bioRxiv preprint doi: https://doi.org/10.1101/318394; this version posted May 9, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Evolution of shell flattening and the loss of coiling in top shells 2 (Gastropoda: Trochidae: Fossarininae) on wave-swept rock reefs 3 4 Luna Yamamori*, Makoto Kato 5 6 Graduate School of Human and Environmental Studies, Kyoto University, Sakyo, 7 Kyoto, Japan 8 9 *Corresponding author 10 E-mail: [email protected] 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 bioRxiv preprint doi: https://doi.org/10.1101/318394; this version posted May 9, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 34 Abstract 35 Flattening of coiled shells has occurred in numerous gastropod lineages, 36 probably as an adaptation to life in narrow protected spaces, such as crevices or the 37 undersides of rocks. While several genera in the top snail family (Trochidae) have 38 flattened shells, two Fossarininae genera, Broderipia and Roya, are unique in having 39 shells that are limpet-like and zygomorphic, lacking any trace of coiling. The sister 40 genera of these two genera are Fossarina and Synaptocochlea, both of which have 41 coiled shells and live in rock crevices or the vacant shells of sessile organisms. 42 Although Broderipia has recently been identified as living symbiotically in the pits of 43 sea urchins, the habitat and biology of Roya are poorly known. After an extensive 44 search for rare Roya snails on rocky shores of the Japanese Archipelago, we found live 45 Roya eximia snails on intertidal/subtidal rock surfaces exposed to strong waves. The 46 Roya snails crawled swiftly over wave-swept rock surfaces at low tide, while they 47 retreated into the vacant shells of barnacles at high tide, where they adhered firmly to 48 the inner wall. A survey of the macrobenthic communities around the snail habitat 49 showed that Roya snails inhabited only wave-swept rocks of exposed reefs, where the 50 substrata was covered by encrusting red algae and barnacles. Despite the abnormal shell 51 morphology, the radula was similar to other species in the subfamily, and the diet of 52 Roya snails was mainly pennate diatoms. The limpet-like shell of Roya caused loss of 53 coiling and contraction of the soft body, acquisition of a zygomorphic flat body, 54 expansion of the foot sole and loss of the operculum. All of these changes improved 55 tolerance of strong waves and the ability to cling to rock surfaces, and thus enabled a 56 lifestyle split between wave-swept rock surfaces and refugia of vacant barnacle shells. 57 58 Introduction 59 Molluscs exhibit a wide range of shell forms as adaptations to surrounding 60 environmental conditions [1], and for defense against predators [2]. In the history of 61 shell-shape evolution, flattening of the coiled shell is one of the most common events; 62 some of these flattened shells subsequently lost their coiling, forming low-conical 63 limpet-shaped shells. Limpet-shaped shells are seen in Patellogastropoda, 64 Cocculiniformia, Lepetodriloidae, Fissurellidae, Phenacolepadidae, Hipponicidae, bioRxiv preprint doi: https://doi.org/10.1101/318394; this version posted May 9, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 65 Calyptraeidae, Umbraculidae, Trimusculidae, Siphonariidae, Ancylidae, some 66 Capulidae, Thyca crystallina (Eulimidae), Amathina (Amathinidae), and a portion of 67 Fossarininae (Trochidae), among other groups [3]. Of these taxa, only the first five 68 lineages listed originally exhibited non-coiled shells. 69 Patellogastropoda is the largest taxon with limpet-shaped shells, which generally 70 attach to rock surfaces with strong adhesive power and feed by grazing on macroalgae 71 and benthic diatoms [4]. Cocculiniformia attach to sunken wood or whale bone in the 72 deep sea, feeding from their adhered foundation [5-6]. Lepetodriloidae live near 73 hydrothermal vents, with occasional grazing and active suspension feeding, and some 74 species host filamentous bacterial episymbionts on their gills [7]. Fissurellidae snails 75 have limpet-shaped shells with a small oval hole on top, or a small cut on the back end, 76 of the shell. Most species of Fissurellidae are herbivorous, feeding on diatoms, 77 cyanobacteria, macroalgae and sea grasses, whereas emarginuline and diodorine species 78 have been reported to feed on sponges and mixed detrital materials [8-9]. 