Recent molecular biological studies have revealed that some photosymbiotic invertebrates dwelling in coral reefs host several genetically different dinoflagellates, Symbiodinium species, as symbionts. However, little is known about the difference in physiologic characteristics among these symbionts living in a single host, because some Symbiodinium strains are difficult to culture in vitro. To isolate some of these Symbiodinium strains, we have developed an agar culture medium plate containing antibiotics and a giant clam tissue homogenate. Using-this medium we isolated two new Symbiodinium strains from two molluscan hosts, Tridacna crocea and Pteraeolidia ianthina, each of which hosted two different Symbiodinium strains belonging to Symbiodinium C and D, respectively. The tissue homogenate was essential for the growth of Symbiodinium D. Although it was not essential for the growth of Symbiodinium C, it did stimulate the initial growth. For the isolation of some Symbiodinium strains, isolation medium containing host homogenate is effective.

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Size-advantage and low-density models have been used to explain how mating systems favor hermaphroditism or gonochorism. However, these models do not indicate historical transitions in sexuality. Here, we investigate the evolution of bidirectional sex change and gonochorism by phylogenetic analysis using the mitochondrial gene of the gobiids Trimma (31 species), Priolepis (eight species), and Trimmatom (two species). Trimma and Priolepis formed a clade within the sister group Trimmatom. Gonadal histology and rearing experiments revealed that Trimma marinae, Trimma nasa, and Trimmatom spp. were gonochoric, whereas all other Trimma and Priolepis spp. were bidirectional sex changers or inferred ones. A maximum-likelihood reconstruction analysis demonstrated that the common ancestor of the three genera was gonochoristic. Bidirectional sex change probably evolved from gonochorism in a common ancestor of Trimma and Priolepis. As the gonads of bidirectional sex changers simultaneously contain mature ovarian and immature testicular components or vice versa, individuals are always potentially capable of functioning as females or males, respectively. Monogamy under low-density conditions may have been the ecological condition for the evolution of bidirectional sex change in a common ancestor. As T. marinae and T. nasa are a monophyletic group, gonochorism should have evolved from bidirectional sex change in a common ancestor.Electronic supplementary materialThe online version of this article (doi:10.1007/s00114-017-1434-z) contains supplementary material, which is available to authorized users.

Summary Intersexual gonads were detected in three clupeiform species, Sardinops melanostictus, Sardinella zunasi and Englaulis japonicus, collected in Tokyo Bay off Yokohama City, Japan, of which appearance rates were 18.2, 34.6 and 33.9 %, respectively. While spermatogenesis proceeded normally in these individuals, previtellogenic stage oocytes were detected outside of the testes in the hermaphrodites, appearing as a single cell or small clusters. The results suggest that these species are non‐functional hermaphrodites. As intersexual gonads have been reported in other clupeiform species collected before significant pollution and/or at sites far from urban influences, such non‐functional hermaphrodites may be common occurrence in the order. However, high rates of hermaphrodite appearance also suggest the possibility that the development of these oocytes were induced by environmental estrogens.