•RESEARCH PAPER• June 2020 Vol.63 No.6: 886–897https://doi.org/10.1007/s11427-020-1679-1

Diverse Asgard archaea including the novel phylum Gerdarchaeotaparticipate in organic matter degradation

Mingwei Cai1,2†, Yang Liu1†, Xiuran Yin3,4, Zhichao Zhou1,5, Michael W. Friedrich3,4,Tim Richter-Heitmann3, Rolf Nimzyk6, Ajinkya Kulkarni3, Xiaowen Wang1,2, Wenjin Li1,

Jie Pan1, Yuchun Yang5, Ji-Dong Gu5 & Meng Li1*

1Shenzhen Key Laboratory of Marine Microbiome Engineering, Institute for Advanced Study, Shenzhen University, Shenzhen 518060, China;2Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic

Engineering, Shenzhen University, Shenzhen 518060, China;3Microbial Ecophysiology Group, Faculty of Biology/Chemistry, University of Bremen, Bremen D-28359, Germany;

4MARUM, Center for Marine Environmental Sciences, University of Bremen, Bremen D-28359, Germany;5Laboratory of Environmental Microbiology and Toxicology, School of Biological Sciences, The University of Hong Kong, Hong Kong 999077,

China;6Department of Microbe-Plant Interactions, Faculty of Biology/Chemistry, University of Bremen, Bremen D-28359, Germany

Received February 27, 2020; accepted March 12, 2020; published online March 16, 2020

Asgard is an archaeal superphylum that might hold the key to understand the origin of eukaryotes, but its diversity and ecologicalroles remain poorly understood. Here, we reconstructed 15 metagenomic-assembled genomes from coastal sediments coveringmost known Asgard archaea and a novel group, which is proposed as a new Asgard phylum named as the “Gerdarchaeota”.Genomic analyses predict that Gerdarchaeota are facultative anaerobes in utilizing both organic and inorganic carbon. Unliketheir closest relatives Heimdallarchaeota, Gerdarchaeota have genes encoding for cellulase and enzymes involved in thetetrahydromethanopterin-based Wood–Ljungdahl pathway. Transcriptomics showed that most of our identified Asgard archaeaare capable of degrading organic matter, including peptides, amino acids and fatty acids, occupying ecological niches in differentdepths of layers of the sediments. Overall, this study broadens the diversity of the mysterious Asgard archaea and providesevidence for their ecological roles in coastal sediments.

Asgard archaea, Gerdarchaeota, coastal sediment, metagenome, metatranscriptome

Citation: Cai, M., Liu, Y., Yin, X., Zhou, Z., Friedrich, M.W., Richter-heitmann, T., Nimzyk, R., Kulkarni, A., Wang, X., Li, W., et al., (2020). Diverse Asgardarchaea including the novel phylum Gerdarchaeota participate in organic matter degradation. Sci China Life Sci 63, 886–897. https://doi.org/10.1007/s11427-020-1679-1

INTRODUCTION

Asgard archaea, proposed as a new archaeal superphylum,are currently composed of five phyla, i.e., Lokiarchaeota(Spang et al., 2015), Thorarchaeota (Seitz et al., 2016),

Odinarchaeota (Zaremba-Niedzwiedzka et al., 2017),Heimdallarchaeota (Zaremba-Niedzwiedzka et al., 2017),and Helarchaeota (Seitz et al., 2019), of which some en-compass lineages formerly named Marine Benthic Group B(MBG-B)(Vetriani et al., 1999), Deep-Sea Archaeal Group(DSAG) (Inagaki et al., 2001), Ancient Archaeal Group(AAG) (Takai and Horikoshi, 1999), and Marine Hydro-thermal Vent Group (MHVG) (Inagaki et al., 2003; Takai

© Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature 2020 life.scichina.com link.springer.com

SCIENCE CHINALife Sciences

†Contributed equally to this work*Corresponding author (email: limeng848@szu.edu.cn)

and Horikoshi, 1999). Asgard archaea contain abundanteukaryotic signature proteins (ESPs, e.g., endosomal sort-ing complex and actin-related proteins) and form a mono-phyletic group with eukaryotes in a phylogenetic treeinferred from 55 concatenated archaeo-eukaryotic riboso-mal proteins. Thus, they are regarded as the closest relativesof Eukarya and have therefore attracted increasing researchinterests (Spang et al., 2015; Zaremba-Niedzwiedzka et al.,2017; Zhou et al., 2018), although the debate about theevolutionary relationship is ongoing (Burns et al., 2018; DaCunha et al., 2017; Da Cunha et al., 2018; Imachi et al.,2020).Metabolic potentials of several Asgard phyla have been

