Marine heatwaves and other direct human impacts threaten global biodiversity, as reefs face a combination of disturbances, including coastal development, pollution, and destructive fishing practices, alongside increasingly frequent extreme warming events that affect critical foundation species such as reef-building corals (Fox and Caldwell, 2006; Smale et al., 2019). While marine heatwave events do not always cause mass coral mortality, their increasing severity and frequency are resulting in increased coral losses and reef degradation (Maynard et al., 2008; Weis, 2010; Gadoutsis et al., 2019; Intergovernmental Panel on Climate Change (IPCC), 2023; Yadav et al., 2023). For instance, marine heatwaves have triggered five mass coral bleaching events on the Great Barrier Reef (GBR) in just nine years (2016 to 2024), reducing coral cover on shallow water coral reefs by as much as 50% (Hughes et al., 2017; Dietzel et al., 2021; Henley et al., 2024). The loss of coral, particularly reef-building species, diminishes the structural complexity of these habitats and disrupts ecological functions, compromising the biodiversity and ecosystem services coral reefs provide (Gratwicke and Speight, 2005; Graham and Nash, 2013; Newman et al., 2015; Souter et al., 2020).

The recovery of coral communities following major disturbances depends on successful reproduction and recruitment, in which the dispersive larval phase replenishes populations (Gilmour et al., 2013; Holbrook et al., 2018; Gouezo et al., 2020) and maintains genetic diversity. While coral communities have an inherent capacity for natural recovery after disturbance events (Baker et al., 2008; Sheppard et al., 2008), this process is increasingly hampered by various biological (declines in fecundity of source corals) and environmental challenges (sedimentation, biofilm, and suitable substrates), and can take a significant period of time free from additional disturbances (Ortiz et al., 2018). One critical factor in determining recovery success is the dispersive larval phase, during which coral larvae supply the new recruits needed to rebuild populations (Harrison and Wallace, 1990; Richmond and Hunter, 1990; Connell et al., 1997; Harrison, 2024a); this process requires successful larval settlement in degraded areas for recovery to occur.

Coral bleaching events and other severe disturbances significantly impact larval recruitment by reducing the availability of mature, breeding corals, which are vital for sustaining reproductive output (Hughes et al., 2019). Recruitment levels often plummet to a fraction of historical averages following such disturbances, with the composition of recruits frequently shifting toward more tolerant taxa (Gilmour et al., 2013; Holbrook et al., 2018; Hughes et al., 2019). Adding to this challenge, frequent disturbances subject many surviving adults to sublethal stressors, causing impaired fecundity and reduced gamete quality, diminishing the supply of healthy larvae and undermining the ability of coral populations to sustain themselves (Michalek-Wagner and Willis, 2001; Ward et al., 2002; Cox, 2007; Gilmour et al., 2013; Piñón-González and Banaszak, 2018; Johnston et al., 2020; Briggs et al., 2024). This decline in larval supply, coupled with altered species composition, hampers natural recovery processes: fewer larvae are available to settle and repopulate degraded reefs, which extends recovery timelines and drives long-term changes in community structure and biodiversity. Moreover, the increasing frequency of mass bleaching events leaves insufficient recovery intervals, often preventing coral communities from regenerating fully before the next disturbance (Babcock et al., 2021). This shortened window for recovery exacerbates the instability of coral populations and contributes to the long-term destabilization of reef ecosystems (Gilmour et al., 2013; van Woesik et al., 2016; Ortiz et al., 2018; Babcock et al., 2021).

Critical gaps exist in monitoring coral reproductive output across assemblages. Current assessment methods are often limited to a few species and rely on resource-intensive techniques such as tissue sampling for fecundity measurements (Howells et al., 2016; Briggs et al., 2024), recruitment data from settlement devices (Mallela and Crabbe, 2009), and benthic surveys (Jonker et al., 2008). These techniques are costly, often restricting their application to small specific sites and target species, and their reliance on morphological identification of recruits can result in low taxonomic resolution; indeed, recruits typically can’t be identified beyond the family level for the first several months of life (Babcock et al., 2003). This narrow focus can overlook broader, community-wide reproductive dynamics, such as potential shifts in the taxonomic composition of spawning slicks towards more tolerant taxa. Understanding these shifts is crucial to monitoring and forecasting the response of coral communities to climate change.

