Research on symbiotic microbial communities of Korean marine sponges remains considerably limited, with existing studies primarily confined to historical investigations conducted over a decade ago using pyrosequencing methods on a restricted number of sponge species collected exclusively from Jeju and Chuja Islands9,13. While our study shares one overlapping species (Cliona celata) with previous Korean research, the distinct geographical location (eastern coastal waters of Korea versus southern islands), different target region (full-length 16S rRNA versus V3-V4 region), and analytical approach (ASV-based versus OTU-based methods) result in substantially different microbial community profiles. Previous studies reported that over 90% of sponge-associated microorganisms belonged to the phylum Proteobacteria9, whereas our results reveal a far more diverse range of phyla and markedly distinct symbiotic microbial patterns for each sponge species, indicating a profound difference from earlier findings. In the present study, eight sponge specimens collected from Korea’s eastern coastline were analyzed using contemporary, high-resolution microbial community analysis techniques, including advanced databases and full-length 16S rRNA gene sequencing, to maximize analytical accuracy and resolution. The use of full-length 16S rRNA sequencing combined with ASV-based analysis represents a significant methodological advancement in sponge microbiome research. This approach provided superior taxonomic resolution compared to traditional OTU-based methods, enabling the detection of fine-scale diversity patterns that would have been missed with shorter sequencing reads.
Diversity of collected sponges and contextualizing HMA-LMA paradigms
The sponge specimens collected for this study represent a broad range of genera and families characteristic of Korean coastal ecosystems: Halichondria dokdoensis (SP1), Mycale sp. (SP3), Geodia reniformis (SP8), Cliona celata (SP21), Halichondria sp. (SP22, SP23), Latrunculia ikematsui (SP32), and Mycale neunggulensis (SP38). This sampling includes both cosmopolitan species, such as Cliona celata and Geodia reniformis, which are frequently featured in global sponge–microbiome research, as well as regionally endemic taxa like Halichondria dokdoensis and Mycale neunggulensis, which have been rarely studied to date. By covering such a diverse taxonomic spectrum, our sampling enables the assessment of both host-specific and environmental factors that shape sponge microbiomes in South Korean temperate coastal waters.
Because the distinction between high microbial abundance (HMA) and low microbial abundance (LMA) sponges is a central paradigm in sponge microbiome research, reflecting fundamentally different holobiont ecologies and functions, we also sought to classify our Korean specimens within this framework. Although we did not directly quantify microbial abundance to formally assign HMA or LMA status, the predominance of Proteobacteria and limited representation of typical HMA-associated taxa in Cliona celata, Halichondria sp. (SP23), Latrunculia ikematsui, and Mycale neunggulensis suggest that these sponges likely belong to the LMA category. In contrast, Halichondria dokdoensis, Mycale sp., Geodia reniformis, Halichondria sp. (SP22) exhibited characteristics typically associated with HMA sponges, such as a lower proportion of Proteobacteria and greater microbial diversity as indicated by Shannon diversity indices, consistent with previous observations14,15.
Distinctive proteobacterial assemblages and symbiotic specificity in sponges
The overwhelming dominance of Proteobacteria, especially Alpha and Gammaproteobacteria, across most sponge samples reflects a global trend in marine sponge microbiomes16,17. In particular, the near-exclusive presence of Gammaproteobacteria in Cliona celata (99.6%) and Latrunculia ikematsui (87.8%) underscores highly specialized host–symbiont relationships, consistent with reports that certain Gammaproteobacterial lineages (e.g., the EC94 clade, now Candidatus Tethybacterales) have evolved to thrive within sponge hosts18. Specifically, one of the most striking features of the microbiome is the dominance of a previously uncharacterized Gammaproteobacterial lineage within Cliona celata. QIIME 2 classification revealed that the single most abundant symbiont in this sponge belongs to the Gammaproteobacteria, a novel order not yet described in existing taxonomies, and is further placed within the Burkholderiales, a group that appears to represent an entirely new family-level clade. Following Gammaproteobacteria, most sponge species harbored abundant Alphaproteobacteria; intriguingly, however, the two sponges, Cliona celata (99.6%) and Latrunculia ikematsui (87.8%), overwhelmingly dominated by Gammaproteobacteria lacked any detectable Alphaproteobacteria. Cases in which certain sponge species are highly specifically associated with Gammaproteobacteria, while other proteobacterial lineages are virtually absent, provide compelling evidence for sponge–microbe coevolution and symbiotic specificity19. Within these dominant classes, functional diversity is apparent in the minimal taxonomic overlap at finer scales, suggesting evolutionary radiation among sponge-associated bacteria20. For instance, the Gammaproteobacterial orders present in C. celata and L. ikematsui share less than 5% of their ASVs, indicating host-specific metabolic adaptations.
