Limited genetic connectivity between gorgonian morphotypes along a depth gradient

RESEARCH ARTICLE Limi ed Gene ic Connec i i y be ween Go gonian Mo pho ypes along a Dep h G adien Fede ica Cos an ini 1 *, And ea Go i 2,6 , Pablo Lopez-González 3 , Lo enzo B aman i 4 , Se gio Rossi 5 , Josep-Ma ia Gili 6 , Ma co Abbia i 1,7 1Dipa imen o di Scienze Biologiche, Geologiche ed Ambien ali (BiGeA) & Cen o In e dipa imen ale di Rice ca pe le Scienze Ambien ali (CIRSA), Uni e si y o Bologna, CoNISMa, Via S. Albe o 163, I-48123, Ra enna, I aly, 2Depa amen d’Ecología, Facul a de Biologia, Uni e si a de Ba celona, A . Diagonal 643, 08028, Ba celona, Spain, 3Biodi e sidad y Ecología de In e eb ados Ma inos, Depa amen o de Zoología, Facul ad de Biología, Uni e sidad de Se illa, A . Reina Me cedes 6, 41012, Se illa, Spain, 4So bonne Uni e si és, UPMC Uni Pa is 06, CNRS, Labo a oi e d'Ecogéochimie des En i onnemen s Ben hiques (LECOB), Obse a oi e Océanologique, 66650, Banyuls su Me , F ance, 5Ins i u de Ciència i Tecnologia Ambien als, Uni e si a Aun ònoma de Ba celona, Ce danyola del Vallés, Spain, 6Ins i u de Ciències del Ma –CSIC, Pg. Ma i im de la Ba celone a 37–49, 08003, Ba celona, Spain, 7Consiglio Nazionale delle Rice che, Is i u o di Scienze Ma ine, ISMAR, Bologna, I aly * ede ica.cos an [email protected] Abs ac Go gonian species show a high mo phological a iabili y in ela ion o he en i onmen in which hey li e. In coas al a eas, pa ame e s such as empe a u e, ligh , cu en s, and ood a ailabili y a y signi ican ly wi h dep h, po en ially a ec ing mo phology o he colonies and he s uc u e o he popula ions, as well as hei connec i i y pa e ns. In opical seas, he exis ence o con- nec i i y be ween shallow and deep popula ions suppo ed he hypo hesis ha he deep co al ee s could po en ially ac as ( ep oduc i e) e ugia os e ing e-coloniza ion o shallow a eas a e mo ali y e en s. Mo eo e , his hypo hesis is no so clea accep ed in empe a e seas. Eunicella singula is is one o he mos common go gonian species in No hwes e n Medi e a- nean Sea, playing an impo an ole as ecosys em enginee by p o iding biomass and com- plexi y o he co alligenous habi a s. I has a wide ba hyme ic dis ibu ion anging om abou 10 m o 100 m. Two dep h- ela ed mo pho ypes ha e been iden i ied, di e ing in colony mo - phology, scle i e size and shape, and occu ence o symbio ic algae, bu no in mi ochond ial DNA haplo ypes. In he p esen s udy he gene ic s uc u e o E.singula is popula ions along a ho izon al and ba hyme ic g adien was assessed using mic osa elli es and ITS1 sequences. Res ic ed gene low was ound a 30–40 m dep h be ween he wo Eunicella mo pho ypes. Con e sely, no gene ic s uc u ing has been ound among shallow wa e popula ions wi hin a spa ial scale o en kilome e s. The b eak in gene low be ween shallow and deep popula ions con ibu es o explain he mo phological a iabili y obse ed a di e en dep hs. Mo eo e , he limi ed e ical connec i i y hin ed ha he e ugia hypo hesis does no apply o E.singula is. Re-coloniza ion o shallow wa e popula ions, occasionally a ec ed by mass mo ali y e en s, should hen be mainly ueled by la ae om o he shallow wa e popula ions. PLOS ONE | DOI:10.1371/jou nal.pone.0160678 Augus 4, 2016 1/20 a11111 OPEN ACCESS Ci a ion: Cos an ini F, Go i A, Lopez-González P, B aman i L, Rossi S, Gili J-M, e al. (2016) Limi ed Gene ic Connec i i y be ween Go gonian Mo pho ypes along a Dep h G adien . PLoS ONE 11 (8): e0160678. doi:10.1371/jou nal.pone.0160678 Edi o : Lo enzo Zane, Uni e si y o Pado a, ITALY Recei ed: Feb ua y 2, 2016 Accep ed: July 24, 2016 Published: Augus 4, 2016 Copy igh : © 2016 Cos an ini 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. Da a A ailabili y S a emen : All ele an da a a e wi hin he pape and i s Suppo ing In o ma ion iles. We ha e ob ained he GenBank Accession numbe s o all he sequences used in he wo k (GenBank om KX051564 o KX051577 and om KX002245 o KX002255). Funding: AG was unded by a I3P con ac o he Consejo Supe io de In es