GENDER VARIATION AND TRANSITIONS BETWEEN SEXUAL SYSTEMS IN
MERCURIALIS ANNUA (EUPHORBIACEAE)
John R. Pannell,1Ma cel E. Do ken, Benoi Pujol, and Regina Be jano2
Depa men o Plan Sciences, Uni e si y o Ox o d, Sou h Pa ks Road, Ox o d OX1 3RB, Uni ed Kingdom
E olu iona y ansi ions be ween he maph odi ism and dioecy ha e occu ed nume ous imes in he land
plan s. We b ie ly e iew he ac o s hough o be esponsible o hese ansi ions, and we p o ide a syn hesis o
wha has been lea ned om ecen s udies o he annual he b Me cu ialis annua, in which dioecy (males and
emales), monoecy ( unc ional he maph odi es), and and odioecy (males and he maph odi es) occu in di e en
pa s o i s geog aphic ange. P e ious esea ch on M. annua has e ealed he impo ance o genome duplica ion
and hyb idiza ion in he o igin o much o he obse ed a ia ion. He e we show, howe e , ha spa ial ansi ions
in he sexual sys em also occu wi hin he same ploidy le el. In pa icula , we p esen an analysis, using low
cy ome y da a, o ploidy a ia ion ac oss a p e iously uns udied ansi ion be ween he maph odi ism and
and odioecy, in which we ind ha he sexual-sys em ansi ion is uncoupled om he shi in ploidy le els. We
e iew ecen esea ch ha shows ha such ansi ions be ween sexual sys ems in M. annua a e consis en wi h
di e en ial selec ion a he egional le el o ep oduc i e assu ance du ing coloniza ion. We also p esen new
expe imen al da a ha highligh bo h he impo ance o he esou ce s a us o plan s and ha o hei local ma ing
con ex in egula ing gende s a egies and sex a ios. The s udies e iewed and he new esul s p esen ed
emphasize he ole ha shi s in he ecological and gene ic con ex o plan popula ions may play in causing
ansi ions be ween sexual sys ems.
Keywo ds: and odioecy, dioecy, gynodioecy, he maph odi ism, ma ing sys em, monoecy.
In oduc ion
By a , he majo i y o lowe ing plan s a e he maph odi ic
(Yampolsky and Yampolsky 1922; Sakai and Welle 1999),
bu ansi ions be ween he maph odi ism and dioecy ha e
been equen (Renne and Rickle s 1995; Weiblen e al.
2000; Vamosi e al. 2003; Case e al. 2008). Mos esea ch has
been di ec ed owa d unde s anding shi s om he maph o-
di ism owa d dioecy, wi h a ange o hypo hesized e olu-
iona y pa hs in oked ( e iewed by Cha leswo h [1999];
Webb [1999]). Indeed, Da win (1877) se he s age o much
o his wo k by no ing he subs an ial ad an ages o he -
maph odi ism and asking why ‘‘he maph odi e plan s should
e e ha e been ende ed dioecious’’ (p. 279). In con as , we
migh simila ly ask why males and emales, which may enjoy
ad an ages o gende specializa ion, should e e be eplaced
by he maph odi es. Al hough p obably less equen han
ansi ions om he maph odi ism o dioecy, he b eakdown
o dioecy owa d he maph odi ism is known o ha e oc-
cu ed in bo h plan s and animals (Des eux e al. 1996; Wol
e al. 2001; Kion ke e al. 2004; Weeks e al. 2006), and a
g owing body o ideas exis s on when such shi s migh occu
(Cha no e al. 1976; Cha no 1982; Mau ice and Fleming
1995; Wol and Takebayashi 2004). Ne e heless, he empi i-
cal ounda ion o unde s anding ansi ions om dioecy o
he maph odi ism emains weak.
F om a heo e ical poin o iew, he b eakdown o dioecy
equi es he in asion and sp ead in a popula ion o ei he
pollen-p oducing emales o seed-p oducing males. Ini ially,
he sp ead o he maph odi es in a dioecious popula ions will
yield ‘‘ ioecy,’’ a sexual sys em in which all h ee gende classes
a e main ained oge he , bu he condi ions unde which such
a gende imo phism can be main ained e olu iona ily ap-
pea o be a he limi ed (Mau ice and Fleming 1995; Wol
and Takebayashi 2004). Mo e likely is he apid displace-
men o ei he he emales o he males om he popula ion,
wi h he in ading he maph odi es main ained in an and odioe-
cious o a gynodioecious popula ion, espec i ely. Al hough
gynodioecy is unde s ood o be an impo an s ep in he e o-
lu ion o dioecy om he maph odi ism, i is no known o
ha e played a majo ole in he b eakdown o dioecy. In con-
as , while and odioecy is exceedingly a e in absolu e e ms
(Da win 1877; Cha leswo h 1984), almos all he known
and odioecious species, bo h in animals and in plan s, appea
o ha e e ol ed om a dioecious a he han a he maph o-
di ic ances o (Pannell 2002; Weeks e al. 2006).
T ansi ions om dioecy o he maph odi ism by way o an-
d odioecy ha e been s udied in only wo plan species in
much de ail. In he wind-pollina ed No h Ame ican pe en-
nial he b Da isca glome a a, he maph odi es appea o be
modi ied (pollen-p oducing) emales wi h he same sex de e -
mina ion as emales in i s dioecious sis e species Da isca
cannabina (Wol e al. 2001). In he Eu opean he b Me cu i-
alis annua ( ig. 1), which is also wind-pollina ed, and odioecy
1Au ho o co espondence; e-mail: john.pannell@plan s.ox.ac.uk.
2Cu en add ess: Depa amen o de Biologı
´a Vege al y Ecologı
´a,
Uni e sidad de Se illa, Apa ado 1095, 41080 Se illa, Spain.
Manusc ip ecei ed Feb ua y 2007; e ised manusc ip ecei ed July 2007.
