Climate-driven introduction of an opportunistic symbiont in the most extensive reef system of the Southwestern Atlantic Ocean

doi:10.21203/rs.3.rs-5020616/v1

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Climate-driven introduction of an opportunistic symbiont in the most extensive reef system of the Southwestern Atlantic Ocean

https://doi.org/10.21203/rs.3.rs-5020616/v1

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Coral symbionts of the Symbiodiniaceae family range from strictly mutualistic to highly opportunistic species. Symbiodinium necroappetens is an opportunistic species and a putative necrophile of dead coral tissue, hitherto reported as endemic to the Caribbean. Symbiodiniaceae were characterized by ITS2 barcoding along the most severe thermal anomaly registered in the Abrolhos Bank, southwestern Atlantic Ocean (SWAO). S. necroappetens relative abundance and prevalence went from undetectable before the anomaly to as much as 73% and 100%, respectively, during and after the heatwave. Sequences were still detectable four years after being initially observed. We are likely observing a climate-driven introduction of S. necroappetens in the SWAO. Given the increasing frequency and intensity of thermal anomalies hitting the global ocean, we can only speculate on the ecological consequences of such invasion for the Abrolhos reefs.

coral bleaching

opportunistic

zooxanthellae

geographic expansion

The dinoflagellate Family Symbiodiniaceae encompasses many symbiotic, photosynthetic forms that live intracellularly in the coral gastrodermis, playing key roles in the health and resilience of coral reefs (Dubinsky and Iluz 2016; LaJeunesse et al. 2018). Extreme thermal anomalies can disrupt symbiosis, leading to coral bleaching and mortality (Brown 1997). Restructuring of Symbiodiniaceae communities has been hypothesized to take place in the thermal acclimation of corals (Buddemeier et al. 2004), although some of the new thriving taxa may not be as mutualistic as the original ones (Berkelmans and van Oppen 2006; Quigley et al. 2022; Buzzoni et al. 2023). Indeed, Symbiodiniaceae exhibits diverse functional niches, ranging from free-living species (Hansen and Daugbjerg 2009) to symbiotic ones, which can vary from strictly mutualistic to highly opportunistic species (Wooldridge 2010; Lesser et al. 2013).

Symbiodinium necroappetens is a putative necrophile of dead coral tissue endemic to the Caribbean and associated with multiple coral host species (LaJeunesse et al. 2015; Lewis et al. 2019). This symbiont opportunistically invades dead coral tissue, especially after bleaching events when necrosis is frequent (LaJeunesse et al. 2015), remaining in the background of restored communities for a while.

The Abrolhos Bank harbors the most extensive reef system in the Southwestern Atlantic Ocean (SWAO), having relatively low coral richness compared to the Caribbean and Pacific reefs (Castro and Pires 2001). These reefs suffered mass coral bleaching in 2016 following a thermal anomaly that led to the third global bleaching event (Hughes et al. 2017; Teixeira et al. 2019). Corals were again affected in 2019 by the hitherto most severe heat wave ever registered for the Brazilian coast (Duarte et al. 2020; Pereira et al. 2022; Corazza et al. 2024). Studies employing NGS have not hitherto reported S. necroappetens in SWAO corals (Silva-Lima et al. 2021; Garrido et al. 2022, 2023, 2024; Varasteh et al. 2022).

This study reports the occurrence and relative abundance of S. necroappetens in scleractinian corals of the Abrolhos Bank at the onset and over four years of the region's most severe positive thermal anomaly ever recorded. These results extend the known distribution range of S. necroappetens and raise essential questions about the symbiont ecological role and impact on coral communities in this region. We discuss the implications of these findings in the context of ongoing climate change and the increasing frequency of thermal stress events.

