First report of Caulerpa taxifolia (Bryopsidales, Chlorophyta) on the Levantine coast (Turkey, Eastern Mediterranean)

Estuarine, Coastal and Shelf Science 74 (2007) 549e556www.elsevier.com/locate/ecss

First report of Caulerpa taxifolia (Bryopsidales, Chlorophyta) on theLevantine coast (Turkey, Eastern Mediterranean)

Cem Cevik a, Mehmet Baki Yokes b,*, Levent Cavas c, Levent Itri Erkol a,Osman Baris Derici a, Marc Verlaque d

a Cukurova University, Faculty of Fisheries, Balcali Campus, Adana, Turkeyb Halic University, Faculty of Arts and Sciences, Department of Molecular Biology and Genetics,

Ahmet Vefik Pasa Cd. Findikzade, 34093 Istanbul, Turkeyc Dokuz Eylul University, Faculty of Arts and Sciences, Department of Chemistry (Biochemistry Division), Izmir, Turkey

d UMR 6540, COM, Universite de la Mediterranee, Campus de Luminy, F13288 Marseille cedex 9, France

Received 22 December 2006; accepted 21 May 2007

Available online 16 July 2007

Abstract

Since its first accidental introduction into the Mediterranean Sea, Caulerpa taxifolia (Vahl) C. Agardh has spread to six Mediterranean coun-tries and has become a major ecological problem. On the basis of morphological and molecular studies (nuclear ribosomal internal transcribedspacer (ITS)), we report for the first time C. taxifolia on the Levantine coast (Gulf of Iskenderun, SE Turkey). Phylogenetic analysis revealed thatthe Iskenderun isolate differs from the invasive aquarium strain first observed in Monaco, and falls in another SW Pacific clade (NE Australia,New Caledonia). The introduction in Turkey by shipping (ballast waters or anchor gears of oil tankers) is possible because C. taxifolia was foundin the Gulf of Iskenderun, which is a major Eastern Mediterranean petrochemical region. On the other hand, C. taxifolia strains are available inaquarium shops in Turkey, suggesting aquarium dumping as another possible cause. As far as the intense maritime traffic of the region is con-cerned, further dispersals of this exotic C. taxifolia strain in the Mediterranean Sea are to be expected.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Caulerpa taxifolia; Bryopsidales; Chlorophyta; introduced species; ITS; rDNA; Turkey; Mediterranean

1. Introduction

Unintentional introductions of non-indigenous species area growing concern in environmental management, especiallyfor marine ecosystems, because of their environmental andeconomic impacts on biodiversity, through habitat alterationand competition with native species (Murphy and Schaffelke,2003; Schaffelke et al., 2006). The number of marine speciesintroductions is increasing everyday; leading to a biologicalpollution recognized as a worldwide problem (Carlton, 1996;Kolar and Lodge, 2001). One of the most publicized marineintroduced species is the tropical green alga Caulerpa taxifolia(Vahl) C. Agardh. It first escaped from an aquarium in Monaco

* Corresponding author.

E-mail address: [email protected] (M.B. Yokes).

0272-7714/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.ecss.2007.05.031

in 1984 (Jousson et al., 1998; Olsen et al., 1998; Meusnieret al., 2001), C. taxifolia has been distributed to six countriesin the Mediterranean Sea: Croatia, France, Italy, Monaco,Spain and Tunisia (Meinesz and Hesse, 1991; Meinesz et al.,2001). In 2000, C. taxifolia was reported for the first time inCalifornia (Dalton, 2000; Kaiser, 2000). Shortly afterwards,new introduced populations of C. taxifolia were recognizedin Australia (New South Wales), approximately 600 km southof the known range for native populations (Millar, 2001;Schaffelke et al., 2002). Molecular studies, based on nuclearrDNA internal transcribed spacer (ITS) and chloroplast markeras well as DNA fingerprints, have revealed that the Mediterra-nean (except for Tunisia), Californian, and SE Australian pop-ulations are of Australian origin (Queensland) and that spreadhas been human-mediated through the aquarium trade. This in-vasive strain is now known as the ‘‘invasive aquarium strain’’

