Ex ensi e Pollen Flow bu Few Pollen Dono s and High
Rep oduc i e Va iance in an Ex emely F agmen ed
Landscape
Ra ael G. Albaladejo
1
*
.
, Bea iz Guzma
´n
1.
, San iago C. Gonza
´lez-Ma ı
´nez
2
, Abela do Apa icio
1
1Depa men o Plan Biology and Ecology, School o Pha macy, Uni e si y o Se ille, Se ille, Spain, 2Depa men o Fo es Ecology and Gene ics, CIFOR-INIA, Mad id,
Spain
Abs ac
Analysing pollen mo emen is a key o unde s anding he ep oduc i e sys em o plan species and how i is in luenced by
he spa ial dis ibu ion o po en ial ma ing pa ne s in agmen ed popula ions. He e we in e pa ame e s ela ed o le els o
pollen mo emen and di e si y o he e ec i e pollen cloud o he wind-pollina ed sh ub Pis acia len iscus ac oss a highly
dis u bed landscape using mic osa elli e loci. Pa e ni y analysis and he indi ec KinDis and Mixed E ec Ma ing models
we e used o assess ma ing pa e ns, he pollen dispe sal ke nel, he e ec i e numbe o males (N
ep
) and hei ela i e
indi idual e ili y, as well as he exis ence o ine-scale spa ial gene ic s uc u e in adul plan s. All me hods showed
ex ensi e pollen mo emen , wi h high a es o pollen low om ou side he s udy si e (up o 73–93%), a - ailed dispe sal
ke nels and la ge a e age pollina ion dis ances (d= 229–412 m). Howe e , hey also ag eed in de ec ing e y ew pollen
dono s (N
ep
= 4.3–10.2) and a la ge a iance in hei ep oduc i e success: 70% o males did no si e any o sp ing among he
s udied emale plan s and 5.5% o males we e esponsible o 50% o pollina ions. Al hough we did no ind educed le els
o gene ic di e si y, he adul popula ion showed high le els o bipa en al inb eeding (14%) and s ong spa ial gene ic
s uc u e (S
p
= 0.012), p obably due o es ic ed seed dispe sal and sca ce sa e si es o ec ui men . O e all, limi ed seed
dispe sal and he sca ci y o success ul pollen dono s can be con ibu ing o gene a e local pedig ees and o inc ease
inb eeding, he p elude o gene ic impo e ishmen .
Ci a ion: Albaladejo RG, Guzma
´n B, Gonza
´lez-Ma ı
´nez SC, Apa icio A (2012) Ex ensi e Pollen Flow bu Few Pollen Dono s and High Rep oduc i e Va iance in an
Ex emely F agmen ed Landscape. PLoS ONE 7(11): e49012. doi:10.1371/jou nal.pone.0049012
Edi o : Gio anni G. Vend amin, CNR, I aly
Recei ed June 1, 2012; Accep ed Oc obe 3, 2012; Published No embe 12, 2012
Copy igh : ß2012 Albaladejo e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s
un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed.
Funding: This s udy has been unded by g an s o he ’Jun a de Andalucı
´a’ (P oyec o de Excelencia P06-RNM-01499) and he Spanish Minis e io de Ciencia e
Inno acio
´n (CGL2008–00938) o AA. The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip .
Compe ing In e es s: The au ho s ha e decla ed ha no compe ing in e es s exis .
* E-mail: [email p o ec ed]
.These au ho s con ibu ed equally o his wo k.
In oduc ion
I is a gene al asse ion ha gene ic d i comp omises he
e olu iona y po en ial and long- e m su i al o agmen ed
popula ions h oughou inb eeding dep ession and educed e-
sponse o selec ion [1,2]. Consequen ly, de e mining in agmen-
a ion s udies he spa ial scale a which gene low is ope a ing
becomes essen ial since ecological and gene ic isola ion o
popula ions may no coincide. Indeed, many empi ical s udies
ha e shown, o example, ha impac s o habi a agmen a ion on
plan ma ing sys ems a e con ex -dependen and ha habi a
agmen a ion can e en inc ease pollina ion dis ances (i.e. gene
dispe sal) unde speci ic ci cums ances [3,4]. Fo he pa icula
case o ees, which a e candida es o long-dis ance dispe sal bo h
in space and ime [5], he ealised lack o conco dance be ween
heo e ical expec a ions and empi ical e idences aised he so-
called ‘pa adox o o es agmen a ion gene ics’ [6] and he claim
o s udies ha ocus gene dispe sal and he p ecise spa ial and
empo al (o sp ing) scale ha in luences he species ep oduc i e
ecology [7].
Pollen low is a main sou ce o gene ic a ia ion among
popula ions. Ne e heless, o small and agmen ed popula ions
no only he sou ce o o igin bu he composi ion (i.e. di e si y) o
con ibu ing pollen pool is equally impo an [7]. Hence, analysing
pollen mo emen s along wi h he di e si y o he e ec i e pollen
cloud is a key o unde s anding shi s in plan ma ing sys ems
associa ed o he pa icula spa ial dis ibu ion o po en ial ma ing
pa ne s bo h wi hin and among agmen ed popula ions [8].
Fac o s such as he numbe o pollen dono s con ibu ing o he
e ec i e pollen cloud and he male ep oduc i e a iance (i. e. how
di e en male ep oduc i e success is ac oss indi iduals) a e highly
ele an o delinea e he gene ic composi ion and pe o mance o
he nex gene a ion. High a iance in he ep oduc i e success o
indi idual plan s can enhance agmen a ion e ec s by u he
educing he numbe o pa ne s a ailable o ma ing [9].
