approximately 10 m gofDNA from each individual was wasnot due solely to poor DNA template quality .PCR results cut with EcoR1, HindIII, PvuII and PstIrestrictionenzymes. fromten females from ¢ vespecies (a putative XX andXY * StandardSouthern hybridization methods and solutions were femalefrom each species) were con¢ rmed by Southern blots. W e used;hybridizations were conducted at 60 8 for24 h, andblots useda secondY -chromosomegene, the sex-determining gene, wereexposed for four to six days. The probewas PCR ampli¢ed Sry asa probefor the Y andY * chromosomes(following the fromtotal genomic Akodonboliviensis DNAusing primers designed protocoldescribed in ½2(f)) andas anindependentlocus on the fromthe conserved HM Gboxregion of thesex-determining gene, Ychromosome.The Southernblot restriction patterns from Sry Sry, in Musmusculus (SRY1 5’-AGCGCCCATGAATGCATT-3 ’ matchedour PCR resultsexactly ,con¢rming our assumption and SRY4 5’-GTTTGGGTATTTCTCTCTG-3 ’).The 197base that the Smcy geneis, in fact,Y -chromosomespeci¢ c andthat pair(bp) product was then cloned and sequenced for veri¢ ca- ourPCR screeningmethod is accurate. tion.The probedid not contain any relevant restriction sites.

(c) Sequencing ofmtDN Acontrol region 3. RESULTS andphylogenetic reconstruction Toreconstructspecies relationships, we sequencedthe entire (a) Frequency ofXY * females across species mtDNAcontrol region of 20 individuals representing 1 6species Results ofthe PCRsurvey show that XY * females are of Akodon.Ampli¢cation primers were designed from £ anking present ineight out of 1 6species tested (table1 ).Three conservedregions in sigmodontinerodents: 12S rRNA (Sullivan species (Akodon mimus , Akodon orophilus and Akodon juninensis ) et al.1995)and the cytochrome b gene(Smith & Patton1993) .The didnot have large enough samples todetermine the status

Proc. R.Soc.Lond. B (2000) Multipleorigins of XYfemale mice H. E.Hoekstraand S. V.Edwards1827

species relationships chromosome relationships single origin multiple origins single origin multiple origins species species (a) XX (b) X Y* (c) Y 1 (d) Y 1

