Genomic Analysis of a New Freshwater Cyanophage Lbo240-yong1 Suggests a New Taxonomic Family of Bacteriophages

A worldwide ecological issue, cyanobacterial blooms in marine and freshwater have caused enormous losses in both the economy and the environment. Virulent cyanophages—specifically, infecting and lysing cyanobacteria—are key ecological factors involved in limiting the overall extent of the population development of cyanobacteria. Over the past three decades, reports have mainly focused on marine Prochlorococcus and Synechococcus cyanophages, while information on freshwater cyanophages remained largely unknown. In this study, a novel freshwater cyanophage, named Lbo240-yong1, was isolated via the double-layer agar plate method using Leptolyngbya boryana FACHB-240 as a host. Transmission electron microscopy observation illustrated the icosahedral head (50 ± 5 nm in diameter) and short tail (20 ± 5 nm in length) of Lbo240-yong1. Experimental infection against 37 cyanobacterial strains revealed that host-strain-specific Lbo240-yong1 could only lyse FACHB-240. The complete genome of Lbo240-yong1 is a double-stranded DNA of 39,740 bp with a G+C content of 51.99%, and it harbors 44 predicted open reading frames (ORFs). A Lbo240-yong1 ORF shared the highest identity with a gene of a filamentous cyanobacterium, hinting at a gene exchange between the cyanophage and cyanobacteria. A BLASTn search illustrated that Lbo240-yong1 had the highest sequence similarity with the Phormidium cyanophage Pf-WMP4 (89.67% identity, 84% query coverage). In the proteomic tree based on genome-wide sequence similarities, Lbo240-yong1, three Phormidium cyanophages (Pf-WMP4, Pf-WMP3, and PP), one Anabaena phage (A-4L), and one unclassified Arthronema cyanophage (Aa-TR020) formed a monophyletic group that was more deeply diverging than several other families. Pf-WMP4 is the only member of the independent genus Wumpquatrovirus that belongs to the Caudovircetes class. Pf-WMP3 and PP formed the independent genus Wumptrevirus. Anabaena phage A-4L is the only member of the independent Kozyakovvirus genus. The six cyanopodoviruses share similar gene arrangements. Eight core genes were found in them. We propose, here, to set up a new taxonomic family comprising the six freshwater cyanopodoviruses infecting filamentous cyanobacteria. This study enriched the field’s knowledge of freshwater cyanophages.


Introduction
With the rapid development of industry and agriculture in recent decades, serious anthropogenic eutrophication and mass developments of cyanobacteria in water, called "water blooms" or "cyanobacterial blooms", have become a common occurrence worldwide [1][2][3]. Water blooms are frequently associated with cyanotoxins, posing a health hazard to other aquatic organisms and drinking waters [4,5]. Planktonic viruses, especially cyanobacterial viruses (cyanophage), are important aquatic ecological factors involving the Here, we isolated a new lytic Leptolyngbya cyanophage, Lbo240-yong1, from freshwater using L.boryana FACHB-240 as the indicator host. The Podovirus-like cyanophage Lbo240-yong1 has a narrow host range. Its genome was sequenced and analyzed, and 92.05% of the genome was predicted to be comprised of coding sequences. Bioinformatics analysis suggested that it is justified to create a new taxonomic family comprising six Podovirus-like cyanophages infecting filamentous freshwater cyanobacteria, which share at least eight core genes (DNA polymerase, DNA primase/helicase, capsid protein, tail protein, tail tubular protein A, tail tubular protein B, murein hydrolase activator, and terminase large subunit).