predicted based on their genomic inventories: hydrogen de-pendency (Sousa et al., 2016) in Lokiarchaeota (which wereoriginally found in deep-sea sediments), mixotrophy (Liu etal., 2018) (i.e., using both inorganic and organic carbon forgrowth) and acetogenesis (Seitz et al., 2016) by Thor-archaeota from estuary sediments, metabolization of halo-genated organic compounds (Manoharan et al., 2019) byLoki- and Thorarchaeota, phototrophy (Bulzu et al., 2019;Pushkarev et al., 2018) in Heimdallarchaeota from coastalsediment and hydrothermal vent samples, anaerobic hydro-carbon oxidation in hydrothermal deep-sea sediment byHelarchaeota (Seitz et al., 2019), and participation in nitro-gen and sulfur cycles (Spang et al., 2019) by all Asgardarchaeal phyla. Metatranscriptomics revealed a few tran-scripts encoding NiFe hydrogenases from deep-sea Lo-kiarchaeota (Huang et al., 2019). Since no comprehensiveinformation about the in situ activity of these Asgard archaeahas been compiled so far, our understanding of these phy-logenetically and evolutionally important archaea is based onprediction, and thus, severely limited.Coastal environments, e.g., mangroves, salt marshes and

seagrass beds, are known sinks of blue carbon (Liang et al.,2019; Mcleod et al., 2011). Although these vegetated coastalecosystems make up less than 0.5% of the seabed, they hold~50% of organic carbon of the surface marine sedimentsglobally (Breithaupt et al., 2012; Kennedy et al., 2010;Mcleod et al., 2011). Here, we reconstructed 15 Asgardmetagenomic-assembled genomes (MAGs) from diversecoastal sediments and analysed them together with tran-scriptomes from mangrove sediments to clarify the ecolo-gical roles of the different Asgard clades in these importantenvironments. Based on phylogenetic analysis of this data-set, we propose a novel Asgard phylum, the “Gerdarchaeo-ta”. Additionally, we recruited all publicly available 16SrRNA sequences to see whether distinct Asgard lineagesshow distinct habitat preferences. Our findings substantiallyextend our knowledge of the lifestyles of these mysteriousarchaea in coastal sediments and their ecological roles oncarbon cycling.

RESULTS AND DISCUSSION

Mining Asgard archaea genomes leads to the proposal ofa new Asgard phylum

Sediments from several coastal sites (mangrove, mudflat andseagrass bed) were collected for deep metagenomic andmetatranscriptomic sequencing (totally 2.3 Tbp, Table S1 inSupporting Information). By combining individual samplesfrom the same site but from different depths of layers forassembly and binning, we recovered 15 Asgard MAGs withcompleteness of >80%. Phylogenetic analyses with a con-catenated set of 122 archaea-specific protein markers and 55archaeo-eukaryotic ribosomal proteins inferred that theseMAGs belong to known Asgard lineages, covering almost allphyla (except Odinarchaeota), i.e., Helarchaeota (n=1), Lo-kiarchaeota (n=2), Thorarchaeota (n=3) and Heimdallarch-aeota (n=9) (Figure 1A; Figure S1, Table S2 in SupportingInformation). Interestingly, the Heimdallarchaeota lineage iscomposed of three robust subclades including two previouslyknown branches, i.e., Heimdall-MHVG and Heimdall-AAG,with high branch support values (Figure 1A). The maximumidentity of 16S rRNA genes from Heimdall-MHVG andHeimdall-AAG to other Asgard groups is 66.6%–77.7% and69.6%–78.5% (Table S3 in Supporting Information), re-spectively, supporting the split of current Heimdallarchaeotainto two sub lineages Heimdallarchaeota-MHVG andHeimdallarchaeota-AAG, which confirms previous results(Spang et al., 2015). The remaining seven Heimdallarch-aeota-like MAGs clustering with B18_G1 formed a mono-phyletic group in a phylogenetic tree of concatenated 122archaea-specific protein markers and 55 archaeo-eukaryoticribosomal proteins (Figure 1A; Figure S1 in Supporting In-formation). The average nucleotide identity (ANI, 62%–65%to other Asgard archaeal phyla), amino acid identity (AAI,43%–45% to other Asgard archaeal phyla), and pan-genomeanalysis agreed on assigning phylum level identity to thismonophyletic group (Figures S2 and S3 in Supporting In-formation). Likewise, phylogenetic analysis of a 16S rRNA(1,152 bp) gene found in those seven MAGs (YT_re_meta-bat2_2.057) showed that it formed a new monophyleticbranch (Figure 1B) with an identity below 74% to 16S rRNAgenes of other Asgard archaeal phyla (Table S3 in Support-ing Information). Thus, we propose this lineage as a newphylum named Gerdarchaeota, after Gerd, the Norse goddessof fertile soil, because these Asgard archaea genomes wereobtained from organic-rich coastal environments (Miyatakeet al., 2013), such as mangrove, mudflat and seagrass (TableS1 in Supporting Information).The presence of eukaryotic signature proteins (ESPs) is a

characteristic feature of Asgard (Figure 1C; Table S4 inSupporting Information). Homologs encoding eukaryotic-type topoisomerase IB and fused RNA polymerase subunit Awere identified in Gerdarchaeota, while neither gene for

