Biogeochemistry and microbial diversity in the marine cavity beneath
the McMurdo Ice Shelf, Antarctica
Trista J. Vick-Majors,1 Amanda Achberger,2 Pamela Santiba~nez,1 John E. Dore,1 Timothy Hodson,3
Alexander B. Michaud,1 Brent C. Christner,2 Jill Mikucki,4 Mark L. Skidmore,5 Ross Powell,3
W. Peyton Adkins,2 Carlo Barbante,6 Andrew Mitchell,7 Reed Scherer,3 John C. Priscu*1
1Department of Land Resources and Environmental Sciences, Montana State University, Bozeman, Montana
2Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana
3Department of Geological and Environmental Sciences, Northern Illinois University, DeKalb, Illinois
4Department of Biology, Middlebury College, Middlebury, Vermont
5Department of Earth Sciences, Montana State University, Bozeman, Montana
6Institute for the Dynamics of Environmental Processes – CNR, Department of Environmental Sciences, University of Venice,
Venice, Italy
7Department of Geography and Earth Sciences, Aberystwyth University, Ceredigion, UK
Abstract
Ice shelves surround  75% of Antarctica’s coastline and are highly sensitive to climate change; several
have recently collapsed and others are predicted to in the near future. Marine waters beneath ice shelves har-
bor active ecosystems, while adjacent seas can be important areas of bottom water formation. Despite their
oceanographic significance, logistical constraints have resulted in few opportunities to directly sample sub-
ice shelf cavities. Here, we present the first data on microbial diversity and biogeochemistry beneath the
McMurdo Ice Shelf (MIS) near Ross Island, Antarctica. Physicochemical profiles obtained via a 56 m deep
borehole through the MIS revealed three vertically layered water masses (Antarctic Surface Water [AASW], Ice
Shelf Water [ISW], and modified High Salinity Shelf Water [mHSSW]). Metabolically active, moderately
diverse (Shannon diversity from 2.06 to 5.74) microbial communities were detected in the AASW and
mHSSW. Heterotrophic bacterial production and dissolved organic matter concentrations were higher (12–
37% and 24%, respectively) in mHSSW relative to AASW. Chemoautotrophic production was 5.3 nmol C L21
d21 and 6.0 nmol C L21 d21 in the AASW and mHSSW, respectively. Phytoplankton cells were more abun-
dant and larger in the mHSSW sample relative to the AASW, which indicates sinking of phytoplankton pro-
duced in surface waters and, together with southerly flowing currents (0.09–0.16 m s21), horizontal
advection of phytoplankton from McMurdo Sound. Advected phytoplankton carbon together with in situ
chemoautotrophic production provide important sources of organic matter and other reduced compounds to
support ecosystem processes in the dark waters in the ice shelf cavity.
Ice shelves, which form where glaciers leave the land and
float on the ocean surface, cover more than 1.5 3 106 km2
of coastal ocean in Antarctica (Rignot et al. 2013). Yet, only
a few studies have directly measured physical or biological
processes beneath Antarctic ice shelves, in the sub-ice shelf
marine waters (e.g., Azam et al. 1979; Riddle et al. 2007;
Robinson et al. 2010; Carr et al. 2013). The Ross Ice Shelf
(RIS) is the most extensive ice shelf on the planet, account-
ing for about one-third of the surface area of all Antarctic ice
shelves (Fox and Cooper 1994; Depoorter et al. 2013). The
RIS covers about half of the Ross Sea, an important area of
Antarctic Bottom Water formation, which is vital to the
global-scale thermohaline circulation (Jacobs et al. 1970;
Budillon et al. 2011). The McMurdo Ice Shelf (MIS) com-
prises the northwestern portion of the RIS, and although
they form a unified sub-ice shelf cavity and are often referred
to as a single entity, the MIS originates from different source
glaciers than the main body of the RIS (Robinson et al.
2010). The RIS front is typically >300 m thick and presents
a significant inflow barrier to the sub-ice cavity, whereas the
*Correspondence: jpriscu@montana.edu
Additional Supporting Information may be found in the online version of
this article.
This article was published online 30 November 2015. A footnote stat-
ing that the change updated in affiliation 2. This notice is included in
the online and print versions to indicate that both have been corrected.
572
LIMNOLOGY
and
OCEANOGRAPHY Limnol. Oceanogr. 61, 2016, 572–586VC 2015 Association for the Sciences of Limnology and Oceanography
doi: 10.1002/lno.10234
thinner ( 20–100 m) MIS front provides an important
hydrologic conduit linking the waters of the Ross Sea to the
sub-RIS cavity (Robinson et al. 2010). Waters beneath the
RIS are also influenced by subglacial water from continental
Antarctica, making the sub-ice shelf waters an important
intermediate between the open Ross Sea and subglacial out-
flow from the Antarctic continent (Horgan et al. 2013).
The Ross Sea is one of the most biologically productive
and best-studied marine regions in Antarctica (Smith et al.
2012). Multiple studies have documented the onset of rapid
phytoplankton growth during spring concurrent with the
loss of winter sea-ice cover (Arrigo and McClain 1994; Smith
and Gordon 1997; Arrigo and van Dijken 2004). The annual
phytoplankton bloom peaks in surface waters between mid-
December and early-January, with chlorophyll a (Chl a) con-
centrations in excess of 10 lg L21 (Smith and Gordon 1997).
The bloom is dominated initially by Phaeocystis antarctica
(Smith and Gordon 1997) followed by a mixed diatom
assemblage (Arrigo 1999). The summer phytoplankton
blooms are sustained by high nutrient levels and shallow
mixed layers initiated by melting sea-ice (Smith and Comiso
2008). Decomposition of phytoplankton biomass in this
region is associated with one of the largest blooms of bacter-
ioplankton recorded, reaching biomass levels of >10 mmol
C m22 (Ducklow et al. 2001). This productive sea comprises
the source of the sub-MIS waters, via McMurdo Sound (Rob-
inson et al. 2010).
In contrast to the relatively well-studied Ross Sea, sub ice-
shelf cavities (marine waters beneath ice-shelves) have
received far less attention, largely due to logistical con-
straints associated with accessing the ocean beneath thick,
floating ice. The first oceanographic data from beneath the
MIS was collected in 2003 and revealed net transport from
McMurdo Sound to the MIS cavity, a diurnal tidal cycle, and
evidence for tidally driven melting near the ice shelf front
(Robinson et al. 2010). The Ross Ice Shelf Project (in 1977;
Clough and Hansen 1979) was the first to make oceano-
graphic measurements (Gilmour 1979; Jacobs et al. 1979;
Williams and Robinson 1979) and detect sub-ice shelf bio-
logical communities (Azam et al. 1979; Lipps et al. 1979)
beneath the RIS (420 m ice thickness) at Station J9, 450 km
from the edge of the RIS. Microorganisms in the water col-
umn and seafloor sediments at Station J9 metabolized added
organic substrates (Azam et al. 1979) and dark carbon diox-
ide (CO2) fixation (1.5 g C m
22 yr21) was attributed to the
activity of nitrifying microorganisms (Horrigan 1981).
