Bivalve-specific gene expansion in the pearl oyster genome: implications of adaptation to a sessile lifestyle
- Takeshi Takeuchi1Email author,
- Ryo Koyanagi2,
- Fuki Gyoja1,
- Miyuki Kanda2,
- Kanako Hisata1,
- Manabu Fujie2,
- Hiroki Goto2,
- Shinichi Yamasaki2,
- Kiyohito Nagai3,
- Yoshiaki Morino4,
- Hiroshi Miyamoto5,
- Kazuyoshi Endo6,
- Hirotoshi Endo7,
- Hiromichi Nagasawa8, 9,
- Shigeharu Kinoshita10,
- Shuichi Asakawa10,
- Shugo Watabe10, 11,
- Noriyuki Satoh1 and
- Takeshi Kawashima1, 12
© Takeuchi et al. 2016
Received: 4 August 2015
Accepted: 11 November 2015
Published: 18 February 2016
Bivalve molluscs have flourished in marine environments, and many species constitute important aquatic resources. Recently, whole genome sequences from two bivalves, the pearl oyster, Pinctada fucata, and the Pacific oyster, Crassostrea gigas, have been decoded, making it possible to compare genomic sequences among molluscs, and to explore general and lineage-specific genetic features and trends in bivalves. In order to improve the quality of sequence data for these purposes, we have updated the entire P. fucata genome assembly.
We present a new genome assembly of the pearl oyster, Pinctada fucata (version 2.0). To update the assembly, we conducted additional sequencing, obtaining accumulated sequence data amounting to 193× the P. fucata genome. Sequence redundancy in contigs that was caused by heterozygosity was removed in silico, which significantly improved subsequent scaffolding. Gene model version 2.0 was generated with the aid of manual gene annotations supplied by the P. fucata research community. Comparison of mollusc and other bilaterian genomes shows that gene arrangements of Hox, ParaHox, and Wnt clusters in the P. fucata genome are similar to those of other molluscs. Like the Pacific oyster, P. fucata possesses many genes involved in environmental responses and in immune defense. Phylogenetic analyses of heat shock protein70 and C1q domain-containing protein families indicate that extensive expansion of genes occurred independently in each lineage. Several gene duplication events prior to the split between the pearl oyster and the Pacific oyster are also evident. In addition, a number of tandem duplications of genes that encode shell matrix proteins are also well characterized in the P. fucata genome.
Both the Pinctada and Crassostrea lineages have expanded specific gene families in a lineage-specific manner. Frequent duplication of genes responsible for shell formation in the P. fucata genome explains the diversity of mollusc shell structures. These duplications reveal dynamic genome evolution to forge the complex physiology that enables bivalves to employ a sessile lifestyle in the intertidal zone.
KeywordsPearl oyster Pinctada fucata Genome Hox ParaHox Heat shock proteins C1q Biomineralization
Bivalves are the second largest group in the Phylum Mollusca, outnumbered only by gastropods, and represent one of the most common animal groups in both marine and freshwater ecosystems. Notably, some bivalve species are abundant in littoral and shallow water environments, where they experience different types of fluctuating stresses, such as temperature, salinity, and oxygen concentration. Most adult bivalves employ a sessile, suspension-feeding life style, the energetic cost of which is lower than that of browsing . Due to their aquatic habitats and filter feeding, they must defend themselves against microbial invasion by means of innate immune systems. In addition, sedentary bivalves cannot escape predation; therefore, they protect themselves with calcareous shells. These biological features of the bivalve adaptation strategy have resulted from expansion of specific gene families related to environmental response , immune defense [2–4], and biomineralization . These findings raise the question of whether these gene expansions occurred in bivalve common ancestor or independently in various lineages.
