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

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.

Fig. 1.

—Phylogeny and morphology of Euhadra quaesita. (A) Phylogeny of molluscs that have been published mantle transcriptome or SMP analysis. Red circles indicate transcriptome analysis using the mantle tissues. Blue circles indicate proteome analysis using the shells. B, Bivalvia; G, Gastropoda; L, Lymnaeoidea; P, Pulmonata; S, Stylommatophora. (B) Reproductive organs. Yellow broken lines indicate the dart sac. ag, accessory glands; ds, dart sac; ga, genital atrium; ps, penial sheath. (C) Morphology of the dart. (D) Morphology of the shell. All scales are 10 mm.

Fig. 2.

—Raman spectra of the shell and dart of Euhadra quaesita. Peaks at 154,205 cm⁻¹ (lattice vibration), 702,706 cm⁻¹ (in-plane bending), and 1,085 cm⁻¹ (CO₃ symmetric stretching) for shell (A) and the peaks at 153, 209, 702, 706, and 1,085 cm⁻¹ for dart (B) are attributed to the Raman shifts specific to aragonite. Enlargements of these peaks are shown in the insets.

Fig. 3.

—Comparisons of the shell- and dart-related transcripts and proteins in Euhadra quaesita. Venn diagrams of transcripts and proteins that were identified by transcriptome (A) and LC-MS/MS analyses (B). Dashed lines indicate the highly expressed transcripts (FPKM >1,000). (C) The ten most abundant SMPs (high abundance index values) in E. quaesita. ASM, acid-soluble matrix; AIM, acid-insoluble matrix.

Fig. 4.

—Phylogeny of the most abundant SMPs identified from Euhadra quaesita. Three of the most abundant SMPs were identified by the abundance index values from LC-MS/MS analysis (fig. 3B). (A) The maximum likelihood tree was inferred from Equ16217–21060 and five genes that were found by BLASTX search against the GenBank database under the rtREV model (using 152 positions). (B) The maximum likelihood tree was inferred from Equ23617–24364 and ten genes that were found by BLASTX search against the GenBank database under the WAG  +  Γ model (using 332 positions). (C) The phylogenetic tree was inferred from Equ26417 and four genes that were found by BLASTX search against the GenBank database under the LG  +  Γ model (using 241 positions). All phylogenic analyses were performed with 100 bootstrap replicates, and bootstrap support values <50% are not shown. Asterisks indicate 100% bootstrap support. Branch lengths are proportional to the expected number of substitutions per site, as indicated by the scale bar. Red circles indicate proteins that have been identified as SMPs in this or previous studies. Aca, Aplysia california; Bgl, Biomphalaria glabrata; CGI, Crassostrea gigas; Cne, Cepaea nemoralis; Equ, Euhadra quaesita; Lgi, Lottia gigantea; Pye, Patinopecten yessoensis; Pfu, Pinctada fucata.

Fig. 5.

—Phylogeny of adipocyte plasma membrane-associated like proteins in various metazoan taxa. The maximum likelihood tree was inferred from 43 APMAP-like gene sequences under the LG  +  Γ + I model (352 positions of the strictosidine synthase domain, 100 bootstrap replicates). Bootstrap support values <50% are not shown. Asterisks indicate 100% bootstrap support. Branch lengths are proportional to the expected number of substitutions per site, as indicated by the scale bar. Red circles indicate proteins that have been identified as SMPs in this or previous studies. Aca, Aplysia california; Bgl, Biomphalaria glabrata; Cgi, Crassostrea gigas; Cne, Cepaea nemoralis; Cte, Capitella teleta; Dre, Danio renio; Equ, Euhadra quaesita; Gga, Gallus gallus; Hsa, Homo sapiens; Lan, Lingula anatina; Lgi, Lottia gigantea; Mmu, Mus musculus; Nge, Notospermus geniculatus; Pau, Phoronis australis; Pfu, Pinctada fucata.

Fig. 6.

—Comparisons of domains of SMPs among four gastropods Euhadra quaesita, Cepaea nemoralis, Haliotis asinina, and Lottia gigantea. Carbonic anhydrase binding motif 14 (CBM_14) is a major domain in SMPs of the four gastropods. Three domains are conserved within three species (except for H. asinina), and four domains are conserved within pulmonate (E. quaesita and C. nemoralis). ALK_phosphatase, alkaline phosphatase; A2M_com, a-macroglobulin compliment component; A2M_recep, a-macroglobulin receptor; Collagen_mid, bacterial collagen middle region; C1q, compliment component 1q; GTP_EFTU, elongation factor Tu GTP binding domain; H_lectin, H-type lectin domain; Polysac_deac_1, polysaccharide deacetylase; Porin_3, eukaryotic porin; Str_syn, strictosidine synthase; Sushi, sushi repeat domain; VWA, von Willebrand factor type A domain.

Fig. 7.

—Phylogeny of alkaline phosphatase (ALP) in various metazoan taxa. The maximum likelihood tree was inferred from 67 alkaline phosphatase gene sequences under the WAG + Γ model (250 positions of the ALK_phosphatase domain, 100 bootstrap replicates). Bootstrap support values <50% are not shown. Asterisks indicate 100% bootstrap support. Branch lengths are proportional to the expected number of substitutions per site, as indicated by the scale bar. Red circles indicate proteins that have been identified as SMPs in this or previous studies. Aca, Aplysia california; Bgl, Biomphalaria. glabrata; Cgi, Crassostrea gigas; Cne, Cepaea nemoralis; Cte, Capitella teleta; Equ, Euhadra quaesita; Hsa, Homo sapiens; Lan, Lingula anatina; Lgi, Lottia gigantea; Mmu, Mus musculus; Nve, Nematostella vectensis; Pfu, Pinctada fucata.

Fig. 8.

—Comparison of pulmonate SMPs and DMPs with other molluscan SMPs. Some SMPs and DMPs in Euhadra quaesita are homologous to other molluscan SMPs (BLASTP comparison, cut-off e-value>1.0e-5, details shown in supplementary table S8, Supplementary Material online), and these SMPs and DMPs are shown in colors. Open stars indicate DMPs that were identified also as SMPs. Black stars indicate DMPs that were identified only from the dart. Cgi, Crassostrea gigas; Cne, Cepaea nemoralis; Eco, Elliptio complanata; Equ, E. quaesita; Has, Haliotis asinina; Lgi, Lottia gigantea; Mco, Mytilus coruscus; Mga, M. galloprovincialis; Pin, Pinctada; Vli, Villosa lienosa.