129
In . J. Plan Sci. 169(1):129–139. 2008.
Ó2008 by The Uni e si y o Chicago. All igh s ese ed.
1058-5893/2008/16901-0011$15.00 DOI: 10.1086/523360
is ound only in polyploid popula ions o a complex in which
dioecy is clea ly he ances al ai (Du and 1963; Du and
and Du and 1992; Pannell 1997d; Pannell e al. 2004). Me -
cu ialis annua is unusual in he ex ao dina y di e si y o
sexual sys ems i displays ac oss i s geog aphic ange; his
a ia ion makes i a use ul s udy sys em in which o add ess
ques ions conce ning e olu iona y ansi ions be ween sexual
sys ems and he o igins and main enance o and odioecy.
In his a icle, we p esen new da a ha ad ance ou unde -
s anding o ansi ions in he sexual sys em o M. annua in
wo ways. Fi s , we ask how closely he spa ial ansi ion be-
ween monoecy and and odioecy in a hi he o poo ly s udied
pa o he species’ ange on he A lan ic coas o Mo occo
co esponds o a shi be ween e aploidy and hexaploidy.
This ques ion is impo an because elsewhe e in he dis ibu-
ional ange o he species complex, spa ial (and pe haps e o-
lu iona y) ansi ions in he sexual sys em and ploidy le els
a e con ounded; ins ances in which sexual-sys em ansi ions
a e uncoupled om hose in ploidy p o ide pa icula ly e ile
g ound o in oking he main enance o di e en sexual sys-
ems by na u al selec ion ( a he han as a esul o he his o i-
cal dis ibu ion o ploidy le els). Second, we p esen esul s o
an expe imen ha asks how he ma ing sys em is a ec ed by
plas ic esponses o esou ce a ailabili y in he ela i e alloca-
ion o male and emale unc ions. We add ess bo h hese
issues wi hin he con ex o a syn hesis o ecen wo k on
M. annua. We begin by conside ing he phylogene ic and phy-
logeog aphic his o y o he species complex in Eu ope. We
hen e iew empi ical es s o a hypo hesis ha in okes di e -
en ial ex inc ion-coloniza ion dynamics in a me apopula ion
o explain he main enance o di e en sexual sys ems. Fi-
nally, we conside he po en ial impo ance o pheno ypic
plas ici y in sex alloca ion in egula ing he selec ion o com-
bined e sus sepa a e sexes. O e all, we highligh he impo -
ance o bo h shi s in he gene ic sys em associa ed wi h
polyploidiza ion and hyb idiza ion and shi s in he ecological
and demog aphic con ex o selec ion o ansi ions be ween
sexual sys ems.
T ansi ions in he Sexual Sys em: The Role
o Polyploidy and Hyb idiza ion
Me cu ialis annua belongs o a small Eu opean genus in
he Eupho biaceae ha comp ises mainly dioecious woody
o he baceous pe ennials. Phylogene ic econs uc ion o he
Fig. 1 Male (A), emale (B), and monoecious (C) indi iduals o Me cu ialis annua. The male lowe s on he male and he monoecious
indi iduals ha e he same mo phology, bu male lowe s o male plan s a e held on e ec peduncles. Also, he emale lowe s o bo h emale and
monoecious plan s ha e he same mo phology and placemen in he lea axils (in he pho og aphs, emale lowe s ha e al eady se ui ). Males o
diploid and hexaploid popula ions ha e he same mo phology. Monoecious plan s a e e ec i ely emales ha p oduce s amina e lowe s a ound
hei pis illa e lowe s.
130 INTERNATIONAL JOURNAL OF PLANT SCIENCES
genus by K ahenbuhl e al. (2002), based on ITS sequence
analysis, indica ed ha dioecy and pe enniali y a e ances al
in he genus and ha monoecy has e ol ed on a leas wo
independen occasions, one o which was in he annual poly-
ploid complex M. annua. This complex comp ises ploidy
le els anging om diploid h ough a leas 12-ploid, wi h di-
oecy con ined o he diploids and monoecy ound only in he
polyploid lineages. He e, he e aploids, oc aploids, and highe
ploidy le els a e exclusi ely monoecious, bu he hexaploids
show ema kable a ia ion in hei sexual sys ems, wi h pop-
ula ions anging om monoecy h ough and odioecy o sub-
dioecy in a ious pa s o hei ange (Du and 1963; Du and
and Du and 1992).
Ea ly s udies o mo phology and meio ic pai ing beha io
sugges ed ha M. annua was an au opolyploid se ies and
ha polyploidiza ion had p ecipi a ed he e olu ion o mono-
ecy (Du and 1963). Howe e , mo e ecen analysis iden i ied
a leas wo di e gen ITS pa alogues in hexaploid popula-
ions o M. annua, only one o which occu ed in diploid and
e aploid indi iduals (Obba d e al. 2006b). The o he ITS
sequence, absen in diploids and e aploids, was ound in
Me cu ialis hue ii, a diploid sis e species o M. annua, which
has he same numbe o ch omosomes and is also annual and
dioecious. Obba d e al. (2006b) in e p e ed hese esul s as
e idence o hyb idiza ion be ween e aploid M. annua and
M. hue ii, yielding iploids and ollowed by polyploidiza ion
o p oduce he hexaploid lineage ( ig. 2). This hypo hesis e-
qui es u he es ing wi h mo e exhaus i e sampling o geno-
ypes and loci, bu p elimina y esul s om mic osa elli e loci,
some o which ampli y only in hexaploid M. annua and M.
hue ii, a e consis en wi h he ITS sequence analysis (P. Ryme ,
H. S one, G. Ko becka, and J. R. Pannell, unpublished da a).
Polyploidy ep esen s a d ama ic shi in he gene ic sys em
o a lineage. No only may i al e he exp ession o inb eeding
dep ession, po en ially allowing he sp ead o sel - e iliza ion
in a popula ion (Ron o 1999; Husband e al. 2008), bu i
may also al e a b oad ange o pheno ypic ai s ha may al-
low a lineage o occupy new and di e en habi a s; i.e., i
can cause ecological shi s ha may also a o a ansi ion in
he sexual sys em o a popula ion (S ebbins 1950; Pannell
e al. 2004). Pannell e al. (2004) e iewed he in e ac ions
ha a e expec ed o occu be ween ploidy and sexual-sys em
e olu ion. Thei main conclusion was ha he complexi ies
in ol ed make gene al p edic ions di icul . Fo example, on
he one hand, polyploidiza ion may allow sel - e ile he -
maph odi ism o eplace dioecy by causing a educ ion in in-
b eeding dep ession (Lande and Schemske 1985; Pannell e al.
