Metapopulation Structure Of Marine Diatom-associated Roseobacter Group

Phycosphere is the direct microenvironment of the marine bacteria-phytoplankton interaction which ultimately shapes ecosystem productivity.The biochemical mechanisms underlying their interactions become increasingly known,yet how these ubiquitous interactions drive bacterial evolution has not been illustrated.Plenty of strains in the Roseobacter group isolated from the same habitat were analyzed based on genomics and phylogenetics in this study.The differentiation of some close related Roseobacter populations in various phycosphere was elucidated.Here are main findings:First,we collected 1 L of seawater from the Pearl River Estuary,isolated 45 diatom cells varying at the level of phylogenetic relatedness,cultivated 861 roseobacters associated with 19 of these diatoms,and sequenced 294 genomes of these roseobacters.All sequenced genomes were used for evolutional and functional analyses at the whole-genome level.Second,these newly sequenced roseobacters comprise eight clades in the phylogenetic tree.Members within each clade share identical 16 S r RNA genes,display whole-genome average nucleotide identity(ANI)over 99.99%,and vary up to 45 non-singleton single nucleotide polymorphisms(SNPs)in which the rare variant occurs in at least two genomes.Clade-2a and clade-2b related to Sulfitobacter pseudonitzschiae,clade-2c related to S.geojensis,clade-2d,clade-2e1 and clade-2e2 related to S.mediterraneus.The remaining clade-1 and clade-3 are related to Marivita cryptomonadis and Ponticoccus sp.LZ-14,respectively,which are distantly related to Sulfitobacter.The top five groups with closed related clades were used to compare the differentiation level and functional differences between clades.Third,a key finding is that closely related roseobacter populations associated with different diatom cells are often genetically differentiated,which is clearly supported by populations diverged at different phylogenetic depths.It also suggests a metapopulation structure of diatom-associated bacteria.The ANI below the boundary(95%)and the higher SNPs density between clades than within clade reflect the differentiation of two clades(clade-2a and clade-2b)related to S.pseudonitzschiae.For the three clades related to S.mediterraneus,clade-2e1 and clade-2e2 have not yet separated into two distinct species,while clade-2d is more divergent than clade-2e1 and clade-2e2.Importantly,these clades each have a distinct host range.The clade-2a is composed solely of members isolated from Skeletonema menzelii 26(abbreviated as ‘SM26'),whereas the clade-2b comprise members from six diatom cells affiliated with four species,suggesting that phycosphere of different species of diatom drive the differentiation of symbiotic bacteria.Members of clade-2e1 are exclusively associated with a single diatom host(SC5),whereas members of clade-2e2 are from three hosts of the same diatom species(SC2,SC4 and SC7)and members of clade-2d are from hosts of two different families(SC1 and MC36),suggesting that phycosphere of different strains of same diatom species also result in the differentiation of symbiotic bacteria.The clustering analysis suggests that diatomdependent differentiation is also evident from the more closely related roseobacters associated with different hosts but sharing membership of the same clade.The differentiation of close related Roseobacter populations isolated from the same species but various strains of diatom suggests the physical barriers of gene flow.Besides,phycosphere colonization represents a new mechanism leading to bacterial metapopulation structure in the pelagic ocean.Finally,the annotation results exhibit clear differentiation between clades at the functional level including secretion systems and motility.A T2 SS and a T6 SS gene clusters are present on the plasmid and chromosome of clade-2d and absent in other clades.A trb cluster is only found on the chromosome of clade-2e2 members.All clades possess the vir B/D4 system but there are one more gene cluster in genomes of clade-2d and clade-2e2 than in clade-2e1,and two more gene clusters in genomes of clade-2a members than in clade-2b.In the co-culture assay of 11 representative strains and a diatom,nine strains significantly promoted the growth of diatom,whereas the remaining two in clade-2e2 did not significantly change the growth rate of the diatom.Motility is also different between clades,among which flagellar gene cluster(FGC)fla2 is present in a plasmid of the S.pseudonitzschiae related clade-2e1,clade-2e2 and clade2 d,whereas fla1 was exclusively found on the chromosome of clade-2e2.However,none of them showed the motility phenotype in the assay.In terms of the S.pseudonitzschiae related clade-2a and clade-2b,they did not possess any type of FGC,but both instead carry homologs of two candidate gene clusters recently hypothesized to be responsible for dendritic motility.The clade-2b members possess an additional copy located within a genomic island compared to clade-2a members,consistent with the greater swimming and dendritic motilities observed in the former.Here,we found the differentiation of the Roseobacter group isolated from the same or different species of diatoms.We revealed that phycosphere of diatom cells may act as an effective physical barrier of gene flow between nearly identical symbiotic roseobacters,thereby conferring a new role of phycosphere in driving the evolution of pelagic marine bacteria.This work is important for revealing the interaction and co-evolution of microalgae and symbiotic bacteria.