Deep-sea Ecosystems Research Articles

Natural warming differentiates communities and increases diversity in deep-sea Ridge Flank Hydrothermal Systems

Ridge Flank Hydrothermal Systems have discrete pockets of fluid discharge that mimic climate-induced ocean warming. Unlike traditional hydrothermal fluids, those discharged by Ridge Flank Hydrothermal Systems have a chemical composition indistinguishable from background water, enabling evaluation of the effect of warming temperature. Here we link temperature and terrain variables to community composition and biodiversity by combining remotely operated vehicle images of vent and non-vent zone communities with associated environmental variables. We show overall differences in composition, family richness, and biodiversity between zones, though richness and diversity were only significantly greater in vent zones at one location. Temperature was a contributing factor to observed greater biodiversity near vent zones. Overall, our results suggest that warming in the deep sea will affect species composition and diversity. However, due to the diverse outcomes projected for ocean warming, additional research is necessary to forecast the impacts of ocean warming on deep-sea ecosystems.

Transcriptome analysis of the bipotential gonads of hermaphroditic bivalve species Tridacna crocea unravels the potential genes responsible for spermatogenesis

Sex is an integral part of the evolution of species and often takes the form of intersex and sexual change in mollusks. As a major member of the deep-sea coral reef ecosystem, Tridacna crocea has complex gonadal development mechanisms and reproductive strategies, and the regulation model of spermatogenesis needs to be further explored. In this study, gonadal transcripts of T. crocea in resting, spermatogonia and sperm stage were evaluated. According to the statistics of bioinformatics analysis, a total of 10,260 DEGs were identified, including 3021 genes shared by three developmental stages. Through dynamic expression analysis, we identified the expression patterns and functional classification of genes that regulate spermatogenesis. These candidate genes were involved in regulatory mechanisms such as nucleotide binding, cell cycle, sexual reproduction and DNA replication. Subsequently, based on functional screening, PPI analysis was performed on the target gene cluster to learn the network relationships among candidate genes and other genes closely related to candidate genes. The results showed that in addition to candidate genes (zmcm6, gins2, mcm9, blm, mcm4, psmc3ip, haus, ccnb3, spice, rad1, rad17, ncapg, bub1b, lin9, tipin, and cks), several genes that have been shown to be associated with gonadal development were present in the network (such as fanci, aurka, kif20a and nek2), suggesting the reliability of the results. Finally, the expression levels of candidate genes between individuals at different developmental stages were compared with transcriptional expression profiles, which further proved that these candidate genes were related to T. crocea gonad development traits. These results make up for the lack of research on T. crocea gametogenesis, spermatogenesis and gonad development, and provide a reference for the research on Tridacna reproductive mechanism.

The spread of the non-indigenous species Bregmaceros nectabanus Whitley, 1941 (Osteichthyes: Bregmacerotidae) in the eastern Mediterranean

This study reports the first record and some features of two individuals of the Lessepsian migrant species smallscale codlet, Bregmaceros nectabanus, found in the stomachs of two individuals of European hake, Merluccius merluccius, sampled in the northern Ionian Sea (eastern Mediterranean) during the MEDITS International Bottom Trawl Survey. The two European hake were collected on muddy bottoms in September 2021 at 63 m depth (39.34950 ºN; 16.52350 ºE) and at 240 m depth (39.07156 ºN; 17.12626 ºE). Their morphological traits together with otolith and scale characteristics allowed the identification of the two specimens found in the stomachs as B. nectabanus. The analysis of the stomach contents for the larger individual is also reported. This finding underlines the utility of stomach content analysis as an indirect method to study the biodiversity of deep-sea ecosystems, including the spread of non-indigenous species as indicators of global warming.

Learning from the history of red shrimp fisheries in the Mediterranean to improve sustainability of deep-water bottom trawling

Abstract As deep-water red shrimp (DWRS) trawl fisheries gain importance in the Mediterranean, their impact on fragile deep-sea ecosystems requires careful management. We reviewed the historical development of DWRS fisheries in the Mediterranean to propose a harvest strategy that would enhance fishery sustainability, while preserving habitat and community integrity. We considered two representative typologies of DWRS fisheries: a domestic fleet operating within the Italian territorial waters of the Ligurian Sea, and a distant fleet operating mostly in international waters of the central-eastern Mediterranean and sharing shrimp stocks with non-European countries. Our proposed management approach is based on three main harvesting measures: (i) preventing the uncontrolled growth in number of trawlers, (ii) adopting a catch control regime, and (iii) identifying a network of deep-water areas designated alternately for fishing and conservation purposes. As with fisheries in other regions, new fishing grounds should be explored using “encounter protocols” to avoid areas hosting species indicators of Vulnerable Marine Ecosystems. Additionally, empirical indicators such as catch rate thresholds derived from fishers’ experience, could be used to move from one fishing ground to another to avoid local depletion of DWRS abundance and a decrease in fishery profitability.