The Genus Pinctada includes the pearl oysters, such as Pinctada fucata, P. margaritifera, and P. maxima, which are distributed in subtropical and tropical portions of the Indo-Pacific Ocean . These species have been commercially farmed for pearl production since Kokichi Mikimoto established the pearl culture industry at the end of 19th century, using P. fucata . Recently, transcriptomics [8, 9], proteomics [10–12], and gene knockdown techniques [13–15] have been used to investigate genetic components of shell and pearl biomineralization. Thus, mechanisms of pearl formation in Pinctada have been actively investigated for their economic potential, as well as their fascinating biology. Now, pearl oysters are becoming experimental model molluscs for biomineralization research.
In 2012, we decoded the draft genome of Pinctada fucata , one of the most important species for cultured pearl production in Asia. The genome of P. fucata has been thoroughly mined to find genes responsible for biomineralization , physiology , and reproduction . A broad range of transcription factors [19–21] and signaling molecules  has also been investigated. These provide valuable information about lophotrochozoans to better understand evolution of Bilaterian body plans. Soon after publication of the P. fucata genome, genomes of the Pacific oyster, Crassostrea gigas  and the limpet, Lottia gigantea  were also published. The growing body of molluscan genome data provides an opportunity to characterize general and unique features among molluscs, for which sequence information has, until recently, been scant.
The present study produced a new version of the P. fucata genome assembly (version 2.0), which provides longer contigs and scaffolds, and more consecutive gene arrays compared to the previous version. To improve the assembly, additional sequence data were generated, and an advanced assembly strategy addressed the heterozygotic nature of the genome. Along with the establishment of a new genome assembly, we also generated gene model version 2.0. Information on gene annotation done manually by the P. fucata research community  was used for the gene model prediction.
In this report, we surveyed bivalve-specific genomic changes. We performed molecular phylogenetic analyses for gene families that have been expanded in bivalves, including heat shock protein 70 (HSP70) and C1q domain-containing proteins (C1qDC). In addition, we thoroughly investigated shell matrix protein (SMP) gene clusters, which were partly described in the previous version of the genome assembly . We also verified conserved gene clusters for Hox, ParaHox, and Wnt genes among bilaterians using the new Pinctada genome assembly.
Genome sequencing and assembly
Genomic DNA, which is identical to that obtained in the previous study , was prepared for paired-end libraries and sequenced with an Illumina MiSeq and a Genome Analyzer IIx (GAIIx) . Raw reads were quality trimmed using Trimmomatic 0.30 . The whole-genome shotgun (WGS) and paired-end reads sequenced by Takeuchi et al. (2012)  and this study were assembled using GS De Novo Assembler, version 2.6 (Newbler, Roche) . After removing redundant sequences from the contig assembly, paired-end and mate-pair sequences were added for scaffolding performed with SSPACE 1.1 . Gaps in scaffolds were filled using GapCloser 1.12 . See Additional file 1: note for more detail.
Transcriptome sequencing and assembly
Transcriptome sequencing used in this study is described in Takeuchi et al. (2012) . Additionally, a cDNA library of early developmental stages and adult tissues, including mantle, was prepared and sequenced with an Illumina GAIIx. All sequences were cleaned and trimmed with Trimmomatic 0.30 , and then assembled using Trinity (version r20140413p1) .
Gene prediction, annotation, and identification of gene families
The resulting genome assembly (P. fucata genome ver. 2.0) and transcriptomic data were used for de novo gene model prediction with PASA (version r20130907)  and AUGUSTUS 3.0.2  platforms, as described previously . Gene annotation information manually confirmed in Pearl Oyster Annotation Jamborees  was added in order to train the gene prediction algorithm and to generate a hint file for AUGUSTUS. Gene models that encoded more than 49 amino acids were retained. Sequences significantly similar to transposable elements were detected with CENSOR 4.2.28  and excluded from the gene model. Gene models of P. fucata, Lottia gigantea , and Crassostrea gigas  were assigned to the ortholog group of the OrthoMCL Database version 5 [34, 35]. Three molluscan gene models that were not assigned to the OrthoMCL ortholog group were then clustered with local OrthoMCL software in order to identify mollusc-specific gene families. Next, gene models that did not cluster with others (“orphan gene models”) were examined with BLASTN against P. fucata transcriptomic sequences. Orphan gene models without transcriptomic evidence were excluded from the final gene model set, named gene model, version 2.0.