Pis acia len iscus L. (Anaca diaceae) is a common sh ub o (mo e
a ely) a small ee ha is abundan in he Medi e anean
scle ophyllous ege a ion. Li e his o y ai s o his species
esemble, o some ex en , hose o la ge ees (i.e. long-li ed,
ou c osse , la ge pollen and seed c ops, high po en ial o pollen
and seed dispe sal). Fu he mo e, he species can be ound om
almos con inuous monospeci ic s ands in o es unde s o ies o
small clumps in open habi a s and e en isola ed indi iduals; hence
P.len iscus cons i u es a sui ed case s udy o analyse pollina ion
PLOS ONE | www.plosone.o g 1 No embe 2012 | Volume 7 | Issue 11 | e49012
biology, ma ing sys em and ine-scale popula ion gene ic s uc u e
in a agmen a ion con ex . In a p e ious s udy pe o med in
a la ge, dense and con inuous s and o P. len iscus, Albaladejo e al.
[10] ound ha he species was cha ac e ized by (i) high gene ic
di e si y o he e ec i e pollen cloud, (ii) low le els o bipa en al
inb eeding, and (iii) no signs o ine-scale spa ial gene ic s uc u e
(SGS), al oge he a ibu able o ex ensi e pollen (and seed)
dispe sal ac oss he s and. Bu , om nume ical simula ions based
on he obse ed da a, he au ho s also o esaw ha popula ion
a i ion and clumping o indi iduals (a common si ua ion o his
and many o he maquis’ species ac oss he Medi e anean) could
e en ually educe he numbe o males a ailable o ma ing and
esul in a ela i ely high p opo ion o ull-sibs wi hin ma e nal
p ogeny a ays (i.e. highe le els o co ela ed pa e ni y).
In he p esen s udy, we ocus on indi idual plan s o P. len iscus
embedded in an in ensi ely managed ma ix. Ou main goal is o
assess he pollina ion biology o P.len iscus by s udying he
pollina ion connec i i y bo h in quan i y and di e si y ac oss his
highly dis u bed landscape and compa e he esul s wi h hose
ob ained in con inuous popula ions. Speci ically, we add ess (i) he
spa ial pa e ns o pollen low in he s udy si e, (ii) he shape o he
e ec i e pollen dispe sal dis ibu ion (i.e. he dispe sal ‘ke nel’)
assessed by bo h di ec and indi ec me hodologies, and (iii) he
sou ce di e si y o success ul ma es ( a es o bipa en al inb eeding,
e ec i e numbe o a he s and male ep oduc i e a iance).
Fu he , we expec o ind, (i ) lowe le els o gene ic di e si y and
a s onge pa e n o spa ial gene ic s uc u e o he adul
popula ion, and ( ) highe le els o bipa en al inb eeding and
wi hin-mo he s co ela ed pa e ni y compa ed o con inuous
popula ion due o no iceable long- e m e ec s o agmen a ion.
Ma e ials and Me hods
S udy Species
Pis acia len iscus is an e e g een long-li ed sh ub ( e y a ely
a small ee up o 4 m in heigh ) ep esen a i e o he woody plan
species in he Medi e anean. The species is dioecious (i.e. obliga e
ou c osse ) and wind-pollina ed, wi h a na ow lowe ing pe iod in
he s udied a ea which spans om mid-Ma ch o la e Ap il (S.
No a, RG. Albaladejo and A. Apa icio, unpublished esul s).
F ui s, which a e small black one-seeded d upes, ipe om
Sep embe o Decembe when hey a e ac i ely consumed by
a wide a ay o small o mid-size bi ds, many o hem mig a o y
[11]. The species is no p o ec ed by law and pe mission o collec
plan ma e ial was ob ained om he Conseje ı
´a de Medio
Ambien e (Andalusian Regional Go e nmen ).
S udy Landscape and Sampling
The lowe ca chmen o he Guadalqui i i e (sou he n Spain)
is a ‘ elic ual’- ype ag icul u al landscape (sensu McIn y e & Hobbs
[12]) cha ac e ized by e y low habi a e en ion (na u al o semi-
na u al woodlands co e s only ca. 1% o i s po en ial a ea), low
connec i i y be ween agmen s and a high deg ee o an h opiza-
ion [13]. He e, as s udy si e we chose a ec angula a ea o ca.
70 ha (1000 6700 m) (c. U e a, 37u11’37’’N, 05u51’31’’W)
embedded in a highly an h opogenic a ea de o ed o ca le ising,
ce eal ields, ineya ds, oli e ees and sca e ed plan a ions o
Eucalyp us globulus Labill. The s udy si e i sel is also a highly
dis u bed si e (Fig. 1A, B) whe e only ca. 9.5 ha (,14% o he
a ea) co espond o emnan s o he o iginal co k-oak (Que cus sube
L.) ege a ion and sca e ed clumps o Pis acia len iscus,My us
communis L., Que cus cocci e a L. and Re ama sphae oca pa (L.) Boiss.
We chose his si e because (1) he a ea is ep esen a i e o he
cu en ege a ion ound in highly-humanized Medi e anean
landscapes, (2) he numbe o bushes o P. len iscus was la ge
enough o gua an ee he ep esen a i eness o he s udy, (3) e e y
indi idual plan could be accessed and iden i ied wi h ease and (4)
ou side he s udy si e no dense clump o s and o P. len iscus exis s
in se e al km a ound (only sca e ed bushes can be ound
elsewhe e). Whe e necessa y, he landowne s au ho ized access
o p i a e a eas.
Wi hin he s udy si e we pe o med a consequen ial sea ch and
iden i ied 514 ep oduc i e plan s o P. len iscus, 350 emales and
164 males ( emale-biased sex a io, x
2
= 34.79, d = 1, P,0.001).
In Oc obe 2007, we selec ed 29 mo he plan s dis ibu ed
h oughou he si e (mean 6SD among-mo he dis ance 425 m
6237 m, ange 5–914 m; Fig. 1B) om which we collec ed a mean
(6SD) numbe o ully ma u e black ui s o 69.6659.3 ( ange
36–306) di ec ly om all o e he plan c owns. O e all, we
collec ed 2014 ui s and he exis ence o a iable seed inside was
e i ied by sinking hem in wa e . Because many black ui s also
con ained abo ed emb yos, he inal p ogeny a ay consis ed o
690 seeds a ailable o gene ic analysis, wi h a mean (6SD)
numbe o seeds pe mo he plan o 24 (64). Young lea es o all
164 P.len iscus ep oduc i e males in he s udy si e and he 29
mo he plan s we e also collec ed and kep d ied in silica-gel un il
DNA isola ion. All he s udied plan s we e geo e e enced.
