Temporal evolution of methane cycling and phylogenetic diversity of archaea in sediments from a ...

17 Pages·2008·0.44 MB·English

TheISMEJournal(2008)2,204–220
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ORIGINAL ARTICLE
Temporal evolution of methane cycling
and phylogenetic diversity of archaea
in sediments from a deep-sea whale-fall
in Monterey Canyon, California
Shana K Goffredi1, Regina Wilpiszeski1, Ray Lee2 and Victoria J Orphan1
1Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena, CA, USA
and 2School of Biological Sciences, Washington State University, Pullman, WA, USA
Whale-falls represent localized areas of extreme organic enrichment in an otherwise oligotrophic
deep-sea environment. Anaerobic remineralization within these habitats is typically portrayed as
sulfidogenic;however,wedemonstratethatthesesystemsarealsofavorablefordiversemethane-
producing archaeal assemblages, representing up to 40% of total cell counts. Chemical analyses
revealedelevatedmethaneanddepletedsulfateconcentrationsinsedimentsunderthewhale-fall,as
comparedtosurroundingsediments.Carbonwasenriched(upto3.5%)inwhale-fallsediments,as
well as the surrounding sea floor to at least 10m, forming a ‘bulls eye’ of elevated carbon. The
diversity of sedimentary archaea associated with the 2893m whale-fall in Monterey Canyon
(California)variedbothspatiallyandtemporally.16SrRNAdiversity,determinedbybothsequencing
and terminal restriction fragment length polymorphism analysis, as well as quantitative PCR of
the methyl-coenzyme M reductase gene, revealed that methanogens, including members of the
Methanomicrobiales and Methanosarcinales, were the dominant archaea (up to 98%) in sediments
immediatelybeneaththewhale-fall.Temporalchangesinthisarchaealcommunityincludedtheearly
establishmentofmethylotrophicmethanogensfollowedbydevelopmentofmethanogensthoughtto
be hydrogenotrophic, as well as members related to the newly described methanotrophic lineage,
ANME-3. In comparison, archaeal assemblages in ‘reference’ sediments collected 10m from the
whale-fall primarily consisted of Crenarchaeota affiliated with marine group I and marine benthic
groupB.Overall,theseresultsindicatethatwhale-fallscanfavortheestablishmentofmetabolically
and phylogenetically diverse methanogen assemblages, resulting in an active near-seafloor
methane cyclein the deepsea.
The ISME Journal(2008)2, 204–220;doi:10.1038/ismej.2007.103;published online24 January2008
Subject Category: microbial ecology and functional diversity of natural habitats
Keywords: archaea; ANME-3; deep sea; methanogenesis; Methanogenium; whale-fall
Introduction and Boetius, 2007). Perhaps the most well known
are hydrothermal vents, emerging along spreading
The deep sea, fed by a trickling input of photo-
centers and subduction zones, fueled by hot
synthetically derived carbon, has historically been
reduced fluids recirculated within the Earth’s crust.
considered a low-energy, oligotrophic environment.
Although less publicized, equally rich areas of
In localized areas, however, oases of elevated
stimulated biomass production and activity also
microbial biomass and activity do exist (Jørgensen
occur in the permanently cold depths of the deep
sea, fueled by methane seepage or large organic
accumulations (that is, food falls). For example,
Correspondence: SK Goffredi, Environmental Science and carcasses of dead whales deliver B20–50ton of
Engineering, MC 100-23, California Institute of Technology,
organicmaterialtoatypicallyfood-limitedseafloor,
1200EastCaliforniaBlvd,Pasadena,CA91125,USA.
E-mail:[email protected] ataratethatisestimatedtobe2000timesfasterthan
VJ Orphan, Division of Geological and Planetary Sciences, MC the supply of planktonic marine snow (Van Dover,
100-23, California Institute of Technology, 1200 East California 2000;SmithandBaco,2003).Themicrobialecology
Blvd,Pasadena,CA91125,USA.
withintheselocallyactivedeep-seahabitatshasthe
E-mail:[email protected]
potential to be unique, particularly with regard to
Received13August2007;revised18October2007;accepted19
October2007;publishedonline24January2008 pathways for carbon degradation and nutrient
Archaealdiversityunderadeep-seawhale-fall
SKGoffredietal
205
regeneration.Thisisespeciallysignificantwhenone matter and the impact of high organic loading on
considers that over half of the sediments covering deep-sea microbial assemblages arose with the
the deep ocean floor are thought to contain at least serendipitous discovery of a decomposing whale at
30% skeletal remains of marine animals (Shepard, 2893m depth in Monterey Bay, California (Goffredi
1973; Kennett, 1982). et al., 2004). In this study, we combine molecular
The anaerobic degradation of organic matter is analyses with geochemical measurements in the
complex, involving many microbial guilds. In sediments underlying this deep-sea whale-fall to
particular, methanogenesis and sulfate reduction characterize changes in the magnitude and distri-
are the terminal steps in the diagenesis of organic bution of the archaeal community, compared to
carbon (Jørgensen, 1982). These processes are the surrounding deep-sea sediments. We assessed
thought to be mutually exclusive in most environ- whether microbial communities were enriched
mental settings and controlled, in large part, by the under the whale-fall, if they changed over time,
amount of available sulfate (Reeburgh and Heggie, andtheextentandimpactofthisenrichmentonthe
1977;WinfreyandZeikus,1977).Archaealmethano- surrounding sea floor. Regular field sampling
genesis is typically considered the dominant was conducted to examine the temporal changes
process in low-sulfate habitats, such as freshwater in the whale-fall-associated microbial assemblages,
lakes,whereasbacterialsulfatereductiondominates an ecological task that is typically difficult to
in the sulfate-replete marine environment. Sulfate- accomplish for deep-sea environments.
