Marine Biology (1997) 129: 425±433 Ó Springer-Verlag 1997 F. A. Monteiro á A. M. Sole -Cava á J. P. Thorpe Extensive genetic divergence between populations of the common intertidal sea anemone Actinia equina from Britain, the Mediterranean and the Cape Verde Islands Received: 18 January 1997 / Accepted: 21 February 1997 Abstract Samples of apparently similar red morphs of (Kola Peninsula) down the Atlantic coast of Europe as the common beadlet sea anemone Actinia equina (L.) far south as West Africa, including the British Isles and were collected from rocky shores on the Isle of Man many islands along the African coast (Azores, Madeira, (Irish Sea), on the French Mediterranean coast near Canaries, Cape Verde and St. Thomas) as well as in the Marseille and on the Cape Verde Island of Sal (o West Mediteranean, Adriatic and Black Seas (Stephenson Africa). For additional comparison an orange morph 1953; Schmidt 1972; Manuel 1988). It is also considered and the green A. prasina were also collected from the Isle to occur in South Africa (Manuel 1988), although of Man. Morphological descriptions were made and the whether there is continuous distribution down the Af- samples were compared by nematocyst analysis and rican coast is unclear. Over this very wide geographical enzyme electrophoresis. The three British samples distribution A. equina is considered to be highly variable showed little genetic divergence I > 0:90 but the in colour pattern, reproductive biology, morphology Mediterranean sample was hugely divergent (I < 0:20) and habitat choice. Not surprisingly this anemone has from the British ones. The Cape Verde Island anemones been the subject of much taxonomic debate. The high were also very dierent (I < 0:60) from all other sam- morphological variability led Gosse (1860) to separate ples. It is concluded that the red morph samples from the the species into several varieties, which were later re- Cape Verde Islands, the Mediterranean and the Isle of duced by Stephenson (1935) to just two varieties: the Man belong to three dierent species. For the new large and essentially monotypic A. equina var. fragacea species from the Mediterranean and Cape Verde Islands and the highly polytypic A. equina var. mesembryanthe- formal descriptions are given, and the names Actinia mum. Schmidt (1971, 1972) in a major study of mor- schmidti sp.n. and Actinia sali sp.n. are proposed. phology, anatomy, nematocysts and reproductive biology of European Actinia equina divided the species into four subspecies: A. equina equina, A. equina atlan- tica, A. equina mediterranea and A. equina fragacea with Introduction most of these subspecies further subdivided into distinct morphs. The common intertidal beadlet sea anemone Actinia The reluctance of these authors to assign speci®c equina (L.) is generally considered to be found on rocky status to the dierent morphotypes was understandable shores over a wide geographical area. A. equina has been because the number of dierent morphs was very great identi®ed from the subpolar coasts of north Russia with several of these often occurring sympatrically on any given shore. Problems were confounded by reports of the existence of intermediates between some of the Communicated by O. Kinne, Oldendorf/Luhe varieties, and the obvious problems of delimitating F.A. Monteiro á A.M. Sole -Cava species boundaries when the morphs present often var- Departamento de Gene tica, Instituto de Biologia, ied greatly between adjacent shores. The availability of Universidade Federal do Rio de Janeiro, more powerful genetic methods, like allozyme electro- Cidade Universita ria, C.C.S., Bloco A, phoresis (reviewed by e.g. Thorpe and Sole -Cava 1994), 21941 Rio de Janeiro, RJ, Brazil presented the objective tools to tackle the systematics of A.M. Sole -Cava á J.P. Thorpe (&) Actinia. In 1981, Carter and Thorpe, using genetic, Department of Environmental and Evolutionary Biology, University of Liverpool, Port Erin Marine Laboratory, ecological and reproductive dierences, were able to ®nd Port Erin, Isle of Man, IM9 6JA, clear diagnostic characteristics that led them to give United Kingdom speci®c status to Stephenson's (1935) varieties fragacea 426 and mesembryanthemum, thus named A. fragacea and A. equina, respectively. Later the green morph of A. equina (possibly Schmidt's A. equina equina morph II) was found to be reproductively isolated from the sym- patric red morph in the Isle of Man, and was therefore also given speci®c status, as A. prasina (Haylor et al. 1984; later con®rmed using samples from a dierent shore, by Sole -Cava and Thorpe 1987). Further work, with other colour morphs from the British Isles, has suggested that even in that region the number of species may be larger still (Sole -Cava and Thorpe 1992; Perrin et al. 1997). Due to their ubiquity and ecological importance, a great deal of work has been carried out on Actinia equina sensu latu) populations from British and Mediterranean