Multidisciplinary Studies of the Genus "Cystodytes" (Ascidiacea): From Molecules to Species

Abstract

[eng] Intraspecies variability is widespread in marine invertebrates. Previous reports of Cystodytes (Ascidiacea, Polycitoridae) in the Mediterranean have been routinely attributed to the nominal species Cystodytes dellechiajei. In the Mediterranean, even though zooid anatomy is remarkably uniform, the general morphology of the colonies varies greatly, especially in terms of color and spicular composition (observed under scanning microscopy). Two different chemotypes, according to their variability in alkaloid chemistry, were defined for 4 different color morphs using both HPLC and RNM techniques and MALDI-TOF analyses. The purple morph displayed a chemotype based on the sulfur-containing pyridoacridines shermilamine B, kuanoniamine D, and their deacetylated forms: deacetylkuanoniamine D and deacetylshermilamine B (new natural product). The blue and green morphs displayed a chemotype based on the C9-unsubstituted pyridoacridines ascididemin and 11-hydroxyascididemin. No major alkaloid was found in the brown form. All of these alkaloids were present in both the tunic and zooids, with the exception of the purple morph, where shermilamine B and kuanoniamine D were found in the tunic, whilst their deacetylated forms were found in both tunic and zooids. Cellular localization of these compounds and ultra structural study of the tunic of the blue, purple and green morphs were attempted by electron microscopy and X-ray microanalysis. The main cell types identified were bladder cells, pigment cells, amebocytes, phagocytes, and morula cells. The sulfur-containing pyridoacridines appeared to be stored in the pigment cells. In addition, the anti-predatory properties of crude extracts, tunic acidity, and spicular contents were assayed in the blue and purple Mediterranean forms, and a purple morph of Guam (USA), using three generalist predators (damselfishes, puffer fishes and sea urchins). Toxicity of crude extracts was also measured by the Microtox bioassay. All crude extracts, as well as ascididemin, were toxic and significantly deterred fish but not sea urchin predation. In contrast, acidity by itself, and spicular shape and concentration did not deter feeding. To determine if the observed variation in the Mediterranean had a genetic basis, a fragment of the mithocondrial gene Cytocrome Oxidase I was sequenced and phylogenetic and population genetic analyses were performed. The results pointed towards the existence of at least 2 species within the 67 samples of 15 colors analyzed. The first well-defined group contained mainly blue colonies with the disc-shaped spicules typical of the genus. The second grouped all samples of a purple morph with sphere-shaped spicules in addition to the disc-shaped ones. When genetic variability was partitioned between color morphs and between geographic locations, the former component explained most of the variance. A study of the biological cycles of the blue and purple Mediterranean morphs was carried out from July 2002 to February 2004. Both the reproductive and growth periods, although partially overlapping, showed significant temporal lags, reinforcing previous genetic and chemical results indicating that these morphs are reproductively isolated and represent distinct species. To determine whether there is a seasonal pattern in the production of chemical and physical defenses, colonies of the blue Mediterranean morph were sampled for a year. Ascididemin was quantified by HPLC and the ash content was calculated as an estimate of the physical defenses. The results showed that when the ascidian invested in reproduction, the energy allocated to other life cycle parameters, such as growth, chemical and physical defenses, was significantly reduced. The multidisciplinary approach used to study the ascidian genus Cystodytes provided an interesting case study through which to develop a better understanding of the biology, ecology and secondary chemistry of marine invertebrates.