reducingbacteriaoutcompetemethanogenicarchaea
for common substrates, including hydrogen and
acetate; thus, it is thought that sulfate reduction Materials and methods
generally precludes methanogenesis as the primary
terminal step in the microbial diagenesis of organic Site description and sampling
carbon in marine sediments (Kristjansson et al., Sediment samples were obtained from a whale-fall
1982; Lovley et al., 1982). in Monterey Canyon, California (Figure 1). The
The quantity and quality of organic carbon in whale-fall was originally discovered in February
sedimentscanalsobeamajorfactordeterminingthe 2002 (Goffredi et al., 2004; 36.781N/122.081W,
distribution and metabolism of microbes (Capone 2893m depth; Figure 1a, star symbol). Samples
et al., 1983). In certain anoxic marine environments were collected by means of the remotely operated
where input of organic carbon is high, including vehicle Tiburon (owned and operated by the
mangroves, eel grass beds, estuaries, swamps and Monterey Bay Aquarium Research Institute).
pollutedintertidalsediments,bothsulfatereduction Sampling occurred over a period of 59 months
and methanogenesis can occur simultaneously (from October 2002 to January 2007) following the
(Oremland and Taylor, 1978; Oremland et al., discovery of the 2893m whale-fall (Table 1 and
1982; Senior et al., 1982). Similarly, in hypersaline Figure 1); 8 months––dive T486; 33 months––dive
environments, the use of non-competitive methy- T769 (Figures 1b and c); 45 months––dive T917
lated substrates by methanogens also enables (Figure 1e); 51 months––dive T991 (Figure 1f); and
co-habitation by both functional guilds (Visscher 59months––diveT1069(notshown).Chemicaldata
et al., 2003). In the marine environment, methano- are shown for two other whale-falls artificially
genesisandsulfatereductionareoftentemporallyor implanted at 385m (36.791N/121.891W) and
spatially separated (Cappenberg, 1974; Mountfort 1018m (36.781N/122.081W) depth in Monterey
and Asher, 1979); for example, methanogenesis is Canyon in April 2005 and October 2004, respec-
typically dominant in deeper sulfate-depleted sedi- tively (Braby et al., 2007).
ment horizons below the zone of active sulfate Sediments were sampled directly beneath the
reduction. The methanogenic conversion of organic whale-fall (0m distance away), and at 1, 3 and
carbon to CO and CH below the depth of sulfate 10m distance from the center of the carcass (the
2 4
depletionpersistsatverylowratesandisthoughtto latterconsideredtobe‘reference’sediment,initially
account for only a small fraction of the carbon outside the influence of the whale-fall; Figure 1d).
catabolism observed in these marine sediments Changes in archaeal community composition were
(Senior et al., 1982). A high surplus of biologically monitored both with increasing distance from the
available organic carbon, however, may have a whale-fall and with increasing depth within
significant influence on the overall habitat distribu- the sediment column. Spatial sampling, thus,
tionofmethanogenicarchaeaandtheircontribution included these two different parameters (Table 1):
to carbon breakdown in these environments. distance from the whale-fall and depth within the
Investigating the diversity and distribution of sediment (referred to as ‘depth horizon’).
natural microbial communities in the permanently Sedimentsampleswerecollectedwithpushcores
low-temperaturesedimentsofthedeepseaiscritical andstoredat41Cuntiltheywereprocessed(usually
to our understanding of global biogeochemical shipboard within 0–5h) and subdivided for
cycles, especially with regard to the breakdown chemical analysis or molecular studies. From the
and recycling of organic carbon. A unique opportu- push core sleeve, samples were extruded upwards
nity in which to study the diagenesis of organic in 3-cm-thick sections and subsampled using cutoff
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Figure1 (a)ShadedreliefmapofthecontinentalmarginoffMontereyBayshowingthewhale-falllocationat2891mdepth(largewhite
star).ModifiedfromGoffredietal.,2004.(b,c,e,f)Photomosaicsofdigitalstillimages.(d)Schematicofthewhaleskeletonat2893m
showingareasofsedimentcollectionsandsamplingstrategy.(b,c)At33months(diveno.T769,November2004),(e)45months(diveno.
T917,November2005)and(f)51months(diveno.T991,May2006).
1 and 5ml syringes. Samples were then subdivided headspace of the bottles was determined with a
for cell counts via fluorescent in situ hybridiza- Shimadzumini-2gaschromatographequippedwith
tion (FISH) (B0.5g sample wet weight in 2–4% a flame ion detector and magnesium perchlorate
paraformaldehdyeasdescribedbelow),nucleicacid trap, with a 2ml injection loop. A 9.93p.p.m.
analyses (approximately 15g of sediment frozen at methane standard was used for calibration.
