Halophila stipulacea (Forsskål) Ascherson, a tropical seagrass, is thought to be a Lessepsian immigrant that entered the Mediterranean Sea from the Red Sea after the opening of the Suez Canal (1869). Up to date, no genetic studies of H. stipulacea from Turkey are available. In order to verify the molecular identity of Turkish isolates of H. stipulacea, a part of the rDNA ITS region was sequenced. Comparisons of the genetic polymorphism of this region between isolates from the Turkish coasts of the Aegean Sea and individuals from putative native (Red Sea) and introduced (Mediterranean) populations deposited previously in GenBank were performed. No intra-individual variability was found in the region considered among the isolates from Turkey

Cavas, L.

Duarte, C. M.

E, Varela-Álvarez

Marrá, N.

Rindi, F.

Serrão, Ester

Sapientia

Reçu le 21 septembre 2010 ; accepté après révision le 17 janvier 2011.Received 21 September 2010; accepted in revised form 17 January 2011.Cah. Biol. Mar. (2011) 52 : 227-232Molecular identification of the tropical seagrass Halophila stipulacea from TurkeyElena VARELA-ÁLVAREZ1, Fabio RINDI2, Levent CAVAS3, Ester A. SERRÃO1, Carlos M. DUARTE4and Nuria MARBÁ4(1) CCMAR-CIMAR, Universidade do Algarve, Gambelas, 8005-139 Faro, PortugalE-mail: evarela@ualg.pt (2) Dipartimento di Scienze del Mare, Universitá Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy (3) Dokuz Eylül University, Faculty of Science, Department of Chemistry, Division of Biochemistry, 35160 Kaynaklar Campus/İzmir, Turkey(4) Department of Global Change Research, IMEDEA, CSIC-UIB, Instituto Mediterráneo de Estudios Avanzados,Esporles, Mallorca, SpainAbstract: Halophila stipulacea (Forsskål) Ascherson, a tropical seagrass, is thought to be a Lessepsian immigrant thatentered the Mediterranean Sea from the Red Sea after the opening of the Suez Canal (1869). Up to date, no genetic studiesof H. stipulacea from Turkey are available. In order to verify the molecular identity of Turkish isolates of H. stipulacea, apart of the rDNA ITS region was sequenced. Comparisons of the genetic polymorphism of this region between isolates fromthe Turkish coasts of the Aegean Sea and individuals from putative native (Red Sea) and introduced (Mediterranean)populations deposited previously in GenBank were performed. No intra-individual variability was found in the regionconsidered among the isolates from Turkey.Résumé : Identification moléculaire de la phanérogame marine tropicale Halophila stipulacea des côtes de Turquie.Halophila stipulacea (Forsskål) Ascherson, une phanérogame marine tropicale, est probablement une espèce lessepsienneoriginaire de Mer Rouge entrée en Méditerranée depuis l’ouverture du canal de Suez (1968). Jusqu’à maintenant, aucuneétude génétique d’Halophila stipulacea n’est disponible. Afin de vérifier l’identité moléculaire d’isolats turcs d’H.stipulacea, une partie de la région ITS de l’ADNr a été séquencée. Le polymorphisme génétique de cette région a étécomparé entre isolats des côtes turques de la Mer Egée et des individus issus de populations de la zone d’origine probable(Mer Rouge) et d’autres populations introduites de Méditerranée et déposés à GenBank. Aucune variabilité intra-individuelle n’a été trouvée parmi les isolats turcs.Keywords: Halophila stipulacea l Seagrass l rDNA ITS l Lessepsian immigrant l Red Sea228 MOLECULAR IDENTIFICATION OF HAloPHIlA STIPUlACEA FROM TURkEyIntroductionHalophila stipulacea (Forsskål) Ascherson is the mostcommon seagrass in the northern Red Sea (Lipkin, 1975a)and it is widely distributed in the warm waters of thewestern Indian Ocean and Red Sea. This species was firstrecorded in the eastern Mediterranean Sea in 1895 (Fritsch,1895), but only 30 years later it was recorded as developingwell-established meadows at Rhodos, Greece (Forti, 1927;Issel, 1928). The species was initially considered a tropicalrelict in the eastern Mediterranean Sea (Pérès, 1967), butlater studies hypothesized that H. stipulacea arrived fromthe Red Sea soon after the opening of the Suez Canal, about130 years ago, in 1869, following the Lessepsianhypothesis (Lipkin, 1975b; Procaccini et al., 1999;Ruggiero & Procaccini, 2004). The species remainedrestricted to the eastern Mediterranean for several decadesfollowed by a progressive expansion towards the westernareas. It was first observed in 1995 at the Island of Vulcano,Sicily (Acunto et al., 1995) and recently on the coast ofSalerno, Italy (Gambi et al., 2008). Genetic and morpholog-ical differentiations of western Mediterranean populationsof H. stipulacea versus putative native populations from theRed sea have been compared using RAPD markers and therDNA ITS region (Procaccini et al., 1999; Ruggiero &Procaccini, 2004). Although the existence of H. stipulaceafrom