Isolation of Cyanophages
The surface water sample was collected from Lake Sunhu (North latitude, 29.982345; East longitude, 121.502455) of Ningbo, Zhejiang province, People's Republic of China, on 20 November 2021. The water sample was centrifuged (12,000× g, 15 min, 4 • C). The supernatant was filtered successively through medium-speed filter paper and 0.45 µm and 0.22 µm polyethersulfone filters (ANPEL Laboratory Technologies, Shanghai, China; product no.14541871). Then, 30 mL of the filtered supernatant of the water sample, 12 mL of a 5 × BG11 medium, and 30 mL of L. boryana FACHB-240 (OD 680 ≈ 0.6) were mixed in a conical flask. The mixtures were cultured in a light incubator under a light: dark cycle of 12: 12 h with a constant illumination of 40 µmol photons m −2 s −1 at 25 • C. The yellowed culture was centrifuged (8000× g, 20 min, 4 • C). The supernatant was co-cultured with fresh L.boryanum FACHB-240 again. The above steps were repeated three times. The double-layer agar method was used to isolate cyanophages with a little modification [22,23]. Lysates were centrifuged (8000× g, 20 min, 4 • C), filtered through 0.45 µm and 0.22 µm polyethersulfone filters, and diluted with BG11 (10 −1 -10 −9 ). Each 200 µL of dilution was mixed with 1.8 mL of concentrated (1:15) FACHB-240 cultures during the logarithmic phase, incubated in a light incubator for 30 min, and then mixed rapidly with 10 mL of molten BG11 agar medium (0.7% agar, pre-incubated at 42 • C), spread onto BG11 agar plates (1.5% agar). Clarified plaques appeared within 3-7 days. A unique plaque was picked, suspended in 1 mL of BG11 medium, and subsequently used for a new round of plaque isolation. The plaque assay was repeated 5 times until plaques of uniform shape and size were obtained. A single plaque of the 5th generation was picked and suspended in 3 mL of FACHB-240 cultures during the logarithmic phase for 1 day. The lysate was centrifuged at 10,000× g for 10 min at 4 • C, and the supernatant was filtered through 0.22 µm polyethersulfone filters. The amplification culture was developed through the co-cultivation of L. boryana FACHB-240 and the filtrate at a volume ratio of 8:1 until the culture turned yellow.

Preparation of Cyanophage Suspensions
Lysates were centrifuged (8000× g, 20 min, 4 • C), and the supernatants were filtered through 0.45 µm and 0.22 µm polyethersulfone filters. The filtrates were layered on top of the sucrose density gradients (20-40%) and then centrifuged (40,000× g, 1 h, 4 • C). The pellets were suspended in 0.01 M of PBS at 1/10 of the original volume of the lysates and dialysed against PBS at 4 • C. Then, BG11 medium was added to make the final volume of the cyanophage suspension equal to reach the original volume of the lysates.

Electron Microscopy Observation
Lbo240-yong1 suspensions were deposited on 400 mesh copper grids for 10 min and negatively stained for 30 s with 2% uranyl acetate (Sigma-Aldrich, St. Louis, MO, USA). Photographs were taken under transmission electron microscopy (Hitachi-7650, Tokyo, Japan) with a magnification of 80,000× at 60 kV. L. boryana FACHB-240 cultures infected with Lbo240-yong1 were negatively stained and observed in a similar way.

Host Range Experiments
Thirty-seven cyanobacterial strains (Table S1) were obtained from the Freshwater Algae Culture Collection at the Institute of Hydrobiology (FACHB), Academy of Sciences, Wuhan, China. The phage suspension was mixed with the cyanobacterial cultures during the exponential growth phase at a volume ratio of 1:2 in 48-well cell culture plates, and incubated in a light incubator under a light:dark cycle of 12: 12 h with a constant illumination of 40 µmol photons m −2 s −1 at 25 • C. In the control groups, phage suspension was replaced with BG11. All cultures were monitored daily for the liquid color, density, integrity, transparency, and boundary clarity of the cells via visual inspection, optical microscopy observation, and OD 680 measurement. Cyanobacterial strains that did not lyse until the 15th day were defined as unsusceptible.

Cyanophage Isolation
The experimental L. boryana FACHB-240 cultures became yellow 18 h after the addition of the filtrate of the water sample collected from Sun Lake. Soon afterwards, the cultures turned colorless and transparent. Contemporaneously, the control groups remained turbid and blue-green. After five successive single-plaque isolations, Lbo240-yong1 developed uniformly big, round, and clear plaques without halos in 3 days ( Figure 1A). A single plaque of the 5th generation made the logarithmic FACHB-240 cultures colorless within 1 day ( Figure 1B). The algal filament of the infected L.boryanum FACHB-240 fractured, and the cells gathered into masses and then died, leading to a sharp decline in the density of living cells ( Figure 1C,D).
(v0.9.13) (10 November 2022), as vConTACT2 is a useful tool for examining measures between pairs of genomes and offers a scalable, robust, system automated means through which to classify virus sequences [44].

