VENUS 78 (1–2): 33–43, 2019 ©The Malacological Society of Japan DOI: http://doi.org/10.18941/venus.78.1-2_33Shell Structures of Pyramidelloid Gastropods December 25, 201933 Comparison of Shell Structures in Pyramidelloid Gastropods (Heterobranchia) Tsuyoshi Takano1,2,3*, Yasunori Kano1 and Takenori Sasaki2 1Atmosphere and Ocean Research Institute, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8564, Japan 2The University Museum, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan 3Meguro Parasitological Museum, 4-1-1 Shimomeguro, Meguro, Tokyo 153-0064, Japan Abstract: The structural composition of shells was observed by scanning electron microscopy for one amathinid and five pyramidellid species in the heterobranch gastropod superfamily Pyramidelloidea. The shells of the six study species consisted of thick crossed-lamellar (CL) layers that were sandwiched between thin outer and inner prismatic layers. Interspecific differences were observed in the number and thickness of the CL layers, the dip angle of the second-order lamellae of each CL layer, and the microstructure of the outer and inner prismatic layers. Of these, the microstructure of the prismatic layers may be useful as taxonomic characters distinguishing the genera and species of Pyramidelloidea. The distributions of observed character states were, however, inconsistent with conventional familial and subfamilial classification, supporting previous anatomical and molecular studies that have challenged the monophyly of pyramidelloid higher taxa. Keywords: Amathinidae, parasite, Pyramidellidae, SEM, shell microstructure, taxonomy Introduction The marine gastropod family Pyramidellidae (subclass Heterobranchia: superfamily Pyramidelloidea) consists at the very least of 5,000 species that belong to approximately 350 genera and subgenera (Schander et al., 1999a). Most pyramidellids are temporary parasites of annelids and of other mollusks. Their functional foot and high-spired shell may facilitate efficient crawling in soft sediment (Vermeij, 1993). Pyramidellids are actually often collected free-living with no information as to their preference for specific hosts (e.g., Hori, 2017). Anatomical traits of the digestive tract, however, seem to confirm a parasitic mode of life for all pyramidellid species. These traits include the absence of a radula and the presence of a long acrembolic proboscis and a buccal pump to suck out body fluids of the host (Fretter & Graham, 1949; Wise, 1996; Ponder & de Keyzer, 1998). The Amathinidae, the only other family of the extant Pyramidelloidea, represents a more specialized group of parasites on bivalve mollusks (Ponder, 1987). Species of the type genus Amathina differ from pyramidellids in having a much higher expansion rate of the aperture that gives the shell a limpet-like (patelliform) appearance (Ponder & de Keyzer, 1998). Amathinids are characterized anatomically in having diffuse salivary glands and a secondary pallial gill, and in lacking the hypobranchial gland, stylet and buccal bulb (Ponder, 1987; Hori & Tsuchida, 1995). Studies on pyramidelloid taxonomy and systematics have been largely based on the shell profile (e.g., Høisæter, 2014; see also Schander et al., 2003 and references therein). Schander et al. (1999a) * Corresponding author: [email protected] 34 T. Takano et al. recognized six distinct families, i.e. Amathinidae, Anisocyclidae, Odostomiidae, Pyramidellidae, Syrnolidae and Turbonillidae, by synthesizing previous conchological studies. In their reviews of gastropod classification, Bouchet & Rocroi (2005) and Bouchet et al. (2017) largely adopted Schander et al.’s (1999a) scheme for Pyramidelloidea, though they downgraded the Odostomiidae, Pyramidellidae, Syrnolidae and Turbonillidae to subfamilies within the Pyramidellidae (s.l.) and synonymized the Anisocyclidae under Turbonillinae; Schander et al.’s (1999a) subfamilies of Pyramidellidae (s.l.) were also downgraded to 11 tribes in the Odostomiinae, Pyramidellinae, Syrnolinae and Turbonillinae. On the other hand, detailed observation of shell morphology and anatomical characters (Wise, 1996; Schander et al., 1999b) has suggested the non-monophyly of the Odostomiinae and Turbonillinae sensu Bouchet & Rocroi (2005). The latter subfamily’s status as a monophyletic clade has been questioned also through analyses of mitochondrial 16S rDNA sequences (Schander et al., 2003). Classification based on gross shell morphology alone, therefore, does not seem to reflect true phylogenetic relationships. However, comprehensive acquisition of anatomical or molecular data is a huge challenge for the Pyramidelloidea, many species of which are represented only by empty shells (Peñas & Rolán, 2010). Comparisons of shell (micro)structures, which can be observed