Extensive Pollen Flow but Few Pollen Donors and High Reproductive Variance in an Extremely Fragmented Landscape

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 PLOS ONE | www.plosone.o g 4 No embe 2012 | Volume 7 | Issue 11 | e49012 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 Pollen Flow in Dis u bed Landscapes PLOS ONE | www.plosone.o g 5 No embe 2012 | Volume 7 | Issue 11 | e49012 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 PLOS ONE | www.plosone.o g 6 No embe 2012 | Volume 7 | Issue 11 | e49012 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 Pollen Flow in Dis u bed Landscapes PLOS ONE | www.plosone.o g 7 No embe 2012 | Volume 7 | Issue 11 | e49012 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. Re e ences 1. Young AG, Boyle T, B own T (1996) The popula ion gene ic consequences o habi a agmen a ion o plan s. T ends Ecol E ol 11: 413–418. 2. Jump AS, Pen˜uelas J (2006) Gene ic e ec s o ch onic habi a agmen a ion in a wind-pollina ed ee. P oc Na Acad Sci USA 103: 8096–8100. 3. Fo e´ SA, Hickey RJ, Nanka JL, Gu man SI, Schae e RL (1991) Gene ic s uc u e a e o es agmen a ion: a landscape ecology pe spec i e on Ace saccha um. 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Ga cı ´a C, Jo dano P, A oyo JM, Godoy JA (2009) Ma e nal gene ic co ela ions in he seed ain: e ec s o ugi o e ac i i y in he e ogeneous landscapes. J Ecol 97: 1424–1435. Pollen Flow in Dis u bed Landscapes PLOS ONE | www.plosone.o g 9 No embe 2012 | Volume 7 | Issue 11 | e49012