Cyanophage Isolation
The experimental L. boryana FACHB-240 cultures became yellow 18 h addition of the filtrate of the water sample collected from Sun Lake. Soon afterw cultures turned colorless and transparent. Contemporaneously, the contro remained turbid and blue-green. After five successive single-plaque isolations yong1 developed uniformly big, round, and clear plaques without halos in 3 day 1A). A single plaque of the 5th generation made the logarithmic FACHB-240 colorless within 1 day ( Figure 1B). The algal filament of the infected L.boryanum 240 fractured, and the cells gathered into masses and then died, leading to a shar in the density of living cells ( Figure 1C,D).

General Features of Lbo240-yong1
TEM observation revealed that the negatively stained Lbo240-yong1 icosahedral head of 50 ± 5 nm in diameter and a short tail when viewed at the corr ( Figure 2A) (white arrow). It is morphologically similar to the Phormidium cyanop WMP4 [45], which possesses an icosahedron head (about 55 nm in diameter) at a short tail. The white particles (arrow head) are intact; the particles with black the center may be depleted of DNA ( Figure 2B). Under electron microscopy number of cyanophage particles (black arrow), adsorbed on the surfaces of host c observed ( Figure 2C,D).

General Features of Lbo240-yong1
TEM observation revealed that the negatively stained Lbo240-yong1 had an icosahedral head of 50 ± 5 nm in diameter and a short tail when viewed at the correct angle ( Figure 2A) (white arrow). It is morphologically similar to the Phormidium cyanophage Pf-WMP4 [45], which possesses an icosahedron head (about 55 nm in diameter) attached to a short tail. The white particles (arrow head) are intact; the particles with black heads in the center may be depleted of DNA ( Figure 2B). Under electron microscopy, a large number of cyanophage particles (black arrow), adsorbed on the surfaces of host cells, were observed ( Figure 2C,D).
In the host range experiments, 37 cyanobacterial strains (Table S1) were co-cultured with Lbo240-yong1 in triplicate. The results showed that only the indicator host, L. boryana FACHB-240, was susceptible to Lbo240-yong1.  (Table S1) w with Lbo240-yong1 in triplicate. The results showed that only the indicator FACHB-240, was susceptible to Lbo240-yong1.

Genomic Analysis of Lbo240-yong1
The complete genome sequence of Lbo240-yong1 was sequenced, w sequencing depth of 89-fold, using next-generation sequencing (NGS) assembly shaped a complete genome with 127 bp kmer located at both analysis showed that the assembled product was a circular molecule. Exce yong1, no other phage was found in NGS. The Lbo240-yong1 genome w length with a G + C content of 51.99%, and 92.05% of the Lbo240-yong occupied by coding sequences. No tRNA gene was found in the Lbo240-y Terminal analysis revealed that the genome of Lbo240-yong1 had preferre terminal redundancy. The complete genome sequence of Lbo240-yong1 w GenBank (https://www.ncbi.nlm.nih.gov/genbank, accessed on 30 March 2 accession number OM897575. The Lbo240-yong1 genome contained 44 predicted open reading which encoded proteins/peptides of 40 to 1544 AA residues in length. Of a 39 (88.6%) had an ATG initiation codon, 3 (6.8%) had a GTG initiation codo had a TTG initiation codon. The best hits of BLASTp scanning with the 44 p of Lbo240-yong1 are summarized in Table 2. In total, 35 ORFs shared the h

Genomic Analysis of Lbo240-yong1
The complete genome sequence of Lbo240-yong1 was sequenced, with an average sequencing depth of 89-fold, using next-generation sequencing (NGS). The SPAdes assembly shaped a complete genome with 127 bp kmer located at both ends. Bandage analysis showed that the assembled product was a circular molecule. Except for Lbo240-yong1, no other phage was found in NGS. The Lbo240-yong1 genome was 39,740 bp in length with a G + C content of 51.99%, and 92.05% of the Lbo240-yong1 genome was occupied by coding sequences. No tRNA gene was found in the Lbo240-yong1 genome. Terminal analysis revealed that the genome of Lbo240-yong1 had preferred termini with terminal redundancy. The complete genome sequence of Lbo240-yong1 was deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank, accessed on 30 March 2022) under the accession number OM897575.
The Lbo240-yong1 genome contained 44 predicted open reading frames (ORFs) which encoded proteins/peptides of 40 to 1544 AA residues in length. Of all the 44 ORFs, 39 (88.6%) had an ATG initiation codon, 3 (6.8%) had a GTG initiation codon, and 2 (4.6%) had a TTG initiation codon. The best hits of BLASTp scanning with the 44 predicted ORFs of Lbo240-yong1 are summarized in Table 2. In total, 35 ORFs shared the highest identity with Pf-WMP4 genes; 8 ORFs had no BLASTp hit. Interestingly, one ORF (ORF5) of Lbo240-yong1 shared the highest identity with a gene (encoding a Gp49 family protein) of a filamentous cyanobacterium (Calothrix sp. strainPCC 7716), which hints at a gene exchange between cyanophage Lbo240-yong1 and cyanobacteria. No ORF was found to be associated with virulence factors and antibiotic resistance genes, which is advantageous for the application development of the cyanophage.   Three matching sequence fragments between the Lbo240-yong1 genome and viral spacer sequences were found within the CRISPRs databases (E-value < 10 −5 ). The three sequences were as follows: CCACCCACACGGGGGGACGGGCGCGCCACCTATATGTA The circular genome map is shown in Figure 3. The predicted Lbo240-yong1 ORFs could be classified into five functional modules: regulation and replication (6 ORFs), structure (4 ORFs), packaging (1 ORF), lysin (1 ORF), and uncharacterized (32 ORFs).