(cid:1)801C) and pore-water chemistry (6–10g for CH Sediment porosity was measured to be 40–60%,
4
and B15g sediment for SO and H S, as described via drying of sediments at 651C for 24h, and was
4 2
below). Additional samples were preserved in takenintoaccountinthefinalmethanecalculations.
ethanol or flash-frozen in liquid nitrogen. Compound specific isotopic values for methane
from eight selected samples representing different
methanogen assemblages were measured via
Sediment chemistry gas chromatography-combustion-isotope ratio mass
Sediments were collected and analyzed for percent spectrometry(GC-C-IRMS;IsotechLaboratoriesInc.,
total carbon and nitrogen, stable isotopes (d13C and Champaign, IL, USA).
d15N)ofbulkcarbonandnitrogen,concentrationand To collect pore waters for both sulfide and sulfate
d13C of methane specifically, as well as concentra- measurements, sediment samples were centrifuged
tions of sulfide and sulfate. For methane analysis, (1380gfor15min)incutoff,stoppered10mlsyringes
6–10g sediment plugs were added (B1:1) to 1M without a headspace (Barry et al., 1996). Pore fluid
NaOHingas-tightserumvials.Greaterthan99.5%of samples were transferred to gas-tight syringes via a
the methane in the sediment is typically present in needle through the side wall of the 10ml syringe,
the gas phase. Methane concentration in the minimizing exposure to oxygen, and preserved
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Table1 Summaryofchemicaldescriptionsforsedimentsamplesinvestigatedinthisstudy
Depth Date(diveno.) Monthsa Descriptionb Sediment CH d13CH SO HS TOC d13C TON d15N
4 4 4 2
(m) (PCno.) depth (mmolg(cid:1)1) (%) (mM) (mM) (%) (%) (%) (%)
(cm)
2893 October2002(T486) 8 0m(PC73) 0–3 — — — — 3.4 (cid:1)19.5 0.5 9.6
November2004(T769) 33 0m(PC18) 0–3 1321 — 11 — 1.5 (cid:1)20.6 0.2 10.5
5–8 4230 — — — — — — —
12–15 2970 — — — 2.2 (cid:1)20.1 0.3 12.6
10m(PC55) 0–3 — — — — 1.9 (cid:1)20.3 0.2 7.5
12–15 — — — — 1.6 (cid:1)21.2 0.2 7.6
November2005(T917) 45 0m(PC43) 0–3 685 (cid:1)50.7 29 7 1.3 (cid:1)20.5 0.2 11.2
9–12 962 (cid:1)50.5 11 0 2.0 (cid:1)20.0 0.3 11.9
1m(PC47) 3–6 39 (cid:1)58.6 28 0 3.6 (cid:1)18.7 0.6 9.9
9–12 43 (cid:1)39.6 25 0 0.7 (cid:1)22.2 0.1 10.8
May2006(T991) 51 0m(PC64) 0–3 33 — 16 0 1.4 (cid:1)20.2 0.2 9.8
12–15 357 (cid:1)54.7 9 60 2.1 (cid:1)20.4 0.2 7.8
January2007(T1069) 59 0m(PC75) 0–3 — — — — 3.2 (cid:1)19.9 0.5 8.9
12–15 — — — — 1.8 (cid:1)19.9 0.2 9.7
Abbreviations:PC,pushcorenumberusedatthetimeofcollectionfromtheseafloor;TOC,totalorganiccarbon;TON,totalorganicnitrogen.
—,notmeasured.
aMonthssincediscoveryforthe2893mwhale-fall.
bDescriptionincludesdistancefromthewhale;0m(sedimentscollectedimmediatelyunderlyingthewhale-fall),1mand10m(consideredtobe
‘reference’sediment,initiallyoutsidetheinfluenceofthewhale-fall).
immediately in either 0.5M zinc acetate or 0.5M paraformaldehyde, rinsed twice with 1(cid:3) phos-
barium chloride (1:1) for sulfide and sulfate, respec- phate-buffered saline (PBS), transferred to an equal
tively. Sulfide was measured via the colorimetric volume of PBS/ethanol and stored at (cid:1)201C.
cline assay (Cline, 1969). A 200mM sodium sulfide Pre-processing for FISH included dilution of sedi-
stock solution was made by dissolving Na S in ments 1:20 (vol/vol) in 1(cid:3) PBS, containing 0.01M
2
N -sparged distilled water (adjusted to pH 10 with pyrophosphate (final concentration), to a final
2
NaOH), and decanted into anaerobic vials. Sulfide volumeof2.0ml,sonicationonicewithasonicating
standards (15–1000mM final concentrations) for the wand (Branson sonifier 150) for two 10s pulses at
Cline assay were made from this stock solution via 8W and shaking at 150r.p.m. for 15min at 351C.
serialdilutionwithN -spargeddistilledwater.Sulfate Samples were briefly centrifuged to pellet large
2
in the pore fluids was determined by turbidometry particles, and the supernatant was collected onto a
using a spectrophotometer (Association APH, 1976; 0.2mm Millipore polycarbonate filter. Catalyzed
Gieskesetal.,1991).Theturbidityofagivenreaction reporter deposition-FISH (or CARD-FISH) using
was measured at 420nm and expressed in units tyramide signaling and probes labeled with heat-
derived from a standard curve prepared by use of stabilized horseradish peroxidase was performed
barium sulfate suspensions (0–28mM). according to the protocol of Pernthaler et al. (2004).
For percent carbon and nitrogen, as well as stable For permeabilization of the target cells, a 40ml
isotopes on bulk sediments, samples were dried at subsample of sonicated sediment was pipetted into
651C for 24h, then milled to a fine powder. Dry 100ml of 1(cid:3) TE (10mM Tris-HCl, 1mM EDTA, pH
material (0.5–2.0mg) was placed into a tin capsule 9.0) in glass dish and microwaved in a histological
and combusted in a Eurovector (Milan, Italy) microwave oven (Microwave Research & Applica-
elemental analyzer. The resulting N and CO gases tions Inc., Laurel, MD, USA) at 651C for 2min.