DNA Isola ion and Geno yping
We isola ed o al genomic DNA om seeds and adul plan s
wi h he In iso b DNA Plan HTS 96 Ki (In i ek, Be lin-Buch,
Ge many) acco ding o he manu ac u e ’s p o ocol. We ampli ied
se en unlinked polymo phic nuclea mic osa elli e loci ollowing
ampli ica ion condi ions and PCR cycle p o iles p o ided in
Albaladejo e al. [14]. Ampli ied p oduc s we e luo escen ly
labelled, wi h 6-FAM, NED, VIC o PET, and analysed on an
ABI 3730 DNA Analyze (PE Applied Biosys ems, Fos e Ci y,
CA, US). Finge p in p o iles we e au oma ically sco ed wi h he
so wa e GeneMappe .3.7 (PE Applied Biosys ems, Fos e Ci y,
CA, US) and isually inspec ed o co ec ions. All seeds we e
success ully geno yped o a leas i e loci each.
Pollen Flow and Shape o he Pollen Dispe sal
Dis ibu ion
The pollen mo emen among indi idual plan s wi hin he s udy
si e was assessed using pa e ni y analyses o assign each seed o i s
mos -likely a he by he maximum likelihood app oach imple-
men ed in Famoz [15]. Con idence in pa e ni y assignmen s was
ob ained by compa ing he dis ibu ion o he loga i hm o he odd
a ios (LOD sco es) o he mos -likely a he s o 50000 syn he ic
seeds wi h hei a he chosen among he 164 po en ial males, o
he dis ibu ion o LOD sco es o he mos -likely a he o 50000
seeds whose pa e nal geno ype was andomly gene a ed acco ding
o e e ence popula ion allele equencies. In wind-pollina ed
species, le els o pollen immig a ion can be high [16], so ex ac ing
allele equencies om he local male popula ion can be mis-
leading since he ac ual b eeding popula ion migh be much
la ge . Then, we ex ac ed he e e ence allele equencies om
he bigge sample o 690 seeds, a e sub ac ing he ma e nal
con ibu ion ollowing Ha dy e al. [17]. Th eshold alue o
ejec ing a candida e male as a ue a he was TF = 5.50 (i.e. he
alue a he in e sec ion o he wo LOD sco e dis ibu ions [15].
Since we a e mos ly in e es ed in desc ibing pa e ns o pollen low
a he han minimizing Type I e o s in he assigning o seeds o
speci ic a he s, he geno yping e o was se o ze o o a oid
inc easing assignmen e o [18]. The inb eeding coe icien (F)
was se o 0.152, he alue es ima ed o he adul popula ion (see
Resul s). A e unning he analysis we placed each analysed seed
Pollen Flow in Dis u bed Landscapes
PLOS ONE | www.plosone.o g 2 No embe 2012 | Volume 7 | Issue 11 | e49012
in o one o he ollowing h ee ca ego ies: (i) no compa ible a he
in he s udy si e (i.e. he minimum bound o incoming pollen low
a e), (ii) a leas one compa ible a he bu wi h a LOD sco e ,TF,
and (iii) one o mul iple candida e a he s wi hin he si e wi h
aLOD sco e .TF. In he la e case, we assigned pa e ni y o he
spa ially closes male. We belie e ha he po en ial bias in oduced
by his p ocedu e is negligible because mos ies in ou da ase
occu ed be ween gene ically ela ed neighbou ing males.
To check whe he ma ing success was a me e unc ion o he
spa ial posi ion o males and emales (i.e. ‘ la ’ dispe sal ke nel), we
compa ed he obse ed equency dis ibu ion o ma ing e en s
wi h i s po en ial dis ibu ion (conside ing all 4756 possible ma ing
e en s be ween he 164 male and 29 emale plan s) using
a Kolmogo o -Smi no es . We also e alua ed he di ec ionali y
(i.e. iso opic s. aniso opic) o his dis ibu ion [19] wi h ega d o
he di ec ion o he p e alen winds du ing he lowe ing pe iod
(15 h Ma ch o 31s Ap il). Wind da a we e aken om he nea es
Figu e 1. Ae ial pho og aphy o he s udy si e (black squa e) embedded in a highly an h opogenic a ea (A). Close up o he s udy si e
showing he 164 ep oduc i e Pis acia len iscus males (whi e ci cles) and he 29 mo he plan s (whi e iangles) sampled o ma ing and pa e ni y
analyses (B). Ne wo k o e ec i e pollina ion e en s de ec ed be ween males (do s) and emales ( iangles) in he pa e ni y analysis (C). Red do s in
panel B ma k he posi ion o males si ing a leas ou seeds (see ex o de ails).
doi:10.1371/jou nal.pone.0049012.g001
Pollen Flow in Dis u bed Landscapes
PLOS ONE | www.plosone.o g 3 No embe 2012 | Volume 7 | Issue 11 | e49012
clima ic s a ion (‘Los Palacios y Villa anca’; 37u10’49’’N,
05u56’15’’W) a simila al i ude and clima ic condi ions (da a
a ailable a h p://www.jun adeandalucia.es/ag icul u aypesca/
i apa). Di ec ional equency his og ams o he dis ibu ion o
obse ed and po en ial ma ing, and p e ailing wind di ec ion we e
compa ed h ough Wa son’s U
2
- es using he R package ci cula
[20].