Underwater camera observations and traps yielded fish and
crustaceans (Lipps et al. 1979), suggesting the existence of
an active food web under the ice shelf, although the benthic
community consisted mainly of scavengers. More recently,
samples from beneath the RIS and MIS near Ross Island led
to the discovery of a new species of sea anemone that
embeds in and inhabits the base of the ice shelf (Daly et al.
2013), and provided information on the microbial commu-
nity composition of marine sediments below this region of
the ice shelf (Carr et al. 2013). Underwater camera observa-
tions showed that a diverse benthic community, unlike that
detected at Station J9 beneath the RIS, exists beneath the
Amery Ice Shelf (Post et al. 2014). These studies suggest that
sub-ice shelf biota are involved in Southern Ocean biogeo-
chemical processes, but that we have a poor understanding
of both the diversity of life and habitats beneath ice shelves.
As Antarctic ice shelves are increasingly threatened with
collapse induced by changing climate (Joughin et al. 2014;
Wouters et al. 2015), the need to develop an understanding
of sub-ice shelf biogeochemical processes becomes progres-
sively more important. Studies following the breakup of the
Larsen A and B Ice Shelves documented changes in biologi-
cal CO2 pumping mediated by water column primary pro-
duction (Bertolin and Schloss 2009) and disruption and
succession of benthic communities beneath the newly open
water (Domack et al. 2005; Fillinger et al. 2013). Still, a pau-
city of baseline data limits the context within which the
ramifications of ice shelf collapse for biogeochemical proc-
esses in the Southern Ocean can be interpreted. Here, we
integrate physical and chemical data with the first microbio-
logical data obtained from the sub-MIS water column. Our
results identify biogeochemical linkages between the Ross
Sea via McMurdo Sound and the dark sub-ice environment
beneath the MIS and RIS.
Methods
Site description
The Ross Ice Shelf stretches  850 km from the Antarctic
coast to the open ocean, with water column depths ranging
from  50 m to  1000 m (Greischar and Bentley 1980). The
MIS comprises the thinnest area of the ice shelf, with ice
thicknesses generally<100 m thick at MIS compared with
up to>800 m thick near the southern extent of the RIS
(most of the RIS is  200–250 m thick; Griggs and Bamber
2011). A coastal current flows along the front of the RIS until
it reaches Ross Island, where some of the water turns north
along the Victoria Land coast, and some flows south,
through the eastern part of McMurdo Sound and beneath
the MIS (Barry 1988).
Our drill site was located on the MIS (77.8902 S, 167.0083
E; Fig. 1) near Ross Island, approximately 8 km from the
edge of the transition from ice shelf to the seasonal and
multi-year fast sea-ice of McMurdo Sound and  2 km from
the ANDRILL HWD-1 site described by Robinson et al.
(2010) and Robinson (2004), which we use for comparison
in this article. At the time of sampling, the transition from
sea-ice to the open water of McMurdo Sound was approxi-
mately 48 km to the north of the sampling site. The 56 m
deep borehole ( 60 cm diameter) that penetrated into the
sub-ice shelf cavity was created with a clean access hot water
drill (Blythe et al. 2014; Burnett et al. 2014; Rack et al. 2014)
Vick-Majors et al. Biogeochemistry under the MCM ice shelf
573
on 18 December 2012. The sub-ice shelf cavity is defined as
the ocean beneath the ice-shelf. The water column depth
below the borehole was 872 m from the ice-water interface
(928 m from the ice surface) to the seafloor. All depths are
reported from the bottom of the ice shelf (i.e., the ice-water
interface). MODIS satellite imagery from around the time of
sampling (not shown; data available from the U.S. Geologi-
cal Survey EarthExplorer) indicated that we did not collect
samples during the Ross Sea phytoplankton bloom.
Physical and chemical parameters
Depth profiles of temperature and conductivity were
measured with a SBE 19plusV2 SeaCAT Profiler CTD (Sea-
bird Electronics) sonde. Potential temperature relative to the
surface was derived according to UNESCO equations (IOC,
SCOR and IAPSO 2010). Water masses observed in the study
area were characterized according to Stover (2006) and
Jacobs and Fairbanks (1985) (see also Celussi et al. 2010) as:
(1) Antarctic Surface Water (AASW) having potential temper-
ature above the freezing point with relatively low salinity
(34.2 and 34.4); (2) Ice Shelf Water (ISW) with potential tem-
perature below the surface freezing point and salinities
between 34.5 and 34.7, and (3) modified High Salinity Shelf
Water (mHSSW) possessing potential temperature near the
surface freezing point with salinities between 34.6 and 34.8.
These water masses were graphically identified using Ocean
Data View 4.5.6 (Schlitzer 2013), as shown in Supporting
Information Fig. S1. Current velocity and direction were
obtained with a Contros Oceanline HS-2X series electromag-
netic current meter and the appearance of the surface sedi-
ments was documented with an attached camera.
Discrete water samples were collected through the bore-
hole with a 10 L General Oceanics Niskin bottle (AASW
[30 m] sample) and a 5 L Go-Flo bottle (mHSSW [850 m]
sample). Both sampling bottles were disinfected with 3%
hydrogen peroxide before deployment. The water was de-
canted immediately through acid leached silicone tubing
into appropriate vessels. Samples for dissolved oxygen and
dissolved inorganic carbon (DIC) were stabilized on site with
a final concentration of sodium azide (0.05% w/v), potas-
sium iodide (0.5% w/v), sodium hydroxide (1.5% w/v) (dis-
solved oxygen) or chloroform (DIC). All water samples were
stored at  48C in the dark until return to McMurdo Station
(MCM) for processing ( 4 h).
Shallow sediment samples were collected from the top
surface of a geothermal probe that penetrated the surface
sediments in an operational test, and the resulting data are
reported qualitatively. Diatom frustules in fine-grained sedi-
ments were processed using standard micropaleontological
techniques (e.g., Scherer 1991), using 15% H2O2 and
mounted as strewn slides in Norland optical adhesive for
light microscopy.
