Insights Into the Evolution of Shells and Love Darts of Land Snails Revealed from Their Matrix Proteins

GBE Insights into the Evolution of Shells and Love Darts of Land Snails Revealed from Their Matrix Proteins Keisuke Shimizu1,2,*, Kazuki Kimura3,4, Yukinobu Isowa5, Kenshiro Oshima6, Makiko Ishikawa1,7, Hiroyuki Kagi8, Keiji Kito9, Masahira Hattori6,10, Satoshi Chiba3, and Kazuyoshi Endo1 1Department of Earth and Planetary Science, The University of Tokyo, Hongo, Japan 2College of Life and Environmental Sciences, University of Exeter, United Kingdom 3Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan 4Research Institute for Ulleungdo and Dokdo Islands, Kyungpook National University, Bukgu, Daegu, Korea 5Organization for the Strategic Coordination of Research and Intellectual Properties, Meiji University, Kawasaki, Kanagawa, Japan 6Center for Omics and Bioinformatics, Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, Japan 7Faculty of Animal Health Technology, Yamazaki University of Animal Health Technology, Hachioji, Tokyo, Japan 8Geochemical Research Center, Graduate School of Science, The University of Tokyo, Hongo, Japan 9Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Kanagawa, Japan 10Cooperative Major of Advanced Health Science, Graduate School of Advanced Science and Engineering, Waseda University, Japan *Corresponding author: E-mail: [email protected]. Accepted: October 31, 2018 Data deposition: This project has been deposited at the DNA Data Bank of Japan (DDBJ) under the accession numbers (The shot-gun sequences; DRA006965-DRA006966, assembled sequences; PRJDB6927: IADG01000001-IADG01059618, the protein sequences; LC375283-LC375300). Abstract Over the past decade, many skeletal matrix proteins that are possibly related to calcification have been reported in various calcifying animals. Molluscs are among the most diverse calcifying animals and some gastropods have adapted to terrestrial ecological niches. Although many shell matrix proteins (SMPs) have already been reported in molluscs, most reports have focused on marine molluscs, and the SMPs of terrestrial snails remain unclear. In addition, some terrestrial stylommatophoran snails have evolved an additional unique calcified character, called a “love dart,” used for mating behavior. We identified 54 SMPs in the terrestrial snail Euhadra quaesita, and found that they contain specific domains that are widely conserved in molluscan SMPs. However, our results also suggest that some of them possibly have evolved independently by domain shuffling, domain recruitment, or gene co-option. We then identified four dart matrix proteins, and found that two of them are the same proteins as those identified as SMPs. Our results suggest that some dart matrix proteins possibly have evolved by independent gene co-option from SMPs during dart evolution events. These results provide a new perspective on the evolution of SMPs and “love darts” in land snails. Key words: biomineralization, evolution, co-option, gastropods. Introduction protection against predators or extreme environments. In the A variety of calcifying organisms have evolved ever since the past decade, “omics” approaches have advanced consider- Cambrian. “Calcification” was a key morphological innova- ably, making it possible to analyze the molecular basis of in- tion that allowed for the diversification of metazoan life, be- teresting phenomena in both model and nonmodel cause mineralized structures play various roles such as support organisms. Recently, many skeletal matrix proteins that may for soft body parts, as devices for feeding or sensing, and as be related to calcification have been identified by integrating ß The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 380 Genome Biol. Evol. 11(2):380–397. doi:10.1093/gbe/evy242 Advance Access publication November 2, 2018 Evolution of Shells and Love Darts of Land Snails GBE transcriptome or expressed sequence tag analysis and mass Hilgers et al. (2018) provided new insights into the genetic spectrometric peptide analysis (e.g., corals, Ramos-Silva et al. basis of radula formation and suggested that the lophotro- 2013; molluscs, Marie et al. 2010; brachiopods, Jackson et al. chozoan hard structures likely evolved by gene co-option. As 2015;seaurchins,Mann et al. 2008). another instance of molluscan hard structures, some land Molluscs are among the most diverse calcifying animals. snails produce a “love dart” that is composed of calcium Most calcifying organisms live in an aquatic environment, carbonate (Tompa 1980). The love dart is a device associated where calcium