shores. This includes ecological (Rees 1984; Chintirog- lou and Koukouras 1992; Perrin 1993), phylogenetic (Sole -Cava et al. 1994a), physiological (Boury-Esnault and Doumenc 1979; Young et al. 1988), behavioral (Brace and Quicke 1986) and more recently toxicological and pharmacological (Macek et al. 1994) studies. For these studies to be of wider applicability it is important that individuals are correctly assigned to a biological species. Studies of British populations of A. ``equina'' indicate that the species may be a complex, but, to date, there is little genetic information available on samples from elsewhere within its range of distribution. In the present work we used allozyme electrophoresis to compare samples of British, Mediterranean and West African (Cape Verde Islands) populations of the red Fig. 1 Location of the three sampling sites in western Europe and A. equina. We have also included in this study individ- northwest Africa. Scale: 1000 km uals of the green A. prasina, and of an orange morph from Britain. The technique of allozyme electrophoresis was chosen because it has proved successful in resolving problems of cryptic speciation in Actinia, as well as kept alive in a sea water aquarium for 1 week, during which wet weight and the precise shade of colour (Kornerup and Wanscher within several other sea anemone genera (e.g. Bunodo- 1978) were determined and nematocyst analysis (Manuel 1988) was soma, McCommas and Lester 1980; Metridium, Bucklin carried out. Subsequently the anemones were cut open to search for and Hedgecock 1982; Urticina, Sole -Cava et al. 1985; brooded juveniles and then stored frozen until required for elec- Sagartia, Shaw et al. 1987; Anthopleura, Smith and Potts trophoresis. 1987). Electrophoresis Tissue samples taken from the oral disc and column of the anem- Materials and methods ones were homogenised with not more than an equal volume of distilled water, and analysed by horizontal 12.5% starch gel elec- Collection of samples trophoresis, using a 0.05 M Tris-citrate buer, pH 8.0 (for further details see Sole -Cava et al. 1985). Thirty enzyme systems were as- Samples of adult red morph Actinia equina were collected from sayed, of which 16 (coding for a total of 19 loci) gave reproducible rocky intertidal areas of Fleshwick Bay (Isle of Man, northern Irish results. Sea) and Marseille (France, Mediterranean Sea) during June 1995, and from the island of Sal (Cape Verde Islands, o West Africa) in October 1994 (Fig. 1). The green A. prasina and a hitherto un- Nematocyst analysis studied orange morph of A. equina were also collected at Fleshwick Bay in June 1995 to provide further comparisons with the Medi- Twenty each of the eight types of nematocysts found in the mes- terranean and Cape Verde samples. The red Actinia collected in enteric ®laments, actinopharynx, acrorhagi, and tentacles were Marseille corresponded closely to Schmidt's (1971, 1972) Actinia measured in three individuals of each morph (a total of 60 nem- equina mediterranea type I. The samples from the Cape Verde Is- atocysts of each type for each tissue). Nematocyst preparation and lands were very similar to Schmidt's (1971, 1972) description of classi®cation followed standard methods (Carlgren 1940; Manuel Actinia equina atlantica type II. 1988; Shick 1991). Nematocyst samples were taken only from adult After collection the anemones were transferred to plastic bags anemones because some authors (Dunn 1981; Fautin 1988) have with wet paper towels and put in an insulated container for argued that the size of the capsules may be related to the size or age transport to the laboratory. No anemones died during transpor- of the anemones. Spirocysts were not measured because they are tation, which took up to 30 h. In the laboratory the anemones were considered to be of little taxonomic value (Manuel 1988). 427 Data analysis counterparts, the Mediterranean anemones did not form aggregations on the shore (Monteiro personal observa- Allozyme data were analysed using the programme BIOSYS-1 (Swoord and Selander 1981). Levels of heterozygosity and genetic tion) a possible further indication that they do not re- distance (Nei 1978) were estimated for all populations analysed. produce asexually. The genetic distances were then used to build neighbour-joining trees (Saitou and Nei 1987), using the programme MEGA (Kumar et al. 1993). Nematocyst analysis Signi®cant dierences (P < 0:0001) were found in the Results average length of some capsule types between the pop- ulations studied (Table 3). The microbasic b-masti- Morphological characteristics gophores of the mesenteric ®laments, one of the classes of type 1 basitrics of the actynopharynx, and the ba- The three red morph samples had red columns, tentacles sitrics of the tentacles appear to be the most useful as and pedal disc. Some red morphs (at least in Britain) diagnostic characters. have a grey or green pedal disc, but we did not sample these in this work.