2004; Husband e al. 2008). On he o he hand, genome du-
plica ion can cause he b eakdown o sel -incompa ibili y in
he maph odi ic popula ions, allowing dioecy o e ol e as an
al e na i e ou c ossing mechanism (Mille and Venable 2000;
bu see Mable 2004; Pannell e al. 2004). Thus, i can be
emp ing o in oke polyploidiza ion as a cause o ansi ions
bo h om he maph odi ism o dioecy and ice e sa. In he
case o M. annua, i would seem no only ha polyploidiza ion
ini ia ed he shi om dioecy o he maph odism bu also ha
i has no p ecluded he main enance o males in hexaploid
popula ions o M. annua o he exis ence o dioecy in he
newly disco e ed e aploid species Me cu ialis cana iensis
(Obba d e al. 2005).
Phylogeog aphy and Regional T ansi ions
in he Sexual Sys em
Spa ial ansi ions in he sexual sys em o Me cu ialis annua
in Eu ope ( ig. 3) co espond, o an impo an ex en ( hough
no exclusi ely; see below), wi h ansi ions in he ploidy
le el. Fully dioecious popula ions a e exclusi ely diploid;
hese a e widesp ead h oughou Eu ope. In con as , popula-
ions con aining he maph odi es (wi h o wi hou males) a e
polyploids; hese a e la gely es ic ed o he wes e n Medi e -
anean Basin and no hwes e n A ica. Te aploids occu
sou h o Raba on he A lan ic coas o Mo occo, and hese
mee wi h hexaploid popula ions o he no h. Te aploids a e
he maph odi ic, whe eas he hexaploids, which a e e y wide-
sp ead in no hwes e n Mo occo and a ound he coas o he
Ibe ian Peninsula, a e a iously he maph odi ic o and odioe-
cious. Hexaploid popula ions mee he diploids a wo con ac
zones in no heas e n and no hwes e n Spain (Du and 1963;
Obba d e al. 2006b).
Pa e ns o allelic ichness and gene ic di e si y a se e al
isozyme loci sugges ha he Spanish diploid-hexaploid an-
si ions a e seconda y con ac zones, wi h diploids ha ing
expanded ac oss Eu ope om an eas e n Medi e anean e u-
gium and he hexaploids ha ing mo ed no h along he coas s
o he Ibe ian Peninsula om sou he n Spain o No h A ica
(Obba d e al. 2006b). This expansion o he geog aphic ange
o diploid M. annua appea s o be con inuing apace, wi h su -
eys sugges ing ha he diploids ha e displaced he hexaploids
by some 80 and 200 km wi hin abou ou decades in no h-
eas e n and no hwes e n Spain, espec i ely, appa en ly bo h
as a esul o diploid supe io i y in hei g ow h (Buggs and
Pannell 2007) and h ough he abili y o diploids o swamp
he monoecious hexaploids wi h he la ge amoun s o pollen
ha diploid males p oduce (Buggs and Pannell 2006).
The highe pollen p oduc ion o diploid males o e he -
maph odi es p o ides a plausible explana ion o he apid
displacemen o he maph odi ic popula ions by he dioecious
lineage. Howe e , wha should we p edic o a con ac zone
be ween dioecy and and odioecy, whe e males co-occu wi h
he he maph odi es? This is a pe inen ques ion because,
should he dioecious lineage con inue i s ad ance down he
coas s o he Ibe ian Peninsula, i will soon encoun e and o-
dioecious popula ions a he sou h. Do ken and Pannell (2007)
add essed his ques ion, using spa ially explici compu e sim-
ula ions. They expec ed ha he p esence o males in and o-
dioecious hexaploid popula ions migh slow he diploid
ad ance by compe ing wi h he diploid males o si e ou -
c ossed p ogeny. Howe e , because ange expansion equi es
ecu en success ul coloniza ion (Bake 1955; Pannell and
Ba e 1998), he occu ence o males wi h monoecious indi-
iduals ac ually dilu ed one o he bene i s ha monoecy has
o e dioecy: he ad an age o ep oduc i e assu ance du ing
coloniza ion. Do ken and Pannell’s (2007) simula ions he e-
o e p edic ha he diploid ad ance migh accele a e when
and odioecious egions a e encoun e ed, and hey ecall he
complexi ies ha can esul when selec ion ac s on plan e-
p oduc i e s a egies a bo h he popula ion and me apopula-
ion le els (Ba e and Pannell 1999; also see nex sec ion).
The ansi ions be ween dioecy and he maph odi ism in
no he n Spain, desc ibed abo e, a e ully con ounded by
131
PANNELL ET AL.—SEXUAL-SYSTEM TRANSITIONS IN MERCURIALIS ANNUA
shi s be ween diploidy and hexaploidy. Howe e , ansi ions
also occu be ween and odioecy and he maph odi ism else-
whe e in he Ibe ian Peninsula and in Mo occo. In he Ibe-
ian Peninsula, hese ansi ions occu wi hin he hexaploid
lineage, bu in Mo occo, he ansi ion occu s along he A -
lan ic coas in a egion b oadly coinciden wi h a ansi ion
be ween hexaploidy in he no h and e aploidy in he sou h
(Du and 1963). To de e mine whe he hese wo ansi ions
coincide p ecisely, we used low cy ome y o assay he ploidy
le el o 22 popula ions a in e als along he A lan ic coas o
Mo occo ( ig. 4). Fo he low cy ome y measu emen s, we
used lea ma e ial om seeds g own in he g eenhouse. Ma e-
ial was p epa ed using he ‘‘LB01’’ me hod o Dolezel e al.
