*2.1. Genomic Hallmarks of a Symbiotic Lifestyle*

#### 2.1.1. Genome Reduction of Sponge-Associate Strains

*Leptothoe kymatousa* TAU-MAC 1615 and *Leptothoe spongobia* TAU-MAC 1115 are not obligate sponge symbionts and can sustain growth in pure cultures. However, these two strains showed considerable smaller genome size (Figure 2a). This pattern of genome reduction was also observed for the obligate sponge cyanobiont *Candidatus Synechococcus spongiarum* [9,31]. Genome reduction is widely observed mainly in obligate bacterial symbionts [11], although signs of genome reduction have been identified in facultative symbionts too [32]. Both cyanobionts are slow growing in culture conditions, and thus they could be considered as facultative symbionts that are generally not essential for the host's survival, although they may contribute to host fitness. Recently evolved symbionts, which have undergone genome reduction, are generally characterized by a proliferation of pseudogenes and mobile elements [11]. The two *Leptothoe* sponge-associated genomes reported here showed lower numbers of those traits compared to the other members of the genus (Table 1); on the basis of these data, we hypothesized that sponges did not recently acquire the *Leptothoe* symbionts.

The number of coding sequences of the sponge-associated strains were also half compared to the remaining strains (Figure 2b). An overview of different subsystems obtained using the RAST and SEED annotation showed that the sponge-associated *Leptothoe* strains harbored considerably fewer genes related to several essential functions (Figure 2c, Table S1). Remarkably, *Le. kymatousa* TAU-MAC 1615 and *Le. spongobia* TAU-MAC 1115 have undergone extreme reduction of the number of genes encoding for cofactors, vitamins, prosthetic groups, pigments, proteins, and amino acid biosynthesis (Figure 2c, Table S1). Sponge-associated strains may be dependent on co-occurring microbes for lost metabolic capacities [33]. A complete loss of genes involved in DNA recombination and fewer genes involved in DNA repair were observed in their genomes, while they retained approximately the same number of genes for DNA replication as the rest of the *Leptothoe* genomes. The sponge-associated strains have lost several genes involved in potassium, sulfur, and phosphorus metabolism (Table S1). They were also found to have fewer genes related to stress response, mainly genes coding for antioxidant enzymes, as well as fewer genes responsible for the biosynthesis of capsular polysaccharide (CPS) and extracellular polysaccharides (Table S1). Our *Leptothoe* genomes were characterized by a complete or near complete lack of chemotaxis and motility traits, which are among the most depleted functions in sponge-associated bacteria genomes [34].

**Figure 2.** Comparison of genome size (**a**), number of coding sequences (**b**), and number of genes in each RAST subsystem [28] (**c**) of the analyzed *Leptothoe* genomes.

The number of COGs (clusters of orthologous groups of proteins) per genome ranged from 6594 in free-living PCC 7375 strain to 2925 in the sponge-associated TAU-MAC 1615 strain. *Leptothoe* strains shared > 80% average amino acid identity (AAI) (Figure S2), while only 1602 COG entries that account for approximately half (for sponge-associated strains) or even lower (≈25% for the rest of the strains) of the total number of COG entries were common in all genomes (Figure 3), likely suggesting specific adaptations for different lifestyles and for different symbiont types. Genome streamlining process forces adaptations of cyanobacterial genomes to specific niches that are also reflected in their different functional capacities [12]. Previously, sponge-associated and free-living *Synechococcus* genomes have also been found to share half of their total number of COGs, suggesting variability and specific adaptations of each member of the genus [9]. Comparisons based on COG categories among the sponge-associated, coral, and/or macroalgae-associated and free-living *Leptothoe* revealed a relative lower abundance of genes belonging to the different functional categories in the sponge-associated strains. This analysis identified an overrepresentation of functional categories 'J' (translation, ribosomal structure, and biogenesis), 'L' (replication, recombination, and repair), 'O' (posttranslational modification, protein turnover, chaperones), and 'P' (inorganic ion transport and metabolism) and was observed in the genomes of the coral and/or macroalgae-associated strains (Figure S3). A uniform distribution of genes belonging to COG functional categories between the two sponge-associated strains was detected.

**Figure 3.** Analysis of homologous protein clusters in the genomes of *Le. kymatousa* TAU-MAC 1615, *Le. spongobia* TAU-MAC 1115, Leptolyngbyaceae sp. CCMR0081, Leptolyngbyaceae sp. CCMR0082, *Leptolyngbya* sp. SIO3F4 *Leptolyngbya* sp. PCC 7375, and *Leptolyngbya* sp. Hero Island J.

The sponge-associated strains possess biosynthetic gene clusters (BGCs) encoding for natural products despite undergoing genome reduction (Figure 4a,b). It has been proposed that maintenance of such clusters sustains the symbiotic interaction [35]. Natural product BGCs were previously detected in another cyanobacterial sponge symbiont, *Hormoscilla spongilae,* suggesting that these biosynthetic capacities to produce metabolically expensive natural products may contribute to host fitness [36].

#### 2.1.2. Eukaryotic-Like Protein (ELP)-Encoding Genes

Genes encoding eukaryotic-like proteins (ELPs) such as ankyrin-like domains (ANKs), tetratricopeptide repeats (TPRs), Leucine-rich repeat (LRR) protein, and pyrroloquinoline quinone (PQQ) were detected in the two sponge-associated *Leptothoe* strains (Table 2); ELPs are often detected in facultative or obligate symbionts and play a key role in the modulation of cellular protein–protein interactions [37,38]. In particular, abundance of ANKs seems to be a major genomic feature of sponge symbionts [6,26] as they have been thought to be involved in preventing phagocytosis by the sponge host. Indeed, the role of ANKs in modulating the amoebal phagocytosis in sponge symbionts was experimentally validated [39]. Genome analyses of sponge-associated Alphaproteobacteria [7,26,30], Deltaproteobacteria [40], and Poribacteria [41] have revealed the presence of ELPs, as well as the particular abundance of ANKs. The obligate sponge symbionts *Hormoscilla spongeliae* and *Candidatus Synechococcus spongiarum* had a great number of ELP repeats, while different free-living cyanobacteria taxa such as *Nodosilinea*, *Leptolyngbya*, *Synechococcus*, *Prochlorococcus*, and *Cyanobium gracile* almost lacked ANK domains, the typical genomic signature of sponge symbionts (Table 2). We also detected the different ELP types in varying proportions in other host-associated and free-living members of the genus *Leptothoe*. Similarly, ELPs have been previously detected in host-associated and free-living members of Alphaproteobacteria, likely suggesting the ability of strains to infect a different range of marine hosts and attach to various marine niches [26,30].

**Figure 4.** Average nucleotide identity heatmap (**a**) and composition of BGCs identified in *Leptothoe* genomes (**b**) and composition of BGCs identified in marine benthic filamentous Oscillatoriales (**c**). The absolute number of BGCs per strain assigned to each BGC class is shown. Triangle, sponge associated strains; polygon, coral and/or macroalgae-associated strains; circle, free-living strains.