The shape o he e ec i e pollen dispe sal cu e was cha ac-
e ized using wo di e en indi ec me hods. Fi s ly, we use he
KinDis app oach [21], which is based on he expec ed decay o
co ela ed pa e ni y among ma e nal pai s wi h dis ance, and
p o ides minimum squa ed-e o es ima es o he scale (a) and
shape (b) pa ame e s o he powe -exponen ial dispe sal unc ion as
well as he a e age e ec i e pollina ion dis ance (d) de i ed om i
[22]. The pa ame e bcon ols he ail o he dis ibu ion so ha
b,1 p o ides a - ailed dispe sal unc ions (which can be e
accoun o long-dis ance pollen dispe sal e en s) while b.1
p o ides hin- ailed unc ions. In ou case, we i s checked ha
among-mo he s co ela ed pa e ni y signi ican ly dec eased wi h
(log) dis ance (Pea son =20.125; P= 0.012, Figu e S1) and hen
se a h eshold dis ance o un ela ed pollen clouds o 300 m. This
analysis was ca ied ou using Poldisp .1.0c so wa e [23].
Secondly, we used he Bayesian Mixed E ec Ma ing model [9]
which es ima es no only he shape pa ame e o he powe -
exponen ial unc ion, bu also join ly assesses sel ing (s) and
immig a ion a es (m), he a io be ween obse ed and e ec i e
male densi y (d
obs
/d
ep
) and, ema kably, he ela i e indi idual
ecundi y o each male (see below) in he s udy si e. We used he
ollowing minimum and maximum alues as p io in o ma ion:
d
obs
/d
ep
= 1, 150; d= 50, 1500; b= 0.1, 10; and m= 0.4, 0.95.
Because P. len iscus is dioecious, we se o ze o he p io alues o s.
Since he analysis equi es om e e ence ou side allele equen-
cies o he calcula ion o he immig a ion a e (see eq. 6 in Klein
e al. [9]), we used he allele equencies ob ained om he se o
569 seeds o which no a he s wi hin he s udy si e could be
con iden ly assigned (LOD sco e ,TF) a e he Famoz analysis.
The analysis was un o 100000 i e a ions plus an ini ial bu n-in
pe iod o 20000 i e a ions. We also included a co a ia e in he
analysis which epo s on he local en i onmen whe e each male
was loca ed in he s udy si e (simila o he neighbou hood densi y
in [24]) ollowing h ee ca ego ies o dec easing neighbou hood
densi y: (1) unde a dense canopy o co k oak o eucalyp us ees,
(2) in spa se sh ublands wi h no ees in he canopy, and (3) in
linea hedges. This analysis was pe o med wi h he so wa e
MEMM 1.1 [9].
Ma ing Sys em, E ec i e Densi y o Pollen Dono s and
Male Rep oduc i e Va iance
Bipa en al inb eeding (i.e. he ma ing be ween close ela i es;
m
-
s
) was calcula ed wi h he so wa e MLTR 3.3 [25] om he
mul ilocus (
m
) and single locus (
s
) ou c ossing a es es ima ed
h ough he New on-Raphson algo i hm. We assessed
m
-
s
s a is ical signi icance by compa ing 1000 boo s ap alues o
m
s.
s
by means o S uden ’s pai ed - es s. Boo s ap eplica es we e
done wi h esampling o amilies. Wi hin-mo he co ela ed
pa e ni y (
p
) was calcula ed as wice he a e age pai wise kinship
coe icien F
ij
[26] be ween pa e nal genes o seed pai s. To check
whe he he composi ion o p ogeny a ays, measu ed as he
p opo ion o ull sibs wi hin ma e nal amilies, was a di ec
consequence o he local en i onmen o emales, we co ela ed
indi idual wi hin-mo he
p
alues agains he dis ance o he
nea es male and o he nea es h ee and i e males, espec i ely.
The e ec i e numbe o pollen dono s o he sampled mo he s in
he s udy si e (N
ep
) was deduced as
p
21
. We compa ed his esul
wi h he N
ep
alue de i ed om he mean alue o he d
obs
/d
e
p obabili y densi y unc ion ob ained om he MEMM analysis.
Male ep oduc i e success in he s udy si e was es ima ed
di ec ly om he pa e ni y analysis wi h Famoz and wi h he
MEMM so wa e. In he Mixed E ec Ma ing model, male
ela i e ecundi ies a e modelled as andom e ec s unde
a Bayesian amewo k. We an wo analyses assuming indi idual
male ecundi y o ollow ei he a log-no mal o a gamma
dis ibu ion [9].
Gene ic Di e si y and Spa ial Gene ic S uc u e (SGS) o
Adul s
Fo he adul plan s (29 emales and 164 males), we also
calcula ed single-locus desc ip i e di e si y pa ame e s using Fs a
.2.9.3.2 [27], including he numbe o alleles (A), he expec ed
he e ozygosi y (H
e
) and he inb eeding coe icien ( ). Depa u e
om Ha dy-Weinbe g (HW) equilib ium was assessed by means o
a Ma ko Chain-based exac es using Genepop .4 [28] unde
de aul se ings (10000 dememo iza ion s eps, 20 ba ches and 5000
i e a ions pe ba ch). Null allele equencies we e also es ima ed
wi h Genepop .4.
We analysed he exis ence o SGS in he adul s by eg essing he
pai wise kinship coe icien s (F
ij
) be ween indi iduals on he
loga i hm o he spa ial dis ance as co esponding o a bidimen-
sional space [29]. The signi icance o SGS was ob ained by
compa ing he obse ed eg ession slope wi h ha ob ained om
andomly pe mu ing (1000 imes) he spa ial loca ion o he adul
indi iduals [30]. Pa e ns o SGS we e isualized by cons uc ing
a co elog am plo ing he a e age F
ij
alues in o dis ance classes.
To allow a di ec compa ison wi h he con inuous popula ion
s udied by Albaladejo e al. [10] we es ic ed he analysis o same
maximal dis ance (460 m) and cons uc ed he same 10 dis ance
classes. App oxima e 95% con idence in e als (CI) we e calcu-
la ed o he F
ij
alues as wice he s anda d e o (SE) ob ained by
jackni ing o e loci. The in ensi y o he SGS was also quan i ied
by he S
p
s a is ic, a dimensionless pa ame e use ul o pe o m
compa isons be ween species and popula ions [30]. S
p
is calcula ed
as b-log/(F
(1)
21), whe e F
(1)
is he mean F
ij
among indi iduals
pai s in he i s dis ance class and b-log is he slope o he
co elog am. All SGS analyses we e pe o med wi h he so wa e
SPAGeDi .1.2 [31].
