Nature Microbiology
| Volume 8 | February
2023 | 231–245
231
nature microbiology
https://doi.org/10.1038/s41564-022-01297-4
Article
Evolutionary diversification of
methanotrophic ANME-1 archaea and their
expansive virome
Rafael Laso-Pérez
1,8
,1 0
, Fabai Wu
2,3,4,5
,1 0
, Antoine Crémière
5
,
Daan R. Speth
6,9
, John S. Magyar
5
, Kehan Zhao
2
, Mart Krupovic
7
&
Victoria J. Orphan
5,6
‘
Candidatus
Methanophagales’ (ANME-1) is an order-level clade of archaea
responsible for anaerobic methane oxidation in deep-sea sediments. The
diversity, ecology and evolution of ANME-1 remain poorly understood.
In this study, we use metagenomics on deep-sea hydrothermal samples
to expand ANME-1 diversity and uncover the effect of virus–host
dynamics. Phylogenetic analyses reveal a deep-branching, thermophilic
family, ‘
Candidatus
Methanospirareceae’, closely related to short-chain
alkane oxidizers. Global phylogeny and near-complete genomes show
that hydrogen metabolism within ANME-1 is an ancient trait that was
vertically inherited but differentially lost during lineage diversification.
Metagenomics also uncovered 16 undescribed virus families so far
exclusively targeting ANME-1 archaea, showing unique structural and
replicative signatures. The expansive ANME-1 virome contains a metabolic
gene repertoire that can influence host ecology and evolution through
virus-mediated gene displacement. Our results suggest an evolutionary
continuum between anaerobic methane and short-chain alkane oxidizers
and underscore the effects of viruses on the dynamics and evolution of
methane-driven ecosystems.
Anaerobic methanotrophic archaea (ANME) is a polyphyletic group of
archaeal lineages that have independently evolved the ability of anaero
-
bic oxidation of methane (AOM), a process that is estimated to remove
more than 80% of the methane produced globally in deep-sea sedi
-
ments
1
by reversing the methanogenesis pathway
2
. Whereas the ANME-2
and ANME-3 lineages share common ancestors with the present-day
methanogens of the
Methanosarcinales
order, ANME-1 archaea form
their own order ‘
Candidatus
Methanophagales’, which is sister to the
non-methane alkane degraders ‘
Candidatus
Syntrophoarchaeales’ and
‘
Candidatus
Alkanophagales’
3
. ANME-1 can grow beyond the cold and
temperate deep-sea habitats that they often share with other ANMEs,
uniquely thriving at higher temperatures within hydrothermal environ
-
ments
2
,
4
,
5
. In marine sediments, ANMEs mostly form syntrophic associa
-
tions with sulfate-reducing bacteria
6
via direct interspecies electron
Received: 13 June 2022
Accepted: 29 November 2022
Published online: 19 January 2023
Check for updates
1
MARUM, Center for Marine Environmental Science, and Department of Geosciences, University of Bremen, Bremen, Germany.
2
ZJU-Hangzhou Global
Scientific and Technological Innovation Center, Hangzhou, China.
3
Ocean College, Zhejiang University, Zhoushan, China.
4
Donghai Laboratory,
Zhoushan, China.
5
Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena, CA, USA.
6
Division of Biology and
Biological Engineering, California Institute of Technology, Pasadena, CA, USA.
7
Institut Pasteur, Université Paris Cité, CNRS UMR6047, Archaeal Virology
Unit, Paris, France.
8
Present address: Systems Biology Department, Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain.
9
Present address:
Max-Planck Institute for Marine Microbiology, Bremen, Germany.
10
These authors contributed equally: Rafael Laso-Pérez, Fabai Wu.
e-mail:
rafael.laso@cnb.csic.es
;
fabaiwu@zju.edu.cn
;
mart.krupovic@pasteur.fr
;
vorphan@gps.caltech.edu
Nature Microbiology
| Volume 8 | February
2023 | 231–245
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Article
https://doi.org/10.1038/s41564-022-01297-4
(Fig.