Active restoration efforts are increasingly recognized as an essential strategy for sustaining coral ecosystems and their services (Bullock et al., 2011; Anthony et al., 2017; Boström-Einarsson et al., 2020; Vaughan, 2021; Banaszak et al., 2023). Among these interventions, two prominent approaches include the use of seeding devices containing settled coral recruits (Chamberland et al., 2017; Randall et al., 2021, 2023; Miller et al., 2022; Whitman et al., 2024) and the collection and deployment of wild coral spawning slicks (Heyward et al., 2002; dela Cruz and Harrison, 2017; 2020; Harrison et al., 2021). Seeding devices often involve controlled breeding and settlement of one or a few species (Chamberland et al., 2017; Randall et al., 2021, 2023; Whitman et al., 2024) to target high-value species, populations, and genotypes of interest, but cannot fully capture the extensive diversity found within broadcast-spawning communities, particularly on Indo-Pacific reefs, including the GBR. In contrast, the use of wild spawning slicks overcomes these limitations by collecting gametes and embryos from diverse coral assemblages and settling them on degraded reefs (Heyward et al., 2002; Doropoulos et al., 2019; Tabalanza et al., 2020). However, the taxonomic composition of these slicks has remained largely uncharacterized, which limits our understanding of the diversity of corals present in the slicks and highlights the risk that other potentially competitive broadcast spawner species (e.g., soft corals, anemones, and sponges) could be inadvertently captured and settled. This underscores the need for monitoring tools to ensure that restoration efforts are inclusive of diverse taxa and aligned to maintain coral ecosystem resilience, as well as for a better understanding of potential species bias within slicks.

Environmental DNA (eDNA) refers to genetic material obtained directly from the environment. Sequencing of eDNA is widely applied in terrestrial and marine ecosystems for biodiversity assessments (Willerslev et al., 2003; Thomsen et al., 2012; Everett and Park, 2018; Cilleros et al., 2019; Alexander et al., 2020; Dugal et al., 2022) and has been shown to produce coral biodiversity assessments comparable to traditional visual surveys (Dugal et al., 2022). Here, we leveraged coral eDNA sequencing to characterize the taxonomic diversity of corals and their relative abundances in natural spawning slicks. We first generated eDNA profiles of known slick mixes containing larvae of 10 coral species from the GBR to assess the accuracy of existing coral eDNA methodology when applied to coral slicks. Following this, we sequenced eDNA of wild slick samples collected from the Cooktown, Townsville, and Whitsunday Islands sectors of the GBR. We show that eDNA profiles accurately recapitulate the taxonomic composition of coral slicks, thus demonstrating the potential of incorporating eDNA assays into wild spawning slick collection activities to identify the diversity of corals in slicks and target coral species most in need of conservation.

This study revealed the community composition of coral spawning slicks for the first time and demonstrated the applicability of using eDNA metabarcoding technology for this purpose. Analysis using the ITS2 region (CoralITS2 and CoralITS2_acro assays) identified 191 ASVs comprising 9 families and 15 genera from wild-collected slick samples on the Great Barrier Reef. The detection capabilities of both assays were validated with samples of known taxonomic diversity, confirming that observed relative abundance values aligned with expected profiles. These findings support further development of eDNA metabarcoding as a cost-effective approach for surveying the community composition of wild-captured spawning slicks, providing valuable insights for monitoring and restoration efforts.

Across the Cooktown and Townsville wild slick collection sites, Acropora was consistently the most abundant genus detected using the Coral ITS2_acro assay, aligning with survey data that shows Acropora as one of the most, and often the most, abundant broadcast spawning hard coral genera in these regions during the collection years (Australian Institute of Marine Science Long Term Monitoring Program ¹ ). In contrast, slicks from the Whitsundays collection sites were dominated by non-acroporid hard corals, particularly members of the Merulinidae family, which is consistent with benthic hard coral composition in this region during the collection years. These results underscore the ability of eDNA to capture region-specific reproductive outputs that mirror known benthic assemblages.

