Towards an in-depth characterization of Symbiodiniaceae in tropical giant clams via 1 metabarcoding of pooled multi-gene amplicons 2

High-throughput sequencing is revolutionizing our ability to comprehensively characterize 28 free-living and symbiotic Symbiodiniaceae, a diverse dinoflagellate group that plays a 29 critical role in coral reef ecosystems. Most studies however, focus on a single marker for 30 metabarcoding Symbiodiniaceae, potentially missing important ecological traits that a 31 combination of markers may capture. In this proof-of-concept study, we used a small set 32 of symbiotic giant clam ( Tridacna maxima ) samples obtained from nine French Polynesian 33 locations and tested a dual-index sequence library preparation method that pools and 34 simultaneously sequences multiple Symbiodiniaceae gene amplicons per sample for in- 35 depth biodiversity assessments. The rational for this approach was to allow the 36 metabarcoding of multiple genes without extra costs associated with additional single 37 amplicon dual indexing and library preparations. Our results showed that the technique 38 effectively recovered very similar proportions of sequence reads and dominant 39 Symbiodiniaceae clades among the three pooled gene amplicons investigated per sample, 40 and captured varying levels of phylogenetic resolution enabling a more comprehensive 41 assessment of the diversity present. The pooled Symbiodiniaceae multi-gene 42 metabarcoding approach decribed here is readily scalable, offering considerable analytical 43 cost savings while providing sufficient phylogenetic information and sequence coverage. 44 45 describes a multi-marker metabarcoding approach using giant clam Tridacna maxima as a model discusses future applications for improving analyses of coral reef This pilot project explored the use of multiplexed amplicons metabarcoding for rapid, cost- effective and in-depth characterization of Symbiodiniaceae dinoflagellates using the giant clam T. maxima as a model. Our results showed that the technique effectively recovered similar proportions of sequence reads and Symbiodiniaceae diversity among the three multiplexed gene amplicons investigated per sample enabling a more comprehensive assessment of the diversity present, while also offering appreciable analytical cost savings. We also found that Symbiodinium (clades A) and Cladocopium (clade C) were the dominant genera in adult giant clams in French Polynesia, with similar sub-generic genotypes ( ITS2 A3, A6, and C1) previously described as commonly associated with giant clams from around the world. Our approach paves the way for more comprehensive surveys of this important yet endangered group of reef invertebrates and its potential role as an important Symbiodinium reservoir for declining coral reefs. Future investigations may also expand on this method to clarify species-level differentiation among Symbiodinium taxa using other markers (e.g. nuclear Actin, chloroplast psbA , mitochondrial COI and 16S ), or simultaneously characterize all organisms (viruses, bacteria, fungi, and other eukaryotes) associated with a more diverse host range. Such holistic diversity assessments will improve our knowledge on the ecology and evolution of tropical holobionts and better predict the adaptation of coral reefs in a rapidly changing environment.