igaciones Cien i icas (Re . I3P-BPD2005). SR wan s o hank he suppo o he Gene ali a de Ca alunya o MERS (2014 SGR - 1356) and he Ma ie Cu ie In e na ional Ou going Fellowship (ANIMAL FOREST HEALTH, p ojec numbe 327845). The s udy was unded by a 2003- 2004 INTERREG p ojec be ween Ca alonia- In oduc ion Ma ine modula o ganisms exhibi a la ge mo phological a iabili y, wi h pheno ypic plas ici y a likely sou ce o his a iabili y (see [1] o a e iew). In co als and go gonians a iabili y has led o conside able con usion ega ding species bounda ies and axonomy. Gene ic in es iga ions can con ibu e o he ecogni ion o c yp ic species bounda ies and popula ion iden i ica ion. Comp ehensi e s udies combining mo phological, ecological and gene ic da a can cla i y whe he gene ic di e en ia ion unde lies mo phological a ia ion. Mo eo e , he in eg a i e app oach can help o esol e and ⁄o e ise axonomic a ini ies among closely ela ed species [2]). Many en i onmen al pa ame e s (e.g. hyd odynamics, salini y, i adiance, ophic esou ces) can in luence o ganisms’mo phology and d i e gene ic di e en ia ion [3]. Each pa ame e exe s i s in luence independen ly, bu hey may ac syne gis ically and hei e ec is mo e e iden along en i onmen al g adien s (e.g. la i ude, dep h). Indeed, i is widely accep ed ha he dep h g adien , in eg a ing se e al en i onmen al pa ame e s (e.g. empe a u e, ligh , hyd odynamics), may a ec popula ion s uc u e, colony mo phology, and connec i i y pa - e ns o co als and go gonians [4–7]. Occu ence o high connec i i y be ween shallow and deep popula ions ga e ise o he hypo hesis ha deep co al ee s can po en ially ac as ( ep oduc i e) e ugia [8], os e ing he e-coloniza ion o shallow a eas a e mo ali y e en s [9]. Recen s udies con lic wi h his hypo hesis, by showing ha dep h ela ed pa e ns o gene ic s uc u ing a y acco ding o spe- cies and si es [10,11]. P ada e al.[7] showed ha colonies o he opical go gonian Eunicea lexuosa exposed o high wa e mo ion in shallow habi a s compa ed o hose li ing in deepe habi a s a e alle , ha e bigge calices, hicke b anches and de eloped on a single plane. Shal- low and deep colonies o E. lexuosa belong o wo gene ic lineages [7], while wi hin lineages high connec i i y was obse ed among popula ions ac oss geog aphic dis ances o housands o kilome e s. Dep h adap i e di e gence gene a es a pa e n whe e neu al gene ic di e gence is p ima ily pa i ioned by habi a s, wi h li le geog aphic s uc u e [12]. Adap a ion o local en i onmen al condi ions ha e been also sugges ed as a d i e o gene ic s uc u ing ac oss habi a s in he opical co als Se ia opo a hys ix [10] and Mon as aea ca e nosa [13]. Go gonian co als o he genus Eunicella a e among he mos ep esen a i e ecosys em engi- nee s p o iding biomass and s uc u al complexi y in subli o al communi ies. Among he 18 ecognized Eunicella species, 6 species a e p esen in he Medi e anean Sea: E.singula is,E. ca olinii.E. e ucosa,E. ili o mis,E.gazella and E.labia a. The dis ibu ion o he las h ee species is limi ed o he S ai o Gib al a and o he Albo an Sea, and hey a e conside ed mainly A lan ic. E.ca olinii and E.singula is occu bo h in he wes e n and eas e n Medi e a- nean Sea, being he la e also p esen in ew loca ions along he A lan ic coas nea he S ai o Gib al a . E.singula is is he only species showing a high mo phological a iabili y along a dep h g adien om 10 o 70 m in he wes e n Medi e anean Sea [14,15](Fig 1;[16]). A shallow and a deep mo pho ype o E.singula is ha e been iden i ied (see [17], [16], [18] o a de ailed desc ip ion o he geog aphical and ba hyme ic dis ibu ion, and he a iabili y o he wo mo pho ypes). The wo mo pho ypes signi ican ly di e in colony/scle i e shape, as well as in ophic ecology [19] and o he p esence/absence o symbio ic algae [16]. Théodo [17] associa ed he deep mo pho ype o he aposymbio ic ( acul a i e symbio ic) o m o E.sin- gula is, aising a ques ion abou i s axonomic s a us. Jus as o he o he go gonians [20–22], mi ochond ial DNA in Eunicella species