XX XX Y 2 Y* 1 XX XX Y 3 Y 2 1 X Y* XX Y * Y* 2 X Y* X Y* Y * 2 Y 3

X Y* X Y* Y * 3 Y* 3

Figure1. Phylogenetic hypotheses of theevolutionary origin(s) of XY * females.Scenarios ( a) and (b)depicthypothetical phylogeneticrelationships of speciesthat either have XY * femalesor donot, represented by XY * andXX, respectively.A single origin of XY* femalespredicts that those species with XY * femalesare each others closest relatives (a monophyleticorigin) as in (a).Multipleorigins of XY * femalesmay produce a patternwhere those species with XY * femalesare scattered throughout the tree as in (b).Apatternsimilar to thatshown in ( a)couldstill be reconstructed by parsimony if therewere multiple origins of XY* femalesif theparsimony assumptions were violated. Scenarios ( c) and (d)showphylogenetic relationships of theY andY * chromosomesfrom three hypothetical species. Species are represented by anumber.A singleorigin of Y * chromosomewould producea patternof clusteringof Y * chromosomesindependent of species.Multiple origins of theY * chromosomeproduces a patternwhere Y andY * chromosomesfrom a singlespecies cluster together. Arrows indicate origins of XY * females. of XY* females, andare thus consideredunknown in the Table 1. Frequency ofXY * females among and within species MP reconstructions. WediscoveredXY * females intwo of Akodon resulting from our PCRgenotyping survey species inwhich they had not yet been reported: Akodon (Ourresults con¢ rm those of previous studies, which torques and Akodon kofordi. Overall,our results extendedbut demonstratedthe presence of XY * females in A. varius and didnot contradict any previously reported dataon the A. mollis andthe absence of XY * females in A. molinae and taxonomicdistribution of XY * females in Akodon. The A. urichi forwhich largesamples in thisstudy were frequencyof XY * females amongall females ineach unavailable.For some species, samples sizes are small and species tested rangedfrom 0 to66% (table1 ),althoughfor resultingfrequencies may be in£ ated.) some species ( A. kofordi and Akodon puer),samplesizes were * * toolow to calculate a reliablefrequency . species n no. XY %XY A. azarae 39 4a,b 10 (b) Phylogenetic analysis A.bolviensis 23 8c 35 Currentlythere areno complete Akodon phylogenies, A.subfuscus 13 5 38 but incompletetrees basedon allozymes (Barrantes et al. A. torques 11 4 36 1993;Rieger et al.1995)or cytochrome b genesequences A. kofordi 3 2 66 (Patton& Smith 1992;Smith &Patton1 993)are avail- A.puer 2 1d 50 able.N oneof these phylogenies,however, are strongly A. varius ö öc ö e resolvedor include all the species of Akodon that contain A. mollis ö ö ö XY* females. Our phylogeny,basedon the mtDNA A. aerosus 10 0 0 controlregion for the 16species of Akodon,includedthe A.a.baliolus 10 0 0 A. cursor 10 0 0 eightspecies that haveXY * females (¢gure 2) .Inseveral A. molinae 4 0f 0 species, twoindividuals were sequenced. In all cases, A. urichi 1 0c 0 mtDNAs fromthe same species clustered togetherwith A.orophilus 3 0 ? greaterthan 95% bootstrap support. The topology A. mimus 2 0 ? upholdspreviously described a¤nities, i.e.between puer, A.juninensis 1 0 ? subfuscus, and juninensis (Myers et al.1990),aswell as torques, mollis and orophilus (Smith &Patton1 993).The a Bianchi& Contreras1 967; b Bianchi et al. 1968; c Bianchi et al. d e f twomajor clades of Akodon shownin ¢ gure2 arevery 1971; Vitullo et al. 1986; Lobato et al. 1982; Bianchi& Merani similar tothe cladesgenerated by the cytochrome b gene 1984. (Smith &Patton1 991),althoughdi¡ ering in the species included.The basal position of Akodon urichi is consistent withthe hypothesisthat A. urichi maynot belong in the topologyis wellsupported by other phylogenetic studies genus Akodon (Smith &Patton1993) .Weaklysupported usingindependent loci. nodesare due primarily to the uncertainposition of A. juniensis andto the `£ip-£ op’ of A. mimus, which also (c) Reconstruction ofXY * origins fallsbasal to Akodon cursor insome trees. Theuncertainty Thephylogenetic tree showsthat those species withXY * in A. mimus’spositionis alsoevidenced by its previous females donot form a monophyleticclade. A likelihood- assignment tothe Microxus genus(Smith &Patton1 993). ratiotest ofmonophyly suggests that atopologywhich Thus,despite lowbootstrap values in some nodes,the forces those species withXY * females tobe monophyleticis

Proc. R.Soc.Lond. B (2000) 1828H. E.Hoekstraand S. V.Edwards Multiple origins of XYfemale mice

(a) ACCTRAN 85 A. puer (b) DELTRAN A. subfuscus ? A. juninensis ? 89 81 A. azarae A. boliviensis 53 A. kofordi A. cursor A. a. baliolus * 96 * A. aerosus 59 63 A. mollis 74 94 A. torques ? A. orophilus ? 100 A. varius A. molinae ? A. mimus ? A. urichi outgroup

