Bacterial Microbiota of Ostreobium, the Coral-Isolated Chlorophyte Ectosymbiont, at Contrasted Salinities

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TL;DR

This study characterized the bacterial microbiota associated with two cultured Ostreobium strains (rbcL clades P1 and P14) at three ecologically relevant salinities (32.9, 35.1, and 40.2 psu). Using CARD-FISH visualization and 16S rDNA metabarcoding, it identified a small core microbiota of seven bacterial ASVs (1.5-1.8% of detected ASVs per strain) persistent across genotypes and salinities, dominated by Alphaproteobacteria including putative intracellular symbionts such as Candidatus Amoebophilus and Rickettsiales_AB1. Microbiota structure was primarily driven by algal genotype rather than salinity, with each genotype responding differently to salinity shifts via altered proportions of bacteria putatively involved in nitrogen metabolism, supporting Ostreobium's broad salinity tolerance and ecological flexibility in reef ecosystems.

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## Background Ostreobium is a genus of siphonous green algae (Ulvophyceae, Bryopsidales) that are major bioeroders of coral reef carbonate, dissolving up to 1 kg of reef carbonate per m² per year. These cryptic algae colonize carbonate skeletons from photic to mesophotic depths and form close associations with their coral hosts, yet the bacterial communities specifically associated with Ostreobium genotypes and their response to abiotic factors such as salinity remain poorly understood. Given that reef salinities range broadly from 20 psu in estuaries to 40-42 psu in the Red Sea, and that climate change and desalination are altering salinity regimes, characterizing Ostreobium-associated microbiota under salinity stress has direct ecological relevance.

## Methods Two genotyped Ostreobium strains (010/rbcL clade P1 and 06/rbcL clade P14; >8% sequence divergence), both originally co-isolated from Pocillopora acuta, were long-term acclimatized (9-13 months) to three salinities: 32.9 psu, 35.1 psu, and 40.2 psu, with n=4 independent cultures per strain-salinity combination (24 cultures total). CARD-FISH with universal bacterial probes (EUB338 mix) was performed on strains at low and high salinity to localize bacteria within algal siphons. Bacterial 16S rDNA V5-V7 amplicons were sequenced via Illumina MiSeq, yielding 7,099,477 sequences and 1564 bacterial ASVs after quality filtering and removal of internal controls. Alpha and beta diversity were analyzed using Kruskal-Wallis tests and PERMANOVA (adonis2). Sparse PLS-DA identified discriminant bacterial ASVs by genotype and salinity.

## Key Results CARD-FISH visualization revealed metabolically active bacteria at the surface (epiphytic), within siphons (endophytic), and in inter-filament mucilage of both strains at both 32.9 and 40.2 psu.

A total of 837 cumulated ASVs were detected in Ostreobium thalli (469 in strain 06; 400 in strain 010), and 809 ASVs in supernatants. Pielou's evenness was significantly higher in strain 010 (0.74-0.79) than strain 06 (0.47-0.60) at 32.9 and 35.1 psu (p = 0.029), but not at 40.2 psu (p = 0.11).

Beta diversity analysis showed that bacterial communities differed significantly between genotypes (F = 3.049, p = 0.001), between salinities (p = 0.001), and between genotype × salinity interactions (F = 1.889, p = 0.002). Each genotype harbored a distinct bacterial profile with different salinity responses.

Only 7 bacterial ASVs (1.5-1.8% of detected ASVs per strain) comprised the core Ostreobium microbiota persistent across both genotypes and all three salinities. Five belonged to Alphaproteobacteria (Rhodospirillaceae ASV16, Hyphomonadaceae ASV57, Rhizobiales/Labrenzia ASV72, Kiloniellales/Fodinicurvata ASV124, Rickettsiales_AB1 ASV76), one to Bacteroidia (Candidatus Amoebophilus ASV166, putative obligate intracellular symbiont), and one to Acidimicrobiia (Ilumatobacter ASV167). These represented 18.6% of cumulative reads in strain 06 and 35.7% in strain 010.

STRAIN-SPECIFIC CORE MICROBIOTA DIFFERED

strain 010 was enriched in Kiloniellaceae/Fodinicurvata (18.9% vs. 1.3% in 06) and Thermoleophilia/Gaiellales; strain 06 was enriched in Rhodospirillaceae (34.8% vs. 5.8% in 010) and Rhodobacteraceae. Strain 010 harbored strain-specific Phycisphaerales SM1A02.

SALINITY RESPONSE WAS GENOTYPE-SPECIFIC

in strain 06, only high salinity (40.2 psu) separated the bacterial community from low/intermediate salinity profiles (PC1, 11.94%); in strain 010, low salinity (32.9 psu) was distinct (PC1, 13.47%). Both strains shared significantly increased proportions of Rhizobiales/ASV597 (Devosia) at high salinity, and strain 010 additionally showed increased Hyphomonadaceae ASV57, Nannocystales ASV170, and Kiloniellales ASV976 at high salinity.

Five of the seven core bacterial taxa were also detected in field-sampled Pocillopora coral skeletons from Guam and Eilat, validating the in vitro findings.

## Clinical Implications Not applicable. This is a marine microbial ecology study without direct clinical relevance. Ecologically, the findings suggest that Ostreobium-associated microbiota, particularly taxa putatively involved in nitrogen metabolism (Rhodospirillaceae, Hyphomonadaceae, Fodinicurvataceae, Devosia, Labrenzia), may support algal adaptation to N-depleted carbonate substrates and broad salinity ranges, contributing to Ostreobium's resilience as a ubiquitous reef bioeroder under climate-driven salinity change.