Turkish coasts was reported previously (Güner et al.,1985; Taskin et al., 2008), neither studies nor informationon the genetic diversity and/or molecular identity of H.stipulacea from any coast of Turkey have been so farpublished.The rDNA ITS region is a multi-copy, tripartite, androughly 650-basepair (bp) segment that combines theadvantages of resolution at various scales (ITS1: rapidlyevolving, 5.8S: highly conserved, ITS2: moderately rapidlyto rapidly evolving; Hillis & Dixon, 1991; Hershkovitz &Lewis, 1996) with the ease of amplification of a multi-copyregion into a readily obtainable product. The ITS has beenwidely used in phylogenetic and phylogeographic studies ina variety of organisms (Baldwin et al., 1995), including thegenus Halophila. Using the ITS, Waycott et al. (2002)inferred relationships among species and biogeographicpatterns in the 13 species of the seagrass genus Halophila.In fact, out of the 137 sequences of Halophila published inGenBank up to date, the rDNA ITS represent the vastmajority, with only 5 sequences belonging to othermolecular markers.In order to verify the molecular identity and possiblyprovide insights into the invasion paths of H. stipulacea inTurkey we compared genetic divergence betweenpopulations from Turkey versus populations from putativenative (Red Sea) and introduced (The Mediterranean Sea)populations at a partial rDNA ITS region. Material and MethodsDescription of material collectedHalophila stipulacea was collected from Gümüldür, Turkeyon the 29th of November of 2006. The seagrass covered asurface of about 250 m2 in a sandy area at the depth of 23m. H. stipulacea formed a homogeneous population, inwhich no other large-sized macrophytes were observed.The meadow showed well-defined boundaries withPosidonia oceanica (Linnaeus) Delile, 1813 and theinvasive green alga Caulerpa racemosa var. cylindracea(Sonder) Verlaque, Huisman & Boudouresque, 2003. H.stipulacea consisted of thin creeping rhizomes, 0.25-0.30cm wide, from which pairs of leaves were issued at regularintervals on the dorsal side. Pairs of leaves were attached tothe rhizomes by petioles. The leaves were 3-5 cm long, 0.7-0.8 cm wide, 1 or 2 at each shoot node, linear-oblong, thinand hairy. The margin of the leaves was spinulose and theirsurface was crossed by 4 to 14 pairs of veins. The rhizomeswere irregularly branched and were fixed to the sandysubstratum by roots issued at each node of the rhizomes.Only non-reproductive plants were observed at the time ofcollection; no male or female flowers were observed. Someblades showed incisions, probably as a consequence ofbites from grazers. Three H. stipulacea shoots connected bya rhizome were collected from Gümüldür, TurkeyTable 1. Halophila stipulacea. Details (collector, date, site, depth, coordinates and primers source) of samples used in this study.Tableau 1. Halophila stipulacea. Détails (récoltant, date, site, profondeur et coordonnées géographiques et origine des primers) deséchantillons utilisés dans cette étude.Collector Location, depth Gene Primersand date and GPS coordinates (Source: White et al., 1990)Levent Cavas Gümüldür, Izmir, Internal transcribedTurkey, -23 m spacer 1 (ITS1) and ITS 1_5´ TCC GTA GGT GAA CCT GCG G 3´38o02’39.51’’ N Partial sequence 5.8S ITS 2_5´ GCT GCG TTC TTC ATC GAT GC 3´27o00’54.07’’ E Length: 325 bpE. VARELA-ÁLVAREZ, F. RINDI, L. CAVAS, E.A. SERRÃO, C.M. DUARTE, N. MARBÁ 229(geographical coordinates of the sampling site38°02’39.51”N-27°00’54.07”E) by SCUBA diving andpreserved in silica gel in a single bag for posterior DNAextraction and PCR amplification (Figs 1 & 2).DNA extraction, PCR amplification and sequencingDNA was extracted using the CTAB method of Doyle &Doyle (1987). The entire ITS1 and a partial region of the5.8S rDNA gene were amplified by PCR using theuniversal primers ITS1 and ITS2 (White et al., 1990) (Table1). PCR cycling conditions were hot start (95°C, 5 min), 40cycles of 95°C for 30 sec, 52°C for 30 sec, 72°C for 1 min30 sec, final extension of 5 min at 72°C. The PCR reactionsFigure 1. Halophila stipulacea. Herbarium specimens fromGümüldür, İzmir, Turkey.Figure 1. Halophila stipulacea. Spécimens d’herbier deGümüldür, İzmir, Turkey.Figure 2. Halophila stipulacea. Map of sampling location atGümüldür, İzmir, Turkey.Figure 2. Halophila stipulacea. Carte de la zone d’échantil-lonnage à Gümüldür, İzmir, Turkey. were performed in a 20 µl volumecontaining buffer (10x), dNPTs (2mM), MgCl2 (50 mM), primers (10mM), 0.3 U AmpliTaq Gold poly-merase and approximately 5 to 10ng of template DNA. 4 replicateswere used. Amplified products werevisualized on a 1% agarose gel andthe excess of primers and dNTPsfrom the PCR product wereremoved using the Amersham PCRPurification kit (Qiagen, Hilden,Germany), following the manufac-turer’s