2 2
were separated by gas chromatography and admitted Subsequently, the slurry was put onto ice and
into the inlet of a Micromass (Manchester, UK) centrifuged for 5min at 1000g at 41C. To inactivate
isoprime isotope ratio mass spectrometer (IRMS) for endogenous peroxidases, samples were first ex-
determination of 15N/14N and 13C/12C ratios. Typical posed to 0.1% H O (in 1(cid:3) PBS for 1min at room
2 2
precisionofanalyseswas±0.5%ford15Nand±0.2% temperature) and centrifuged at 1000g for 4min.
for d13C where d¼1000(R /R )(cid:1)1%, where Hybridization buffer was made as described
sample standard
R¼15N/14N, the standard for d15N is atmospheric previously, using 35% formamide concentration
nitrogen and PeedeeBelemnite for d13C. Eggalbumin (vol/vol) and 0.5ngml(cid:1)1 of a horseradish peroxi-
was used as a reference material during each run. dase-labeled oligonucleotide probe (Pernthaler
et al., 2004). The universal bacterial probe set
EUB338 I-III (Daims et al., 1999) and a universal
Microscopy archaeal probe, Ar915 (Stahl and Amann, 1991),
Samples for microscopy were preserved shipboard both labeled with horseradish peroxidase (Biomers.
for 4–12h at 41C in phosphate-buffered 4% net, Ulm, Germany), were used for cell counts.
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Independent checks for nonspecific hybridization PCR, clone library construction and sequencing
by the CARD-FISH procedure were negative. SSUrRNA(16S)andmethyl-coenzymeMreductase
Hybridizations were conducted at 461C for 2–3h, (mcrA) genes were amplified by PCR from purified
followed by a 15min wash in 1(cid:3) PBS (at room DNAsamples.PCRmixtures(25ml)contained0.4mM
temperature) and a 10min amplification step at of either Archaea-specific 16S rRNA primers, 20F
371C.Amplificationbuffercontained0.0015%H O and958R(Delong,1992),ormcrA-FandmcrA-Rfor
2 2
and 0.5mg of tyramide labeled with Alexa (green) the mcrA analysis (Luton et al., 2002). Reactions
488
orAlexa (red)specifically.Cellswerepost-stained also contained 2.5ml of 10(cid:3) PCR buffer (containing
546
with 40,60-diamidino-2-phenylindole (5mgml(cid:1)1) for 2mM MgCl ), 0.2mM each of deoxynucleotide
2
1min and counted by epifluorescence microscopy triphosphates and 0.025U of Eppendorf HotMaster
using a DeltaVision restoration microscopy system Taq (Westbury, NY, USA). Thermal cycling condi-
(Applied Precision LLC, Issaquah, WA, USA). tions included 45s each of denaturation at 941C,
annealingat54and501C,respectivelyfor16SrRNA
and mcrA, elongation at 721C (25–30 cycles) and a
final6minofelongationat721C.PCRproductswere
Nucleic acid extraction cloned using the Topo TA cloning kit (Invitrogen,
For 16S rRNA analysis, total nucleic acids were Carlsbad, CA, USA). Clone libraries of PCR-ampli-
extracted from 3-cm-depth sediment intervals, with fiedarchaeal16SrRNAgeneswereconstructedfrom
each representing a 3-cm-depth interval. Cell lysis 2 to 3 depth horizons from each core, with 35–119
and DNA extraction from B0.5g (wet weight) of clones analyzed for each library, depending on the
sediment was conducted using the Ultra-Clean Soil diversity of recovered ribotypes. A summary of
DNA kit (MoBio Laboratories Inc., Carlsbad, CA, the samples analyzed is included in Table 2. To
USA). The protocol was modified by two initial 5- group similar clone types for subsequent sequence
min incubations at 701C followed by beadbeating analysis, 16S rRNA clones were subjected to
(using a Bio101 Fastprep machine, speed 5.5 for restriction fragment length polymorphism (RFLP)
45s; Thermo Electron Corporation, Whaltham, MA, analysis using the restriction enzyme HaeIII (New
USA). The remainder of the extraction procedure England Biolabs, Beverly, MA, USA). Inserts from
was carried out according to the manufacturer’s clones chosen for sequencing were amplified with
instructions, with the exception of a second 41C M13 primers, as above except with Taq polymerase
incubation in IRS (Inhibitor Removal Solutions from Promega (Madison, WI, USA). M13 amplicons
(MoBio Laboratories Inc., Carlsbad, CA, USA)) were cleaned prior to sequencing with MultiScreen
solution between solutions S2 and S3. DNA HTS plates (Millipore Corporation, Bedford, MA,
concentrations were determined using a Nano-Drop USA). Sequencing reactions were performed on
1000 Spectrophotometer (Nano-Drop Technologies, these products using the Genome Lab DTCS Quick
Wilmington, DE, USA). Start Kit (Beckman Coulter, Fullerton, CA, USA),
Table 2 Summary of samples used in the construction of 16S rRNA libraries from the 2893m whale-fall, including results of the
dominantmethanogensrecoveredfromeach
Date Monthsa Descriptionb Sediment Library No.of Total% Dominant
(diveno.) (PCno.) depth namec clones methanogens methanogentaxa
(cm) screened (%abundance)d
October2002(T486) 8 0m(PC73) 0–3 R8_0s 39 93 Methanococcoides(93)
November2004(T769) 33 0m(PC18) 0–3 R33_0s 89 36 Methanogenium(31)
5–8 R33_0m 65 98 Methanogenium(75)
12–15 R33_0d 70 96 Methanogenium(96)
10m(PC55) 0–3 R33_10s 88 0 None
12–15 R33_10d 62 0 None
November2005(T917) 45 0m(PC43) 0–3 R45_0s 36 88 Methanogenium(80)
9–12 R45_0d 66 89 Methanogenium(56)
1m(PC47) 3–6 R45_1m 32 36 Methanococcoides(36)e
9–12 R45_1d 90 64 ANME-3(64)
May2006(T991) 51 0m(PC64) 0–3 R51_0s 46 97 Methanogenium(85)
12–15 R51_0d 31 80 Methanogenium(80)
Abbreviation:PC,pushcorenumberusedatthetimeofcollectionfromtheseafloor.