has a e y low mu a ion a e and is no a use ul ma ke o disc imina e be ween he wo E.singula is mo pho ypes as well as among he di e - en Medi e anean species o Eunicella [16,21]. To da e, a de ailed phylogene ic analysis wi hin he Eunicella genus is missing, and he axonomic s a us o he wo E.singula is mo pho ypes has no been cla i ied ye [16]. The objec i e o he p esen s udy is o es whe he along he Dep h-Rela ed Gene ic Pa e ns in Eunicella singula is PLOS ONE | DOI:10.1371/jou nal.pone.0160678 Augus 4, 2016 2/20 Languedoc Rouissillon ''Pi ineus Medi e ànis: La mun anya que uneix'' o he CSIC and CNRS, o he 2009-2013 Eu opean p ojec LIFE+ INDEMARES ''In en a io y designación de la ed na u a 2000 en á eas ma inas del es ado español'' (LIFE07/NAT/E/ 000732) o he Eu opean Union, and by a PRIN 2010-2011 p ojec (p o . 2010Z8HJ5M) on 'Coas al biocons uc ions: s uc u e, unc ion and managemen ' I alian Minis y o Educa ion, Uni e is y and Resea ch. 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 . dep h g adien mo phological di e ences obse ed in E.singula is ma ch wi h gene ic di e en- ia ion. To his aim a popula ion gene ic app oach using mic osa elli es and ITS1 sequence polymo phism was ca ied ou . Mo eo e , di e gence pa e n wi hin he genus we e in es i- ga ed by add essing he phylogene ic ela ionships among he Eunicella species. To assess he le el o e ical connec i i y, and he a ia ion o he gene ic pa ame e s wi h dep h samples o E.singula is we e collec ed e e y 10 m om 10 o 60 m dep h in he only si e whe e i occu s on a e ical cli (Cap de C eus, No hwes e n Medi e anean Sea). Eigh u he samples we e collec ed in he same a ea along abou 15 km o coas a 15–20 m dep h o assess he ho izon al pa e n o gene ic connec i i y, and o be able o compa e he 2 pa e ns. Finally, he phyloge- ne ic ela ionships be ween E.singula is, he Medi e anean species E.ca olinii and E. e u- cosa, and he A lan ic E. acemosa, we e analyzed o scale hei gene ic dis ances. Fig 1. Colonies o Eunicella singula is om 10 o 60 me e dep h sampled in Cap de C eus; Eunicella ca olinii om Elba Island a 18–20 m dep h, E. e ucosa om Ta agona a 13–17 m dep h, and E. acemosa along he Mo occo coas . Rep in ed om [16], unde a CC BY license, wi h pe mission om SPRINGER, o iginal copy igh 2012 doi:10.1371/jou nal.pone.0160678.g001 Dep h-Rela ed Gene ic Pa e ns in Eunicella singula is PLOS ONE | DOI:10.1371/jou nal.pone.0160678 Augus 4, 2016 3/20 Ma e ials and Me hods E hic s a emen Pe missions o collec he go gonian samples we e eques ed o he local au ho i ies a each si e: he Pa c Na u al de Cap de C eus o he Cap de C eus MPA; he Socie a d'Explo acions Subma ines de Ta agona o Ta agona; he Pa co Nazionale dell’A cipelago Toscano o Elba Island, and he Uni e si é Ibn Zoh Agadi -Ma oc o Taghazou . The ield s udies did no in ol e endange ed o p o ec ed species. S udy a ea and sampling design The s udy was planned a Cap de C eus (42°18’44’N; 003°19’05”E), whe e Eunicella singu- la is is he mos common and abundan go gonian species occu ing a high densi ies in sub- li o al ocky bo oms om 10 o 70 m dep h [18]. Cap de C eus is cha ac e ised by e ical cli s. Howe e , a e a ca e ul sea ch, only in one accessible si e (Els Fo ca s; CCR3) E.singu- la is occu ed along a e ical ansec om 10 o 60 m dep h. In his si e 16 b anch ag- men s we e collec ed om di e en colonies e e y 10 m dep h (Fig 2,Table 1). Fu he mo e, in eigh loca ions along 15 km o Cap de C eus coas , b anch agmen s we e collec ed by SCUBA di ing om abou 30 colonies o E.singula is a 18–20 m dep h (Fig 2;Table 1). Finally, o analyse he gene ic di e si y and phylogene ic ela ionship in he Eunicella genus, a small b anch agmen was sampled om 5 di e en colonies o : E.ca olinii a Elba Island (42°49’18”N; 010°09’52”E, Cen al Medi e anean) a 18–20 m dep h, E. e ucosa a Ta a- gona (41°06’07”N; 001°15’12”E, No hwes e n Medi e anean) a 13–17 m dep h, and E. acemosa along he Mo occo coas (30°32’51”N; 9°43’59”W, Taghazou , Eas A lan ic Ocean) a 15 m