Data Analysis in Deep-Sea Mineral and Organism Interplay: A Review

The deep sea, a realm characterized by extreme environmental conditions and remarkable biodiversity, has become a focal point for both scientific exploration and industrial exploitation. This comprehensive review aims to elucidate the data analysis techniques that are instrumental in studying the complex relationship between deep-sea mineral resources and the organisms that inhabit these extreme environments. The paper begins by outlining the types of deep-sea mineral resources and their distribution, followed by an overview of the methods and technologies used for exploration and extraction. It then delves into the diversity and classification of deep-sea organisms, their unique survival mechanisms, and their ecological roles. The core of the review focuses on data analysis techniques, covering traditional methods, advancements in machine learning and AI, and the inherent challenges and limitations in analyzing intricate deep-sea data. The paper concludes by discussing the direct and indirect impacts of mineral extraction on deep-sea ecosystems, supported by case studies and potential long-term effects on biodiversity and ecosystem health.

High-quality draft genome of Gammaproteobacterial SUP05 cluster from non-buoyant hydrothermal plumes of ultraslow spreading Gakkel Ridge (Central Arctic Ocean).

Hydrothermal plumes are an important yet understudied component of deep-sea microbial ecosystems. We report metagenome-assembled genomes (MAGs) of three Bacteria belonging to the Gammaproteobacterial SUP05 cluster (family Thioglobaceae), assembled from the metagenomes of two non-buoyant hydrothermal plumes in the ultraslow spreading Gakkel Ridge.

Deep sea treasures - Insights from museum archives shed light on coral microbial diversity within deepest ocean ecosystems

Deep sea benthic habitats are low productivity ecosystems that host an abundance of organisms within the Cnidaria phylum. The technical limitations and the high cost of deep sea surveys have made exploring deep sea environments and the biology of the organisms that inhabit them challenging. In spite of the widespread recognition of Cnidaria's environmental importance in these ecosystems, the microbial assemblage and its role in coral functioning have only been studied for a few deep water corals. Here, we explored the microbial diversity of deep sea corals by recovering nucleic acids from museum archive specimens. Firstly, we amplified and sequenced the V1–V3 regions of the 16S rRNA gene of these specimens, then we utilized the generated sequences to shed light on the microbial diversity associated with seven families of corals collected from depth in the Coral Sea (depth range 1309 to 2959 m) and Southern Ocean (depth range 1401 to 2071 m) benthic habitats. Surprisingly, Cyanobacteria sequences were consistently associated with six out of seven coral families from both sampling locations, suggesting that these bacteria are potentially ubiquitous members of the microbiome within these cold and deep sea water corals. Additionally, we show that Cnidaria might benefit from symbiotic associations with a range of chemosynthetic bacteria including nitrite, carbon monoxide and sulfur oxidizers. Consistent with previous studies, we show that sequences associated with the bacterial phyla Proteobacteria, Verrucomicrobia, Planctomycetes and Acidobacteriota dominated the microbial community of corals in the deep sea. We also explored genomes of the bacterial genus Mycoplasma, which we identified as associated with specimens of three deep sea coral families, finding evidence that these bacteria may aid the host immune system. Importantly our results show that museum specimens retain components of host microbiome that can provide new insights into the diversity of deep sea coral microbiomes (and potentially other organisms), as well as the diversity of microbes writ large in deep sea ecosystems.

