Evolution of Sexes from an Ancestral Mating-Type Specification Pathway

16 Pages·2014·3.58 MB·English

Evolution of Sexes from an Ancestral Mating-Type
Specification Pathway
Sa Geng1, Peter De Hoff2¤, James G. Umen1*
1DonaldDanforthPlantScienceCenter,St.Louis,Missouri,UnitedStatesofAmerica,2TheSalkInstituteforBiologicalStudies,LaJolla,California,UnitedStatesof
America
Abstract
Maleandfemalesexeshaveevolvedrepeatedlyineukaryotesbuttheoriginsofdimorphicsexesandtheirrelationshipto
matingtypesinunicellularspeciesarenotunderstood.VolvocinealgaeincludeisogamousspeciessuchasChlamydomonas
reinhardtii, with two equal-sized mating types, and oogamous multicellular species such as Volvox carteri with sperm-
producing males and egg-producing females. Theoretical work predicts genetic linkage of a gamete cell-size regulatory
gene(s)toanancestralmating-typelocusasapossiblestepintheevolutionofdimorphicgametes,butthisideahasnot
beentested.Hereweshowthat,contrarytopredictions,asingleconservedmatinglocus(MT)geneinvolvocinealgae—
MID,whichencodesaRWP-RKdomaintranscriptionfactor—evolvedfromitsancestralroleinC.reinhardtiiasamating-type
specifier,tobecomeadeterminantofspermandeggdevelopmentinV.carteri.TransgenicfemaleV.carteriexpressingmale
MID produced functional sperm packets during sexual development. Transgenic male V. carteri with RNA interference
(RNAi)-mediated knockdowns of VcMID produced functional eggs, or self-fertile hermaphrodites. Post-transcriptional
controls were found to regulate cell-type–limited expression and nuclear localization of VcMid protein that restricted its
activitytonucleiofdevelopingmalegermcellsandsperm.Crosseswithsex-reversedstrainsuncoupledsexdetermination
from sex chromosome identity and revealed gender-specific roles for male and female mating locus genes in sexual
development, gamete fitness and reproductive success. Our data show genetic continuity between the mating-type
specificationandsexdeterminationpathwaysofvolvocinealgae,andrevealevidenceforgender-specificadaptationsinthe
male and female mating locus haplotypes of Volvox. These findings will enable a deeper understanding of how a master
regulatorofmating-typedeterminationinanancestralunicellularspecieswasreprogrammedtocontrolsexuallydimorphic
gametedevelopment inamulticellular descendant.
Citation:GengS,DeHoffP,UmenJG(2014)EvolutionofSexesfromanAncestralMating-TypeSpecificationPathway.PLoSBiol12(7):e1001904.doi:10.1371/
journal.pbio.1001904
AcademicEditor:NickH.Barton,InstituteofScienceandTechnologyAustria,Austria
ReceivedMarch24,2014;AcceptedMay30,2014;PublishedJuly8,2014
Copyright: (cid:2) 2014 Geng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
DataAvailability:Theauthorsconfirmthatalldataunderlyingthefindingsarefullyavailablewithoutrestriction.Allrelevantdataarewithinthepaperandits
SupportingInformationfiles.
Funding:ThisworkwassupportedbygrantNIHR01GM078376toJ.G.U.Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisionto
publish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
Abbreviations:BFP,bluefluorescentprotein;DIC,differentialinterferencecontrast;HA,hemagglutinin;IF,immunofluorescence;MT,matingtypelocus;RNAi,
RNAinterference;RT,reversetranscription;SVM,standardvolvoxmedium.
*Email:[email protected]
¤Currentaddress:SyntheticGenomics,Inc.,LaJolla,California,UnitedStatesofAmerica
Introduction proposed by Parker and colleagues [7] modeled the evolution of
anisogamy (asymmetric-sized gametes) from a starting population
In many unicellular and simple multicellular eukaryotes sexual of isogametes (i.e., mating types) and identified the evolutionary
interactions are governed by mating types. Among sexually forces that might cause a mating-type system to evolve into
reproducing organisms mating types are thought to have evolved anisogamy (large and small gamete types) or oogamy (eggs and
before gamete size differences and separate sexes evolved [1,2]. sperm).Additionaltheoriesandmodificationstotheoriginalideas
Mating types (defined below) control sexual differentiation and of Parker and colleagues have been proposed (reviewed in [1,8–
specializedrolesofcellsthatfunctionasgametesinadiverserange 10]), but very little attention has been given to the mechanism
oftaxaincludingfungi,algae,ciliates,andcellularslimemolds[3– throughwhichnaturalselectionmightactonamating-typesystem
6]. In mating-type systems gametes can be isomorphic but can to drive the transition to anisogamy or oogamy. One model
onlymatewithpartnersthatexpressadifferentmatingtypethan involves the establishment of genetic linkage between a polymor-
their own. Male and female gametes, on the other hand, are a phic locus that affects gamete size and a mating-type locus [11].
hallmark of multicellular organisms such as metazoans and land However, the genetic basis for the evolution of anisogamy/
plants. Males and females have developmentally specialized oogamyhasnotbeendeterminedinanyexperimentalsystem,and
gamete types: large immotile eggs that are produced by females it is not known whether it requires the addition of size control
or female reproductive organs, and small motile sperm produced genes or other genes to an ancestral mating locus as the model
bymalesormalereproductiveorgans.Thegroundbreakingtheory proposes.
