| Abstract | Background: Freshwater ecosystems are inhabited by members of cosmopolitan bacterioplankton lineages despite the disconnected nature of these habitats. The lineages are delineated based on > 97% 16S rRNA gene sequence similarity, but their intra-lineage microdiversity and phylogeography, which are key to understanding the ecoevolutional processes behind their ubiquity, remain unresolved. Here, we applied long-read amplicon sequencing targeting nearly full-length 16S rRNA genes and the adjacent ribosomal internal transcribed spacer sequences to reveal the intra-lineage diversities of pelagic bacterioplankton assemblages in 11 deep freshwater lakes in Japan and Europe.
Results: Our single nucleotide-resolved analysis, which was validated using shotgun metagenomic sequencing, uncovered 7-101 amplicon sequence variants for each of the 11 predominant bacterial lineages and demonstrated sympatric, allopatric, and temporal microdiversities that could not be resolved through conventional approaches.
Clusters of samples with similar intra-lineage population compositions were identified, which consistently supported genetic isolation between Japan and Europe. At a regional scale (up to hundreds of kilometers), dispersal between lakes was unlikely to be a limiting factor, and environmental factors or genetic drift were potential determinants of population composition. The extent of microdiversification varied among lineages, suggesting that highly diversified lineages (e.g., Iluma-A2 and acI-A1) achieve their ubiquity by containing a consortium of genotypes specific to each habitat, while less diversified lineages (e.g., CL500-11) may be ubiquitous due to a small number of widespread genotypes. The lowest extent of intra-lineage diversification was observed among the dominant hypolimnionspecific
lineage (CL500-11), suggesting that their dispersal among lakes is not limited despite the hypolimnion being a more isolated habitat than the epilimnion.
Conclusions: Our novel approach complemented the limited resolution of short-read amplicon sequencing and limited sensitivity of the metagenome assembly-based approach, and highlighted the complex ecological processes underlying the ubiquity of freshwater bacterioplankton lineages. To fully exploit the performance of the method, its relatively low read throughput is the major bottleneck to be overcome in the future. |
| Text | 443318 2021 10.1186/s40168 020 00974 y Freshwater bacterioplankton Long read amplicon sequencing Microdiversity PacBio Phylogeography Ribosomal internal transcribed spacers Microdiversity and phylogeographic diversification of bacterioplankton in pelagic freshwater systems revealed through long read amplicon sequencing Yusuke Okazaki, Shohei Fujinaga1, Michaela M. Salcher, Cristiana Callieri6 Atsushi Tanaka, Ayato Kohzu, Hideo Oyagi, Hideyuki Tamaki and Shin ichi Nakano Yusuke Okazaki Center for Ecological Research, Kyoto University, 2 509 3 Hirano, Otsu, Shiga 520 2113, Japan; Shohei Fujinaga Center for Ecological Research, Kyoto University, 2 509 3 Hirano, Otsu, Shiga 520 2113, Japan; Michaela M. Salcher Limnological Station, Institute of Plant and Microbial Biology, University of Zurich, Seestrasse 187, 8802 Kilchberg, Zurich, Switzerland; Cristiana Callieri CNR IRSA Verbania; Atsushi Tanaka National Institute for Environmental Studies, 16 2 Onogawa, Tsukuba, Ibaraki 305 8506, Japan; Ayato Kohzu National Institute for Environmental Studies, 16 2 Onogawa, Tsukuba, Ibaraki 305 8506, Japan; Hideo Oyagi Faculty of Policy Studies, Nanzan University, 18 Yamazato cho, Showa ku, Nagoya, Aichi 466 8673, Japan; Hideyuki Tamaki Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Central 6, Higashi 1 1 1, Tsukuba, Ibaraki 305 8566, Japan; Shin ichi Nakano Center for Ecological Research, Kyoto University, 2 509 3 Hirano, Otsu, Shiga 520 2113, Japan Background Freshwater ecosystems are inhabited by members of cosmopolitan bacterioplankton lineages despite the disconnected nature of these habitats. The lineages are delineated based on > 97% 16S rRNA gene sequence similarity, but their intra lineage microdiversity and phylogeography, which are key to understanding the ecoevolutional processes behind their ubiquity, remain unresolved. Here, we applied long read amplicon sequencing targeting nearly full length 16S rRNA genes and the adjacent ribosomal internal transcribed spacer sequences to reveal the intra lineage diversities of pelagic bacterioplankton assemblages in 11 deep freshwater lakes in Japan and Europe. Results Our single nucleotide resolved analysis, which was validated using shotgun metagenomic sequencing, uncovered 7 101 amplicon sequence variants for each of the 11 predominant bacterial lineages and demonstrated sympatric, allopatric, and temporal microdiversities that could not be resolved through conventional approaches. Clusters of samples with similar intra lineage population compositions were identified, which consistently supported genetic isolation between Japan and Europe. At a regional scale up to hundreds of kilometers , dispersal between lakes was unlikely to be a limiting factor, and environmental factors or genetic drift were potential determinants of population composition. The extent of microdiversification varied among lineages, suggesting that highly diversified lineages e.g., Iluma A2 and acI A1 achieve their ubiquity by containing a consortium of genotypes specific to each habitat, while less diversified lineages e.g., CL500 11 may be ubiquitous due to a small number of widespread genotypes. The lowest extent of intra lineage diversification was observed among the dominant hypolimnionspecific lineage CL500 11 , suggesting that their dispersal among lakes is not limited despite the hypolimnion being a more isolated habitat than the epilimnion. Conclusions Our novel approach complemented the limited resolution of short read amplicon sequencing and limited sensitivity of the metagenome assembly based approach, and highlighted the complex ecological processes underlying the ubiquity of freshwater bacterioplankton lineages. To fully exploit the performance of the method, its relatively low read throughput is the major bottleneck to be overcome in the future. 9 Published version Articolo Okazaki_et_al_2021.pdf Articolo in rivista BioMed Central 2049 2618 Microbiome Microbiome Microbiome cristiana.callieri CALLIERI CRISTIANA |
