Molecular evidence for the coexistence of two sibling species in littoralis (, Phaeophyceae) along the Brittany coast Alexandre Geoffroy, Stéphane Mauger, Aurélien de Jode, Line Le Gall, Christophe Destombe

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Alexandre Geoffroy, Stéphane Mauger, Aurélien de Jode, Line Le Gall, Christophe Destombe. Molecular evidence for the coexistence of two sibling species in (Ectocarpales, Phaeophyceae) along the Brittany coast. Journal of Phycology, Wiley, 2015, 51 (3), pp.480-489 ￿10.1111/jpy.12291￿. ￿hal-01147191￿

HAL Id: hal-01147191 https://hal.sorbonne-universite.fr/hal-01147191 Submitted on 29 Apr 2015

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Accepted Article Roscoff, 29688 Roscoff, France CNRS Algae,of EcologyStation Biologique Roscoff, de CS 90074, 29688Roscoff, France Sorbonne Universités, UPMCUniversité06, ParisUMI 3614 Biology Evolutionary and Aurélien de Jode Roscoff, 29688 Roscoff, France CNRS Algae,of EcologyStation Biologique Roscoff, de CS 90074, 29688Roscoff, France Sorbonne Universités, UPMCUniversité06, ParisUMI Biology 3614Evolutionary and Mauger Stéphane Roscoff, 29688 Roscoff, France CNRS Ecology of Algae, Station Biologique de Roscoff, CS 90074, 29688 Roscoff, France Sorbonne Universités, UPMCUniversité06, ParisUMI Biology 3614Evolutionary and Alexandre Geoffroy (Ectocarpales, Phaeophyceae) alongtheBrittany coast Molecular evidence forcoexistence the oftwo siblingin species , , , UMI 3614 Evolutionary Biology and Ecology of Algae, Station Biologique de UMI 3614 Evolutionary Biology and Ecology of Algae, Station Biologique de UMI 3614 Evolutionary Biology and Ecology of Algae, Station Biologique de 1 Pylaiella littoralis Pylaiella

Accepted Article Brittany coast.sampled We 140individuals fro 10 microsatellite markers.addition, Inspatio-temporalsampling was conducted thealong 1 2 Christophe Destombe postale N° 57rueCuvier, 39, 75231Cedex05 Paris, France 7205UMR CNRS-EPHE-MNHN-UPMC, Equipe E M Gall Le Line chloroplastic ( using a DNA barcoding multilocus approach with mitochondrial ( ofthe diversity render the systematic studythis genus of probl phenotypicThe great variability the and lackof diagnostic characters in genus the Abstract Responsibility:Clerck O. De Editorial (Associate Editor) Sibling species in Phone: +33 (0)2 98 29 23 20 Roscoff, 29688 Roscoff, France CNRS Algae,of EcologyStation Biologique Roscoff, de CS 90074, 29688Roscoff, France Sorbonne Universités, UPMCUniversité06, ParisUMI Biology 3614Evolutionary and date ofsubmission and acceptance corresponding author Email: [email protected] uséum National d’HistoireNaturelle, Institutde Systématique, Evolution,Biodiversité, , UMI 3614 Evolutionary Biology and Ecology of Algae, Station Biologique de rbc Pylaiella littoralis Pylaiella L and atp L and Pylaiella littoralis Pylaiella 2

B) markers associated with a populationusinggeneticsmarkers a approach associatedB) with (Linnaeus) Kjellman along the Brittany (France) coast

