A metabarcoding framework for facilitated survey of endolithic phototrophs with tufA

Abstract

Background: In spite of their ecological importance as primary producers and microbioeroders of marine calcium carbonate (CaCO 3 ) 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 Illuminasequenced marine CaCO 3 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. Results: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. Conclusions: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.

Eukaryotic Life Inhabits Rhodolith-forming Coralline Algae (Hapalidiales, Rhodophyta), Remarkable Marine Benthic Microhabitats

“…The endolithic life history stages in microalgae may be much more widespread than is currently understood and require further attention to assess the universality of endolithic stages within bloom-forming microalgae across multiple algal phyla in marine ecosystems worldwide. For example, Ochrosphaera verrucosa has been detected by tuf A metabarcoding in reef substrata from geographically isolated reef habitats of Okinawa, Japan and the Florida Keys, USA27. In the Pacific and Atlantic oceans, respectively, and thus appears universally present in the CaCO 3 endolithic niche in the tropics.…”

“…and haptophyte-specific 18S primers43 and degenerate tufA primers27. The resulting PCR products were then gel-purified followed by gene cycle sequencing with the BigDye ® .…”

“…Many taxa subsequently reached sexual maturity and life cycle completion. It was newly hypothesized that since macroalgae emerged from the surface of the bare rhodoliths, the latter may serve as seedbanks2526, a hypothesis subsequently confirmed by metabarcoding27.…”

Evolutionary Dynamics of Chloroplast Genomes in Low Light: A Case Study of the Endolithic Green Alga Ostreobium quekettii

“…Although the possibility that this gene has been transferred to the nucleus in Ostreobium cannot be completely ruled out, cemA was neither found in other assembled contigs nor in a recently sequenced Ostreobium transcriptome (see section “Searching for cemA ” in the Supplementary Materials). Once the capacity to tolerate high light is lost, there would be strong constraints on subsequent transitions to higher-light habitats, providing a plausible explanation for why Ostreobium lineages have diversified abundantly within the endolithic niche (Marcelino and Verbruggen 2016; Sauvage et al 2016) but are not known to have diversified out of it (i.e., given origin to nonendolithic species). Endolithic algal species are often light saturated at low light intensities but some experimental studies show that they are able to photoacclimate to light levels approaching full solar irradiance (see Tribollet 2008 for a review).…”

“…Endolithic algal species are often light saturated at low light intensities but some experimental studies show that they are able to photoacclimate to light levels approaching full solar irradiance (see Tribollet 2008 for a review). There are high levels of cryptic diversity within endolithic green algae (Marcelino and Verbruggen 2016; Sauvage et al 2016) and it is not known which species are able to cope with higher levels of light, raising the question of whether cemA has been lost in other lineages of Ostreobium and whether they acquired other mechanisms to tolerate high light.…”