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Communi y ecology and unc ional
po en ial o bac e ia, a chaea,
euka ya and i uses in Gue e o
Neg o mic obial ma
P. Maza‑Má quez
1,2*, M. D. Lee
1,3 & B. M. Bebou
1
In his s udy, he mic obial ecology, po en ial en i onmen al adap i e mechanisms, and he po en ial
e olu iona y in e linking o genes be ween bac e ial, a chaeal and i al lineages in Gue e o Neg o
(GN) mic obial ma we e in es iga ed using me agenomic sequencing ac oss a e ical ansec a
millime e scale. The communi y composi ion based on unique genes comp ised bac e ia (98.01%),
a chaea (1.81%), euka ya (0.07%) and i uses (0.11%). A gene‑ ocused analysis o bac e ia a chaea,
euka ya and i uses showed a e ical pa i ion o he communi y. The g ea es co e ages o genes
o bac e ia and euka ya we e de ec ed in i s laye s, while he highes co e ages o genes o a chaea
and i uses we e ound in deepe laye s. Many genes po en ially ela ed o adap a ion o he local
en i onmen we e de ec ed, such as UV adia ion, mul id ug esis ance, oxida i e s ess, hea y
me als, salini y and desicca ion. Those genes we e ound in bac e ial, a chaeal and i al lineages wi h
6477, 44, and 1 genes, espec i ely. The e olu iona y his o ies o hose genes we e s udied using
phylogene ic analysis, showing an in e linking be ween domains in GN ma .
Mic obial ma s a e one o he mos ancien ecosys ems known, ha ing pe sis ed h ough a ound 85% o he
Ea h’s his o y1 and played a key ole in he e olu ion o Ea h’s a mosphe e2,3. Today’s ma s a e mode n ana-
logues o hese i s ecosys ems on he Ea h. As mic obial ma s we e likely he loca ions a which oxygen was
i s p oduced in an o he wise anoxic wo ld, hey may o e an ecological model o unde s and bo h he e olu-
ion o biochemical cycles and mic obial adap a ion o d as ic en i onmen al changes, including he ad en o
an oxygena ed a mosphe e. Mic obes ound in mic obial ma s ha e been shown o exhibi a numbe o adap i e
esponses o ex eme en i onmen al condi ions. In one o he ew me agenomics s udies o mic obial ma s, genes
in ol ed in adap i e esponses and esilience agains high-UV i adia ion, ele a ed salini y condi ions, oxida i e
s ess and hea y me al esis ance we e desc ibed om mic obial ma s om Sha k Bay4– one o he mos ex ensi e
ma ine mic obial ma sys ems in he wo ld.
Gue e o Neg o mic obial ma is one o he bes s udied mic obial ma ecosys ems, and he use o molecula
analysis based on sequencing o small-subuni RNA genes and 454 sequencing ha e e ealed he high complex-
i y o his s a i ied ecosys em5,6. Ma s loca ed in sal e ns managed o he p oduc ion o sal a e pe manen ly
subme ged by a hype saline wa e column which se es o p o ec he e ical s uc u e o he ma s— ela i e o
ma s ound in in e idal en i onmen s subjec ed o mo e p o ound en i onmen al dis u bance7. Ea lie wo k
de ec ed a e ical s a i ica ion o oxygen and sul ide as well as a su p ising occu ence o sul a e educ ion in he
oxic zone o he ma 8. A ecen s udy e ealed a e ical pa e ns o ni ogen cycling genes wi h espec o e ical
a ia ions in oxygen concen a ion9. The e ical o ganiza ion o o he unc ions in hese communi ies has been
less well s udied. GN mic obial ma s a e cha ac e ized by ex eme e ical chemical g adien s a mic ome e o
millime e spa ial scales, and hese e ical g adien s ha e been shown o co espond o changes in mic obial
communi y composi ion6. Bac e ia and a chaea ha e been well documen ed in GN, as well as ungi in a ecen
s udy (wi hin he euka ya domain)10. Howe e , he composi ion and a iabili y o i uses and hei unc ions a
ine scale esolu ions in hese communi ies is less known. Th ough me agenomics, he cu en s udy p o ides
a axonomic desc ip ion o he e ical axonomic o ganiza ion as well as a unc ional o ganiza ion delinea ed
be ween bac e ia, a chaea, euka ya and i uses in a GN mic obial ma — e ealing new insigh s in o he ecology
o hese communi ies. The goals o he p esen s udy we e o cha ac e ize he communi y s uc u e and hei
unc ional po en ial in his mic obial ma h ough: (I) analyzing he unc ional anno a ions and axonomic
OPEN
1Exobiology B anch, NASA Ames Resea ch Cen e , Mo e Field, CA, USA. 2Uni e si y o G anada, G anada,
Spain. 3Blue Ma ble Space Ins i u e o Science, Sea le, WA, USA. *email: [email p o ec ed]
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classi ica ion o assembled genes spanning bac e ial, a chaea, euka ya and i uses; (II) examining po en ial
gene ic mechanisms o adap a ion in mic obial ma ; and (III) explo ing he in e linkages be ween genes p esen
in bac e ia, a chaea, euka ya, and i uses h ough gene-le el phylogene ic analysis.