aMonthssincediscoveryforthe2893mwhale-fall.
bDescriptionincludesdistancefromthewhale;0m(sedimentscollectedimmediatelyunderlyingthewhale-fall),1mand10m(consideredtobe
‘reference’sediment,initiallyoutsidetheinfluenceofthewhale-fall).
cLibrarydesignationsareR(Ruby,2893mwhale-fall),no.(monthsfromdiscoveryorimplantation)_0,1or10mdistancefromwhale,ands,mord
forshallow(0–3cm),mid(3–6or5–8cm)ordeep(9–12or12–15cm)depthhorizons.
dInsomecases,othermethanogenswerepresentatlowerabundances(o10%).
eANME-3methane-oxidizersalsopresentatB10%.
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thenprecipitatedwithglycogenandsodiumacetate, size 600; Beckman Coulter). Fluorescently labeled
resuspended in 40ml of formamide and run on a fragments were separated by capillary electro-
CEQ 8800 Genetic Analysis System (Beckman phoresis and analyzed on a CEQ 8800 Genetic
Coulter).Sequencesacquiredduringthisstudywere Analysis System (Beckman Coulter). Fragment sizes
deposited in GenBank (accession numbers wereparsedtothenearesttwowholebasepairsand
EU084511–EU084526, EU220725 and EU201175– relative abundances were estimated using the CEQ
EU201182). Library designations are R (Ruby, 8800 Fragment Analysis software. Clones positively
2893m whale-fall)_no. (months post discovery)_0, identified via sequencing were used as controls
1 or 10m distance from whale, and s, m or d for for amplification, restriction and sizing during the
shallow (0–3cm), mid (3–6cm) or deep (9–12 or T-RFLP analysis.
12–15cm) depth horizons, respectively. Additional
sequences were obtained from GenBank and
compiledandalignedwithour16SrRNAsequences Quantitative PCR analysis of archaeal mcrA genes
using ARB Fast Aligner (Ludwig et al., 2004) and The copy number of mcrA genes in each DNA
Sequencherv4.5(GeneCodes,AnnArbor,MI,USA). extract was determined using real-time PCR. Pri-
Phylogenetic trees were generated using PAUP mers, modified from Luton et al. (2002), were used
v4.0b10 (Swofford, 1998). The Kimura two-para- to amplify an B470-bp fragment from the archaeal
meter model was used to estimate evolutionary mcrA gene (mcrA-f, 50GGTGGTGTMGGATTCA-CA
distance. Maximum-parsimony and neighbor-join- CAR-30, T 56.51C, and mcrA-r, 50-TTCATTGCR
m
inganalyseswereconductedwithheuristicsearches TAGTTWGGRTAG-30, T 50.01C). Triplicate real-
m
using 100 and 5000 non-parametric bootstrap repli- time PCR reactions (20ml) contained 0.5mM of each
cates, respectively, to assign confidence levels to primerand1(cid:3) PCRbuffercontainingMgCl ,dNTPs
2
nodes.mcrAgenesequences werefirsttranslatedin and Amplitaq Gold in the SYBR green master
Sequencher v4.6 and then aligned manually in mix (Applied Biosystems, Foster City, CA, USA).
MacCladev4.08.Totalcharacterdifferencewasused Purified DNA (1ml) was added as template (DNA
to estimate evolutionary distance and a neighbor- extracts ranged from 10 to 160ngml(cid:1)1 but were
joining analysis was conducted using 5000 normalized to 10ngml(cid:1)1 and diluted 100(cid:3)). All
non-parametric bootstrap replicates, to assign reagents were RNase- and DNase-free. A standard
confidence levels to nodes. curve was generated using serial dilutions of a
pBAD-derived plasmid (Invitrogen) containing a
known partial mcrA sequence. This template was
Terminal restriction fragment length polymorphism CsCl-purified to reduce the presence of inhibitory
analysis compounds,andlinearizedtomorecloselyapproxi-
Terminal RFLP (T-RFLP) was used to further mate environmental DNA. The slope of this
characterize the diversity and relative proportion standard curve (regression lines of C versus logN,
t
of methanogen-associated 16S rRNA genes in sedi- the log of initial DNA concentration in standard
ment samples. 16S rRNA genes from purified DNA templates) was used to estimate amplification
sampleswerePCR-amplifiedusingarchaealprimers efficiency for these particular primers in our
20F (fluorescently labeled with WellRED dye D4, quantitative PCR (QPCR) assays (B82%, slope of
Sigma-Proligo, St Louis, MO, USA) and 958R using C versus concentration¼3.86). Reactions were
t
the same PCR conditions as described above for carried out using the Sequence Detector System
unlabeled PCR reactions. For a few samples that ABI Prism 7300 (Applied Biosystems). Cycling
were particularly difficult to amplify, 20 cycles of parameters included an initial denaturation of
PCR were performed using unlabeled primers, 951C for 3min, followed by 45 cycles of 951C
followed by a subsequent reaction using 1ml of the (30s) and 60s of annealing/extension at 58.51C,
unlabeled PCR product for an additional 25 cycles determined empirically. A dissociation curve from
using the fluorescently labeled forward primer. For eachQPCRreactionwasexaminedtofurtherensure
each sediment sample, duplicate PCR amplifica- proper target sequence amplification.