ematic. Inpresent the study, we investigated m locatedfour sites between Saint-Malo and xploration, Espèces,xploration, Evolution, case cox1, nad 1 and atp Pylaiella 9) and 9) and Accepted Article lack ofdiagnosticlack characters insomegroups, speciesdelimitation using conventional morphological(Mayden characters 1997). However, due to phenotypic plasticityand the andthe delimitation ofspecies is thereforeessentially basedon discontinuitiesin Most species indescriptions brown algal groups follow the morphological species concept INTRODUCTION atp atp9, ATP synthase subunit 9 nad1, NADH dehydrogenasesubunit 1 rbcL, ribulose-1,5-biphosphate carboxylaseoxygenase largesubunits cox analysiscorrespondence factorial FCA, principalPCA, component analysis nt, nucleotide List ofabbreviations Keywords: gene flow, microsatellite,barcode, multilocus genetics approaches to shedlight onthe evol is not complete. Ourstudybarcoding highlighted the of complementarity andpopulation occurrence ofgenetic admixturesin some popul oftwo mitochondrial clades Microsatellite genotypic congruentlydata revealed twowell-supported clustersmatching the them. between divergence 2.4% of minimum a with species, sibling two of occurrence (380Concarneau km)ApriltoOctober. from Mitochondrial sequence data revealedthe B, ATPasebetaB, subunit 1, cytochrome c oxidase subunit I Pylaiella . Although. gene flowlimited is betweenspecies, utionary processes that utionarytolead processes speciation. ations suggested that reproductive isolation Pylaiella littoralis , sibling species. species. sibling , Accepted Article haveauthors suggested that these various forms habitat and seasonal distribution characteristics (GuiryandGuiry 2014).A ofplethora 1999). More than50or varietiesforms have beendescribed onthebasis morphology, of cold and cold temperate coastsof both hemispheres (Müller andStache 1989, Silva et al. the most common accepted species and has beenreported worldwide inthe littoralzones of accepted(Guiry taxonomically Guiry2014). and Pylaiella littoralis currently are seven only which of genus, this for reported been have names infraspecific delimitation very problematic (Fritch 1952, BoltonMore 1979a). than 90 specific and disconcerting phenotypic plasticity and their centered on morphological species However,concept. Pylaiella variousbranchingwith patterns. As in most algal groups, systematics in The brown genus algal estimate theextent of genebetween flow closely relatedspecies. Populationet al.2005). genetic data acquiredmicrosatellite fromloci are usefulto extremely (Tellieramong taxa et al. 2011)and to hybridizationidentify or introgression events (Engel geneticpopulation approach has been used in to detect gene flow discontinuity toalgae aggregateintodifferentindividuals closely species related (Leliaert et al. 2014). The tool delineatingfor species(Sites and Marshall 2003)andhas been successfully appliedin discontinuity insequence variationas diagnostic character —hasproven to be an effective between closely related species.As phylogenetic a approach,barcoding DNA based — on genetics population approaches helpcan determine algaldiversity studyand boundaries delimitationspecies (for Leliaertreview, et al. 2014). The combination of phylogenetic and 2009, Tronholm etal. 2010). Analysesof molecula often is methods problematicand has led to erroneous Pylaiella includes species characterizedby a filamentous structure lack of diagnostic characters, making species and varietiesand possibly can be attributed to r datar become have the closestproxy of taxonomic assignme speciesknown are for their Kjellman(Linnaeus) is Pylaiella nts (Tellier et al. has been been has Accepted Article Ruprecht, (Ruprecht 1850), chloroplastic geneschloroplastic assessgene flow between populations. Mitochondrial sequences combined withthe approach, we used a population genetics approach based on 10 microsatellite markers to Although Pylaiella useful alternative to morphological observation for clarifying the systematics of the genus delimitation within M phenotypicplasticity (i.e., KnightNewton1923, 1931, Russell 1963, 1967, Bolton 1979b, species inHelgoland-Northern( Sea LehvoOctober (KiirikkiandKornmann 1997). and Sahling (1977)two reported different degradation detachmentand of filamentsand one growthwith ina September- period growth in earlyspring with a maximum cover beginningat theMay-June of followed by Baltic Sea, two different seasonalhave cohorts been observed,one characterizedby rapid substratesand as an epiphyteof seaweed perennial species(Feldmann 1954). Inthenorthern at the specieslevel in Brittany,France.this Inis region, the both species rockyfound on different effective sizes: the mitochondrial genes combination of five molecularmarkers characterized bydifferent mutation rates and ofamong populations The aimsofthepresentwere studyto spatio-temporal thediversity pattern assess and its macrocarpa Pylaiella that üller andStacheTherefore, 1989).no clear consensus has ever beenon adopted species P. littoralis .

various species, such as have been reported is found throughouttheyear whereas

Feldmann (1954), andmost authorsrecognized after him,only Pylaiella rbcL and , Kornmannobserved 1990),is onlyin spring. P. littoralis (Siemer and Pedersen 1995). Molecular tools appear as a

atp in England and in south-western France, respectively B (Silberfeld et(Silberfeld Bthe al.2010).Inadditionto multilocus Pylaiella anglica in Brittany. Toward theseobjectives, wefirstused a P. littoralis and cox Ruprecht and Pylaiella 1, 1, the second species as (later identified 1 and nad1 and sp.). These authors observed 9, and the two two the 9, atp and Pylaiella aquitanica P. littoralis