550 C. Cevik et al. / Estuarine, Coastal and Shelf Science 74 (2007) 549e556

(Jousson et al., 1998, 2000; Olsen et al., 1998; Meusnier et al.,2001, 2002, 2004; Wiedenmann et al., 2001; Murphy andSchaffelke, 2003; Stam et al., 2006; Walters et al., 2006).On the other hand, molecular studies showed that the Tunisianstrain is different from the invasive aquarium strain, highlight-ing a second introduction event in the Mediterranean Sea(Jousson et al., 2000; Fama et al., 2002; Meusnier et al.,2004; Stam et al., 2006).

Caulerpa taxifolia has not yet been reported from the Le-vantine coast (Middle East, Eastern Mediterranean). But in2006, a small feather-like Caulerpa species has appeared forthe first time in the Gulf of Iskenderun (SE Turkey, observa-tion: Cem Cevik). In the present study, we carried out morpho-logical and molecular analyses (ITS1-5.8S-ITS2 rDNAsequences) to identify the taxon as well as its possible originand vector of introduction.

2. Materials and methods

2.1. Specimen collection and herbariums

Specimens of Caulerpa were hand-collected by SCUBAdiving in June 2006 in the Gulf of Iskenderun, SE Turkey,(Fig. 1) and preserved in buffered 4% formaldehydeeseawa-ter. The characteristics of the colonized area and different en-vironmental parameters (temperature, salinity, dissolved O2

and pH) were registered during the field study by using YellowSprings Instruments (YSI) 6600 Multiparameter Water QualitySonde. Secchi depth was also measured. The material wascompared with a Caulerpa taxifolia strain kindly providedby an aquarium shop in Izmir, Turkey.

The following voucher specimens were deposited in theHerbarium Verlaque, Centre d’Oceanologie de Marseille(COM), Marseille, France: H7741, Caulerpa taxifolia (Vahl)C. Agardh, leg. C. Cevik, Gulf of Iskenderun, Turkey, 15 June2006, 11 m depth, silted sand; H7742, C. taxifolia (Vahl) C.Agardh, leg. MB Yokes, aquarium shop, Izmir, Turkey, 10September 2006.

It was compared with the following dried specimens depos-ited in the Herbarium Verlaque: H5393, Caulerpa taxifolia(Vahl) C. Agardh, invasive aquarium strain, Cap Martin,France, 15 September 1992, 8 m depth; H5420, C. taxifolia(Vahl) C. Agardh, invasive aquarium strain, Porquerolles,France, September 1997, reared for 2 months in an aquariumin low light conditions; H5421, C. taxifolia (Vahl) C. Agardh,invasive aquarium strain, Cap Martin, France, September2000, reared for 3 months in an aquarium, as well as a largeset of introduced and native specimens of Caulerpa spp. Mea-surements were made on wet specimens.

2.2. DNA isolation, polymerase chain reactionamplification and sequencing

Fresh material was used for DNA extraction. Approxi-mately 0.5 cm of fronds were rinsed with sterilized sea waterand were directly added to 1 ml preheated (60 �C) 2% w/vCTAB buffer (with 2 ml b-mercaptoethanol) and incubatedfor 1 h at 60 �C. Repeated extractions with 0.5 ml chloroformiso-amyl alcohol (24:1 v/v) were performed until complete re-moval of the interphase was achieved. DNA was precipitatedwith 0.7 volumes of cold isopropanol for 1 h at 4 �C, pelletedby centrifugation at 10,000 � g for 20 min. The pellets were

Fig. 1. Gulf of Iskenderun with the sampling locality (>). The colonies of Caulerpa taxifolia were found on a transect line of 36� 49.4090 N, 35� 53.3500 E and 36�

50.0490 N, 35� 53.9380 E. The gulf is one of the major industrial regions of Turkey with intense ship traffic due to the presence of ports and terminals, especially the

BakueCeyhan pipeline, and thermal power plant.