2b
), where ANME-1c were the dominant lineage according to
genome coverage (Fig.
2a
). These putative ANME-1c cells exhibit the
typical cylindrical shape previously reported for other ANME-1 popula
-
tions
6
and were loosely associated with bacterial cells in an extracellular
polymeric substances matrix, or found as single cells.
Physiological differentiation of diverse ANME-1 archaea
The deep-branching position of ANME-1c led us to examine the
genomic patterns of emergence and differentiation of ANME-1 from
the sister orders Alkanophagales and Syntrophoarchaeales. Like all
ANME-1, ANME-1c encode a complete reverse methanogenesis path
-
way including a single operon for the methyl coenzyme M reduc
-
tase enzyme (MCR), responsible for the activation of methane, and
the replacement of F
420
-dependent methylene-H
4
MPT reductase
by 5,10-methylenetetrahydrofolate reductase characteristic for
ANME-1
2
,
8
. Similar to other ANME clades, ANME-1c encodes several
multiheme cytochromes, which likely mediate the transfer of electrons
during syntrophic AOM to sulfate-reducing bacteria
2
,
7
,
8
.
Notably, ANME-1c exhibit distinct features compared to the
ANME-1a and ANME-1b in the operon encoding the MCR enzyme.
This enzyme consists of six subunits with the structure α
2
β
2
Υ
2
and
the unique nickel-containing cofactor coenzyme F
430
(ref.
26
). In the
maturation of this cofactor, McrC and McrD, two additional pro
-
teins encoded by the MCR operon in methanogens, are involved
27
,
28
.
Although
mcrD
is not present in ANME-1a and ANME-1b, both genes
are present in ANME-1c, where
mcrD
forms an operon with
mcrABG
(Fig.
1
). Previous analysis suggested that ANME-1 acquired the
mcr
genes from distant H
2
-dependent methylotrophic methanogens of
the class Methanofastidiosa
2
, whereas they lost the divergent MCRs
present in Syntrophoarchaeales and Alkanophagales, which seem to
use larger alkanes. Likewise, we found that the ANME-1c McrD is closely
related to the McrD of Methanofastidiosa but only distantly related
to the McrD of Syntrophoarchaeales and Alkanophagales that form
a different cluster (Extended Data Fig. 3). These results suggest that
during the emergence of ANME-1, a full operon of methane-cycling
mcr
(including
mcrCD
) was acquired by horizontal gene transfer from
a Methanofastidiosa-related methylotrophic methanogen, and
mcrD
was later lost in both ANME-1a and ANME-1b clades. The ANME-1c also
exhibit several additional genomic features that are distinct, high
-
lighted in Fig.
1
and described in Supplementary Information and
Supplementary Table 5.
Shared origin and differential loss of hydrogenases
Hydrogen was proposed as one of the first candidate intermediates in
syntrophic AOM, but fell out of favour after several genomic studies
showed that the majority of ANME genomes do not encode hydroge
-
nases. However, recent studies have reported NiFe-hydrogenases in
subclades of larger ANME groups, including an ANME-1b subclade
‘
Candidatus
Methanoalium’ and from select ANME-1a genomes
(Fig.
1
)
2
,
29
. Interestingly, the genomes of the sister orders Syntropho
-
archaeales and Alkanophagales encode a NiFe hydrogenase (Fig.
1
),
but physiological experiments did not support a role of this hydroge-
nase in syntrophic alkane oxidation
21
. Our expanded phylogenomic
analysis of ANME-1 confirm that genomes associated with three distinct
subclades of the ANME-1a, ANME-1b and now ANME-1c each encode a
NiFe hydrogenase operon (Fig.
1
). Phylogenetic analysis of the large
subunit of these hydrogenases revealed a monophyletic group of
ANME-1-affiliated hydrogenases clustering with those of Syntropho
-
archaeales and Alkanophagales (Fig.
3a
and Supplementary Table 6).
