**2. Results and Discussion**

Two new sponge-associated cyanobacterial draft genomes belonging to the newly described *Leptothoe* genus were recovered from the isolates TAU-MAC 1615 and 1115 (Figure S1, Table 1). To date, most microbial isolates derived from sponges have been mainly affiliated with Proteobacteria [7,25,26]. A limited number of cyanobacterial strains have been isolated from sponges [24]. Indeed, only one genome assembly from a spongeassociated cyanobacterial strain is available in public databases for a *Myxosarcina*-like cyanobacterium isolated from *Terpios hoshinota* [27]. Recently, a considerable number of novel genera and species of cyanobacteria were found to be associated with Aegean Sea sponges [21,22].

The size of the assembled sponge-associated *Leptothoe* genomes ranged between 4.06 Mb for *Le. kymatousa* TAU-MAC 1615 and 5.24 Mb for *Le. spongobia* TAU-MAC 1115, with G+C contents of 50.5% and 47.3%, respectively (Figure S1, Table 1). Table 1 summarizes the basic genome features of sponge-associated *Leptothoe* genomes, *Leptothoe* genomes associated with other macro-organisms (isolated from turfs), and free-living *Leptothoe* genomes used for comparative genomic analyses in this study after verification of genome-wide relatedness using phylogenomic approaches (Figure 1). The draft assemblies of sponge-associated strains showed the smallest genome sizes (Table 1). Although the genome coverage of sponge-associated strains was high (approximately ×450 and ×90 for TAU-MAC 1615 and TAU-MAC 1115, respectively), the genome completeness estimated by the marker sets used by CheckM (encode essential functions) was substantially complete (≈70%) (Table 1). The GC% contents of the strains were quite similar, varying from 47.3% (TAU-MAC 1115, CCMR0081) to 50.5% (TAU-MAC 1615). Concerning the number of scaffolds, the SIO3F4 metagenome showed the highest number (1508) and the lowest N50 (7412). The number of coding sequences (CDSs) of sponge-associated strains was considerably lower (3638–4790) than the rest of the *Leptothoe* strains, while the amount of CDSs categorized in RAST subsystems (13–17%) was quite similar (Table 1). The remaining 84–87% of CDSs were not classified in any subsystem.


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> **Table 1.** Genome properties and quality metrics of *Leptothoe* strains used for comparative

> genomic analyses. Subsystem statistics were obtained using the RAST server [28]

**Figure 1.** Maximum likelihood phylogenomic tree based on 120 conserved proteins in cyanobacterial genomes. The sponge-associated *Leptothoe* strains sequenced in the present study are shown in bold. Accession numbers of the sequences are presented in parentheses.

> The phylogenomic reconstruction based on 120 bacterial single-copy conserved marker genes indicated that our two sponge-associated strains were placed inside *Leptothoe* clade and grouped together with 5 of the 35 public cyanobacterial genomes identified as *Leptolyngbya* or Leptolyngbyaceae (Figure 1). *Leptothoe* is a thin marine filamentous cyanobacterium with benthic lifestyle being epilithic, epizoic, or epiphytic, recently separated from the polyphyletic filamentous genus *Leptolynbya* by a taxonomic re-evaluation [21].

> The remaining 30 cyanobacteria, identified as *Leptolyngbya* or Leptolyngbyaceae, were grouped in different clades spread across a variety of cyanobacteria orders (Figure 1). This analysis further supported the evolutionary divergence of *Leptothoe* from *Leptolyngbya* sensu

stricto and other thin filamentous genera separated from *Leptolyngbya* such as *Nodosilinea* and *Elainella* (Figure 1). The analysis also supported the evolutionary distinction of the two sponge-associated strains, TAU-MAC 1115 and TAU-MAC 1615, which belong to different species *Le. spongobia* and *Le. kymatousa* (Figure 1). The free-living strains PCC 7375 and Heron Island J were placed separately within a subclade along with two strains (CCMR0081 and CCMR0081) isolated from turf samples growing over corals, while the metagenome-assembled genome SIO3F4 isolated from turfs assembles in Panama was placed in the same subclade with *Le. spongobia* TAU-MAC 1115 (Figure 1). Further, our phylogenomic analysis showed that the members of genus *Leptothoe* (seven in total) were not grouped on the basis of the isolation source (host-associated or free-living), indicating the lack of host-specific clustering. Other marine bacterial genera, including free-living and host-associated members such as *Pseudovibrio*, have shown a lack of host-specific clustering [30]. However, the sponge-associated *Le. kymatousa* TAU-MAC 1615 was placed separately in the phylogenomic tree, likely suggesting its independent evolution from other host-associated strains of the genus. These results might indicate distinct patterns of evolution among members of genus *Leptothoe*.