tions were performed and pooled prior to digestion
witheitherHaeIIIorSau961(for6–8hat371C;New
England Biolabs). Digest reactions contained 3ml Results
DNA, 0.5ml of enzyme and 2ml 10(cid:3) buffer diluted
with purified H O to a final volume of 20ml. HaeIII Chemistry
2
and Sau961 were chosen based on in silico analysis Sulfatewasdepletedbeneaththewhale-fall,relative
of known methanogen 16S rRNA sequences using tobothseawaterandreferencecores(B28mM),with
the restriction enzyme cutting program in the lowest measured value of 9mM SO at a depth
4
Sequencher v4.6 (Gene Codes). Digested products horizon of 12–15cm (Table 1). Sulfide concentra-
were precipitated using glycogen and sodium tions at the 2893m whale-fall were generally low
acetate as carriers (as above for direct sequencing) (from undetectable levels to only 60mM; Table 1);
and resuspended in 40ml of formamide containing however,thistrendappearsuniquetothisparticular
0.5ml of an internal dye standard (GenomeLab DNA whale-fall as significant sulfide levels were
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observed at other whale-fall sites in Monterey while a second core (PC43) appeared to have more
Canyon (up to 2.5mM, data not shown). constant d13CH values (B(cid:1)50%; Table 1).
4
Methane concentrations were elevated in sedi- High levels of carbon (total organic carbon; TOC)
ments directly beneath the whale (up to were observed in sediments under the 2893m
4230mmolCH g(cid:1)1 sediment; Table 1) relative to the whale-fall for all time points investigated. This
4
reference sediments. Two reference cores taken enrichment in organic carbon was established
approximately 450m from the whale-fall (and relativelyearly,withnonoticeablechangeinsurface
B60m apart) had methane values of B0.2mM sediments from 8 (3.4% TOC) to 59 months (3.2%
(Bill Ussler, personal communication, Monterey TOC) post-discovery (Table 1). Spatial relationships
Bay Aquarium Research Institute). Spatial (lateral) (vertical and lateral) in TOC were investigated for
heterogeneity in methane levels was recorded at 45 cores collected directly beneath the whale-fall (0m
months, with higher values observed in sediments distance), and at 3 and 10m distances from the
immediately underlying the whale-fall (up to center of the carcass (Figure 3). At 59 months,
1455mmol CH g(cid:1)1 sediment at 6cm depth, closed the greatest enrichment was observed in shallow
4
circles in Figure 2a), compared to a core (PC47) depthhorizons(0–3cm),averaging2.5±1.1totalwt
taken at a distance of 1m away (129mmolCH g(cid:1)1 % (n¼7), compared to deeper (9–15cm) sediment
4
sediment, closed diamonds; Figure 2a). Associated depths (1.8±0.5 total wt %,n¼8; Figure 3a). There
stable carbon isotope values for methane were was a significant decrease in total carbon occurring
measured for a subset of samples beneath, or at 3cm depth (decreasing by 44±12 relative %),
adjacent to, the whale-fall. The greatest difference indicating a very shallow and localized zone of
in d13CH was observed between different depth enriched carbon immediately under the whale-fall
4
horizons (3–6cm versus 9–12cm) within a single (Figure 3a, open squares and circles). A similar
core (PC47), taken 1m from the whale-fall, with sharp decrease in carbon was observed in sedi-
d13CH ranging from (cid:1)39.6% to (cid:1)58.6% (see 45 ments collected at a distance of 3m from the
4
months, Table 1; closed diamonds in Figure 2a), whale-fall(Figure3a,opentrianglesanddiamonds).
a b
0
-50.7 -20.5 -18.7
(Methanogenium)
2
-58.6
(Methanooccoides)
m) 4
c
n (
o
z
ori 6 -54.2
h
h
pt
e
d
8
-39.6 -50.5 -22.2 -19.9
(ANME-3) (Methanogenium)
10
12 -43.5 a -20.2 -19.6 b
0 500 1000 15000 1.5 3 4.5
Methane ((cid:1)mol CH4 / g) TOC (% wt)
T917_PC43 (0 m core)
T917_PC47 (1 m core)
T917_PC52 (10 m reference core)
Figure 2 Pore-water profile of (a) total CH (mmol per g sediment) under the 2893m whale-fall (0m, T917_PC43), and at 1m
4
(T917_PC47) and 10m (T917_PC52) distance away. d13CH (%) are noted next to each depth horizon, along with the predominant
4
methanogendetectedin16SrRNAlibraries.(b)Totalorganiccarbon(%TOC)forthesamesedimentsamplesanddepthhorizonswith
d13Cvalues(%)notednexttoeachdepthhorizon.Allsamplesweretakenat45months.
