Phylogenetic comparisons reveal mosaic histories of larval and adult shell matrix protein deployment in pteriomorph bivalves

Abstract

Molluscan shells are organo-mineral composites, in which the dominant calcium carbonate is intimately associated with an organic matrix comprised mainly of proteins and polysaccharides. However, whether the various shell matrix proteins (SMPs) date to the origin of hard skeletons in the Cambrian, or whether they represent later deployment through adaptive evolution, is still debated. In order to address this issue and to better understand the origins and evolution of biomineralization, phylogenetic analyses have been performed on the three SMP families, Von Willebrand factor type A (VWA) and chitin-binding domain-containing protein (VWA-CB dcp), chitobiase, and carbonic anhydrase (CA), which exist in both larval and adult shell proteomes in the bivalves, Crassostrea gigas and Pinctada fucata. In VWA-CB dcp and chitobiase, paralogs for larval and adult SMPs evolved before the divergence of these species. CA-SMPs have been taken as evidence for ancient origins of SMPs by their presumed indispensable function in biomineralization and ubiquitous distribution in molluscs. However, our results indicate gene duplications that gave rise to separate deployments as larval and adult CA-SMPs occurred independently in each lineage after their divergence, which is considerably more recent than hitherto assumed, supporting the "recent heritage and fast evolution" scenario for SMP evolution.

Figure 1

Phylogenetic analyses of CB domains of VWA-CB dcps of molluscan shells. (a) Schematic representations of the domain structures of shell-specific VWA-CB dcps. (b) A Bayesian tree based on LG model and 84 amino acid residues. Polychotomy is generated if the posterior probability value of the node is < 50. Posterior probability values are shown if ≥ 50, and marked with black dots if ≥ 80. The same symbols denoting different proteins in panel a are used in panel b. Larval SMPs are marked by “L”, and Pfu-BMSP identified in both larval and adult shells of P. fucata is marked by “L&A”. Cgi, Crassostrea gigas; Pfu, Pinctada fucata; Mga, Mytilus galloprovincialis; Lgi, Lottia gigantea; Lan, Lingula anatina.

Figure 2

Phylogenetic analyses of VWA domains of VWA-CB dcps of molluscan shells. (a) Schematic representations of domain structures of shell-specific BMSPs. (b) A Bayesian tree based on the LG model and 187 amino acid residues. Polychotomy is generated if the posterior probability value of the node is < 50. Posterior probability values are shown if ≥ 50, and marked with black dots if ≥ 80. The same symbol marks as in Fig. 1 are used to denote different SMPs. Larval SMPs are marked by “L”, and Pfu-BMSP identified from both larval and adult shells of P. fucata is indicated by “L&A”. BMSP family proteins are indicated by bold characters. Cgi, Crassostrea gigas; Pfu, Pinctada fucata; Mga, Mytilus galloprovincialis; Lgi, Lottia gigantea; Lan, Lingula anatina; Dp, duplication.

Figure 3

Phylogenetic analyses of Laminin G domains on 210 amino acids and the concatenated sequence of a CB domain and the Laminin G domain on 282 amino acids of VWA-CB dcps of molluscan shells. (a) A Bayesian phylogenetic tree based on Laminin G domains. (b) A Bayesian phylogenetic tree based on concatenated sequences of the CB and Laminin G domains. Polychotomy is generated if the posterior probability of the node is < 50. Posterior probabilities are shown if ≥ 50, and marked with black dots if ≥ 80. Cgi, Crassostrea gigas; Pfu, Pinctada fucata; Mga, Mytilus galloprovincialis; Lgi, Lottia gigantea; Lan, Lingula anatina. The group formed by BMSPs is indicated.

Figure 4

Phylogenetic analyses of chitobiases in molluscs on 1066 amino acid residues. (a) Schematic representations of the domain structures of chitobiases. (b) Bayesian phylogenetic estimation for chitobiases in molluscs using the concatenated sequences of CHB_HEX domain (IPR004866), Glyco_hydro_20b domain (IPR015882), Glyco_hydro_20 domain (IPR015883) and CHB_HEX_C domain (IPR004867). (c) Stage-specific expression of the larval chitobiase during development in C. gigas (blue) and P. fucata (orange)^,,. Polychotomy is generated if the posterior probability value of the node is < 50. Posterior probability values are shown if ≥ 50, and marked with black dots if ≥ 80. SMPs are indicated by blue (C. gigas) and orange (P. fucata) arrowheads. The question mark indicates that whether or not the gene is encoding an SMP is uncertain. Cgi, Crassostrea gigas; Pfu, Pinctada fucata; Lgi, Lottia gigantea; Obi, Octopus bimaculoides. Lan, Lingular anatina; Dp, duplication.

Figure 5

Bayesian phylogenetic analysis based on 935 amino acid residues of CA domains in molluscs. SMPs are indicated by blue (C. gigas), orange (P. fucata) and green (L. gigantea) arrowheads. Polychotomy is generated if the posterior probability of the node is < 50. Posterior probabilities are shown if ≥ 50, and marked with black dots if ≥ 80. Cgi, Crassostrea gigas; Pfu, Pinctada fucata; Lgi, Lottia gigantea; Obi, Octopus bimaculoides. Ath, Arabidopsis thaliana.