Occurrence of the thermotolerant zoochlorellae Symbiochlorum hainanense associated with hydrocorals in the Southwestern Atlantic Ocean

Sampling, algal isolation, and culturing

Sampling was conducted in May 2021 at Parcel dos Abrolhos (17°57.703′ S, 38°41.741′ W) in the Abrolhos Bank. Fragments of one specimen of the coral Millepora alcicornis were collected with a hammer and chisel by SCUBA, rinsed with 0.22 μm filtered seawater and stored in plastic containers with 0.22 μm filtered seawater during transportation to the laboratory. These fragments had their tissue scratched and homogenized with f/2 medium (Guillard and Lorenzen 1972) containing 1 μg mL⁻¹ of GeO₂. The obtained cell suspension was serially diluted in 96-well clear polycarbonate microtitration plates.

Plates were kept in a culture room at 50 μmol s⁻¹ m⁻², 24°C and photoperiod of 12-h light / 12-h dark. Wells that showed algal growth with the least amounts of contaminants were inoculated in 50 mL flasks. Dense, homogeneous cultures were filtered through a 70 μm nylon mesh to remove large cell clumps and subjected to single-cell sorting in a flow cytometer (MoFlo DakoCytomation, Carpinteria, CA, USA) equipped with an electrostatic droplet deflection system, and using a detection and sorting strategy as described elsewhere (Silva-Lima et al. 2015). Putative algal cells were individually sorted into the wells of 96-well microtitration plates containing 150 μL of sterile f/2 medium per well. The plates were monitored in an inverted microscope for algal growth. Several cultures established in the microtitration plates were transferred to fresh f/2 medium in 50 mL flasks. Four cultures were incorporated in the Culture Collection of Microalgae of the Universidade Federal do Rio de Janeiro, Brazil (CCMR) with strain codes CCMR0224, CCMR0235, CCMR0236, and CCMR0237. The strains are maintained by successive transfers (ca. four weeks) to fresh f/2 medium. Genetic and phenotypic characterization was done with the isolate CCMR0224 and compared to the holotype of S. hainanense CCTCC M2018096^T, CCTCC M2018096. These two Symbiochlorum strains will be referred to in the text as CCMR0224 and CCTCC M2018096.

Sampling permit was provided by Ministério do Meio Ambiente (MMA), Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio), Sistema de Autorização e informação da Biodiversidade (SISBIO) (No. 65055-8).

Phenotypic characterization

Two milliliters of cultures in the late stationary phase were transferred to flasks containing fresh f/2 medium in the same growth conditions described above. Zoospores were collected from swarms in the flasks’ corners with a micropipette and then sieved twice through a 5 μm nylon mesh to remove vegetative cells. The filtered suspension was transferred in small drops to a Petri dish and exposed to 900 μmol s⁻¹ m⁻², accumulating swarms opposite to the light source. Then, they were collected with a micropipette, diluted with fresh medium, distributed into the wells of 96-well microtitration plates and kept in the light and temperature regime as above. Growth was monitored and quantified daily from inoculation until the onset of sporogenesis (after 4 d) using photographs taken in an Olympus IX70 (Tokyo, Japan) microscope at 40× magnification. Images of the cells were obtained by tracing longitudinal transects in three wells. Cell length and width (max and min Feret, respectively) were measured with Fiji (Schindelin et al. 2012). Cell volume was estimated as a prolate ellipsoid. Biovolume increase over time was used to estimate cell growth rate by the slope of the linear regression fit between Ln of the average biovolume versus time. Graphs were built using the RStudio version 2023.12.0+369 (R Core Team 2021).

TrackMate (Olympus) was used for zoospores’ swimming speed calculation and swimming path tracking, employing a Hessian detector set to detect 7 μm particles with gap and linking distances of up to 30 μm (Ershov et al. 2022). The quality threshold was kept at 0.001. Videos of zoospores are available in the supplementary material (Supplementary Videos S1–S4). Newly released spores were photographed in an Olympus BX51 microscope at 200× magnification.

