Hidden diversity and host specificity of Cladocopium symbionts revealed by phylotranscriptomics in giant sea anemones.

Abstract

Giant sea anemones form an intricate, metabolically linked symbiosis with anemonefish (genus Amphiprion) and dinoflagellates (family Symbiodiniaceae). While the association between fish and anemone is well documented, the identity and distribution of resident Symbiodiniaceae remains poorly characterized. Here, we used RNA-sequencing of anemone tentacles and post-assembly filtering to recover Symbiodiniaceae sequences from Japanese anemones. Using transcriptome-wide phylogenetic approaches, we identified distinct Symbiodiniaceae strains in seven sea anemone species and four cryptic lineages of Entacmaea quadricolor. Phylogenies of 1300 and 214 nuclear genes resolved relationships among these symbionts with greater sensitivity than traditional molecular markers such as the Internal Transcribed Spacer 2 (ITS2) or partial 28S ribosomal DNA. Marker-based assessments indicate these Symbiodiniaceae as novel and specific to sea anemone hosts. Our transcriptome-wide phylogenies reveal previously undescribed Cladocopium (Clade C) strains associated with giant sea anemones, with symbionts clustering according to host species and lineage. Cophylogenetic analysis revealed significant phylogenetic congruence between anemone and Symbiodiniaceae, indicating a nonrandom association between host and symbiont evolutionary histories. Two sympatric cryptic lineages of E. quadricolor previously identified through host transcriptomics harbor distinct, specific Symbiodiniaceae assemblages. Analysis of genes under positive selection indicates functional divergence between these Symbiodiniaceae; three-dimensional reconstructions of symbiont cells using serial block-face scanning electron microscopy show consistent differences in cellular organization between two strains, further supporting their distinct functional identity. Together, our results uncover a hidden diversity of host-specific Symbiodiniaceae and suggest that cryptic diversification in giant sea anemones is mirrored by diversification of their algal symbionts within this tripartite symbiosis.