instructions. Cloning wascarried out using the pGEMT-EasyVector system II (Promega,Madison, USA) as vector accordingto the manufacturer’s instructions,with JM109 competent cells (highefficiency, > 108 cfumg1). Bacteriawith cloned PCR products wereincubated overnight at 37°C. Threeclones were selected and sent forminiprep preparation and sequenc-ing to MACROGEN (Seoul,korea). Sequence alignments wereconducted with alignment explorerin MEGA4 (Tamura et al., 2007).Results and DiscussionThe three sequences produced in thisstudy were compared with 41published rDNA ITS sequencesavailable in GenBank using the BasicLocal Alignment Search Tool(BLAST) to find regions of localsimilarity and then deposited at theNCBI database (accession numbersshown in Table 2). The amplificationsgave a PCR product of 325 bp: from 1to 271 bp corresponding to the ITS1,and from 272 to 325 bp correspondingto a partial region of the 5.8S gene.The three clones sequenced hereproduced identical sequences with thehighest significant sequencealignment with H. stipulacea(AJ012307) (Evalue < e-15).The final dataset was composedby 43 sequences from 6 differentlocalities. In all analyses, all230 MOLECULAR IDENTIFICATION OF HAloPHIlA STIPUlACEA FROM TURkEyReferenceH. stipulaceaNumber of Accession numbers of rDNA ITS AveragesAverage nucleotide localitiesindividualsH. stipulaceasequences used in this studyintra-individual diversity per localitynucleotide diversityRuggiero &  Vulcano Island, Italy4Ay352637, Ay352636, Ay352634, Ay352633, 1.3-3.6%2.0%Procaccini, 2004 Ay352632, Ay352631, Ay352630, Ay352629, Ay352628, Ay352627, Ay352626, Ay352625, Ay352624, Ay352623, Ay352622, Ay352621, Ay352620, Ay352619, Ay352618, Ay352617Waycott et al., 2002 Sicily, ItalyNo infoAF366436--Ruggiero & Rhodes Island, 1Ay352635--Procaccini, 2004Greece Ruggiero &  Ras Mohammed, Egypt2Ay352616, Ay352615, Ay352614, Ay352613,3.4-53.5%Procaccini, 2004Ay352612, Ay352611. Ay352610, Ay352609,Ay352608, Ay352607, Ay352606 Ruggiero &  Small Creek2Ay352605, Ay352603, Ay352604, Ay3526021.5-4.4%1.9%Procaccini, 2004Egypt,Ay352601, Ay352600Ficca et al., 2000Unknown locationNo infoAJ0123070%0%This studyIzmir, Turkey1GU987099, GU987100, GU9871010%0%Table 2.Halophila stipulacea. rDNA ITS nucleotide divergence at intra-individual and intra-population level within complex, calculated comparing our data with sequencesfor all known sequences deposited in the NCBI data base up to date.Tableau 2.Halophila stipulacea. Divergence nucléotidique de l’ITS de l’ADNr au niveau intra-individuel et intra-population du complexe, calculé en comparant nos donnéesà toutes les séquences connues déposés dans la banque de données NCBI. positions containing gaps and missing data were eliminatedfrom the dataset (complete deletion option). We used forthe analyses the common region for all the sequences.There were a total of 271 positions unambiguously alignedin the final dataset. ITS sequences were considerablydivergent within the H. stipulacea complex (Table 2), bothat intra-individual and intra-population level. At intra -individual level, isolates from Ras Mohammed and SmallCreek in Egypt, and from Vulcano Island in Italy presentedranges of average sequence divergence within individualsfrom 1.3 to 5% (the highest at Ras Mohammed in Egypt).Conversely, no intra- individual nucleotide diversity wasfound in our isolate from Turkey. Table 3 shows ITSnucleotide divergence at inter population level between ourisolate from Turkey and the other isolates from differentlocations. Divergences were quite high, with the highestpairwise uncorrected divergence observed between Turkeyand Ras Mohammed Egypt (11.2%), and the lowest with asample from an unknown location that was 100% identicalto our sequences. The geographical distance in km did notseem to have any relation with intra or interspecificdiversity among isolates.Using a partial nuclear ribosomal DNA region (rDNAITS), we were able to confirm the molecular identity of H.stipulacea from İzmir, Turkey, but also to infer the lack ofintraindividual diversity in this partial region of the ITS, inisolates from Turkey. This result is rather unexpected ifcompared with previous published results for other parts ofthe Mediterranean coasts (Ruggiero & Procaccini, 2004).The ITS sequences of H. stipulacea in İzmir, Turkey seemto be one single variant. It has still to be verified if thispattern is common to other H. stipulacea samples and/orpopulations of this region.AcknowledgementsThis work was financed through the project AXA Aliens,founded by the AXA Foundation to NM and the PortugueseScience Foundation (FCT), with co-funding by ESF andFEDER to EVA. We also thank Liam Cronin for readingand improving the English in the manuscript and MarHuertas and Vincent Laizé for their help with thetranslation of the