**4. Conclusions**

Here, we report the first two draft genomes of sponge-associated filamentous Synechococcales. Our comparative genomic analyses revealed symbiosis signatures of spongeassociated *Leptothoe* such as reduction of their gene content, functional dissimilarities to other host-associated and free-living members of *Leptothoe*, and presence of ELP repeats. Moreover, genome-mining analysis revealed the unique biosynthetic potential of *Leptothoe* with more than 100 natural product BGCs, emerging as an unexplored source of potent marine natural products. Additional studies are needed to identify and characterize these produced compounds. Future research to address the actual functioning of sponge-associated cyanobacteria should include metagenomics, metabolomics, and metatranscriptomics.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/md19060298/s1, Table S1. Number of genes in subsystems (obtained by RAST server and SEED tool) per *Leptothoe* strain. Table S2. Biosynthetic gene clusters per *Leptothoe* genome (antiSMASH results). Table S3. Compounds produced by *Leptothoe* cyanobacteria extracted from the CyanoMetDB (Jones et al., 2020). Figure S1. Circular view of the genomes of two sponge-associated *Leptothoe* species, generated with CGview (Stothard and Wishart, 2005). Circles from interior to exterior represent GC content and GC skew. Blue circles denote the coding sequences on forward and reverse strands. Figure S2. Average amino acid identity heatmap of *Leptothoe* genomes. Figure S3. Distribution of the categories of functional genes present in seven *Leptothoe* strains on the basis of the cluster of orthologous groups (COGs) of proteins. The alphabetic code for the COG categories is as follows: C, energy production and conversion; D, cell cycle control, cell division, chromosome partitioning; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; J, translation, ribosomal structure, and biogenesis; K, transcription; L, replication, recombination, and repair; M, cell wall/membrane/envelope biogenesis; N, cell motility; O, posttranslational modification, protein turnover, chaperones; P, inorganic ion transport and metabolism; Q, secondary metabolite biosynthesis, transport, and catabolism; R, general function prediction only; S, function unknown; T, signal transduction mechanisms; U, intracellular trafficking, secretion, and vesicular transport; V, defense mechanisms.

**Author Contributions:** Conceptualization, D.K. and S.G.; methodology, D.K., R.V.P., and D.P.F.; validation, D.K., D.P.F., and S.G.; formal analysis, D.K. and R.V.P.; investigation, D.K.; resources, S.G., D.P.F., and K.S.; data curation, D.K.; writing—original draft preparation, D.K.; writing—review and editing, all authors.; visualization, D.K.; supervision, S.G. and D.P.F.; project administration, S.G.; funding acquisition, S.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the General Secretariat for Research and Technology (GSRT) and the Hellenic Foundation for Research and Innovation (HFRI), grant number 938 (http://www. elidek.gr/en/homepage/, accessed on 12 March 2021) to D.K. S.G. would like to acknowledge co-funding of this work by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH—CREATE— INNOVATE (project code: T1EDK-02681). R.V.P. received funding from the Doctoral Program in Microbiology and Biotechnology from the University of Helsinki.

**Data Availability Statement:** The *Leptothoe kymatousa* TAU-MAC 1615 Whole Genome project was deposited at DDBJ/ENA/GenBank under the accession number JADOER000000000. The *Leptothoe spongobia* TAU-MAC 1115 Whole Genome Shotgun project was deposited at DDBJ/ENA/GenBank under the accession number JADOES000000000.

**Acknowledgments:** We thank Lyudmila Saari for her kind assistance in purifying the spongeassociated strains.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