Samples for DOC and three dimensional spectrofluoro-
metric characterization of organic matter (excitation-emis-
sion matrix spectroscopy; EEMS) were decanted into 1%
hydrochloric acid and deionized water rinsed fluorinated
high density polyethylene (HDPE) carboys (Thermo Scien-
tific, Nalgene, Waltham, Massachusetts) and filtered through
acid-leached and combusted (> 4 h at 4508C) 25 mm glass
fiber filters (GF/F, effective retention size 0.7 lm). The filtrate
was collected in acid washed and combusted (> 4 h at
4508C) 125 mL amber borosilicate glass bottles fitted with
polytetrafluoroethylene (PTFE) lined caps and stored at 48C
until analysis. DOC concentrations were determined using a
Shimadzu TOC-V Series TOC analyzer following acidification
with hydrochloric acid to pH2 to remove inorganic carbon
as CO2.
EEMS were determined with a Horiba Jobin Yvon
Fluoromax-4 Spectrophotometer (Horiba, Ltd., Japan) equipped
with a Xe light source using a 1 cm path length quartz cuvette.
Excitation data were measured every 10 nm from 240 nm to
450 nm, and emission data every 2 nm from 300 nm to
560 nm. Measurements were corrected for background (0.2 lm
filtered Milli-Q water) and Raman scattering, and for inner-filter
effects using absorbance spectra collected between 190 nm and
1100 nm with a Genesys 10 Series Spectrophotometer (1 cm
path length, Thermo Scientific) as described by McKnight et al.
(2001). The following indices were derived to reveal the charac-
ter of the DOC: (1) Fluorescence Index (the ratio of the emission
at 470 nm to the emission at 520 nm at an excitation of
370 nm; McKnight et al. 2001; Cory and McKnight 2005), (2)
Freshness Index (the emission at 380 nm divided by the maxi-
mum emission between 420 nm and 435 nm at an excitation
170°E165°E
77
°S
78
°S
Ross Sea
McMurdo
Sound
Ross
Ice Shelf
Ice Shelf
McMurdo
Ross  Island
McMurdo
Dry Valleys
McMurdo
Station Sampling Site
77.8902º S
167.0083º E 
40 km
Antarctica
N
Fig. 1. Sampling site location. Map by Bradley Herried, Polar Geospa-
tial Center.
Vick-Majors et al. Biogeochemistry under the MCM ice shelf
574
370 nm; Huguet et al. 2009), and (3) Humification Index (the
area of the peak under emission 435–480 nm divided by the
peak area under emission 330–345 nm; Zsolnay et al. 1999). To
compare fluorescence intensities in the two samples, standar-
dized maximum fluorescence was calculated as follows: (value-
min)/(max-min), where “value” is the maximum fluorescence
in the sample, and “max” and “min” are the maximum and
minimum fluorescence for the two samples combined.
Suspended matter collected on the filters from DOC and
EEMS filtration was stored frozen (2208C) in the dark until
acid-fuming (to remove inorganic C) and analysis of particu-
late organic C (PC) and nitrogen (PN) on a CE Instruments
Flash EA 112 (ThermoQuest, San Jose, California). Dissolved
oxygen and DIC were measured in MCM using the azide
modification of the mini-Winkler titration and infrared gas
analysis of acid sparged samples, respectively, according to
the limnological methods used by the McMurdo LTER
(http://www.mcmlter.org/data_methods2.htm).
Samples for dissolved inorganic nutrients (soluble reactive
phosphorus [SRP], nitrate1nitrite [N1N], and ammonium
[NH14 ] were filtered through combusted GF/F filters into 1%
hydrochloric acid leached and deionized water rinsed HDPE
bottles and frozen at 2208C until analysis. The concentra-
tions of these compounds were determined colorimetrically
according to Strickland and Parsons (1968) and Solorzano
(1969).
Biological parameters
Chl a concentrations were determined on 200 mL samples
filtered onto 25 mm diameter GF/F filters; the filters were
stored at 2208C in the dark immediately on return to MCM.
The samples were extracted in 90% acetone (v:v; acetone:
water) for  24 h in the dark at 48C and analyzed with a cali-
brated fluorometer (Turner 10-AU-10) as described by
Welschmeyer (1994).
Dark carbon fixation (chemoautotrophy) was determined
on quadruplicate live and trichloroacetic acid [TCA;  5%
final concentration] killed 40 mL samples (Christner et al.
2014). The samples were incubated in the dark in glass bot-
tles filled to the top (no head-space) and capped with PTFE
lined caps. Each sample was amended with 14C-bicarbonate
(stock solution5114.41 lCi mL21) to a final radioactive con-
centration of 10 lCi mL21 and incubated in the dark at 48C
for 94 h. Long incubation times and high specific activity
substrates were used in anticipation of low rates of biological
activity in the sub-ice shelf cavity, and were similar to those
used in other studies aimed at detecting chemoautotrophic
carbon fixation (48 h, Horrigan 1981; 168 h, Yakimov et al.
2011). Incubations were terminated by the addition of ice-
cold 100% TCA (2.5% final concentration) and size fractio-
nated onto GF/F filters (to capture larger, aggregated and fila-
mentous cells) and 0.2 lm polycarbonate filters (to capture
smaller or non-aggregated cells). Filters were placed in 20 mL
scintillation vials, acidified with 1.5 mL of 3N HCl and dried
at 608C. Radioactivity incorporated into cellular carbon
retained on the filters was counted on a calibrated scintilla-
tion counter after addition of 10 mL Cytoscint ES scintilla-
tion cocktail (MP Biomedicals).
Heterotrophic bacterial (where “bacterial” can include
bacteria and archaea throughout the text) production was
determined using [3H]methyl-thymidine incorporation into
DNA (Fuhrman and Azam 1982) and [3H]L-leucine incorpo-
ration into protein (Kirchman et al. 1985). Samples (1.5 mL;
five live and five TCA killed) were incubated with 20 nM
radio-labeled thymidine (specific activity 20 Ci mmol21) or
leucine (specific activity 84 Ci mmol21) at 48C in the dark
for  20 h. Incubations were terminated by the addition of
100% cold TCA (5% final). Following centrifugation, a series
of extractions with cold ( 48C) 5% TCA and cold ( 48C)
80% ethanol were performed and the residual pellet was
dried overnight at room temperature. One milliliter of Cyto-
scint ES scintillation cocktail was added to each vial and the
samples were counted on a calibrated scintillation counter.