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Fig. 2. Morphological features of the Iskenderun isolate (H7741). (A) Habit: scale bar 1 cm. (B) Details of the stolon and rhizoids: scale bar 1 mm. (C) Details of

the pinnules: scale bar 1 mm.

washed with 70% ethanol, air-dried and dissolved in 0.5 mlddH2O. RNA was removed by incubation (30 min, 37 �C)with RNase (Roche Diagnostics). DNA was precipitated with0.7 volumes of cold isopropanol in the presence of 1/10 vol-ume of 4 M NaAc, washed with 70% ethanol, air-dried andre-dissolved in TE buffer. DNAs were checked on 1% agarosegel.

The partial rDNA and complete ITS DNA sequences wereamplified from the isolated genomic DNA by PCR, using ther-mocycler (Techne TC-312). The PCR mixture included 1 ml ofgenomic DNA, 1 unit of Taq Polymerase (Fermentas), 0.2 mMdATP, dCTP, dTTP, dGTP, reaction buffer with 2 mM MgCl2,0.1 mM each of the forward primer (50-GAGGCAATAAC-AGGTCTGTGATGC-30) and reverse primer (50-TCCTCCGCTTATTGATATGC-30) and ddH2O to a final volume of 25 ml.

The PCR steps were as follows: initial denaturation at95 �C for 2 min; 35 cycles of denaturation at 95 �C for1 min; annealing at 51 �C for 1 min and 30 s; extension at72 �C for 2 min. The PCR was completed with a final exten-sion step at 72 �C for 5 min. After amplification the PCRproducts were checked on 1.5% agarose gel. The products

were purified with High Pure PCR Product Purification Kit(Roche Diagnostics). Cycle sequencing was performed using80e100 ng of purified PCR product with ABI Prism BigDyeTerminator v1.1 Cycle Sequencing Kit (Applied Biosystems)and PCR primers. Band separation was carried out on anABI PRISM 377 Automated Sequencer (Applied Biosystems).

2.3. Sequence alignment and phylogenetic analysis

The sequence covering the ITS1, 5.8S and ITS2 sequenceswas used for phylogenetic analysis. Nucleotide sequenceswere aligned automatically using MEGA version 3.1 (Kumaret al., 2004). A set of 29 additional sequences (27 Caulerpataxifolia, one Caulerpa mexicana Sonder ex Kutzing and oneCaulerpa prolifera (Forsskal) Lamouroux) obtained from Gen-Bank were also included (Table 1). Alignments were subjectedto a phylogenetic analysis using both Bayesian (MrBayes 3.1;Ronquist and Huelsenbeck, 2003) and parsimony (PAUP4.0b10; Swofford, 2003) frameworks. Optimal model for se-quence evolution for the Bayesian analysis was determinedwith ModelTest 3.7 (Posada and Crandall, 1998) using

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Table 1

Caulerpa taxa used for phylogenetic analysis, the locality where the isolate was collected, reference and the EMBL/GenBank accession no. of the ITS1-5.8S-ITS2