Foraging behavior and age affect maternal transfer of mercury to northern elephant seal pups

Deep ocean foraging northern elephant seals (Mirounga angustirostris) consume fish and squid in remote depths of the North Pacific Ocean. Contaminants bioaccumulated from prey are subsequently transferred by adult females to pups during gestation and lactation, linking pups to mercury contamination in mesopelagic food webs (200–1000 m depths). Maternal transfer of mercury to developing seal pups was related to maternal mercury contamination and was strongly correlated with maternal foraging behavior (biotelemetry and isotopes). Mercury concentrations in lanugo (hair grown in utero) were among the highest observed worldwide for young pinnipeds (geometric mean 23.01 μg/g dw, range 8.03–63.09 μg/g dw; n = 373); thus, some pups may be at an elevated risk of sub-lethal adverse health effects. Fetal mercury exposure was affected by maternal foraging geographic location and depth; mercury concentrations were highest in pups of the deepest diving, pelagic females. Moreover, pup lanugo mercury concentrations were strongly repeatable among successive pups of individual females, demonstrating relative consistency in pup mercury exposure based on maternal foraging strategies. Northern elephant seals are biosentinels of a remote deep-sea ecosystem. Our results suggest that mercury within North Pacific mesopelagic food webs may also pose an elevated risk to other mesopelagic-foraging predators and their offspring.

DECIPHERING THE DEEP: MACHINE LEARNING APPROACHES TO UNDERSTANDING OCEANIC ECOSYSTEMS

This paper presents a detailed exploration of the transformative role of Machine Learning (ML) in oceanographic research, encapsulating the paradigm shift towards more efficient and comprehensive analysis of marine ecosystems. It delves into the multifaceted applications of ML, ranging from predictive modeling of ocean currents to in-depth biodiversity analysis and deciphering the complexities of deep-sea ecosystems through advanced computer vision techniques. The discussion extends to the challenges and opportunities that intertwine with the integration of AI and ML in oceanography, emphasizing the need for robust data collection, interdisciplinary collaboration, and ethical considerations. Through a series of case studies and thematic discussions, this paper underscores the profound potential of ML to revolutionize our understanding and preservation of oceanic ecosystems, setting a new frontier for future research and conservation strategies in the realm of oceanography.

Short-terms variability of water properties and phytoplankton blooms along the central eastern Florida shelf edge

The central eastern Florida shelf edge is a highly dynamic area that supports an important yet vulnerable deep-sea coral reefs ecosystem, the Oculina Coral Habitat Area of Particular Concern. Rapid and large short-terms (days to weeks) changes of bottom temperature (up to 9 °C) and pCO2 (up to 180 μatm) were observed at the shelfbreak during a two-month (May 13-July 10, 2017) deployment of a lander package. An analysis suggests that these changes are the combined results of tides, the Gulf Stream meandering, and submesoscale eddies and filaments. The processes responsible for sub-tidal variability may include 1) the Gulf Stream frontal movements, 2) upwelling/downwelling of slope waters in association with the Gulf Stream variability, and 3) submesoscale processes and associated vertical movements. Satellite images also frequently show a narrow plume of elevated chlorophyll concentration that stretches from the coast northward up to >200 km along the Gulf Stream front during late spring and early summer. Our analysis indicates that these phytoplankton blooms in the plume are likely supported by the nutrient supply from the nutrients-rich slope waters to the shelf edge and subsequent local vertical mixing. Carbon export associated with these blooms can be an important food source to the Oculina corals. Upwelling of slope waters, on the other hand, will lead to increased CO2 and reduced pH and aragonite saturation state along the shelf edge. Therefore, these dynamic processes may have strong impacts on the health and sustainability of the Oculina coral ecosystem.

On the genetic diversity of Chimaera monstrosa Linnaeus, 1758 (Chordata, Chondrichthyes, Holocephali) in the Mediterranean Sea

The sustainable management and conservation of deep-sea species may be hampered by the paucity of data on their population structure and connectivity, in the face of ever-increasing fisheries pressure and other forms of impacts on deep-sea ecosystems. The rabbit fish, Chimaera monstrosa, is a deep-sea cartilaginous fish, reported worldwide in the past, but currently distributed only in the North-Eastern Atlantic Ocean and Mediterranean Sea. It is currently experiencing high levels of mortality associated with by-catch. Its slow growth, low fecundity and late maturity make this species particularly vulnerable to anthropogenic impact, although little is known about processes of connectivity between ecoregions. This study utilized DNA sequencing of the mitochondrial cytochrome c oxidase subunit I (COI) gene to investigate the population structure and demography of C. monstrosa both at the small (around the coasts of Sardinia, western Mediterranean Sea) and at larger spatial scales (at the pan-Mediterranean level, and between the Atlantic Ocean and the Mediterranean Sea). A total of 100 new sequences were obtained from specimens of Mediterranean origin, identifying 15 new haplotypes out of the 30 known so far for the species. Evidence of feeble but significant differentiation was detected among locations within the Tyrrhenian basin. Bayesian clustering analyses indicated the occurrence of three distinct haplogroups: the most common spread all over the Mediterranean, and the other two limited to the Western basin. Greater levels of genetic differentiation were found between the Atlantic and Mediterranean populations which constituted two main genetic clusters, with no shared haplotypes. The two populations became separated at the end of the Middle Pleistocene, with a clear sign of demographic expansion during the same period. The rabbit fish constitutes an important exception to a general paradigm of deep-sea species being connected by high levels of gene flow and such results could be useful to implement current management strategies to conserve this vulnerable by-caught species.