French abstract.ReferencesAcunto S., Maltagliati F., Rindi F., Rossi F. & Cinelli F. 1995.Osservazioni su una prateria di Halophila stipulacea (Forssk.)Aschers. (Hydrocharitaceae) nel Mar Tirreno meridionale. Attidella Società Toscana di Scienze Naturali, Memorie, Serie B,102: 19-22. Baldwin B.G., Sanderson M.J., Porter J.M., WojciechowskiM.F., Campbell C.S. & Donoghue M.J. 1995. The ITS regionof nuclear ribosomal DNA: A valuable source of evidence onangiosperm phylogeny. Annals of the Missouri BotanicalGarden, 82: 247-277.Doyle J.J. & Doyle J.L. 1987. A rapid DNA isolation procedurefor small quantities of fresh leaf tissue. PhytochemicalBulletin, 19: 11-15.Ficca A.G., De Dominicis R.I., Gregori C. & Tucci G.F. 2000.Phylogenetic relationship in the subclass Alismatidae based onITS sequence analysis. GenBank Accession # AJ012307.1,submitted 27 October 2000. Available online at:http://www.ncbi.nlm.nih.gov, 7 March 2006.Forti A. 1927. La propagazione dell’ Halophila stipulacea (Forsk)Asch. anche nel Mediterraneo. Nuovo Giornale BotanicoItaliano n.s., 34: 714-716. Fritsch C. 1895. Uber die auffindung einer marinenHydrocharidee im Mittelmeer. Verhandllungen der kaiserlich-kongiglichen zoologish -botanischen Gesellschaft in Wien, 45:104-106.Gambi M.C., Barbieri F. & Bianchi C.N. 2008. New record ofthe alien seagrass Halophila stipulacea (Hydrocharitaceae) inthe western Mediterranean: a further clue to the changingMediterranean Sea biogeography. JMBA2 BiodiversityRecords. DOI 10.1017/S175526720900058X.Güner H., Aysel V., Sukatar A. & Öztürk M. 1985. Türkiye EgeDenizi florası. I. Mavi-yeşil, yeşil, Esmer Algler ve kapalıTohumlular. Doğa Bilim Dergisi, 9: 272-279.Hillis D.M. & Dixon M.T. 1991. Ribosomal DNA: Molecularevolution and phylogenetic inference. The Quarterly Review ofE. VARELA-ÁLVAREZ, F. RINDI, L. CAVAS, E.A. SERRÃO, C.M. DUARTE, N. MARBÁ 231Table 3. Halophila stipulacea. Ranges of rDNA ITS nucleotide divergence and geographical distances of the Turkish isolated in rela-tion to each locality studied by other authors.  Tableau 3. Halophila stipulacea. Gamme de divergence nucléotidique et distances géographiques des isolats turcs pour chaque siteétudié par d’autres auteurs.Halophila stipulacea localities Distances in km rDNA ITS nucleotide(aprox.) divergence ranges Gümüldür, Turkey vs. Sicily, Italy 1000 1.1%Gümüldür, Turkey vs. Vulcano Island, Italy 1000 0.4-10.1%Gümüldür, Turkey vs. Rothes Island, Greece 250 0.4%Gümüldür, Turkey vs. Small Creek, Egypt 1000 0.7-4.3%Gümüldür, Turkey vs. Ras Mohammed, Egypt 1000 0.4-11.2%Gümüldür, Turkey vs. unknown location - 0%Biology 66: 411-453.Hershkovitz M.A. & Lewis L.A. 1996. Deep-level diagnosticvalue of the rDNA-ITS region. Molecular Biology andEvolution, 13: 1276-1295.Issel R. 1928. Cenni sui risultati ottenuti dalla MissioneZoologica nel Dodecaneso (1926) per quanto concerne la faunae la flora marine, con alcune osservazioni generali. ArchivioZoologico Italiano, 12: 259–271. Lipkin Y. 1975a. Halophila stipulacea in Cyprus and Rhodes,1967-1970. Aquatic Botany, 1: 309-320.Lipkin Y. 1975b. Halophila stipulacea, a review of a successfulimmigration. Aquatic Botany, 1: 203-215. Pérès J.M. & Picard J. 1958. Recherches sur les peuplementsbenthiques de la Méditerranée nord-orientale. Annales del’Institut océanographique, Paris, 34, 213-291.Pérès J.M. 1967. The Mediterranean benthos. oceanography andMarine Biology: An Annual Review, 5: 449-553 Procaccini G., Acunto S., Famà P. & Maltagliati F. 1999.Structural, morphological and genetic variability in Halophilastipulacea (Hydrocharitaceae) populations in the westernMediterranean. Marine Biology, 135: 181-189.Ruggiero M.V. & Procaccini G. 2004. The rDNA ITS region inthe Lessepsian marine angiosperm Halophila stipulcea(Forssk.) Aschers. (Hydrocharitaceae): Intragenomic variabili-ty and putative pseudogenic sequences. Journal of MolecularEvolution, 58: 115-121. Tamura K., Dudley J., Nei M. & Kumar S. 2007. MEGA4Molecular Evolutionary Genetics Analysis (MEGA) softwareversion 4.0. Molecular Biology and Evolution, 24: 1596-1599.DOI: doi: 10.1093/molbev/msm092.Taskin E., Öztürk M., Kurt O. & Öztürk M. 2008. The check-list of the marine algae of Turkey. pp. [i-ii]-[1]-87. Manisa,Turkey: Ecem kirtasiye.Waycott M., Freshwater D.W., York R.A., Calladine A. &Kenworthy W.J. 2002. Evolutionary trends in the seagrassgenus Halophila (Thouars): insights from molecularphylogeny. Bulletin of Marine Science, 71: 1299-1308.White T.J., Bruns T., Lee S. & Taylor J. 1990. Ampliﬁcationand direct sequencing of fungal ribosomal RNA genes forphylogenetics. In: PCR protocols: A guide to methods andapplications (M.A. Innis, D.H. Gelfand, J.J. Sninsky, T.J.White eds), pp 315-322. Academic Press: New york, 232 MOLECULAR IDENTIFICATION OF HAloPHIlA STIPUlACEA FROM TURkEy

Molecular identification of the tropical seagrass Halophila stipulacea from Turkey

Universidade do Algarve

Reçu le 21 septembre 2010 ; accepté après révision le 17 janvier 2011.
Received 21 September 2010; accepted in revised form 17 January 2011.
Cah. Biol. Mar. (2011) 52 : 227-232
Molecular identification of the tropical seagrass 
Halophila stipulacea from Turkey
Elena VARELA-ÁLVAREZ1, Fabio RINDI2, Levent CAVAS3, Ester A. SERRÃO1, Carlos M. DUARTE4
and Nuria MARBÁ4
(1) CCMAR-CIMAR, Universidade do Algarve, Gambelas, 8005-139 Faro, Portugal
E-mail: evarela@ualg.pt 
(2) Dipartimento di Scienze del Mare, Universitá Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy 
(3) Dokuz Eylül University, Faculty of Science, Department of Chemistry, Division of Biochemistry, 
35160 Kaynaklar Campus/İzmir, Turkey
(4) Department of Global Change Research, IMEDEA, CSIC-UIB, Instituto Mediterráneo de Estudios Avanzados,
Esporles, Mallorca, Spain
Abstract: Halophila stipulacea (Forsskål) Ascherson, a tropical seagrass, is thought to be a Lessepsian immigrant that
entered the Mediterranean Sea from the Red Sea after the opening of the Suez Canal (1869). Up to date, no genetic studies
of H. stipulacea from Turkey are available. In order to verify the molecular identity of Turkish isolates of H. stipulacea, a
part of the rDNA ITS region was sequenced. Comparisons of the genetic polymorphism of this region between isolates from
the Turkish coasts of the Aegean Sea and individuals from putative native (Red Sea) and introduced (Mediterranean)
populations deposited previously in GenBank were performed. No intra-individual variability was found in the region
considered among the isolates from Turkey.
Résumé : Identification moléculaire de la phanérogame marine tropicale Halophila stipulacea des côtes de Turquie.
Halophila stipulacea (Forsskål) Ascherson, une phanérogame marine tropicale, est probablement une espèce lessepsienne
originaire de Mer Rouge entrée en Méditerranée depuis l’ouverture du canal de Suez (1968). Jusqu’à maintenant, aucune
étude génétique d’Halophila stipulacea n’est disponible. Afin de vérifier l’identité moléculaire d’isolats turcs d’H.
stipulacea, une partie de la région ITS de l’ADNr a été séquencée. Le polymorphisme génétique de cette région a été
comparé entre isolats des côtes turques de la Mer Egée et des individus issus de populations de la zone d’origine probable
(Mer Rouge) et d’autres populations introduites de Méditerranée et déposés à GenBank. Aucune variabilité intra-
individuelle n’a été trouvée parmi les isolats turcs.
Keywords: Halophila stipulacea l Seagrass l rDNA ITS l Lessepsian immigrant l Red Sea
228 MOLECULAR IDENTIFICATION OF HAloPHIlA STIPUlACEA FROM TURkEy
Introduction
Halophila stipulacea (Forsskål) Ascherson is the most
common seagrass in the northern Red Sea (Lipkin, 1975a)
and it is widely distributed in the warm waters of the
western Indian Ocean and Red Sea. This species was first
recorded in the eastern Mediterranean Sea in 1895 (Fritsch,
1895), but only 30 years later it was recorded as developing
well-established meadows at Rhodos, Greece (Forti, 1927;
Issel, 1928). The species was initially considered a tropical
relict in the eastern Mediterranean Sea (Pérès, 1967), but
later studies hypothesized that H. stipulacea arrived from
the Red Sea soon after the opening of the Suez Canal, about
130 years ago, in 1869, following the Lessepsian
hypothesis (Lipkin, 1975b; Procaccini et al., 1999;
Ruggiero & Procaccini, 2004). The species remained
restricted to the eastern Mediterranean for several decades
followed by a progressive expansion towards the western
areas. It was first observed in 1995 at the Island of Vulcano,
Sicily (Acunto et al., 1995) and recently on the coast of
Salerno, Italy (Gambi et al., 2008). Genetic and morpholog-
ical differentiations of western Mediterranean populations
of H. stipulacea versus putative native populations from the
Red sea have been compared using RAPD markers and the
rDNA ITS region (Procaccini et al., 1999; Ruggiero &
Procaccini, 2004). Although the existence of H. stipulacea
from Turkish coasts was reported previously (Güner et al.,
1985; Taskin et al., 2008), neither studies nor information
on the genetic diversity and/or molecular identity of H.
stipulacea from any coast of Turkey have been so far
published.
The rDNA ITS region is a multi-copy, tripartite, and
roughly 650-basepair (bp) segment that combines the
advantages of resolution at various scales (ITS1: rapidly
evolving, 5.8S: highly conserved, ITS2: moderately rapidly
to rapidly evolving; Hillis & Dixon, 1991; Hershkovitz &
Lewis, 1996) with the ease of amplification of a multi-copy
region into a readily obtainable product. The ITS has been
widely used in phylogenetic and phylogeographic studies in
a variety of organisms (Baldwin et al., 1995), including the
genus Halophila. Using the ITS, Waycott et al. (2002)
inferred relationships among species and biogeographic
patterns in the 13 species of the seagrass genus Halophila.
In fact, out of the 137 sequences of Halophila published in
GenBank up to date, the rDNA ITS represent the vast
majority, with only 5 sequences belonging to other
molecular markers.
In order to verify the molecular identity and possibly
provide insights into the invasion paths of H. stipulacea in
Turkey we compared genetic divergence between
populations from Turkey versus populations from putative
native (Red Sea) and introduced (The Mediterranean Sea)
populations at a partial rDNA ITS region. 
Material and Methods
Description of material collected
Halophila stipulacea was collected from Gümüldür, Turkey
on the 29th of November of 2006. The seagrass covered a
surface of about 250 m2 in a sandy area at the depth of 23
m. H. stipulacea formed a homogeneous population, in
which no other large-sized macrophytes were observed.
The meadow showed well-defined boundaries with
Posidonia oceanica (Linnaeus) Delile, 1813 and the
invasive green alga Caulerpa racemosa var. cylindracea
(Sonder) Verlaque, Huisman & Boudouresque, 2003. H.
stipulacea consisted of thin creeping rhizomes, 0.25-0.30
cm wide, from which pairs of leaves were issued at regular
intervals on the dorsal side. Pairs of leaves were attached to
the rhizomes by petioles. The leaves were 3-5 cm long, 0.7-
0.8 cm wide, 1 or 2 at each shoot node, linear-oblong, thin
and hairy. The margin of the leaves was spinulose and their
surface was crossed by 4 to 14 pairs of veins. The rhizomes
were irregularly branched and were fixed to the sandy
substratum by roots issued at each node of the rhizomes.
Only non-reproductive plants were observed at the time of
collection; no male or female flowers were observed. Some
blades showed incisions, probably as a consequence of
bites from grazers. Three H. stipulacea shoots connected by
a rhizome were collected from Gümüldür, Turkey
Table 1. Halophila stipulacea. Details (collector, date, site, depth, coordinates and primers source) of samples used in this study.
Tableau 1. Halophila stipulacea. Détails (récoltant, date, site, profondeur et coordonnées géographiques et origine des primers) des
échantillons utilisés dans cette étude.
Collector Location, depth Gene Primers
and date and GPS coordinates (Source: White et al., 1990)
Levent Cavas Gümüldür, Izmir, Internal transcribed
Turkey, -23 m spacer 1 (ITS1) and ITS 1_5´ TCC GTA GGT GAA CCT GCG G 3´
38o02’39.51’’ N Partial sequence 5.8S ITS 2_5´ GCT GCG TTC TTC ATC GAT GC 3´
27o00’54.07’’ E Length: 325 bp
E. VARELA-ÁLVAREZ, F. RINDI, L. CAVAS, E.A. SERRÃO, C.M. DUARTE, N. MARBÁ 229
(geographical coordinates of the sampling site
38°02’39.51”N-27°00’54.07”E) by SCUBA diving and
preserved in silica gel in a single bag for posterior DNA
extraction and PCR amplification (Figs 1 & 2).
DNA extraction, PCR amplification and sequencing
DNA was extracted using the CTAB method of Doyle &
Doyle (1987). The entire ITS1 and a partial region of the
5.8S rDNA gene were amplified by PCR using the
universal primers ITS1 and ITS2 (White et al., 1990) (Table
1). PCR cycling conditions were hot start (95°C, 5 min), 40
cycles of 95°C for 30 sec, 52°C for 30 sec, 72°C for 1 min
30 sec, final extension of 5 min at 72°C. The PCR reactions
Figure 1. Halophila stipulacea. Herbarium specimens from
Gümüldür, İzmir, Turkey.
Figure 1. Halophila stipulacea. Spécimens d’herbier de
Gümüldür, İzmir, Turkey.
Figure 2. Halophila stipulacea. Map of sampling location at
Gümüldür, İzmir, Turkey.
Figure 2. Halophila stipulacea. Carte de la zone d’échantil-
lonnage à Gümüldür, İzmir, Turkey. 
were performed in a 20 µl volume
containing buffer (10x), dNPTs (2
mM), MgCl2 (50 mM), primers (10
mM), 0.3 U AmpliTaq Gold poly-
merase and approximately 5 to 10
ng of template DNA. 4 replicates
were used. Amplified products were
visualized on a 1% agarose gel and
the excess of primers and dNTPs
from the PCR product were
removed using the Amersham PCR
Purification kit (Qiagen, Hilden,
Germany), following the manufac-
turer’s instructions. Cloning was
carried out using the pGEMT-Easy
Vector system II (Promega,
Madison, USA) as vector according
to the manufacturer’s instructions,
with JM109 competent cells (high
efficiency, > 108 cfumg1). Bacteria
with cloned PCR products were
incubated overnight at 37°C. Three
clones were selected and sent for
miniprep preparation and sequenc-
ing to MACROGEN (Seoul,
korea). Sequence alignments were
conducted with alignment explorer
in MEGA4 (Tamura et al., 2007).
Results and Discussion
The three sequences produced in this
study were compared with 41
published rDNA ITS sequences
available in GenBank using the Basic
Local Alignment Search Tool
(BLAST) to find regions of local
similarity and then deposited at the
NCBI database (accession numbers
shown in Table 2). The amplifications
gave a PCR product of 325 bp: from 1
to 271 bp corresponding to the ITS1,
and from 272 to 325 bp corresponding
to a partial region of the 5.8S gene.
The three clones sequenced here
produced identical sequences with the
highest significant sequence
alignment with H. stipulacea
(AJ012307) (Evalue < e-15).
The final dataset was composed
by 43 sequences from 6 different
localities. In all analyses, all
230 MOLECULAR IDENTIFICATION OF HAloPHIlA STIPUlACEA FROM TURkEy
R
ef
er
en
ce
H
. 
st
ip
u
la
ce
a
N
u
m
b
er
 o
f 
A
cc
es
si
on
 n
u
m
b
er
s 
of
 r
D
N
A
 I
T
S
 