Ma e ial and me hods
Mic obial ma sampling
The sampled mic obial ma s a e loca ed in sal e ns managed by he wo ld’s la ges sal -p oducing company
(Expo ado a de Sal SA ESSA), si ua ed on he Paci ic Ocean side o he Baja peninsula. Expo ado a de Sal has
minimum human ac i i y and consis s o a se ies o 13 concen a ion a eas wi h an app oxima e ex ension o
28,184 hec a es. The concen a ion a eas we e cons uc ed app oxima ely 2m abo e sea le el in he low lands
adjacen o he ponds “Ojo de Lieb e” and “Gue e o Neg o” (Fig.S1). The mic obial ma s we e collec ed in June
2019 om hype saline ponds concen a ion a ea 4 (Fig.S1), as p e iously desc ibed10. A he ime o ma col-
lec ion, salini y o b ine was 125 pp , empe a u e 24.4 C, pH 8.3, ammonium concen a ion 0.12μM, dissol ed
oxygen 7mg/L, and ni a e concen a ion was below he limi o de ec ion (< 0.5μM). Samples om he mic o-
bial ma s we e collec ed wi h a s ainless-s eel co e o 1cm diame e as p e iously desc ibed10. Th ee eplica e
co es we e placed in o s e ile cen i uge ubes (Falcon®, Co ning, Co ning, NY, USA), capped, and immedia ely
ozen in liquid ni ogen. To ge he e ical laye s a one-millime e in e als o he i s ou laye s (0–1, 1–2,
2–3 and 3–4mm om he op o he ma ), he ma was sec ioned using s e ile scalpels. Th ee eplica es we e
pooled o me agenomic analysis, esul ing in a single pooled me agenome o each dep h o lib a y p epa a-
ion and sequencing.
DNA ex ac ion
To al DNA ex ac ion ( om app oxima ely 0.20g pe sample) was pe o med om each mic obial ma laye ,
using a DNeasy Powe Bio ilm Ki , (Qiagen, Venlo, The Ne he lands), acco ding o manu ac u e ’s ins uc ions.
A nanopho ome e (Implen GmbH, München, Ge many) was used o checking he quali y (A260/A280) and
quan i y (A260) o ex ac ed genomic DNA. Lib a y p epa a ion and me agenomic sequencing we e pe o med
a Molecula Resea ch (MR DNA, Texas, USA,h p:// www. m dna. o g/ con a c . h ml). Lib a ies we e p epa ed
using he Nex e a DNA Flex lib a y ki (Illumina) ollowing he manu ac u e ’s ins uc ions, and sequencing
was pe o med on he No aSeq 6000 pla o m (2 × 150 nucleo ides).
Me agenomic da a p ocessing
Me agenomics p ocessing wi h anno a ed code is documen ed a ou open-Science Founda ion si e, h ps://
os . io/ 9kwn3/ wiki 11. Conda (2020; www. anaco nda. com (accessed on 10 Ma ch 2021)) was u ilized o p o-
g am ins alla ion and en i onmen managemen . Read quali y was scanned wi h Fas QC 0.11.912 and eads
immed/ il e ed wi h immoma ic 0.3913. A co-assembly o all 4 dep hs was pe o med wi h SPAdes 3.14.014,
he assembly was il e ed and summa ized wi h bi 1.8.1615 (see TableS1 o assembly summa y s a is ics),
and each indi idual samples’ eads we e mapped o he il e ed co-assembly wi h bow ie2 2.3.5.116 and so ed
and indexed wi h sam ools 1.917. Me agenomic sequence da a om he 4 dep hs a e a ailable h ough NCBI’s
Sequence Read A chi e a BioP ojec PRJNA688760. NCBI accession numbe s: SRX9761389, SRX9761388,
SRX9761387 and SRX9761386.
Taxonomic classi ica ion and unc ional anno a ion
Co-assembly and ead-mapping iles we e in eg a ed in o an i’o 6.218 o anno a ion wi h he KEGG da abase19
and pa sing and ex ac ion o he gene-le el co e age and de ec ion da a (wi h “de ec ion” being he p opo ion
o a gene ha ec ui ed any eads o i ). Gene-le el axonomic classi ica ion was pe o med wi h CAT 5.1.220
agains he NCBI n da abase. No maliza ion and analyses we e pe o med wi h R 3.6.3 (Co e Team 2017) in
Rs udio 1.1.456 (www. s ud io. com). To mi iga e non-speci ic ead- ec ui men , gene-le el co e age in o ma-
ion was il e ed based on de ec ion (p opo ion o a gene ha ec ui ed any eads o i ), such ha hose wi h
a de ec ion less han 50% had hei co e age se o 0. This had a ne e ec o emo ing less han 3% o he p e-
il e ed o al co e age.
In o ma ion on KEGG’s Me abolism pa hway was accessed wi h KEGGREST 1.26.0, de ining he KEGG
O hology (KO) e ms. The gene able co e ages we e no malized ac oss he 4 samples by di iding each alue
by i s sample’s o al co e age and mul iplying by 1 million o gene a e alues o Co e age pe Million (he ein
e e ed o as CPM). En ez Di ec 13.9, www. ncbi. nlm. nih. go / books/ NBK17 9288/) was u ilized o sea ch and
e ie e e e ence sequences om NCBI. Hea map and clus e analyses based on B ay Cu is simila i ies we e
calcula ed o all genes wi h a mean co e age o >= 9 in when summed ac oss he 4 samples.
T ee cons uc ion
Fo making phylogene ic ees, sequences we e aligned wi h Muscle 3.8.155121, and hen immed using imal
1.4.1. T ees we e gene a ed using he open-sou ce so wa e IQ-TREE 2.2.022. We in e ed he maximum-like-
lihood ee wi h au o-model selec ion ia he buil -in ModelFinde (op ion `-m MFP`) using 1000 boo s aps23.
The ees we e edi ed h ough he In e ac i e T ee o Li e web-in e ace24. Accu a ely oo ing is essen ial o he
co ec in e p e a ion o he gene ic changes be ween sequences since he oo gi es he di ec ionali y o e olu ion
wi hin he ee25. Howe e , due o he span o di e si y we we e conside ing (ac oss all domains), and o ha e
a consis en me hod o oo ing ac oss all he gene a ed gene ees, we u ilized mid-poin oo ing26. All ees in
Newick o ma a e a chi ed wi h h ps:// doi. o g/ 10. 6084/ m9. igsh a e. 25018 007 and a ailable he e: h ps:// doi.
o g/h ps:// doi. o g/ 10. 6084/ m9. igsh a e. 25018 007.
