Green Alga Caulerpa Taxifolia Introduced To

MARINE ECOLOGY PROGRESS SERIES Vol. 172: 275-280, 1998 Published October 22 Mar Ecol Prog Ser

Molecular evidence for the aquarium origin of the green alga Caulerpa taxifoliaintroduced to the Mediterranean Sea

0. ~ousson~~~~*,J. ~awlowski'~~, L. Zaninetti', A. ~einesz~,C. F. ~oudouresque~

'Departement de Zoologie et Biologie animale, Universite de Geneve, CH-1224 Chene-BougeriesIGenBve. Switzerland 'Centre d'Oceanologie de Marseille. UMR CNRS 6540, F-13288 Marseille, France 3Mus6um d'Histoire Naturelle. CH-1211 Geneve 6, Switzerland "aboratoire Environnement Marin Littoral, Universite de Nice-Sophia Antipolis, Parc Valrose. F-06108 Nice Cedex 2, France

ABSTRACT. Here, we present the first molecular evidence that thetrop~cal green alga Caulerpa taxifolia, which is quickly spreading in the Mediterraneanand out-competing natlve species, escaped to the sea from a public or private equarium. Our data show that this alga is genetically identicalto the strain cultivated in western Europeanaqudria since the early 1970s The facillty with which this strain is obtained world-widerepresents a potential danger of additional biological invasions.

KEYWORDS: Caulerpa taxifolja Mediterranean . Aquaria . ITS rDNA

INTRODUCTION In the mid-1980s, an alga similar to the Caulerpa taxifolia strain cultivated in public aquaria was observed The genus Caulerpa (Ulvophyceae) comprises about for the first time in the Mediterranean Sea, off the coast 70 species ubiquitous in coastal marine environments. of Monaco (Meinesz & Hesse 1991). Since then, the One of these species, C. taxifolia (Vahl) C. Agardh, is species has rapidly spread in the Northwestern common in tropical seas and has been reported along Mediterranean, invading most of the sublittoral envi- the Atlantic American coast (from Brazil to the ronments and competing with native benthic species Caribbean), in the African Atlantic (Gulf of Guinea), (Verlaque & Fntayre 1994, Villele & Verlaque 1995, the Indian Ocean and the Pacific Ocean (Taylor 1977, Bellan-Santini et al. 1996, Bartoli & Boudouresque Meinesz & Boudouresque 1996). Since the early 1970s, 1997). Spectacular progression of this alga was ob- a straln assigned to this species, of unknown geo- served on the French and Italian Riviera, where the graphical origin, has been cultured to be used as a nat- affected areas increased from 1 m2 in 1984 to 3 ha in ural display in the tropical marine aquarium of the Wil- 1990 and 3000 ha in 1996 (Meinesz et al. 1997). Iso- helma Zoologischbotanischer Garten (Stuttgart, lated colonies were also discovered in French Catalo- Germany). Between 1980 and 1983, this strain was nia (1991), Tuscany, the Balearic Islands (1992), Sicily given to the tropical aquarium of Nancy (Northern (1993), and Croatia (1994) (Meinesz & Boudouresque France) and subsequently to the aquarium of Monaco, 1996) (Fig. l), possibly resulting from vegetative dis- located on the Mediterranean shore (Meinesz & semination by pleasure boats and/or fishing nets (Sant Boudouresque 1996). As a rapidly growing and deco- et al. 1996). Morphological, ecological and physiologi- rative alga, it is particularly appreciated by aquario- cal studies have demonstrated that the Mediterranean philes (Artaut 1987). C. taxifolla differs from known tropical populations, exhibiting larger size, vlgorous growth and resistance to low temperatures (Meinesz et al. 1995, Komatsu et

O Inter-Research1998 Resale of full article not permitted 276 Mar Ecol Prog Ser 172: 275-280, 1998

al. 1997). Its competitive success has also been attrib- cies belonging to the Ulvophyceae (Bakker et al. 1995, uted to a number of other factors such as a lack of Leskinen & Pamilo 1997) and to examine the intraspe- severe nutrient limitation, heterotrophy, and the pro- cific variations in Caulerpa (Pillman et al. 1997). duction of toxic and/or repellent secondary metabo- lites (Guerriero et al. 1993, Boudouresque et al. 1996. Chisholm et al. 1996, Delgado et al. 1996, Lemee et al. MATERIALS AND METHODS 1996, Pesando et al. 1996, Boudouresque 1997, Chisholm & Jaubert 1997). It has been proposed that C. DNA extraction, PCR amplification, cloning and taxifolia was accidentally released into the Mediter- sequencing. Living specimens of 10 Mediterranean ranean from a public aquarium (Meinesz & Hesse populations, 5 aquarium isolates and 3 tropical popula- 1991, hleinesz & Boudouresque 1996). Alternatively, tions of Caulerpa taxifolia were isolated, together with the alga may have originated from a Caulerpa species 6 specimens belonging to 4 other Caulerpa species (C. (identified as C. mexicana Sonder ex Kiitzing) that mexicana, C. prolifera [ForsskAl] Lamouroux, C. race- migrated through the Suez Canal from the Red Sea to mosa [Forssk&l] J Agardh and C. sertularioides the Eastern Mediterranean, and then to the Western [Gmelin] Howe), between November 1997 and Febru- Mediterranean (Chisholm et al. 1995). ary 1998 (Table 1). DNA was extracted in guanidine In order to test the hypothesis of an aquarium origin lysis buffer, precipitated with isopropanol and dis- for the Mediterranean Caulerpa taxifolia, we analysed solved in distilled water. PCR amplifications were per- the ribosomal DNA sequences from isolates of C. taxi- formed in a total volume of 50 p1 with an amplification folia obtained from several public aquaria and corn- profile consisting of 40 cycles of 30 s at 94"C, 30 S at pared them to the algae collected in different localities 52°C and 120 S at 72"C, followed by 5 rnin at 72OC for of the Mediterranean. Additionally, we analysed rDNA final extension. The ITS rDNA region was amplified sequences of a number of tropical populations of C. using a Caulerpa-specific primer located near the 3' taxifolia as well as other species of the genus Caulerpa, end of the 18s rDNA (5'-CCTCTGAACCTTCGGGAG including 3 populations of C. mexicana, of which the -3') and a universal primer located near the 5' end of possible delineation from C. taxifolia remains contro- the 28s rDNA (5'-TTCACTCGCCATTACT-3'). Ampli- versial (Taylor 1977, Coppejans & Prud'homme van fied PCR products were purified using the High Pure Reine 1992, Meinesz et al. 1994, Chisholm et al. 1995, PCR Purification Kit (Boehringer), and were subse- Meinesz & Boudouresque 1996). In the present study, quently either sequenced directly or after cloning (3 the internal transcribed spacer (ITS) rDNA was chosen clones for each C. taxifolia specimen), using the AB1 as an appropriate marker to distinguish between spe- 377 automatic sequencer (Perkin Elmer).

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Fig. 1. Geographical map of studied Caulerpa from European aquana and the Mediterranean Jousson et a1 . Molecular evidence for the aquarium ongin of Caulerpa taxifol~a 277