Resulting thymidine and leucine incorporation rates (nM
TdR d21or nM Leu d21, respectively) at the incubation tem-
perature (48C) were converted to rates at in situ temperatures
(21.78C to 21.98C) using energy of activation of 12,600 kcal
mol21 determined from Antarctic lakes (Takacs and Priscu
1998). Temperature corrected rates were converted to hetero-
trophic bacterial production (BP TdR and BP Leu) using pub-
lished conversion factors of 2.0 3 1018 cells mol21
thymidine (Bell 1993) and 1.42 3 1017 cells mol21 leucine
(Chin-Leo and Kirchman 1988) and a cellular carbon content
of 11 fg C cell21 (Kepner et al. 1998). Cell specific activity
(mg C cell21 d21) was determined by dividing heterotrophic
bacterial production by bacterioplankton cell abundance (see
below) for each sample.
Heterotrophic bacterial respiration was measured in the
850 m sample by adding 60 mL of sample water to an auto-
claved amber HDPE bottle (Nalgene) followed by the addi-
tion of uniformly labeled 14C-L-leucine (specific
activity>300 mCi mmol21; final leucine concentration
60 nM; final activity 0.0180 lCi mL21) (del Giorgio et al.
2011). Five-milliliter aliquots of the radiolabeled sample
were added to autoclaved 25 mL glass side arm flasks (six
live and six TCA killed controls; 250 lL of cold 100% TCA).
The top of the flask was sealed with a butyl rubber septum
holding a small basket containing a folded GF/C filter sus-
pended above the aqueous phase (Christner et al. 2006); the
sidearm was sealed with a butyl rubber septum. Following
incubation in the dark for 39 h at 2–48C, the reactions in
live incubations were terminated by injecting cold 100%
TCA (final concentration 5%) into the sample through the
sidearm which lowered the pH to 2. b-phenylethylamine
(100 lL; Sigma, catalog number P2641) was added to the GF/C
filter through the septum with a needle and syringe to trap
respired CO2. Killed samples were maintained at 48C for 5 d
with occasional gentle swirling to liberate and trap all respired
Vick-Majors et al. Biogeochemistry under the MCM ice shelf
575
CO2 from the aqueous phase. Cellular
14C incorporation was
determined on the liquid fraction following filtration onto 0.2
lm polycarbonate filters. The GF/C and polycarbonate filters
were placed in 20 mL scintillation vials followed by the addi-
tion of 10 mL of Cytoscint-ES and the 14C activity was deter-
mined using a calibrated scintillation counter. Bacterial growth
efficiency was calculated using the leucine respiration data
(described above) as ((BP Leu)/(BP Leu1Leu respiration)) 3
100. Leu: TdR ratios are based on molar incorporation rates.
Samples for phytoplankton (autofluorescent cells) and
bacterioplankton (non-autofluorescent bacteria and archaea)
enumeration were preserved with sodium borate buffered
formalin (5% v/v) and stored at 48C in the dark. Samples
were filtered through 30 lm mesh using a sterile BD Falcon
12 3 75 mm tube with cell strainer cap to eliminate large
particles. Cell density was determined with a PhytoCyt flow
cytometer (Turner Designs) at a flow rate of 50 lL min21
and core size of 12 lm. Unstained seawater samples were
used for the detection of phytoplankton. Bacterioplankton
were enumerated using seawater samples stained with SYBRV
R
Green I (SGI; Molecular Probes; supplied at 10,000X) at a
final concentration of 1X original (Marie et al. 2001).
Bacterioplankton cells were identified as a distinct popula-
tion on a density plot of SGI emission-height (excitation
488 nm, emission 515–545 nm) vs. forward light scatter-area
(FSC-A: 086158). High nucleic acid (HNA) and low nucleic
acid (LNA) bacterioplankton were identified according to the
intensity of their SGI emission (Gasol and del Giorgio 2000;
Lebaron et al. 2001; Bouvier et al. 2007). Because FSC-A and
SGI emission from small phytoplankton cells may overlap
with those of stained bacterioplankton, we also examined
small phytoplankton (< 2 lm Chl a autofluorescing cells) in
stained samples by plotting FSC-A vs. orange fluorescence
(excitation 488 nm, emission 565–605 nm), blue laser-
dependent red fluorescence (excitation 488 nm, emission
>670 nm; Chl a) and red fluorescence (excitation 640 nm;
emission 650–700). Small phytoplankton cells determined
using these criteria were subtracted from the total counts in
this region to obtain bacterioplankton cell counts. Bacterio-
plankton cell counts were verified on selected samples by
epifluorescence microscopy of SYBRV
R
Gold-stained cells using
the protocol of Lisle and Priscu (2004).
Larger phytoplankton cells (> 2 lm) were enumerated on
a density plot of FSC-A vs. Chl a using 2 mL of unstained
sample. The larger phytoplankton population was divided
into subpopulations (a, b, and c) based on density plots of
FSC vs. Chl a. The presence of each distinct population was
verified by measuring the mean emission ratios of red fluo-
rescence/Chl a and orange fluorescence/red fluorescence. To
determine cell sizes, the larger phytoplankton were also
examined with an epifluorescence microscope (Nikon Eclipse
80i) equipped with a Retiga-2000R Fast 1394 camera under
blue excitation cube filter (excitation 450–490 nm; emission
>515 nm) following the method of Hillebrand et al. (1999).
Samples were prepared for microscopy by filtering 1.6 mL
(30 m sample) and 2.2 mL (850 m sample) through 0.2 lm
13 mm hydrophilic PTFE membrane filters (Millipore, cata-
log number JHWP013000) and mounting the filters on glass
slides with 1:1 (v:v) glycerol/water. Sixty photographs were
taken of random fields at 1000X magnification and the
diameter of  200 cells were measured in each sample
(standard error<2% of the mean) using ImageJ software
(Schneider et al. 2012).
Gating strategies for the different classes of planktonic
cells are available in the MIFlowCyt-Compliant Items (Lee
et al. 2008; Supporting Information) and archived with the
data at http://www.flowrepository.org under the ID number
FR-FCM-ZZK3.
A discrete water sample for nucleic acid extraction was
collected in situ at 850 m with a Large Volume Water Trans-
fer System (WTS-LV; McLane Research Laboratories, East Fal-
mouth, Massachusetts). This system was fitted with a stacked
143 mm diameter filter housing that allowed the sample to
be size fractionated into 10 lm, 3 lm, and 0.2 lm classes
(Supor Membrane filters were used; Pall). This system
allowed us to concentrate 295 L of seawater on 142 mm fil-
ters during a 4 h in situ deployment. An additional discrete
water sample (300 mL) from 30 m was obtained for nucleic
acid extraction from a 10 L Niskin bottle sample that was fil-
tered through a 47 mm 0.2 lm Supor membrane filter (Pall).