887Cai, M., et al. Sci China Life Sci June (2020) Vol.63 No.6

Figure

1Phylogeneticpositions

andES

Psof

Asgardarchaeareconstructed

from

coastalsediments.A

,Maximum

likelihoodtreeof

GerdarchaeotaMAGsbuilt

onaconcatenated

alignm

entof1

22archaea-

specificproteinmarkers.The

treewasinferred

with

LG+F+R

10mixturemodeinIQ-TREE

androoted

with

DPA

NNandEuryarchaeota.AsgardarchaeaMAGso

btainedinthisstudyaremarkedinboldface.B

,PhylogeneticpositionofAsgardarchaeal16SrRNAgenes.Red

colorrepresents1

6SrRNAgenesfromnewlydiscovered

AsgardMAGs,andblue

colorrepresentssequencesfrom

references.The

treewasbuilt

usingtheIQ-TREE

softw

arewith

GTR

+I+G

4mixturemodeandrooted

with

Crenarchaeota.C

,ESPsidentifiedinGerdarchaeotaandotherAsgardarchaea.AsgardarchaeaMAGsobtained

inthisstudyare

markedbold.C

olorsindicatephylum

-levelassignm

ent(see1A

).

888 Cai, M., et al. Sci China Life Sci June (2020) Vol.63 No.6

eukarya-specific DNA polymerase epsilon nor DNA-direc-ted RNA polymerase subunit G was detected (Figure 1C;Table S5 in Supporting Information). Gerdarchaeotal topoi-somerase IB clustered into a group with Thorarchaeota, andthese two lineages together were monophyletic with highsupporting values (Figure S4A in Supporting Information).The fused RNA polymerase A genes of Gerdarchaeotacluster into a group with Heimdallarchaeota, while those ofHelarchaeota branch with Loki- and Thorarchaeota (FigureS4B in Supporting Information). In both cases, eukaryoticgenes do not cluster with Asgard homologs. Besides, Ger-darchaeota also comprise expressed homologs of ribophorinI and STT3 subunit and lack OST3/OST6 homologs in mostgenomes except YT_bin5.010. The phylogenetic tree of ri-bophorin I showed that Gerdarchaeota is monophyletic andbranches with Heimdallarchaeota, and the eukaryotes branchwithin the cluster of Thor-, Hel- and Lokiarchaeota (FigureS4C in Supporting Information). In addition to the reportedESPs, we identified DAD/OST2 homologs within Ger-darchaeotal MAGs, which is a component of the N-oligo-saccharyl transferase for N-linked glycosylation (Silbersteinet al., 1995).

Metabolic potential of the new phylum Gerdarchaeota

Gerdarchaeota harbor the gene set for oxidative phosphor-ylation, including V/A-type ATPs, succinate dehydrogenase,NADH-quinone oxidoreductase, the key enzyme cyto-chrome c oxidase (three of seven Gerdarchaeotal MAGs)(Figure 2; Figure S5, Tables S6–S8 in Supporting Informa-tion), and enzymes for the non-typical cytochrome bc1complex (i.e., the Rieske iron-sulfur protein SoxL) (Mar-reiros et al., 2016) but are lacking genes for other respiratorycomplexes of type III (e.g., SoxN and CbsA), suggesting thatGerdarchaeota most likely perform aerobic respiration.Gerdarchaeotal cytochrome c oxidases are phylogeneticallyseparated into two lineages (Figure S6 in Supporting In-formation), one of which clusters closely with the facultativeanaerobic Crenarchaeotal Acidianus brierleyi (Konishi et al.,1999; Segerer et al., 1986) and the other one groups with thebacterial Synechocystis sp., which is capable of aerobic re-spiration in the dark (De Rosa et al., 2015). Meanwhile,Gerdarchaeotal MAGs harbor genes encoding heliorho-dopsins (Figure S7 in Supporting Information), which mightsense light in the top layers of sediment (Bulzu et al., 2019;Pushkarev et al., 2018). A complete tricarboxylic acid cycle(e.g., citrate synthase, malate dehydrogenase, fumarate hy-dratase, succinate-CoA ligase, isocitrate dehydrogenase, andaconitate hydratase; Figure 2; Table S6 in Supporting In-formation) further supports aerobic respiration as importantdissimilatory pathway. Besides, Gerdarchaeota are equippedwith enzymes for removal of As(V) and As(III), which candisrupt oxidative phosphorylation and inhibit respiratory