Table 1. Analysed material: Caulerpa species, geographical or~ginand collector

Specimen Species Locality Collector number

C. taxifolia Majorca, Balearic I..Spain A. Meinesz C. taxifolia Saint-Cyprien, French Catalonia J de Vaugelas C. taxjfolia Le Brusc, Provence, France V Gravez C. taxifolia Toulon, Provence, France A. Meinesz C. taxifolia Port-Cros I.,Provence, France J M. Cottalorda C. taxifolia Le Lavandou, Provence, France A. Meinesz C. taxifolia Villefranche, French Riviera L. Burtaire C. taxifolia Messina, Sicily, Italy C. Orestano C. taxifolia Krk I., Malinska, Croatia A. Meinesz C. taxifolia Hvar I., Starigrad, Croatia A. Meinesz C. taxifolia Nancy aquarium, France B. Conde C. taxifolia Stuttgart aquarium, Germany Mrs Koch C. tax~folia Geneva aquarium shop, Switzerland 0. Jousson C. taxifolia Enoshima aquarium, Japan T. Komatsu C. taxifolia Oahu aquarium, Hawaii, USA P. Arnade C. taxifolia Guadeloupe I.,Canbbean J. Blachier C. taxifolia Martinique I.,Canbbean J. Blachier C. taxifolia Ryu-Kyu I.,Japan T. Komatsu C. prolifera Martinique I., Canbbean J. Blachier C. sertulanoides Martinique I., Caribbean J. Blachier C. mexicana Jedda, Red Sea, Saudi Arabia A. Meinesz C. mexicana Stot-Yam, Israel, Mediterranean M. Fine C. mexicana Martinique I., Caribbean J. Blachier C. racemosa Marseille, Provence, France S. Ruitton

Sequence analysis. The sequences were manually similarity contrasts with the high divergence observed aligned using the GDE 2.2. (Larsen et al. 1993) and among the tropical specimens of C. taxifolia, which can analysed using the following methods: the neighbor- attain values of up to 7.3 %. The interspecific sequence joining (NJ) method (Saitou & Nei 1987), the maximum divergence within Caulerpa ranges from 9 to 14 %. The parsimony (MP) method, using the heuristic search aquarium and Mediterranean specimens of C. taxifolia option included in PAUP 3.1.1 (Swofford 1993), and the appear to be closely related to their Caribbean coun- maximum likelihood (ML) method using the fast terparts (100% bootstrap support). In contrast, the DNAml program (Olsen et al. 1994). The reliability of single Indo-Pacific specimen of C. taxifolia branches internal branches in the NJ and MP trees was assessed as a sister group to the aquarium-Mediterranean- using the bootstrap method (Felsenstein 1988). The Caribbean clade. Direct sequencing of PCR products Phylo-win program (Galtier & Gouy 1996) was used for was only possible for the aquarium and Mediterranean distance computations, NJ and ML tree building and specimens of C. taxifolia. For the tropical C. taxifolia bootstrapping. and the other Caulerpa species examined, sequencing of several cloned PCR products showed ITS rDNA polymorphism wlthin all specimens. The sequence RESULTS divergence within a given individual, null for aquarium and Mediterranean C. taxifolia, ranges from 0.5 % The aligned data set of ITS1, 5.8s and ITS2 rDNA in tropical C. taxifolia specimens to 3.7 % in C. race- consisted of 665 sites, including 86 variable and 48 par- m osa. simony informative positions. The phylogenetic analysis of Caulerpa ITS sequences using different phylogenetic methods generated similar results (Fig. 2). DISCUSSION Comparison of sequences revealed the presence of a striking similarity between all of the Mediterranean The ITS rDNA sequence identity of all aquarium and and aquarium C. taxifolia. Very few insertions or dele- Mediterl-anean specimens of Caulerpa taxifolia is con- tions were observed between specimens, and no sistent with the hypothesis that all these specimens nucleotide substitutions were revealed. Such sequence form a single strain. Furthermore, the lack of ITS poly- 278 Mar Ecol Prog Ser 172: 275-280, 1998

I BalcaricIslands 1 specimens of C. rnexicana appears to be related to the I French Catalonia I aquarium-Mediterranean C. taxifolia, invalidating the BruxProvcnce hypothesis of Chisholm et al. (1995), who tentatively ToulonProvence derived Mediterranean C. taxifolia from Eastern Port-CrosProvence 3 Mediterranean C. mexicana in order to support a Red LirradouProvencc Sea origin of the former. Phylogenetic analysis demon- F& Uirlcra strates, with 100% bootstrap support, that the aquarium-Mediterranean strain of C. taxifolia is related to the Caribbean populations. However, accurate identi- fication of its origin necessitates the study of populations covenng the entire geographical range of C. taxifolia.