dep h. All he collec ed ma e ial was p ese ed in 80–100% e hanol and s o ed a 4°C pending analysis. Mic osa elli e geno yping and ITS1 ampli ica ion To al genomic DNA was ex ac ed om h ee o i e polyps pe colony agmen s ollowing he ce yl ime hyl ammonium b omide (CTAB) p ocedu e [23]. A e DNA ex ac ion, all he indi iduals we e geno yped using 6 mic osa elli e loci de eloped by [24]: C21, C30, C40 and [25]: EVER1, EVER3, EVER9 ollowing hei p o ocols. Geno yping o indi iduals was ca ied ou on an ABI 310 Gene ic Analyse (Applied Biosys ems), using o wa d p ime s labelled wi h FAM, HEX, TAMRA, ROX (Sigma) and LIZ HD500 (Applied Biosys ems) as in e nal size s anda d h ough MACROGEN INC. Se ice. Allele sizing was de e mined using PEAK SCANNER .1.0 so wa e om Applied Biosys ems, Inc. Fo ITS1 sequence analysis, only indi iduals belonging CCR1, CCR2, CCR3 (10–60) and CCR4 we e ampli ied. Polyme ase chain eac ion (PCR) ampli ica ions o he ITS1 egion we e ca ied ou using he p ime s ITS1-new-F and ITS1-new-R [26]. Each 25 μL PCR eac ion con- ained app oxima ely 20 ng DNA, 1× PCR bu e (P omega), 2 mm MgCl 2 , 0.5 μm o each p ime , 0.4 mm dNTPs and 1 U o Taq polyme ase (P omega). Ampli ica ions we e pe o med on a GeneAMP PCR Sys em 2700 (Applied Biosys ems) as ollows: an ini ial dena u a ion a 95°C o 3 min, 30 cycles including 95°C o 30 s, 57°C o 30 s and 72°C o 60 s. A inal ex en- sion a 72°C o 7 min was added. PCR p oduc s we e sen o Mac ogen (Sou h Ko ea) o pu i- ica ion and sequencing since some sequences con ained se e al he e ozygous si es, ITS1 sequence ypes we e es ima ed using PHASE 2.1 [27] on DNASP . 5 [28], which implemen s a coalescen -based Bayesian me hod o in e hem. Bo h alleles o all indi iduals we e included in he alignmen (286 e e ence indi iduals). Alignmen was made manually using he biologi- cal sequences alignmen edi o BIOEDIT . 7.2.5. Dep h-Rela ed Gene ic Pa e ns in Eunicella singula is PLOS ONE | DOI:10.1371/jou nal.pone.0160678 Augus 4, 2016 4/20 Gene ic a iabili y: mic osa elli e and ITS Linkage disequilib ium analysis o pai s o loci was based on he likelihood a io es wi h he EM algo i hm [29] h ough 10,000 pe mu a ion p ocedu es (numbe o ini ial condi ions o EM, 100) using ARLEQUIN . 3.5 [30]. Indi iduals sha ing he same mul ilocus geno ype (MLG) we e checked using GENALEX . 6.1 [31]. Iden ical MLGs can be he esul o wo di - e en geno ypes o igina ed by wo dis inc sexual ep oduc ion e en s bu sha ing he same alleles o all geno yped loci. The unbiased p obabili y o iden i y (P ID )[32] was compu ed o es his possibili y ha wo sampled indi iduals sha e iden ical MLG by chance h ough sexual Fig 2. Map o he sampling a eas whe e he Eunicella species whe e collec ed, and posi ion o he 8 loca ions whe e Eunicella singula is colonies we e sampled along he Cap de C eus coas . In Els Fo ca s (CCR3) colonies we e collec ed a 6 dep hs along a sloping ocky bo om om 10 o 60 m dep h (named CCR3_10 o CCR3_60). Rep in ed om [16], unde a CC BY license, wi h pe mission om SPRINGER, o iginal copy igh 2012. doi:10.1371/jou nal.pone.0160678.g002 Dep h-Rela ed Gene ic Pa e ns in Eunicella singula is PLOS ONE | DOI:10.1371/jou nal.pone.0160678 Augus 4, 2016 5/20 ep oduc ion and no o clonal ep oduc ion o sampling e o s due o he collec ions o he same colony. Gene ic di e si y wi hin si e o each locus and o e all loci was es ima ed as obse ed (H O ) and expec ed (H S ) he e ozygosi y using he GENETIX so wa e package . 4.05 [33]. Allelic ichness (A ) and p i a e allelic ichness (Ap) we e calcula ed wi h a a e ac ion p o- cedu e using he HP-Ra e so wa e [34]. Single- and mul ilocus F IS alues we e es ima ed using Wei & Cocke ham’sF[35], and signi ican depa u es om he Ha dy–Weinbe g equilib ium we e es ed using “Fishe ’s exac es ”in GENEPOP . 3.4 [36] as implemen ed o online use (h p://genepop.cu in.edu.au/), wi h he le el o signi icance de e mina e by a Ma ko -chain andomiza ion. The p esence o null alleles was es ima ed using he expec a ion maximiza ion (EM) algo i hm o [29]inFREENA[37]. BOTTLENECK . 