Figure2. Phylogenetic pattern of XY * femaleevolution. The presenceof XY * femaleswas mapped onto the ML topologyof Akodon species(based on 1085 bp ofmtDNAcontrol region) using equal-weighted parsimony reconstruction. Solid lines indicate presenceof XY * females.Boxes at tips indicated absence (empty box), presence (¢ lled box) or unknown state (?) ofXY * females inthespecies. Gain or loss of XY * females(steps) are indicated by arrows.Numbers above the lineages represent bootstrap values(1000 replicates). ( a)Reconstructionusing ACCTRAN (acceleratedtransitions) to resolvebranch assignments. ( b) Recon- structionusing DELTRAN (delayed transitions). The relativeareas in thepies represent the relative ML supportfor the presence(black) or absence(grey) of XY * femalesat thestarred node ( *)usingthe one-rate model. signi¢cantly worse than the `best’ phylogeny( p 5 0.01) ML analysisestimated the rate ofgain and loss ofXY * (Huelsenbeck et al.1996). females asapproximately equal, thus atwo-rate model Thepresence orabsence of XY * females wasthen that allowsrates ofgain and loss todi¡ er wasnot mappedonto this topology,usingACCTRAN (acceler- statistically better thana one-ratemodel (Mooers & ated)and DEL TRAN(delayed)options to resolve unre- Schulter 1999).Over alleight trees, the MLestimate of solvedbranches (¢ gure 2) .Toevaluatethe phylogenetic rate variedfrom 0.0056 to 0.01 37.ML wasalso used to labilityof the XY * system across species, the number of estimate the ancestralcharacter state atthe nodethat is characterstate changes(steps, asshown by arrows in expectedto have XY * females inthe singleorigin ¢gure2) was calculated on the MLtopologyand scenarioand no XY * females inthe multiple origin comparedto the number ofsteps required by10 00recon- scenario(the asterisk in¢ gure2) .When unknowntips structions after randomlyshu¥ ing the characters atthe werereconstructed usingACCTRAN, ML estimated the tips (Maddison& Slatkin1 991).Thenumber ofsteps on relativesupport for XY * females present as0.67 . the best topology( x 3)is notsigni¢ cantly di¡ erent to However,when A. orophilus wascoded as havingno XY * ˆ the averagenumber ofsteps ontrees withrandom char- females, the ML supportfor XY * females atthe acter distributions ( x 4.227; p 4 0.05),implyingthat the ancestralnode dropped to 0.42, and the multiple origin ˆ observeddistribution of XY * females isnotdi¡ erent from hypothesiswas favoured. When allthe possiblepermuta- arandomscattering ofXY * females. Thisresult also tionsof the unknowntips wereaveraged (using a one- suggests that XY * females aregained and lost withlittle rate model),the ML estimate was0. 70for having XY * phylogeneticinertia or historicalcontingency . females and0.30 for the absenceof XY * females atthe Thus,instead of a singleevent, the phylogenyimplies nodeof interest. multiple gainsor losses ofXY * females (¢gure 2) .The accelerated(ACCTRAN) reconstruction suggests asingle (d) Yand Y* chromosome relationships across ancientorigin of XY * females withloss ofXY * females in species A. cursor and the ^Akodon aerosus balious Todetermine relationshipsbetween Y andY * chromo- lineage.On the otherhand, the delayed(DEL TRAN) somes withinand between species, restriction pattern reconstruction showsthree independentorigins of XY * variationin XY males andXY * females wasexamined females andno losses. Thesetwo reconstructions di¡er in usinga Y-chromosome-speci¢c probeconsisting of a assigningone unknown state in A. orophilus . The uncer- portionof the Akodon sex-determining gene, Sry. Southern taintyin phylogeneticposition of A. juniensis and A. mimus blotsreveal that YandY * chromosomepatterns arealways doesnot a¡ ect the characterreconstruction becauseboth similar withina species, but alwaysdi¡ er betweenspecies, species areof unknown state andare assigned the same forall six species tested (¢gure 3) .Restriction patterns state inbothreconstructions. di¡ered inbothband number (range1^3) and band size When allpossible assignments ofthe three unknown betweenspecies. Thispattern holdswhen additional states weretried ( n 8),multiple originswere supported restriction enzymes ( PstI and PvuII) areused (datanot ˆ infour cases, andin the remainingfour cases multiple shown).DNA fromXY * females of Akodon mollis and originswere as likelyas asingleorigin reconstruction. Akodon varius wasunavailable, but XY malerestriction

Proc. R.Soc.Lond. B (2000) Multiple origins of XYfemale mice H.E.Hoekstraand S. V.Edwards1829

phenotype: M F M F M F M F M F M F genotype: X X X X X X X X X X X X X X X X X X X Y Y X Y Y X Y Y Y X Y Y X Y Y X Y Y X