Taxonomic Analysis
A GenBank BLASTn search with the Lbo240-yong1 genome showed that
In March of 2022, ICTV made a huge update to the phage classification system, which abolished the Caudovirales order and three morphologic-based taxa (Siphoviridae, Myoviridae, and Podoviridae) that have been repeatedly shown not to be monophyletic (2021.001B, https://ictv.global/taxonomy/taxondetails?taxnode_id=20171285, accessed on 30 March 2022) [46]. The abolished Siphoviridae family once harbored all the phages with long non-contractile tails; the abolished Myoviridae family once harbored all the phages with straight contractil tails; the abolished Podoviridae family once harbored all the phages with short noncontractile tails. In the updated classification system of the ICTV, Podovirus-like phages containing short noncontractile tails were classified in different orders, families, and genera. The order Caudovirales was replaced by the class Caudoviricetes to group all tailed bacterial and archaeal viruses with icosahedral capsids and a double-stranded DNA genome. The new Caudoviricetes class comprises 4 orders (Crassvirales, Kirjokansivirales, Methanobavirales, and Thumleimavirales), 33 independent families, 37 independent subfamilies, and 493 independent genera directly to the class Caudoviricetes [46]. The new taxonomy release (#37) can be found on the ICTV website (https://ictv.global/, accessed on 30 March 2022). A proteomic tree, based on the genome-wide sequence similarities of 73 reference sequences (including all 12 reported Podovirus-like cyanophages with sequenced genomes and all 3 sequenced cyanophages capable of infecting Leptolyngbya cyanobacteria (Figure 4), was established. In the tree, Lbo240-yong1 and five freshwater cyanopodoviruses form a monophyletic group that is more deeply diverging than other families. The evolutionary distances between this family and all other sequences were maximal. We propose the creation of a novel family, Filumcyanopodoviridae, within the class Caudoviricetes, that comprises the six freshwater cyanopodoviruses (Leptolyngbya phage Lbo240-yong1; Phormidium phages Pf-WMP4, Pf-WMP3, PP; Arthronema phage Aa-TR020; and Anabaena phage A-4L) infecting filamentous freshwater cyanobacteria [47][48][49]. In the proposed new family, Leptolyngbya cyanophage Lbo240-yong1 and Phormidium cyanophage Pf-WMP4 belong to the Wumpquatrovirus genus, Phormidium cyanophage Pf-WMP3 and PP form the Wumptrevirus genus, and Anabaena phage A-4L represents the Kozyakovvirus genus, while Arthronema cyanophage Aa-TR020 has not been classified. These freshwater cyanopodoviruses, except for the lysogenic cyanophage Aa-TR020, are lytic. Except for Lbo240-yong1, the five cyanophage genomes all contain long terminal repeats (107-234 bp). The absence of the genome terminal repeats of Lbo240-yong1 may be due to recombination, the jumping of mobile elements, or other factors. The genome sizes of the six cyanophages are approx. 40 kb to 45 kb (Table 3). In total, there are seven Podovirus-like freshwater cyanophages with sequenced genomes. The proposed new family harbors six of them. The exception is Synechococcus cyanophage S-SRP01, which was reported to have a high degree of similarity with marine cyanophages [50]. In the proteomic tree (Figure 4), S-SRP01 and two marine Synechococcus cyanopodoviruses form a monophyletic clade.    A genome comparison of Lbo240-yong1 and the closest relative, Phormidium cyanophage Pf-WMP4, is shown in Figure 5. Lbo240-yong1 shares 35 homologous ORFs with Phormidium cyanophage Pf-WMP4. The arrangements and orientations of these homologous ORFs are essentially the same. At the proteomic level, high sequence identity existed between their terminase large subunit (~95%) and structural proteins, such as major capsid protein (~98%), tail tubular protein (~98%), and portal protein (~98%). Conversely, ORF2, ORF16, ORF23, ORF24, ORF35, ORF37, ORF38, ORF39, ORF41, and ORF44 of Lbo240-yong1 shared low identity (<70%) with Pf-WMP4. The other dissimilar sequences were located at the C-terminal coding region of a large hypothetical protein (ORF 12) in the middle of the genome, next to the ORF predicted to encode a putative murein hydrolase activator.