Leveraging eDNA methods for monitoring the community composition of coral spawning slicks offers several significant advantages for understanding coral spawning patterns and coral restoration applications. Spawning slicks represent a critical life-history stage for corals, acting as a source of larval dispersal and recruitment that ultimately shapes reef population dynamics (Harrison and Wallace, 1990; Jones et al., 2009). Understanding the taxonomic composition of these slicks allows for improved monitoring of reproductive success, assessment of species-specific contributions, and evaluation of how environmental stressors may influence reproductive output. This study makes key advancements in this area by providing the first taxonomic characterization of wild-collected spawning slicks, validating the use of eDNA for slick monitoring, and highlighting the potential for applying this approach in restoration efforts.

eDNA enhances detection capabilities by allowing for the simultaneous identification of multiple taxa, including those that are difficult to observe directly, such as cryptic or rare species. This approach also enables differentiation between reef-building corals and other marine mass synchronized spawners—such as soft corals, anemones, worms, and sponges (see Babcock et al., 1986, 1992) that may have contributed to the spawning slick and might otherwise be mistaken for corals based on visual observations alone. Moreover, eDNA reduces the need for specialized training and extensive night diving to accurately record observations of coral spawning on the reef during nocturnal spawning periods (Harrison et al., 1984; Babcock et al., 1986). This makes it an accessible and scalable option for monitoring coral reefs, especially for sites that are expensive and/or difficult to reach, facilitating frequent and widespread surveys at a lower cost. Additionally, eDNA-based methods provide a valuable tool for tracking changes in spawning patterns in response to environmental stressors, such as bleaching events, and offer insights into the health and reproductive output of coral spawning communities over time. Furthermore, emerging eRNA approaches, such as those described by Ye et al. (2025), may offer additional benefits by providing more immediate assessments of metabolically active organisms. These methods could enhance monitoring efforts during the culture of slicks for restoration, offering insights into coral larval viability and early-stage development.

Despite its advantages, the resolution of eDNA-based identification is currently constrained by inherent challenges in taxonomic assignment. The ITS2 region captures both inter- and intra-specific variation, describing diversity within and across coral species. Historical and contemporary hybridization events contribute to the intragenomic variation observed in the ITS2 region (Coleman and Van Oppen, 2008; Chan et al., 2018, 2019; Lamb et al., 2024) and can lead to ambiguous or incorrect taxonomic assignments, resulting in errors in sequence-based identification (Willis et al., 2006). Additionally, multiple potential matches may arise, leading to lower resolution in taxonomic assignments. Here we observed a reduction in the overall explained diversity when resolving taxa from the family to the genus level, likely due to these complexities.

Taxonomic resolution is further constrained by limitations in available sequence databases, which may lack comprehensive or up-to-date references for coral taxa (Dugal et al., 2022). These database gaps introduce uncertainties in eDNA-based identifications, increase the likelihood of unresolved classifications, and result in trade-offs between detailed resolution (genus-level) and the completeness of diversity representation (family-level). To address these issues, future work could enhance reference databases with better-curated and updated taxonomy and/or include internal spiked datasets containing known samples to improve detection depth and classification accuracy (Dugal et al., 2022). Moreover, differences in the coral taxa recovered between the CoralITS2 and CoralITS2_acro assays underscore the necessity of integrating multiple or alternative DNA markers, especially to capture Acropora and non-Acropora taxa.

ITS2 community profiling could be biased by variation in DNA copy number and fragment length, with shorter amplicons preferentially amplified during PCR, and species with more ITS2 copies potentially overestimated. To account for this, mock communities composed of known coral taxa were included in this study. A Bayesian beta-binomial model was applied to compare observed and expected taxonomic compositions within these mock communities. The model showed no evidence that expected and recovered relative abundances differed. This quality control step lends support that the taxonomic profiles recovered from wild slick samples reflect true biological community composition rather than technical artifacts introduced during amplification or sequencing.