(1989) and Lycope sicon esculen um c . Ga dene ’s Deligh
Fig. 2 Hypo heses o he ela ionships be ween he annual lineages o Me cu ialis. Hea y lines indica e he phylogene ic ela ionships
be ween diploid species, and hin a ows show polyploidiza ion o hyb idiza ion e en s; Mindica es p oposed ma e nal pa en age, and Pindica es
p oposed pa e nal pa en age. A, Diploid M. annua p oposed as he pa en o polyploid M. annua. The he e ogeneous ITS ypes p esen in
hexaploid M. annua show ha i has an allopolyploid o igin h ough hyb idiza ion be ween M. annua and M. hue ii, and he hexaploid
ch omosome complemen is consis en wi h hyb idiza ion be ween a e aploid and a diploid lineage, ollowed by ch omosome doubling. ITS da a
also show M. cana iensis o be allopolyploid in o igin, p obably a hyb id be ween M. annua s.l. and an unknown axon (dashed box). Chlo oplas
sequence simila i y o M. annua s.l. sugges s ha M. annua s.l. was he ma e nal pa en , and ch omosome numbe s a e consis en wi h bo h
pa en s being diploid. B, Gi en he g ea e simila i y be ween ITS sequences om M. cana iensis and e aploid and hexaploid M. annua, he
diploid p ogeni o o hese axa may ha e be an ea lie wes e n lineage o M. annua, di e gen om he lineage ha has ecen ly expanded om
he eas . F om Obba d e al. (2006a).
132 INTERNATIONAL JOURNAL OF PLANT SCIENCES
lea ma e ial as a s anda d. Each sample was assessed on a
Bec on Dickinson FACScan low cy ome e and analyzed us-
ing Bec on Dickinson CellQues so wa e (BD BioSciences,
F anklin Lakes, NJ).
Ou esul s indica e ha he ansi ion be ween e aploidy
and hexaploidy in Mo occo occu s some 100 km sou h o
he monoecy-and odioecy ansi ion, which is loca ed a Ra-
ba ; males a e ound only in popula ions no h o his poin
( ig. 4). (No e ha wo e aploid popula ions we e ound
no h o he majo ploidy ansi ion wi hin he hexaploid
zone. Such popula ions a e likely o be he esul o ea h-
mo ing oadwo ks, al hough hey migh also be emnan
popula ions ollowing a hexaploid ad ance sou h; see Buggs
and Pannell 2006.) The geog aphic shi in he sexual sys em
is hus uncoupled om he shi in ploidy in Mo occo, and i
occu s wi hin he hexaploid lineage jus as i does in he
o he monoecy-and odioecy ansi ions in he Ibe ian Penin-
sula. This poses he ques ion o why he sexual sys em should
change o e geog aphical space, gi en ha popula ions on
bo h sides o hese ansi ions ha e he same gene ic sys em
and appea o exchange genes (Obba d e al. 2006b).
Mechanisms o T ansi ions among Sexual Sys ems:
The Role o Me apopula ion Dynamics
To explain he main enance o he maph odi ism e sus di-
oecy o and odioecy in di e en egions o he ange o hexa-
ploid Me cu ialis annua, Pannell (1997c) hypo hesized
a me apopula ion model ha in okes di e en ial selec ion
o ep oduc i e assu ance du ing coloniza ion. Because only
sel - e ile he maph odi es, and no males, can colonize un-
occupied habi a on hei own, we should expec ex inc ion-
coloniza ion dynamics o educe he equency o males a
he me apopula ion (i.e., egional) le el. A he same ime,
he maph odi es wi h emale-biased sex alloca ion should be
a o ed by selec ion du ing coloniza ion o e hose wi h
mo e equal sex alloca ion because hei popula ions will g ow
mo e quickly (no e ha selec ion o emale-biased sex alloca-
ion a he me apopula ion le el can be seen equi alen ly in
e ms o local ma e compe i ion du ing sel ing o in e ms o
deme-le el selec ion; see F ank 1986). As popula ions g ow
dense a e coloniza ion, he selec i e ad an age o maleness
inc eases, bo h because o he a ailabili y o la ge numbe s
o o ules o e ilize and because o he low numbe s o pol-
len g ains p oduced by he maph odi es wi h which hey mus
compe e. Thus, whe eas males a e dis a o ed du ing coloni-
za ion, hey may be a o ed as mig an s (o , p obably much
mo e a ely, as new mu an s) in o es ablished demes. Unde
his model, whe he males a e ound a a egional le el de-
pends la gely on he balance be ween hese wo opposing se-
lec i e o ces (Pannell 1997a; see also Pannell 2001).
The me apopula ion model makes a numbe o p edic ions
ha ha e ecen ly been es ed. One demog aphic p edic ion is
ha , i he egional absence o males is he esul o apid
Fig. 3 Dis ibu ion o Me cu ialis annua in Eu ope and a ound he Medi e anean Basin. In he no h and eas o his ange, M. annua is dioecious
and diploid, whe eas in Ibe ia and No h A ica, i is monoecious (and and odioecious) and polyploid. Ci cles indica e he loca ions o seed collec ions
used o es ima e pa e ns o di e si y by Obba d e al. (2006b). Regions ma ked ‘‘Dioecy,’’ ‘‘Monoecy,’’ and ‘‘And odioecy’’ deno e zones occupied by he
co esponding sexual sys ems (see ‘‘Phylogeog aphy and Regional T ansi ions in he Sexual Sys em’’). Modi ied om Obba d e al. (2006b).