Resul s
Pollen Flow and Shape o he Pollen Dispe sal
Dis ibu ion
The cumula ed exclusion p obabili y (when he mo he is
known) o he se en mic osa elli e loci used was 0.995, suppo ing
he sui abili y o ou da ase o conduc a pa e ni y analysis (see
Table 1). The es ima ed equencies o null alleles we e on a e age
low, only mode a e ($0.150) o he loci Pislen 114 and Pislen R05
(Table 1). Ou o he 690 geno yped seeds, 121 (17.5%) had a
leas one candida e a he wi h a LOD sco e .TF. Addi ionally,
63 seeds (9.1%) had a leas a compa ible a he bu wi h a LOD
sco e below he h eshold equi ed o a con iden assignmen .
This esul means ha a minimum o 73.3% and a maximum o
82.4% o he seeds we e si ed by a he s loca ed ou side ou s udy
si e.
The pa e ni y analysis also de ec ed high connec i i y wi hin he
s udy a ea (Fig. 1C). The mean (6SD) dispe sal dis ance o he
121 con iden ly assigned male-o sp ing pai s was 412 m (6276)
( ange 9–970 m) wi h abou 50% o he seeds being si ed by
a he s loca ed mo e han 370 m apa . The success o ma ing
e en s was no a me e unc ion o he spa ial dis ibu ion o males
Pollen Flow in Dis u bed Landscapes
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and emales since he obse ed ma ing equency dis ibu ion was
signi ican ly di e en om he po en ial dis ibu ion (K-S
d= 0.280; P,0.001). In spi e o he la ge dis ance o he obse ed
ma ings, hese occu ed a dis ances sho e han expec ed (Fig. 2).
Thus, si ing e en s we e no p oduced a andom wi h ega d o
among-ma es dis ance.
Mos pollina ion e en s ook place in he S-N di ec ion (Fig. 3A)
acco ding o he ela i e spa ial a angemen o males and emales
sampled in he plo (Fig. 3B). Du ing he blooming phase,
p e ailing winds occu ed in N-E and S-W di ec ions (Fig. 3C).
The obse ed di ec ional dis ibu ion o ma ing e en s was
signi ican ly di e en om bo h he expec ed andom dis ibu ion
conside ing spa ial posi ion o males and emales alone (U
2
-
es = 0.432; P,0.001) and he one o p e ailing winds du ing he
lowe ing pe iod (U
2
- es = 0.252; P,0.05). In e es ingly, he
obse ed dis ibu ion was a somewha combined dis ibu ion
conside ing bo h he spa ial posi ion and wind di ec ion.
The KinDis analysis success ully i ed a powe -exponen ial
unc ion o he da a and p o ided poin es ima es o he scale and
shape pa ame e s o a= 2.8 10
24
and b= 0.16, as well as an
a e age e ec i e dispe sal dis ance o d= 268.5 m. No mal,
geome ic o exponen ial unc ions showed a poo e i based on
he leas -squa e esiduals ( esul s no shown).
The mean condi ional log-likelihood o he wo uns wi h
MEMM, ei he assuming a log-no mal o gamma dis ibu ion o
indi idual male ecundi y, we e 28145 and 28138 espec i ely;
an app oxima e Bayes ac o , compu ed as he a io o he
es ima ed likelihoods, s ongly suppo ed he model wi h he
gamma dis ibu ion (BF <1097; alues abo e 100 a e usually
conside ed as decisi e o suppo one model agains he al e na i e
[32]). Consequen ly, we show he e only he esul s o he second
un (gamma dis ibu ion). Rega ding he i ed unc ion, he Mixed
E ec s Ma ing model p o ided simila esul s o he KinDis
app oach, wi h mean alues (and 95% c edibili y in e als) o he
pos e io dis ibu ion o b= 0.19 (0.10–0.46) and d= 229 m (52–
1069 m). Addi ionally, he immig a ion a e es ima ed by MEMM
was e y high, m= 0.937 (0.915–0.949), which suppo s he esul s
o he di ec pa e ni y analysis.
Ma ing Sys em, E ec i e Densi y o Pollen Dono s and
Male Rep oduc i e Va iance
In he s udy si e, we ound a highly signi ican di e ence
be ween mul ilocus and single-locus ou c ossing a es (
m
= 0.999
s.
s
= 0.856; S uden ’s = 94.466, P,0.001), indica ing ha abou
14% o ma ings occu ed be ween gene ically ela ed plan s.
Indi idual wi hin mo he co ela ed pa e ni y showed a signi ican
nega i e co ela ion wi h he (log)dis ance o he nea es male
(Pea son =20.382, P= 0.041) bu his end anished and
became non-signi ican when e e ed o he a e age dis ance o
he nea es h ee and i e males ( =20.308, P= 0.104 and
=20.297, P= 0.118). The a e age wi hin mo he co ela ed
pa e ni y calcula ed om kinship coe icien s was also high
(
p
= 0.231) indica ing ha in a e age abou 23% o seeds in
a gi en mo he plan we e si ed by he same a he . This alue
ansla es in o an e ec i e numbe o a he s N
ep
= 4.3. The Mixed
E ec s Ma ing model p o ided a pos e io mean alue (95%
c edibili y in e al) o he a io d
obs
/d
e
= 16 (2.7–45) which
Table 1. Gene ic di e si y pa ame e s o Pis acia len iscus
adul plan s geno yped a se en mic osa elli e loci.