enzymes (Reitner and Thiel, 2011).Within Gerdarchaeota MAGs, complete gene sets for the

acetogenesis pathway (e.g., acetyl-CoA synthase) are present(Figure 2), showing their ability to transfer CO2 to acetate.We further identified all subunits of [NiFe]-hydrogenaseheterodisulfide reductase hdrABC. Since this communitydoes not contain the key enzymes mcrABC for methano-genesis, hdrABCmight function in both directions, hydrogenoxidation (supported by hydrogenases of [NiFe] Group 3c)and hydrogen formation (supported by hydrogenases of[NiFe] Group 3b), though it needs more evidence to confirm(Greening et al., 2016; Hua et al., 2018; Peters et al., 2015;Spang et al., 2019). Additionally, we identified homologs forferric reductase but their capability to use Fe(III) as electronacceptor remains open. Notably, the canonical nitrate re-ductase (previously identified in Heimdallarchaeota (Spanget al., 2019)) was not detected in Gerdarchaeota MAGs.Gerdarchaeota appear to use diverse organic compounds

(e.g., formaldehyde, amino acid, peptide, lipid and ethanol)as electron donors (Figure 2). Serine peptidases encodinggenes are over-represented (~44.1% of total peptidase, Fig-ure S8A in Supporting Information). Besides, we identifiedgenes for cellulose degradation (e.g., GH5 and GH9, FigureS8B, Table S9 in Supporting Information) and a near-com-plete gene set (except genes encoding glucokinase or hex-okinase), which may be further degraded through theEmbden–Meyerhof–Parnas (EMP) pathway (Figure 2). Thelack of genes encoding glucokinase might be due to genomeincompleteness, considering that it widely exists in otherAsgard archaeal phyla. Different from the facultative anae-robic relatives Heimdallarchaeota, Gerdarchaeota containthe complete genes for the reversable tetrahydromethan-opterin Wood–Ljungdahl (THMPT_WL) pathway and thekey enzyme acetyl-CoA decarbonylase/synthase (Figure 2).The presence of the genes for groups 3b and 3c [NiFe]-hydrogenases (Figure S9 in Supporting Information) impliesthat these archaea may grow using H2 as electron donors asalso reported for Lokiarchaeota (Sousa et al., 2016) or mayform hydrogen as reported for fermenting microorganisms(Müller et al., 2018). Meanwhile, we identified other po-tential CO2 assimilation pathways in Gerdarchaeota MAGs.For example, Gerdarchaeota have the potential to fix carbonvia the reductive citric acid cycle (e.g., citrate synthase(Nunoura et al., 2018), 2-oxoglutarate:ferredoxin oxidor-eductase, and fumarate reductase); and they harbor genescoding pyruvate:ferredoxin oxidoreductase required forgeneration of pyruvate from acetyl-CoA and CO2, whichunderpins the importance of inorganic carbon for biomasssynthesis (Yin et al., 2019). Different from other Asgardphyla, we did not find type III or type IV ribulose 1,5-bi-sphosphate carboxylase (RuBisCO) (Figure S10 in Sup-porting Information), which might function in the nucleotidesalvage pathway (Burns et al., 2018; Liang et al., 2019;

889Cai, M., et al. Sci China Life Sci June (2020) Vol.63 No.6

Figure

2Key

potentialm

etabolicpathwaysofGerdarchaeota.Solidlines

representpathw

aystepspresentin8MAGsassigned

toGerdarchaeota.D

ashedlines

representpathw

aystepsnotidentified.C

ircles

representtranscriptsfrom

surfacesedimentsandrectanglesrepresenttranscriptsfrom

subsurface

samples.T

herelativeabundanceofthetranscripts(transcriptsperm

illionreads,TP

M)foreach

gene

ismarked

with

differentcolors.Pathway

abbreviations:PPP,

pentosephosphatepathway

(archaea);

CBB,Calvin–Benson–Bassham

cycle;

EMP,

Embden–M

eyerhof–Parnas

pathway,TH

MPT

WL,

tetra-

hydrom

ethanopterin-dependent

Wood–Ljungdahlpathway;TC

Acycle,

tricarboxylic

acid

cycle.

Detailedmetabolic

informationfortheMAGsisavailablein

Figure

S5,ablesS6

andS7

inSupporting

Information.

