308 Sperm morphology of Echinotriton andersoni (Caudata: Salamandridae) Mitsuru Kuramoto1, Satoshi Tanaka2 1 Departmentof Biology,Fukuoka University of Education,Munakata, Fukuoka, 811-41 Japan 2 Nago Senior High School,Nago, Okinawa,905 Japan Shape and size of urodele spermatozoa show significant differences (Picheral, 1979; Wortham et al., 1977, 1982). In an earlier study, 11Japanese salamanders belong- ing to three families were examined (Kuramoto, 1995). The two salamandrid species, Cynops pyrrhogaster and C. ensicauda, have long spermatozoa with a distinct acro- somal barb, and their axial rod and flagellum unite at the end of the tail. In con- trast, spermatozoa of hynobiid and cryptobranchid species do not have an acrosomal barb and the flagellum does not fuse with the axial rod at the end of the tail, leav- ing a relatively long end piece. The sperm heads of hynobiid species are covered with trifoliate acrosomal sheath and a thin perforatorium appears by detachment of the sheath. Spermatozoa of the salamandrids appeared to have a number of derived characters, compared with those of hynobiid and cryptobranchid species (Kuramoto, 1995). Echinotriton andersoni is a terrestrial salamander that is endemic to the Okinawa and Amami island groups of the Ryukyu Islands. It lays eggs on land and the larvae develop in the near-by waters (Matayoshi et al., 1978; Utsunomiya et al., 1978). There is morphological, palaeontological, and biochemical evidence to suggest that Echinotriton, Tylototriton and Pleurodeles constitute a cluster in the phylogenetic relationships of Salamandridae (Wake and Ozeti, 1969; Hayashi and Matsui, 1989; Titus and Larson, 1995). The present study was undertaken to see whether the spermatozoon of E. andersoni would conform to the general salamandrid pattern and to describe its specific features in detail. Sperm morphology appears to contain phylogenetically significant informa- tion (Kuramoto, 1995, 1996). Spermatological data may be useful for elucidating the taxonomic status of Echinotriton versus Tylototriton and other genera (Nussbaum and Brodie, 1982; Hayashi and Matsui, 1989). Five males of E. `andersoni were collected from Motobu, Okinawa Island, in November and December 1995. Snout-vent lengths ranged from 74 to 83 mm and body weight from 14.2 to 20.6 g. This salamander is a protected species, so all specimens were released at the collecting sites after extracting sperm samples. Fresh sperm fluid was squeezed out from the cloaca by lightly pressing the trunk with fingers. By mixing the fluid with Holtfreter's solution, sperm suspensions were prepared 309 individually. Preparations for scanning electron microscopy followed van der Horst and Cross (1978), with minor modifications. Sperm suspension was put on a cover slip, and spermatozoa were fixed with 2.5% glutaraldehyde, dehydrated with ethanol and n-amyl acetate, coated with gold, and observed with a scanning electron microscope JSM-T200 (JEOL). For light microscopic preparations, spermatozoa were fixed on slide glasses and stained with Giemsa. Lengths of sperm head, tail (axial rod), and end piece were mea- sured on enlarged photomicrographs, and widths of head, axial rod, and flagellum were measured on enlarged scanning electron photomicrographs, using a digitizer KD4300 (Graphtec). Sequential Bonferroni technique was applied to Student's t-tests for mean values (Rice, 1989). From the tip to the end of a spermatozoon, an acrosome (or perforatorium), a head proper, a neck piece, an axial rod, and an end piece were clearly identifiable (fig. la). In scanning electron microscopy, the acrosome and neck piece were not so distinct from the head proper (fig. 2a), whereas under light microscopy, dark-stained acrosome and neck piece became clear as the focus was slightly shifted. Such a heterochromatic change was never observed for the head proper and the axial rod. The acrosome is slender, with a sharply pointed tip. At the tip of the acrosome, an acrosomal barb is present (fig. 2b). The barb is about 1 Jlm in length and projects backward at an angle of 40-50°. The head proper is thick, about 1.0 pm in width and about 28% of total sperm length. Unlike for instance in many hynobiid spermatozoa (Kuramoto, 1995), no protoplasmic droplets were observed on the sperm head including acrosome and neck piece. The neck piece was distinct from the head proper not only in its heterochromatic nature but also in its thickness in the Giemsa-stained specimens. The axial rod was about a half of total sperm length, and its width about 0.6 Am at the proximal part. From the base of the axial rod, a flagellum extended along the axial rod. Width of the flagellum was about 0.3 p,m. The flagellum was much longer than the axial rod, and extended further beyond the end of axial rod, leaving a long end piece (fig. la, 2a, and 2c). The five males used in this study differed in their sperm sizes (table 1). Although mean head lengths differed significantly (P < 0.01) only between males nos. 2 and 3 and between nos. 3 and 5, total lengths differed in all combinations excepting nos. 1 and 4. Length of the axial rod was most variable, and the mean values differed significantly in all combinations. Sperm head length was the least variable, with a coefficient of variation of 3.2. Sperm size and morphology of two Cynops species, C. pyrrhogaster and C. ensicauda, were described by the senior author (Kuramoto, 1995). Because spermatozoa of Cynops species had been observed under scanning electron microscopy, we prepared Giemsa- stained sperm specimens of C. pyrrhogaster. Under light microscopy, the acrosome and the neck piece, which had not been descernible in the previous study, were clearly identifiable for C. pyrrhogaster. From both light and scanning electron microscopic .