Accumulation of polychlorinated biphenyls in the ovaries of deep-sea chemosynthetic clam Phreagena okutanii

Polychlorinated biphenyls (PCBs) persistently pollute marine ecosystems even though their production has been discontinued decades ago. Deep-sea organisms have been shown to accumulate PCBs and other persistent organic compounds; however, the extent of contamination and its effects remain poorly understood. Here, we measured PCB concentrations in separated organs of non-feeding bivalve clams belonging to Phreagena, a taxon representative of deep-sea chemosynthetic ecosystems. The highest concentrations of PCBs were detected in the ovaries. We also examined surface sediments of the clam habitat and observed concentrations comparable to those in the sediments of the seas around Japan, including shallow waters. The results showed that PCBs from the environment penetrate Phreagena clams through the body surface and accumulate in the female reproductive system. This raises concerns about a potentially severe direct impact on the survival of vulnerable and highly endemic deep-sea chemosynthetic bivalves.

Bioturbation and faunal-mediated ecosystem functioning in a deep-sea benthic community recovering from a severe seabed disturbance

Kaikōura Canyon, offshore Aotearoa/New Zealand, is a hotspot for deep-sea benthic biology with globally high faunal abundance. The Mw7.8 Kaikōura earthquake in 2016 triggered a severe disturbance that reshaped the canyon, evacuating an estimated 850 metric megatonnes of sedimentary material down canyon. The Kaikōura Canyon habitat is now recovering from this removal of both seafloor substrate and associated organisms.We measured post-event benthic macrofauna density together with biomass and sediment properties and related these to infaunal bioturbation activity, sediment community oxygen consumption (SCOC) and associated benthic macronutrient biogeochemical fluxes. Three legacy sites were used as reference along a depth transect of the canyon axis at 917, 1249, and 1480 m water depths. We found distinct differences in benthic functioning between sites.While many parameters measured followed the expected relationships of decreasing with depth some benthic biological activity metrics did not. Our data suggests that the sites differ based on the relative impact and type of disturbance experienced during the earthquake-triggered, canyon-flushing event. We find SCOC was linked to bioturbatory behaviour and sediment organic matter content, rather than taxa density or biomass. While the greatest bioturbation activity was measured at the shallowest site, the greatest SCOC was measured at the middle depth site due to elevated sediment organic matter content.These observations provide a wider benchmark for infaunal bioturbation and faunal-mediated biogeochemical processes in deep-sea canyon environments and provide rare insights of recovery trajectories for deep-sea benthic communities and ecosystem function after large seafloor disturbances.

Comparative proteomic analysis of cold seep clam Archivesica marissinica and shallow water shellfish Ruditapes philippinarum provides insights into the adaptation mechanisms of deep-sea mollusks