890 Cai, M., et al. Sci China Life Sci June (2020) Vol.63 No.6

Marreiros et al., 2016; Segerer et al., 1986).Gerdarchaeota contain genes (e.g., [acyl-carrier-protein]

S-malonyltransferase and medium-chain acyl-[acyl-carrier-protein] hydrolase) for fatty acid synthesis, which are quitecommon among Asgard archaea. Most reported Asgardgenomes contain genes for glycerol-1-phosphate dehy-drogenase (G1PDH) involved in ether-bound phospholipidsynthesis (Da Cunha et al., 2017; Hua et al., 2018). A recentstudy reported a co-existence of enzymes for both ether-(G1PDH) and ester-bound (bacterial/eukaryal type, glycerol-3-phosphate dehydrogenase (G3PDH)) phospholipid synth-esis in some Lokiarchaeotal genomes, providing a hint thatAsgard archaea might produce chimeric lipids (Manoharanet al., 2019). Gerdarchaeota MAGs lack the key enzymeG1PDH for archaeal lipid biosynthesis, but contain thebacterial/eukaryal-type G3PDH for synthesis of bona fidebacterial lipids (Figure 2). Thus, Gerdarchaeota might haveevolved a bacteria-like membrane predating eukaryogenesis.The Asgard archaeal G3PDH is encoded by glpA (re-sponsible for the reverse reaction catalyzed by GpsA) insteadof GpsA, suggesting that G3PDH may participate in organiccarbon degradation rather than lipid synthesis (Villanueva etal., 2017; Yokobori et al., 2016), leaving the mechanism oflipid synthesis to be further explored.

Actively expressed genes of Asgard archaea in differentniches of coastal sediments

Gene expression patterns derived from metatranscriptomicanalysis have been used in a number of recent studies todeduce active microbial processes in marine sediments,especially in the deep sea (Li et al., 2015; Lloyd et al., 2018;Orsi et al., 2013). This technique might be constrained by thelargely unknown pool size of mRNAs maintained in en-dospores and dormant cells (Bergkessel et al., 2016), al-though the former seems to be irrelevant for Asgard archaea.Through recruiting 16S rDNA/rRNA sequences (n=10,448,read length >600 bp) belonging to Asgard archaea frompublic databases, we found that based on 16S rRNA ex-pression, all Asgard phyla are likely active under certaincircumstances (circle II in Figure S11 in Supporting In-formation) and diversely distributed (with ~92% of AsgardOTUs originating from sediment samples, Table S10 inSupporting Information). Thus, to better uncover their ac-tivities, we used 818,479 transcripts (mRNA) belonging toAsgard archaea including Loki-, Thor-, Hel-, Heimdall- andGerdarchaeota from mangrove sediments to elucidate theirecological roles in coastal sediments.Organic carbon in coastal sediments is mainly composed

of carbohydrates, amino acids, and lipids (Burdige, 2007;Miyatake et al., 2013). Accordingly, we detected high ex-pression levels of genes encoding extracellular peptidases,ABC transporter, and the enzyme sets (e.g., urocanate hy-

dratase and ornithine carbamoyltransferase) for the conver-sion of amino acids to acetyl-CoA in both surface andsubsurface coastal sediments, implying that Asgard archaeamight be essential participants in the degradation of thesesubstrates (Figure 2; Figure S5, Tables S6, S7 and S11 inSupporting Information). This notion is supported by thehigh proportion of peptidases in Asgard archaea MAGs(4.1%–6.3% of the functional genes, Figure S8A and TableS12 in Supporting Information). We also detected transcriptsfor ethanol metabolism (alcohol dehydrogenase, aldehydeferredoxin oxidoreductase), suggesting that ethanol might beanother substrate or product.Due to its higher yield in energy conservation compared to

fermentative processes, aerobic respiration contributes to50% or more of the total organic matter decomposition inoffshore marine sediments (Bergkessel et al., 2016). Theexpressed gene set for aerobic respiration in Gerdarchaeota,Heimdallarchaeota-AAG and Heimdallarchaeota-MHVG,including the key transcript of cytochrome c oxidase (be-longing to Gerdarchaeota) indicates that these Asgard ar-chaea might participate aerobically in organic matterdegradation in surface sediments (Figure 3). Although theAsgard archaea phyla co-inhabit the same sediment layers,distinct ecological roles are played by a given phylum. Forexample, unlike Heimdallarchaeota-AAG and Heimdal-larchaeota-MHVG, Gerdarchaeota contain and expressedgenes for autotrophy and cellulose degradation (Figure 3).Like Gerdarchaeota, other Asgard archaea have the potentialto perform anaerobic metabolisms (e.g., acetogenesis) underanoxic conditions (subsurface layers) (Sørensen et al., 1979).Notably, Helarchaeota-like mcrA transcripts found in un-binned scaffolds (e.g., SZ_4_scaffold_203331_2, Figure S12in Supporting Information) highlight the involvement ofHelarchaeota in alkane oxidation in coastal sediments, inwhich ethane and butane might originate from oil-gas see-page or human activities (Zhang and Zhai, 2015), and arepreferentially used as revealed by molecular modelling anddynamics studies (Figure S13 and Supplementary Results inSupporting Information).Previous studies suggested that Asgard archaea could ac-

count for up to 50% of the total prokaryotes in some marinesediments (Jørgensen et al., 2013), and attribute up to 40% ofthe total archaeal sequences in coastal sediments (Burdige,2007; Sørensen et al., 1979). The discovery of novel linea-ges, as well as the discovery of co-occurring diverse lineagesin one vertical biosphere in this study, may elevate theirrelative abundance and ecological roles in natural environ-ments. Therefore, we propose that Asgard archaea might beessential archaeal lineages for organic carbon degradation incoastal sediments, similar to previously proposed roles forBathyarchaeota (Pan et al., 2019) and Thermoprofundales(Zhou et al., 2019), which also support the notion that Asgardarchaea are critical participants for organic matter utilization