Accepted Article in Silberfeld et al. (2010). wereamplifiednt) and sequenced.used Primers PCRand reactions performedare described nad1 (800nt)and than heating to 65°C for 30 min. Sequences of three mitochondrial genes thefor cell lysis bufferinwhich were samples left at roomtemperature for ratherone hour (Macherey-Nagelkit GmbH and Co. KG) accordingthe to manufacturer’s protocolexcept DNA was extracted from 5-10 mg of dry alga DNA extraction,and amplification sequencing 1970 and 2002, wereincluded inthe analysis. the CultureCollection of Algae andProtozoa (CCAP) isolatedrespectively in Roscoff in To complete sampling,the two strainsof (1330/1 reference 1330/2) and of Naturelle d’Histoire anatomical features (vouchers were deposited in the herbarium of the specimen from each site was pressed andmounted onherbarium a sheet to preserve wasindividual preserved in silica gel molecularfor analysis. In addition, at least one compare spring and autumn samples. In the km(about 380 coastline). Samplingwas conducte specimens per site)alongthe coast of Brittany,France (Fig. 1), fromDinardto Concarneau littoralis Pylaiella of specimens 140 total, In Sampling MATERIALS ANDMETHODS under then geneticspopulation approach revealed the occurrence of two sibling species in Brittany ame of ame atp P. littoralis , Paris, France,nos. PC0533607-PC0533613). 9 (200nt), ofthe two and genes plastid cox 1 and 1 and . rbc L genes were sequenced for specimensall (i.e.,140 laboratory, a fragment of tissue from each were sampled at four different localities (16-62 l tissuethe plant usingNucleospin® Multi-96 d from April d from to October 1)to (Table rbc L (1300 nt)and Museum National cox P. littoralis 1 (658 nt), atp B (630 from Accepted Article Germany), 2.5 mM MgCl of mM 2.5 Germany), Operon, Ebersberg,Germany), pmol 30 revers YakimaFAM, Yellow ATTO550 (VIC), MA,pmol 30 primer USA), ScientificInc.,forward Waltham, Fisher fluorescent-labelled of (Thermo dNTPs DNA of diluted1:25, template 150µM the following 2µL components: P. littoralis 2).These (Table 10were markers used for thepopulation analysison the 140specimens of positive repeatableshowed amplification,butonly10legible asignal after sequencing Microsatellite developed markers for Population genetic approach between intraspecific interspecific and divergences(Puillandreet al. 2011). rbcLgenes. Pairwise comparisons were calculated aligned using Codoncode Aligner v. 3.7 (www.codoncode.com, sequencedby LGCgenomics (Berlin, Germany).obtainedsequences The edited were and used to confirmresults obtained with the pr 2010) were totransferred version 4.2.0.1version(Bandelt etal. 1999). Mismatch distributionwere analyzed for chloroplast genes ( individuals of ( Phylogenetic relationships among mitochondrialDNA( Phylogenetic approach USA). cp DNA) haplotypesDNA) were reconstructed using according to the following method.volume of PCR was carried out in 10a µL P. littoralis nad1, atp strains). twoCCAPand For the other mitochondrial and P. littoralis 9 andB) atp a subsample of45 individuals of 2 (Promega Corp.), 1 X GoTaq® Flexi Buffer (Promega Corp.), Corp.), (Promega Flexi Buffer 1GoTaq® X Corp.), (Promega . Of the60 microsatellitesanalyzed, 30showed siliculosus (NED) or ATTO565(PET) (Eurofins MWG MWG (Eurofins (NED) orATTO565(PET) evious evious genes. PCR products were purified and e (Eurofins MWGprimer Operon, Ebersberg, median-joiningnetworks with Network each genefor theto determinethreshold mt DNA) andchloroplastic DNA (Dillwyn)Lyngbye (Cock et al.