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Vi al analysis
Fo i al speci ic iden i ica ion, con igs om he ini ial co-assembly we e sc eened o i al sequences using VIR-
So e 2 3, hen checkV was passed o quali y con ol o he Vi So e 2 esul s, ollowing p e ious p o ocols27.
Vi al ead o each sample we e mapped using bow ie2 2.3.5.116 and so ed and indexed wi h sam ools 1.917.
An i’o 6.218 was used o anno a e i al gene and calcula e co e age p o iles. Fo i us gene le el axonomic
classi ica ion, wo da abases we e used: Re Seq8428 and IMGVR29. To a oid alse posi i e i al gene iden i ica-
ion, he genes we e il e ed based on wo c i e ia: genes axonomic classi ied in he Re Seq84 and/o IMGVR
da abases; and unc ional i al p o ein anno a ed in KEGG da abase.
Resul s
A gene‑ ocused iew o bac e ia, a chaea, euka ya, and i uses.
As desc ibed in Me hods, me agenomes om all 4 dep hs we e co-assembled oge he , genes we e iden i ied, and
no malized co e age alues we e a ained by ec ui ing he indi idual sample eads o he assembled con igs.
Mean co e age alues o genes we e ex ac ed, and o explo a o y pu poses hese mean co e age alues o each
sample we e no malized o be ou o 1 million (co e age pe million (CPM)). Genes iden i ied we e axonomi-
cally classi ied and unc ionally anno a ed, and he e we b eak down hose esul s. We also include in o ma ion
on ead-based classi ica ion, as a means o po en ially iden i y i any la ge biases migh ha e been in oduced
h ough he assembly- o-gene-calling p ocess, bu he esul s did no la gely a y in any cases.
A gene‑ ocused iew o Bac e ia
Taxonomically classi ied wi hin he bac e ial domain, 764,307 unique genes we e assembled and iden i ied, wi h
461,332, 679,559, 656,264 and 677,165 o hem ha ing a co e age g ea e han 0 in Laye 1, Laye 2, Laye 3,
and Laye 4, espec i ely (TablesS2 and S3; Fig.1A1). Howe e , he o al no malized co e ages o he bac e ial
genes dec eased wi h dep h (Fig.1A1). Laye 1 con ained he g ea es co e age o bac e ial genes, wi h 901,325.3
CPM, while Laye s 3 and Laye 4 con ained he lowes , 864,618.79 and 861,536.72 CPM, espec i ely. Hea map
and clus e analyses based on B ay Cu is esemblance o he genes a e shown in Fig.S2A1. The global simila -
i y was > 40%, wi h Laye 1 being he leas simila and clus e ing ou sepa a ely; he simila i y be ween Laye
2, Laye 3, and Laye 4 was > 80%. SIMPROF analysis based on B ay Cu is esemblance a 5% o signi icance
le el de ec ed a signi ican di e ence be ween Laye s 3 and Laye 4 (p = 0.001) and Laye 3-Laye 4, and Laye 2
(p = 0.001), and be ween Laye 3-Laye 4- Laye 2 and Laye 1 (p = 0.001) 32.8% o bac e ial genes we e success-
ully unc ionally anno a ed wi h KO e ms, comp ising 357,984 ± 13,375 CPM ac oss he 4 laye s (mean ± 1SD;
TableS2). Acco ding o KEGG’s g oupings, genes wi h >= 9 a e age CPM ac oss he 4 laye s we e asc ibed o
42 me abolic pa hways, wi h he mos abundan being ela ed o gene ic in o ma ion p ocessing, signaling and
cellula p ocesses, ca bohyd a e me abolism, and ene gy me abolism (TableS2).
Gene-le el axonomic classi ica ion wi hin he Bac e ia de ec ed 44 phyla, 106 amilies, and 430 species
(Fig.1A2, S3, TableS3). The dominan phyla we e Cyanobac e ia, ollowed by Chlo o lexi, P o eobac e ia, Fi -
micu es and Bac e oide es. Cyanobac e ia we e dominan in Laye 1, while Chlo o lexi was dominan in Laye
4. A amily le el, Mic ocoleaceae was he mos p e alen amily, ollowed by Coleo asciculaceae and Oscilla o i‑
aceae. A species le el, he dominan species we e Chlo o lexi bac e ium, Anae olineae bac e ium, Anae olineales
bac e ium, Gei le inema sp. PCC 9228 and Cyanobac e ia bac e ium J055. Chlo o lexi bac e ium, Gei le inema
sp. PCC 9228, Cyanobac e ia bac e ium J055 and Coleo asciculus ch honoplas es we e he p edominan species
in Laye 1, while Chlo o lexi bac e ium, Anae olineae bac e ium, Anae olineales bac e ium and The mo lexia
bac e ium we e dominan species in Laye 4.
A gene‑ ocused iew o A chaea
Fo he a chaeal domain, 14,148 unique genes we e iden i ied wi h 2,762, 8,338, 13,250, 13,793 ha ing co e -
ages > 0 o Laye 1, Laye 2, Laye 3, and Laye 4, espec i ely. The highes co e ages o genes we e de ec ed in
Laye 3 and 4 (Fig.1B1), which we e > 7 imes mo e abundan han in Laye 1. The dend og am in Fig.S2A2
shows he hea map and clus e analyses o samples based on B ay Cu is simila i ies o he a chaeal composi-
ion o he communi y. Laye s 3 and 4 we e mo e simila o e all, a > 70% simila i y, while Laye 1 was he leas
simila (Fig.S2A2). Acco ding o SIMPROF analysis, he e was a signi ican di e ence in he a chaeal gene
co e ages (based on an alpha alue o 0.05) be ween Laye 3 and Laye 4 (p = 0.022), Laye s 3- Laye 4 and Laye
2 (p = 0.001), and Laye s 3- Laye 4- Laye 2 and Laye 1 (p = 0.001). 25.7% o a chaeal genes we e success ully
unc ionally anno a ed wi h KO e ms, wi h hose unanno a ed accoun ing o 7,792.4 ± 5,215.8 CPM ac oss
he dep hs (TableS4). The 14,148 genes we e classi ied in o 26 KEGG me abolic pa hways (TableS4). Gene ic
in o ma ion p ocessing was he mos abundan pa hway, while he lowes abundan pa hways we e ela ed o
amino acids, cellula p ocesses, cell mo ili y, and sul u me abolisms (TableS4).