For life cycle characterization, 1 mL of dense cultures were inoculated in 20 mL of fresh medium and maintained under the same growth conditions as above. After 3 d, cells were fixed in 1% glutaraldehyde and treated with a 1% polysorbate solution (Tween 80; Sigma-Aldrich, St. Louis, MO, USA) to permeabilize cell walls. Nuclei were stained with 0.3% SYBR Green DNA dye and visualized under blue light in epifluorescence microscopy at 40× magnification (Vítová et al. 2005). The classification of the multiple fission type followed the method described by Bišová and Zachleder (2014).

To evaluate the thermotolerance of the strain, we exposed cultures to two different temperatures during one week. Chlorophyll a concentration was measured in the beginning and at the end of the exposure. Initially, cultures were acclimated to 24°C and a photon flux of 50 μmol s⁻¹ m⁻² for 3 d. The experiment consisted of a control (24°C) and treatment (32°C) using four replicates. The cultures were manually mixed, and cells collected on glass fiber filters (47 mm Whatman GF/F) were transferred to glass test tubes and frozen and thawed to help break the cell walls. Afterward, 5.4 mL of 100% acetone (considering the water absorbed by the filter to obtain extracts in 90% acetone) were added to the filters that were macerated with a glass rod and kept at 4°C in the dark for 12–16 h. Following extraction, the fluorescence of the acetonic extracts was measured in a VARIAN Cary Eclipse fluorescence spectrophotometer (Melbourne, Victoria, Australia) and pigment concentrations were determined using a modified version of the method described by Neveux and Lantoine (1993), as detailed in Tenório et al. (2005).

DNA extraction and sequencing

DNA extraction followed the cetyltrimethylammonium bromide protocol. Briefly, cells were collected by centrifugation (5,000 rpm, 5 min), heated to 56°C for 1 h and lysed. The DNA was isolated with chloroform–isoamyl alcohol, rinsed with ethanol, and eluted in MilliQ water. DNA was quantified by spectrophotometry (NanoDrop One; Thermo Fisher, Wilmington, DE, USA) and the concentration was adjusted to 15 ng μL⁻¹ with MilliQ water for subsequent purification with a ProNex Promega size-selective purification system (Madison, WI, USA) following the manufacturer’s instructions.

The molecular markers 18S nuclear rDNA (18S), plastid elongation factor Tu (tufA), rbcL and internal transcribed spacer cistron (ITS1-5.8s-ITS2) were selected for PCR amplification based on primer universality, internal variability and barcode gap (Saunders and Kucera 2010), and sequence availability in public databases. PCR cycles diverged in their annealing temperature and extension time, depending on the marker, having an initial denaturation at 95°C (5 min), followed by 35 cycles of denaturation at 95°C (30 s), annealing at variable temperatures (30 s), extension at 72°C (variable time), and a final extension of 72°C (5 min). PCR cycle details and primer sequences are shown in Supplementary Table S1. The reaction mixture in a final volume of 10 μL included 5 μL of Green Master Mix with GoTaq G2 DNA polymerase (Promega), 2 μL of water, 2 μL of purified DNA, and 0.5 μL of each forward and reverse primers at 10 mM. Amplification of the rbcL gene was performed with Taq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s recommendations.

PCR products were purified using the commercial system PuriLink quick gel extraction (Thermo Fisher) and / or the Exo-SAP-IT (Thermo Fisher). Sequencing of PCR products was performed using the Big Dye Terminator Cycle Sequencing Ready Reaction Kit v. 3.1 (Thermo Fisher) by the PSEQDNA-UFRJ facility, at Biophysics Institute, Federal University of Rio de Janeiro. Contig sequences were assembled and manually verified by checking electropherograms in BioEdit (Hall 1999).

