The Ecological Functions And Evolutionary Characteristics Of Marine Heterotrophic Archaea And Their Viruses In The Pearl River Estuary

Marine microbes and viruses with tremendous abundance and diversity serve as keystones in the evolution of marine life and ecological processes,playing an important role in the carbon and nitrogen cycles in the ocean.Marine group Ⅱ archaea(abbreviated as MGⅡ,Candidatus Poseidoniales)are the most abundant archaea among global surface waters,capable of degrading a variety of organic matter.Intimately,MGⅡ viruses have a significant impact on the abundance,diversity,ecological functions,and evolution of MGⅡ.The genomic studies of MGⅡ and their viruses in coastal and open ocean environments have been well studied,and highthroughput sequencing-based research has demonstrated the high abundance of MGⅡ in estuaries,which are the critical transition zone linking the material cycles of land and ocean.However,the genomic diversity,community structure,ecological relationships,and evolutionary characteristics of MGⅡ and their viruses in the estuaries are poorly understood.The ecological and evolutionary characteristics of MGⅡ and their viruses in the estuarine environments have become an important bottleneck for understanding the role of archaea play in material cycling and the evolution of life at the land-ocean interface.The main scientific question of this thesis is lineage divergence of MGⅡ in estuarine brackish environments and the ecological and evolutionary characteristics of their viruses.A systematic study on the genomes of the prokaryotes and viruses of seawater from the Pearl River estuary(PRE)along a salinity gradient was conducted using metagenomics,and the genomic diversity,distribution characteristics and driving factors of MGⅡ community were investigated.The evolutionary history of species differentiation and salinity adaptation mechanism of MGⅡ was further investigated by integrating with global ocean metagenomic data and combining the methods of molecular biology,bioinformatics,and evolutionary analysis,and the evolutionary history of species differentiation of MGⅡ was discussed in the context of geological history.In addition,the genomic diversity,abundance distribution and community structure characteristics of MGⅡ viruses in estuarine environments were clarified,and a systematic classification of MGⅡ viruses was performed,and the evolutionary features of estuarine and marine MGⅡ viruses were revealed at the genomic level.A large number of genomes of prokaryotes and viruses in the PRE were obtained,and the MGⅡ community structure,distribution characteristics and its main driving factors were systematically analyzed.Overall,1,205 non-redundant mediumand high-quality prokaryotic genomes and 78,502 non-redundant viral-like genomes were generated from PRE metagenomes.Among the estuarine prokaryotic genomes,86.8% were novel at the species level according to public databases and among the viral genomes only 15.3% could be assigned at the family level,indicating that the diversity and ecological functions of microorganisms and viruses in the PRE remain a "black box".MGⅡ was the main archaeal group in the PRE,consisting of MGⅡa and MGⅡb.A total of nine genera were found in the MGⅡ community of PRE,two of which were new genera,revealing the substantial diversity of brackish MGⅡ.The diversity and distribution of MGⅡ were mainly affected by salinity,and the number of MGⅡ genomes and reads increased with the increasing salinity.In the phylogenomic tree,genomes from brackish water formed several clusters in the subclade of MGⅡa and encoded the new type of proteorhodopsin genes,suggesting there was species-and gene-level evolution of MGⅡ in the PRE,which may be driven by adaptation to salinity change.By integrating with global ocean metagenomic data,this thesis revealed the species divergence of MGⅡ between brackish and marine waters,of which the salinity adaptation mechanism was triggered by the cor A gene,and the time of species divergence was during the Pangea period.This study contributed 89 highquality novel MGⅡ genomes to the updated non-redundant MGⅡ genome dataset and thus has filled a significant gap in species diversity of MGⅡ from global low-salinity environments.In the phylogenomic tree,genomes from global estuaries and enclosed seas formed six clades in the subclade of MGⅡa.Abundance mapping of MGⅡ in global coastal and pelagic surface waters with distinct salinities showed that the MGⅡ of six clades were enriched in salinity between 6.6 to 23.In addition,the MGⅡ of six clades had a lower ratio of acidic proteins than that of their marine counterparts.Together,with the above comprehensive results,six monophyletic brackish-specific clades can be identified.By comparative genomic analysis and ancestral gene reconstruction,we showed that the divergence of MGⅡ between brackish and marine waters was triggered by the insertion,inversion,and possibly regulatory coupling of a key gene cor A in a highly conservative stress-response gene cluster,which was followed by metabolic acclimation and diversification that were characterized by proteome acidity change.Molecular clock analysis revealed that genus-level subgroups divergence of MGⅡ was from 0.834 to 0.28 Ga,while the divergence of the brackish and the marine subgroups was from 0.299 to 0.094 Ga.According to the geological events,we speculated that rapid environmental salinity change caused by drastic landform changes on the continental margins during the Pangea period(0.335-0.175 Ga)was the key selective force.Based on these discoveries,we proposed that the MGⅡ habitat expansion and lineage divergence at the land-ocean interface were caused by sudden change of primary and qualitative niche traits,which were followed by the gradual changes in secondary and accumulative traits.The ecological characteristics of MGⅡ viruses in global estuarine environments were elucidated and systematic classification was performed,and the evolutionary features of estuarine and marine MGⅡ viruses were revealed at the genomic level.A total of 234 MGⅡ viral contigs were obtained from brackish surface waters by using homology searches for viral signature proteins.Phylogenetic analysis classified MGⅡ viruses into six families and nine genera,highlighting the vast diversity of this novel viral group.Phylogenetic analysis also revealed their close evolutionary connection to archaeal head-tailed viruses,suggesting the common origin among them.The frequent adaptive evolution of MGⅡ viruses between brackish and marine environments resulted in the differentiation of the community structure of the MGⅡ viruses between the two environments.However,abundance analysis showed that MGⅡ viruses were abundant both in brackish and open ocean environments and most MGⅡ viruses in the brackish environments were widely distributed in both brackish and open ocean environments.Genome annotation suggested MGⅡ viruses may be involved in multiple metabolic pathways of MGⅡ archaea and play an important role in the biological activities and ecological processes of MGⅡ.Discoveries and hypotheses in this study provided important research data and foundation for evaluating the important roles of MGⅡ and their viruses in the carbon and nitrogen cycles of estuaries,as well as for revealing the trajectories and mechanisms of microbial evolution at the interface of land and ocean.