and carbonate ions are easily available, with reproductive behavior and is formed in a dart sac, which whereas one group of molluscs, the gastropods, and only a has muscle cells and different types of secretory cells (Koene few other calcifying animal taxa, including vertebrates, have et al. 2013)(fig. 1B and C). Snails pierce the body wall of a adapted to terrestrial ecological niches. Thus, molluscs are a partner with the dart and transfer bioactive substances cov- good model to study various aspects of the evolution of bio- ering the dart during mating: this curious behavior is known mineralization. In particular, gastropods provide a unique op- as “dart shooting” (Adamo and Chase 1988; Chase 2007). portunity to study the evolution of shell matrices in relation to Although the substances do not contain sperm, they induce the transition from aquatic to terrestrial habitats. Since terres- physiological changes in the mating partner and increase fer- trial environments differ from aquatic ones in various aspects tilization success of the sperm donated by the dart shooter including humidity, pH, temperature, and the availability of (Chase 2007). Interestingly, this reproductive trait has evolved calcium ions, land snails have acquired novel features such as repeatedly in land snails (Davison and Mordan 2007), and its lungs instead of gills. Thus, it is possible that they have also evolution is an example of a coevolutionary arms race (Koene changed their shell matrix proteins (SMPs) accordingly in the and Schulenburg 2005). Several studies on the functional process of adapting to terrestrial environments. aspects of dart shooting have been performed on species The pulmonate land snails represent one of the most di- within the families Helicidae and Bradybaenidae (Chase verse groups of gastropods (Tillier et al. 1996). Although SMPs 2007; Baur 2010; Kimura et al. 2014). Euhadra quaesita, or shell-related genes have already been reported from a which belongs to the Bradybaenidae, is one of the species number of molluscan species (abalone, Haliotis asinina, used in those works (Kimura et al. 2013; Kimura and Chiba Marie et al. 2010; limpet, Lottia gigantea, Mann et al. 2015) and our preliminary investigations have revealed that 2012; oyster, Crassostrea gigas, Zhang et al. 2012; pearl oys- this snail discards its dart after a single mating event and ter, Pinctada margaritifera and P. maxima, Marie et al. 2012; produces a new one (N > 30: Kimura K, unpublished data). mussel, Mytilus galloprovincialis, Gao et al. 2015,andM. Preliminary observations have also revealed that it takes 6– edulis, Liao et al. 2015,clam,Mya truncata, Arivalagan 7 days to complete dart formation in E. quaesita (Kimura K, et al. 2016; king scallop, Pecten maximus, Arivalagan et al. unpublished data). Although their curious morphological evo- 2017; fresh water mussels, Elliptio complanata and Villosa lution has attracted attention in the field of behavioral ecol- lienosa, Marie et al. 2017), reports for terrestrial snails remain ogy, especially as a sexually selected behavior in relatively scarce, with only two studies having reported the hermaphrodites (Chase 2007; Baur 2010; Kimura et al. SMPs of land snails for Helix aspersa (Pavat et al. 2012)and 2014), the genes or matrix proteins that correlate with dart Cepaea nemoralis (Mann and Jackson 2014)(fig. 1A). Pavat formation have been ignored. et al. (2012) reported the biochemical properties of SMPs and Here, we performed a combined transcriptome and prote- 14 partial peptides (4–11 amino acid residues) from H. ome analysis in order to identify the SMPs in the terrestrial aspersa, showing that the repertoire of these proteins differs snail Euhadra quaesita. Comparisons of SMPs between two greatly from that of marine molluscan shell proteins. In addi- terrestrial snail species (E. quaesita and Cepaea nemoralis)and tion, Mann and Jackson (2014) performed both proteome other marine molluscs have made it possible to investigate the and transcriptome analyses for the grove snail Cepaea kinds of SMPs that evolved in the common ancestor of these nemoralis and reported 59 major SMPs. Interestingly, more two terrestrial snails. Furthermore, we analyzed the dart ma- than half of these proteins (52.5%) were classified as unchar- trix proteins (DMPs) of E. quaesita, and compared them with acterized and/or novel proteins (Mann and Jackson 2014). molluscan SMPs to infer whether DMP genes have evolved by However, it is uncertain whether some of these novel SMPs gene co-option or represent

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