sequences

Isolate Locality Reference Accession no

Caulerpa taxifolia Monaco Aquarium, Monaco Olsen et al., 1998 AJ007822

C. taxifolia Sicily, Italy Olsen et al., 1998 AJ007819

C. taxifolia Townsville, Australia Olsen et al., 1998 AJ007823

C. taxifolia Stuttgart Aquarium, Germany Jousson et al., 1998 AJ228976

C. taxifolia Sousse, Tunisia Jousson et al., 2000 AJ299753

C. taxifolia Fraser Island, Australia Jousson et al., 2000 AJ299771

C. taxifolia Noumea, New Caledonia Jousson et al., 2000 AJ299784

C. taxifolia Safaga, Egypt, Red Sea Jousson et al., 2000 AJ299792

C. taxifolia Carlsbad, California, USA Jousson et al., 2000 AJ299742

C. taxifolia Puerto Rico, USA Jousson et al., 2000 AJ299804

C. taxifolia Tahiti, French Polynesia Meusnier et al., 2001 AF259579

C. taxifolia Bolinao, Philippines Meusnier et al., 2001 AF259589

C. taxifolia Majorca, Spain Meusnier et al., 2001 AF259585

C. taxifolia Port Alassio, Italy Meusnier et al., 2001 AF259588

C. taxifolia Cangalayan, Philippines Fama et al., 2002 AJ316278

C. taxifolia Kelso Reef, Australia Meusnier et al., 2002 AF401316

C. taxifolia Kissing Point, Australia Meusnier et al., 2002 AF401319

C. taxifolia Kissing Point, Australia Meusnier et al., 2002 AF460994

C. taxifolia Arlington Reef, Australia Schaffelke et al., 2002 AF316353

C. taxifolia Careel Bay, Australia Schaffelke et al., 2002 AY034868

C. taxifolia Lake Conjola, Australia Schaffelke et al., 2002 AF316356

C. taxifolia Gladstone Harbour, Australia Schaffelke et al., 2002 AF316355

C. taxifolia Port Hacking, Australia Schaffelke et al., 2002 AF316358

C. taxifolia Michaelmas Reef, Australia Schaffelke et al., 2002 AF323601

C. taxifolia Moreton Bay, Australia Schaffelke et al., 2002 AF323597

C. taxifolia Myrmidon Reef, Australia Schaffelke et al., 2002 AY034873

C. taxifolia Kissing Point, Australia Meusnier et al., 2004 AY314762

Caulerpa mexicana Haifa, Israel Olsen et al., 1998 AJ007815

Caulerpa prolifera Martinique, Caribbean Jousson et al., 1998 AJ228988_Izmir isolate Izmir, Turkey (aquarium shop) Present study EF190262

Iskenderun isolate Iskenderun, Turkey Present study EF190263

hierarchical likelihood ratio tests and K80 þ G was found tobe the best model. The MCMC search was run for 5 � 106

generations. The trees obtained from Bayesian analysis werehandled with PAUP to construct a phylogenetic tree. Parsi-mony analysis was performed using heuristic search withTBR branch swapping algorithm and random addition oftaxa (10 replicates). Gaps were coded as ‘‘missing data’’ andthe three indels defined by Meusnier et al. (2004) were re-corded in a gap matrix as independent events with equalweights. Bootstrap resampling (Felsenstein, 1985) with 1000replicates was performed on a reduced data set in whichidentical sequences were only represented once. Caulerpa

Table 2

Comparison of the characteristics of Caulerpa taxifolia from Turkey with the

invasive aquarium strain

Strain

Gulf of

Iskenderun

Aquarium

shop, Izmir

Invasive aquarium

strain

Specimen H7741 H7742 H5393eFrance

Diameter of stolons (mm) 0.9e1.1 1.6e2.0 2.0e2.5

Width of fronds (mm) 1.9e3.5 10.0e13.0 11.0e22.0

Maximal length of

pinnules (mm)

0.8e1.9 8.0e9.0 11.0e15.0

Width of pinnules (mm) 0.2e0.5 1.0e1.2 1.0e1.5

prolifera and Caulerpa mexicana sequences were used asoutgroup taxa.

3. Results

3.1. Distribution and ecology

The colonies of Caulerpa taxifolia were found on a singletransect line beginning from 36� 49.4090 N, 35� 53.3500 Eand ending at 36� 50.0490 N, 35� 53.9380 E. The total lengthof the transect was 150 m with a constant depth of 11 mthroughout the line. The sediment was silted sand and theenvironmental characteristics of the sampling day were: tem-perature 26.12 �C; salinity 38.37; dissolved O2 7.15 mg l�1;pH 8.31; and Secchi depth 4 m.