Gene model version 2.0 was BLASTN-searched against version 1.0 models and the best match model was regarded as synonymous. Hox, ParaHox, Wnt, and SMP genes were annotated by referring to previous studies [5, 21, 22]. SMP genes were also searched with BLASTP against SMPs of P. margaritifera and P. maxima  and were annotated manually. BLASTP searches against the UniProtKB and NCBI non-redundant (nr) databases were also conducted in order to confirm annotations. Results of BLASTP searches (E-value threshold 1e-5) against protein sequences of the UniProtKB database, were analyzed with PANTHER  for GO annotation. GO enrichment of genes conserved between P. fucata and C. gigas, but not L. gigantea, was investigated by calculating a P value based on hypergeometric distribution.
Molecular phylogeny of expanded gene families
Conserved domains of proteins predicted in the Pinctada fucata genome were searched against the Pfam database (Pfam-A.hmm, release 24.0; http://pfam.xfam.org/) using HMMER 3.0 . Representative animal genomes were also surveyed for comparison and phylogenetic analysis. The following protein sequences from public databases were retrieved for this study; Acropora digitifera (http://marinegenomics.oist.jp/), Lottia gigantea, Helobdella robusta, Capitella teleta, Daphnia pulex, and Nematostella vectensis (http://genome.jgi.doe.gov/), Crassostrea gigas (http://gigadb.org), Hydra magnipapillata, Amphimedon queenslandica (http://www.ncbi.nlm.nih.gov) and Caenorhabditis elegans, Drosophila melanogaster, Branchiostoma floridae, Strongylocentrotus purpuratus, and Homo sapiens (http://www.genome.jp). Amino acid sequences of Mytilus galloprovincialis C1q domain-containing proteins were downloaded from NCBI. Amino acid sequences were aligned with Muscle , then maximum likelihood trees were constructed with RAxML 8.1.3 . The best-fit model of amino acid evolution for each tree was selected using ProteinModelSelection.pl script provided in the RAxML package. One hundred bootstrap replicates were generated.
Results and discussion
New genome assembly
Summary of the P. fucata genome assembly version 2.0
Number of contigs
Total length of sequences
Average contig length
Contig length N50
Number of scaffolds
Total length of sequences
Total gap length
Average scaffold length
Scaffold length N50
New gene models
Comparison of gene model versions of P. fucata
Ver. 1.0 Takeuchi et al. 
Ver. 2.0 This study
Number of gene models
Number of full length gene models
23257 (53.1 %)
23516 (80.1 %)
Number of exons per transcript
Average range per gene model (bp)
In order to predict functions of genes conserved between the two bivalves or among all three molluscs, GO annotation was conducted (Fig. 2). Genes that were related to “receptor activity,” “response to stimulus,” “immune system process,” and “extracellular region” were more abundant in bivalves compared to those of genes shared by all three molluscs. This suggests that several gene families related to environmental response and immune system are expanded in bivalves. Similarly, bivalve-specific gene expansion corresponding to “extracellular region” and “extracellular matrix” was evident, and these events were responsible, at least in part, for evolution of bivalve biomineralization-related genes. In addition, genes assigned to “biological adhesion” are significantly enriched in the bivalve lineage, possibly reflecting their sedentary lifestyles.
Gene expansion of heat shock protein 70 and C1q domain-containing proteins
Recent genomic and transcriptomic surveys have shown that some gene families involved in responses to environmental change or microbial attack, are greatly expanded in bivalves, including Crassostrea  and Mytilus [3, 40, 41]. However, it has been unclear whether gene expansion events are lineage-specific or common to all bivalves.