Sampling and Symbiodiniaceae community characterization

Specimens of Montastraea cavernosa (Linnaeus, 1766), Mussismilia braziliensis (Verrill, 1868), Mussismilia harttii (Verrill, 1902), and Favia gravida (Verrill, 1868) were sampled in four reef sites in the Abrolhos Bank, Southwestern Atlantic Ocean (SWAO), at nine occasions from November 2018 to April 2023 (Supplementary Material, S1). Colony fragments were collected by SCUBA using a chisel and hammer, rinsed with 0.22 µm-filtered seawater water, and either directly frozen in liquid nitrogen or had their tissue first collected with an air gun and then frozen. Tissue was lysed in a chaotropic saline buffer (Fukami et al. 2004) and then CTAB was added. DNA was separated with chloroform-isopropanol followed by 75% ethanol rinses and purified with a ProNex® Size-Selective system. The internal transcribed spacer II region (ITS2) was amplified with primers Sym_VAR_5.8SF and Sym_VAR_REV (Hume et al. 2018) as follow: 20 cycles using the enzyme kit UPC hot start (Qiagen); PCR products run in agarose gel and cut off for extraction with PuriLink™ quick gel kit (Thermofisher); 15 more thermal cycles. Sequencing was done in an Illumina MiSeq platform with 20,000 to 50,000 paired-end 250bp-long reads per sample. Sequence analysis was done in the SymPortal framework (Hume et al. 2019) with ITS2 types inferred following Villela et al. (2024). Sea surface temperature (SST) and cumulative thermal stress (Degree Heating Week - DHW) for the Abrolhos region was retrieved from the NOAA Coral Reef Watch (CRW) database (https://coralreefwatch.noaa.gov). A generalized linear model with Gaussian error and log-link function was used to evaluate the relationship between the proportion of S. necroappetens and DHW.

Establishment of S. necroappetens cultures and strain characterization

Coral fragments for Symbiodiniaceae isolation have been sampled in the Abrolhos Bank as part of a monitoring program, including, but not exclusively, during the sampling periods described above. After rinsing with 0.22 µm-filtered seawater coral tissue was removed from polyps with a spatula, suspended in seawater-based f/2 medium (Guillard and Rythe 1962) containing 1 µg mL^-1 GeO₂, and aliquoted in 96-well clear polycarbonate microtitration plates and were kept at 24ºC and 50 µmol photons s^-1m^-2 in a 12h:12h light:dark cycle. Cultures that grew on the plate were subjected to single-cell sorting in a flow cytometer (MoFlo DakoCytomation®, USA) (Silva-Lima et al. 2015). Cultures established in this way are maintained by successive transfers to fresh f/2 medium. For strain characterization DNA was extracted from cell pellets with chloroform washings (Fukami et al. 2004). The ITS2 marker was amplified and sequenced (Sanger) with the same primer pair used in the metabarcoding approach. Sequences were identified by Blastn similarity search against the NCBI-nt database and in Symportal. Mastigote and coccoid (vegetative) cell types were photographed in an Olympus BX51 (Shinjuku-ku, Tokyo, Japan) microscope at 100X and 200X magnification. Cell maximum linear dimension was measured with Fiji (Schindelin et al. 2012).

S. necroappetens was undetectable in corals collected before the onset of the 2019 thermal anomaly. Conversely, the symbiont was present in three coral species - M. cavernosa, M, hartti, and M. braziliensis - and all reef sites during and after the anomaly. The relative abundance of S. necroappetens increased during thermal anomaly and peaked at 0.73 four months after the maximum DHW, decreasing in the next sampling and remaining < 0.1 until April 2023 (Fig. 1a). All coral specimens collected in 2019 had detectable S. necroappetens whereas 65% of the specimens still had the symbiont at the last sampling in April 2023 (Fig. 1b). There was a significant correlation between the proportion of S. necroappetens and DHW (Fig. 1c).

Symbiodinium necroappetens was successfully established in culture from three specimens of M. cavernosa collected at the ESQ in May 2021 (one strain, CCMR0229) and September 2021 (two strains, CCMR0231 and CCMR0233). Cells of all three strains have similar morphology, bright yellow to brown, with coccoid (average length: 9.0 µm, sd: 0.9 µm, N: 86) and flagellated mastigote (average length: 8.8 µm, sd: 1.0 µm, N: 71) types (Fig. 2). The strains' ITS2 sequences (Genbank accession number PQ238075) were 100% identical to S. necroappetens (AF333504).