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SexesEvolvedfromAncestralMating-TypeSpecificationPathway
andrepressionoftheminusprogram.AsecondMT2gene,MTD1,
Author Summary
alsocontributestoMT2gameticdifferentiationbutisnotessential
Sexual differentiation in eukaryotes is manifested in two forit[26].MIDisarapidlyevolvinggene[27],butorthologshave
fundamentallydifferentways.Unicellularspeciesmayhave been found in MT2 strains or in males of all volvocine algae
matingtypeswheregametesaremorphologicallyidentical examined to date including VcMID in V. carteri (Figure S1A)
but can only mate with those expressing a different [20,21,27–30].However,theroleofMIDinsexdeterminationhas
mating type than their own, while multicellular species not beeninvestigated outside ofChlamydomonas.
suchasplantsandanimalshavemaleandfemalesexesor
Volvox carteri f. nagariensis (hereafter V. carteri) is a spheroidal
separate reproductive structures that produce sperm and
multicellularalgawhosevegetativeformisidenticalformalesand
eggs. The relationship between mating types and sexes
females (Figure 1A). Each vegetative spheroid contains ,2,000
and whether or how an ancestral mating-type system
sterile flagellated somatic cells on the periphery that provide
could have evolved into a sexually dimorphic system are
motility, while inside the spheroid are ,16 large immotile
unknown.Inthisstudyweinvestigatedsexdetermination
reproductive cells called gonidia. All of the cells are embedded
inthemulticellulargreenalgaVolvoxcarteri,aspecieswith
within a clear extracellular matrix that comprises most of the
geneticsexdetermination;weestablishedtherelationship
of V. carteri sexes to the mating types of its unicellular spheroid volume. The two-day vegetative reproductive cycle
relative, Chlamydomonas reinhardtii. Theoretical work has begins with mature gonidia undergoing embryogenesis to form
suggested that sexual dimorphism could be acquired by new miniature juvenile spheroids. During embryogenesis a pro-
linkage of gamete size-regulatory genes to an ancestral grammed series of symmetric and asymmetric cleavage divisions
mating-type locus. Instead, we found that a single occurs to produce a hollow ball of 2,000 small cells with 12–16
ancestral mating locus gene, MID, evolved from its role large cells on the anterior surface. The process of inversion then
in determining mating type in C. reinhardtii to determine turns the embryo inside out so that the large cells end up on the
either spermatogenesis or oogenesis in V. carteri. Our interior of the spheroid where they will differentiate into new
findings establish genetic and evolutionary continuity gonidia,andthesmallcellsenduporientedwiththeirbasalbodies
between the mating-type specification and sex determi- facing outward, and will begin to grow flagella as they undergo
nation pathwaysof unicellularand multicellular volvocine somaticdifferentiation.Overthenext1.5daysthejuvenilesgrow,
algae, and will enable a greater understanding of how a
mature intoadults, hatch,andbegin thecycleagain(reviewed in
transcriptional regulator, MID, acquired control over a
(Figure1B)[31,32]).UnlikeC.reinhardtiithatusesanutrienttrigger
complex developmental pathway.
forgametogenesis,sexualdifferentiationinV.carteriistriggeredby
adiffusibleglycoproteinhormonecalledsex-inducerthatisactive
Volvocine algae are an excellent model for investigating the on both sexes [33–35]. In response to sex-inducer, gonidia from
evolutionofsexualdimorphism.Theyformamonophyleticclade vegetative females and males undergo modified embryogenesis
encompassing a progression from unicellular species to multicel- programs to produce sexual spheroids (Figure 1C) [36,37].
lular forms with increasing organismal size and cell-type special- Sexually induced female spheroids have ,2,000 somatic cells
ization [12,13]. Volvocine algae all have a haploid vegetative similar to vegetative females, but inside contain 32–48 large egg
reproductivecycle,butunderspecificconditionscanbeinducedto cellsthatareformedduringembryogenesisthroughalteredtiming
undergo sexual differentiation and mating to form dormant of asymmetric cell divisions. Sexually induced male spheroids
diploid zygospores. Zygospores undergo meiosis and produce develop with 128 somatic cells and 128 large cells called
haploid progeny that reenter the vegetative phase [4,14]. androgonidia that are also produced through modification of
Chlamydomonas and smaller colonial volvocine genera are isoga- asymmetric embryonic division patterning. The day after male
mous,whilelargercolonialformsareanisogamousoroogamousas sexualembryogenesiseachandrogonidialcellundergoesaddition-
is the case with the genus Volvox [15,16]. Some species of Volvox al cleavage divisions to form a packet of 64 or 128 sperm cells.
and other anisogamous volvocine algae are heterothallic with Spermpacketshatchandswimtogethertoasexualfemalewhere
genetically determined male and female sexes, while others are they break apart into individual sperm that enter the female
homothallic with a single clone producing a mixture of all-male through a fertilization pore. Sperm swim within the female until
andall-femalecolonies(dioecy),orhomothallicwithasingleclone theyfindaneggandthenfusewithittoformadiploidzygospore.
producing colonies containing both male and female gametes Upon germination a single vegetative meiotic progeny is formed
(monoecy) (reviewed in [16]). Previous studies have made use of whiletheremainingthreemeioticproductsarediscardedaspolar
volvocine algae to evaluate theories relating to the evolution of bodies (Figure 1C)[38].
anisogamy and oogamy [13,17–19], but the genetic basis for Sexual differentiation in V. carteri is controlled by a dimorphic
sexual dimorphism inthisclade isstill unclear[4,20,21]. sex-determining locus (MT) with haplotypes designated MTM
In C. reinhardtii, the two genetically determined mating types, (male) and MTF (female). V. carteri MT occupies an equivalent
plus and minus, are morphologically similar, but express mating- chromosomal position to C. reinhardtii MT based on flanking
related genes that allow fusion with a partner of the opposite syntenicgenecontent,butisatleast5-foldlarger.Comparedwith
mating type [14,22]. Gametic differentiation in C. reinhardtii is C.reinhardtii MTV.carteri MT contains moresequence rearrange-
triggeredbyabsenceofnitrogen(2N)andisgovernedbyamating ments between haplotypes, more repeat sequences, and has
locus (MT) whose two haplotypes, MT+ and MT2, are large, gametologpairs(geneswithanalleleinbothMThaplotypes)that
rearranged multigenic regions, which are suppressed for recom- arefarmoredifferentiatedfromeachother[20,24].V.carteriMTF
binationandthereforesegregateasMendelianalleles[23,24].The and MTM haplotypes can thus be considered a UV sex
C. reinhardtii gene MID (CrMID) is present only in the MT2 chromosomepair[39].Asdescribedabove,ithasbeenproposed
haplotype and encodes a putative RWP-RK family transcription thatanisogamyoroogamycouldevolvethroughasize-regulatory
factor whose expression is induced by 2N and that governs gene becoming linked to an ancestral mating locus [11]. Both
gametic differentiation [25]. The presence of MID activates the MTMandMTFhaplotypescontainaputativecell-sizeregulatory
minus differentiation program and represses the plus program, gene,MAT3,whoseallelesarehighlydimorphicinsequenceand
while the absence of MID causes activation of the plus program expression between the sexes [20]. However, it is now apparent
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Figure1.V.carterivegetativeandsexualcycles.(A)ColorDICimageofvegetativeV.carterispheroidwithlargereproductivecells(gonidia)on
theinteriorandsomaticcellsontheexterior.Scalebar=50mm.(B)Keystagesofthetwo-dayvegetativereproductivecyclearedepictedwithrelative
timingindicatedbytheinteriorclockdiagramshowingthe16hlightand8hdarkphases.Startingfrom,6:00andgoingclockwisevegetative
gonidiaundergoembryoniccleavagefollowedbyinversiontomakenewjuvenilespheroids.Juvenilesgrowandeventuallyhatchandmatureinto
thenextgenerationofparentalspheroids.Thevegetativecycleisidenticalformalesandfemales.(C)Keystagesofthesexualcyclearedepictedtop
to bottomstarting with vegetative male or female gonidia that have been exposedto sex-inducer. Sexually induced gonidia undergomodified
embryogenesistoproducesexualmaleswith128largeandrogonidiaand128somaticcells,orsexualfemaleswith32–48eggs.Subsequentcleavage
ofandrogonidiaproducesspermpacketsthatarereleasedandswimtoafemalewhereupontheydissolveintosinglespermandenterthefemale
spheroid to fertilize eggs. Diploid zygotes differentiate into environmentally resistant, orange-pigmented, dormant zygospores that when
germinatedundergomeiosisandproducethreepolarbodiesplusasinglehaploidvegetativeprogenythatcanreenterthevegetativereproductive
cycle.