Hence, the occurrence of hydrogenases appears to be an ancient
trait of the class
Syntrophoarchaeia
that was vertically inherited by
the common ancestor of ANME-1 and later differentially lost during
ANME-1 clade diversification. Strikingly, the occurrence of hydroge
-
nase has an apparent mosaic distribution among MAGs even within
the hydrogenase-containing clades. For instance, within ANME-1c,
transfer
7
,
8
. However, some ANME-1 have been observed as single cells
or as monospecific consortia without partner bacteria
5
,
9
–
11
, and have
been proposed to perform hydrogenotrophic methanogenesis
10
–
12
,
although physiological experiments have thus far failed to support
this hypothesis
13
,
14
. Overall, it remains largely unclear what factors
have contributed to the physiological and ecological diversification
of ANME-1 from their short-chain alkane relatives and other ANME
lineages.
Despite the dominance of ANME archaea in many methane-rich
ecosystems, viruses targeting ANME lineages are largely unex
-
plored
15
–
17
. By exploiting and spilling host cellular resources through
their replication and lytic cycles, viruses play a major role in the eco
-
logical dynamics and nutrient cycling in diverse microbial systems
18
.
In deep-sea ecosystems, viral lysis has been estimated to cause annual
archaeal mortality that releases up to around 0.3–0.5 gigatons of car-
bon globally
19
. Characterizing the distributions and functions of viruses
of ANMEs is thus one of the most important tasks for quantitatively
linking ANME physiology to the elemental and energy flows in deep-sea
methane-driven ecosystems, and understanding the drivers of ANME
evolution.
Results
A unique ANME-1 clade from hydrothermal vents
In this study, we recovered 13 metagenome-assembled genomes (MAGs)
of ANME-1 in native and laboratory-incubated mineral samples from the
Southern Pescadero Basin hydrothermal vent system
20
in the Gulf of
California, Mexico (Supplementary Tables 1 and 2). These samples
not only expanded the known diversity within the ANME-1a clade,
particularly the ANME-1 G60 group, but also contained five MAGs
and one 1.6 Mb circular genome scaffold of a previously uncharacter-
ized deep-branching clade phylogenetically positioned at the base
of the ANME-1 order (Fig.
1
, Extended Data Fig. 1 and Supplementary
Tables 2 and 3). We name this family-level clade ‘
Candidatus
Methano
-
spirareceae’, or ANME-1c. Given its basal position, it is the phylo
-
genetically closest ANME-1 to the sister orders of non-methane alkane
degraders Alkanophagales and Syntrophoarchaeales
21
.
Our ANME-1c MAGs represent two different genera, ‘
Candidatus
Methanoxibalbensis’ and ‘
Candidatus
Methanospirare’ within the
same family with an average nucleotide identity of 76%, represented
by species ‘
Candidatus
Methanoxibalbensis ujae’ (species 1) and
‘
Candidatus
Methanospirare jalkutatii’ (species 2, Methods). Based
on genome coverage, these two ANME-1 species were the most abun-
dant organisms in rock samples 12,019 and NA091.008, whereas they
were hardly detected in rocks 11,868 and 11,719 and in hydrothermal
sediments (Fig.
2a
).
So far, all ANME-1c MAGs and 16S rRNA gene sequences from the
National Center for Biotechnology Information (NCBI;
https://www.
ncbi.nlm.nih.gov/
) and SILVA (
https://www.arb-silva.de/
) databases
have originated from hydrothermal environments, specifically the
sediments of Guaymas
11
,
22
and Southern Pescadero basins
23
. These
hydrothermal habitats are 400 km apart along the same fault sys
-
tem in the Gulf of California and exhibit 20% species-level overlap in
the microbial community
23
. This distribution suggests a strong ther-
mophilic physiological specialization of ANME-1c to hydrothermal
environments. Indeed, genome-based prediction
24
suggested a high
theoretical optimal growth temperature (OGT; Supplementary Table
4 and Extended Data Fig. 2) for both ANME-1c species (>70 °C) that
was higher than the average predicted OGT for both ANME-1a (62 °C)
and ANME-1b (52 °C). Such high temperature adaptation by ANME-1c
could be related to their reduced estimated genome size (‘
C
andidatus
Methanoxibalbensis ujae’: 1.81 Mb; ‘
C
andidatus
Methanospirare jalku
-
tatii’: 1.62 Mb) as previously observed in other thermophilic bacteria
and archaea
25
.
