key: cord-0255628-u3f17do0 authors: Pasella, Marisa M.; Lee, Ming-Fen Eileen; Marcelino, Vanessa R.; Willis, Anusuya; Verbruggen, Heroen title: Ten Ostreobium (Ulvophyceae) strains from Great Barrier Reef corals as a resource for algal endolith biology and genomics date: 2021-08-17 journal: bioRxiv DOI: 10.1101/2021.08.16.453452 sha: 45591378bd6dbee45d936c7b710af2c0d13dc55b doc_id: 255628 cord_uid: u3f17do0 Ostreobium is a genus of siphonous green algae that lives as an endolith in carbonate substrates under extremely limited light conditions and has recently been gaining attention due to its roles in reef carbonate budgets and its association with reef corals. Knowledge about this genus remains fairly limited due to the scarcity of strains available for physiological studies. Here, we report on 10 strains of Ostreobium isolated from coral skeletons from the Great Barrier Reef. Phenotypic diversity showed differences in the gross morphology and in few structures. Phylogenetic analyses of the tufA and rbcL put the strains in the context of the lineages identified previously through environmental sequencing. The chloroplast genomes of our strains are all around 80k bp in length and show that genome structure is highly conserved, with only a few insertions (some containing putative protein-coding genes) differing between the strains. The addition of these strains from the Great Barrier Reef to our toolkit will help develop Ostreobium as a model species for endolithic growth, low-light photosynthesis and coral-algal associations. Corals are the results of a symbiotic association between animals, algae, and prokaryotes. The In the calcium carbonate skeleton beneath the coral tissue, a green layer containing 52 the green alga Ostreobium can often be seen. Ostreobium is the second major photosynthetic 53 organism in the coral holobiont, with its biomass often exceeding that of Symbiodiniaceae 54 (Odum & Odum 1955 ). Due to its peculiar niche burrowing into limestone substrates, studies 55 of Ostreobium sp. have been few in comparison with Symbiodiniaceae. Until very recently, only two Ostreobium strains were available from public 76 repositories (SAG strains 6.99 and 7.99). The former was isolated 30 years ago as an epiphyte 77 on a red alga from the Philippines and the latter 20 years ago as an epiphyte of Jania sp. from 78 southern Australia. In a recent paper, 9 closely related strains isolated from the coral The scarcity of available Ostreobium strains slows progress in understanding the 84 biology of this genus and its interactions with coral and functions in reef decalcification. 85 Here, we present a collection of 10 Ostreobium strains obtained from the skeletons of corals 86 from the Great Barrier Reef, deposited in the Australian National Algal Culture Collection 87 (ANACC, Hobart). Our aims for the paper are to describe the collection, isolation, and 88 culturing procedures of these strains, provide their phylogenetic context and describe and 89 compare their completely sequenced chloroplast genomes. Coral fragments were collected with hammer and chisel at different sites and depths at Heron 94 Island (Great Barrier Reef; Fig.1 and Table 1) Once the filaments covered more than 50% of an individual well, they were transferred to 105 plates with larger wells until enough biomass was reached for them to be moved to a 200mL 106 glass culturing flask. Initially, some cultures were seen to contain diatoms, coccolithophores, 107 prasinophytes, and cyanobacteria that were initially kept at very low levels by subculturing. At that stage, we avoided using antibiotics to allow Ostreobium to retain more of its natural 109 microbiome for an ongoing research project. Substrains were also processed to obtain unialgal axenic strains at the Australian (Table 1) . 143 The phylogenetic affiliation of the strains was inferred using two different (Table S1 ). For rbcL, we 157 We initially isolated 11 strains of Ostreobium from coral skeleton fragments collected on 158 Heron Island (Australia, GBR). Strain VRM647 died soon after we sequenced it, so ten 159 unialgal strains were deposited in the Australian National Algal Culture Collection (ANACC, 160 CSIRO, Hobart; Table 1 ). After several months of growth at low illumination, the free-living 161 thalli showed apparent variation, with gross morphology varying from very compact, dark 162 green thalli to relatively diffuse structures with filaments that appear less pigmented despite 163 growing in identical conditions. Strain VRM650 (Fig. 4) presented very dark compact thalli, 164 while strains VRM605, VRM609, VRM623, VRM633, VRM644 and VRM646 shared the Ostreobium, occurring at the ends of some filaments. We also observed these in all of our 172 strains, and we noticed new filament growth on the glass culture flasks for several strains. While we could not make microscopic observations to confirm whether these filaments grew 174 from spores that were released from the sporangia-like structures, this is the most likely 175 explanation for our observations. Chloroplasts were not observed to be reticulated as reported by Lukas (1974) . Instead, 177 they were large and ovoid and often nearly as wide as the filaments for all the strains (Fig. 3) . The chloroplasts appeared to be homogeneously distributed along all the filaments, in The phylogeny based on the tufA gene (Fig. 6) showed that the newly established 189 strains belong to Ostreobium lineages 3 and 4, following the classification by Marcelino et al. 190 (Table S2 ). The ribosomal protein L19 contributes to bridging the two ribosomal In all the newly sequenced chloroplast genomes, we found group II introns in rpoB, 236 rpl23, rpl5 and rpoC1, as had previously been reported for other Ostreobium strains (Table S3) . Group II introns were also found in rps4 and ycf3 for 239 strains VRM650, VRM647, VRM646 and VRM642. The GenBank accession numbers are listed in Table 1 and Table S1 . Note that the phylogeny also includes 394 VRM647, an isolate for which we have molecular data available but that has died. Table 1 and Table S1 . 406 Reference sequences Genbank accession number used to reconstruct the green algae tufA 407 phylogeny and outgroup of the rbcL phylogeny. Table S2 . 410 Features of the chloroplast genome in the Ostreobium strains. Table S3 . 413 Comparison of the Group II introns distribution in the genes of the Ostreobium sp strains. 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