doi:10.1371/journal.pbio.1001904.g001
that anisogamy and oogamy in volvocine algae predate the (Figure2B).When wild-typevegetative malegonidiaareexposed
appearance of MAT3 allelic dimorphism in the lineage meaning tosexinducertheyundergomodifiedembryogenesisanddevelop
that other mating locus genes probably underlie the origins of with128spermpacketsand128somaticcells(Figure2C).When
anisogamy and oogamy [40]. Although MTM contains a MID vegetativegonidiafromEve::VcMID-BHlineswereexposedtosex
homolog, VcMID (Figure S1A), its role in sexual differentiation is inducer they developed into progeny spheroids with a novel
unclearbecauseVcMIDmRNAisexpressedconstitutivelyinboth pseudo-male sexual phenotype: They produced ,2,000 somatic
vegetative and sexual stages of males [20]. The apparent cellsand32–48sexualgerm-cellprecursorsinapatternsimilarto
uncoupling of VcMID expression from the sexual cycle suggests thatoffemaleeggs;buteachofthe32–48germ-cellprecursorsin
thattheVcMidproteinmighthaveafunctionoutsideofthesexual the Eve::VcMID-BH lines underwent additional cleavage divisions
cycleorthatitsfunctionmightberegulateddifferentlythanthatof like male androgonidia to produce sperm packets (Figure 2D).
CrMIDwhose expression isinduced by 2N. Moreover, the sperm produced in Eve::VcMID-BH lines were
In this study, we tested the role of VcMID in V. carteri sex capable of fertilizing wild-type female eggs to produce character-
determination by making transgenic females that express VcMid istically orange-pigmented, thick-walled zygotes (Figure 2E).
proteinorbyknockingdownitsexpressioninmalesusingRNAi. However, male function was incomplete as fertility defects were
We found that expression of VcMID in females is sufficient to noted (see next section). Similar to wild-type sperm (and unlike
convert eggs to sperm packets, while its absence in males causes wild-type female eggs) the Eve::VcMID-BH sperm cells were
androgonidial cells to differentiate into eggs. However, alteration terminally differentiated and could not revert back to vegetative
ofVcMidexpressiondidnotaffectfemaleormaleearlyembryonic growth if left unfertilized. Another male-specific phenotype
patterning during which the number and location of germ-cell exhibited by Eve::VcMID-BH lines was frequent spontaneous
precursors is established. We found that VcMID mRNA is occurrence of sexual differentiation in vegetative cultures [36], a
expressed in all cell types, but VcMid protein accumulation is traitwhoseunderlyingbasisisnotclear,butwhichappearstobe
underthecontrol of VcMID.
regulatedbycelltypeanditssubcellularlocalizationisrestrictedto
nuclei of differentiating and mature male gametes. Swapping
experiments with CrMid demonstrated that the VcMid DNA Fertility Defects in Females Expressing VcMid
bindingdomainandN-terminaldomainarebothrequiredforits Although some of the Eve::VcMID-BH sperm were functional
functionindirectingspermatogenesisinV.carteri.Crosseswithsex- and could fertilize wild-type eggs, the sperm packets and sperm
reversed strains revealed sexually antagonistic interactions be- cells from these strains had multiple defects including hetero-
tween genes in MT and the sexual development pathway chronic delays in maturation and hatching defects (Figure S3A).
controlledbyVcMidthatnegativelyimpactedreproductivefitness The sperm packets were four times larger than those from wild-
whengamete typedid not match thematinglocusgenotype. type males and contained about four times as many sperm cells
(256 sperm/packet) (Figure 2F and 2G), some of which were
Results aberrantlyformedincontrastwithwild-typespermcellsthathad
uniformmorphology(Figures2H,2I,andS3B–S3F).Nonetheless
VcMid Controls Spermatogenesis the crosses between Eve::VcMID-BH pseudo-males and wild-type
WetestedtheroleofVcMidproteininsexualdifferentiationby females produced MTF/MTF diploid zygotes (Figure 2E) that
generatingfemaletransgeniclineswithanautosomallyintegrated couldgerminateandproducehaploidprogeny.Forty-oneprogeny
VcMID transgene (pVcMID-BH) expressed under its own from one such cross were genotyped, half of which (19/41)
promoter and fused to a blue fluorescent protein (BFP) and a inherited the VcMID-BH transgene and developed as pseudo-
hemagglutinin (HA) epitope tag at its C-terminus to detect males, and half of which lacked the transgene and developed as
expression (Eve::VcMID-BH) (Figures 2A and S1B). Female normalfemales (22/41).