23

9.4

6.6

Akodon species: kofordi boliviensis azarae puer subfuscus torques

Figure3. Molecular comparison of theY andY * chromosomes.For each of six Akodon species,DNA froman XYmale,XY * femaleand XX femalewas probed with aY-chromosomefragment. Phenotype, male (M) orfemale (F). Genotype,XY (XY male or XY* female)or XX(XXfemale).Arrows highlight strongly hybridizing fragments shared between XY malesand XY * females.Scale in kilobasesis indicatedat theleft. patterns fromthese species arealso unique (data not alwaysdi¡ er betweenspecies, suggestinga neworigin of shown). Y* chromosomes withineach species. Thisanalysis suggests that there areat least sixindependent origins of XY* females, rather thanthree aspredicted byequally 4.DISCUSSION weightedparsimony reconstruction usingDEL TRANor Thephylogenetic distribution of XY * females across oneorigin as predicted byACCTRAN .Theserestriction Akodon species is itself unableto distinguish between pattern dataare particularly relevant because they singleor multiple originsof XY * females. While the parsi- provideindependent evidence that doesnot rely as monyresults suggestthat singleand multiple evolutions stronglyon the assumptions associatedwith character of XY* females areequally likely ,these analysesrely on mapping.Additionally ,ourparticular results arerobust to the traditionalassumptions ofparsimony reconstruction, discordancesbetween gene and species trees (e.g.hybridi- speci¢cally equal weighting of character loss versus gain zationand incomplete lineage sorting) or uncertainties in (Ree &Donoghue1998) .ML reconstructions relyon esti- characterscoring. These restriction fragmentlength poly- mates ofrate that arebased on the distributionof charac- morphism (RFLP) dataalso show that it wouldbe incor- ters onthe tips, rather thanindependent biological data rect tosuggest the presence ofXY * females incommon (Schultz &Churchill1 999).Relativerates ofgain and ancestors ofeven closely related pairs of Akodon species loss candramatically in£ uence results. Forexample,if we that bothpossess XY * females, since RFLPpatterns in weightcharacter gain (the appearanceof XY * females in such pairs(e.g. azarae and boliviensis )aredi¡ erent. The aspecies) asmore likelythan character loss (the extinc- presence ofdi¡erent patterns inbothYandY * chromosomes tion of XY* females froma species),ascenariowith inthese species suggests independentorigins even among multiple originsof XY * females inseveral species closerelatives, although this doesnot exclude the possibility becomes more likely.Additionally,ifrates ofcharacter of XY* femaleextinctions in species orcommonancestors. changeare rapid, this canalso complicate ancestral state Thesesix origins of XY * females triples the number ofstable reconstruction (Cunningham1 999).Ultimately then, a XYfemalesystems described inmammals andis the ¢rst species tree approach,although useful, is notde¢ nitive time multiple species withina genushave been shown to without a priori informationabout the relativeprobability sharethis trait. Theestimate ofsixindependent origins of ofloss versus gainof XY * females ina species. XY* females isaminimum estimate, asthere arestill many Tocomplement the phylogeneticreconstruction ofXY * species of Akodon tosurvey .Additionaldetailed sampling females, wefurther tested the hypothesesby direct exam- mayalso be able to detect temporaland spatial variation inationof the genealogicalrelationships of Y andY * in XY* femalefrequencies. haplotypes.The predicted patterns in¢ gure1 (clustering Why can XY* females evolverepeatedly in Akodon? byspecies orclustering bysex) stem fromthe factthat Y While XYfemales occurin several mammalian species, chromosomes areinherited paternallyand Y * chromo- includinghumans and house mice ( M. musculus), they somes areinherited maternally.Thus,Y andY * chromo- experiencedramatic declines in¢ tness. HumanXY somes arenever found in a singleindividual and therefore females areinfertile, su¡ering from 46,XY gonadal donot have an opportunity to recombine, thereby dysgenesis(Kent &Watchel1989) and house mice are avoidingpossible Y ^Y* conversionor recombination sub-fertile, havingsigni¢ cantly smaller litters ifany withinor between species. Unlikethe phylogenetic (Eicher et al.1982).First, unlikehumans and house mice, mapping,direct analysisof relationships between Y and Akodon XY* females areboth viable and fertile. The Y* usingSouthern blots can di¡ erentiate betweenthe mechanisms involvedin Akodon XY* femalefertility are singleand multiple originhypotheses and suggests that currently beingexplored (A. Vitullo, personal communi- XY* females arosemultiple times. Inevery case, restric- cation).Second,XY * female Akodon producemore eggs tionpatterns areidentical within each species tested but thancan implant and therefore donot su¡ er froma