1.2.02 [38] was used o es o ecen demog aphic changes. Signi ican di e ences be ween He and Heq–he e ozygosi y unde mu a- ion-d i equilib ium calcula ed using wo-phase model (TPM) wi h 95% o he s epwise mu a- ion model (SMM) and a iance among mul iple s eps equal o 12 [38] we e es ed using Wilcoxon’s signed ank- es . A one-way ull- ac o ial pe mu a ion mul i a ia e analysis o a i- ance (PERMANOVA, [39]) was used o es he null hypo hesis o no di e ence in gene ic di e - si y indexes (obse ed he e ozygosi y, allelic ichness and p i a e allelic ichness) be ween dep hs using he 6 loci as eplica es. Da a we e no malized (a e ans o ma ion o p i a e allelic ich- ness as loga i hm plus one), and he Euclidean dis ance calcula ed be ween each pai o samples. A o al o 9,999 pe mu a ions we e used unde a educed model. The analysis was ollowed by pai wise compa isons o es o di e ences be ween dep hs. Sequence gene ic di e si y wi hin samples was es ima ed as numbe o haplo ypes (h), haplo ype and nucleo ide di e si y (Hd and π, espec i ely). All hese pa ame e s we e calcula ed h ough DNASP. To display e olu iona y ela ionships be ween sequence ypes (ST), ITS1 sequences we e ep esen ed in a haplo ype ne - wo k calcula ed by Median Joining wi h he so wa e Ne wo k . 4.6.1.1 [40]. Popula ion gene ic s uc u e Fo bo h molecula ma ke s, he gene ic di e gence among popula ions was es ima ed using Wei & Cocke ham [35]F ST es ima o in ARLEQUIN. Geno ypic di e en ia ion among popu- la ions was es ed wi h an exac es implemen ed in GENEPOP (Ma ko chain pa ame e s: 1,000 dememo iza ions, ollowed by 1,000 ba ches o 1,000 i e a ions pe ba ch). Since he p esence o null alleles, gene ic di e gence among samples a mic osa elli e loci was es ima ed Table 1. Geog aphical coo dina es o he sampling loca ions and dep h ange a which E.singula is colonies we e collec ed. N: numbe s o ana- lysed colonies. Loca ion Code La . Long. Dep h (m) N Fa allons CCR1 423.247 33.111 18–21 30 Es Cama allis CCR2 423.230 33.186 18 30 Els Fo ca s CCR3_10 423.144 33.186 10 16 Els Fo ca s CCR3_20 ““ 20 16 Els Fo ca s CCR3_30 ““ 30 16 Els Fo ca s CCR3_40 ““ 40 16 Els Fo ca s CCR3_50 ““ 50 16 Els Fo ca s CC3_60 ““ 60 16 Ses Ielles CCR4 423.119 32.977 17–20 30 Es Caials CCR5 422.841 32.977 15–17 30 Es Cucu ucú CCR6 422.733 32.844 15–17 30 La Repa ada CCR7 422.511 32.808 17 25 Cap No eu CCR8 422.391 32.675 20 30 doi:10.1371/jou nal.pone.0160678. 001 Dep h-Rela ed Gene ic Pa e ns in Eunicella singula is PLOS ONE | DOI:10.1371/jou nal.pone.0160678 Augus 4, 2016 6/20 in FREENA using he F ST es ima es o Wei [41] and ollowing he so-called ENA me hod desc ibed in [37] which p o ides unbiased F ST es ima es, compu ed excluding null alleles. To e alua e he numbe o clus e s (K) in he da ase wi hou p io in o ma ion ega ding he geo- g aphic dis ibu ion o he samples, a Bayesian me hod implemen ed in STRUCTURE . 2.3.4 [42,43] was used unde he admix u e model and choosing he assump ion o co ela ed allele equencies and he op ion o ecessi e alleles o cope wi h null alleles, as sugges ed by Falush [43]. Mean and a iance o log likelihoods o he numbe o clus e s o K = 1 o K = 10 we e in e ed om mul ilocus geno ypes. Ten uns we e pe o med o each K wi h 500,000 i e a- ions and a bu n-in pe iod o 50,000. In o de o iden i y he numbe o clus e s ha bes i he da a, he esul ing ou pu iles we e hen analysed using he E anno me hod in STRUCTURE HARVESTER . 0.6.94, as implemen ed o online use (h p:// aylo 0.biology.ucla.edu/ s uc u eHa es e /)[44]. Mo eo e , he STRUCTURE esul s we e summa ized using CLUM- PAK [45] o ob ain he p obabili y o each indi idual o belong o each clus e . A disc imina - ing analysis o p incipal componen s (DAPC) as implemen ed in he ADEGENET so wa e . 