133
PANNELL ET AL.—SEXUAL-SYSTEM TRANSITIONS IN MERCURIALIS ANNUA

popula ion u no e compa ed wi h egions in which males
occu , hen popula ions in monoecious egions ough o be
smalle han hose in and odioecious egions (because hey
a e, on a e age, younge ). Ano he p edic ion is ha a es
o habi a occupancy should be lowe in monoecious han in
and odioecious egions i ex inc ion-coloniza ion a es a e
highe in he o me (Gas on e al. 2000; F eckle on e al.
2005). Pa e ns o occupancy and abundance obse ed ac oss
se e al clines in he sexual sys em o M. annua in Spain we e
consis en wi h bo h hese p edic ions (Eppley and Pannell
2007b).
F om a popula ion gene ics pe spec i e, he me apopula-
ion model p edic s ha monoecious popula ions should
ha e lowe gene ic a ia ion and should be mo e s ongly di -
e en ia ed om one ano he han and odioecious popula-
ions. This is because monoecious popula ions a e expec ed
o display gene ic signa u es o mo e ecen coloniza ion bo -
lenecks ha subsequen mig a ion has no had ime o e ase
(e.g., Sla kin 1977; Wade and McCauley 1988; Pannell and
Cha leswo h 2000). To es hese p edic ions, Obba d e al.
(2006b) measu ed gene ic di e si y in hexaploid monoecious
and and odioecious popula ions ac oss hei ange in he Ibe-
ian Peninsula and No h A ica and in dioecious popula ions
ac oss hei Eu opean ange. They ound ha , e en a e he
e ec s o he hypo hesized ange expansion had been ac-
coun ed o (see ‘‘Phylogeog aphy and Regional T ansi ions
in he Sexual Sys em’’), hexaploid and odioecious popula-
ions we e s ikingly mo e di e se han hei monoecious
coun e pa s, as expec ed. Mo eo e , whe eas pai wise ge-
ne ic di e en ia ion among hexaploid monoecious popula ions
was highly a iable and o en la ge, pai s o and odioecious
popula ions we e in a iably e y simila , as we e dioecious
popula ions. These pa e ns sugges a his o y o homogenizing
gene low among and odioecious popula ions and among di-
oecious popula ions ( hough no be ween and odioecious and
dioecious popula ions, owing o hei di e en ploidy le els;
Obba d e al. 2006b). Al hough popula ions o M. annua wi h
sepa a e sexes migh be linked by mo e gene low because hey
con ain males ha dispe se mo e pollen han he maph odi es,
such an explana ion would no accoun o he low di e si y
o monoecious popula ions occu ing wi hin and odioecious
egions (Obba d e al. 2006b).
A key assump ion o he me apopula ion model is ha sel -
ing a es a e densi y dependen : he maph odi es sel - e ilize
hei p ogeny when ma es a e absen , bu oppo uni ies o
ou c ossing inc ease when popula ions g ow. Eppley and Pannell
(2007a) es ed his assump ion by es ima ing sel ing a es in
popula ions g owing a di e en densi ies. Sel ing a es we e
high when indi iduals we e mo e han ca. 30 cm apa bu quickly
d opped in dense s ands, as p edic ed by he model. Eppley
and Pannell (2007a) used hei esul s o p edic he h eshold
densi y below which males should be excluded om a popula-
ion and abo e which hei main enance was assu ed. This
h eshold densi y was much lowe han commonly measu ed
in pu ely monoecious popula ions, indica ing ha he p esence
o males in a popula ion is indeed mig a ion limi ed (Eppley
and Pannell 2007a).
E olu ion o And odioecy: Sex Alloca ion and
In lo escence S uc u e
We ha e seen ha dense popula ions o Me cu ialis annua
a e la gely ou c ossing, easing he po en ial in asion o males
in o he maph odi ic popula ions. Bu how do males su pass
he wo old h eshold in pollen p oduc ion equi ed o hei
main enance a equencies g ea e han ze o (Lloyd 1975;
Cha leswo h and Cha leswo h 1978; Cha leswo h 1984)?
The me apopula ion hypo hesis sugges s one mechanism by
in oking selec ion o emale-biased sex alloca ion in he he -
maph odi es. Thus, i he maph odi es alloca e a p opo ion
x<0:5 o hei ep oduc i e esou ces o hei male unc-
ion, hen emale-s e ile indi iduals ha di e all hei e-
sou ces o he wise in es ed in seeds o pollen p oduc ion will
be able o dispe se 1=x>2 uni s o pollen ela i e o he pol-
len p oduced by he maph odi es. Full ‘‘compensa ion’’ in e-
sou ce alloca ion be ween male and emale unc ions would
he e o e be su icien o male in asion as long as he he -
maph odi es a e emale biased in hei sex alloca ion.
Ano he way in which males migh c oss he wo old in a-
sion h eshold is by dispe sing he pollen hey p oduce be e
han he maph odi es. Assume again ha he maph odi es al-
loca e a p opo ion xo hei esou ces o pollen p oduc ion
and ha males alloca e a p opo ion 1.0. Assume u he
ha pollen g ains dispe sed by males a e g imes mo e likely
o ind a ecep i e s igma han hose dispe sed by he maph-
odi es. Then males should enjoy a ela i e si ing success o
g=x. Clea ly, i g>1:0, hen males migh in ade a popula-
ion o he maph odi es e en i x¼0:5, i.e., he popula ion is
unbiased in i s sex alloca ion. Because we expec he sex allo-
ca ion o ou c ossing he maph odi es o be ca. 0.5, i he
male and emale i ness gain cu es a e no e y di e en
Fig. 4 Map showing he loca ions o 22 popula ions o Me cu alis
annua o which he ploidy le el was de e mined along he A lan ic
coas o Mo occo. Open and illed ci cles ep esen e aploid and
hexaploid popula ions, espec i ely. The solid line nea Raba indi-
ca es he poin o ansi ion be ween monoecious popula ions ( o he
sou h) and and odioecious popula ions ( o he no h). Two e aploid
popula ions we e ound wi hin he a ea occupied p incipally by hexa-
ploid popula ions.