Proc. R.Soc.Lond. B (2000) 1830H. E. Hoekstraand S. V.Edwards Multiple origins of XYfemale mice decrease inlitter size dueto the loss ofYY * zygotes regionessential for spermatogenesis and expression of male- (Lizarralde et al.1982;Espinosa & Vitullo1 996).Finally, speci¢c minorhistocompatibility antigens. Hum.Mol. Genet . 3, XY* females producemore o¡spring than Akodon XX 873^878. females overequal lifetimes (Hoekstra &Hoekstra2000) . Barrantes,G. E., Ortells, M. O.& Reig,O .A. 1993New studies Theseunique characteristics give Akodon the potentialto onallozyme genetic distance and variability in akodontine () and their systematic implications. Biol. J. maintain XY* females oncethey have evolved. Linn. Soc. 48, 283^298. Bianchi,N. O. & Contreras,J. R. 1967The chromosomesof the Tissueswere kindly provided by J. L. Patton, A. L.Gardner, B. ¢eld mouse Akodonazarae (Cricetidae,Rodentia) with special Pattersonand T .Yates.The authorsthank members of the referenceto sex chromosome anomalies. Cytogenetics 6, 306^312. Edwardslaboratory ,J.J. Bull, B. J.Haeck, J. C. Herron,J. M. Bianchi,N. O. & Merani,M. S. 1984Cytogenetics of South Hoekstra,R. T.Paine,S. C. Stearnsand two anonymous Americanakodont rodents ( Cricetidae).X.Karyological reviewersfor helpful discussion and comments on the manu- distancesat generic and intergeneric levels. J. Mamm. 65, script.M. Pagelkindly provided the program DISCRETE 1 .01b. 206^219. A.Mooersprovided helpful advice and interpretation of the Bianchi,N. O .,Dulout,F .N.& Contreas,J. 1 968Sex chromo- ML reconstructiondata. A. Minn, C. Galliariand G. Diaz somereplication and sex chromatin in Akodonazarae providedinvaluable assistance in the¢ eld.Special thanks to the (Rodentia,Cricetidae) . Theor.Appl.Genet . 38, 343^347. Diazand Galliari families for their hospitality .H.E.H.was Bianchi,N. O., Reig, O. A., Molina,O .J.& Dulout,F .N.1 971 supportedby a HowardHughes Predoctoral F ellowship.The MurrayL. JohnsonMemorial A wardfrom the Burke Museum Cytogeneticsof the South American akodont rodents toH.E.H. helped fund ¢ eldwork.This workwas funded in part (Cricetidae).I.A progressreport of Argentinian and byN ationalScience F oundationgrants DEB-94 19738and DEB- Venezuelanforms. Evolution 25, 724^736. 9707549to S. V.E. Bianchi,N. O., Bianchi, M. S., Baillet,G. & Chapelle,A. D.L. 1993Characterization and sequencing of thesex determining regionY gene( Sry) in Akodon (Cricetidae)species with sex APPENDIX A reversedfemales. Chromosoma 102, 389^395. List ofspecies, ¢eldnumbers, museum sources and Bull, J.J. 1983 Evolutionof sex determining mechanisms .MenloPark, CA:Benjamin-Cummings. generallocation of individuals used inreconstructing the Bull, J.J.1985 Sex determining mechanisms: an evolutionary Akodon phylogeny.Abbreviationsfor sources: Universityof perspective. Experimentia 41,1285^1296. WashingtonBurke Museum (UWBM),NationalMuseum Burgos,M., Jimenez,R. &dela Guardia, R. D.1988 XY ofN aturalHistory (USNM),Museum ofSouthwestern females in Microtuscabrerae (Rodentia:Microtidae): a caseof Biology(MSB) andMuseum ofV ertebrate Zoology possiblyY -linkedsex reversal. Cytol.Cell Genet . 49, 275^277. (MVZ). Cunningham,C. W.1999Some limitations of ancestral character-statereconstruction with testingevolutionary hypotheses. Syst. Biol. 48, 665^674. speciesnumber sourcelocality Eicher,E.M., Washburn,L. L.,Whitney ,J.B. &Morrow,K.E. 1982 Musp oschiavinus Ychromosomein theC57BL /6Jmurine A. azarae HEH373 UWBM Argentina, genomecauses sex-reversal. Science 217, 535^537. P.BuenosAires Espinosa,M. B. &Vitullo,A. D.1996O¡ springsex-ratio HEH391 UWBM Argentina, andreproductive performance in heterogameticfemales of P.BuenosAires theSouth American ¢ eldmouse Akodonazarae . Hereditas 124, A. molinae HEH236 UWBM Argentina,P. Mendoza 57^62. HEH214 UWBM Argentina,P. 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