Genomic Insights into the Origin and Evolution of Molluscan Red-Bloodedness in the Blood Clam Tegillarca granosa

Abstract

Blood clams differ from their molluscan kins by exhibiting a unique red-blood (RB) phenotype; however, the genetic basis and biochemical machinery subserving this evolutionary innovation remain unclear. As a fundamental step toward resolving this mystery, we presented the first chromosome-level genome and comprehensive transcriptomes of the blood clam Tegillarca granosa for an integrated genomic, evolutionary, and functional analyses of clam RB phenotype. We identified blood clam-specific and expanded gene families, as well as gene pathways that are of RB relevant. Clam-specific RB-related hemoglobins (Hbs) showed close phylogenetic relationships with myoglobins (Mbs) of blood clam and other molluscs without the RB phenotype, indicating that clam-specific Hbs were likely evolutionarily derived from the Mb lineage. Strikingly, similar to vertebrate Hbs, blood clam Hbs were present in a form of gene cluster. Despite the convergent evolution of Hb clusters in blood clam and vertebrates, their Hb clusters may have originated from a single ancestral Mb-like gene as evidenced by gene phylogeny and synteny analysis. A full suite of enzyme-encoding genes for heme synthesis was identified in blood clam, with prominent expression in hemolymph and resembling those in vertebrates, suggesting a convergence of both RB-related Hb and heme functions in vertebrates and blood clam. RNA interference experiments confirmed the functional roles of Hbs and key enzyme of heme synthesis in the maintenance of clam RB phenotype. The high-quality genome assembly and comprehensive transcriptomes presented herein serve new genomic resources for the super-diverse phylum Mollusca, and provide deep insights into the origin and evolution of invertebrate RB.

Keywords: blood clam; genome sequencing; heme biosynthesis; hemoglobin evolution; red-bloodedness.

Fig.1.

(A): Global genome landscape of T. granosa. From outer to inner circles: the 19 chromosomes at the Mb scale (a), GC content (b), depth of coverage of Illumina reads (c), depth of coverage of PacBio reads (d). (B):Oxford dot plot of orthologous genes between T. granosa and P. yessoensis. The horizontal axis represents the chromosomes of T. granosa, and the vertical axis represents the chromosomes of P. yessoensis.(C): Phylogenetic tree and number of shared orthologs among T. granosa and other animal species. Numbers of gene families undergoing rapid expansion and contraction for each lineage are shown in red and green, respectively. (D): Significantly enriched GO terms of the HREGs unique to blood clam, including tetrapyrrole biosynthesis, heme binding, and ferrochelatase activity regulation, which involved in the conventional RB-related functions. MF: molecular function; BP: biological process; CC: cellular component. (E): Gene co-expression network of the hemolymph-related module. The top 200 genes with the highest intramodular connectivity are chosen for network display. Gene names or IDs of top 100 genes are noted. Node size represents the intramodular connectivity of a given gene.

Fig.2.

Morphology of blood clam T. granosa and diversity of respiratory proteins in Mollusca.(A): (a) External morphology of blood clam T. granosa. (b) A blood clam with an open shell. (c) The blood traits of three molluscs: 1. T. granosa (red, hemoglobin); 2. Haliotis discus hannai (blue, hemocyanin), 3. S. constricta (colorless, unknown). (B): Diversity and distribution of respiratory proteins in molluscs. (C): Classification and distribution of globin family in molluscs. Abbreviations for globins: androglobin (Adgb), neuroglobin (Ngb), globin X (Gbx), myoglobin (Mb), Red blood cell hemoglobin (RB Hb), extracellular hemoglobin (Extra Hb).

Fig.3.