Phylogenetic analysis

Sequences of phylogenetic neighbors were retrieved from GenBank (Benson et al. 2012), except for the 18S gene sequences, which were retrieved from the SILVA database considering sequence identity, alignment, and pintail quality scores (Pruesse et al. 2007). Sequences were aligned in Molecular Evolutionary Genetics Analysis (MEGA) version 7 (Kumar et al. 2016) using the ClustalW algorithm (Thompson et al. 1994). Current knowledge concerning Chlorophyta molecular evolution, systematized by Leliaert et al. (2012), was considered when choosing representative sequences of each order.

Firstly, we allocated the new lineage within the Chlorophyta phylogenetic space using the 18S sequences. The tree was built using MEGA7 (Kumar et al. 2016) with the Maximum likelihood (ML) method. The model finder function was used to select the best fit evolutionary rate model, the Tamura-Nei, with a discrete Gamma distribution and with a proportion of invariable sites (TN93 + G + I). There were 28 sequences with 1,577 aligned positions, of which 615 were parsimony-informative, in the final dataset. A subsequent inner placement in the Ulvophyceae class was confirmed with a concatenated 18S, tufA, and rbcL gene sequences phylogeny. ML and Bayesian inference (BI) were used to build the concatenated tree. For tufA and rbcL genes, the first and second codon positions were used due to the saturation levels in the third codon position. Removing fast-evolving sites, such as the third codon position, was effective for phylogenetic reconstructions of the Ulvophyceae class (Cocquyt et al. 2010, Škaloud et al. 2012). There were 34 sequences with 2,619 aligned positions, of which 1,124 were parsimony-informative, in the final dataset. The ML phylogeny was built using IQ-TREE (Nguyen et al. 2015), admitting the DNA model selected by ModelFinder (Kalyaanamoorthy et al. 2017). The TIM3e + I + R3 model was chosen according to the BIC criterion for the 18S alignment, while TVM + F + G4 was applied to the coding genes tufA and rbcL in the concatenated alignment. In addition, both ultrafast bootstrap (Hoang et al. 2018) and SH-like approximate likelihood ratio test (Guindon et al. 2010) were used to evaluate branch support. For BI, IQ-TREE ModelFinder was used to infer the best substitution model (GTR + I + G model for the three genes was chosen based on Akaike information criterion), which was subsequently applied for phylogeny reconstruction in MrBayes v 3.2.7.a (Ronquist et al. 2012). Three heated and one cold parallel chain were run for 5 million generations, with sampling every 1,000 generations. Since the architecture of the concatenated tree diverged from the 18S phylogeny shown in the description of S. hainanense (Gong et al. 2018), we employed the same dataset they used to build 18S gene sequence trees. We built the trees with and without Symbiochlorum using ML and BI methods. Trees were built using IQ-TREE (Nguyen et al. 2015) and MrBayes (Ronquist et al. 2012) with the substitution model GTR + I + G. BI was run as for the concatenated tree with 10 million generations. Phylogenetic trees were visualized in FigTree v 1.4.4 (Rambaut 2009). Sequence accession numbers for the Ulvophyceae representatives are shown in Supplementary Table S2. The percentage identities of the 18S, rbcL, tufA, and ITS cistron gene sequences were calculated for the Ignatiales members (S. hainanense [CCMR0224 and CCTCC M2018096], Ignatius tetrasporus, and Pseudocharacium americanum), using the alignment built with MEGA7 (Kumar et al. 2016).

ITS2 secondary structure modeling

ITS cistron sequence (CCMR0224) was aligned in MEGA7 with that of I. tetrasporus (HE610121.1). ITS2 and flanking regions were identified and annotated using ITS2 database (Merget et al. 2012), preserving the flanks needed for secondary structure modeling. Afterward, this sequence was aligned with that of S. hainanense CCTCC M2018096 (MH061388.1), and polymorphisms were identified. The adopted workflow for secondary structure prediction was the same as that used by Darienko and Pröschold (2017, 2024). Secondary structures were built and visualized in the RNAfold web server (Gruber et al. 2008). Structures of CCTCC M2018096 (MH061388.1) and CCMR0224 were compared by searching for Compensatory Base Changes (CBCs) and hemi-CBCs.

Phenotypic characterization of strain CCMR0224

Vegetative cells are green and spherical, with visible cell walls, a single parietal reticulated chloroplast and a central vacuole up to 13 μm in diameter (Fig. 1A). Zoospores have a visible eyespot (Fig. 1B) and are quadriflagellated (Fig. 1C), with dimensions ranging from 3.6–7 × 3.1–5.1 μm just after being released (Supplementary Table S3). They exhibited a negative phototaxis when exposed to light, with a mean speed of 249 ± 77 μm s⁻¹ (Supplementary Table S4). We tracked and analyzed 154 independent zoospores, yielding a mean total distance of 228 μm and a confinement ratio 0.88. Most cells randomly settled on the well’s bottom a few hours later. Usually, zoospores kept moving slower, spinning in the same place until losing their flagella and stopping to move. They maintained their elongated shape, slowly becoming rounder. We measured 1,471 cells over 4 d, starting with a mean cell volume and diameter of 572 μm³ and 5.8 μm, respectively, and reaching 5,902.5 μm³ and 11.1 μm on the fourth measurement, respectively, until they released both flagellated and non-flagellated spores (Supplementary Table S3). Aplanosporangium synchronously releases spores as soon as the light period starts (Fig. 1E). The growth rate based on biovolume increase was 0.8 d⁻¹ (Fig. 2A). Another reproductive strategy was identified throughout many culture inspections: cells could undergo multiple fission and remain attached, forming coenobia with multiple cells (Supplementary Fig. S1). The cell cycle matches a clustered type of multiple fission, with no multinuclear stages observed (Supplementary Fig. S2). When subjected to 32°C, no difference in culture growth accessed by chlorophyll a concentration was observed relative to the control at 24°C (0.3 d⁻¹) (Fig. 2B). The proportion of phaeophytin to chlorophyll a was insignificant (<5%) and similar in both temperatures.

Fig. 1

Cells under bright-field (A & C–J), differential-phase contrast microscopy (B) and epifluorescence (K–N). (A) Vegetative cells. (B) Aplanosporangium with visible cell wall. (C) Cells in different rounds of cytokinesis. (D) Zoospore just before settling on the chamber bottom with visible flagella. (E) Spores clump. (F) Vegetative cells with a single cup-shaped parietal chloroplast. (G–N) Same vegetative cells with the parietal chloroplast at bright-field and epifluorescence microscopy. P, pyrenoid; CW, cell wall; V, vacuole; CH, chloroplasts; F, flagellum; ES, eyespot. Scale bars represent: A–G & K, 20 μm.

Fig. 2

Growth of Symbiochlorum hainanense CCMR0224. (A) Increase in mean cell volume (μm³) measured on 1,471 cells over 3 days. (B) Increase in chlorophyll a concentration in cultures grown at 24°C and 32°C over a 6-day period. Mean ± standard deviation (SD; whiskers). Some SD whiskers are smaller than the symbols. Dashed line: exponential fit. Note that the y-axis is on a log scale; thus the exponent of “e” represents the instantaneous growth rate.

Molecular taxonomy and phylogeny

The overall tree topology of Chlorophyta (Supplementary Fig. S3) was congruent with published phylogenies of the phylum (Leliaert et al. 2012). Chlorophyceae and Ulvophyceae are allocated in the bifurcations of the same major branch. CCMR0224 clustered in the Ulvophyceae branch, with S. hainanense as a sister strain with high support (100) and the Ignatiales representatives as their closest neighbors (99).

BI and ML analyses of the 18S-tufA-rbcL alignment of Ulvophyceae sequences yielded identical tree topologies. Most of the nodes were highly supported (>70) according to both methodologies. Only two basal nodes and one inside the clade TCBD were exceptions. TCBD was recovered, as well as Oltmannsiellopsidales and Ulvales-Ulotrichales (OUU), in line with previous findings (Watanabe and Nakayama 2007, Cocquyt et al. 2010). CCM0224 was placed as a sister strain of CCTCC M2018096 in the order Ignatiales (Fig. 3).