Filters from the WTS-LV were preserved as described previ-
ously (Christner et al. 2014) while the entire 47 mm filter
from the 30 m sample was placed in a 7 mL cryovial and
preserved with 7 mL of a solution of 40 mM EDTA pH 8.0,
50 mM Tris pH 8.3, and 0.73 M Sucrose to prevent cell lysis
during storage.
Nucleic acids were extracted from the 10 lm, 3 lm, and
0.2 lm filters from 850 m and the 0.2 lm filter from 30 m
using a MO BIO PowerWater DNA Isolation Kit according to
the manufactures instructions. The V4 hypervariable region
of the small subunit (SSU) rRNA gene was amplified using
the primers 515F and 806R (Caporaso et al. 2012) and
sequenced on an Illumina MiSeq as described by Christner
et al. (2014). Sequence reads were assembled and quality fil-
tered using the Mothur (v1.33.2; Schloss et al. 2009) phylo-
genetic pipeline following the recommended MiSeq standard
operating procedure. Sequences were clustered into opera-
tional taxonomic units (OTUs; cluster.split command) based
on a pairwise distance matrix (dist,seq) calculated using
default settings. OTUs were defined based on 97% sequence
similarity cutoff. Preliminary classification of resulting SSU
rRNA gene sequences was accomplished using the SILVA
database as implemented within the SILVA Incremental
Aligner (SINA). Poorly classified sequences were further
examined using NCBI Blastn. Statistics were calculated using
Mothur (v1.33.2; Schloss et al. 2009) according to Chao
(1984), Good (1953), and Magurran (1988). Estimates of
diversity and richness include singletons; singletons were
Vick-Majors et al. Biogeochemistry under the MCM ice shelf
576
removed from other analyses. SSU sequences obtained from
chloroplasts were removed from the analyses. Sequences
were uploaded to SRA under accession number PRJNA278982.
We note that recent efforts have shown that the primers used
in our study underestimate the abundance and diversity of the
ubiquitous SAR11 clade of bacteria. A comprehensive discus-
sion of the primers can be found in Appril et al. (2015).
Results
Water column structure
The upper  95 m of the water column (relative to the
bottom of the ice shelf) was characterized by relatively
warm, less saline water (21.708C to 21.898C, Salinity 34.45–
34.61) (Fig. 2a). A colder layer (21.908C to 21.938C) of inter-
mediate salinity (34.60–34.70) was present from  95 m to
315 m. Water below  315 m had temperatures ranging
from 21.888C to 21.908C and was relatively higher in salin-
ity (34.66–34.75). These water masses were similar to those
described previously in this area (Jacobs and Fairbanks 1985;
Stover 2006; Celussi et al. 2010): AASW, ISW and mHSSW
(Supporting Information Fig. S1). Current flow was generally
to the south-southeast with true headings (declination cor-
rected) of 1408, 1618 and 1628 for the AASW, ISW and
mHSSW layers, respectively (Fig. 2b). Current velocities for
these respective layers averaged 0.21 m s21, 0.11 m s21, and
0.14 m s21. Current velocity in the mHSSW layer showed a
clear demarcation at 500 m with average velocity in the
upper part of this water layer averaging 0.09 m s21 while
that below 500 m averaged 0.14 m s21.
Inorganic chemistry
Concentrations of NH14 , N1N, and SRP were 40%, 9%,
and 7%, respectively, higher in the AASW (30 m sample) rel-
ative to the mHSSW (850 m sample). Dissolved oxygen con-
centrations were 330 lmol L21 and 370 lmol L21,
representing 92% and 97% of air saturation, respectively
(Table 1). The concentration of DIC was 2.2 mmol L21 in
both samples. The molar ratio of dissolved inorganic N
(N1N1NH14 ) to SRP was 16.1 in the AASW and 15.7
mHSSW water layers.
Microbiological characteristics
Bacterioplankton cell densities were 1.2 3 108 cells L21 in
the AASW and 1.1 3 108 cells L21 in the mHSSW water
layers (Table 2), and similar counts were determined via epi-
fluorescent microscope (data not shown). High-nucleic acid
and low-nucleic acid (HNA and LNA) bacterioplankton cells
Temperature (oC)
-2.0 -1.9 -1.8 -1.7 -1.6
D
ep
th
 (m
)
0
200
400
600
800
1000
Salinity (PSU)
34.4 34.5 34.6 34.7 34.8
Ice cover
Sample depths
0
200
400
600
800
1000
Temperature
Salinity
Current
    Velocity
Current Velocity (ms-1)
0.0 0.1 0.2 0.3
B
AA
SW ISW
m
H
SSW
A
Surface freezing temperature
Direction
0 50 100 150 200
Current Direction (oE)
a b
Fig. 2. Profiles of salinity (practical salinity units; PSU) and temperature (a), current velocity and direction relative to true north (b). Sidebar in panel
(a) designates three water layers: Antarctic Surface Water (AASW), Ice Shelf Water (ISW), and modified High Salinity Shelf Water (mHSSW).
Table 1. Water column inorganic chemistry from the AASW (30 m) and mHSSW (850 m) water masses sampled.
Sample DIC mmol L21 NH14 lmol L
21 N1N lmol L21 SRP lmol L21 DO lmol L21
AASW 2.2 0.7 25.1 1.6 333
mHSSW 2.2 0.5 23.0 1.5 367
DIC, dissolved inorganic carbon; N1N, nitrate 1 nitrite; SRP, soluble reactive phosphorus; DO, dissolved oxygen.
Vick-Majors et al. Biogeochemistry under the MCM ice shelf
577
comprised  35% and  64% of total bacterioplankton cells
in both the AASW and mHSSW, respectively.
Small (< 2 lm) phytoplankton were more abundant in
the surface water layer than the deep layer (1.5 3 106 cells
L21 in AASW, 1.0 3 104 cells L21 in mHSSW), while the den-
sities of larger (> 2 lm) phytoplankton were 33% higher at
depth (Table 2). Microscopic enumeration showed that 83–
85% of phytoplankton cells in both samples were Phaeocystis
antarctica; the remainder consisted of diatoms. Total phyto-
plankton (large plus small) were 10% higher in the mHSSW
relative to densities in the AASW.