Centerville, Massachusetts, P. littoralis cox 1 and 1 and was was Accepted Article (Table 1).(Table Expected heterozygosity (H to corresponding cluster ofindividuals assignedtodifferent cytotypes for sampleeach andSmouse 2012):meannumber of alleles per locus (N (Peakall 6.5 version GenAlEx using estimated were diversity genetic of measures Standard between sampled populations was computed by calculating an estimate of versionweb GENEPOP 4.0.10 (Raymond and Rousset1995) the and geneticstructure disequilibrium using Markova approxima chain cluster washigher than10. Moreover, allpairsof loci across sites weretested forlinkage was run under the admixture and independent allele frequency model using a Markov chain chain Markov a using model frequency allele independent and admixture the under run was STRUCTURE version2.2 et (Pritchard 2000) al. In a separate analysis, the most likely number of genetic clusters was inferred using statisticalsignificance associated with each analysis (PCA)performed using PCA-GEN software (version1.2; Goudet 1999). The using (Bel performed (FCA) GENETIXsoftware dataThese were summarized and statisticallytested with a factorial correspondence analysis Cockerham 1984). For each population, allele frequencies were calculated at all 10 loci. anddilutedpooled (1:10) and 2 s and, 72°C 30sfor with a final elongation stepof 72°Cfor 10 min. PCR products were dena initial an included cycling PCR Corp.). (Promega polymerase DNA GoTaq® U 0.35 and (Applied Biosystems (Applied Analyzer (Life Technologies 2012).Thisal. loading mix was (95ºCrunmin) and for5 denatured in a 3130XLGenetic Technologies(Life formamide Corporation) and 0.5 turingstep at95°C for5min, followedby 35 cycles at 95°Cfor 30 s, 50or 54°C30 for TM ). Corporation). Alleles were scored using GeneMapper μ Lwereto added a mixture containing 9.5 e ) was) when estimated thenumber of individuals per axis was evaluated after 10,000 randomizations. tion ofan exact test as implemented in on the seven sampled sites. This program khir et al. 2003) and a principal component μ L of SM594 Lof SM594 size standard (Mauger et a )was calculatedin 10populations F μ ST L of Hi-Di Hi-Di L of (Weir & (Weir ® v. 4.0 4.0 v. Accepted Article andM4a),withtwo mitotypes found in only one individual (M8aandM9a) and the 0fromGroup to 0.45%. was MtA mostly represented by mitotypes four (M1a, M2a, M3a found in only oneIn individual (M5b). MtB, the intragroup haplotypic divergence ranged representedby mainly mitotypesthree (M1b, M2b M3b) and oneand mitotypewas unique, nucleotides (2.4%) was observed betweenmitotypesof these twogroups. MtB was Group betweendifferences) cytoplasmic clusters MtA andMtB. A divergenceofminimum 16 confirmed the existence of a barcode gap (i.e., intergroup differences greater than intragroup distribution (first peak centered on 2 differences and the second on 18 differences) bimodal The (inset). 2 Figure in shown is individuals analyzed the all considering KM027333).KM027317to Analysis ofmismatchof distributions the MtA and MtB, withrespectively 12and5 mitotypes (GenBankaccession number: structure patterns in the data was determined as the smallest K at which average log Pr (X 10, averagedover20 independent runs. Thevalueof K that mostlikely captured the major was Carlo lengthof250000Monte steps after aburn-in of50,000. Thenumber (K) ofclusters mitotypes (Fig. 2). These mitotypes were clearl revealed a totalof 30polymorphicsites (up to3.2%divergence) 17 corresponding to Sequenceof comparisons the 5’endof the Haplotypic diversity RESULTS DISTRUCT 1.1 version (Rosenberg2004). CLUMPP version 1.1.2 (Jakobsson and Rosenberg 2007) and displayed graphically using estimates leveledFor out. the selected K,results fromreplicate were runs averaged with determined by theestimating probability of the data [log Pr (X cox 1 gene1(658 nt)obtained specimens for 142 y separated into two di | K)]for runs with K= 2- stinct clusters, named clusters, stinct cox 1 sequence, 1 sequence, | K) K) Accepted Article aOctober), seasonal shift wasobserved in their frequencies. The maximum frequency of Althoughboth mitotype clustersMtB MtA or were found together in the field(except in distributionTemporal of twothe mitochondrial clusters sharedby MtA and MtB. accession number: KM057707) were congruent with those of mostly byrepresented a haplotypesingle (CL1).Results for the mostly represented by two chlorotypes unique tothegroup MtB CL10,(CL3, CL4, uniquewere tothe MtA group(CL8, CL9, CL14, CL13, CL16 andCL17) and seven were chlorotypesof the (7of the 15haplotypes f 3). Thetwo cytoplasmic groups and MtA MtB, resultingfrom 0.7% divergence); nevertheless these chlorotype GenBank accession number:with12 KM057683 toKM057702) polymorphicsites(up to Analysis of rbcL sequences (1321 nt) showed high diversitychlorotype (20 chlorotypes; individuals analyzed only had MtA mitotypes. except in Concarneau (site at which sampling was done only once, in spring) where all MtAof thecluster.Individuals with