A
ve
ra
ge
s
A
ve
ra
ge
 n
u
cl
eo
ti
d
e 
lo
ca
li
ti
es
in
d
iv
id
u
al
s
H
. 
st
ip
u
la
ce
a
se
q
u
en
ce
s 
u
se
d
 i
n
 t
h
is
 s
tu
d
y
in
tr
a-
in
d
iv
id
u
al
 
d
iv
er
si
ty
 p
er
 l
oc
al
it
y
n
u
cl
eo
ti
d
e 
d
iv
er
si
ty
R
ug
gi
er
o 
&
  
V
ul
ca
no
 I
sl
an
d,
 I
ta
ly
4
A
y
35
26
37
, A
y
35
26
36
, A
y
35
26
34
, A
y
35
26
33
, 
1.
3-
3.
6%
2.
0%
Pr
oc
ac
ci
ni
, 2
00
4 
A
y
35
26
32
, A
y
35
26
31
, A
y
35
26
30
, A
y
35
26
29
, 
A
y
35
26
28
, A
y
35
26
27
, A
y
35
26
26
, A
y
35
26
25
, 
A
y
35
26
24
, A
y
35
26
23
, A
y
35
26
22
, A
y
35
26
21
, 
A
y
35
26
20
, A
y
35
26
19
, A
y
35
26
18
, A
y
35
26
17
W
ay
co
tt 
et
 a
l.,
 2
00
2 
Si
ci
ly
, I
ta
ly
N
o 
in
fo
A
F3
66
43
6
-
-
R
ug
gi
er
o 
&
 
R
ho
de
s 
Is
la
nd
, 
1
A
y
35
26
35
-
-
Pr
oc
ac
ci
ni
, 2
00
4
G
re
ec
e 
R
ug
gi
er
o 
&
  