transformants carrying an untagged version of VcMID (Figure
S1C) had identical phenotypes as those carrying the tagged Expression and Localization of VcMid Are under Post-
version,andallsubsequentworkwasdonewithtaggedstrainsand transcriptional Control
untagged transformants as a negative control for detection of The absence of a vegetative phenotype in Eve::VcMID-BH
VcMidprotein.Eve::VcMID-BHlinesshowedanormalvegetative transgenic lines despite constitutive expression of the VcMID-BH
phenotypeandconstitutivelyexpressedthemRNAfortheVcMID- mRNA (Figure S2) suggested that VcMid protein expression or
BH transgene, an expression pattern identical to the endogenous localization might be under posttranscriptional control. Although
VcMID mRNA in males (Figure S2) [20]. As described above, we could not detect BFP fluorescence in Eve::VcMID-BH strains,
when wild-type vegetative female gonidia are exposed to sex we could detect the HA epitope tag by Western blotting at all
inducer they undergo modified embryogenesis and develop into stages ofvegetative andsexual development (Figures 3Aand S4).
sexualspheroidswith32–48eggsand,2,000sexualsomaticcells Using immunofluorescence (IF) a nuclear-localized signal was
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Figure2.EctopicExpressionofVcMIDconvertseggprecursorstospermpackets.(A)DiagramofVcMID-BHexpressionconstruct.Darkblue
boxesdepictVcMIDexons;blacklinesdepictintronsandintergenicregions;lightblueboxdepictsBFP;orangeboxdepictsHAepitope.Start(ATG)
andstop(TAA)codonpositionsareshownalongwithscalebarinblack.(B)Wild-typematuresexualEve(female)spheroidwith,32largegreen
eggs.(C)Wild-typematuresexualAichiM(male)spheroidwith,128spermpackets.(D)MaturesexualEve::VcMID-BHpseudo-malewith,32sperm
packets,twoofwhicharemagnifiedinexpandedboxtotheright.Scalebarsfor(B–D)=50mm.(E)MaturezygotefromEve::VcMID-BH6Eve.(F)Sperm
packetfromwild-typeAichiM.(G)Spermpacketfrompseudo-maleEve::VcMID-BH.Scalebarsfor(E–G)=25mm.(H)Spermcellfromwild-typemale
withasingleeyespotindicatedbyablackarrowhead.(I)AberrantspermcellfromEve::VcMID-BHpseudo-malewithtwoeyespotsindicatedbyblack
arrowheads.Scalebarsfor(H)and(I)=5mm.
doi:10.1371/journal.pbio.1001904.g002
detected for VcMid-BH protein in cleaving androgonidia and in that restricts its accumulation to somatic cells during vegetative
maturespermpackets(Figures3B–3J,S5A–S5R,andS6C–S6H), growth (Figures 3K and S6B). However, unlike the case for
a result similar to earlier findings of Mid protein localization in androgonidia and sperm cells, the VcMid-BH protein signal in
spermnucleiofPleodorina[21].However,nuclearVcMid-BHwas vegetative somatic cells was excluded from the nucleus and was
not detected during early stages of sexual male embryogenesis instead observed only in the cytosol and peri-nuclear region
prior toandrogonidia cleavage (Figure S7). (Figures 3L–3O, S6I–S6P, and S9C–S9J). Together these data
We also examined VcMid-BH expression and localization in suggest that cell-type–limited expression and regulated nuclear
gonidia and somatic cells from vegetative spheroids. RNA was localization of VcMid restrict its function to developing and
prepared from purified gonidial or somatic cells and reverse mature sexual male germcells inV. carteri.
transcription and PCR (RT-PCR) detected VcMID-BH mRNA
at similar levels in both cell types from transgenic females VcMid Is Required for Spermatogenesis and Represses
(Eve::VcMID-BH) and males (AichM::VcMID-BH) (Figure S8B and Oogenesis in Males
S8D).TheendogenousVcMIDtranscriptfromwild-typemaleswas In order to test whether VcMID is necessary for sexual
alsoexpressedinbothvegetativecelltypes(FigureS8CandS8D). differentiation of males we developed a new strategy for gene
Whole cell extracts were prepared from purified Eve::VcMID-BH knockdownonthebasisofRNAi-inducinghairpinconstructs.The
or AichM::VcMID-BH somatic cells and gonidia and subjected to hairpin-formingportionoftheconstructcorrespondingtoVcMID
SDS-PAGE and Western blotting to detect VcMid-BH protein sequences was inserted directly into the 39 UTR of the nitA
(Figures3K,S9A,andS9B).IncontrasttoVcMID-BHmRNAthat selectable markergene toallow direct andsimultaneousselection
was present in both vegetative cell types, VcMid-BH protein was of both NitA+and hairpin expression (Figures4A andS10;Text
only detected in vegetative somatic cells indicating that there is S1).ThisstrategyhasbeensuccessfulforotherlocibesidesVcMID
cell-type–specificregulationofVcMidproteinsynthesisorstability (unpublished data), but only the results for VcMID are presented
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Figure 3. Cell-type restricted expression and sex-regulated nuclear localization of VcMid. (A) Immunoblot of SDS-PAGE fractionated
proteinextractsfromHA-taggedpseudo-malestrainEve::VcMID-BH(lanes1–9),untaggedpseudo-malestrainEve::VcMid(lanes10,11),andwild-type
malestrainAichiM(lanes12,13).Lanes1–3containextractsfromvegetativespheroidsatadultstage,mid-cleavagestage,andunhatchedjuvenile
stage respectively. Lanes 4–9 contain extracts from spheroids undergoing sexual development with pre-cleavage stage, mid-cleavage stage,
unhatchedjuvenilestage,cleavingandrogonidiastage,andmaturesexualadultstage,respectively.SeealsoFigureS2BandS2C.Lanes10and12
containextractsfromadultvegetative-stagespheroids.Lanes11and13containextractsfrommaturesexual-stagespheroids.Thebandsintheupper
panelareVcMid-BHproteindetectedwithananti-HAantibody.Thebandsinthelowerpanelcomefromthesameblotre-probedwithananti-
tubulinantibodyasaloadingcontrol.(B–J)DIC(B,E,H)orfalse-coloredIFimagesofcleavingandrogonidiafromEve::VcMID-BHsexualgermcellsat
thetwo-cellstage(B–D),sixteen-cell(E–G)stage,andfromamaturespermpacket(H–J).IFsampleswerestainedwithDAPIshowninblue(C,F,I)or
withanti-HAshowningreen(D,G,J).Arrowsandarrowheadsshowlocationsofarepresentativenucleusfromeachimage.Scalebars=10mm.(K)
Upperpanel,anti-HAimmunoblotofSDS-PAGEfractionatedproteinextractsofpurifiedvegetativegonidia(G)orsomatic(S)cellsfromEve::VcMID-BH
and Eve::VcMID spheroids. Lower panel, Coomassie-stained gel used as a loading control. (L–O) IF detection of VcMid-BH protein from a
representativevegetativesomaticcellofEve::VcMID-BHstainedasin(B–J).Thearrowsin(M,O)showthenucleus,whilethechloroplastnucleoids
(smallerDAPI-stainedspots)areunlabeled.TheVcMid-BHsignalin(N,O)isexcludedfromthenucleusasevidentinthemergedimage(O).Scale
bar=7.5mm.