3.2. Morphological data

The specimens of Caulerpa collected in the Gulf of Isken-derun exhibit neither the typical robust morphology of the in-vasive aquarium strain of Caulerpa taxifolia, nor the usualmorphology of the species. The feather-like thallus is delicatewith narrow stolons, fronds, and pinnules (Fig. 2A). The sto-lons are slender, 0.9e1.1 mm in diameter, with short rhizoidalpillars, 1.0e3.0 mm long (Fig. 2B). The fronds are green,

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Fig. 3. Morphological features of the Izmir aquarium shop isolate (H7742). (A) Specimen reared in aquarium in low light conditions. (B) Specimen reared in

aquarium in normal light conditions. (C) Details of the stolon and rhizoid pillars: scale bar 1 cm. (D) Details of the pinnules: scale bar 1 cm.

simple to 1e2 times laterally branched, 2.0e10.0 cm high and1.9e3.5 mm broad. The number of fronds is 10e12 fronds per10 cm of stolon. The rachis is terete below becoming com-pressed above, 0.5e0.8 mm broad, bearing opposite, disti-chously arranged, closely adjacent pinnules. The pinnulesare compressed, 0.8e1.9 mm long, slightly upwardly curved,broadest (0.2e0.5 mm) just above the base, and tapering toa distinct spinous tip (Figs. 2C and 4C; Table 2). This mor-phology is similar to some varieties of Caulerpa cupressoides(Vahl) C. Agardh.

Conversely, the Caulerpa taxifolia strain provided by theIzmir aquarium shop looks like the invasive aquarium strainof C. taxifolia. The feather-like thallus is more robust, withbroader stolons, fronds, and pinnules (Fig. 3B). The stolons,1.6e2 mm in diameter, bear regularly spaced long rhizoid pil-lars (Fig. 3C). The fronds are green, frequently branched, up tosecond order ramifications, 1.5e15.0 cm high and 10.0e13.0 mm broad, bearing distichously arranged, closely adja-cent pinnules. The pinnules are sickle shaped, 8.0e9.0 mmlong, broadest (1.0e1.2 mm) just above the base, and taperingto a distinct spinous tip (Figs. 3D and 4A; Table 2). In cultivation

Fig. 4. Comparison of the pinnules: scale bar 1 cm. (A) Specimen H7742,

Izmir isolate reared in normal aquarium conditions. (B) Izmir isolate reared

in low light conditions. (C) Specimen H7741, Iskenderun isolate.

554 C. Cevik et al. / Estuarine, Coastal and Shelf Science 74 (2007) 549e556

under low light conditions, morphological variations wereobtained consisting in the production of slender stolons bearingshorter rhizoidal pillars and more delicate fronds (up to 5.5 cmhigh and 7 mm broad) (Figs. 3A and 4B).

3.3. Sequence data

PCR produced single well-defined bands and direct se-quencing resulted in sequences of good quality. No ambiguousalleles were found in any sequence. The phylogenetic analysisrevealed that, in spite of its particular morphology, the isolateof the Gulf of Iskenderun is Caulerpa taxifolia. However, itdiffers from the invasive aquarium strain observed in the Med-iterranean, and falls in another SW Pacific clade (NE

Australia, New Caledonia) (Fig. 5). The Izmir aquarium iso-late falls neither with the invasive aquarium strain, nor withthe Iskenderun strain, but in a third C. taxifolia clade alongwith isolates from Philippines (Fig. 5).

4. Discussion

Hitherto, six taxa of Caulerpa have been reported from Tur-key in the early twenty-first century (Aysel et al., 2002; Cirikand Akcali, 2006): Caulerpa ollivieri Dostal, Caulerpa prolif-era (Forsskal) J.V. Lamouroux, Caulerpa racemosa var.cylindracea (Sonder) Verlaque, Huisman et Boudouresque,C. racemosa var. lamourouxii f. requienii (Montagne) Weber-van Bosse, Caulerpa scalpelliformis var. denticulata (Decaisne)

Fig. 5. Bayesian phylogenetic tree based on ITS1-5.8S-ITS2 sequences from Caulerpa taxifolia. The analysis was run under K80 þ G model of sequence evolution

for 5 � 106 generations. Numbers above branches are Bayesian posterior probabilities (�90%) and the numbers below branches are bootstrap values (�50%).

Caulerpa prolifera and Caulerpa mexicana are used as outgroup taxa. The scale bar is expected changes per site.