Gene-le el axonomic classi ica ion o a chaea is summa ized in Figs.1B2, S4 and TableS5. A o al o 20 phyla,
36 amilies and 496 species we e iden i ied. The dominan phyla we e Eu ya chaeo a, ollowed by Candida us
Lokia chaeo a, Candida us Mic a chaeo a, Candida us Woesea chaeo a, Candida us Tho a chaeo a. A amily
le el, Me hanosa cinaceae we e he dominan amily, ollowed by Me hano ichaceae, Me hanobac e iaceae and
Candida us Me hanope edenaceae. A species le el, Candida us Lokia chaeo a a chaeon we e he dominan spe-
cies, ollowed by The moplasma a a chaeon, Candida us Mic a chaeo a a chaeon and Candida us Woesea chaeo a
a chaeon.
A gene‑ ocused iew o Euka ya
Fo he euka yal domain, 547 genes we e de ec ed, wi h 403, 396, 298, 318 o hem ha ing co e ages > 0 o Laye
1, Laye 2, Laye 3 and Laye 4, espec i ely. The g ea es co e ages we e iden i ied in Laye 1, wi h 1,696.08 CPM
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(Fig.1C1). The co e ages in Laye s 2–4 we e > 6 imes lowe han in Laye 1, wi h 241.6, 188.6, 257.7 CPM o
Laye 2, 3 and 4, espec i ely. The hea map and clus e analysis ep esen ing he simila i y o he 547 genes wi h
dep h is show in Fig.S2A3. The global simila i y o he euka yal communi y was low, < 5%. Laye 1 ep esen ed
Figu e1. Summa ies o no malized gene-le el co e ages b oken down by domain; no e he x-axes a y
be ween A1-D1. (A1) Ba plo s o he genes iden i ied in bac e ia ac oss he uppe s 4 laye s examined [Laye 1
(0-1mm om su ace), Laye 2(1–2mm om su ace), Laye 3 (2–3mm om su ace), and Laye 4 (3-4mm
om su ace)]. (A2) Hea map showing he ead-based axonomic classi ica ion o bac e ia a species le el wi h
co e ages >= 9 ac oss he uppe s 4 laye s examined. (B1) Ba plo s o he CPM o he 14,184 genes iden i ied in
a chaea ac oss he uppe s 4 laye s examined [Laye 1 (0–1mm om su ace), Laye 2(1–2mm om su ace),
Laye 3 (2–3mm om su ace), and Laye 4 (3–4mm om su ace)]. (B2) Hea map showing he ead-based
axonomic classi ica ion o a chaea a species le el ac oss he uppe s 4 laye s examined. (C1) Ba plo s o he
CPM o he 547 genes iden i ied in euca yo e ac oss he uppe s 4 laye s examined [Laye 1 (0–1mm om
su ace), Laye 2(1–2mm om su ace), Laye 3 (2–3mm om su ace), and Laye 4 (3–4mm om su ace)].
(C2) Hea map showing he ead-based axonomic classi ica ion o euca yo es a species le el ac oss he uppe s
4 laye s examined. (D1) Ba plo s o he CPM o he 394 genes iden i ied in i uses ac oss he uppe s 4 laye s
examined [Laye 1 (0–1mm om su ace), Laye 2(1–2mm om su ace), Laye 3 (2–3mm om su ace), and
Laye 4 (3–4mm om su ace)]. (D2) Hea map showing he ead-based axonomic classi ica ion o i uses a
species le el ac oss he uppe s 4 laye s examined.
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he leas simila one, while he simila i y be ween Laye s 3 and 4 was > 80%. Acco ding o SIMPROF analysis,
he e was a signi ican di e ence in he co e age (based on an alpha alue o 0.05) be ween Laye 1 and Laye
2-Laye 3-Laye 4 (p = 0.001), and be ween Laye 2-Laye 3 and Laye 4 (p = 0.001), bu no be ween Laye 3 and
Laye 4 (p = 1), whe e he simila i y was > 80%. 26.7% o euka yal genes we e success ully unc ionally anno a ed
wi h KO e ms, wi h hose unanno a ed accoun ing o 377.6 ± 352.6 CPM ac oss he dep hs (TableS6). Acco d-
ing o KEGG o hology classi ica ion, he 547 genes we e g ouped in 11 me abolic pa hways (TableS6). The mos
abundan pa hways we e gene ic in o ma ion p ocessing and pho osyn hesis. O e wise, he mino co e ages o
genes we e ela ed o he pa hways sul u elay sys em and quo um sensing (TableS6).
Gene-le el axonomic classi ica ion o euka ya iden i ied 19 phyla, 119 amilies and 15 species (Fig.1C2, S5,
TableS7). Bacilla iophy a we e he dominan phyla, ollowed by S ep ophy a and Ascomyco a. A amily le el,
Thalassiosi aceae we e he mos p edominan amily, ollowed by Symbiodiniaceae and Bacilla iaceae. A species
le el, Thalassiosi a pseudonana was he p edominan species, ollowed by Symbiodinium mic oad ia icum and
Fis uli e a sola is.