Locus A H
e
Null Exc
Pislen 21 14 0.820 0.204*** 0.090 0.684
Pislen 114 6 0.704 20.067** 0.178 0.497
Pislen 333 18 0.883 0.249*** 0.112 0.745
Pislen 501 10 0.774 0.210*** 0.097 0.587
Pislen 510 15 0.779 0.189*** 0.085 0.609
Pislen 526 3 0.432 0.010
ns
0.006 0.180
Pislen R05 3 0.212 0.205** 0.150 0.079
O e all 69 0.658 0.152*** – 0.995
**P,0.01;
***P,0.001;
ns
no signi ican .
Numbe o alleles (A), expec ed he e ozigosi y (H
e
), Wei and Cocke ham’s
(1984) inb eeding coe icien ( ), null allele equency (Null) and exclusion
p obabili y o pa e ni y analysis (Exc).
doi:10.1371/jou nal.pone.0049012. 001
Figu e 2. F equency his og ams o e ec i e pollina ion dis ances (black ba s) es ima ed ia pa e ni y analysis and pai wise
dis ances be ween males and he sampled mo he plan s (whi e ba s).
doi:10.1371/jou nal.pone.0049012.g002
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co esponded o a N
ep
alue o 10.2 (3.6–60.7) acco ding o he
obse ed census o males (N
obs
= 164) in he s udy si e.
Based on he pa e ni y analysis, male ep oduc i e success was
highly lep oku ic since 114 ou he 164 males (70%) did no si e
any seed in any o he 29 emales sampled wi hin he s udy a ea.
Only nine a he s (5.5%) we e esponsible o nea ly 50% o he
assignmen s ( ed do s in Fig. 1B), each o hem si ing ou o mo e
seeds and one a he si ing 15 seeds. These esul s we e
conco dan wi h hose ob ained om he MEMM analysis,
es ima ed join ly wi h he dispe sal pa ame e s, which e ealed
e y ew males wi h a e y high ecundi y ela ed o a majo i y o
males wi h a poo ep oduc i e con ibu ion. Fecund males we e
spa ially loca ed in spa se sh ublands and linea hedges while none
occu ed unde a dense ee canopy (Fig. 4).
Gene ic di e si y and ine-scale SGS o adul s.
Gene ic di e si y o P. len iscus was ela i ely high (A= 69,
H
e
= 0.658). Howe e , mos loci showed a signi ican excess o
homozygo es (Table 1).
The o e all slope o he co ela ion be ween pai wise kinship
and (log) dis ance was highly signi ican (b-log = 20.0117;
P,0.001) indica ing ha gene ically ela ed adul indi iduals we e
agg ega ed ac oss he space. The co elog am e ealed a sha p
decline in he kinship coe icien s wi h dis ance, wi h signi ican
posi i e a e age F
ij
in he i s h ee dis ance classes, which
ex ended h ough a dis ance o app oxima ely 85 m (Fig. 5). The
calcula ed alue o he S
p
s a is ic was 0.0122.
Discussion
Long e m iabili y o popula ions elies on main aining
adequa e le els o gene ic a ia ion and gene low [1]. Conse-
quen ly, a conside able esea ch ac i i y on he mechanisms o
dispe sal o pollen and seeds and i s gene ic and demog aphic
consequences has been de eloped in ecen decades [33]. F om
a conse a ion and landscape gene ics pe spec i e, analyzing
pa e ns o pollen dispe sal in dispa a e landscapes should allow
isualizing hose shi s in he pollina ion biology pa e ns o species
ha could ul ima ely os e inb eeding dep ession, gene ic
impo e ishmen and popula ion di e gence.
Pollen Flow
Ou analyses p o ided compelling e idence ha pollen mo e-
men in he s udied a ea was ex ensi e, no only wi hin he s udy
si e, bu also om ou side: be ween 73–93% (depending on he
me hod) o he sampled seeds we e si ed by incoming pollen low.
Admi edly, we igno e he ac ual loca ion o he male plan s ha
con ibu ed so ema kably as pollen dono s, bu we belie e ha
hey mus p obably be loca ed in p i ileged posi ions (see below) in
he icini y o he s udy si e, despi e only sca e ed bushes and e y
small clumps o P. len iscus can be ound in he su oundings. The
closes ela i ely la ge popula ion o he species is loca ed en
kilome es eas wa ds bu can p obably be uled ou as a sou ce o
pollen in ou da a se gi en he es ima ed low numbe s o e ec i e
pollen dono s ound o single mo he s. Indeed, i incoming pollen
was o igina ed in a la ge popula ion, hen, many di e en a he s
would be expec ed o con ibu e o single mo he s, inc easing N
ep
es ima es. The e o e, despi e he s udy si e appea s emo e and
Figu e 3. Wind ose pe cen age equency his og ams o (A)
he di ec ion o ma ing e en s de ec ed in he pa e ni y
analysis, (B) he di ec ion o andom po en ial ma ing e en s
(i.e. jus condi ioned by he spa ial loca ion o males and
emales), and (C) he di ec ion o winds du ing he lowe ing
season.
doi:10.1371/jou nal.pone.0049012.g003
Pollen Flow in Dis u bed Landscapes
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disconnec ed, ou esul s highligh he cohesi e ole ha neglec ed
small ege a ion pa ches o isola ed indi iduals may play in highly
agmen ed landscapes [34], pa icula ly i high e ec i e pollen
dispe sal is inhe en o he ep oduc i e biology o he species.
In ag eemen , bo h he di ec and indi ec me hods ha we used
indica ed la ge a e age pollina ion dis ances, al hough he
es ima es a ied somehow ac oss he me hods. The pa e ni y
analysis, which is cons ic ed by he p ecise spa ial dis ibu ion o
sampled emale plan s (gi en ha no all emale plan s we e
sampled), p o ided an es ima e o abou 400 m, well abo e he
mean dispe sal dis ance in e ed h ough di ec me hods o wind-
pollina ed species [35]. Bo h indi ec ma ing models (KinDis and
Klein’s mixed e ec model) showed an a e age dispe sal dis ance
abo e 200 m, which is also a compa a i ely high es ima e (e.g.