134 INTERNATIONAL JOURNAL OF PLANT SCIENCES
(Lloyd and Bawa 1984), his easoning shows ha he condi-
ion g>1:0 ough o be su icien o he e olu ion o
maleness ai ly gene ally. Eppley and Pannell (2007a) es ed
his idea and es ima ed a alue o g¼1:6.
Tha males a e so much be e han he maph odi es a dis-
pe sing hei pollen is almos ce ainly due o he di e ences
be ween he wo mo phs in hei in lo escence a chi ec u es.
Al hough monoecious plan s ha e a emale mo phology, wi h
male lowe s held a ound a subsessile emale lowe in he
lea axils, males dispe se hei pollen om lowe s held abo e
he plan on long, e ec peduncles. In lo escences such as
hose o M. annua males a e widely ound in wind-pollina ed
he bs and a e expec ed o inc ease he si ing success o pollen
by allowing i o a el a he a e elease (Le in and Ke s e
1974; Niklas 1985; Bu d and Allen 1988). Such adap a ions
almos ce ainly gi e ise o an accele a ing male i ness gain
cu e, and hey should hus s abilize he main enance o sep-
a a e sexes (Cha no e al. 1976). I is poo ly unde s ood
whe he he e olu ion o such in lo escences is mo e likely o
p ecede, and hus o p ecipi a e, a ansi ion om he maph-
odi ism o dioecy o o ollow he e olu ion o dioecy and
wind pollina ion. Howe e , ansi ions be ween wind pollina-
ion and dioecy o en go hand-in-hand (Renne and Rickle s
1995; Wallande 2001; F iedman and Ba e 2008), and
hey may coincide wi h seconda y sexual adap a ions such
as hose ound in M. annua (see Welle e al. 1998, 2006; Ka -
enbe g e al. 2002; F iedman and Ha de 2004; Golonka
e al. 2005).
Main enance and B eakdown o And odioecy:
The Role o Pheno ypic Plas ici y
Sex exp ession is no o iously labile in plan s, wi h males
and, less o en, emales o dioecious popula ions equen ly
p oducing lowe s o he opposi e sex (Lloyd and Bawa
1984; Ko pelainen 1998). In gynodioecious and and odioe-
cious species, he he maph odi es oo may a y in hei sex
alloca ion in esponse o en i onmen al cues ( e iewed by
Delph and Wol [2005]). Whe eas he implica ions and unc-
ional signi icance o comple e gende swi ches emain poo ly
unde s ood, mo e sub le exp essions o sexual labili y may
ha e impo an consequences o sexual-sys em e olu ion,
e.g., by con e ing upon a lineage an abili y o sel - e ilize
a e long-dis ance dispe sal (Bake and Cox 1984). Plas ici y
in he sex alloca ion o he maph odi es is p obably d i en by
a ia ion in he ma ginal cos o esou ces needed o ep o-
duc ion o in changes o he shapes o i ness gain cu es
(Lloyd and Bawa 1984; Klinkhame e al. 1997), and i has
impo an implica ions o sexual-sys em e olu ion h ough
i s e ec on he main enance o males o emales.
The pheno ypic plas ici y in sex alloca ion o Me cu ialis
annua he maph odi es should ha e impo an implica ions
o he equency o males ha can be main ained in and o-
dioecious popula ions. Indeed, along wi h me apopula ion
dynamics, i migh be a u he ac o unde lying he la ge
among-popula ion a ia ion obse ed in male equencies o
he species. As a gued by Delph (2003), plas ici y in sex allo-
ca ion by he maph odi es can change he ela i e e ili y o
unisexual and he maph odi e plan s (see also Delph and
Lloyd 1991). Because he ela i e pollen o seed e ili y o uni-
sexuals and he maph odi es egula es hei equilib ium e-
quencies (Lloyd 1976), such plas ici y can cause a ia ion
in unisexual equencies ac oss ecological g adien s (Delph
1990; Delph and Lloyd 1991; Asikainen and Mu ikainen
2003; Ba 2004). Fo example, males always alloca e all o
hei ep oduc i e esou ces o pollen p oduc ion (i.e., xm¼1).
I he maph odi es change he p opo ion o esou ces alloca ed
o pollen in esponse o an ecological g adien (i.e., x
h
a ies
ac oss he g adien ), hen his also changes he ela i e pollen
e ili ies o males and he maph odi es (i.e., ¼xm=xh). Hal
o he p ogeny si ed by males a e, on a e age, male, ia he seg-
ega ion o dominan male-de e mining alleles (Pannell 1997b).
Thus, di e ences in ac oss en i onmen al g adien s will a -
ec he ela i e equency o male-de e mining pollen in he
popula ion and he equency o males a equilib ium. In e -
es ingly, such a scena io would concu wi h Da win’s (1877)
easoning ha un a o able en i onmen al condi ions can lead
o an inc eased sepa a ion o he sexes and ollows simila pa -
e ns shown by o he species ( e iewed by Delph and Wol
[2005]).
In a p e ious expe imen , M. E. Do ken and J. R. Pannell
(unpublished manusc ip ) ound ha plan densi y go e ns
e olu iona y ajec o ies in male equencies by a ec ing he
magni ude o . Unde high plan densi ies, was mo e han
h ee imes highe han unde low densi ies (and see Pannell
1997c). In he nex gene a ion, male equencies we e 38%
highe among he p ogeny o plan s g own unde high densi-
ies (M. E. Do ken and J. R. Pannell, unpublished manu-
sc ip ). Densi y can a ec male si ing success in wo ways.