the MtA cytoplasm were MtBand found in all locations MtB cluster, whereas the sequence ofthe1330/2corresponded strain tothe haplotypeM5a of one CCAP strain of 20 andsitespolymorphic S1 4 S2 (Figs. and in Thesequence theSupporting Information). atp GenBank accession number:KP058512, KP058513 andKM057703to and KM057706) othe intragroup haplotypic divergence ranged from 0 to 0.9%. Similar results were obtained for 9 (1.5–2.0%;GenBank9 accessionrespectively number:KM057708 to KM057710) with 1 (1.9–2.1% divergence between mitotypes; mitotypes; between divergence r sequencedmitochondrial genes nad1(1.9–2.1% Pylaiella littoralis (CL3 and CL4), whereas Group MtA was was MtA Group whereas CL4), and (CL3 (1330/1) corresponded (1330/1) to haplotype M1b ofthe oundin atleast twoindividuals), whereas six CL11, CL15, CL18CL11, CL15, and s notdid cluster in conspicuousgroups (Fig. rbc cox1clustering, shared 47% atp L, showing onehaplotype B gene (GenBank CL20). GroupCL20). MtB was Accepted Article The results ofThe results thePCA (Fig. 4a) showed a significant overall significant after sequential Bonferroni correction. loci (datanotshown) except one for pair of lociremained (M105-1/M194),which genotypes were detected. Thereno was evidence from 0.608 to 0.741. No repeat multilocus genotypes and no multilocus monomorphic in St. Malo and in clustered Roscoff together, whereas individuals sampled in spring in these date,whateverpartitioned according to sampling theirorigin. sampled Individuals in autumn MtAmitotypes andMtB 4b). (Fig. theWithin MtB mitotype group, individualswere thegroupsconfirmed partitioningintotwo of main individuals along 1toaxis corresponding separated MtA samples MtB samples. Thefrom analysis(FCA) correspondencefactorial accounted50.8% for of the variance and 2 axis accounted for of 13.6% the variance.Axis1 The first two PCA axes were significant and explained 64%of the variance:axis 1 (N locus per Table 2).respectively, For withlocalities morethan 10samples, the mean number ofalleles ranged 18tofrom the 52fromleast to the polymorphiclocus most (M028-1 andM194, two groups, MtA MtB and (Table 1). Thetotal numberof alleles observed samplesover all microsatellitelocus. Based onthe mitochondrial clusters, were individuals assignedtothe All the microsatellite genotypes observed in th Structure ofpopulations (October; Table 1). ma individuals with MtA cytoplasm (83.6%) was found in spring (April), whereas the alleles) with frequencies oscillating between 0.014 and 0.208. Gene diversity (H diversity Gene 0.208. and 0.014 between oscillating frequencies with alleles) found only in the MtB cluster 94(26%)were and found only in the MtA cluster (i.e. private ximum frequencyof with individuals wasMtB (63.3%) observed in cytoplasm autumn a ) ranged from6.7 to14 (Table1). Overtotal a of353 alleles, 153(43%)were is studyis heterozygous wereat least for one of linkage disequilibrium between pairs of F ST valueof 0.22= (P 0.0001). e ) ranged ) ranged Accepted Article 2014) and Ectocarpales2014) and (Peters et 2015al. in press) thatestimated species-level cut-off at Thus, our results in are agreementwiththerecent proposals onDesmaretiales (Yangetal. (2.4%–3.2%), was substantially greater than values detected within a (0.0%–0.9%). cluster observed betweendivergence two the mitochondrial ofclusters (MtA MtB)and the existence oftwogeneticunder entitiesname the of BothFeldmann (1954). phylogeneticandpopulation inferences genetic studies highlighted far been overlooked in Brittany, since only one had been recognized after the work of Our findings revealed that DISCUSSION 5). (Fig. cluster (MtA-dominant) first to the assigned were mitotypes 3) MtB with (sample Dinard from individuals Five observed. was admixture 6), 3 and (2, location same the in together sampled whichgroupedthese samples along axis 1 (Fig. 4). However, when individualswere second cluster includes MtB samples.Thisre clustersgiven is inFigure 5. The first clus samples into two “clusters”The(K=2). proportion (Table 3) from ranged(Table 0.07 (samples 4 the variance ( Regarding the analysis of structure, the the structure, of analysis the Regarding to mito assigned samples for observed were whereasto thesamebetween mitotype, observedthewere assigned samples highestvalues accounted28% for variance ofthe ( Again, the twoFCAaxeswere significantand explained45% ofthe variance: 1axis sites wereintermediate between the MtB individuals theMtAand group 4b).mitotype (Fig. F ST = 0.03, P = 0.815). Genetic differentiation between pairs of samples ( Pylaiella littoralis Pylaiella F ST vs. = 0.11,P =0.001) andaxis 2 accounted17% offor Δ Kcriterion separated thesevenspatiotemporal 1) to 0.24 (samples 4 tervirtually allthesamples and includedMtA the types sampled inspringin and autumn. comprises two crypticspecies that have thus sult confirms sultthe PCA confirms (andresults, FCA) ofgenotypes assignedto each ofthe two P. littoralis P. vs. . The level of of level The . 7). The lowest P. littoralis cox1 F ST