79 Phenacolepadidae inhabit the bottom surfaces of deeply-embedded rocks or decaying 80 wood, which can be described as a dysoxic, sulfate-rich environment. Phenacolepadid 81 gastropods are thought to feed on chemosynthetic bacteria [10]. Hipponicidae attach to 82 hard inorganic or organic substrata such as rocks, dead corals, and the shells of large 83 gastropods by extracting calcareous substances, and are filter feeders [11]. 84 Calyptraeidae normally attach to solid organic substrates such as dead bivalves, shells 85 occupied by hermit crabs, and the undersides of horseshoe crabs. Calyptraeids are filter 86 feeders that consume particulate foods in water, including small plankton, detritus, and 87 excrement of their host [12]. Umbraculidae have vestigial flat shells that are deeply 88 coated with mantle, feeding on sponges [13]. Trimusculidae gather particles of 89 phytoplankton using a mucus curtain secreted from glands on the head, taking 90 advantage of the turbulence of water [14]. Siphonariidae is an air-breathing family with 91 asymmetrical limpet-shaped shells, which feed on microalgae such as cyanobacteria and 92 diatoms [15]. Ancylidae are limpet-shaped freshwater snails that attach to rocks or 93 aquatic plants, feeding on diatoms on their substrates [16]. The limpet-shaped Capulid 94 genus Capulus, which have top-curled limpet-like shells, attach mainly to bivalves and 95 exploit the feeding currents of their host bivalves or steal the bivalves’ accumulated 96 food using a pseudoproboscis [17-18]. Thyca crystallina is an obligate parasite of sea 97 star Linckia spp., feeding on its host’s hemal and perihemal fluids [19]. Amathina snails bioRxiv preprint doi: https://doi.org/10.1101/318394; this version posted May 9, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 98 with top-curled limpet-like shells attach to large bivalves, such as the fan shell Pinna 99 bicolor, and collect waste food materials from their host bivalves by inserting their 100 proboscis inside the host’s shell [20]. 101 Branch (1985) presented some major advantages of the limpet-shaped shell [3]. 102 First, conical shells greatly reduce water resistance, such that limpets can venture into 103 strongly wave-exposed areas where most coiled gastropods cannot maintain adherence. 104 Second, the large aperture of the limpet-shaped shell allows development of a large 105 foot. Limpets cannot withdraw into their shells or protect themselves with an 106 operculum, instead utilizing a strong clinging force with hard substrata, which is 107 derived from the large foot, as protection from predation. However, the factors that 108 promote the evolution of limpet-like shells have not yet been identified, because most 109 members of the aforementioned families have limpet-like shells and species in transition 110 from coiled to non-coiled shells are rare. 111 The top-shelled family Trochidae is characterized by conical, coiled shells and 112 an alga-grazing habit, although some linages are filter feeders (Umboniinae) and others 113 have flattened shells (Alcyninae, Fossarininae and Stomatellinae). In particular, a 114 completely limpet-like shell is seen only in two genera of Fossarininae, Broderipia and 115 Roya. The sister genera of Broderipia and Roya are Fossarina, with a round-spiral shell 116 and Synaptocochlea with a loosely coiled auriform shell like abalone [21]. Because the 117 four genera of Fossarininae are currently at various stages in the evolutionary process of 118 shell flattening, comparisons of their biology and habitats may help clarify the selection 119 pressures driving shell flattening and loss of coiling. Broderipia, which has an 120 extremely flat shell, was recently revealed to be symbiotic in the pits of sea urchins, and 121 its flat limpet-like shell is apparently adaptive to life in the narrow open space of the 122 pits [22]. On the other hand, the habitat and biology of Roya are poorly understood. 123 To detect selective pressures acting on shell flattening and the loss of coiling in 124 Fossarininae, we first conducted an extensive search for habitats of the key genus, Roya. 125 Because Roya snails are found in low intertidal areas of wave-swept rocky reefs, we 126 carried out a field survey of the macrobenthic and macrophytic communities on various 127 types of rocky reefs surrounding Roya snails, and observed the diurnal behavior of the 128 snails. To determine their feeding biology, radula and gut contents of Roya snails were 129 also examined. By superimposing the data thus obtained on a phylogenetic tree of bioRxiv preprint doi: https://doi.org/10.1101/318394; this version posted May 9, 2018.