A gene‑ ocused iew o Vi uses
835 genes we e assembled and axonomically classi ied as i al, wi h 451, 474, 791 and 759 o hem ha ing a
co e age g ea e han 0 in Laye s 1, 2, 3, and 4, espec i ely. The co e ages o i al genes inc eased wi h dep h,
wi h Laye s 3 and 4 ha ing co e age alues > 3 imes highe han in Laye 1 (Fig.1D1). Figu e S2A4 shows he
hea map and clus e analysis based on he co e ages o he 835 genes iden i ied. The global simila i y be ween he
laye s was < 20%, wi h Laye 1 again being he leas simila . A SIMPROF es a 5% o signi icance le el e ealed
no signi icance di e ence in he co e age o he genes be ween Laye 3 and Laye 4 (p = 0.35), bu de ec ed sig-
ni ican di e ences be ween Laye s 3-Laye 4 and Laye 2 (p = 0.001), and be ween Laye 2-Laye 3- Laye 4 and
Laye 1 (p = 0.001). 17.9% o iden i ied i al genes we e success ully unc ionally anno a ed wi h KO e ms, wi h
hose unanno a ed accoun ing o 1531.0 ± 1095.9 CPM ac oss he dep hs (TableS8). KEGG g ouped hese genes
in o 10 pa hways (TableS8), he highes co e ages we e ela ed o gene ic in o ma ion p ocessing and phage
e minase la ge subuni / phage eplica ion ini ia ion p o ein.
Figu eS6 shows he ead-based classi ica ion o he iden i ied i ome a phyla and amily le els: 3 phyla, 16
amilies and 149 species we e iden i ied (TableS9). The dominan phyla we e U o i ico a, ollowed by Nucleo-
cy o i ico a and Ho nei i ico a. A species le el, he mos abundan species was ela ed o uncul u ed Caudo i i‑
ce es phage, ollowing by P oka yo ic dsDNA i us sp., uncul u ed Medi e anean phage u MED, uncul u ed
Medi e anean phage, S ep omyces phage B ock, Cellulophage phage phi17:1, Podo i idae sp. c y5g4, Ma seille i‑
us LCMAC101 (Fig.1D2).
Li ing in Gue e o Neg o mic obial ma : bac e ia, a chaea, and i us genes ela ed o po en‑
ial adap a ion mechanisms
He e we ocus on eco e ed genes ela ed o an ibio ic and mul id ug esis ance, hea y me al oxici y, oxida i e
damage genes, cold, hea and phage shock p o eins, UV- adia ion s ess genes, salini y and desicca ion s ess
condi ions. We eco e ed 6477 unique genes anno a ed wi h hose unc ions ha we e classi ied wi hin he
bac e ial domain and 44 wi hin he a chaeal domain (Fig.2, TablesS10, S11, S12, S13, S14, S15, S16 and S17).
Mo eo e , one gene ( ela ed o UV-DNA damage endonuclease TableS18) was eco e ed ha was classi ied
as o igina ing om a i us. Acco ding o KEGG, hose genes we e classi ied wi hin memb ane anspo (76),
signaling and cellula p ocesses (488), an imic obial esis ance genes (245), signal ansduc ion (394), gene ic
in o ma ion p ocessing (4220), me abolism (758), anspo and ca abolism (24), eplica ion and epai (10)
and ene gy me abolism (301) (Fig.2). The p esence o hese genes may ha e implica ions o adap abili y and
esilience o s ess condi ions, as has been p e iously desc ibed in ano he mic obial ma 4.
Genes ela ed o po en ial adap a ion mechanism in Bac e ia
Fo Bac e ia, 6477 genes ela ed o po en ial en i onmen al adap a ion we e de ec ed (Fig.2A and supplemen a y
TablesS10–S16). Acco ding o KEGG o hology classi ica ion, he 6477 genes we e classi ied wi hin memb ane
anspo (76), signaling and cellula p ocesses (480), an imic obial esis ance genes (245), signal ansduc ion
(393), gene ic in o ma ion p ocessing (3834), me abolism (749), anspo and ca abolism (24), eplica ion and
epai (370) and ene gy me abolism (301): 564 unique genes we e ela ed o mul id ug esis ance p o ein/mul i-
d ug e lux pump (TableS10); 86 unique genes we e de ec ed o mul iple an ibio ic esis ance p o ein; 280 genes
we e de ec ed o luo oquinolone, ca echol, e acycline, osmidomycin, qua e na y compound, e acycline
and ancomycin esis ance p o ein; 92 unique genes we e iden i ied o esis ance o a senical, coppe , me cu ic,
ellu i e and zinc (TableS11); 25 genes we e ela ed o hea y me al (TableS12); 410 in ol ed in dealing wi h
oxida i e s ess (TableS13); 237 genes we e ela ed o cold shock p o eins (TableS14); 283 genes we e iden i ied
as hea shock p o eins (TableS14); 281 genes o phage shock p o ein (TableS14); 997 genes we e ela ed o
UV damage: excinuclease ABC subuni ABC, DNA helicase II /ATP-dependen DNA helicase Pc A, UV DNA
damage endonuclease, and ATP-dependen DNA helicase U sW (TableS15).; and 3222 genes we e associa ed
wi h desicca ion and salini y condi ions (TableS16): 1329 genes we e anno a ed as RNA polyme ase sigma-
70 ac o , ECF sub amily ( poE), 301genes o F- ype H + /Na + - anspo ing ATPase subuni be a (ATPF1B,
a pD), 281 genes o chape onin G oEL (g oEL, HSPD1), 251 genes o chape onin G oES (g oES, HSPE1), 259
genes o molecula chape one G pE (GRPE), 121 genes o molecula chape one DnaJ (dnaJ), 116 genes o
molecula chape one DnaK (dnaK, HSPA9), 15 genes o ehalose 6-phospha e syn hase (o sA), 35 genes o
ehalose 6-phospha e phospha ase (o sB), 81 genes o osmop o ec an anspo sys em subs a e-binding
p o ein (opuC), 113 genes o glycine be aine/p oline anspo sys em ATP-binding p o ein (p oV), 106 genes
o glycine be aine/p oline anspo sys em pe mease p o ein (p oW), 206 genes o glycine be aine/p oline

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anspo sys em subs a e-binding p o ein (p oX) and 8 genes o L-ec oine syn hase (ec C). Summa y o hese
genes and hei no malized co e ages a e showed in Fig.2A.