Large phytoplankton populations a, b, and c had dis-
tinctly different FSC-A intensities, where greater intensity is
related to greater cell size. The mean FSC-A per cell in popu-
lation b was approximately twice that of population a. The
mean FSC-A per cell for population c was approximately
twice that of b (Fig. 3). The AASW water sample was domi-
nated by population b (56% of total phytoplankton), while
the mHSSW was dominated by populations b and c (43%
and 40%, respectively; Table 2; Fig. 3). The increase in popu-
lation c relative abundance indicates an increase in cell size
(FSC-A) at depth. Microscopic examination of the AASW and
mHSSW>2 lm phytoplankton showed that the between
water mass size difference was small (mean 53.27 and 3.44
lm for AASW and mHSSW, respectively) but significant
(t524.06, n5410, p<0.001), supporting the increase in
size indicated by the shift toward population c dominated
phytoplankton at depth. The mean emission ratios of red
fluorescence/Chl a fluorescence and orange fluorescence/Chl
a fluorescence remained constant within populations indi-
cating a similar phytopigment composition was present at
each depth and only the phytoplankton abundances and
sizes changed.
Rates of both Leu and TdR incorporation were higher in
the deep mHSSW waters but the difference was proportion-
ally greater for TdR, leading to a lower Leu:TdR molar ratio
in the deep waters (14 in the AASW, 9 in the mHSSW; Table
3). In terms of bacterial carbon production, BP-TdR and BP-
Leu ranged from 34.5 nmol C L21 d21 (AASW Leu) to 63.6
nmol C L21 d21 (mHSSW TdR). Cell specific activity was also
higher at depth (3.5 3 10212 mg C cell21 d21 (Leu) and 4.0
3 10212 mg C cell21 d21 (TdR) in the AASW vs. 4.8 3 10212
mg C cell21 d21 (Leu) and 6.9 3 10212 mg C cell21 d21
(TdR) in the mHSSW). Increased activity at depth was also
associated with relatively high bacterial growth efficiency
(70%). Dark 14C-bicarbonate incorporation ranged from 1.6
nmol C L21 d21 (AASW>3 lm size fraction) to 3.1 nmol C
L21 d21 (mHSSW 0.2–3 lm size fraction) and was higher in
the 0.2–3 lm than the>3 lm size fraction (12% greater in
AASW and 6% greater in mHSSW). The summed size fractio-
nated dark 14C-bicarbonate incorporation was greater in the
mHSSW (11% difference) and ranged from 9% (mHSSW BP-
TdR) to 15% (AASW BP-Leu) of heterotrophic bacterial
production.
The molar ratio of particulate organic C:N was
slightly higher in the mHSSW (10.5 vs. 10.0), while DOC
concentrations were higher in the AASW (42.1 lmol L21
Table 2. Bacterioplankton and phytoplankton cell counts (cells L21) determined by flow cytometry for samples collected in the
AASW (30 m sample) and mHSSW (850 m sample). Bacterioplankton cell counts are the sum of low nucleic acid (LNA) and high
nucleic acid (HNA) cells. Phytoplankton include autofluorescent cells of <2 lm and >2 lm diameter. Mean intensity is fluorescence
intensity (relative units) per cell excited with the blue laser and read at emission of >670 nm (Chl a). Phytoplankton >2 um were
divided into three populations: Phyto a, Phyto b, and Phyto c based on size (FSC-A). Total phytoplankton are the sum of all auto-
fluorescing organisms.
Sample
Bacterio-
plankton
(3108)
LNA
(3107)
HNA
(3107)
Phyto-
plankton
<2 lm
(3106)
Phytoplankton
>2 lm
Phyto a
(3106)
Phyto b
(3106)
Phyto c
(3106)
Total
Phytoplankton
(3106)3106
Mean
intensity
AASW 1.2 7.5 4.1 1.5 4.5 41836 0.92 2.5 0.59 6.0
mHSSW 1.1 7.2 3.9 0.0010 6.7 54147 0.41 2.9 2.7 6.7
ch
l a
 fl
uo
re
sc
en
ce
  
105
104
105 106 105 106
FSC-A
a b
a b c
cba
a=79337
b=141065
c=328730
a=90677
b=172312
c=370583
Fig. 3. Density plots of Chl a fluorescence (relative intensity units) vs.
forward scatter-area (FSC-A; relative intensity units) in the AASW water
(30 m) (a) and mHSSW water (850 m) (b). Both samples show three
distinct populations (a, b, and c) based on differences in FSC-A mean
emission intensity for each population. Mean emission intensity for each
group is given in the lower right corner of each plot.
Vick-Majors et al. Biogeochemistry under the MCM ice shelf
578
vs. 31.8 lmol L21). Chl a was 2.4 lg L21 in the shallow
water and 2.9 lg L21 in the deep water (Table 4), and
PC:Chl a (lg C L21: lg Chl a L21) was 109 in the AASW
and 91 in the mHSSW.
The fluorescence and freshness indices for DOM were 2%
and 21% higher, respectively, in the mHSSW sample than
the AASW sample while the humification index was 55%
higher in the AASW sample (Table 4). The maximum excita-
tion/emission of fluorescent DOM (Exmax/Emmax) occurred
in the range of tryptophan-like fluorescence (240/322-324 in
the AASW and 240/334-352 in the mHSSW), and standar-
dized maximum fluorescence revealed 1.7 times greater
tryptophan-like fluorescence in the mHSSW sample com-
pared with the AASW (Table 4).
The seafloor at the sample site was characterized by a
rocky lag deposit, indicative of continuous or episodic strong
bottom currents. Fine-grained sediment, including diatom
frustules, was present interstitially between rocks and peb-
bles. The diatom assemblage recovered from near surficial
sediments was characterized by Southern Ocean and Ross
Sea pelagic species including Eucampia antarctica, Thalassio-
sira oliverana, Thalassiosira tumida, Stelarima microtrias, Tha-
lassiosira lentiginosa, Thlassiosira gracilis, Actinocyclus
actinochilus, and Fragilariopsis obliquicostata. Taxa characteris-
tic of the northern polar frontal zone and lower latitudes,
including Thalassionema nitzschioides, Thalassiosira oestrupii
and Actinocyclus octinarius, were also represented. Fragilariop-
sis curta accounted for less than 1% of the diatom flora in
the MIS sediment.
Bacterial and archaeal diversity and community structure
Microbial diversity, as indicated by the Shannon diversity
index (H0) and the inverse Simpsons diversity index (1/k),
was greatest in the >10 lm size fraction of the mHSSW
sample (Table 5). Good’s coverage, a non-parametric cover-
age estimator, indicated that the majority of the OTUs in
the samples were identified through sequencing, (> 99%;
Table 5) with projected 140 to 500 (calculated as 1/(1-
Good’s coverage)) more sequencing reads required to detect
an additional OTU from each sample (Fig. 4). The total
numbers of OTUs observed in the SSU sequence libraries
represented between 60% (mHSSW 0.2–3 lm size fraction)
and 75% (mHSSW 3–10 lm size fraction) of the expected
number of OTUs predicted by the Chao1 richness estimator
(Table 5).