(A): Maximum likelihood phylogram of two blood clam Hbs and bivalve Mbs. Numbers at the nodes correspond to support from the aBayes test and 10,000 pseudoreplicates of the ultrafast bootstrap procedure. The tree was rooted using blood clam Ngbs as outgroup sequences. (B): Structure of Hb gene clusters of T. granosa, S. broughtonii and P. yessoensis. The direction of arrows indicates that of gene transcription. (C): Alignment of T. granosa Hb and Mb sequences. The percentages of identity of Hb to Mb1 and Mb2 is showed ahead of the alignment. The globin alpha-helical structure is illustrated according to the HbI of blood clam S. inaequivalvis. The two intron positions shared by all the genes are shown on top of the alignment. Functional residues related to metal coordination, heme and oxygen binding are indicated at the bottom. (D): Expression patterns of Hb and Mb genes among the 8 different tissues in T. granosa. (E): Oxygen binding curves of T. granosa HbI (black diamond, solid line) and HbII (red circle, dashed line). Hb-O₂ affinity was indexed by P50 values (i.e., the PO₂ in mmHg at which Hb becomes half-saturated). (F): A faded blood color was observed after interference of Hb genes by RNAi. The Hb concentrations were verified with decreased levels. Vertical bars are reported as means ± standard error. Significant differences between group PBS and group interference (HbI and HbIIA) are indicated with the different alphabet for P < 0.05.

Fig.4.

Maximum likelihood phylogenetic reconstruction reveals possible orthologous relationshipsamong globin genes from representative mollusks and deuterostome taxa. Numbers on the nodes represent support from the aBayes test and 10,000 pseudoreplicates of the ultrafast bootstrap procedure. The tree was rooted using plant leghemoglobins as outgroup sequences. Abbreviations of species, Callorhinchus milii (Cmi), Xenopus tropicalis (Xtr), Gallus gallus (Gga), Meleagris gallopavo (Mga), H. sapiens (Hsa), Ciona intestinalis (Cin), B. floridae (Bfl), Lepisosteus oculatus (Loc), Saccoglossus kowalevskii (Sko), T. granosa (Tgr), S. broughtonii (Sbr), P. yessoensis (Pye), Medicago sativa (Msa), Lupinus luteus (Llu).

Fig.5.

(A): Macro-synteny of the 19 T. granosa chromosomes to contemporary chicken and human genomes. The conserved syntenic blocks are shown by the local fraction of genes from each T. granosa chromosomes. The HBβ clusters in chicken and human reveled a higher level of conservation compared with Hbα. (B): Micro-synteny of conserved Hb cluster flanking genes among blood clam, chicken, and human. Genes in the ancestral chordate linkage group F (CLG-F) was indicated in orange.

Fig.6.

(A): Numbers of enzyme-encoding genes within the heme biosynthetic pathway in the blood clams T. granosa (Tgr) and S. broughtonii (Sbr) and representative mollusks and deuterostomes invertebrates and vertebrates. Bgl, B. glabrata; Lgi, L. gigantea; Pye, P. yessoensis; Cfa, C. farreri; Sgl, Saccostrea glomerata; Cgi, C. gigas; Obi, O. bimaculoides; Dme, Drosophila melanogaster; Bfl, Branchiostoma floridae; Gga, Gallus gallus; Hsa, Homo sapiens; Dre, Danio rerio; Cpb, Chrysemys picta bellii. (B): Maximum likelihood phylogenetic tree of ALAS genes. Numbers above the nodes correspond to support from the aBayes test and 10,000 pseudoreplicates of the ultrafast bootstrap procedure. The tree was rooted using the ALAS of yeast Schizosaccharomyces pombe (Spo) as outgroup sequence (UniProtKB accession O14092). (C): Relative expression levels of ALAS and other heme biosynthetic key genes in the hemolymph compared with other tissues/organs in T. granosa and four other bivalves. Note that ALASII exhibits the strongest hemolymph-specific expression in T. granosa and is selected for illustration. (D): Hb concentrations in hemolymph of T. granosa after interference of ALASII by RNAi. Vertical bars are reported as means ± standard error. Statistical significance is indicated with different letters.