1.3 [46] was pe o med. This echnique ex ac s in o ma ion om gene ic da ase s (mul i a i- a e in na u e) by i s pe o ming a p incipal componen analysis (PCA) on p e-de ined g oups o popula ions, and hen using he PCA ac o s as a iables o a disc imina ing analysis (DA), which seeks o maximize he in e g oup componen o a ia ion. The op imal numbe o clus- e s (popula ions) was p edic ed using he k-means clus e ing algo i hm, ind clus e s, e ain- ing all p incipal componen s. In all analyses, 20 p incipal componen s o PCA we e e ained as inpu o DA, which accoun ed o app oxima ely 96% o he o al gene ic a iabili y. Signi i- cance le els o mul iple compa isons o loci ac oss samples we e adjus ed using a alse disco - e y a e (FDR) co ec ion o mul iple es s [47]. Hie a chical analysis o molecula a iance (AMOVA) was conduc ed in ARLEQUIN in o de o quan i y gene ic a ia ion be ween wo g oups depending on he dep h: shallow popula ions and deep popula ions (CCR3_40, CCR3_50 and CCR_60). Signi icance o F-s a is ics was achie ed using 99,999 pe mu a ions. Indi idual assignmen es s we e pe o med using he p og am GENECLASS . 2 [48]. This Bayesian p ocedu e com- pu es he likelihood o a geno ype in a gi en popula ion assuming an equal p io p obabili y densi y o he allelic equencies o each locus in each popula ion. Bayesian me hod [49] and he e-sampling algo i hm o Co nue e al.[50] se a 1,000 indi iduals we e used. An indi id- ual was excluded om a gi en candida e popula ion i i s p obabili y o belonging o a pa icu- la popula ion was lowe han 5%. To e alua e he ex en o con empo a y (ecological ime- scale) dispe sal o E.singula is among si es, he numbe s o i s -gene a ion mig an we e also calcula ed h ough he p og am GENECLASS . 2. Phylogene ic analyses To pe o m a p elimina y phylogeny o he genus Eunicella, a da ase wi h all he indi idual ITS1 sequences om he di e en species he e examined was c ea ed. Dis ance ma ixes o sequence di e gence among he species we e calcula ed as p dis ance (Dp) in MEGA . 5.05. The bes - i subs i u ion model o he da ase was calcula ed using JMODELTEST . 1.1 [51] Mac so wa e, conside ing 88 subs i u ion models by hLRT calcula o wi h 4-gamma ca e- go y. Phylogene ic ela ionship among species we e ca ied ou using a Bayesian app oached implemen ed in M Bayes . 3.1.2 so wa e using he Hasegawa, Kishino and Yano subs i u ion model. The analysis was ca ied ou o 2,000,000 gene a ions, sampling e e y 1,000 gene a- ions. The i s 500 o sampled gene a ions we e disca ded as he bu n in. Phylogene ic ee we e isualized as pos e io p obabili y using FIGTREE . 1.4.0. ITS1 sequences o Co allium ub um (GenBank accession numbe : FJ87608282 and FJ87608283) we e used as ou g oup. Dep h-Rela ed Gene ic Pa e ns in Eunicella singula is PLOS ONE | DOI:10.1371/jou nal.pone.0160678 Augus 4, 2016 7/20 Resul s Gene ic a iabili y: mic osa elli e and ITS 301 indi iduals we e geno yped o he 6 loci. Tes s o linkage disequilib ium be ween loci wi hin popula ions indica ed no signi ican associa ion o alleles (all p>0.05), con i ming ha all loci a e independen ma ke s. Six sha ed mul ilocus geno ypes (MLGs) be ween colonies we e ound. Fi e MLGs we e encoun e ed wice in CCR3_40, CCR3_50 and CCR3_60. Wi hin CCR3_50 one MLG was encoun e ed i e imes. In CCR3_50 he p obabili y ha each o hese geno ypes was p oduced by chance alone was high (P ID = 4.6 e-02). The allelic ichness (A ) based on a minimum sample size o 22 genes, anged om 2.24 (in CCR3_50) o 4.25 (in CCR7), while he p i a e allelic ichness (Ap) anged om 0 (in CCR3_20 and CCR3_30) o 0.46 (in CCR7). Expec ed he e ozygosi y (H E ) and obse ed he - e ozygosi y (H O ) we e lowe in CCR3_50 (H E = 0.25, H O = 0.28) and highe in CCR4 (H E = 0.58, H O = 0.57). Th ee (CCR2, CC3_10 and CCR3_30) ou o 13 samples showed signi ican de ia ions om HW equilib ium wi h alues o F IS posi i e and highe han 0.173 (Table 2). Es ima ed null allele equencies anged be ween loci om 0.022 ± 0.035 in EVER9 o 0.0748 ± 0.087 in C21. The Wilcoxon’s signed ank- es , pe o med in BOTTLENECK showed no demog aphic popula ion changes in he ecen pas o all samples (da a no shown). The one-way PERMANOVA showed ha he o al gene ic a iabili y di e ed by dep h (MS = 6.68; F = 2.65; d = 