The dominance of the EC94 lineage in both Latrunculia ikematsui and Mycale neunggulensis (49.3 and 56.4%, respectively) represents a fascinating example of convergent evolution in sponge-associated bacteria21. This pattern suggests that the EC94 lineage has evolved specific adaptations that make it particularly suited for certain sponge host environments, possibly related to nutrient processing or host defense functions18,22.
No ASVs were shared between seawater and sponge samples, highlighting the distinctiveness and specificity of sponge-associated microbiomes. This pattern aligns with established paradigms in sponge microbiology, each species harbors its own dominant bacterial classes with little interspecies overlap, demonstrating that host identity rather than ambient environment shapes these communities. Both Bray–Curtis and weighted UniFrac analyses further confirm clear clustering by host taxonomy and separation from seawater communities, reinforcing the idea that sponges actively select and maintain particular microbial partners likely through complex immune-recognition mechanisms and metabolic complementarity23.
Genus-level associations and Entotheonella symbiosis in Halichondria sponges
The clustering of Halichondria specimens (SP1, SP22, and SP23) represents one of the most compelling findings of this study, demonstrating both the stability and specificity of genus-level associations in sponge microbiomes. The presence of both Entotheonellaceae and Endozoicomonadaceae exclusively in the three Halichondria samples suggests that these bacterial families form unique and metabolically intimate associations with sponges of this genus. In particular, in Halichondria dokdoensis (SP1), genome-resolved metagenomic analysis of the recovered Entotheonella MAG revealed the potential to produce a wide range of secondary metabolites, including bioactive halicylindramides12. This finding supports the notion that these microorganisms make significant metabolic contributions to their host sponges. The morphological differences observed between Halichondria dokdoensis and the morphologically similar Halichondria sp. SP22 and SP23, despite their shared microbial taxa, highlight the complex interplay between host genetics, morphology, and microbiome composition. This pattern suggests that while host phylogeny is a primary driver of microbiome composition, additional factors such as local environmental conditions or host developmental stage may fine-tune these associations24.
In a previous study of Halichondria dokdoensis, Candidatus Entotheonella halido comprised 41% of the total microbial community, indicating that this symbiont may play a crucial role in the biological functions of its host. Its predominant presence in Halichondria sponges mirrors the well-characterized symbiotic association between Theonella swinhoei and Entotheonella25, suggesting that members of the genus Entotheonella have co-adapted with specific sponge genera. Notably, Entotheonella was also detected at 1.3% abundance in Geodia reniformis (SP8), pointing to a possible expansion of its host range and challenging the previous notion that Entotheonella is restricted to Theonella species.
Analysis of ASVs assigned to Entotheonella from both Halichondria and Geodia sponge samples (Table 2) reveals substantial phylogenetic diversity within the genus. While Candidatus Entotheonella halido predominates in H. dokdoensis, other Halichondria specimens (SP22, SP23) primarily harbor distinct, sponge-derived Entotheonella lineages (e.g., AY897123.1). The Entotheonella ASVs identified in G. reniformis are also closely related to other sponge-associated Entotheonella taxa (OX639990.1, FJ900569.1) documented in public databases. This intrageneric diversity suggests that Entotheonella has undergone genetic differentiation in response to adaptation to various host environments.
The genus Entotheonella possesses mixotrophic metabolic capabilities enabling it to construct complex metabolic networks through the utilization of diverse carbon sources, sulfate reduction, and anaerobic respiration26. These diverse metabolic capabilities make crucial contributions to nutrient cycling and energy supply within sponges, supporting the ecological success of their hosts. Moreover, the high genetic uniqueness of biosynthetic gene clusters (BGCs) harbored by each Entotheonella variant (with almost no overlapping BGC patterns suggests that they perform distinct biochemical functions specialized for their respective host environments27. This demonstrates that sponge-microbe symbioses transcend mere spatial coexistence to achieve functional integration as holobiont systems.