doi:10.1371/journal.pbio.1001904.g003
here. We also note that the VcMID knockdown phenotype was MaterialsandMethodsfordetails).Alltransgenicmalestrainshad
genespecificanddidnotoccurwhenhairpinstargetingotherloci normal vegetative phenotypes, and both hairpin constructs
were introduced into V. carteri (unpublished data). Two hairpins reduced VcMID expression; but AichiM::VcMID-hp1 lines had
targeting VcMID (VcMID-hp1 and VcMID-hp2) were introduced lower VcMID transcript levels than AichiM::VcMID-hp2 lines
into a V. carteri wild-type male strain AichiM to generate (Figure S2). We note that unlike wild-type males or pseudomales
AichiM::VcMID-hp1 and AichiM::VcMID-hp2 transgenic lines (see (seeabove),vegetativeculturesofAichiM::VcMID-hp1linesdidnot
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Figure4.ConversionofmalegermcellstoeggsbyknockdownofVcMID.(A)DiagramofVcMIDhairpinconstruct.Greenboxes,NitAexons;
medium green line, intron; thick black lines, UTRs; VcMID hairpin structure inserted in 39 UTR is shown in its approximate location. (B) Sexually
inducedAichiM::VcMID-hp1pseudo-femalewitheggsinplaceofspermpackets.Blackarrowheadshowsanegg.(C)AichiM::VcMID-hp1pseudo-female
fertilizedbywild-typemalesperm.Blackarrowheadshowsanormal-appearingdevelopedzygote;whitearrowheadshowsanabortedzygote.(D)
HermaphroditephenotypeofmaturesexuallyinducedAichiM::VcMID-hp2spheroidwitheggsandspermpackets.Blackarrowheadshowsegg;boxed
regionhasthreespermpacketsthataremagnifiedintheexpandedviewtotheright.Scalebarsfor(B–D)=50mm.(E)Zygoteproducedbyself-
fertilizationofAichiM::VcMID-hp2spheroid.Scalebar=10mm.
doi:10.1371/journal.pbio.1001904.g004
undergo spontaneous sexual induction. Sexually induced Ai- Partial VcMID Knockdown Generates Self-Fertile
chiM::VcMID-hp1 lines with strong knockdowns showed a novel Hermaphrodites
phenotype:Theirearlysexualdevelopmentproceededasitwould
AichiM::VcMID-hp2lineswerenotasseverelyknockeddownfor
forawild-typemalestrainandresultedinspheroidsthatcontained
VcMIDexpressionasAichiM::VcMID-hp1lines(FigureS2),andhad
128 small somatic cells and 128 large cells that resembled
a distinct hermaphrodite phenotype in which sexual spheroids
uncleaved androgonidia (Figure 4B), but the large cells never
developed with a mixture of normal-looking male sperm packets
underwent further cleavage into sperm packets. Instead, many of
and pseudo-female eggs (Figure 4D). The hermaphrodite lines
themcouldbesuccessfullyfertilizedwithwild-typemalespermto
exhibited self-fertility as evidenced by zygospores that formed in
makeMTM/MTMdiploidzygospores(Figure4C).Theabilityto
sexuallyinducedmonocultures(Figure4E).Theseresultsindicate
differentiate as zygospores when fertilized indicates that the
thatV.carterisexdeterminationishighlysensitivetoVcMiddosage
presumptive androgonidia in AichiM::VcMID-hp1 strains were
whereeitheramaleorfemalefateisestablisheddependingonthe
convertedtofunctionaleggsandthatthesestrainswerebehaving
levelof VcMid.
aspseudo-females.However,unlikenormalzygotesfromawild-
type cross, 30%–50% of the zygotes from AichiM6AichiM::Vc-
Volvox and Chlamydomonas Mid Proteins Are
MID-hp1 pseudo-female crosses died and bleached shortly after
fertilization(Figure4C),aphenotypethat dependedon addition Functionally Distinct
of exogenous sperm. The surviving zygotes from these crosses InseveralinstancesgenesfromC.reinhardtiihavebeenshownto
produced some viable meiotic progeny, but germination and function interchangeably with their V. carteri orthologs in
survival of the progeny were reduced compared with normal developmental processes such as inversion and asymmetric cell
wild-typezygotes(TableS1).29/43viableprogenyinheritedthe division (reviewed in [42,43]). Mid proteins have at least two
VcMID-hp1 transgene and developed as pseudo-females, while domains:TheC-terminalregionhasapredictedRWP-RKmotif
14/43lackedthetransgeneanddevelopedasnormalmales.The DNAbindingdomain,whiletheN-terminalregiondoesnotshow
apparent deviation from a 1:1 inheritance pattern of the similarity tocharacterized proteindomains fromother organisms
transgene was noted but was not pursued further in this study. (Figure S1) [28]. We tested whether either of the domains from
The high mortality of pseudo-female eggs—whose mating loci CrMid could substitute for those of VcMid to control spermato-
are genetically male—suggest that MTF contains genes that genesis in V. carteri. To do so we generated three constructs in
promote female gamete and/or zygote fitness that are absent which all or part of the CrMID genomic coding region was
fromMTM.Ifleftunfertilized,theeggsfromAichiM::VcMID-hp1 substituted for VcMID sequences in pVcMID-BH. One construct
lines could de-differentiate and reenter the vegetative reproduc- containedtheentireCrMIDgene(pCrMID-BH)(Figure5A)while
tive cycle as do unfertilized female eggs. A similar phenotype as the other two contained the CrMID N-terminal domain fused to
our pseudo-male strain was reported previously for a male theVcMIDDNAbindingdomain(pMID-V C -BH)(Figure5B),
N C