5; p= 0.005). The pai wise es loca ed a h eshold o gene ic a iabili y a 40 m dep h (10–50 = 2.42, p= 0.0097; 20–50 = 3.86, p= 0.0001; 30–50 = 2.88, p= 0.0054). Samples collec ed in 3 shallow si es (CCR1, CCR2, CCR4) and along he e ical g adien in CCR3 (CCR3_10 o CCR3_60) we e analyzed using ITS1 sequence polymo phism. In o al, 286 ITS1 sequences co esponding o 143 indi iduals we e analyzed. The leng h o he ampli- ied ITS1 agmen was 200 bp wi h 9 a iable si es a he posi ions 14, 21, 114, 119, 158, 159 and 173, co esponding o 9 sequence ypes (ST1 o ST9; Table 3). The mo e equen sequence ype was ST1 occu ing in all popula ions, while he second mo e equen sequence ype was ST7 occu ing in CCR3_40 and CCR3_60 wi h 9 and 5 indi- iduals, espec i ely (Table 3;Fig 3). Haplo ype di e si y anged om 0 in CCR3_50 o 0.653 in CCR3_60 (Table 3). No signi ican di e ences be ween CCR3_10, CCR3_20, CCR3_30 s. CCR3_40, CCR3_50, CCR3_60 we e obse ed in he haplo ype di e si y (S uden ’s - es , Table 2. Gene ic di e si y o Eunicella singula is a six mic osa elli e loci; H O : obse ed he e ozygosi y, H E : unbiased expec ed he e ozygosi y, A : allelic ichness, Ap: p i a e allelic ichness, F IS : Wei and Cocke ham’s[35] es ima e ixa ion index wi h signi ican alues in bold (0.05 h esh- old a e FDR co ec ion). Si e H O H E A Ap F IS CCR1 0.50 0.57 3.68 0.18 0.115 CCR2 0.47 0.57 3.85 0.02 0.173 CCR3_10 0.44 0.55 3.43 0.06 0.205 CCR3_20 0.52 0.52 3.21 0.00 0.07 CCR3_30 0.47 0.59 3.42 0.00 0.203 CCR3_40 0.54 0.53 3.39 0.15 -0.028 CCR3_50 0.28 0.25 2.24 0.05 -0.098 CCR3_60 0.49 0.51 3.46 0.13 0.045 CCR4 0.57 0.58 3.70 0.07 0.011 CCR5 0.55 0.58 4.29 0.21 0.053 CCR6 0.52 0.54 3.62 0.05 0.043 CCR7 0.52 0.57 4.25 0.46 0.096 CCR8 0.53 0.58 3.98 0.15 0.083 doi:10.1371/jou nal.pone.0160678. 002 Dep h-Rela ed Gene ic Pa e ns in Eunicella singula is PLOS ONE | DOI:10.1371/jou nal.pone.0160678 Augus 4, 2016 8/20 p= 0.07; da a no shown). The sequence ype ne wo k showed a s a -shape pa e n wi h a high equency dominan haplo ype wi h a he cen e . De i a i es wi h lowe equencies we e con- nec ed o he dominan haplo ypes by only one mu a ion s ep (Fig 3). Popula ion gene ic s uc u ing Geno ypic di e en ia ion be ween samples and F ST es ima es based on he ENA me hod ga e simila esul s o hose ob ained when p esence o null alleles was no aken in o accoun . Pai - wise F ST alues anged om -0.002 (CCR2 s. CCR5) o 0.482 (CCR3_50 s. CCR3_60). A e FDR co ec ion, all he pai wise compa ison wi h CCR3_40, CCR3_50 and CCR3_60 we e signi - ican (Table 4). Du ing he i s ound o STRUCTURE, wo gene ic clus e ing we e iden i ied as mo e plausible, K = 2 (ΔK = 74.71) (Fig 4A). When K = 2 is conside ed, he i s clus e includes CCR3_40, CCR3_50 and CCR3_60, while he o he clus e includes all he emaining samples. When K = 4 is conside ed, STRUCTURE ou line a new gene ic pool cha ac e ized co esponding o CCR3_50 (Fig 4B). The DAPC con i med he gene ic di e en ia ion obse ed using S uc u e. In he da a ans o ma ion s ep o PCA analysis, 20 p incipal componen s (PCs) we e e ained, accoun ing o app oxima ely he 95% o he o al gene ic a iabili y. The eigen alues o he DAPC indica ed ha he i s wo componen s explained mos o he a ia ion. Along he i s axis CCR3_40, CCR3_50 and CCR3_60 we e sepa a ed om he o he s samples; he second axis con as s CCR3_50 wi h CCR3_40 and CCR3_60 (Fig 5). In ac , 93% o indi iduals we e eas- signed o hei o iginal clus e s CCR3_50 and CCR3_40, while 75% o he indi iduals we e eas- signed o CCR_60. The alues o he p opo ions o success ul eassignmen we e below 30% in all he shallow popula ions sugges ing admix u e among hem. The AMOVA suppo ed he clus e ing s uc u e showing a signi ican di e ence be ween shallow and deep g oups: 15.39%, p<0.05 (Table 5). O he 301 indi iduals included in he assignmen analysis, 38.2% we e assigned o hei ac ual sampling loca ion. In pa icula , all indi iduals om CCR3_40, CCR3_50 and CCR3_60 we e assigned o hei popula ion o o igin. The analysis o i s gene a ion mig an s, based on he Bayesian compu a ion o Rannala e al. [49] implemen ed in GENECLASS . 