Here we used three technical replicates per sample of briefly mixed samples. This recommended approach (Ficetola et al., 2015; Fonseca, 2018) produced consistent averages in our curated mix, however, more variable relative abundance ratios were observed in the wild slick samples. We suspect this is due to biological sample heterogeneity as gametes and larvae may clump together, particularly for slicks collected shortly after spawning when not all gamete bundles have dissociated. Yet, we cannot exclude the inconsistencies in DNA extraction efficiency and PCR amplification as contributors to variation among technical replicates. In future studies, we recommend implementing a more thorough mixing process to fully homogenize samples and exploring the effect of various levels of replication in quantifying the abundance of taxa in wild samples.

To further enhance the robustness of future analyses, we suggest that biological replicates from the same spawning slick are also collected to provide more precise estimates of community composition. Pairing this information with comprehensive environmental data such as hydrodynamic models (Gouezo et al., 2025), water temperature, and other relevant conditions during spawning (Keith et al., 2016; Lin and Nozawa, 2023; Nozawa, 2012; van Woesik, 2010) and with the context of disturbance history (e.g. bleaching events) would enable a deeper understanding of how environmental factors influence community-level reproductive output. Resampling the same regions over multiple spawning seasons can also shed light on the trajectories of community recovery following bleaching events, where surviving corals are expected to experience reduced fecundity (Ward et al., 2002). Once temporal variability of taxa in slicks at a given reef site has been characterized, shifts in the relative abundance of taxa within spawning slicks can be linked to changes on the reef benthos; these data would provide important insights into the long-term dynamics of coral populations and assemblages.

Importantly, sampling limitations arise from the timing of surface-collected spawning slick material, which may not capture the full diversity of coral taxa. For example, certain groups, such as taxa that release negatively or neutrally buoyant gamete bundles, and species that spawn during the day rather than at night, and at different times after sunset or on different lunar phases may be missed (Babcock et al., 1986; Harrison and Wallace, 1990; Harrison, 2024a). The timing of sampling relative to the full moon also influences what species may be captured due to differences in reproductive timing across lunar phases and months (Baird et al., 2021). Furthermore, brooding coral species, which release fully formed swimming larvae will not be captured in spawning slicks. Improving the temporal resolution of eDNA sampling around the timing of spawning to collect over multiple days and nights around the full moon would increase our understanding of the diversity of species reproducing during these periods (Ip et al., 2023).

Lastly, in the context of restoration activities that use wild slicks as material for restoring degraded reefs (Heyward et al., 2002; Harrison et al., 2021; Banaszak et al., 2023; Harrison, 2024a), the current processing times for PCR and sequencing of eDNA samples can exceed the time available before settlement begins. Understanding the species composition and diversity within larval cohorts is essential for predicting settlement and recruitment dynamics. More importantly, identifying potential contamination by soft corals, which are major competitors for reef-building corals, is crucial before larvae are introduced to target reef sites. A potential solution to this challenge is the use of lateral flow assay (LFA) dipstick methods, as demonstrated by Doyle and Uthicke (2020). LFA enables rapid, field-based detection of eDNA within minutes, rather than the days required for traditional sequencing-based workflows. Incorporating LFA into spawning slick monitoring could facilitate real-time taxa verification, ensuring that the collected spawn produces larval cohorts that are appropriate for larval restoration, including target taxa before larval enhancement occurs.

This study provides significant advances in our understanding of the taxonomic composition of wild coral spawning slicks and demonstrates the feasibility of using eDNA metabarcoding for their characterization. While current limitations in taxonomic resolution and database completeness remain challenges, the integration of additional markers, database improvements, and methodological refinements can further enhance eDNA-based monitoring. The successful validation of this approach in both mock communities and wild slicks establishes eDNA as a valuable tool for assessing coral community reproductive output, monitoring ecosystem health, and informing restoration strategies.