891Cai, M., et al. Sci China Life Sci June (2020) Vol.63 No.6

in coastal sediments (Biddle et al., 2006; Jørgensen et al.,2013).

CONCLUSION

Currently, the research of the diversity and ecology of As-gard archaea is still in its infancy. Contrasting previousstudies showed that Asgard archaeal MAGs covered notmore than three phyla in one sampling site (Bulzu et al.,2019; Seitz et al., 2019; Zaremba-Niedzwiedzka et al.,2017). Here, we demonstrate an expanded taxonomic spec-trum, as we obtained almost all known Asgard phyla in theorganic rich and diversified coastal sediment column (Bur-dige, 2007; Miyatake et al., 2013), and additionally found anew Asgard archaea phylum, the Gerdarchaeota. Metaboliccomparison and transcriptomic evidence suggest divergentecological roles and niches for different Asgard phyla butthey share key transcripts involved in the degradation ofspecific compartments of organic matter (e.g., peptides andamino acids). Thus, considering their high relative abun-dance (Burdige, 2007; Sørensen et al., 1979) and ubiquitous

distribution in coastal areas, we infer that Asgard archaea areimportant players for organic matter utilization (Pan et al.,2019; Zhang et al., 2019). However, their contribution to thecoastal sediment carbon budget remains to be further ex-amined. Overall, the metabolic features, transcript evidence,and their global distribution imply that Asgard archaea areessential players in carbon cycling of coastal sediments.

METHODS

Sediment sample collection and processing

Samples for metagenome analysis were collected from thecoastal sediment (i.e., mangrove, mudflat and seagrass se-diments) of China and Helgoland coastal mud area during theRV HEINCKE cruise HE443 (Table S1 in Supporting In-formation). They were sampled using custom corers, sealedin plastic bags in duplicates, stored in sampling box with icebags, and transported to the lab within 4 h. The physio-chemical parameters of the samples were determined aspreviously described (Zhou et al., 2017). Samples for RNAextraction were preserved in RNALater (Ambion, Life

Figure 3 Ecological niches of Asgard archaea in coastal sediments. Dashed lines represent pathways with no transcript for the key genes. Detailedinformation is available in Figure S5, Tables S6 and S7 in Supporting Information.

892 Cai, M., et al. Sci China Life Sci June (2020) Vol.63 No.6

Technologies). For each sample, 10 g of sediment each wasused for DNA and RNA isolation with the PowerSoil DNAIsolation Kit (MO BIO) and RNA Powersoil™ Total RNAIsolation Kit (QIAGEN), respectively. The rRNA genes wereremoved from the total RNA using the Ribo-Zero rRNAremoval kit (Illumina, Inc., USA) and the remaining mRNAwas reverse-transcribed. DNA and cDNA were sequencedusing an Illumina HiSeq sequencer (Illumina) with 150 bppaired-end reads at BerryGenomics (Beijing, China). Meta-transcriptomic reads were quality-trimmed using Sickle(version 1.33) (Joshi and Fass, 2011) with quality score ≥25,and the potential rRNA reads were removed using Sort-MeRNA (version 2.0) (Kopylova et al., 2012) against boththe SILVA 132 database and the default databases (E-valuecutoff ≤1×10–5).