We reconstructed a molecular phylogenetic tree of HSP70 proteins of five lophotrochozoan (three molluscs and two annelids) and fly genomes (Fig. 3b). The tree clearly shows two distinct groups: one is ancestral and the other is almost completely composed of bivalve genes (Fig. 3b). HSP70 genes of a polychaete, Capitella teleta, are also included in the latter group. Although the evolutionary relationship between these bivalve and polychaete genes is indeterminate because of the low bootstrap value, we speculate that the bivalve-dominant gene group was derived from the ancestral gene shared by molluscs and polychaetes. It then expanded in the bivalve lineage while being lost in the gastropod lineage. In the bivalve-dominant gene group, nine pairs of P. fucata and C. gigas genes are closely associated (Fig. 3b). This topology suggests that they are orthologous pairs and that these nine bivalve-specific HSP70 genes were present prior to the divergence of the Pinctada and Crassostrea lineages, which date back approximately 400 million years . Furthermore, species-specific gene expansions are also observed (Fig. 3b). At least six gene families in the tree suggest independent gene expansion events in P. fucata, while eleven more suggest the same in C. gigas. Pearl oysters are mainly found in the subtidal zone, which is a more stable environment than the intertidal zone because it is always inundated. However, the subtidal and intertidal zones are still very stressful environments, and sessile bivalves (the pearl oyster and the Pacific oyster) need to cope with adverse conditions in shallow water, such as salinity and temperature changes caused by weather. Nutrient and oxygen concentrations are also changed by plankton blooms. Therefore, the independently expanded HSP70 gene family in both P. fucata and C. gigas may underlie adaptation to a sessile lifestyle in such fluctuating and stressful conditions. Moreover, aside from the bivalve-dominant group, eight groups that include HSP70 genes of four or more protostome species, are recognized (Fig. 3b), six of which are supported by high bootstrap values (≥80 %). Therefore, it is likely that the common ancestor of protostomes had approximately eight or nine HSP70 genes, and evolutionary radiation of this gene family has occurred in each lineage.
These phylogenetic analyses demonstrate that gene duplication events occurred prior to the divergence of the bivalve species examined here, and that lineage-specific gene expansion is also common in bivalve genomes. This flexible expansion of gene families has generated an immense gene repertoire associated with stress responses and immune defense. By contrast, the limpet, Lottia, did not acquire enlarged gene families, despite its similar habitat in the littoral zone. We suggest that because bivalves cannot escape from the adverse conditions, they must respond to the variable environment by having expanded HSP70 gene family. Their suspension-feeding system is protected from wide range of invading microorganisms. Gene expansion allows bivalves to settle in dynamic marine environments, such as the intertidal and subtidal zones.
Tandem duplication of genes responsible for shell formation
Shell formation is one of the unique features of molluscs. The process is highly controlled by the organism by secretion of an organic shell matrix, which generates an organic framework that regulates calcification of the shell [54, 55]. Shell matrix proteins (SMPs) are considered the major components of the organic shell matrix, and SMP evolution is implicated in diversification of mollusc shell characters, including morphology, microstructure, and crystal polymorphism [10, 56].
Orthologous genes that encode SMPs, reported from P. margaritifera and P. maxima , were also investigated. Interestingly, a number of SMP genes are tandemly arranged in the scaffolds (Fig. 5 and Additional file 1: Table S5). Pairs of genes encoding alveolin-like and MP10, chiobiase, chitinase-like protein (Clp), EGF domain-containing protein (EGF-like), and tyrosinase are present (Fig. 5e-i). Likewise, more than two tandem gene clusters of fibronectin domain-containing protein (fn), Kunitz/BPTI serine protease inhibitors (SPI), and peroxidase-like (pl) proteins are identified (Fig. 5j-l). These results show that SMP genes were frequently duplicated in the Pinctada genome. Tandemly arranged genes that encode SMPs (EGF domain-containing protein, peroxidase, and uncharacterized proteins) are also evident in the Lottia genome , indicating that tandem duplication of SMP genes is a common feature of bivalve and gastropods. Although the precise function of these genes in shell formation remains unknown, duplication and rapid molecular evolution [9, 62] of SMP genes may be a key feature for understanding the diversification of mollusc shell structure. It is also possible, however, that molecular functions of duplicated SMPs are redundant, and that increased gene copy number results in larger numbers of transcripts , to accelerate shell formation. Indeed, we reported an example in which tandem arrays of SMP genes were expressed in coordinated fashion . In order to resolve the complexity of molecular evolution and functional diversification of SMPs, proteomic analysis of the P. fucata shell and a genome-wide gene expression survey of mantle are underway.