Discussion
In the present work, a novel freshwater cyanophage Lbo240-yong1 was isolated. In the phylogenetic tree, Lbo240-yong1 and five cyanopodoviruses infecting filamentous freshwater cyanobacteria formed a monophyletic group. We propose setting up a new family within the class Caudoviricetes comprising the six cyanopodoviruses (Lbo240-yong1, Pf-WMP4, Pf-WMP3, PP, A-4L, and Aa-TR020) infecting filamentous freshwater cyanobacteria. Lbo240-yong1, Pf-WMP4, Pf-WMP3, PP, A-4L, and Aa-TR020 share common features (Table 3). Their hosts are all filamentous freshwater cyanobacteria. They are similar in morphology and size, all being Podovirus-like, having icosahedral heads (50 nm to 55 nm in diameter) and short tails. Their genomes are similar in size (40 kb to 45 kb), architecture, and gene content ( Table 3, Figure 6A). The genes (associated with phage packaging, structure, and bacteriolysis) located in the first half of the genome of Lbo240-

Discussion
In the present work, a novel freshwater cyanophage Lbo240-yong1 was isolated. In the phylogenetic tree, Lbo240-yong1 and five cyanopodoviruses infecting filamentous freshwater cyanobacteria formed a monophyletic group. We propose setting up a new family within the class Caudoviricetes comprising the six cyanopodoviruses (Lbo240-yong1, Pf-WMP4, Pf-WMP3, PP, A-4L, and Aa-TR020) infecting filamentous freshwater cyanobacteria. Lbo240-yong1, Pf-WMP4, Pf-WMP3, PP, A-4L, and Aa-TR020 share common features (Table 3). Their hosts are all filamentous freshwater cyanobacteria. They are similar in morphology and size, all being Podovirus-like, having icosahedral heads (50 nm to 55 nm in diameter) and short tails. Their genomes are similar in size (40 kb to 45 kb), architecture, and gene content (Table 3 and Figure 6A). The genes (associated with phage packaging, structure, and bacteriolysis) located in the first half of the genome of Lbo240-yong1 were predicted to transcribe in the same orientation, while the other genes (associated with replication and regulation) located in the remaining half of the genome were predicted to transcribe in the opposite direction. Similar gene arrangements exist in the other five freshwater cyanopodoviruses ( Figure 6A). metagenomes were downloaded and assembled, a gene-sharing network was inferred using vContact2 software, and the contigs related to Lbo240-yong1 were predicted usingVirSorter2 software.
The homologues of Lbo240-yong1 genes were found in all four metagenomes ( Figure  S5). Five viral contigs sharing significant similarities with Lbo240-yong1 were found. Four of them were cyanopodoviruses (Pf-WMP4, A-4L, Pf-WMP3, and PP) having been isolated. The other uncharacterized one (32,719 bp) was from Israel's freshwater ponds and Lake Kinneret. The contig and the six related cyanopodoviruses were used for proteomic tree construction ( Figure S4C). Compared with the six related cyanopodoviruses, the unidentified contig is only distantly related, sharing five core genes (capsid protein, tail protein, tail tubular protein B, murein hydrolase activator, and terminase large subunit) with the other six cyanopodoviruses.  (Table S2, Figures S1 and S2) contained genomes much smaller than the six cyanopodoviruses ( Figure S3) [51,52].
VirClust was used to group orthologous proteins into protein clusters (PCs). Eight core proteins (DNA polymerase, DNA primase/helicase, capsid protein, tail protein, tail tubular protein A, tail tubular protein B, murein hydrolase activator, and terminase large subunit) were found within the six related cyanopodoviruses using VirClust. The eight hallmark genes of the six cyanopodoviruses were then used to build maximum likelihood phylogenetic trees ( Figure S4A). Another maximum likelihood phylogenetic tree, based on the terminase large subunit, was constructed using outgroups, demonstrating that the proposed family clearly forms a monophyletic clade ( Figure S4B). All phylogenetic trees, whether based on the whole genome (Figures 4 and 6A,C), the core genes ( Figure S4A), or the terminase large subunit, were broadly similar, i.e., the six related cyanopodoviruses clustered in the same way and formed a monophyletic clade.