mutant [41], but the mutant strain is no longer available for or the VcMID N-terminal domain fused to the CrMID DNA
characterization. binding domain (pMID-C V -BH) (Figure 5C). All three
N C
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SexesEvolvedfromAncestralMating-TypeSpecificationPathway
Figure5.ChlamydomonasMIDcannotsubstituteforVolvoxMID.(A–C)DiagramsofconstructspCrMID-BH(A),pMid-V C -BH(B),andpMid-
N C
C V -BH(C).DarkblueboxesdepictVcMIDexons;blacklinesdepictVcMIDintronsandintergenicregions;greenboxesdepictCrMIDexons;brown
N C
linesdepictCrMIDintrons;lightblueboxesdepictBFP;OrangeboxdepictsHAepitope.Start(ATG)andstop(TAA)codonsareshownalongwithscale
barinblack.(D–F)ImmunoblotsofSDS-PAGEfractionatedproteinextractsfromEvetransformantsorcontrolstrainsprobedwithanti-HAantibodies
(upperpanels)orre-probedwithtubulinantibodies(lowerpanels).Numberedlanesindicateindependenttransformants,someofwhichexpressthe
taggedMidconstructs.Controlstrainsarewild-typemaleAichiM,EvetransformedwithuntaggedVcMID,andEvetransformedwithtaggedVcMID-BH
showninFigure1A.(D)Eve::Mid-CrMID-BHtransformants.ArrowheadindicatespredictedfulllengthCrMid-BHandasterisksrepresentbreakdownor
alternativeprocessing products.(E) Eve::Mid-V C -BHtransformants. (F) Eve::Mid-C V -BHtransformants. (G)ColorDIC imageof sexuallyinduced
N C N C
Eve::Mid-CNVC-BHtransformantnumber6showingtypicalarrangementofeggssimilartoawild-typefemale.Scalebar=200mm.
doi:10.1371/journal.pbio.1001904.g005
constructsaswellaspVcMID-BHwereintroducedintowild-type Discussion
females, and transformants that expressed the different predicted
proteins were identified (Figures 5D–5F). Unlike Eve::VcMID-BH Deep Homology between Mating Types and Sexes
transformantsthatshowedapseudo-malephenotype(Figure1D), Although it was reasonable to predict that the route for
noneofthetransformantsthatexpressedCrMidorMidchimeras evolving sexual dimorphism would be through addition of new
had this phenotype, but instead always developed as wild-type genetic functions and pathways to a core mating locus as
females (Figure 5G; Table S2). The CrMid-BH protein was proposed originally by Charlesworth for the evolution of
expressed as a full-length form and two shorter isoforms anisogamy [11], we found instead that a single conserved
(Figure 5D), possibly due to proteolytic cleavage or incorrect volvocinealgalmatinglocusgene,MID,islargelyresponsiblefor
pre-mRNA processing. However, lines with either of the two controllingmaleversusfemalesexualdifferentiationinV.carteri.
chimericconstructsexpressedonlyfull-lengthpredictedproteinsat ItisnotablethatVcMIDcontrolsmultiplesexualtraitsinV.carteri
levelscomparabletoVcMid-BH(Figure5Eand5F).Weconclude that have no analogs in C. reinhardtii. The male traits controlled
from these experiments that CrMid and VcMid are not byVcMIDincludespecializedcleavagedivisionsofandrogonidia,
functionally interchangeable, and that both the N-terminal and sperm packet inversion, sperm packet hatching, specialized
RWP-RK domains of VcMid are required to activate spermato- spermcellmorphology,andgameterecognitionwithoutflagellar
genesisin sexualgerm cells. adhesion.
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SexesEvolvedfromAncestralMating-TypeSpecificationPathway
The term deep homology is used to describe ancient and Evolution of Mid and Sexual Cycles in the Volvocine
conserved genetic mechanisms that control traits that, on the Lineage
surface, appear disparate or have no obvious homology relation- As described in the Introduction, volvocine algae exhibit wide
ship[44].Metazoaneyedevelopmentisaclassicexample;across diversityintheirsexualcycles:Thereareisogamous,anisogamous,
phyla with very different eye architecture, it is controlled by the andoogamousspecieswithsexualcyclesthatcanbeheterothallic
conserved transcription factor, eyeless/Pax6 [45]. In the case of or homothallic. Among homothallic species some are dioecious
volvocine algae Mid proteins control two very different manifes- (producingamixtureofallmaleandallfemalesexualoffspring)or
tations of sexual reproduction in C. reinhardtii and V. carteri whose monoecious (producing sexual offspring containing gametes of
MIDorthologshavebeendivergingforaslongas200millionyears bothsexes inoneindividual) [16,55].
[46]. Sexually dimorphic gametes have evolved from isogamous V. carteri is a heterothallic species with genetically determined
ancestorsseveraltimesinindependentmulticellulartaxa,andthe
sexes; but, a remarkable phenotype was produced by a partial
mechanismcouldbesimilarto,ordifferentfrom,thatinvolvocine RNAi knockdown of VcMID in males (Figure 4D). Rather than
algaewhereamasterregulatorygeneacquiredtheabilitytodirect developing as male or female, the partial-knockdown male
sperm-eggdimorphism.Oogamywaslikelyestablishedveryearly
spheroids became self-fertile monoecious hermaphrodites that
in the Streptophyte lineage before the split between Charophyte produced sperm and eggs in a single individual. On the basis of
algae and land plants, but the origins and bases of oogamy in thisobservation,itcanbeinferredthatsexualdifferentiationinV.
Charophytesremainunclear[47].Therecentidentificationofan carteriisbi-stableandhighlysensitivetoinitialMIDdosage.Once
anisogamous sexual cycle in a Choanoflagellate—a unicellular
the Mid-sensitive step of development is initiated (which may
relative of metazoans—introduces the potential for investigating coincide with nuclear translocation of VcMid protein), positive
theearly evolution ofgamete sizedimorphism inanimals [48]. and negative feedback loops may be used to lock the sex
determination program into a male or female state. This state
MID Is a Master Regulator of Sex Determination in could be achieved either independently of VcMid concentration,
Volvocine Algae or through positive/negative reinforcement of initial expression
Our results demonstrate for the first time a function for Mid states. The phenotype of hermaphroditic development by partial
protein in a volvocine algal sexual cycle outside of the genus VcMID knockdown may be relevant to the evolution of homo-
ChlamydomonasandsuggestthatMidcouldbethemasterregulator thallism,whichappearstohaveariseninallthreemajorcladesof
ofmatingtype,gametesize,andgenderthroughoutthevolvocine Volvox[16,55,59].Wespeculatethatnaturallyevolvedhomothallic
lineage where MID genes have been identified in most genera volvocinealgaepossessaMIDgenewhoseexpressionisinsufficient
[21,28,30]. Although MID sequences were previously shown to tospecify100%malegameteproduction—muchlikeourVcMID-
evolve rapidly, the finding that Mid protein from C. incerta (now hp2strains.Moreover,thetimingofMIDexpressioninhomothallic
reclassified as C. globosa [49]) can substitute for C. reinhardtii Mid speciesofVolvoxcouldplayaroleindeterminingmonoeciousversus
indicates that functional conservation can be retained after dioecious reproductive development:Ifthe Mid-sensitivedevelop-
speciation [27]. However, C. reinhardtii Mid protein could not mentalswitchistriggeredrelativelylateindevelopmentaftergerm-
substitutefortheV.carteriortholog,whichappearstorequireboth cell precursors are formed then a mixture of male and female
itsnativeDNAbindingandN-terminaldomainstofunctioninsex gametes could develop within a single spheroid as we found with
determination(Figure5).Futureworkusingcross-speciescomple- VcMID-hp2strains(i.e.,homothallism,monoecy).Incontrast,ifthe
mentation will help clarify whether Mid protein function co- Mid-sensitive step occurred very early in development before
evolved with sexual dimorphism in volvocine algae as our data individualgermcellswereestablished,thenthefateofallgermcells
suggest might bethecase. within a mature spheroid might be locked into a male or female
Transcriptional regulatory network evolution has been studied programandtheresultingpopulationwouldproduceamixtureof
invariousdevelopmentalcontexts[50–53],butverylittleisknown all-femaleorall-malespheroids(i.e.,homothallism,dioecy).
about how regulatory networks are modified or coopted during The bi-stability of sex determination at intermediate levels of
unicellular to multicellular transitions [54]. The Mid system in MID expression in V. carteri isalso reminiscent of the iso1 mutant
volvocine algae represents a new opportunity for understanding phenotypeinC.reinhardtiiwhereMT2iso1cellsdifferentiateintoa
how a cell-type specification pathway in a unicellular ancestor mixture of plus and minus gametes that iso-agglutinate but cannot
evolved to control a complex developmental program in a self-fertilize [60].Theself-infertility ofiso1MT2strainsisdue to
multicellular descendant. While mating-type differentiation in the absence of FUS1, a gene from the MT+ haplotype that is
Chlamydomonasappearstoinvolvedifferentialexpressionofasmall requiredforgametefusion[61].Incontrast,therearenoessential
number of genes between the plus and minus gametes [14,22], a genes in MTF of V. carteri that are absolutely required for
larger set of differentially expressed genes might be expected to fertilizationandsubsequentgerminationofprogenyfrommatings
specify spermand eggs,whichare developmentally verydifferent between pseudo-females (VcMID knockdown strains) and males.
from each other [37,55]. An important future goal will be to This lack of essential female MT genes for completing the sexual
identify and compare the direct and indirect targets of Mid cycle may have facilitated transitions from heterothallism to
proteinsinbothC.reinhardtiiandV.carterithatarepredictedtobe homothallism involvocine algae.
more numerous anddiverse inV.carteri.
AnotherinterestingquestioniswhetherMID-likegenesfunction V. carteri Has Evolved New Regulatory Inputs for Mid
in sex determination in green algae outside of the volvocine In volvocine genera other than Volvox—including the anisoga-
lineage.Themolecularbasesforsexdeterminationinmostgreen mousgenusPleodorina—sexualdifferentiationandMIDexpression
algae and protists are poorly understood, but RWP-RK family arebothtriggeredbytheabsenceofnitrogen(2N)[21,25,28,62].
proteins are found throughout the green eukaryotic lineage [56], In contrast, V. carteri f. nagariensis and other Volvox species use
including smallMid-likeproteinsinPrasinophyte algae [57],and species-specific pheromones called sex inducers to trigger sexual
evenindistantlyrelatedCryptophytealgae[58].Itremainstobe differentiation[16,55,63].Aseeminglyparsimoniousevolutionary
seen whether these Mid-like proteins in non-volvocine species route for rewiring input into the Mid pathway in V. carteri would
functionin sexdetermination. simplyplaceVcMIDtranscriptionunderthecontrolofsexinducer
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SexesEvolvedfromAncestralMating-TypeSpecificationPathway
instead of nitrogen availability. Unexpectedly, however, VcMID sex, but harm the other [39,65], and should accumulate repeat
mRNAandVcMidproteinarebothexpressedconstitutivelyatall sequences, as appears to have occurred in both Volvox and in the
lifecyclestages(Figures3AandS4)[20],andunlikethecaseinC. bryophyteMarchantia[20,66].Accumulationofsexuallyantagonistic
reinhardtii, VcMid appears to be under at least three types of alleles has not been tested for haploid sex chromosomes, but the
posttranscriptional control:(i)AlthoughVcMIDmRNA ispresent extensivedivergencebetweenV.carteriMTgametologssuggeststhat
in both vegetative cell types (somatic and gonidial cells), VcMid thisphenomenonmaycontributetothedevelopmentalandfitness
proteinisonlytranslatedorstablyproducedinsomaticcellsandis defectsweobservedinpseudo-maleandpseudo-femalestrains.