Fi s , densi y may a ec he local a ailabili y o esou ces by
inc easing compe i ion among plan s. Second, because pollen
is dispe sed locally (Eppley and Pannell 2007a), densi y di-
ec ly a ec s male si ing abili y. We a emp ed o dissec
hese con ounded e ec s by con olling plan densi y and ma-
nipula ing esou ce a ailabili y. Does esou ce a ailabili y
a ec he magni ude o ? I so, does a ia ion in esou ce
a ailabili y egula e male equencies in M. annua?
To add ess hese ques ions, we mixed seeds om i e
nea by si es in Mo occo, each wi h high equencies o males
(i.e., be ween 25% and 47%). We g ew plan s om seed in
12 3 33-m aised beds in s anda dized, low-nu ien soil mix-
u es a he Wy ham Field Lab be ween July and Oc obe
2004. We added slow- elease nu ien e ilize pelle s (Os-
moco e, Sco s, Ma ys ille, OH) a he ecommended dosage
o hal o he beds, using a andomized block design wi h
h ee blocks. On a e age, he e we e 566 697 (SD) plan s in
he high- esou ce plo s and 566 642 (SD) plan s in he low-
esou ce plo s. We main ained high male equencies in each
plo by ansplan ing males om addi ional simila plo s no
included in his analysis he e (a e age male equency in
high- esou ce plo s ¼52:8% 61:4% [SE], in low- esou ce
plo s ¼52:5% 68:2% [SE]). A he end o he expe imen ,
we ha es ed a s anda dized sample o 20 he maph odi e
and 10 male plan s om each plo by sampling indi iduals a
egula in e als along ou e enly spaced ansec s. Follow-
ing Pannell (1997c), we calcula ed he maph odi e alloca ion
o male unc ion ( ) as he abo eg ound p opo ion o bio-
mass alloca ed o pollen o he maph odi es (p
h
) ela i e o
135
PANNELL ET AL.—SEXUAL-SYSTEM TRANSITIONS IN MERCURIALIS ANNUA
ha o males (p
m
; i.e., ¼pm=ph). Thus, lowe alues o
indica e highe he maph odi e alloca ion o pollen.
We ound ha he maph odi es g own unde high- esou ce
condi ions (a e age ¼11:260:9 [SE]) had nea ly double
he alloca ion o pollen o plan s g own unde low- esou ce
condi ions (a e age ¼21:662:3 [SE]; ig. 5a; wo- ailed
- es wi h equal a iances: ¼4:28, d ¼10, P<0:005).
A he end o Sep embe , we ha es ed all he maph odi es in
a plo and collec ed hei seed in bulk, and we sowed an a -
e age o 1500 seeds (es ima ed by weighing ou s anda d
amoun s o seeds) back in o he plo in June 2005. Because
he maph odi es had highe alloca ion o pollen in high-
esou ce condi ions, he p opo ion o p ogeny si ed by males
should ha e been lowe han ha unde low- esou ce condi-
ions. Mo eo e , because maleness is go e ned by he seg e-
ga ion o a dominan Mendelian allele (Pannell 1997b), we
p edic ed ha he equency o males among he p ogeny o
plan s g own unde high- esou ce condi ions should be lowe
han ha among hose om low- esou ce condi ions. Ou
esul s a e consis en wi h his p edic ion ( ig. 5). The a e age
equency o males om high- esou ce plo s was 40:9% 6
1:1% (SE; a e age numbe o plan s pe plo ¼720 674
[SE]), compa ed wi h 45:8% 61:6% (SE) o low- esou ce
plo s (a e age numbe o plan s pe plo ¼501 683 [SE];
one- ailed - es wi h equal a iances: ¼1:92, d ¼10,
P<0:05).
Ou esul s indica e ha plas ici y in sex alloca ion egu-
la es he equency o unisexual plan s in gende -dimo phic
popula ions, suppo ing he p edic ion made by Delph (2003).
E en hough ou expe imen was conduc ed ac oss a single
gene a ion, we ound conside able changes in male equen-
cies in esponse o en i onmen al condi ions. Speci ically, we
ound ha male equencies no only can espond o densi y,
which egula es male si ing abili y (as p edic ed by Eppley
and Pannell 2007a), bu also can espond di ec ly o a ia ion
in imposed by a ia ion in he esou ce s a us o he maph-
odi es. These esul s demons a e he impo ance o pheno-
ypic plas ici y in he maph odi e sex alloca ion o egula ing
male equencies in and odioecious M. annua.
Gene ics o Sex De e mina ion and Sex Alloca ion
in Me cu ialis annua
The gene ic basis o sex exp ession in dioecious Me cu ia-
lis annua was s udied in ensi ely by Du and and co-wo ke s
(Louis and Du and 1978; Dauphin-Gue in e al. 1980; Du-
and and Du and 1991a, 1991b). On he basis o c ossing
expe imen s using a small sample o o iginal geno ypes, hey
concluded ha sex was de e mined by epis a ic in e ac ions
be ween alleles seg ega ing a h ee independen ly seg ega ing
loci. The ac ha amily sex a ios a e o en s ongly male
o emale biased, despi e a 1 : 1 sex a io a he popula ion
le el (J. R. Pannell, pe sonal obse a ions), would seem o
con i m ha mo e han one locus is in ol ed, bu esul s
o ecen gene ic analysis based on sequence-cha ac e ized
ampli ied- egion (SCAR) ma ke s we e consis en wi h a sin-
gle locus (Khadka e al. 2002). Pannell (1997b) concluded
ha maleness in an and odioecious popula ion was de e -
mined by he p esence o a single dominan allele, bu his
analysis also poin ed o he impo ance o a plas ic compo-
nen o sex exp ession. Clea ly, much emains o be lea ned
abou sex de e mina ion in M. annua and abou he e olu-
ion o sex de e mina ion in he complex h ough i s sexual-
sys em ansi ions.
I is clea om he s udies e iewed abo e ha sex alloca-
ion in he maph odi es o M. annua is also highly a iable,
albei as a con inuous ai . Much o his a ia ion is e i-
den ly due o pheno ypic plas ici y, bu compa isons be ween
Fig. 5 Pheno ypic plas ici y in he maph odi e sex alloca ion a -
ec s e olu iona y ajec o ies in male equencies. a, The ela i e
pollen p oduc ion ( ) o males, compa ed wi h ha o he maph odi es,
di e s be ween low- and high- esou ce condi ions. b, Because he
pollen e ili y o males ela i e o ha o he maph odi es egula es
he equency o males, di e en alues o be ween low- (ci cles) and
high- esou ce condi ions (squa es) lead o di e gence in male e-
quencies in he nex gene a ion. In b, e o ba s ep esen s anda d
e o s.
136 INTERNATIONAL JOURNAL OF PLANT SCIENCES
Mo occan and Spanish popula ions sugges an impo an ge-
ne ic componen , oo (J. R. Pannell, R. Be jano, and S. M.
Eppley, unpublished manusc ip ); howe e , he a chi ec u e
o his gene ic a ia ion in M. annua is unknown. I a mo e
comple e pic u e o pas e olu iona y ansi ions be ween
sexual sys ems in he species complex is o eme ge, we need
u he in o ma ion abou he quan i a i e gene ics o sex al-
loca ion.
Two speci ic ques ions a e cu en ly he ocus o a en ion
o ou esea ch, and bo h ela e o he so-called b eede ’s
equa ion, R¼h2S, which ela es he po en ial esponse o a
popula ion o selec ion, R, o he p oduc o he selec ion co-
e icien , S, and he na ow-sense he i abili y o he ai , h
2
.
Because h2¼VA=VP, i.e., he ac ion o o al pheno ypic
a iance ha is due o addi i e gene ic a iance, he po en ial
o esponse o selec ion on sex alloca ion will depend on
bo h he absolu e amoun o addi i e gene ic a iance, V
A
,
and he size o V
P
, which will be in la ed by pheno ypic plas-
ici y (Falcone and Mackay 1996). The i s ques ion hus
conce ns how pheno ypic plas ici y in sex alloca ion in M.
annua will a ec esponses o selec ion on pollen p oduc ion.
This ques ion is o gene al impo ance o ou unde s anding
o ansi ions om gynodioecy and and odioecy o dioecy
(Delph and Wol 2005).
The second ques ion conce ns he size o V
A
: how much
addi i e gene ic a iance in sex alloca ion is p esen in popu-
la ions o M. annua wi h di e en sexual sys ems, and how
migh his a y geog aphically? The geog aphic pe spec i e
on his ques ion is impo an because ange expansions,
which a e known o ha e occu ed in M. annua (see ‘‘Phylo-
geog aphy and Regional T ansi ions in he Sexual Sys em’’),
ypically in ol e epea ed popula ion bo lenecks ha a e ex-
pec ed o educe quan i a i e gene ic a ia ion (Lande 1992).
Indeed, ange expansion in M. annua was in e ed on he ba-
sis o educed gene ic a ia ion a isozyme loci wi h dis ance
om pu a i e e ugia (Obba d e al. 2006b). Wha we do
no know is whe he he hypo hesized ange expansions a -
ec ed gene ic a ia ion in sex alloca ion, which is unde
equency-dependen selec ion, di e en ly. Cu en wo k is
add essing his ques ion h ough an assessmen o he e-
sponse o selec ion on pollen p oduc ion in he maph odi es
sampled om popula ions a di e en la i udes in Spain and
No h A ica.
Conclusions
Me cu ialis annua displays unusual a ia ion in i s sex ex-
p ession a se e al le els in he genealogical hie a chy, in-
cluding wi hin and among geno ypes, among popula ions in
pu a i e me apopula ions, among egions in di e en pa s o
Eu ope, and be ween ela ed species in a clade ep esen ed by
lineages wi h e icula e phylogene ic ela ionships. This a ia-
ion e lec s a complex his o y o di e gence, mig a ion, and
gene low, which ha e all con ibu ed o ansi ions be ween
sexual sys ems. Resea ch on M. annua has h own ligh on o
a numbe o hemes in sexual-sys em e olu ion. These include
he impo ance o ploidy, hyb idiza ion, and ange expan-
sions in egula ing sexual-sys em a ia ion and hus se ing
he s age on which na u al selec ion hen ac s; he impo ance
o pheno ypic plas ici y unde lying much o he a ia ion ob-
se ed be ween indi iduals and i s esponsibili y in egula ing
de ails o sex a io e olu ion; he ole o con ex -dependen
ma ing in species cha ac e ized by demog aphic luc ua ions
and possible me apopula ion dynamics; he adap i e alue o
sexual specializa ion o pollen dispe sal unde wind pollina-
ion; and he po en ial ole o and odioecy in e olu iona y
pa hs be ween dioecy and he maph odi ism, a pa hway abou
which e y li le was hi he o known. Fu u e wo k aims o
cha ac e ize he demog aphic p ocesses hypo hesized o ha e
played a ole in d i ing he obse ed sexual-sys em ansi-
ions a egional and local-pa ch scales as well as o desc ibe
he quan i a i e gene ic a chi ec u e o sex de e mina ion and
sex alloca ion ac oss he phylogene ic and geog aphic ange
o he species complex.
Acknowledgmen s
We hank Spence Ba e o in i ing us o con ibu e o
his special issue o he jou nal, Anne Sakai and wo anony-
mous e iewe s o use ul commen s on he manusc ip , and
he Wellcome T us and F. M. Pla o low cy ome y acili-
ies. J. R. Pannell and B. Pujol we e suppo ed by g an s
om he Na u al En i onmen Resea ch Council (NERC) o
he Uni ed Kingdom; M. E. Do ken was suppo ed join ly
by NERC and he Na u al Sciences and Enginee ing Re-
sea ch Council o Canada; and R. Be jano was suppo ed by
a Spanish g an o he Minis e io de Educacio
´n y Ciencia.
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