Depth-driven symbiotic specialization in Geodia reniformis microbiomes
Geodia reniformis (SP8), collected from a relatively deeper habitat (35 m) compared to the other sampled sponges, exemplifies pronounced host- and habitat-specificity of sponge-associated microbiomes. Microbial community analyses such as Bray–Curtis and weighted UniFrac demonstrate that SP8 harbors a highly distinct symbiotic assemblage, dominated by unique phylogenetic clades, including SAR202, PAUC34f, Nitrospinaceae, UBA10353_marine_group, and . Intriguingly, several of these lineages, particularly SAR202 (Chloroflexi) and PAUC34f, are widely recognized as characteristic members of deep-sea or low-light, low-nutrient marine environments28. SAR202, for example, is known for its metabolic versatility, including the degradation of recalcitrant dissolved organic carbon under oligotrophic and low-oxygen conditions29,30, while PAUC34f is associated with unique carbon and sulfur cycling capabilities, also favoring deeper or more extreme environments31.
The prevalence of these metabolically versatile and environmentally adapted taxa in G. reniformis (SP8), in contrast to their absence or low abundance in sponges from shallower waters, strongly suggests that environmental filtering associated with depth has played a key role in structuring the sponge’s symbiotic microbiome32. Such specific associations may confer functional advantages to the host, such as enhanced nutrient transformation or access to otherwise inaccessible carbon and sulfur sources, which are capabilities that could underpin G. reniformis’s ecological success in deeper habitats33. Moreover, the enrichment of lineages like Nitrospinaceae, which are relevant in nitrogen and sulfur cycling34, and the unique presence of Dadabacteriales35 and UBA10353_marine_group36, further underline the distinct metabolic landscape afforded by these symbionts37, possibly enabling the sponge holobiont to better exploit deep-sea ecological niches. The clear divergence of G. reniformis’s microbial community from those of other sympatric sponge species echoes the broader view that sponge-microbe symbioses are shaped by a combination of host selection and environmental pressures, resulting in “nested ecosystems” defined by both phylogenetic and ecological specificity38. Thus, the unique and functionally rich microbiome of G. reniformis (SP8), strongly linked to its deeper collection site, underscores the evolutionary and ecological significance of depth- and environment-driven symbiotic specialization in sponges.
Microbial contributions to nutrient cycling within sponge holobionts
The microbial community associated with sponges (Fig. S2) includes a diverse array of taxa that play critical roles in nutrient cycling, maintaining the metabolic balance within the holobiont. Photosynthetic cyanobacteria, such as members of Prochloron, contribute organic carbon through photosynthesis, serving as primary producers and energy sources for the host39. Bacteroidota, including Chitinophagales and Cryomorphaceae, aid in the degradation of complex organic matter, facilitating carbon and nitrogen recycling40, a function that is also performed by Dadabacteriales35. Key groups involved in nitrogen transformation include Alphaproteobacteria like Rhodobacterales (Amylibacter)41 and Parvibaculales (PS1_clade)42, which participate in ammonia oxidation and denitrification, thus regulating nitrogen availability and preventing accumulation of nitrogenous wastes43. Proteobacteria, especially Burkholderiales, further contribute to denitrification and nitrogen removal44, mitigating nitrogen overload and greenhouse gas emissions. Also, deep-sea-adapted clades such as SAR202 (Chloroflexi) and Entotheonella are involved in breaking down recalcitrant organic compounds and producing bioactive secondary metabolites, which may influence host defense and chemical interactions12,29. Lastly, Endozoicomonas and other uncultured lineages may facilitate host-microbe communication and metabolic exchange, supporting sponge resilience in fluctuating environments45. Collectively, these microbial taxa not only sustain nutrient cycling but also contribute to the ecological success and chemical defense strategies of the sponge holobiont, emphasizing their fundamental role in marine nutrient turnover and host health.
In conclusion, this study of eight Demospongiae specimens from Chuksan Harbor illuminates the rich sponge biodiversity and host-specific microbial assemblages in South Korea’s coastal waters. Our findings reinforce that sponge–microbe partnerships are ancient, co-evolved systems under strong evolutionary pressures, with both vertically and horizontally transmitted symbionts enabling adaptive flexibility. The striking functional diversity, particularly in nitrogen and sulfur cycling, positions sponge holobionts as nested ecosystems driving key marine biogeochemical processes. Furthermore, the biosynthetic potential of these specialized microbiomes, including novel lineages like Entotheonella with unique secondary-metabolite gene clusters, demostrates their promise as genetic resources for natural-product discovery and ecological restoration. Together, these insights establish a foundational framework for harnessing Korean sponges in ecosystem-based resource management for future marine biotechnology.