even in empty shells, might thus shed new light on pyramidelloid systematics. Previous studies have reported variation in shell structures within a family or a higher taxonomic group of the Gastropoda. These include Taylor & Reid (1990) for the Littorinidae (Caenogastropoda), Falniowski & Szarowska (1995) and studies cited therein for the Truncatelloidea (Caenogastropoda), Hedegaard (1997) for various Vetigastropoda, Sasaki (2001) for the Neritoidea (Neritimorpha), and Kiel (2004) for various gastropod taxa from the deep-sea hydrothermal vents and cold seep environments. Notably, Fuchigami & Sasaki (2005) have shown that the structure is almost stable within each patellogastropod genus while differences exist between genera. However, previous studies for the Heterobranchia have focused on the identification of observed microstructures and comparison with other gastropod taxa, rather than structural variation among heterobranch families and genera (e.g., Bandel, 1979, 1990). Here we investigate the shell structure for the Pyramidelloidea by scanning electron microscopy (SEM) for the first time and evaluate its usefulness for the generic and (sub) familial classification of the superfamily. Materials and Methods We selected ten specimens for the observation of shell structures (Fig. 1; Table 1). These include five pyramidellid species that cover all four subfamilies of Pyramidellidae (sensu Bouchet et al., 2017) as well as a single species of Amathinidae. Specimen identification followed Hori (2017). Sayella? sp. (= Gen. aff. Sayella sp. sensu Hori, 2017) belongs to the subfamily Pyramidellinae, provided that the species is phylogenetically allied to the type of Sayella (S. hemphilli). The samples were collected from intertidal and shallow subtidal waters. Brachystomia bipyramidata and Leucotina sp. were found on their bivalve hosts (Crassostrea gigas and Ruditapes philippinarum, respectively; Table 1). The other species were collected while autonomous and thus there is no information as to the host. All animals were treated with hot water and preserved in pure ethanol for DNA extraction and molecular phylogenetic analysis (Takano, Kano et al., in prep.). The study material of Leucotina sp. and B. bipyramidata might represent immature forms of the species (see Hori, 2017 for fully-grown shells). The structural composition of the shell was observed on the fractured faces of both penultimate and body whorls to ensure examination of all shell layers of the specimens. The shell was broken with a vise into pieces, which were then treated with half-diluted commercial bleach for 1–3 h. The shell pieces were cleaned with an ultrasonic cleaner for 1 min, rinsed in double distilled water for more than 2 h, dried, and coated with osmium for 15 s using a plasma coater (Vacuum Device Shell Structures of Pyramidelloid Gastropods 35 Fig. 1. Study specimens of amathinid (A) and pyramidellid (B–F) gastropods. A. Leucotina sp. (UMUT RM33113, specimen T37, shell height: 3.7 mm). B. Brachystomia bipyramidata (UMUT RM33114, T23–2, 3.3 mm; subfamily Odostomiinae). C. Pyramidella maculosa (UMUT RM33117, T40, 29.6 mm; Pyramidellinae). D. Sayella? sp. (UMUT RM33118, T35, 2.6 mm; Pyramidellinae). E. Syrnola serotina (UMUT RM33119, T22–2, 6.7 mm; Syrnolinae). F. Cingulina circinata (UMUT RM33120, T32, 8.5 mm; Turbonillinae). HPC-1SW). The fractured face and outer and inner surfaces of the shell were observed with a SEM (Keyence VE-8800) at The University Museum, The University of Tokyo (UMUT). Multiple shell pieces were examined to characterize fractured faces in directions parallel and perpendicular to growth lines. Other pieces were used for the observation of the outer and inner surfaces of the shell. Voucher material has been deposited at UMUT (see Table 1). The identification and terminology of microstructures follow previous studies; microstructures equivalent to those observed in this study have been precisely described and illustrated for other molluscan groups (e.g., Carter, 1990; Taylor & Reid, 1990; Fuchigami & Sasaki, 2005). We therefore highlight differences in the structural composition among the study species rather than the details of the respective microstructures. To avoid any confusion, these are our definitions of four technical terms: - dip angle: the angle of the growth axis of crystal units to the plane of the inner shell surface - microstructure: the morphology of crystal units and their mode of aggregation - shell layer: a sheet-like component consisting of a single microstructure - structural composition: the total composition of microstructures and shell layers constituting the shell. T. Takano et al. 3 Table 1. Species used in present study with collection sites and habitats of specimens. Abbreviation: