Short Communication Pheromone evolution in the protozoan ciliate, Euplotes: The ability to synthesize diffusible forms is ancestral and secondarily lost Adriana Vallesi a, * , Graziano Di Giuseppe b , Fernando Dini b , Pierangelo Luporini a a Dipartimento di Biologia Molecolare Cellulare Animale, University of Camerino, Italy b Dipartimento di Biologia, University of Pisa, Italy Received 4 September 2007; revised 26 November 2007; accepted 29 November 2007 Available online 7 January 2008 1. Introduction Ciliates synthesize cell type-specific chemical signals, designated ‘‘pheromones” (earlier ‘‘mating-type factors”, or ‘‘gamones”), that in association with their mating type systems control the switching between the reproductive (mitotic growth) and mating (non-reproductive, sexual) stages of their life cycles (Dini and Nyberg, 1993; Vallesi et al., 1995). Whereas all the species of some genera (e.g., Paramecium and Tetrahymena) retain these signal mole- cules bound to the cell surface and the species of others (e.g., Blepharisma and Dileptus) release them into the extra- cellular environment, the most cosmopolitan and speciose genus Euplotes (Borror and Hill, 1995), is apparently unique because it includes both pheromone-secreting and pheromone-retaining species (reviewed in Miyake, 1981, 1996). Pheromone-secreting and pheromone-retaining species are usually identified by bioassay of clonal cell cultures (i.e., expanded asexually from a single isolated individual) for their capacity to form mating pairs (of self, or homo- typic nature) after having been washed-free of their culture medium and re-suspended in media obtained from other conspecific cell cultures (Beale, 1990). Mating pair forma- tion implies that pheromone activity is present in the cul- ture medium and provides direct evidence that a given species constitutively secretes its pheromones into the extracellular environment. The inability to form mating pairs implies that pheromone activity is absent in the cul- ture medium and is usually taken as evidence (albeit indi- rect) that a species retains its pheromones bound to the cell surface. Four Euplotes species have been identified as phero- mone-secreting, beginning with E. patella more than half century ago (Kimball, 1942) and followed by E. raikovi (Luporini et al., 1983), E. octocarinatus (Schulze-Dieckhoff et al., 1987) and E. nobilii (Alimenti et al., 2002). With the exception of E. patella, several pheromones have been iso- lated and characterized, either by direct chemical analysis of the purified molecules or by molecular cloning of the rel- evant genes, from the other three species (reviewed in Luporini et al., 2005). In E. raikovi and E. nobilii, in partic- ular, NMR spectroscopy (Luginbu ¨hl et al., 1994; Placzek et al., 2007 and references therein) and crystallographic analysis (Weiss et al., 1995) have revealed that the Euplotes pheromones are formed of species-specific families of struc- turally homologous proteins of 40–62 amino acid residues. They share three strictly conserved intra-chain disulfide bonds, as well as a three-helix bundle (core) that is largely responsible for their capacity to elicit varied, context- dependent cell responses according to whether they bind to their cell receptors in autocrine (or self) or paracrine (non-self) manner (Luporini et al., 2005). By contrast, three popular and closely allied species, E. crassus, E. minuta, and E. vannus, are usually regarded as pheromone-retaining species because strenuous efforts to isolate and characterize their pheromones in a soluble form have met with frustrating results (reviewed in Miyake, 1981, 1996). In these species, heterotypic cell/culture-med- ium combinations have never been observed to induce mat- ing pair formation. Rather, pair formation has always been observed to require heterotypic cell/cell combinations and physical interactions between genetically distinct cells (Miyake and Nobili, 1974). By studying the biology of a variety of Euplotes species isolated during several years of sampling marine polar (Antarctic and Arctic) habitats, we have identified two additional pheromone-secreting species that represent 1055-7903/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2007.11.025 * Corresponding author. Fax: +39 0737 403290. E-mail address: adriana.vallesi@unicam.it (A. Vallesi). www.elsevier.com/locate/ympev Available online at www.sciencedirect.com Molecular Phylogenetics and Evolution 47 (2008) 439–442