Metagenomic assembly, genome binning and geneannotation

Raw metagenomic DNA reads of the coastal sediments weretrimmed and dereplicated (identical reads) using Sickle(version 1.33) (Joshi and Fass, 2011) with the option “-q 25”and the script “dereplicate.pl”. More than 89% of the readswere retained for each sample after quality control (Table S1in Supporting Information). Paired-end Illumina reads foreach sample were assembled de novo using IDBA-UD(version 1.1.1) (Peng et al., 2012) with the parameters“-mink 65, -maxk 145, -steps 10”. Scaffolds were binned intogenomic bins through MetaBAT (Kang et al., 2015) with 12sets of flags inducing different sensitivity and specificitycombinations followed by dereplication with DAS_Tool(Sieber et al., 2018) and manual curation (see SupplementaryMaterial) with trimmed reads for each sample. Briefly, 12sets of parameters (see Supplementary Methods) were set forMetaBAT binning (Zhou et al., 2020), and Das Tool wasfurther applied to obtain an optimized, non-redundant set ofbins. To improve the quality of the bins (e.g., scaffold lengthand bin completeness), each Asgard-related bin was re-mapped with the short-read mapper BWA and re-assembledusing SPAdes (version 3.0.0) (Bankevich et al., 2012) orIDBA-UD (version 1.1.1) (Peng et al., 2012), followed byMetaBAT and Das Tool binning (Zhou et al., 2020). AsgardMAGs with high contamination were further refined withAnvi’o software (version 2.2.2) (Delmont and Eren, 2018).The completeness, contamination and strain heterogeneity ofthe genomic bins were estimated by CheckM (version 1.0.7)software (Parks et al., 2015). Anvi’o software (version 2.2.2)(Delmont and Eren, 2018) was applied for pan-genomeanalysis of Asgard MAGs with the option “–min-occurrence3”.Protein-coding regions were predicted using Prodigal

(version 2.6.3) with the “-p meta” option (Hyatt et al., 2010).The KEGG server (BlastKOALA) (Kanehisa et al., 2016),

eggNOG-mapper (Huerta-Cepas et al., 2017), InterProScantool (V60) (Jones et al., 2014), and BLASTp vs. NCBI-nrdatabase searched on December 2017 (E-value cutoff≤1×10–5) were used to annotate the protein-coding regions.Archaeal peptidases were predicted against the MEROPSdatabase (Rawlings et al., 2015), and the extracellular pep-tidases were further identified using PRED-SIGNAL(Bagoset al., 2008) and PSORTb (Yu et al., 2010).

Phylogenetic analyses of Asgard MAGs

The 16S rRNA gene sequences and a concatenated set of 122archaeal-specific protein markers (Rinke et al., 2019; Van-wonterghem et al., 2016) and a concatenated set of 55 ar-chaeal genes (Jay et al., 2018) were used for phylogeneticanalyses of Asgard archaea. Ribosomal RNA genes in theAsgard MAGs were extracted by Barrnap (version 0.3,http://www.vicbioinformatics.com/software.barrnap.html).An updated 16S rRNA gene sequence dataset from referencepapers (Durbin and Teske, 2012; Spang et al., 2017) withgenome-based 16S rRNA genes were aligned using SINA(version 1.2.11) (Pruesse et al., 2012). The 16S rRNA genesequences maximum-likelihood tree was built with IQ-TREE (version 1.6.1) (Nguyen et al., 2014) using the GTR+I+G4 model (recommended by the “TESTONLY” model),with option “-bb 1000”. Marker genes for protein tree wereidentified using hidden Markov models (HMMs) and werealigned separately using hmmalign from HMMER3 (Mistryet al., 2013) with default parameters. The 122 archaea-spe-cific protein markers and 55 archaeo-eukaryotic ribosomalproteins were identified using hidden Markov models. Eachprotein was individually aligned using hmmalign (Eddy,2011). The concatenated alignment was trimmed by BMGEwith flags “-t AA -m BLOSUM30” (Criscuolo and Gribaldo,2010). Then, maximum-likelihood trees were built using IQ-TREE with the best-fit model of “LG+F+R10” followed byextended model selection with FreeRate heterogeneity and1,000 times ultrafast bootstrapping. The final tree was rootedwith the DPANN superphylum and Euryarchaeota.

Metabolic pathway construction

Potential metabolic pathways were reconstructed based onthe predicted annotations and the reference pathways de-picted in KEGG and MetaCyc (Caspi et al., 2007). Meta-transcriptome data from mangrove and mudflat sediments ofShenzhen Bay (Table S1 in Supporting Information) wereanalyzed to clarify the transcriptomic activity of Asgard ar-chaea. The abundance of transcripts for each gene was de-termined by mapping all non-rRNA transcripts to predictedgenes of all available Asgard archaeal MAGs (Table S2 inSupporting Information) using BWAwith default setting (Liand Durbin, 2009; Li et al., 2015). Normalized expression

893Cai, M., et al. Sci China Life Sci June (2020) Vol.63 No.6

was expressed in transcript per million units (TPM), fol-lowed by normalization by genome number of each phylum.

ESP identification

As predicted by prodigal (v2.6.3) with default parameters,genes of Gerdarchaeota were searched against InterPro andeggNOG databases to gain the IPRs and arCOGs. The list ofthose annotations in Zaremba-Niedzwiedzka et al. (2017)was searched for in the Gerdarchaeota bins. We also manu-ally inspected the IPRs and arCOGs only present in Ger-darchaeota. MAFFT-linsi and trimAl (-gappyout) were usedto align and trim the protein sequences. IQ-TREE (version1.6.1) (Nguyen et al., 2014) was used to infer phylogeny ofunder best-fit models with 1,000 ultrafast bootstraps withSH-aLRT test values.