[36]). This la e is pe haps a mo e ealis ic es ima e because he
i ing o dispe sal ke nels p o ides a mo e gene al pic u e o
pollen dispe sal pa e ns, as i pollen could land e e ywhe e
[18,34]. A di ec compa ison wi h he pollen dispe sal ke nel in
a p e iously-s udied con inuous popula ion [10] was no possible
due o he weak gene ic s uc u e o he e ec i e pollen cloud in
ha case, which did no allow o con e gence o model i ing
algo i hms. Howe e , his esul sugges s pollen dispe sal ke nels in
un agmen ed si ua ions o be e en la e han in agmen ed
ones.
Al hough we ha e shown ex ensi e pollen low in his
agmen ed landscape he e is also an impo an componen o
Figu e 4. Indi idual ela i e ecundi ies o he 164
Pis acia len iscus
male plan s es ima ed using Klein’s Mixed E ec Ma ing model.
Di e en shades e e o male plan s occu ing in di e en landscape ypologies: dense ee canopy (black), spa se sh ublands (g ey), and linea
hedges (whi e). Ba s ep esen he 95% c edibili y in e als.
doi:10.1371/jou nal.pone.0049012.g004
Figu e 5. A e age pai wise kinship coe icien s (
F
ij
) o adul plan s plo ed agains spa ial dis ance in he s udied agmen ed
landscape (ci cles) and in a p e iously-s udied la ge con inuous popula ion (squa es; eanalysed om [10]). E o ba s ep esen
app oxima e 95% con idence in e als and emp y symbols mean hey a e signi ican ly di e en om he null hypo hesis o no spa ial s uc u e
assessed ough pe mu a ion p ocedu es (1000 pe mu a ions). Symbols ha e been sligh ly sc olled o acili a e isualiza ion.
doi:10.1371/jou nal.pone.0049012.g005
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es ic ed pollen dispe sal a local scales. Below 500 m, he
pa e ni y analysis e eals mo e ma ings han expec ed a andom
(see Fig. 2), which suppo he iew o pollen dispe sal in wind-
pollina ed plan s o be cha ac e ized by a ac ion o sho dis ance
pollina ions and ano he ac ion o pollina ions occu ing a la ge
dis ances enla ging he ail o he pollen dispe sal dis ibu ion [35].
Assessing he di ec ional dis ibu ion o success ul ma ings is also
impo an because i may de e mina e he numbe o si es
a ailable o emale plan s. Ou sampling scheme esul ed in
he e ogenei y in he di ec ional dis ibu ion o males and he
sampled emales (wi h no hwa ds ma ings being clea ly a ou ed),
which su ely is esponsible o he high p opo ion o ma ings in
he S-N di ec ion we de ec ed (see Figs. 3A & B). In e es ingly, we
also de ec ed ela i ely high le els o success ul ma ing e en s in
he hi d (S-W) quad an , p obably in luenced by he S-W
p e ailing winds du ing he blooming pe iod (see Figs. 3A & C).
Many s udies ha e no de ec ed e ec s o egional winds on pollen
dispe sal pa e ns (e.g. [37,38]). This disco dance migh indica e
lack o po en ial males in a ou able wind di ec ions and/o ha
pollen dispe sal is in luenced by localized wind pa e ns, only
de ec able by placing me eo ological s a ions wi hin he s udy plo
(an issue, howe e , ha seems no ele an in he la lands o ou
s udy a ea). In addi ion, de ec ing clea aniso opic pollen dispe sal
pa e ns in he sp ead o pollen clouds may be also a echnical
issue since i equi es a g ea e geno yping e o (he e 29 mo he
plan s and 690 o sp ing) han usual [19].
Ma ing Sys em and Male Rep oduc i e Success
One aspec o he ep oduc i e ecology o plan s ha becomes
c ucial in agmen ed popula ions is co ela ed pa e ni y ( he
di e si y o a he s si ing p ogeny in a mo he ) because whe e
spa ial connec i i y and he a ailabili y o sui able places o seed
a i al and seedlings es ablishmen is educed, p ogeny a ays
composed o ull-sibs can pe o m poo ly compa ed o hose
composed o hal - o un ela ed sibs [39]. Mo eo e , i is expec ed
ha popula ion a i ion and he spa ial clumping o pollen sou ces
inc eases he p opo ion o ull-sibs in he annual c op o a emale
plan [40], an ou come ha Albaladejo e al. [10] p edic ed o
his species h ough nume ical simula ions. The e o e, i is e y
impo an o s ess ha in ou s udy si e, we ha e ound abou six-
old highe alues o co ela ed pa e ni y compa ed o he la ge,
dense and con inuous s and o he same species s udied by
Albaladejo e al. [10] (23% s. 3–8%, espec i ely).
Besides high co ela ed pa e ni y, we ha e also ound a s ong
a iance in male ela i e ecundi y since jus a ew males ha e
copped a high p opo ion o all ma ing e en s. To explain his
unbalance in male con ibu ions se e al easons ha e been
adduced, among hem di e en ial quan i y o quali y o he
pollen eleased [24], lack o synch ony in he blooming pe iod [41]
and/o densi y depended e ec s. In ou case, males ha si ed
a high numbe o o sp ing we e loca ed in a eas o low ege a ion
densi y (see ed do s in Fig. 1) on spa se sh ublands o hedges, bu
none unde a dense ee canopy (Fig. 2). In wind-pollina ed species
pollen g ains eleased a he edge o ege a ion pa ches ha e less
ae odynamic impedimen s o a el han hose eleased wi hin
closed s ands [42]. In ag eemen wi h his obse a ion, densi y
dependen ac o s and he a he ecological neighbo hood seem o
be de e minan in ou s udy si e; ne e heless, we canno ule ou
some e ec s om blooming synch ony and pollen quali y ac ing in
conce .
We ound signi ican le els o inb eeding in all bu one locus,
which in his dioecious species can be explained by signi ican
le els o bipa en al inb eeding. A ele an impac o null alleles
was uled ou because hei es ima ed equency was in a e age
low and because signi ican inb eeding was also de ec ed in he
same popula ion wi h o he nuclea ma ke s [43].