absentfromvegetativegonidiaandvegetativeembryos(Figures3K Although none of the sex-limited MT genes besides VcMID
and S6B). (ii) The VcMid protein produced in somatic cells is appear to be essential for V. carteri sex determination and
excluded from the nucleus (Figures 3L–3O, S6I–S6P, and S9C– completion of the sexual cycle, they clearly impact sexual
S9J).(iii)Inresponsetothepresenceofsexinducer,VcMidprotein development and reproductive fitness. A striking phenotype for
accumulatesinthenucleiofcleavingandrogonidialcellsandinthe both the pseudo-male and pseudo-female strains was the
nucleiofspermcellswhereitispresumedtofunctioninspecifying patterning of their germ-cell precursors formed during sexual
sperm development (Figure 3B–3J,S5,and S6C–S6H). development (Figures 2B–2D and 6A–6C). It is clear from these
In depth study will be required to determine how cell-type– phenotypesthatsexualgermcellpatterning(i.e.,thenumberand
regulatedproductionandlocalizationofVcMidareachieved,but distribution of germ-cell precursor cells and ratio of germ-cell
itseemsreasonabletoinferthatoneormorefactorsareproduced precursors to sexual somatic cells) is separable from germ cell
insexuallyinducedspheroidsthatpromotethetranslationand/or differentiation and is not controlled by the VcMid pathway.
stabilityofVcMidanditsnuclearlocalizationinandrogonidiaand Instead, this sexual patterning trait must be controlled by other
sperm. The factor mayinteract directly withVcMidas a partner MTgenes(Figure6).Onecandidateforthismale-femalepatterning
andmayhelpspecifyitslocalizationatpromotersoftargetgenes, difference is the MAT3 gene that encodes the retinoblastoma-
butthisidearemainstobetested.TheabsenceofVcMidprotein relatedhomologinV.carteri[20,67].InChlamydomonas,Mat3protein
invegetativegonidiadespiteitsmessageaccumulatingtothesame controlsthemultiplefissioncellcyclebyestablishingthethreshold
extentasinsomaticcellsseemspuzzlingatfirstglance.However, sizeatwhichdivisioncanoccurandbycouplingtheextentofcell
theblockinstabilityortranslationofVcMidingonidiamayhave divisiontomothercell-size[68,69].Althoughnotdirectlyinvolved
evolved as a failsafe mechanism to prevent sexual differentiation indetermininggametecell-sizeaswehadoriginallypredicted[40],
during vegetative embryogenesis. Such a mechanism would be itispossiblethatthemaleandfemaleallelesofVcMAT3dictatethe
unnecessaryinvegetativesomaticcellsthatarealreadyterminally timing of asymmetric cell divisions by coupling embryonic
differentiated, but could potentially be important for vegetative blastomere cell-size to the asymmetric division machinery. Future
gonidialcellsbecausemalesexualdifferentiationisirreversibleand workwillbeaimedtowardsdeterminingtheroleofmaleandfemale
would be fatal if it occurred at the wrong time. However, why VcMat3gametologsinsexualcelldivisionorotherpartsoftheV.
vegetative somatic cells express VcMid remains a mystery. As carterisexualcycle.
noted in Results, we observed no obvious vegetative phase In addition to defects in germ cell patterning in Volvox pseudo-
phenotypes in AichiM::VcMID-hp strains whose somatic cells were
males and pseudo-females, these strains show other reproductive
missing VcMid, or in Eve::VcMID-BH strains that contained
defects.Theseincludeabnormalspermcellshapeandmorphology
VcMidinsomaticcells,butadefinitiveconclusionaboutwhether
(Figures 2H, 2I, and S3B–S3F), low efficiency of sperm packet
cytoplasmic VcMid has a role in vegetative somatic cells awaits
hatching (Figure S3D), and delayed timing of androgonidial
more indepth examination.
cleavage into sperm packets (Figure S3A). In depth study may
reveal other defects in pseudo-male sperm related to cytoskeletal
Uncoupling of Gender from Sex Chromosome Identity organization,gameterecognition,motility,andfertilizationdynam-
Uncovers Possible Sexually Antagonistic Interactions in ics. Although egg cells lack distinct morphological features like
the MT Locus sperm,wenotedaveryhighmortalityrateinpseudo-femaleeggs
Our resultsshowthat thepresenceorabsence ofVcMIDisthe that occurred when we attempted fertilization. The mortality we
keydeterminantofdifferentiationinV.carterisexualspheroids;yet observedundertheseconditionscouldbeduetopre-zygoticdefects
theMTlocusofthisalgahasaround70additionalgenes,manyof intheeggscausedbytheirsmallersizethanwild-typefemaleeggsor
which show sex-biased gene expression [20]. Some of the MT byamismatchbetweenthemalematinglocusandthefemalesexual
genes are present only in the male or only in female haplotype, differentiationpathway.Themortalitymightalsobeduetozygotic
while the majority are male and female gametolog pairs that are defectsthatoccurwhenacopyofthefemalematinglocusisabsent
highly diverged in sequence and expression pattern [20]. Our fromthezygoteimmediatelyafterfertilization.Futureworkaimed
ability to uncouple gender from sex chromosome identity by atdevelopingquantitativeassaysfordistinctstepsoffertilizationwill
manipulationofVcMIDexpressionallowedustouncoverpotential allowustodocumentinmoredetailthefitnesscontributionsofmale
contributionsofmaleandfemaleMTgenestosexualdimorphism andfemaleMTgenestothesexualcycle.
and reproductive fitness. Haploid sex chromosome systems have
received less attention than diploid systems, but there are several Materials and Methods
predictions about them that our results begin to address. Under
haploid dioecy, recessive mutations in sex-linked genes are not Detailed Materials and Methods are provided in Text S1.
sheltered from selection as they are in the heterogametic sex of Materials used in this study will be made available upon request
diploidsystems,andarethereforeexpectedtodegenerateequally with the completion of a Materials Transfer Agreement from
andloseonlygenesrequiredfortheoppositesexandnottheirown Donald DanforthPlant ScienceCenter.
[64].Wenote,however,thatinV.carteriMTthereisnoevidence
foranalleleofagametologpairhavingbeeneliminatedfromone Volvox Strains and Culture Conditions
mating haplotype and retained in the other [20]. Haploid sex Eve (Volvox carteri. f. nagariensis UTEX 1885) and AichiM (Volvox
chromosomesarepredictedtobesimilartodiploidsystemsinthat carteri. f. nagariensis NIES 398) were obtained from stock centers
bothshouldaccumulatesexuallyantagonisticallelesthatbenefitone http://web.biosci.utexas.edu/utex/ and http://mcc.nies.go.jp/,
PLOSBiology | www.plosbiology.org 10 July2014 | Volume 12 | Issue 7 | e1001904