The three size fractions of mHSSW were dominated by
phylotypes that classify within the Gammaproteobacteria
and Bacteroidetes, with greater proportions of Bacteroidetes
(predominantly Flavobacteria) occurring in the>10 lm
and 3–10 lm fractions relative to the 0.2–3 lm fraction
(Fig. 4). Together, the Gammaproteobacteria and Bacteroi-
detes accounted for  53% of the OTUs in the mHSSW
sample (size fractions pooled), with the Gammaproteobac-
teria alone accounting for nearly 75% of the OTUs
observed at the sampling depth in the AASW. The most
abundant operational taxonomic unit (OTU; 3.7% of
sequence reads) in the mHSSW sample was most closely
related (96% identity) to Candidatus Vesicomyosocius okuta-
nii, a sulfide-oxidizing symbiont of a deep-sea clam. An
OTU most closely related (98% sequence similarity) to the
ammonia oxidizer Nitrosopumilus maritimus, was the third
most common in the mHSSW sample, comprising 3.5% of
Table 3. Water column organic chemical composition and microbial activity (6 propagated standard error) for samples collected in
the AASW (30 m) and mHSSW (850 m) water masses. Particulate organic carbon and nitrogen (PC and PN), the molar PC to PN
ratio (C:N), dissolved organic carbon (DOC), Chl a, bacterial growth efficiency (BGE) rates of 3H-leucine (Leu) and 3H-thymidine
(TdR) incorporation and bacterial production (nmol C L21 d21) the molar ratio of Leu: TdR (nmol Leu L21 d21: nmol TdR L21 d21),
rate of 14C-bicarbonate incorporation in the dark (0.2–3 lm and>3 lm size fractions and the sum of the two size fractions).
Sample
PC
lmol L21
PN
lmol L21 C:N
DOC
lmol L21
Chl a
lg L21 BGE
Leu TdR
Leu:
TdR
14C-bicarb
nmol C L21 d21
nmol
L21 d21
nmol
C L21 d21
nmol
L21 d21
nmol
C L21 d21
0.2–3
lm
> 3
lm Sum
AASW 28.4 2.2 11.3 42.1 2.4 ND 0.27 (0.02) 35.1 (2.8) 0.022 (0.003) 39.5 (6.0) 12 2.8 (0.2) 2.5 (0.5) 5.3 (0.5)
mHSSW 26.1 1.9 12.0 31.8 2.9 0.7 0.36 (0.08) 47.0 (10.0) 0.035 (0.0003) 63.6 (9.0) 10 3.1 (0.6) 2.9 (1.0) 6.0 (1.0)
ND, not determined.
Table 4. Fluorescence characteristics of dissolved organic mat-
ter (DOM) in the two water masses sampled. A higher fluores-
cence index indicates DOM of more microbial character, a
higher humification index indicates a higher degree of humifica-
tion, and a higher freshness index indicates a higher proportion
of recently produced DOM. Standardized maximum fluores-
cence (Max Fluor) occurred in the region of tryptophan-like flu-
orescence for both samples (excitation 240 nm and emission
334–352 (30 m) and 322–334 (850 m)).
Sample
Fluorescence
index
Humification
index
Freshness
index
Max
fluor
AASW 1.65 0.31 1.35 0.56
mHSSW 1.70 0.14 1.72 0.97
Vick-Majors et al. Biogeochemistry under the MCM ice shelf
579
total sequencing reads. Members of the Thuamarchaeota
comprised a total of 4.1% of the mHSSW community
(10.9%, 2.6%, and 1.3% of reads from the 0.2 lm to 3 lm,
3 lm to 10 lm and >10 lm size fractions, respectively).
The majority of sequences in the AASW were related to Oce-
anospirillales sp. (most abundant OTU, 42.4% of reads) and
members of the SAR92 clade (second most abundant OTU,
28.9% of reads). OTUs most closely related to the SAR11
clade were rare, comprising 0.13% of the total library in
the AASW and 1.2% in the mHSSW. A total of  9.0% of
the sequence reads from the AASW were closely related to
Polaribacter sp., a marine genus reported to possess gas
vacuoles.
Approximately 10% of the OTUs identified in all samples
were present in both the AASW and the mHSSW (Fig. 4).
Eighteen of these OTUs were present at relative
abundances>0.1% in both water masses. In general, OTUs
that were common in one water mass ( 1%) were rare in
the other water mass. Exceptions to this were in OTUs
related to the Oceanospirillaceae, which comprised 42.4%
and 4.1% of the AASW and mHSSW populations, respec-
tively, an OTU related to the SAR92 cluster (28.9% and 2.3%
in the AASW and mHSSW, respectively), and Flavobacteria-
ceae (2.6% and 1.0% in the AASW and mHSSW,
respectively).
Discussion
Sub-ice shelf habitats are among the least-studied ecosys-
tems in the world’s oceans. Our results show an active
microbial ecosystem under the MIS, supported in part via
chemoautotrophic activity and in part via advection of
nutrients and biomass from eastern McMurdo Sound. Thick
ice prevents the penetration of sunlight, leaving sub-ice shelf
waters devoid of the primary production typical of open
ocean photic zones, therefore horizontal advection from
adjacent open waters should play a proportionally greater
role in sub-ice shelf biogeochemistry. Sub-ice shelf habitats
AASW
>0.2µm
mHSSW
0.2-3µm
mHSSW
3-10µm
mHSSW
>10µm
R
el
at
iv
e 
ab
un
da
nc
e
0.0
0.2
0.4
0.6
0.8
1.0
Gammaproteobacteria Bacteroidetes Alphaproteobacteria
Planctomycetes Thaumarchaeota 
Deltaproteobacteria
Deferribacteres 
Unclassified and Others 
c
Number of Sequences (x105)
1
N
um
be
r o
f O
TU
s
0
500
1000
1500
2000
2500
3000
3500
0 2 3 4 5
AASW >0.2µm
mHSSW 0.2-3µm
mHSSW 3-10µm
mHSSW >10µm
a
AASW
381 OTUs
mHSSW
2814 OTUs
Shared
354
 OTUs
b
Fig. 4. (a) Rarefaction curves for OTU richness of samples from AASW (30 m) and mHSSW (850 m), (b) comparison of microbial communities from
the AASW water mass and mHSSW water mass samples showing a 10% overlap in community composition at the OTU level (97% SSU sequence iden-
tity), (c) and relative abundance data for AASW and mHSSW.
Table 5. Prokaryotic diversity estimates for the different water masses sampled. OTUs were calculated based on 97% similarity.
