In spite of their ecological importance as primary producers and microbioeroders of marine calcium carbonate (CaCO₃) substrata, endolithic phototrophs spanning both prokaryotic (the cyanobacteria) and eukaryotic algae lack established molecular resources for their facilitated survey with high throughput sequencing. Here, the development of a metabarcoding framework for the elongation factor EF-Ttu (tufA) was tested on four Illumina-sequenced marine CaCO₃ microfloras for the characterization of their endolithic phototrophs, especially the abundant bioeroding Ostreobium spp. (Ulvophyceae). The framework consists of novel tufA degenerate primers and a comprehensive database enabling Operational Taxonomic Unit (OTU) identification at multiple taxonomic ranks with percent identity thresholds determined herein.

The newly established tufA database comprises 4057 non-redundant sequences (from 1339 eukaryotic and prokaryotic phototrophs, and 2718 prokaryotic heterotrophs) including 27 classes in 10 phyla of phototrophic diversity summarized from data mining on GenBank^®, our barcoding of >150 clones produced from coral reef microfloras, and >300 eukaryotic phototrophs (>230 Ulvophyceae including >100 ‘Ostreobium’ spp., and >70 Florideophyceae, Phaeophyceae and miscellaneous taxa). Illumina metabarcoding with the newly designed primers resulted in 802 robust OTUs including 618 phototrophs and 184 heterotrophs (77 and 23 % of OTUs, respectively). Phototrophic OTUs belonged to 14 classes of phototrophs found in seven phyla, and represented ~98 % of all reads. The phylogenetic profiles of coral reef microfloras showed few OTUs in large abundance (proportion of reads) for the Chlorophyta (Ulvophyceae, i.e. Ostreobium and Phaeophila), the Rhodophyta (Florideophyceae) and Haptophyta (Coccolithophyceae), and a large diversity (richness) of OTUs in lower abundance for the Cyanophyta (Cyanophyceae) and the Ochrophyta (the diatoms, ‘Bacillariophyta’). The bioerosive ‘Ostreobium’ spp. represented four families in a large clade of subordinal divergence, i.e. the Ostreobidineae, and a fifth, phylogenetically remote family in the suborder Halimedineae (provisionally assigned as the ‘Pseudostreobiaceae’). Together they harbor 85–95 delimited cryptic species of endolithic microsiphons.

The novel degenerate primers allowed for amplification of endolithic phototrophs across a wide phylogenetic breadth as well as their recovery in very large proportions of reads (overall 98 %) and diversity (overall 77 % of OTUs). The established companion tufA database and determined identity thresholds allow for OTU identification at multiple taxonomic ranks to facilitate the monitoring of phototrophic assemblages via metabarcoding, especially endolithic communities rich in bioeroding Ulvophyceae, such as those harboring ‘Ostreobium’ spp., Phaeophila spp. and associated algal diversity.