Bipa en al inb eeding accoun ed o a mode a e po ion (14%)
o he ma ing e en s, being p obably a s aigh consequence o he
exis ence o signi ican SGS (see below) and o he ac ha ma ing
we e mo e likely a sho dis ances han a andom (discussed
abo e). Fu he , he p opo ion o ull-sibs wi hin mo he p ogeny
a ays is signi ican ly highe as he dis ance o he nea es male
dec eases which sugges some deg ee o monopoliza ion o some
emales by hei closes male pollen cloud. Howe e , his in luence
is spa ially e y es ic ed since only he ela ionship wi h he
dis ance o he nea es male was signi ican . This nega i e
ela ionship (albei weake ) was also ound in a p e iously-s udied
con inuous popula ion bu only in one o he s udied seasons [10].
Gene ic Va ia ion and Fine-scale SGS o Adul s
Despi e he ex eme dis u bance o he s udied landscape,
gene ic di e si y o P. len iscus was ela i ely high and simila o ha
o o he Medi e anean woody plan s, ei he in agmen ed
popula ions o no [2], including ha ob ained in con inuous
popula ions o he same species [10]. Indi idual longe i y, obliga e
ou c ossing, high a es o pollen gene low and low popula ion
gene ic di e en ia ion a he egional scale [43] seem o make his
species esis an o an immedia e loss o allelic di e si y, simila ly
o o he long-li ed woody plan s [44].
Finally, he S
p
alue (0.0122) is indica i e o a s ong pa e n o
SGS, since i is oughly wice he ypical o wind-pollina ed
(0.0064) o animal-dispe sed (0.0088) plan species [30], and mo e
impo an , i is mo e han i e imes highe han he one ound o
he same species in a p e iously-s udied la ge and con inuous
popula ion whe e no signi ican pa e n o SGS was de ec ed
(S
p
= 0.0022; ecompu ed om [10]). Besides he sca ci y o
success ul pollen dono s, pa e ns o spa ial gene ic s uc u e a e
also dependen on he e ec i eness o seed dispe sal. Despi e
a wide a ay o bi d species eeds on ui s o P. len iscus [11], in ou
pa icula s udy sys em he ecological condi ions a e p obably
limi ing seed dispe sal and ec ui men (i.e. i he e a e e y ew
sui able sa e si es, dispe se s would no each dis an sa e si es, and
mos seeds would be dispe sed om only a ew mo he plan s
[45]). Ex eme an h opogenic p essu e can es ic sa e mic osi es
o ec ui men jus below he canopy o mo he plan s, os e ing
he o ma ion o gene ic pedig ees and ma e nal and pa e nal
co ela ions (i. e. he p obabili y o sha ing pa en al plan s) in he
seed ain.
Conclusions
The eal impac o an h opiza ion on he iabili y and
pe o mance o na u al plan popula ions is s ill con o e sial. So
a , ideal expe imen al designs unde a agmen a ion gene ics
pe spec i e a e e y di icul o mee in na u e. Mo eo e , o assess
he ac ual scale o gene dispe sal (pollen and/o seed) wi hin and
among plan popula ions (especially o ees and long-li ed
sh ubs) become essen ial o econcile he heo e ical and empi ical
e idence [7].
Keeping his idea in mind, his is one o he sca ce s udies
de o ed o assess pollen dispe sal and he shi s imposed by se e e
an h opiza ion in he ma ing sys em o a long-li ed woody plan
species. Speci ically, ou s udy illus a es he pa amoun impo -
ance o small clumps o isola ed indi idual plan s in keeping
gene ic connec i i y e en in, a i s glance, emo e and isola ed
popula ions. Indeed, we ha e ound gene ic di e si y (numbe o
alleles and he e ozygosi y) a le els ha , in he ligh o he
ecological cha ac e is ics o he s udy si e, we e unexpec ed.
Pollen Flow in Dis u bed Landscapes
PLOS ONE | www.plosone.o g 8 No embe 2012 | Volume 7 | Issue 11 | e49012
Ne e heless, we also ound ha mos male plan s did no si e
a single seed om he s udied emale plan p ogenies and ha he
ma ing sys em o P. len iscus was se e ely impac ed by agmen-
a ion: compa ed o a la ge and con inuous popula ion [10], he
highly-dis u bed popula ion s udied he e had inc eased bipa en al
inb eeding, inc eased co ela ed pa e ni y (and dec eased numbe
o pollen dono s) and highly signi ican spa ial gene ic s uc u e.
No only he pollen cloud pe cei ed by emales is less di e se, bu
also, due p obably o he sca ci y o sui ed places o ec ui men ,
he species is expe iencing he o ma ion o local pedig ees and
inc eased inb eeding, he p elude o gene ic impo e ishmen .
Suppo ing In o ma ion
Figu e S1 Co ela ion be ween among mo he co ela ed
pa e ni y (i.e. p opo ion o hal -sib among mo he s) agains he
dis ance (a loga i hmic scale) among mo he s pai s.
(PDF)
Acknowledgmen s
We hank he landowne s o pe mission o wo k in he a ea, E. Rubio and
M. Leo´n o assis ance du ing ield and lab wo k, C. de Vega o help ul
commen s on di e en e sions o he manusc ip and A. Se ano o
e iewing he English s yle. Lab wo k was pa ially conduc ed a he
Se icio de Biologı
´a Molecula o he Cen o de In es igacio´n, Tecnologı
´a
e Inno acio´n (CITIUS) o he Uni e si y o Se ille.
Au ho Con ibu ions
Concei ed and designed he expe imen s: AA RGA. Pe o med he
expe imen s: BG RGA. Analyzed he da a: SCGM BG RGA. W o e he
pape : RGA BG AA.
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Pollen Flow in Dis u bed Landscapes
PLOS ONE | www.plosone.o g 9 No embe 2012 | Volume 7 | Issue 11 | e49012