850 J. Phycol. 34, 850–856 (1998) MEDITERRANEAN CAULERPA TAXIFOLIA AND C. MEXICANA (CHLOROPHYTA) ARE NOT CONSPECIFIC 1 Jeanine L. Olsen 2 Department of Marine Biology, Centre for Ecological and Evolutionary Studies, University of Groningen, Biological Centre, Postbus 14, 9750 AA Haren, The Netherlands Myriam Valero, Isabelle Meusnier Laboratoire de Ge ´ne ´tique et Evolution des Populations Ve ´ge ´tale, URA-CNRS 1185, Bat. SN2, Universite ´ de Lille 1, 59655 Villeneuve D’Ascq Cedex, France Stella Boele-Bos and Wytze T. Stam Department of Marine Biology, Centre for Ecological and Evolutionary Studies, University of Groningen, Biological Centre, Postbus 14, 9750 AA Haren, The Netherlands ABSTRACT In 1984, Caulerpa taxifolia (Vahl) C. Agardh was reported along the coast of Monaco. Over the past decade it has spread along 60 km of the Mediterranean coastline and presently represents a potential risk to biodiversity. Sev- eral explanations have been advanced regarding the pres- ence of C. taxifolia in the Mediterranean. One hypothesis maintains that the alga was introduced accidentally into the sea at Monaco, where it has been used as a decorative alga in aquaria. Caulerpa taxifolia has not been reported in earlier marine floras of the Mediterranean, and its sud- den appearance has suggested that it may be a recent in- troduction. Another hypothesis proposes that C. taxifolia and Caulerpa mexicana Sonder ex Ku ¨tzing are mor- phological variants of one another and hence conspecific taxa. Caulerpa mexicana has been found in the eastern Mediterranean since at least 1941. In order to establish the taxonomic identities of these taxa, individuals from five populations of C. taxifolia and four populations of C. mexicana were collected from within and outside of the Mediterranean. Comparative DNA sequence analysis of the nuclear ribosomal cistron, including the 3'-end of the 18S, ITS1, 5.8S, and ITS2 regions, show clear phylogenetic sep- aration of the two taxa using parsimony and maximum likelihood analyses. Separation is maintained whether the analyses are based on just the more conserved 18S data or just the fast- evolving spacers. The two species are thus not conspecific. For specimens of uncertain identity (i.e. taxi- folia–mexicana intermediates), a PCR diagnostic ampli- fication can easily be performed because the ITS1 in C. taxifolia is 36 nucleotides shorter than the ITS1 in C. mexicana. Whether or not C. taxifolia has been present for a longer period of time in the marine flora, either as a cryptic endemic species or as the result of one or more in- troductions, represents an additional hypothesis that will require identification of biogeographic populations from throughout the world, as well as a population-level study of the Mediterranean region. Key index words: Caulerpa mexicana; Caulerpa taxi- 1 Received 29 April 1998. Accepted 23 June 1998. 2 Author for reprint requests; e-mail olsenjl@biol.rug.nl. folia; introduced species; ITS; Mediterranean invasions; rDNA Abbreviations: CIA, chloroform-isoamyl alcohol; CTAB, ce- tyltrimethylammonium bromide; ITS, internal transcribed spacer; NaAc, sodium acetate; TE, Tris-EDTA buffer More than 60 species of exotic macrophytic algae have been documented in the Mediterranean Sea— more than 50% of them since 1970 (Boudouresque and Ribera 1995). Since the beginning of the 19th century, seven species of Caulerpa have been record- ed, including C. mexicana Sonder ex Ku ¨tzing (= C. crassifolia (C. Ag.) J. Ag.), C. ollivieri Dostal, C. prolif- era (Forsska ˚l) Lamour., C. racemosa (Forsska ˚l) J. Ag., C. scalpelliformis (Brown ex Turn.) C. Ag., C. sertular- ioides (S.G. Gmelin) Howe, and C. taxifolia (Vahl) C. Agardh (Gallardo et al. 1993). Most of these species, however, are restricted to the warm waters of the east and southeastern Mediterranean and are con- sidered to have entered the Mediterranean since the opening of the Suez Canal in 1869. The only excep- tions are C. prolifera, which is found throughout the Mediterranean and recorded by Lamouroux (1809), and the 1984 appearance of C. taxifolia off the coast of Monaco (Meinesz and Hesse 1991). Over the past decade, C. taxifolia has spread between Toulon, France, and Genoa, Italy, occurring at 70 sites and covering nearly 3000 hectares of subtidal area (De Ville `le and Verlaque 1995). About 60 km of coast- line are currently affected and there is concern about potential reduction in biodiversity (Boudour- esque et al. 1995, Romero 1997), demise of seagrass beds (Posidonia oceanica (L.) Delile) (De Ville `le and Verlaque 1995), effects on local fisheries (Francour et al. 1995), and general negative effects on the coastal ecosystem (Acade ´mie des Sciences–Paris 1997). The mode of origin of C. taxifolia along the coasts of France, Monaco, and Italy is disputed (Acade ´mie des Sciences–Paris 1997, Olsen 1997a). A widely ex- pressed hypothesis is that C. taxifolia was released accidentally from the Monaco Aquarium, where it