Information about isolated and sequenced freshwater cyanophage is very limited. To find out whether the cyanophages related to Lbo240-yong1 are prevalent, four freshwater metagenomes were downloaded and assembled, a gene-sharing network was inferred using vContact2 software, and the contigs related to Lbo240-yong1 were predicted us-ingVirSorter2 software.
The homologues of Lbo240-yong1 genes were found in all four metagenomes ( Figure S5). Five viral contigs sharing significant similarities with Lbo240-yong1 were found. Four of them were cyanopodoviruses (Pf-WMP4, A-4L, Pf-WMP3, and PP) having been isolated. The other uncharacterized one (32,719 bp) was from Israel's freshwater ponds and Lake Kinneret. The contig and the six related cyanopodoviruses were used for proteomic tree construction ( Figure S4C). Compared with the six related cyanopodoviruses, the unidentified contig is only distantly related, sharing five core genes (capsid protein, tail protein, tail tubular protein B, murein hydrolase activator, and terminase large subunit) with the other six cyanopodoviruses.
The CRISPR/Cas immune system is a defense strategy against extrinsic nucleic acids, such as the virus genomes of bacteria and archaea [55]. The CRISPR loci generally consist of non-continuous direct repeats separated by short stretches of DNA sequences called spacers, and these were related to cas genes [56]. Three sequence fragments of Lbo240-yong1 were found to match with the spacers of the Leptolyngbya genus via scanning the viral spacer database of IMG/VR (E-value < 10 −5 ). These sequence fragments match the viral spacers of the Leptolyngbya genus, including L.boryana FACHB-402 (Bit Score, 57.2; E-value, 10 −6 ; identity, 90%). The results suggest that this Leptolyngbya spp. may have once been infected by related phages in the past and has formed immune resistance [57]. In the host range test in this study, two L. boryana strains (FACHB-402 and FACHB-240) were tested. As a result, it was shown that FACHB-402 is resistant, while FACHB-240 is the susceptible indicator host. The presence of the matching CRISPR spacersin, FACHB-402, may provide immune protection against infection.
In summary, based on the morphology and sequence characteristics, we propose that the Lbo240-yong1 should be classified as a novel species of the Wumpquatrovirus genus in the Caudoviricetes class. A novel family was proposed to harbor all the reported freshwater cyanopodoviruses with sequenced genomes-except for S-SRP01, which reveals a high degree of similarity with marine cyanophages. The isolation and genome analysis of Lbo240-yong1 enriches the field's knowledge of cyanophages and provides basic useful information for further research and application development. Due to the limited information in the freshwater cyanophage database, it is vital to isolate and identify more cyanophages from freshwater environments.
Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/v15040831/s1, Figure S1: Genome map of phage Claudivirus aurora; Figure  S2: Genome map of phage Claudivirus claudi; Figure S3: Genome comparison of three podoviruses (Brucesealvirus CPS2, Claudivirus claudi, Lbo240-yong1); Figure S4: Maximum likelihood phylogenetic trees generated using MEGA X and proteomic tree generated using ViPTree online. (A) Maximum likelihood phylogenetic tree based on eight core genes of the six related freshwater cyanopodoviruses by using MEGA X (B) Maximum likelihood phylogenetic tree based on terminase large subunits of the six relative freshwater cyanopodoviruses and outgroups of four phages. (C) Proteomic tree for Lbo240 yong1 genome and the homologues including an uncharacterized contig and four freshwater cyanopodoviruses (Pf WMP4, A 4L, Pf WMP3, and PP) found in the assembled sequences of the metagenomes and the freshwater cyanopodoviruses Aa-TR020; Figure S5: Result of gene-sharing network analysis of Lbo240-yong1 and four freshwater metagenomes; Table S1: Results of the host range analysis of Lbo240-yong1 against 37 cyanobacterial strains; Table S2: Basic characteristics of four podoviruses of Guelinviridae and Northropvirinae.