Fig. 3

Phylogeny inferred from concatenated 18S, tufA, and rbcL gene sequences of 32 Ulvophyceae members (2,619 aligned nucleotide positions). All nodes were recovered by both Maximum likelihood (ML) and Bayesian inference methods. Bayesian posterior probabilities and ML bootstrap support values (>70) are shown at nodes. The scale bar represents the expected number of substitutions per site. TCBD stands for Trentepohliales, Cladophorales, Bryopsidales, and Dasycladales. The symbols in the map indicate habitat: circle, marine; star, freshwater.

The 18S phylogenies built with the Symbiochlorum genus reproduced the multigene tree architecture, with Ignatiales and Scotinosphaerales bifurcating from the same node and Ulvales-Ulotrichales branching from the same node of Oltmannsiellopsidales. When Symbiochlorum was included, Scotinosphaerales branched earlier and, from the same node, another branch further bifurcated in the Ignatiales and the OUU clades (Supplementary Fig. S4).

The percentage identities and coverages of the 18S, ITS2, rbcL, and tufA gene sequences amongst the Ignatiales members are summarized in Table 1. The 18S gene sequence’s identity between CCMR0224 and CCTCC M2018096 is slightly lower than that between I. tetrasporus and P. americanum. For the protein-coding genes, rbcL and tufA, the identities between S. hainanese CCM0224 and S. hainanense CCTCC M2018096 are low, 96.3 and 97.1%, respectively, while these gene sequences are identical when I. tetrasporus and P. americanum are compared.

Table 1

Identity and coverage (% identity / % coverage) of four gene sequences (18S, ITS2, rbcL, and tufA) amongst the Ignatiales members

ITS2 secondary structure

Both secondary structures obtained from CCTCC M2018096 and CCMR0224 were compared, and a deletion was identified in helix 3, as shown in Fig. 4.

Fig. 4

ITS2 secondary structure of CCMR0224 depicting helices and splicing processing sites (C1 and C3) found in Chlorophyta. Insertion of an adenine in helix 3 of CCTCC M2018096 is shown in the inset (top right corner).

Taxonomic aspects

Our work agrees with the previous placement of Symbiochlorum genus in Chlorophyta and Ulvophyceae (Gong et al. 2018) using a concatenated matrix of one nuclear (18S rRNA) and plastidial markers (tufA and rbcL). The single gene trees based on the 18S rRNA gene sequences differed depending on including the genus Symbiochlorum in the phylogeny. When absent, the architecture resembled those built before the description of this genus, with Scotinosphaerales branching earlier than the OUU clade. When Symbiochlorum was included, Scotinosphaerales and Ignatiales bifurcated from the same early node, and the OUU clade further bifurcated from the same node of the TCBD clade. Regardless of the dataset, CCMR0224 and CCTCC M2018096 remained as sister strains, having members of the other Ignatiales as closest neighbors. The divergences in tree topologies are not unexpected since they occur at basal, low-supported nodes, which anchor ancient and fast evolving groups (Del Cortona et al. 2020). Moreover, there are only a few Ignatiales sequences available. These results highlight the importance of characterizing more Ignatiales strains, especially those from poorly represented and deeply rooted branches, to accurately delineate the evolutionary history of the group.

S. hainanense CCMR0224 was recovered as a sister strain of S. hainanense CCTCC M2018096. S. hainanense is highly distinct from its phylogenetic neighbors within Ignatiales, namely I. tetrasporus and P. americanum. This strain (Gong et al. 2018) exhibits smaller spherical cells (3.6 to 13.6 μm), multiple chloroplasts, and spores released by a big sporangium (up to 20 μm); while the previously described species of Ignatiales displays larger saccate cells (20–40 × 12–25 μm), a single parietal cup-shaped chloroplast, and spores released by tetrads to octads aggregated cells; moreover, they are not marine inhabitants (Bold and MacEntee 1974, Lee and Bold 1974, Watanabe and Nakayama 2007, Gong et al. 2018).