values values F ST )

Accepted Article mitotypes (Fig. 4). These results suggest that there is some contemporary gene flow sampled in thegenotypically spring wereintermediate to the MtA grouptheand MtBother admixtureswas found (Fig. 5).FCA The showed that individuals withMtB mitotypes MtA and individualsMtB were sampledsimultan their distribution spatio-temporal intersects (i.e populationsallopatric (1,4, 5, 7)that were assigned todifferent clusters. However, when maternally aremitochondria inherited(butsee Hoarau et al. 2009in by the difference in effective population size (Palumbi et al. 2001). In Phaeophyceae, Difference in resolution found between mitochondrial and plastid markers can be explained (e.g., forbrown et 2006,markers Engel Presting al.2008,Tellier algae: 2009). et al. Our results confirm that have markerschloroplastic lowerratesmutation than mitochondrial species (Bickford et al. 2007). these twoclosely related species of the obs divergence of about 1.2% and 1.8% respectively. Furthermore, the common chlorotypes observed in widespreadbe in isogamousbrownalgae. Forexample, biparental chloroplast inheritance is suggested that the biparental chloroplast inheritance observed in plastid may be biparentallypartly inherite Nuclearmicrosatellite markers consistently recovered the twowell-supported isavailableinformation on Pylaiella of genus the taxa. Likewise,taxa. we observed high values ofgenetic differentiation ( mitochondrial clusters(i.e.,MtAandMtB), suggesting that genebetween flowis limited erved in the two Scytosiphon lomentaria P. littoralis is phylogenetically related is phylogenetically to chloroplast inheritance. species suggest that differentiation is recent. Therefore, (Lyngbye) Link (Kato (Lyngbye)Link et al. 2006). However, although P. littoralis d (Peters et Peters2004).al. etal. (2004) ., sympatric populations in which individuals eously in spring ineously andat the site), same shouldbecomplex regarded siblingas Ectocarpus and Ectocarpus siliculosus Ectocarpus F Scytosiphon ST ) between pairs of species), whereas cox1 , no

may may Accepted Article environment independent) barriersExtrinsiccan and greatly reproductiveextrinsic barriers. hybridizationFurthermore, in natural populations depends on both(i.e., intrinsic consequences for reproductive isolation andsympatricspeciation. duringtheir peakof abundance. Suchdiffer grazers, competition, host) and parameters abiotic inphenologydifference boththat implies species probablyface different biotic(e.g., maydifferences act as a favoring barrier prezygotic reproductiveisolation. Moreover,this more accurate informationon reproductionnot is available,these apparent phenological other speciesby characterized MtB the mitotype, was mainlyobserved in autumn. Although characterized bythe MtA mitotype waspresent in segregation may promote reproductive isolation. study,In our although thetwo sibling specie major prezygoticbarrier hybridization to (L fertilizingspecies, asynchronous gamete release between relatedtaxa isconsidered the as wellas ecological factors reinforcing thedivergence betweenspecies. In externally Pearson isto due a combination of limited gamete dispersal capacities,reproductive system F. spiralis F. et Coyer(2011) al. suggest thatdespiteintrogre 2005, 2010, Engel etal. 2005, Coyer et al. 2007, 2011, Zardi et al. 2011). Fucus geneInterspecific flowwell isdocumented insome otherbrown algae, such as in the genus geneeffective flowlow istoothetwothese tointegrity genetic of compromise entities. reflecting thefact that reproductive isolationis not complete. However, we arguethat sympatric natural populations of etEngel al. (2005), usingmicrosatellite markers, identifiedabout 13%of hybridsin resulting from hybridization and introgression (Wallaceresultingand hybridization from et introgression al. 2004, Billard et al. , F. vesiculosus and Fucus guiryi Linnaeus and spiralis Fucus G.I. Zardi, K.R. Nicastro, E.S. Serrão & G.A. & G.A. Serrão E.S. Nicastro, K.R. Zardi, G.I. enceshave probably important evolutionary evitan etal. Monteiro 2004, et al. 2012). s arebroadlysympatric, a temporal niche ssion, theof maintenance species integrityof Our observations showedthat the species (e.g. temperature,day length,irradiance) spring and absent in autumn whereas, the the whereas, in autumn absent and spring