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a 0 and Figure 2b). With the exception of PC47,
2893 m whale (at 59 months) however, bulk d13C TOC values beneath the
3 whale-fall were not significantly different from
m) the 10m sediments ((cid:1)20.2±0.7% (n¼5) versus
h (c 6 (cid:1)20.3±0.7% (n¼4), respectively, for all depth
pt horizons at both 33 and 51 months; Table 1, not all
de 9 data are shown).
ent Total nitrogen ranged from 0.2% to 0.5%
dim12 0m_PC74 immediately underlying the carcass and from
se 0m_PC75 0.2% to 0.3% for sediments collected at 3 and
3m_PC54
15 3m_PC58 10m distance from the whale-fall. d15N values were
10m_PC47 variable and ranged from 7.5% to 12.6%, with no
0
b obvious spatial or temporal pattern (Table 1).
1018 m whale (at 24 months)
3
m)
h (c 6 ACercllhaceoaulnctosmrmevuenailteidesaanssioncciareteadsewiinthttohtealwmhaicler-ofbalilal
dept 9 abundance (B1.6(cid:3)). Under the whale-fall, with
nt archaea representing 10–40% of the total cells
e
m12 detected by FISH microscopy. Total microbial
di
se 0m_PC80 counts(bacterialandarchaea)weregenerallyhigher
15 0m_PC71 (1.5–2.2(cid:3)) in shallow (0–3cm) versus deep depth
3m_PC53
horizons (9–15cm), a trend that was consistent
mat_PC50
c 0 385 m whale (at 20 months) in both 0 and 10m cores. Similarly, a decrease in
the yield of genomic DNA with increasing depth
3 in the sediment was also observed, from 4 to
m) 5mgDNAg(cid:1)1 sediment recovered from the 0–3cm
h (c 6 depth horizon and 0.5–1mgDNAg(cid:1)1 sediment
pt recovered from the 9–15cm horizon.
de 9
nt At 33 months, a comparison was made between
e archaeal assemblages in sediment immediately
m12 0m_PC44
di 0m_PC80 underlying the whale-fall and at a ‘reference’ site
se 0m_PC71 10m away. The majority of phylotypes recovered
15 0m_PC49
3m_PC47 from ‘reference’ sediments (T769_PC55) were
10m_PC64
18 related to uncultured marine Crenarchaeota,
0 1 2 3 4 including phylotypes associated with groups pre-
%C viouslyrecoveredfromdeep-seasediments(Lietal.,
Figure 3 Total carbon (%) in sediments under the (a) 2893m 1999; Vetriani et al., 1999; Knittel et al., 2005;
whale-fall(0m),andat3and10mdistancesaway(sampleswere Inagaki et al., 2006). Specifically, archaeal assem-
taken at 59 months). For comparison, similar measurements are blages in the reference core from the 2893m whale-
shownforwhale-fallsat1018m(b)and385m(c)depthatearlier
fallprimarilyconsistedofmarinegroupI(MGI)and
stagesintheirdecomposition(24and20monthspost-deposition
ontheseafloor,respectively). marine benthic group B (MBGB), representing up to
98% and 27% of the libraries from ‘reference’
sediment, respectively (Table 2 and Figure 4). The
At 59 months, elevated levels of TOC were also dominant Crenarchaeota MGI phylotypes in both
observed for sediments collected 10m from the depth horizons within reference sediments were
whale-fall; however, the decrease in % TOC with related to an uncultured archaeon from a fish gut
sediment depth was much less (decreasing by only (R33_10d_G7;vanderMaareletal.,1998)aswellas
13%; Figure 3a, closed diamonds). For two arti- an uncultured Crenarchaeote from sediments in the
ficially implanted whale-falls with known dates of Japan Trench (R33_0s_B10; Li et al., 1999). These
origin, we observed slightly less elevated carbon MGI phylotypes fell within the MGI-a microcluster,
in underlying sediments at 24 and 20 months which contains both surface and deep-water taxa
after deposition on the sea floor (1.4–2.1%; (Massana et al., 2000). The dominant Crenarchaeota
Figures 3b and c). MBGB phylotype (R33_10d_E11), present only in
The lowest TOC value (0.7%) was measured 1m thedeeper12–15cmlayerofthereferencecore,was
from the whale-fall (PC47, Table 1) in a sediment 98% similar to an uncultured archaeon from deep-
horizonwithad13Cvalueof(cid:1)22.2%(Figure2b)and sea sediments in the Atlantic Ocean (Table 3 and
heterogeneous d13CH . This particular core showed Figure 5; Vetriani et al., 1999). Phylotypes related
4
the greatest difference in d13C TOC among different to the Euryarchaeota were also found only in
depthhorizonswithad13Cof(cid:1)18.7%inthe0–3cm the 12–15cm layer (representing up to 24% of
horizonand(cid:1)22.2%inthe9–12cmhorizon(Table1 the recovered phylotypes; Table 3 and Figure 4),
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including a phylotype (R33_10d_G1) related to example,R33_0s_F10),relatedtofreshwaterandsoil
uncultured Euryarchaeota from sediments in Skan archaea (Bintrim et al., 1997; Stein et al., 2002),
Bay, Alaska and other members of the Thermo- made up the remainder of the sequence diversity
plasmatales-related marine benthic group D, (11%; Table 3 and Figure 4). No methanogen-
(Girguis et al., 2003; Kendall et al., 2007). affiliated sequences were recovered from the 10m
Additional, as yet unclassified Crenarchaeota (for reference core.