IntroductionThe deep-sea environment is always characterized by high hydrostatic pressure, fluctuating temperatures, heavy metals, darkness, and others. The Vesicomyidae inhabit cold seep zones, hydrothermal vents, and other chemically reduced environments.MethodsTo enhance the understanding of the adaptation mechanisms of clams in extreme environments, a comprehensive proteomic study was conducted on the cold seep clam Archivesica marissinica and shallow water clam Ruditapes philippinarum.ResultsA total of 4,557 proteins were identified from the comparative groups. The Gene Ontology results indicated that the differentially expressed proteins (DEP) for the comparative group Rpgill vs. Amgill were enriched in the nitrogen compound metabolic process, and others. The comparative analysis for Amfoot vs. Amgill and Ammantle vs. Amgill revealed significant enrichment of the differential proteins that were involved in metal ion transport, divalent inorganic cation transport, and so on. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis for the comparative group Rpgill vs. Amgill was significantly enriched in the regulation of actin cytoskeleton, lysosome, and others. The proteins that exhibited differential expression in the cold seep clam’ different tissues were also enriched in important pathways, such as lysosome, fatty acid degradation, nitrogen metabolism.DiscussionThe further analysis identified crucial response proteins involved in various biological pathways. For example, the pattern recognition receptors, such as galectin and peptidoglycan recognition protein, participated in recognition of symbiotic microorganisms. The lysosome pathway members, such as cathepsin and saposins, were engaged in the degradation process of symbiont proteins during symbiont digestion. Profilin and gelsolin from actin cytoskeleton pathway might be pressure-related proteins. Furthermore, carbonic anhydrases from nitrogen metabolism KEGG pathway provide inorganic carbon for symbiotic bacteria. Additionally, Mn superoxide dismutase plays a role in the scavenging of superoxide anion radicals and antioxidant activity. Then, arginine kinases facilitate the low temperature adaptation of deep-sea shellfish with its cold adaptation characters. These findings offer novel perspectives on the proteins that are implicated in A. marisica’s response to cold seep environments, thereby contributing to the understanding of deep-sea biological adaptation and the preservation of deep-sea ecosystems.

Exploring the hidden treasures: Deep-sea bacterial community structure in the Bay of Bengal and their metabolic profile

Deep sea bacterial communities demonstrate remarkable adaptability to high-pressure environments coupled with low temperatures which has sparked curiosity about their diversity and exceptional metabolic pathways. Additionally, bacteria in the deep sea exert a substantial influence over various biogeochemical processes. To date, we have relatively very little information about the deep-sea bacterial communities and, they remain largely unexplored. We investigated the variability in the physicochemical conditions, heavy metals and their influence on deep-sea bacterial community structure across three different depths in the Bay of Bengal. The structural and metabolic diversity of deep-sea sediment microbial communities were examined through culture-based sequencing of 16S rRNA genes, ecto-enzymatic studies, and community-level physiological profiling. Bacillota was the most dominant phylum representing 61% of the cultured bacterial isolates, while the remaining belonged to Actinomycetota and Pseudomonodata. Five potential novel species belonging to the genera Fictibacillus, Lysinibacillus, Salinicola, Robertmurraya and Blastococcus were identified. The extracellular enzymatic activity was positive for >50% of the bacterial isolates, wherein the genera Bacillus and Micromonospora exhibited versatile profiles. High metabolic diversity was recorded through the carbon substrate utilization profiles indicating that microbial communities are active participants in biogeochemical cycles in the deep sea. The most prominently utilized carbon substrates were α-cyclodextrin, glucose-1-phosphate, D-xylose, glycogen, and 2-hydroxy benzoic acid which serve as organic substrates for microbial metabolism, facilitating the decomposition of organic matter and, recycling carbon in deep-sea ecosystems. Multivariate statistical analyses confirmed that the environmental variables had a profound influence on the bacterial community. The findings shed light on spatial variability in the bacterial community structure, enzyme activity and metabolic profiles, and enhance our understanding of Bay of Bengal deep-sea sedimentary microbial ecology.

A new deep-sea species of golden gorgonian (Octocorallia: Scleralcyonacea: Chrysogorgiidae) from Antarctic waters

The Southern Ocean harbours rich deep-sea ecosystems with local hotspots of benthic biodiversity. Still, many species, including deep-water octocorals, remain undescribed despite the fact that the exploration of the deep-sea has improved thanks to recent technological advances. In this context, a new species of golden gorgonian has been collected at 1407–1581 m depth during a recent cruise to Dronning Maud Land in the Eastern Weddell Sea. Currently, after recent changes in Chrysogorgia taxonomy, Chrysogorgia lunae sp. nov. (Octocorallia: Scleralcyonacea) is the only known representative of the genus in Antarctic waters. It corresponds to the "Squamosae typicae" and is characterized by a bushy colony with branching sequence of 2/5L and bell-shaped polyps. A molecular comparison with published sequences of Chrysogorgia species is provided based on a concatenated sequence of two mitochondrial genes, mtMutS and COI, as well as the 28S nuclear ribosomal gene.

A high-quality chromosomal genome assembly of the sea cucumber Chiridota heheva and its hydrothermal adaptation.