Using fluorescence in situ hybridization with an ANME-1-targeted
16S rRNA probe, we detected ANME-1 cells in rock NA091.008
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only two out of five MAGs of ‘
C
andidatus
Methanospirare jalkutatii’
(FW4382_bin126 and NA091.008_bin1) encode hydrogenases, whereas
the complete ‘
Candidatus
Methanospirare jalkutatii’ MAG FWG175,
assembled into a single scaffold, does not contain them. To verify that
this distribution is caused by the intraspecies variation rather than
incomplete genome assembly, we conducted independent metagen-
omic analyses that confirmed the differential presence of hydrogenase
genes within ANME strains of different rock samples (Fig.
3b
, Extended
Data Fig. 4 and Supplementary Information). Hydrogenases thus appear
to be a part of the pangenomic repertoire of certain ANME-1 subclades
and species, likely preserved in the ANME-1 pangenome as an envi
-
ronmental adaptation rather than as an absolute requirement for the
methanotrophic core metabolism.
The potential role of these hydrogenases in ANME-1 is still unclear.
Their phylogenetic position, next to hydrogenotrophic enzymes
30
of
the NiFe groups 1g and 1h (Fig.
3a
; only a few affiliated to NiFe Group
3 and 4, Extended Data Fig. 5), suggest a possible involvement in
hydrogenotrophic methanogenesis, as previously proposed based
on biochemical
31
, environmental
11
,
12
, isotopic
10
and metagenomic
data
29
, although enrichment cultivation attempts with hydrogen
have been unsuccessful
13
,
14
. Recently, the genomic analysis of the
hydrogenase-encoding ANME-1b group ‘
C
andidatus
Methanoalium’
showed the presence of distinct electron-cycling features (Rnf complex,
cytochrome b) and the absence of multiheme cytochromes suggest-
ing a methanogenic metabolism for this group
2
. By contrast, ANME-1c
encodes multiheme cytochromes and lacks these electron-cycling
features. Hence, the physiological utility of hydrogenases may vary
between lineages. Whereas hydrogen is likely not feasible as the sole
intermediate for syntrophic AOM
7
,
13
, it could be produced by ANME-1c
as an additional intermediate, as proposed in a mixed model involving
direct electron transfer and metabolite exchange
2
,
32
.
CRISPR-based discovery of an expansive ANME-1 mobilome
ANME-1 genomes recovered in this study contained various CRISPR–Cas
loci (Extended Data Fig. 6a), enabling the analysis of ANME-1-hosted
mobile genetic elements (MGEs) through CRISPR spacer-based
ANME-1 GLR107 (GCA_013139985.1)
ANME-1 AG-394-G21(GCA_009903435.1)
ANME-1 S3_bin35
ANME-1 S5_bin17
ANME-1 AG-394-G06 (GCA_009903405.1)
ANME-1 GoMg3.2 (GCA_013180565.1)
ANME-1 FP565147.1
ANME-1 CONS3730B06UFb1 (GCA_003336485.1)
ANME-1 WJOV01 (GCA_009618475.1)
ANME-1 GoMg4 (GCA_012979255.1)
ANME-1 S7_bin35
ANME-1 S6_bin16
ANME-1 Co_bin222