O e all, laye 1 con ained he highes co e ages o genes in ol ed in an ibio ic and mul id ug esis ance
(pumps ATP-binding casse e, sub amily B, mul id ug e lux pump and memb ane usion p o ein, mul id ug
e lux sys em), esis ance o me als (a senic, coppe esis ance p o ein, ellu i e, ellu ium and e acycline genes),
hea y me al, oxida i e s ess genes (supe oxide dismu ase, Cu–Zn amily, supe oxide dismu ase Fe, Mn amily
and supe oxide oxidase) and phage and hea shock p o eins. On he o he hand, deepe laye s con ained he g ea -
es co e ages o genes ela ed o mul id ug esis ance genes (MFS anspo e , ACDE amily, mul id ug esis ance
p o ein, mul id ug esis ance p o ein, MATE amily and ou e memb ane p o ein, mul id ug e lux sys em), zinc
esis ance gene, supe oxide educ ase gene, cold shock p o ein and genes associa ed wi h UV- esis ance/ epai
(u A, u B, u C and u D genes). Genes in ol ed wi h desicca ion and salini y s ess condi ions had simila
Figu e2. (A) Hea map showing he co e ages o po en ially adap a ion- ele an genes p esen in bac e ia
ac oss he uppe s 4 laye s examined. (B) Hea map showing he co e ages o po en ially adap a ion- ele an
genes p esen in a chaea ac oss he uppe s 4 laye s examined. (C) Hea map showing he UV gene p esen in
i us ac oss he uppe s 4 laye s examined.
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co e ages ac oss laye s, de ec ing he g ea es co e ages o genes o encoded he p oduc ion o exopolysac-
cha ides ( poE) and molecula chape ones (G oES, G oEL, DnaJ and DnaK). Deepe discussion o explain hei
ele ance in he con ex o po en ial ecological adap a ions o mic obial ma s is de ailed in Sec ion“Discussion”.
Genes ela ed o po en ial adap a ion mechanism in A chaea
In he a chaeal domain, 44 genes iden i ied in he me agenome da a we e ela ed o s ess condi ions: 2 o mul i-
d ug esis ance, 4 o mul iple an ibio ic esis ance, 8 o in e ac ions wi h oxida i e genes, 16 shock p o eins and
14 o UV genes (Fig.2B, TableS17). Laye 3 and 4 con ained he g ea es co e ages o mul id ug esis ance genes,
an ibio ic, oxida i e genes (supe oxide educ ase and supe oxide dismu ase), cold and phage shock p o eins and
UV genes. On he o he hand, an ibio ic esis ance genes we e no eco e ed in Laye 1 o 2; while hea shock
p o ein hsIJ was highe han h pX and he g ea es co e ages we e ound in Laye 2. Acco ding o KEGG o hol-
ogy classi ica ion, he 44 genes we e classi ied wi hin signaling and cellula p ocesses (8), signal ansduc ion (1),
gene ic in o ma ion p ocessing (18), me abolism (9), eplica ion and epai (8). Deepe discussion o explain hei
ele ance in he con ex o po en ial ecological adap a ions o mic obial ma s is de ailed in Sec ion“Discussion”.
In e linking be ween he genes p esen in bac e ia, a chaea and i uses using phylogene ic
analysis
To u he examine he dis ibu ion o genes ela ed o po en ial adap a i e mechanisms in e ms o hei e o-
lu iona y ela edness, we buil phylogene ic ees. A o al o 12 ees we e buil o he genes: MFS anspo e ,
ACDE amily, mul id ug esis ance p o ein (KO ID K08221), small mul id ug esis ance pump (KO ID K03297),
supe oxide dismu ase, Fe–Mn amily (KO ID K04564), supe oxide educ ase (KO ID K05919), hea shock p o ein
(KO ID K03799), cold shock p o ein (KO ID K03704), phage shock p o ein (KO ID K03973), excinuclease ABC
subuni A (KO ID K03701), excinuclease ABC subuni B (KO ID K03702), excinuclease ABC subuni C (KO
ID K03703), DNA helicase II ATP dependen DNA helicase (KO ID K03657) and UV DNA damage endonu-
clease (KO ID K13281). These genes we e p esen in bac e ia, a chaea and i uses domains as ollow: o KO ID
K08221: 14 sequences we e p esen in bac e ia and 1 in a chaea; o KO ID K03297: 21 sequences we e de ec ed
in bac e ia and 1 in a chaea; o KO ID K04564: 171 sequences we e p esen in bac e ia and 1 in a chaea; o
KO ID K05919: 107 sequences we e ound in bac e ia and 7 in a chaea; o KO ID K03799: 106 sequences we e
p esen in bac e ia and 4 in a chaea; o KO ID K03704: 221 sequences we e ound in bac e ia and 7 in a chaea;
o KO ID K03973: 114 sequences we e p esen in bac e ia and 2 in a chaea; o KO ID K03701: 362 sequences
we e p esen in bac e ia and 5 in a chaea; o KO ID K03702: 193 sequences we e p esen in bac e ia and 1 in
a chaea; o KO ID K03703: 203 sequences we e ound in bac e ia and 3 in a chaea; o KO ID K03657: 214 we e
iden i ied in bac e ia and 1 in a chaea; o KO ID K13281: 17 sequences we e de ec ed in bac e ia, 2 in a chaea
and 1 in i uses. Figu es3, 4, 5 and 6 show he phylogene ic ela ionships be ween domains; he comple e ees
a e in he supplemen a y ma e ial Figs.S7–S15. And see Table1 and he co esponding supplemen al ables o
gene IDs and ull amino-acid sequences.