Chiridota heheva is a cosmopolitan holothurian well adapted to diverse deep-sea ecosystems, especially chemosynthetic environments. Besides high hydrostatic pressure and limited light, high concentrations of metal ions also represent harsh conditions in hydrothermal environments. Few holothurian species can live in such extreme conditions. Therefore, it is valuable to elucidate the adaptive genetic mechanisms of C. heheva in hydrothermal environments. Herein, we report a high-quality reference genome assembly of C. heheva from the Kairei vent, which is the first chromosome-level genome of Apodida. The chromosome-level genome size was 1.43 Gb, with a scaffold N50 of 53.24 Mb and BUSCO completeness score of 94.5%. Contig sequences were clustered, ordered, and assembled into 19 natural chromosomes. Comparative genome analysis found that the expanded gene families and positively selected genes of C. heheva were involved in the DNA damage repair process. The expanded gene families and the unique genes contributed to maintaining iron homeostasis in an iron-enriched environment. The positively selected gene RFC2 with 10 positively selected sites played an essential role in DNA repair under extreme environments. This first chromosome-level genome assembly of C. heheva reveals the hydrothermal adaptation of holothurians. As the first chromosome-level genome of order Apodida, this genome will provide the resource for investigating the evolution of class Holothuroidea.

Discovery of Prevalent Ciliophora, Discoba, and Copepoda Protists in Deep Sea Water by In Situ Nucleotide Extraction

Deep-sea eukaryotic microorganisms play a vital role in degrading organic matter and geochemically cycling elements in the deep ocean. However, the impact of sampling methods on detection of these microorganisms under high hydrostatic pressure remains uncertain. In this study, we compared a traditional water sampling method using a Niskin bottle, an in situ microbial filtration and fixation method (ISMIFF), and a multiple in situ nucleic acid collection (MISNAC) method to exhibit differences in the community structures that were collected at ~590–3100 m in the South China Sea (SCS). The classification and biodiversity indices of 18S rDNA Illumina sequencing reads from the V9 variation region revealed higher diversity for MISNAC DNA absorption column samples compared to others. Importantly, the relative abundance of Ciliophora (19.49%), Copepoda (23.31%), and Diplonemea (10.67%) was higher in MISNAC adsorption column samples, while Retaria (48.86%) were dominant in the MISNAC membrane samples. This indicates that MISNAC columns might collect more DNA in situ for the naked protists, while Retaria with a carbonate shell were more likely retained on the membrane. In conclusion, MISNAC is an effective method for DNA collection of deep-sea eukaryotic microorganisms and provides valuable materials for studying deep-sea microbial ecosystems.

The BBNJ Agreement: Through the Prism of Deep-Sea Vulnerable Marine Ecosystems

On 19 June 2023, member States of the United Nations adopted, by consensus, the Agreement under the United Nations Convention on the Law of the Sea on the Conservation and Sustainable Use of Marine Biological Diversity of Areas Beyond National Jurisdiction (BBNJ Agreement). The BBNJ Agreement seeks to address one of the greatest challenges of the 21st century—managing our deep ocean environment, including its ecosystems and resources, in a way that does not lead to its long-term decline and maintains its potential to meet the needs of present and future generations. This article assesses the BBNJ Agreement through an analysis of its application to deep-sea vulnerable marine ecosystems (VMEs). As VMEs represent a key component of marine biodiversity and are particularly susceptible to anthropogenic disturbance, they provide a useful medium for undertaking a legal analysis of the provisions, rules, and procedures of the recently adopted instrument.

A sequential one-pot approach for rapid and convenient characterization of putative restriction-modification systems.

The elucidation of the molecular basis of virus-host coevolutionary interactions is boosted with state-of-the-art sequencing technologies. However, the sequence-only information is often insufficient to output a conclusive argument without biochemical characterizations. We proposed a 1-day and one-pot approach to confirm the exact function of putative restriction-modification (R-M) genes that presumably mediate microbial coevolution. The experiments mainly focused on a series of putative R-M enzymes from a deep-sea virus and its host bacterium. The results quickly unveiled unambiguous substrate specificities, superior catalytic performance, and unique sequence preferences for two new restriction enzymes (capable of cleaving DNA) and two new methyltransferases (capable of modifying DNA with methyl groups). The reality of the functional R-M system reinforced a model of mutually beneficial interactions with the virus in the deep-sea microbial ecosystem. The cell culture-independent approach also holds great potential for exploring novel and biotechnologically significant R-M enzymes from microbial dark matter.