Incontrast,archaealassemblagesdirectlybeneath
the whale-fall (T769_PC18), at the same time point
(33 months), were distinct from the surrounding
communities,withmethanogen-affiliatedsequences
dominating the archaeal diversity. In both shallow
and deep sediment horizons, methanogen phylo-
types represented between 36% and 98% of the
archaealdiversityobservedatalltimepoints(Table2
and Figure 5). Vertical heterogeneity in methanogen
abundancewasobservedatthe33-monthtimepoint
with Methanogenium-related phylotypes account-
ing for approximately 31% of the archaeal diversity
in the 0–3cm depth, yet dominating in the deeper
5–8and12–15cmlayers (representingup to98%of
the diversity; Table 2 and Figure 4). This trend in
vertical methanogen abundance was confirmed by
QPCR analysis of the mcrA gene with 30(cid:3) greater
mcrAcopynumbersdetectedat12–15cmcompared
to 0–3cm.
Most of the methanogenic phylotypes in sedi-
ments underlying the whale-fall (for example,
R33_0s_H2) were highly related (99% similarity
in 16S rRNA) to Methanogenium marinum from
SkanBay,Alaska(Table3andFigure5;Chongetal.,
Figure 4 Differences in the abundance of archaeal subgroups 2002). Additional methanogenic phylotypes in-
recovered from sediments immediately underlying the 2893m
cludedthoserelatedtoMethanococcoidesalaskense
whale-fall, at two different depth horizons (0–3 and 12–15cm)
(99% similarity, R33_0s_B2), present in both sedi-
versusthoserecoveredfrom‘reference’sedimentscollected10m
fromthewhale-fall(samplesweretakenat33months). ment horizons, and Methanosarcina baltica (98%
Table3 Summaryofarchaealribotypes(basedon16SrRNA)indifferentsedimentdepthhorizons,inboth0m(whale-fall)and10m
(reference)coresassociatedwiththe2893mwhale-fallat33months
Representative GenBank No.ofclonesatthefollowingsedimentdepthsa Group %
ribotypeIDb accessionno. Closestrelative similarity
(accessionno.)
0mcore(whale-fall) 10mcore(reference)
0–3cm 12–15cm 0–3cm 12–15cm
Euryarchaeota
Methanomicrobiales
R33_0s_H2 EU084515 28 67 0 0 Methanogenium,DQ177344 99
R45_0s_A9 EU084512
Methanosarcinales
R33_0s_B1 EU084513 0 1 0 0 Methanosarcina,AY663809 98
R45_0d_C4 EU084523
R8_0s_B2 EU084526 4 1 0 0 Methanococcoides,AY941802 99
R33_10d_G1 EU084518 0 0 0 15 Euryarchaeota––other,AY592255 97
Crenarchaeota
R33_10d_G7 EU084520 47 1 86 24 MarinegroupI,AF052948 98
R33_10d_E11 EU084517 0 0 0 17 MarinegroupB,AF119137 98
R33_0s_F10 EU220725 10 0 2 6 Crenarchaeota––other,AF418939 99
89 70 88 62 Total
aSediment depth horizons include shallow (0–3cm) and deep (12–15cm) layers, and correspond to samples collected during dive T769
(November2004)underthewhale-fall(0m,PC18)andinreferencesediments(10m,PC55)asshowninTable1.
bRibotypeID’scorrespondtothoseshowninFigure5.
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Figure 5 Phylogenetic relationships of archaea associated with a 2893m whale-fall in Monterey Canyon (CA), based on sequence
divergence within a 938-bp region of the 16S rRNA gene, to selected cultured and environmental sequences in public databases.
Thermotoga maritima (AJ401021) and Aquifex aeolicus (AE000709) were used as outgroups (not shown). Numbers next to nodes
correspond to bootstrap values based on neighbor-joining distance (top number, 5000 replicates) and maximum-parsimony (bottom
number,100replicates)methods(–indicatesnobootstrapsupportavailable).GenBankaccessionnumbersforsequencesobtainedinthis
studyareEU084511–EU084526).TaxondesignationscorrespondtothoselistedinTable3.Forlibrarydesignations,refertoTable2.
similarity, R33_0s_B1), present only in the deeper beneath the whale-fall (n¼4 time points; Figure 6).
12–15cm layer (Table 3 and Figure 5; Singh et al., Specific notable changes in methanogen diversity
2005). Intheuppersedimenthorizon (0–3cm),MGI includedatransitionfromdominanceofmembersof
made up much of the remainder of the recovered the Methanococcoides at 8 months (in the 0–3cm
phylotpes (53%, R33_0s_B10; Figure 5), including horizon beneath the whale-fall) to the later
similar MGI phylotypesasrecovered from reference establishmentofMethanogenium,detectedthroughout
sediments (R33_10d_G7; Table 3 and Figure 5). The the sediment column, at all subsequent time points
relative increase in abundance of methanogens (Table 2 and Figure 6). Methanogenium represented
under the whale-fall was additionally supported 31%, 80% and 85% of the recovered ribotypes in
by QPCR analysis of the methyl-coenzyme M surface sediments (0–3cm) collected at 33, 45 and
reductase (mcrA), with 40–120(cid:3) greater mcrA 51 months, respectively (Figure 6). Similarly,
signal in sediments underlying the whale-fall Methanogenium represented 56–96% of the
compared to reference sediments (data not shown). recovered ribotypes in deeper (9–15cm) sediment
layers (Table 3 and Figure 6). We believe the whale-
fall was well into the process of degradation
Changes over time (duration on the sea floor was estimated to be 6–12
Over a period of 43 months, methanogen-affiliated months at the time of discovery; Goffredi et al.,
phylotypes dominated the archaeal diversity directly 2004); thus, characterization of earlier archaeal
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