The strain characterized herein produces zoospores, exhibits a single parietal reticulated chloroplast and forms coenobia, retaining non-flagellated autospores. Zoospore production and single chloroplasts were also reported by Darienko and Pröschold (2024) in a Symbiochlorum strain isolated from the ascidian Lissoclinum patella from Palau island. These characteristics were amended in the formal description of the genus and species (Darienko and Pröschold 2024) and differ from S. hainanense isolated from the South China Sea, whose formal description stated the presence of multiple chloroplasts and that no zoospores were observed. Zoospores are found in other Ignatiales members (Bold and MacEntee 1974, Lee and Bold 1974, Gong et al. 2018), albeit much smaller flagellated cells are found in S. hainanense from Palau and from the Southwestern Atlantic than in other Ignatiales representatives (3.6–7 × 3.1–5.1 μm against 12–16 × 4–6 μm in I. tetrasporus) (Bold and MacEntee 1974, Lee and Bold 1974).

ITS2 sequences have been widely used in Chlorophyta taxonomy to erect new species (Caisová et al. 2013), and even though their primary sequence is not easily alignable, their secondary structure has shown to be phylogenetically informative. CBCs have been searched in the least ITS2 variable regions, as their presence between two different haplotypes strongly suggests these strains are unable to cross and, thus, can be recognized as different biological species (in organisms with sexual reproduction) (Müller et al. 2007). The comparison between the ITS2 secondary structures of CCTCC M2018096 and CCM0224 revealed one difference, consisting of a nucleotide insertion in a bulge loop in Helix III in the latter, thus not configuring a taxonomic diagnostic CBC. Finally, we tried to deduce the consensus structures for the order Ignatiales using the framework suggested by Caisová et al. (2013), but sequences between S. hainanense and Ignatius were so divergent that their alignments yielded many equally possible results, not phylogenetically informative (results not shown).

Furthermore, taxonomic-relevant genes (tufA, rbcL) identities between both Symbiochlorum species are much bigger than those between I. tetrasporus and P. americanum. Thus, we suspected that I. tetrasporus and P. americanum belong to the same species. Moreover, Family and Order affiliation of the Symbiochlorum genus is incertae sedis, according to the algal database AlgaBase (https://www.algaebase.org/). Indeed, gene identities (Table 1) between both Symbiochlorum strains and the other Ignatiales members (82.4–89.1%) are low enough to raise the question of whether the genus could be placed in a new family or even a new order, as previously suspected by Gong et al. (2018). The plastome and zoospores’ ultrastructural features could help further refine the taxonomic status of this singular, hitherto exclusively marine Ulvophyceae genus.

Life cycle characterization

The life cycle type Cn was identified in CCMR0224, characterized by multiple fission of the mother cell. Cycle type Cn of vegetative reproduction is common in Chlorophyta, found in many genera such as Chlorella, Desmodesmus, Scenedesmus, and Chlamydomonas (Zachleder et al. 2016). However, no multinuclear stage was observed, contrasting the Scenedesmus and Chlorella models (Bišová and Zachleder 2014). Indeed, it was possible to verify that mitosis still happens after the first rounds of cytokinesis (e.g., cells with double, less-fluorescent nuclei together with cells with one, more-fluorescent nucleus in the same coenobia), indicating both processes are interspersed at the end of the cell cycle. Thus, it shows a cell cycle organized as the one found in Chlamydomonas, having a clustered type of multiple fission cell cycle, a long G1 phase, and a DNA replication-division sequence just at the very end of the cell cycle in the dark period. However, just-released zoospores do not seem to have a cell wall (as observed in optical microscopy) in contrast to Chlamydomonas zoospores (Bišová and Zachleder 2014, Zachleder et al. 2016). In truth, many fast-growing microalgae undergoing multiple fission synchronize their cell cycles with circadian rhythms, harnessing the light phases for maximum energy exploitation and growth and dark phases for independent light processes such as DNA replication and cell division (Bišová and Zachleder 2014, Zachleder et al. 2016). CCM0224 culture were synchronized both on cell division (during the dark period) and zoospores release (at the onset of the light period). This growth strategy for better resource exploitation only makes sense in organisms that can more than double their cell volume per day (Bišová and Zachleder 2014), such as CCMR0224 which exhibits a biovolume intrinsic growth rate of 0.8 d⁻¹, which translates into doubling the volume every 20 h. Moreover, multiple fission represents a good strategy for fast-growing algae, as they maximize the number of daughter cells instead of enlarging daughter cell size, responding faster to improving environmental conditions than cells that undergo binary fission (Rading et al. 2011, Zachleder et al. 2016).