ANME-1 LC (GCA_014061035.1)
ANME-1 SpSt-1198 (GCA_011049045.1)
ANME-1 THS (GCA_004212135.1)
ANME-1 SA
ANME1_FWG146
ANME1_12019_bin9
ANME1_11719_bin3
MANV_001 (GCA_021159465.1)
ANME-1 G60 (GCA_003194435.1)
MANV_002 (GCA_021159475.1)
ANME-1 B48_G6 (GCA_003661165.1)
ANME1_FW4382_bin054
ANME-1 CONS3730MDAH03UFb1 (GCA_013374565.1)
ANME-1CONS3730F07p2b1 (GCA_013374555.1)
ANME-1 CONS3730H04p2b1 (GCA_013374505.1)
ANME-1 GoMg2 (GCA_013180585.1)
ANME-1 S3_bin12
ANME-1 ERB6
MBMC_171 (GCA_021161765.1)
ANME-1 S3_bin4
ANME-1 SCGC AAA252-L18 (GCA_020793595.1)
ANME-1 GoMg1 (GCA_013180605.1)
ANME-1 M5.MMPM (GCA_003160755.1)
ANME1_FWG148
ANME-1 B64_G16 (GCA_003661125.1)
ANME-1 WYZ-LMO12 (GCA_003601545.1)
ANME-1 WYZ-LMO13 (GCA_003601795.1)
ANME1_FWG147
ANME-1 ex4572-4 (GCA_002254785.1)
ANME-1 G37 (GCA_003194425.1)
ANME1_11719B_bin4
ANME1_NA091.008_bin1
ANME1_FW4382_bin126
ANME1_12019_bin1
ANME1_FWG175
ANME1_FW4382_bin044
ANME1_NA091.008_bin2
ANME-1 B22_G9 (GCA_003661195.1)
Alkanophagales B39_G2 (GCA_003661185.1)
Ca.
Syntrophoarchaeum butanivorans (LYOR00000000)
Syntrophoarchaeum HyVt-185 (DQZR00000000)
Syntrophoarchaeum Hyvt-386 (DRIE00000000)
Syntrophoarchaeum WYZ-LMO15 (PIXX00000000)
Syntrophoarchaeum_12019_bin3
Ca.
Syntrophoarchaeum caldarius (LYOS00000000)
Environment
Anoxic seawater/marine sediment
Cold seep
Hydrothermal sediment/rock
Mud volcano
Terrestrial/fresh water systems
OGT (°C)
33
42
46
50
55
66
69
72
77
63
60
Environment
Location
OGT
ANME-1c
ANME-1a
ANME-1b
Syntrophoarchaeales
Alkanophagales
flaB/FlgA
flaCDE
flaF
flaG
flaH
flaI
flaJ
flaK
pilA
pilA arch
cheA
cheB
cheC
cheD
cheF
cheR
cheW
cheY
MCP
comA
comB
comC
mdh
comD
comE
comDE
comF
MA_3297
mcrA
mcrB
mcrC
mcrD
mcrG
hupD/hyaD
hycI
hypA
hypC
hypD
hypE
hypF
hyd large
hyd small
glnA
nifH
nifD
nifI1
nifI2
pdaD(speA)
speB(arg)
speD
speE
argG
argH
CPS1
OTC/argF/argI
csdA
Transferase
proA
proB
proC
cdsB
nifK
thyX
thyA
Archaellum/Pili
Chemotaxis
CoM synthesis
Hydrogenase
N cycling
Amino acid/nucleotide
metabolism
MCR
0.5
>80%
Location
Aarhus Bay
California, USA
Crimean Sea
Eel River
Guaymas
Gulf of Cadiz
Gulf of Mexico
New Zealand
Hydrate Ridge
Lost City
Pescadero
Scotian Basin
South African Mine
Tibet
1
2
Fig. 1 | Phylogenomic tree and lineage differentiation of the ANME-1 order.
In bold, genomes retrieved from the South Pescadero Basin. The colour bars
indicate from left to right: environment, location, predicted OGT (in °C) and a
genomic comparison of some metabolic features (main text and Supplementary
Information). Numbers after the ANME-1c names indicate the two species of
ANME-1c and the stars after the names denote MAGs containing at least the large
subunit of a NiFe hydrogenase. Black circles indicate bootstrap support values
over 80%. The scale bar represents the number of nucleotide substitutions per
site. CoM, Coenzyme M.