R
as
 M
oh
am
m
ed
, E
gy
pt
2
A
y
35
26
16
, A
y
35
26
15
, A
y
35
26
14
, A
y
35
26
13
,
3.
4-
5
3.
5%
Pr
oc
ac
ci
ni
, 2
00
4
A
y
35
26
12
, A
y
35
26
11
. A
y
35
26
10
, A
y
35
26
09
,
A
y
35
26
08
, A
y
35
26
07
, A
y
35
26
06
 
R
ug
gi
er
o 
&
  
Sm
al
l C
re
ek
2
A
y
35
26
05
, A
y
35
26
03
, A
y
35
26
04
, A
y
35
26
02
1.
5-
4.
4%
1.
9%
Pr
oc
ac
ci
ni
, 2
00
4
E
gy
pt
,
A
y
35
26
01
, A
y
35
26
00
Fi
cc
a 
et
 a
l.,
 2
00
0
U
nk
no
w
n 
lo
ca
tio
n
N
o 
in
fo
A
J0
12
30
7
0%
0%
T
hi
s 
st
ud
y
Iz
m
ir
, T
ur
ke
y
1
G
U
98
70
99
, G
U
98
71
00
, G
U
98
71
01
0%
0%
T
ab
le
 2
.H
al
op
hi
la
 s
tip
ul
ac
ea
. r
D
N
A
 I
T
S 
nu
cl
eo
tid
e 
di
ve
rg
en
ce
 a
t i
nt
ra
-i
nd
iv
id
ua
l a
nd
 in
tr
a-
po
pu
la
tio
n 
le
ve
l w
ith
in
 c
om
pl
ex
, c
al
cu
la
te
d 
co
m
pa
ri
ng
 o
ur
 d
at
a 
w
ith
 s
eq
ue
nc
es
fo
r 
al
l k
no
w
n 
se
qu
en
ce
s 
de
po
si
te
d 
in
 th
e 
N
C
B
I 
da
ta
 b
as
e 
up
 to
 d
at
e.
T
ab
le
au
 2
.H
al
op
hi
la
 s
tip
ul
ac
ea
. D
iv
er
ge
nc
e 
nu
cl
éo
tid
iq
ue
 d
e 
l’
IT
S 
de
 l’
A
D
N
r 
au
 n
iv
ea
u 
in
tr
a-
in
di
vi
du
el
 e
t i
nt
ra
-p
op
ul
at
io
n 
du
 c
om
pl
ex
e,
 c
al
cu
lé
 e
n 
co
m
pa
ra
nt
 n
os
 d
on
né
es
à 
to
ut
es
 le
s 
sé
qu
en
ce
s 
co
nn
ue
s 
dé
po
sé
s 
da
ns
 la
 b
an
qu
e 
de
 d
on
né
es
 N
C
B
I.
 
positions containing gaps and missing data were eliminated
from the dataset (complete deletion option). We used for
the analyses the common region for all the sequences.
There were a total of 271 positions unambiguously aligned
in the final dataset. ITS sequences were considerably
divergent within the H. stipulacea complex (Table 2), both
at intra-individual and intra-population level. At intra -
individual level, isolates from Ras Mohammed and Small
Creek in Egypt, and from Vulcano Island in Italy presented
ranges of average sequence divergence within individuals
from 1.3 to 5% (the highest at Ras Mohammed in Egypt).
Conversely, no intra- individual nucleotide diversity was
found in our isolate from Turkey. Table 3 shows ITS
nucleotide divergence at inter population level between our
isolate from Turkey and the other isolates from different
locations. Divergences were quite high, with the highest
pairwise uncorrected divergence observed between Turkey
and Ras Mohammed Egypt (11.2%), and the lowest with a
sample from an unknown location that was 100% identical
to our sequences. The geographical distance in km did not
seem to have any relation with intra or interspecific
diversity among isolates.
Using a partial nuclear ribosomal DNA region (rDNA
ITS), we were able to confirm the molecular identity of H.
stipulacea from İzmir, Turkey, but also to infer the lack of
intraindividual diversity in this partial region of the ITS, in
isolates from Turkey. This result is rather unexpected if
compared with previous published results for other parts of
the Mediterranean coasts (Ruggiero & Procaccini, 2004).
The ITS sequences of H. stipulacea in İzmir, Turkey seem
to be one single variant. It has still to be verified if this
pattern is common to other H. stipulacea samples and/or
populations of this region.
Acknowledgements
This work was financed through the project AXA Aliens,
founded by the AXA Foundation to NM and the Portuguese
Science Foundation (FCT), with co-funding by ESF and
FEDER to EVA. We also thank Liam Cronin for reading
and improving the English in the manuscript and Mar
Huertas and Vincent Laizé for their help with the
translation of the French abstract.
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232 MOLECULAR IDENTIFICATION OF HAloPHIlA STIPUlACEA FROM TURkEy


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Molecular identification of the tropical seagrass Halophila stipulacea from Turkey

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