Figu e3 shows he phylogene ic ee o MFS anspo e , ACDE amily, mul id ug esis ance p o ein (yi G,
ym D, y mO; 3A) and small mul id ug esis ance pump (em E, qac, mm , sm ; 3B). Fo ACDE amily, mul id ug
esis ance p o ein (yi G, ym D, y mO) (Fig.3A), he gene ID 57669 p esen in a chaea belonging o Candida us
Me hanolli ie a hyd oca bonicum was be ween clades holding genes classi ied as sou ced om he ollowing
bac e ial lineages: Spi ochae es bac e ium (gene ID 391435), Candida e di ision Zixibac e ia bac e ium (gene
ID 13873), Bac e oide es bac e ium (genes IDs 219193, 511031), Tang ei ania di e sio iginum (gene ID 127482)
and he genes IDs 158770, 316573, 83331 and 254423. Wi h espec small mul id ug esis ance pump (em E,
qac, mm , sm ) (Fig.3B), he gene ID 419991 p esen in A chaeoglobales a chaeon o med a clade wi h 4 genes
p esen in bac e ia: he genes IDs 424272, 737623, gene ID 683720 p esen in Desul onemais himo onii and gene
ID 212872 p esen in Ola ius sp. associa ed p o eobac e ium Del a1.
Figu e4 and Fig.S7–S8 show he phylogene ic ees o supe oxide dismu ase, Fe–Mn amily (SOD2) (Fig.4A
and S7) and supe oxide educ ase (d x) (Fig.4B and S8). Fo SOD2, he gene ID 550693 p esen in a chaea
o med a clade wi h a gene ID 201256 p esen in bac e ia. Fo d x gene, he gene ID 612886 p esen in a chaea
o med a clade wi h 3 genes p esen in bac e ia lineages: wo p esen in Del ap o eobac e ia (genes IDs 56860,
26015) and one p esen in bac e ia gene ID 612887. The gene ID 571069 p esen in Candida us Mic a chaeo a
was closely ela ed o genes p esen in Del ap o eobac e ia (gene ID 59597) and Desul obac e aceae (gene ID
241743). Finally, 5 genes p esen in a chaea wi h genes IDs 323416, 494295, 151464, 732097, 733106 p esen s in
The moplasma ales and The moplasma a o med a clade. This clade was closely ela ed o a clade o med by he
bac e ial lineages Candida us Cloacimonas sp. (411781), Pep oclos idium li o ale (685247), Clos idia (847882),
Planc omyce es bac e ium (315242) and he gene IDs 40055, 525801, 693963, 411780, 6209.
Figu e5 and Figu esS9–S11 show he phylogene ic ees co esponding o hea , cold and phage shock p o ein.
Fo hea shock p o ein H pX (h px) (Fig.5A and S9), he gene IDs 443088 and 222562 om he a chaea lineages
o The moplasma a and Candida us Mic a chaeo a o med a clade wi h a gene eco e ed om a Del ap o eobac‑
e ia (gene ID 17526). The genes IDs 350980 and 60713 p esen in a chaea deeply b anched in be ween clades
holding bac e ial genes om Planc omyce es (563474, 523502, 102322), Phycisphae ales (754540), candida e
di ision Zixibac e ia (73164) and Desul o us is sp (446773). Rega ding o cold shock p o ein (cspA) (Fig.5B and
S10), he genes ID 806860, 245087, 649029, 199423 and 224539 de ec ed om he a chaeal lineages o The mo‑
plasma a, Candida us Aenigma chaeo a a chaeon and The moplasma ales o med a clade wi h wo genes p esen
in Chlo o lexi bac e ium (genes IDs 641093, 589131, 623971, 155751). The genes ID 454595 and 191917 p esen s
in Candida us Woesea chaeo a and Eu ya chaeo a we e in a deeply b anching clade wi h genes om Planc omy-
ce o a (gene ID 76796) and Bac e oide es (genes IDs 20000, 628969). Fo phage shock p o ein C (pcpc) (Fig.5C
and S11), he gene ID 259327 p esen in a chaea o med a clade wi h genes om Bac e oide es bac e ium (gene
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ID 585054). The gene ID 254336 p esen in Me hanomas siliicoccalesa o med a clade wi h bac e ial genes om
Alphap o eobac e ia bac e ium (gene ID 125844) and Bac e oide es bac e ium (gene ID 585054).
The phylogene ic ees ep esen ing he UV genes (u A, u B, u C, u D, pc A, u sE, UVE1) a e shown
in Fig.6 and in he supplemen a y Figs.S12–S15. Fo u A gene (Fig.6A and S12), h ee genes classi ied as
coming om he a chaeal lineages o Eu ya chaeo a and The moplasma a (genes IDs 848457, 361714, 636321)
o med a clade wi h a bac e ia gene ID 519469 om Candida us Buchananbac e ia bac e ium. One gene clas-
si ied as coming om Me hano he mobac e (gene ID 727168) was wi hin a deep clade wi h bac e ial genes
om Phycisphae ae (gene ID 701638) and B achyspi a (gene ID 577760). Fo u B gene (Fig.S13), he gene ID
653100 axonomically classi ied as The moplasma a b anched nea genes classi ied as Planc omyce o a (genes IDs
728796, 54766). Fo u C gene (Fig.6B and S14), he genes IDs 699307, 905297, 647629 co esponding o a chaea
o med a clade wi h 6 genes p esen in bac e ial lineages: Pseudomonado a (gene ID 873573), Spi ochae es (gene
ID 664616), Spi ochae ia (gene ID 391558), Spi ochae ia (gene ID 399873), Spi ochae ia (gene ID 179402) and
Gemma imonade es bac e ium (gene ID 82391). Fo u D, pc A gene (Fig.S15), he gene ID 181004 p esen
in Candida us Ba hya chaeo a o med a clade wi h a gene ID 297131 p esen in Bac e oidales bac e ium. Fo
u sE, UVE1 gene (Fig.6C), wo genes p esen in The moplasma a (genes IDs 807563 and 510363) and one gene
p esen in uncul u ed Caudo i ice es phage (gene ID 667218) o med a clade wi h 3 genes p esen in bac e ia
(genes IDs 572443, 553365 and 74783, ep esen ing a Chlo o lexi bac e ium, an unclassi ied bac e ium, and a
Del ap o eobac e ia bac e ium, espec i ely).