Among the SMP gene families discussed above, genes homologous to MSI60, Shematrin, and N19 are absent in the C. gigas genome. Likewise, another tandemly duplicated gene family, N16 , is not found in the oyster genome. In other words, these SMP gene families are unique to the P. fucata lineage. As mentioned before, abundant lineage-specific gene families are a feature of molluscan genomes (Fig. 1b). These gene families emerged and became duplicated in P. fucata lineage after the split of the pearl oyster and Pacific oyster lineages. Alternatively, it is possible that these two bivalves share an ancestral SMP gene, and that the gene evolved so rapidly that, at present, SMP genes in two bivalve genomes are significantly different from each other. In either case, rapid molecular evolution and a diverse repertoire of SMPs made possible the great variety of molluscan shell structures.
Conserved clusters of Hox, ParaHox, and Wnt genes in the P. fucata genome
ParaHox genes, Gsx, Xlox, and Cdx are found in a single scaffold (Fig. 6b), which is the first indication of close linkage of the three ParaHox genes in molluscan genomes. These genes are separated by non-ParaHox gene models, which are also observed in the Lottia and Crassostera genomes (Fig. 6b and Additional file 1: Table S7). We also found a cluster of Wnt gene family genes (Fig. 6c and Additional file 1: Table S8). The tandem arrangement of wnt9, 1, 6, and 10 genes is identical to that of the limpet genome ; therefore this arrangement is considered the ancestral state in bivalves and gastropods. These results demonstrate that Hox, ParaHox, and Wnt gene clusters in the P. fucata genome are comparable to those of other molluscan genomes.
We performed comparative genomic analyses using accessible molluscan genomes with our newly updated genome assembly of the pearl oyster, Pinctada fucata. Genes common to the two bivalves include a larger number of genes potentially relevant to extracellular matrix, environmental responses, and immune systems than are seen in a gastropod and other protostomes (Fig. 2). Consistently, protein-domain surveys and molecular phylogenetic analyses reveal extensive gene duplication of stress response genes (HSP70 in Fig. 3, C1qDC in Fig. 4). A survey of gene arrangements confirmed that frequent gene duplication of shell matrix proteins has occurred in bivalves (Fig. 5). All of these results suggest that adaptive changes in extant bivalve genomes have occurred in a species-specific manner. We also confirmed relatively conserved clusters of Hox, ParaHox, and Wnt genes among protostomes (Fig. 6). The revised pearl oyster genome provides insights into the molecular basis of oyster physiology and radiation.
Availability of supporting data
All reads generated by this study have been deposited in the Sequence Read Archive (SRA) database (http://trace.ncbi.nlm.nih.gov/Traces/sra/) under the accession IDs DRP000496 and DRP000497 .
C1q domain-containing protein
fibronectin domain-containing protein
heat shock protein
pathogen-associated molecular pattern
shell matrix protein
serine protease inhibitor
We thank Dr. Steven D. Aird for editing the manuscript. We acknowledge all participants of the Pearl Oyster Genome Jamborees for their enthusiasm and contributions. Super-computing was supported by the IT Section of OIST. This study was supported by Japan Society for the Promotion of Science KAKENHI (no. 23780209) to T. T., and the Japanese Association for Marine Biology (JAMBIO) to K. E. We also gratefully acknowledge support from OIST Graduate University to members of the Marine Genomics Unit.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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