Sponges are known for hosting diverse communities of microbial symbionts, but despite persistent interest in the sponge microbiome, most research has targeted marine sponges; freshwater sponges have been the focus of less than a dozen studies. Here, we used 16 S rRNA gene amplicon sequencing and shotgun metagenomics to characterize the microbiome of the freshwater sponge Ephydatia muelleri and identify potential indicators of sponge-microbe mutualism. Using samples collected from the Sooke, Nanaimo, and Cowichan Rivers on Vancouver Island, British Columbia, we show that the E. muelleri microbiome is distinct from the ambient water and adjacent biofilms and is dominated by Sediminibacterium, Comamonas, and unclassified Rhodospirillales. We also observed phylotype-level differences in sponge microbiome taxonomic composition among different rivers. These differences were not reflected in the ambient water, suggesting that other environmental or host-specific factors may drive the observed geographic variation. Shotgun metagenomes and metagenome-assembled genomes further revealed that freshwater sponge-associated bacteria share many genomic similarities with marine sponge microbiota, including an abundance of defense-related proteins (CRISPR, restriction-modification systems, and transposases) and genes for vitamin B12 production. Overall, our results provide foundational information on the composition and function of freshwater sponge-associated microbes, which represent an important yet underappreciated component of the global sponge microbiome.

海绵以拥有不同的微生物共生群落而闻名，但尽管人们对海绵微生物组一直很感兴趣，但大多数研究都针对海绵。淡水海绵一直是不到十二项研究的重点。在这里，我们使用 16 S rRNA 基因扩增子测序和鸟枪法宏基因组学来表征淡水海绵Ephydatia muelleri的微生物组，并确定海绵-微生物共生的潜在指标。使用从不列颠哥伦比亚省温哥华岛的 Sooke、Nanaimo 和 Cowichan 河收集的样本，我们表明E. muelleri微生物组不同于环境水和相邻的生物膜，并且以Sediminibacterium、Comamonas为主和未分类的红螺菌目。我们还观察了不同河流之间海绵微生物组分类组成的系统型水平差异。这些差异并未反映在环境水中，这表明其他环境或宿主特定因素可能会推动观察到的地理变化。鸟枪宏基因组和宏基因组组装基因组进一步揭示了淡水海绵相关细菌与海绵微生物群具有许多基因组相似性，包括丰富的防御相关蛋白（CRISPR、限制性修饰系统和转座酶）和维生素 B12 生产基因。总体而言，我们的研究结果提供了有关淡水海绵相关微生物的组成和功能的基础信息，这些微生物代表了全球海绵微生物组中一个重要但未被充分认识的组成部分。