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Weber-van Bosse, and Caulerpa sertularioides (S.G. Gmelin)M. Howe.

The present work constitutes the first report of Caulerpataxifolia along the Levantine coast. Molecular studies clearlyshowed that the Caulerpa isolate from Turkey does not belongto the invasive aquarium strain. The sequence divergence be-tween the Iskenderun and Egypt (Safaga) isolates excludesthe possibility of a Red Sea origin and thus introduction viathe Suez Canal. The Iskenderun isolate carries the indelotypeof the Tunisian isolate, defined by Meusnier et al. (2004). Con-trary to the claim of Langar et al. (2002), which said ‘‘the Tu-nisian isolate is genetically the same strain as the one existentin the NW Mediterranean Sea (i.e. invasive aquarium strain)’’,the only Tunisian material sequenced until now is the isolateAJ299753 that differs from the invasive aquarium strain, andtestifies to a second introduction event in the MediterraneanSea (Jousson et al., 2000; Meusnier et al., 2004). Due to thedifferences in sequences other than indels, the Iskenderun iso-late falls into a different clade on the phylogenetic tree.(Fig. 5). The Izmir aquarium shop isolate differs both fromthe invasive aquarium and Iskenderun strains, and belongs tothe ‘‘wild type’’ as defined in Stam et al. (2006), confirmingthat several aquarium strains are purchased and exchangedon the aquarium trade (Stam et al., 2006; Walters et al., 2006).

The Iskenderun isolate is much smaller in size compared tothe invasive aquarium strain and wild Caulerpa taxifolia.However, it was found that high misidentification rates(>12%) may be reached with Caulerpa species if the identifi-cation is only based on the morphological features, showingthat morphological identification is unreliable compared tothe molecular methods (Stam et al., 2006). It is well knownthat C. taxifolia exhibits morphological variations dependingon depth, light and season (Meinesz et al., 1995). In aquarium,decreases in size and morphological changes are easily ob-tained under low light conditions (Figs. 3A,4B,6). Morpholog-ical variations have also been highlighted in the native eastern

Fig. 6. Morphological variations of the invasive aquarium strain of Caulerpataxifolia; scale bar 1 cm. (A) Herbarium specimen H5393, Cap Martin, France,

8 m depth. (B) Herbarium specimen H5421, Cap Martin, France, reared for

3 months in aquarium. (C) Herbarium specimen H5420, Porquerolles, France,

reared for 2 months in aquarium in low light conditions.

Australian populations, with small and delicate strains innorthern, tropical regions, and large and robust strains insouthern cold regions (Benzie et al., 2000; Meusnier et al.,2002; Schaffelke et al., 2002). It is worth noting that the Isken-derun isolate falls in a clade along with several Australian iso-lates native to northern, tropical regions (Kissing Point,Townsville, Gladstone) (Fig. 5). The very small size of the Is-kenderum strain can also result from a degeneration or a ge-netic selection of the transported and thrown out fragmentsdue to the hard conditions in the donor site (e.g. turbid har-bour), during the transit (e.g. by ship) and in the new site.At each step, the mortality rate and so the genetic selectioncan be high.

The genetic and morphological data suggest a new primaryintroduction from NE Australia is possible. The Gulf of Is-kenderun is a major Eastern Mediterranean petrochemical re-gion with intense ship traffic due to the presence of severalcrude oil pipelines, especially the BakueCeyhan pipeline,and a thermal power plant (Fig. 1). Consequently, an intro-duction from Australia by shipping is possible. Another pos-sible scenario of introduction of Caulerpa taxifolia in theGulf of Iskenderun is aquarium dumping. The Izmir isolateobtained from an aquarium shop proves the presence of C.taxifolia strains other than the invasive Mediterranean onesthat are available on the market in Turkey. As far as the in-tense maritime traffic of the Gulf of Iskenderun is concerned,further dispersals of this exotic C. taxifolia strain in the Med-iterranean Sea are to be expected.

Acknowledgement

This study is partially funded by the Cukurova UniversityResearch Fund (project no: SUF2003BAP6).

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