Discussion
Gue e o Neg o mic obial ma is one o he bes s udied mic obial ma ecosys ems; howe e , he e ical unc-
ional o ganiza ion has been less well s udied. In his s udy, 922,765 unique gene-copies we e eco e ed (mean-
ing assembled and p edic ed), wi h 84.51% o hose being classi ied o a leas he domain le el, lea ing 15.49%
unclassi ied (TableS19). The g ea es co e ages o bac e ia and euka ya genes we e de ec ed in Laye 1, while he
highes co e ages o i uses and a chaea genes we e ound in Laye s 3 and 4 (Fig.1). The uppe one-millime e s
254423
Tang ei aniadi e sio iginum(127482)
Bac e oide esbac e ium (511031)
83331
316573
158770
Bac e oide esbac e ium (219193)
Candida usMe hanolli ie a hyd oca bonicum(57669)
Spi ochae esbac e ium(391435)
candida edi ision Zixibac e iabac e ium(13873)
Gemma imonade esbac e ium(161363)
6839
625332
130037
240999
T eescale: 0.1
135285
Halieaceae bac e ium (692358)
333319
Xan homonadales bac e ium (165873)
15297
360116
654474
498441
Rhodo ulum sp.12E13 (803754)
Roseicyclus(115830)
Rhodobac e aceae bac e ium HLUCCO18 (404976)
789305
209865
392516
572799
26137
A chaeoglobales a chaeon (419991)
424272
Desul onemaisishimo onii (683720)
737623
Ola iussp.associa ed p o eobac e iumDel a1(212872)
Roseospi a isakhapa namensis (440177)
T eescale:0.1
A. MFS anspo e , ACDE amily, mul id ug esis ance p o ein (yi G, ym D, y mO)
B.
Small mul id ug esis ance pump (em E, qac, mm , sm )
Bac e oido a
Candida e di ision Zixibac e ia
Gemma imonado a
Pseudomonado a
Spi ochae o a
A
A
c
c
h
h
a
a
e
e
a
a
p
p
h
h
y
y
l
l
a
a
Bac e ia phyla
Eu ya chaeo a
75
14
47
57
55
54
62
97
47
17
100
33
83
99 32
26
29
90
68
35
98
72
59
82
45
100
26
63
77
70
87
Figu e3. (A) Mid-poin oo ed phylogene ic ee based on amino-acid sequences de ec ed in ou s udy om
MFS anspo e , ACDE amily, mul id ug esis ance p o ein (yi G, ym D, y mO). (B) Phylogene ic ee based
on amino-acid sequences om small mul id ug esis ance pump (em E, qac, mm , sm ). Amino-acid sequences
om bac e ia (black colo ), amino acid sequences om a chaea (pu ple colo ). Fo buil he ee, 37 sequences
we e included in he phylogene ic analysis (15 sequences o yi G, ym D, y mO genes and 22 sequences o em E,
qac, mm , sm genes). The sequences we e aligned wi h Muscle and he ee was gene a ed wi h IQTREE2 wi h
1000 boo s aps, wi h au o-model selec ion ia he buil -in ModelFinde .
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Figu e4. (A) Mid-poin oo ed phylogene ic ee based on amino-acid sequences de ec ed in ou s udy
om supe oxide dismu ase, Fe–Mn amily (SOD2). (B) Phylogene ic ee based on amino-acid sequences
om supe oxide educ ase (d x). Amino-acid sequences om bac e ia (black colo ), amino acid sequences
om a chaea (pu ple colo ). Fo buil he ee, 286 sequences we e included in he phylogene ic analysis (172
sequences o SOD2 gene and 114 sequences o d x gene). The sequences we e aligned wi h Muscle and he ee
was gene a ed wi h IQTREE2 wi h 1000 boo s aps, wi h au o-model selec ion ia he buil -in ModelFinde .
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Acknowledgemen s
This s udy was suppo ed by a g an om NASA’s Exobiology P og am. The au ho s would like o hank he Bay
A ea En i onmen al Resea ch (BAER) Ins i u e o managing he pos doc o al ellowships awa ded o P.M.M
and M.D.L. We also hank ep esen a i es o Expo ado a de Sal o Gue e o Neg o, Baja Cali o nia Su . México
o access o si es and assis ance. We also hank ou colleagues om México: José Q. Ga cía Maldonado, Jacob
Albe o Valdi ieso Ojeda, San iago Cadena Rod iguez, Alejand o López Co és and He e La isne e Ba agán
o ield suppo and logis ical assis ance.
Au ho con ibu ions
B.M.B., P.M.M. and M.D.L. concep ualized he s udy and/o suppo ed he da a analysis. P.M.M. pe o med
he nucleic acid ex ac ions. M.D.L. pe o med he bioin o ma ic analysis and P.M.M. d a ed he manusc ip .
P.M.M., M.D.L. and B.M.B. all pa icipa ed in w i ing, e iewing and edi ing he d a . All au ho s ha e ead
and app o ed he inal manusc ip .
Funding
This s udy was unded by NASA’s Exobiology P og am. Re . 17-EXO17-2-0134.
Compe ing in e es s
The au ho s decla e ha he esea ch was conduc ed in he absence o any comme cial o inancial ela ionships
ha could be cons ued as a po en ial con lic o in e es .
Addi ional in o ma ion
Supplemen a y In o ma ion The online e sion con ains supplemen a y ma e ial a ailable a h ps:// doi. o g/
10. 1038/ s41598- 024- 52626-y.
Co espondence and eques s o ma e ials should be add essed o P.M.-M.
Rep in s and pe missions in o ma ion is a ailable a www.na u e.com/ ep in s.
Publishe ’s no e Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in published maps and
ins i u ional a ilia ions.

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