Materials and correspondence

Archaeal 16S rRNA gene sequences were retrieved fromNCBI database, SILVA SSU r132 database, and a referencepaper as described in Supplementary Methods. Public As-gard MAGs were from NCBI database and MG-RAST. Thenewly obtained Asgard MAGs and metatranscriptomic dataare available in NCBI database under the projectPRJNA495098 and PRJNA360036. Supplementary tablesare available in figshare with the identifier https://figshare.com/articles/Supplementary_Material/11905680. Supple-mentary Data 1 and Data 2 are available with the identifierhttps://figshare.com/articles/Supplementary_Data_1/11906793 and https://figshare.com/articles/Supplementar-y_Data_2/11906814, respectively.

Compliance and ethics The author(s) declare that they have no conflictof interest.

Acknowledgements We thank Dr. Nidhi Singh for her suggestions inmolecular modeling. We thank the captain, crew and scientists of R/VHEINCKE expeditions HE443. This research was financed by the NationalNatural Science Foundation of China (91851105, 31622002, 31970105,31600093, and 31700430), the Shenzhen Science and Technology Program(JCYJ20170818091727570 and KQTD20180412181334790), the KeyProject of Department of Education of Guangdong Province(2017KZDXM071), the China Postdoctoral Science Foundation(2018M633111), the DFG (Deutsche Forschungsgemeinschaft) Cluster ofExcellence EXC 309 “The Ocean in the Earth System - MARUM - Center forMarine Environmental Sciences” (project ID 49926684) and the Universityof Bremen.

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SUPPORTING INFORMATION

Methods

Results

Figure S1 Maximum likelihood tree of Gerdarchaeota MAGs built on a concatenated alignment of 55 archaeal genes.

Figure S2 Average nucleotide identity (ANI) (a) and amino acid identity (AAI) of Asgard (b) archaeal MAGs.

Figure S3 Pan-genome analysis of protein clusters within all Asgard MAGs using the Anvi’o software.

Figure S4 Maximum likelihood phylogenetic analyses of Topoisomerase IB, ribophorin I, and DNA-directed RNA polymerase A.

Figure S5 Key potential metabolic pathways of Asgard archaea.

Figure S6 Phylogenetic position of the Gerdarchaeota cytochrome c oxidases subunit II.

Figure S7 Phylogenetic position of the Gerdarchaeota rhodopsins.

Figure S8 Abundance of (a) peptidases and (b) carbohydrate-active enzymes in Asgard MAGs.

Figure S9 Phylogenetic position of the Gerdarchaeota [NiFe]-hydrogenases.

Figure S10 Phylogenetic maximum likelihood tree of RuBisCO amino acid sequences (large subunit).

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Figure S11 Diversity and biotope of Asgard archaea.

Figure S12 Protein tree based on mcrA gene sequences as identified in the scaffolds.

Figure S13 Molecular modelling and dynamics of MCR complex.

Figure S14 Phylogenetic position and evolution of Asgard archaea mcrA genes.

Figure S15 Protein trees of Asgard archaea (a) McrB and (b) McrG.

Figure S16 Phylogenetic position of the Gerdarchaeota nifH.

Table S1 Information of sedimental samples analysed in this study

Table S2 Overview of Asgard archaea genomic bins

Table S3 The 16S rRNA gene identity (%) of Asgard groups Asgard lineage 1 to Asgard lineage 5 with other Asgard groups

Table S4 Accession numbers list of INTERPRO (IPR) domains and UniProtKB protein sequences related to ESPs

Table S5 Expression of ESPs in the novel Asgard archaea

Table S6 Gene annotation of Asgard archaea based on the NCBI-nr database, KEGG database, and InterProScan tool

Table S7 Overview of the transcript abundance (TPM) in surface and subsurface sediments at the phylum level

Table S8 ORFs of the scaffolds with cytochrome c oxidases

Table S9 Carbohydrate active enzymes numbers for each phylum

Table S10 Overview of 170 archaeal libraries that include Asgard archaeal 16S rRNA sequences

Table S11 Amino acid degradation pathways in Asgard archaea MAGs

Table S12 Summary of intracellular and extracellular peptidases from Asgard archaea MAGs.

Table S13 Characteristics of Asgard phyla

Supplementary Data 1 Phylogenetic position of Gerdarchaeota cytochrome c oxidases (newick format)

Supplementary Data 2 Phylogenetic position of Gerdarchaeota rhodopsins (newick format)

The supporting information is available online at http://life.scichina.com and https://link.springer.com. The supportingmaterials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and contentremains entirely with the authors.

897Cai, M., et al. Sci China Life Sci June (2020) Vol.63 No.6