For example, For example,

Linnaeus. Accepted Article cryptic species showed high values of gene diversity (H diversity gene of values high showed species cryptic littoralis microsatelliteresults obtained instudy our that suggest the two sibling species of (Pacific), species this reproducesonly asexually Stache (Müllerand 1989).The Chile and (North Sea) Helgoland from strains different in However, (IrishSea). of Man demonstrated the sporophytesalternation and game of sporophytes andgametophytes innatural patterns have been observed (Müller and Stac historyThe life of the genus se frequency-dependentnegative synchronyof spawning) by or the limiting su affect thesuccess of interspecific crosses, either byfavoring prezygotic barriers (phenology, materials, whichmaterials, prevents bridging thegap diagnosticputatively speciescharacters for either uncovered study in this and or inthe type plants fungi, (McNeill et2012).and Unfortunately, wepresently areal. identifyto unable algae, for Nomenclature of Code International the of 11 of Article application in species and Guiry 2014), all of which would have to because the genus gene flow. neverthelessWe didnot assign names tothetwo species uncoveredinthis study Two sibling species were detected in genetics is apowerful approach understanding for and delimiting species status in seaweed. In conclusion, ourstudy illustratesthat theof combination phylogenetic andpopulation similar to that of microsatellite data in kelps (Valero et al. 2011). characterized bylarge populations. Therangeof genetic diversity withinwas populations reproduce mainly sexually. Moreover, itis noteworthy in ourstudy thatthetwo Pylaiella encompasses roughlyspecific 90 intraspecificand (Guiry taxa Pylaiella lection (Tellierlection et al.2011). P. littoralis remains poorly known and controversial, as several betweenthe classical taxonomical approach and populations inSweden Knight and (1923) ccess of hybrids via habitat-dependent or be regarded possibleas names ofthe two he 1989). Kylin (1933)noticedoccurrence the as distinct genetic entities with limited tophytes intophytesin culture, material from Isle e ), suggesting that these species are Pylaiella Accepted Article (ANR-10-BTBR-04) ‘Investisse(ANR-10-BTBR-04) par devoie subvention – Fondsdecompétitivité desentreprises AQUACTIFS “ Economy, Industry and Employment and the Brittany Regional Council through the project technical support. This study was partially supported by grants from the French Ministry for comments. areWe also most grateful to the BiogenouestGenomics core facility for its helpful for Engel C. and Reviers de B. Goer, de S. A. Peter, Valero, M. thank We Acknowledgments ecological tolerance. todocumentexperiments sexual reproductionand torevealdifferences the intheir of thesedifferent speciesphenological, requiresbiological, ecological and genetic distances. However, understanding of speciationfurther of and themaintenance ofintegrity andpopulationgenetics analysescanhelp solvetaxonomic questions atshort phylogenetic speciesmethods.modern delimitation Ourstudy highlights complementaryhow barcoding littoralis ASSEMBLE no. 227799. grant Special thanks tofor CCAP providing strainsof . Convention de soutien de l‘état à des actions de recherche et d‘innovation ments d’avenir, Biotechnol ogies-Bioressources’ and ”, the project IDEALG IDEALG project the ”, Pylaiella Accepted Article sequences, N individuals, N:number of individuals assigned to MtBmitotype MtA or according to expected heterozygosity;—:nodata. Ta mean numberof alleles per microsatellite locus; H ocf R) 62 (RO) Roscoff iad(I 16 (DI) Dinard ocreu(O 21 Concarneau (CO) Location N

an ao(T 41 Saint Malo (ST)

ble 1. Characteristics of of 1. Characteristics ble

cox 1: ofmitotypes number cox1, N

T

name name Sample Pylaiella littoralis Pylaiella 7 6 26October 2008 5 4 3 2 15October 2008 1 Date Mitotype Date Mitotype N 1Arl21 MA 2 36 — 3.6 3 2 4 MtA 21 April 2010 9Arl21 MA 12 96 0.617 9.6 1 2 21 0.608 MtA 9.6 9 19 April 2011 6 26 0.694 6.7 MtA 6 29 April 2009 2 12 MtB 21 April 2010 5Jn 09 t 4 4 . — 3.9 4 4 4 MtA 15 June 2009 4My21 MA 3 44 — 4.4 1 3 6 MtA 14 May 2010 4My21 MB 23 85 0.741 8.5 4 3 12 MtB 14 May 2010 — 4.8 2 2 5 MtB 15 June 2009

samples; N samples; rbc e : microsatellite multilocus non-biased L: number of chlorotypes

t 82 1 2 9.6 0.658 MtB 18 6 4 14 0.672 MtB 32 T: total number of sampled cox N 1 rbcL N

rbc N L, N L, a H cox a : e 1

Table 2. List and characteristics of microsatellite makers used for population analysis. Na: number of alleles detected at each locus.

Marker name Gene Length Left primer (5'-3') Right primer (5'-3') Tm Product Na Location (°C) size (bp) M-168 No Gene 76 AAACTACACCAACGAAATCAA AACCAACCTCCTCCAATC 50 279 37 M-239-3 No Gene 66 ATAAGACTCAGCAAGAGGCAG CATTGAACGACAAATCCC 50 241 42

Article M-383-1 Intron 76 GGGTGAGACAGTAGTAGCAGG CTCGGAACAGGAAGGTTT 50 240 50 M-392 Intron 137 CTTGTGTGCGTGCTGTTG GACTTAGATTTCCGTGTCCTC 54 304 21 M-199-3 No Gene 42 GCAGTAGTAGCAGTAGTCGG TTCATTGTGTTCTTCTTCGTC 50 227 18 M-231 Exon 91 CGGTGACAGATAGACAGACAG CCCTCAGTTGTTCCTCAAGT 50 193 38 M-028-1 Intron 126 ATTACAGCAAGGACAAATGG TAAGAAGGACTGGTGGAGG 54 262 18