This is the first report of Symbiodinium necroappetens for the SWAO and, as far as we are concerned, also outside the Caribbean, the hitherto known distribution of this species. Noteworthily, S. necroappetens was dominant (up to 73%) in Abrolhos corals at the onset of a massive thermal anomaly that later on in the year translated into severe coral bleaching and mortality (Pereira et al. 2022; Corazza et al. 2024). Also noticeable is that only five months before the first observation of S. necroappetens, in November 2018, the symbiont was undetectable even though as many as 64 specimens were screened then. The observed decline in relative abundances with time after bleaching and the alleviation of thermal stress suggests it can not outcompete other Symbiodiniaceae lineages under non-stressful conditions (LaJeunesse et al. 2015). The timing and environmental conditions in which this dominance was observed agree with the opportunistic life strategy attributed to S. necroappetens in Caribbean reefs (LaJeunesse et al. 2015).

We hypothesize that S. necroappetens have expanded its geographical distribution in response to the increased frequency and intensity of thermal anomalies, which could have opened favorable niches and ecological corridors along the Brazilian coast. Alternatively, S. necroappetens may have been at background abundance but have not been detected due to insufficient sampling and low sequencing depth. The Brazilian coast experienced a thermal anomaly and a mass bleaching event in 2016 (Teixeira et al. 2019). No S. necroappetens sequence was reported on that occasion in Symbiodiniaceae ITS2 libraries from two common SWAO corals (M. harttii and Millepora alcicornis), including an area in the northernmost part of the Abrolhos Bank (Garrido et al. 2023). Likewise, no S. necroappetens sequences were reported after in M. hispida and Siderastrea stellata sampled during the 2019 thermal anomaly in an area ca. 540 km south of the ESQ reef site studied here (Garrido et al. 2024). Given that we observed up to 73% dominance of S. necroappetens in 2019, outbursts may be limited to the Abrolhos area due to environmental filters that hinder its establishment/augmentation in other places. The herein-reported occurrence and high proportion of S. necroappetens in three common coral species in Abrolhos highlight the importance of detailing its geographical distribution along the Brazilian coast.

Our results and previous observations suggest a recent introduction of S. necroappetens in the SWAO. Despite the decline in relative abundances, the high prevalence of coral specimens containing S. necroappetens indicates a long persistence - at least four years - of this opportunistic Symbiodinidaceae after an outbreak. Following global trends (Reimer et al. 2024), a recent thermal anomaly was observed in the northern region of the SWAO in 2024. With the expected increase in the frequency and intensity of marine heatwaves, it is still premature to predict the ecological consequences of the detection and persistence of S. necroappetens in the region.

Competing interests

The authors declare no conflicts of interest.

Ethics approval and consent to participate

Sampling permits: NGI Abrolhos/ICMBio 65055-8.

Funding

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Fundação Renova, Conselho Nacional de Desenvolvimento Científico e Tecnológico (grant 310057/2022-1) and PELD Abrolhos (Grant 441418/2020-1).

Author Contribution

LBV, RLM, AWSL and PSS did the experimental design. LBV, YRA, AWSL, PSS, wrote the main manuscript text. LBV, AW and YRA prepared figures. YRA conducted culture isolation, cell measurements, genetic identification and pictures. LBV prepared the ITS2 gene libraries and the bioinformatic analysis. AWSL did the statistical analysis. All authors reviewed the manuscript.

Acknowledgement

L.B.V thanks Programa de Pós graduação em Genética (UFRJ), FAPERJ, CAPES, and FEST/RENOVA for her doctoral scholarship and resources. The authors thank all lab and boat support teams for assistance with sampling during the expeditions. R.L.M. and P.S.S. acknowledge long-term individual grants from CNPq.

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No competing interests reported.

SupplementaryMaterial.docx

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Climate-driven introduction of an opportunistic symbiont in the most extensive reef system of the Southwestern Atlantic Ocean

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Abstract

Figures

Introduction

Material and Methods

Sampling and Symbiodiniaceae community characterization

Establishment of S. necroappetens cultures and strain characterization

Results

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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