2, classi ied 6 colonies as o igina ing om a si e di e en o he one hey we e collec ed a . None o he 6 col- onies we e iden i ied as immig an s coming om deepe popula ions (da a no shown). Fo he ITS1 sequence da a se , F ST anged om 0 (CCR3_10 s. CCR3_20; CCR3_20 s. CCR3_30 and CCR3_50 s. CCR3_10, CCR3_20, CCR3_30) o 0.548 (CCR3_40 s. Table 3. Sequence ypes equencies, including he e ozygous indi iduals in each popula ion o E.singula is.H: numbe o haplo ypes (in he sense o sequence ype); Hd: haplo ype di e si y; πd: nucleo ide di e si y. CCR1 CCR2 CCR£_10 CCR3_20 CCR3_30 CCR3_40 CCR3_50 CCR3_60 CCR4 ST1 26 22 31 31 31 14 32 16 26 ST2 4 6 ST3 7 ST4 3 ST5 1 1 ST6 1 ST7 18 10 ST8 4 ST9 2 H232222142 Hd 0.024±0.092 0.486±0.085 0.063±0.058 0.063±0.058 0.006±0.058 0.508±0.031 0 0.653±0.057 0.315±0.087 πd 0.00120 0.00398 0.00124 0.00063 0.00031 0.00254 0 0.00415 0.00157 doi:10.1371/jou nal.pone.0160678. 003 Dep h-Rela ed Gene ic Pa e ns in Eunicella singula is PLOS ONE | DOI:10.1371/jou nal.pone.0160678 Augus 4, 2016 9/20 ex eme he mal s ess in shallow wa e s has ecen ly esul ed in mass mo ali ies o ben hic in e eb a e species, including go gonians (e.g. [69]). Howe e , e-coloniza ion and popula- ion’s eco e y a e a mass mo ali y e en canno be suppo ed by la ae om deepe colo- nies, bu can ely only on la ae p oduced by nea by shallow wa e popula ions. Conce ning he axonomy o he genus Eunicella in he Medi e anean Sea, low le els o mi ochond ial genome e olu ion [20], and low polymo phism o nuclea ma ke s, do no allow o alida e he species s a us nei he o he wo mo pho ypes, no o he h ee Eunicella species. The la ge mo phological a iabili y o Eunicella genus, as in o he Oc oco allia gene a, leads unce ain y ega ding species bounda ies and classi ica ion. Me hods based on nex -gene a ion sequencing (e.g. RAD sequencing, [70]) may p o ide u he insigh s in o he e olu iona y ela ionships in ecalci an axa such as Eunicella. Suppo ing In o ma ion S1 Da ase . Mic osa elli e da a se . The allele sizes in 6 loci s udied in 13 E.singula is popula- ions. (XLS) S1 Ma e ials and Me hods. Ma e ials and Me hods and Resul s. DNA ex ac ion o he Euni- cella species and PCR ampli ica ion o he h ee mi ochond ial ma ke s and hei gene ic a i- abili y. (DOCX) S1 Table. Pai wise gene ic p dis ance among Eunicella species. Pai wise gene ic p dis ance (Dp) among Eunicella species including he sampled popula ions o Eunicella singula is. (DOCX) Acknowledgmen s Au ho s a e g a e ul o L. Te azzoni o he collabo a ion in lab wo k, and o N Vilad ich, I Fio illo and T Ga cia o hei help du ing sampling. A Go i was unded by a I3P con ac o he Consejo Supe io de In es igaciones Cien i icas (Re . I3P-BPD2005). S Rossi wan s o hank he suppo o he Gene ali a de Ca alunya o MERS (2014 SGR—1356) and he Ma ie Cu ie In e na ional Ou going Fellowship (ANIMAL FOREST HEALTH, p ojec num- be 327845). This wo k is pa o he 2003–2004 INTERREG p ojec be ween Ca alonia-Lan- guedoc Rouissillon ‘‘Pi ineus Medi e ànis: La mun anya que uneix”o he CSIC and CNRS, o he 2009–2013 Eu opean p ojec LIFE+ INDEMARES ‘‘In en a io y designación de la ed na u a 2000 en á eas ma inas del es ado español”(LIFE07/NAT/E/000732) o he Eu opean Union, and o he PRIN 2010–2011 p ojec (p o . 2010Z8HJ5M) on ‘Coas al biocons uc- ions: s uc u e, unc ion and managemen ’I alian Minis y o Educa ion, Uni e is y and Resea ch. Au ho Con ibu ions Concei ed and designed he expe imen s: FC AG JMG PLG SR LB. Pe o med he expe imen s: FC AG LB SR. Analyzed he da a: FC MA. Con ibu ed eagen s/ma e ials/analysis ools: MA JMG. W o e he pape : FC AG PLG LB SR JMG AM. Dep h-Rela ed Gene ic Pa e ns in Eunicella singula is PLOS ONE | DOI:10.1371/jou nal.pone.0160678 Augus 4, 2016 16 / 20 Re e ences 1. Todd PA. Mo phological plas ici y in scle ac inian co als. Biol Re . 2008; 83: 315–337. PMID: 18979594 2. Pinzón JH, LaJeunesse TC. 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