M-105-1 Intron 106 CTACTCTCCGCAATAAAGACA AGGTATGAACACGACGACAG 50 298 45 M-138 No Gene 69 TGTCTGAATGTTGATGTGTGT AGGGTAGGCAAAGAATGG 50 271 33 M-194 Intron 91 GTCGGATGACGAGGACTT AATGTTTATTTAGGCTTTGCT 50 296 51

Accepted Accepted Article (N>10). AllF (N>10). pairsof samples assigned to different mitotypes. location. Perpendicular bars indicate additional mutations. Inset shows a mismatch frequency. Lines joining haplotypes indicatemutation events.Colors are coded bysampling sequence from 140 Pylaiella littoralis 2.Median-joiningofFigure network the relationships haplotypesamong ofthe 1). Table (see name s e the indicate 3°59.260’W) andConcarneau 2°1.819’W),(48°38.912’N, Dinard (48°37. 593’N 2°02.600’W),Roscoff (48°43.658’N, 1.Mapthe Figure of sampled locationsalongfour theBrittany Saint coast (France), Malo Legends 3.Table Pairwise population genetic differentiation (pairwise The values of (P < significant Thenumbers (1 0.001). were to 7) to refer the 1). sample Table name (see

MtA MtA MtA MtB MtB MtB MtB MtB MtB 1 3 6 4 7 5

MtA MtA 0 0.09 0.09 0 5 MtA MtA ST 0 7 F ST values were significant (P < 0.001). In grey, the highest highest the grey, In 0.001). < (P significant were values werecalculated only forthan populationswithmore 10 individuals MtB MtB 0.21 0.21 0.20 0 0.07 0.07 0 0.09 0.11 1 MtB MtB 0.24 0.24 0.22 0 0.08 0.08 0 0.11 4 MtB MtB 0.14 0.14 0.15 (47°51.193’N3°55.055’W). Numbers inparentheses (1–7) 0 0.08 0.08 0 6 MtB 0.15 0.15 0.15 0 3 individuals. Circle size is proportional to haplotype F ST estimates). All estimates). F ST values between 1 coding cox1 coding F ST values values Accepted Article 1). black;(MtA, MtB, white). Theseven samples are separated byvertical gray lines (see Table Circles atthe topor the bottomof eachindividual bar indicates the individual’s mitotype black(in white)representingand the individua represented10 loci).Eachindividual by into thin vertical barpartitionedis a K =2 clusters, 5.BayesianFigure clusteringmicrosatellite of data obtained for allsamples (considering all 1. Table see names, sample to key a For parentheses. in given is axis mitotype MtAMtB or are indicated by triangles and circles, respectively. Inertia of each Samplinglocations arecolor-coded.PopulationsFrance. and individuals assigned to analyses were conducted on 10microsatellite markers for 10subsamples from Brittany, sequence from 140 individuals of 3.Median-joiningofFigure network the relationships haplotypesamong ofthe observed number of pairwise differences between distribution histogram for the factorial correspondence analysis (FCA) Figure 4. (a) Principal component analysis between chloroplastic haplotypes, with the frequency of theobserved of number pairwise differences withnetwork maximum parsimony. Insetshow which is require (often ancestral) sequence location. Perpendicular bars indicate additional mutations. frequency. Lines joining haplotypes indicatemutation events.Colors are coded bysampling Pylaiella littoralis Pylaiella individuals. individuals. cox 1 mitochondrial haplotypes, withthe frequencyof the Pylaiella littoralis Pylaiella

on individual multilocus genotypes. These two d to connect sequences existing within the (PCA) populationon allele frequencies and(b) l’s estimated nuclear admixture coefficient. s the mismatch distribution for histogram Pylaiella littoralis . Circle size is proportional to haplotype mv : median vector, hypothesized individuals. rbc L coding Accepted Article logiciel sous TMWindows pour la génétique deswww.genetix.univ–montp2.fr. populations. Belkhir, K., Borsa, P.,L., Chikhi, Raufaste,N. & Bonhomme, F. 2003. GENETIX4.05, intraspecific phylogenies. Bandelt, H. J., Forster,P.& Röhl,1999. A. networks Median-joining inferring for References littoralis Nwithin the networknumber parsimony. with of represents maximum hypothetical sequence (often ancestral) which is required to connect existing sequences sampling location. Perpendicularbarsindicate additional mutations. haplotypefrequency. Lines haplotypesjoining indicate mutationevents. Colors are coded by coding sequence from 45individualsof S2. Median-joiningFigure network ofthe results. individuals. littoralis MtA Pylaiella and MtBto refergroup the with defined the location. Perpendicular bars indicate additional mutations. N represents the number of frequency. Lines joining haplotypes indicatemutation events. Colors are coded bysampling sequence from 45 S1. Median-joiningFigure network ofthe Supplementary material individuals. MtA MtBto and refergroup the with defined the

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