i HawaiiUSA CONCLUSIONS Guadeloupe Maniniquc Our results confirm that the Caulerpa species which Ryu-Kyu(lapan) C. Martiniquc is invading the Mediterranean belongs to C. taxifolia sertularioides ~~~~i~i~~~and originates from an aquarium strain accidentally --lL C. Jedda(Rcd SLYL) introduced to the sea. This is consistent with the fact lsracl (blcd~tcrrancan) that C. taxifolia was cultivated in a Northern European Martinique public aquarium long before its first appearance in the Provence(Mediterranean) Mediterranean. Further support for this hypothesis is provided by the fact that this alga was then introduced to a number of aquaria, including one present on the Fig. 2 Maximum likelihood tree of Caulerpa spp. inferred from ITS rDNA sequences. Numbers at nodes are percent- Mediterranean coast, just before being actually ob- ages of 100 bootstrap replicates. The same tree topology was served in the sea, off of this aquarium. The biological obtained using neighbor-joining and maximum parsimony invasions resulting from species introduction are today phylogenetic methods a cause of growing concern. In the marine environ- ment, several vectors of introduction have been sug- gested: transport on ship hulls by fouling and clinging, morphism provides an additional argument to support ballast waters, deliberate introductions for aquaculture the hypothesis that we are dealing with a population purposes and unintentional introductions associated comprising few individuals which has undergone pro- with aquaculture practices (Carlton & Geller 1993, Rib- longed confinement under aquarium conditions. era & Boudouresque 1995, Carlton 1996). In addition, a Indeed, such isolation could be responsible for the few introduced species are suspected to have escaped complete homogenization of ribosomal DNA, in agree- from aquaria (Russell 1992). The case of C. taxifolia ment with the observed loss of heterogeneity in labo- described here is the first demonstration of the intro- ratory stocks (Linares et al. 1994). The sequence- duction of an aquarium selected species. As the aquar- homogenization mechanisms of multigenic families, ium-Mediterranean strain of C. taxifolia is appreciated referred to as concerted evolution, have been observed as an ornamental, robust, and fast growing seaweed, it both in sexual (Schlotterer & Tautz 1994) and can be purchased in many aquarium shops and is actu- parthenogenetic lineages (Hillis et al. 1991).Complete ally present world-wide in a number of public and pn- sexual reproduction has never been observed in C. vate aquaria. This represents a potential danger, as taxrfolia in either aquaria or the Mediterranean; as a aquarium specimens of C. taxifolia may be acciden- matter of fact, only male gametes have been reported tally released into other seas (see also Gillespie et al. (Meinesz & Boudouresque 1996). This would suggest 1997). Given the potentially invasive character of the that the aquarium-Mediterranean strain of C. taxrfolia aquarium-Mediterranean strain of C. taxifolia, it would represents a clone. Despite their unusual morphologi- appear that a stricter control of its sale is warranted. cal and physiological characteristics, aquarium and Mediterranean specimens of C. taxifolia branch within Acknowledgements. This study was supported by the Euro- the tropical C. taxifolia group. It can be noted that the pean Community (Life-DGXI: 'Control of the spread of Caulerpa taxjfolia in the Mediterranean'), the French Min- C. taxifolia group is clearly distinct from related spe- istere de 1'Environnement and the Swiss National Science cies of the section Filicoidae, C. sertularioides and C. Foundation. We thank aIl collectors (see names in Table 1) for mexicana. Our results show that none of the studied field assistance and 3 anonymous reviewers for helpful com- Jousson et al.: Molecular evidence for the aquarium origin of Caulerpa taxifolla 279

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Editorial responsibility, Otto Kinne (Editor), Submjtted: May 29, 1998; Accepted September 8, 1998 OldendorfILuhe, Germany Proofs received from authoris): September 23, 1998