Furthermore, zoospores release at the beginning of the light phase, and its fast flagella detachment from the cell is also in accordance with the proposed benthic lifestyle of S. hainanense, using light stimulus just for reaching the substrate, fastly halting its movement after cell attachment. Indeed, the high confinement ratio found on zoospores’ swimming tracks reveals a highly oriented movement away from the light source. In addition, multiple fission is also commonly found in benthic algae, which relies on good dispersal strategies and invests in cell size enlargement when reaching a favorable substrate for growing (Cavalier-Smith 1980, Bell and Koufopanou 1991). This cell strategy is also found in microeukaryotes that rely on hosts for survival (e.g., parasites), which are strongly constrained by natural selection on dispersal ability (eased by small and numerous propagules) and growth investment when reaching a food source (Cavalier-Smith 1980, Bell and Koufopanou 1991). These life cycle features corroborate the hypothesis that S. hainanense is a benthic alga that can also be symbiotically associated with corals.

Ecological implications

All Symbiochlorum hainanense strains isolated until now were from benthic organisms known for their associations with photosymbionts, suggesting its participation in the homeostasis of coral holobiont communities. Although most studies have focused on the ecophysiology of Symbiodiniaceae dinoflagellates and their roles in meta-organisms (Gong et al. 2019, Russnack et al. 2021), other microeukaryotes have been recognized as important players on coral physiology, such as photoendosymbiotic and endolithic microalga Ostreobium, which supplies photosynthates and alleviates high light stress in bleached corals (Galindo-Martínez et al. 2022). Gong et al. (2019) reported the presence of S. hainanense in healthy and bleached coral samples, albeit much more abundant in the latter ones. The heat tolerance of CCMR0224 (present work) and CCTCC M2018096 (Gong et al. 2020, Xiao et al. 2024), together with the prevalence of CCTCC M2018096 when compared to the thermotolerant Symbiodiniaceae genus Durusdinium in bleached corals (Gong et al. 2019), suggests a niche as zoochlorellae on coral holobionts subjected to heat stress. Remarkably, CCMR0224 could withstand higher and faster temperature increases (up to 32°C) than those recorded in Abrolhos Bank during heat waves, which peaked at 29°C in 2019 (Duarte et al. 2020). Not only CCM0224 survived this challenge but its growth rate did not seem to be impaired. Noteworthily, strain CCMR0224 was isolated in 2021, after the 2019 heatwave, which was the most severe ever recorded for the area. This ecological particularity suggests niche partition or competition with other members of the coral microbiome, even posing the risk of overwhelming the population of at least some Symbiodiniaceae members of coral holobionts in the increasingly frequent warmer oceans.

In conclusion, employing a multilocus phylogenetic framework, this new strain of coccoid green algae associated with corals was identified as a S. hainanense strain. This recently established species was initially found in bleached corals in the South China Sea, displaying a notable thermotolerance. The Southwestern Atlantic strain also exhibited thermotolerance and was isolated from a major reef-building hydrocoral species in the Abrolhos Bank. In times of increased frequency of seawater thermal anomalies, it is paramount to investigate the occurrence and diversity of this green algae in Abrolhos corals to better understand its ecological roles and implications for reef resilience.