H05 Shannon diversity index, 1/k5 inverse Simpsons diversity index. Good’s coverage is a non-parametric coverage indicator. Chao1
gives the expected number of OTUs.
Sample # of reads # of OTUs Good’s coverage Chao1 H0 1/k
AASW 238448 1475 99.8% 2140 2.06 3.79
mHSSW 0.2–3 lm 193331 1805 99.6% 2995 4.26 23.37
mHSSW 3–10 lm 101768 2208 99.3% 2960 5.27 75.10
mHSSW>10 lm 391658 3442 99.7% 4880 5.74 117.10
Vick-Majors et al. Biogeochemistry under the MCM ice shelf
580
can be oligotrophic, such as the J9 site beneath the RIS,
(Lipps et al. 1979) where chemoautotrophically produced
new carbon may be an important food source (Horrigan
1981). Conversely, the diverse assemblage that comprised
the benthic community beneath the Amery Ice Shelf indi-
cated a nutrient rich environment likely sustained by advec-
tion of phytoplankton produced organic matter (Riddle et al.
2007; Post et al. 2014). The J9 and Amery sites differ in their
distance to open water ( 400 km for J9 and  100 km for
Amery). These results, along with those of our study, indi-
cate that biogeochemical processes beneath an ice shelf are
controlled by the proximity to open water, the trophic state
of the source waters and new (chemoautotrophic) carbon
production beneath the ice shelf.
Source of sub-MIS waters
The currents observed at our sampling site (Fig. 2b) are in
agreement with previous observations of a strong southward
flow component from the Ross Sea to the MIS via the eastern
side of McMurdo Sound (Robinson et al. 2010). Our biologi-
cal and physicochemical measurements support an eastern
McMurdo Sound source for the sub-MIS waters at our sample
site. Bacterioplankton abundances in eastern McMurdo
Sound were similar to those at our site (Table 2;  108 cells
L21, Hodson et al. 1981; Rivkin 1991). In contrast, the oligo-
trophic western side, which is a mixture of sub-MIS outflow
and water circulating from the Ross Sea, contained an order
of magnitude fewer cells (Hodson et al. 1981). Nutrient con-
centrations (SRP and DIN; Table 1) were within a few lmol
L21 of those measured during December in eastern McMurdo
Sound (Rivkin 1991). These data imply that the transport
time from the open waters of McMurdo Sound to our sample
site ( 2 d; Robinson 2004) did not result in major changes
to the concentrations of inorganic nutrients or microbial
cells. Given the similarity between the biological and physi-
cochemical character of our samples and eastern McMurdo
Sound waters and the prevailing current direction we con-
clude that nutrient and biomass-rich water advected from
eastern McMurdo Sound likely plays an important role in
sub-MIS biogeochemical processes.
Sedimentary diatoms can also provide tracers of water col-
umn advection, however, strong bottom currents (Fig. 2b) at
our sample site suggest that little deposition occurs there at
present. The most common diatoms we observed (multiple
species of Thalassiosira, A. actinochilus, and F. obliquicostata)
are also found in sediments from the eastern side of
McMurdo Sound (Leventer and Dunbar 1988), however the
near absence of F. curta, which is commonly observed in
McMurdo Sound, is striking. No viable diatoms were recov-
ered in our surface sediment samples, suggesting that the
diatoms we observed may represent earlier deposition, or
that they are the result of advection from pelagic sources.
Together, these data indicate that diatom deposition may
not be an accurate tracer of modern water sources at this
site.
Organic carbon sources
Sub-ice shelf systems are unique from open ocean envi-
ronments in their dependence on horizontal advection of
food sources or in situ chemoautotrophic production, rather
than on vertical fluxes of phytoplankton-derived food sour-
ces (Gutt et al. 2011). To determine whether intact phyto-
plankton cells are advected beneath the MIS, and consider
their potential importance as a food source, we examined
Chl a concentrations and phytoplankton abundances in the
AASW and mHSSW. Chl a concentrations in both samples
were similar to values measured in McMurdo Sound during
mid-December (Rivkin 1991), suggesting that phytoplankton
loss rates (i.e., due to sedimentation, cell lysis, grazing) were
low by the time water from McMurdo Sound reached our
sampling site. Our CTD data revealed the presence of three
distinct water masses beneath the MIS, rather than a well-
mixed water column that would result in even distribution
of Chl a, so the simplest explanation for the similar concen-
trations of Chl a we measured in the AASW and mHSSW
(2.4 lg L21 and 2.9 lg L21, respectively) is settling of Chl a
containing cells into the aphotic mHSSW during the  2 d
transport time to our sample site.
Indeed, our flow cytometry and microscopy data showed
that Chl a containing phytoplankton cells were present in
both water masses. Phytoplankton cells were larger in the
mHSSW than in the AASW and had higher Chl a emission
per cell, suggesting that intact cells or colonies were trans-
ported to depth, a phenomenon that has been previously
reported for P. antarctica (DiTullio et al. 2000). The PC:Chl a
(mg L21: mg L21) measured in our samples (109 in the AASW
and 91 in the mHSSW) was similar to that measured in
active populations from the Ross Sea (92; Ditullio and Smith
1996), supporting our contention that cells transported to
depth were intact. Intact phytoplankton cells can be
advected great distances under ice from open water (Holm-
Hansen et al. 1978), where they may later be decomposed as
a source of nutrition for heterotrophic growth of sub-ice
shelf microorganisms.
To our knowledge, no measurements of P. antarctica
decomposition rates have been made for the MIS/RIS cavity,
although DiTullio et al. (2000) tracked P. antarctica export in
the Ross Sea and showed that intact cells are rapidly
exported to depth. Based on a phytoplankton decomposition
rate of 0.032% d21 for a cold saline Antarctic lake near
McMurdo Sound (Priscu 1992), 100% of the phytoplankton
particulate carbon we observed would be decomposed after
10 yr. This potential decadal scale decomposition suggests
that sinking and advected phytoplankton are important
sources of organic matter beneath the ice shelf, and may
continue to be as they are advected farther from the ice shelf
front.
Vick-Majors et al. Biogeochemistry under the MCM ice shelf
581
Bulk DOC concentrations in our samples were relatively
low, similar to Ross Sea wintertime background concentra-
tions (e.g., Ducklow 2003). Based on our determinations of
bacterial growth efficiency and bacterial carbon production,
the heterotrophic bacterial carbon demand (bacterial carbon
production1bacterial carbon respiration) in our samples
was  50–90 nmol C L21 d21. Assuming that the entire DOC
pool is bioavailable and the system in steady-state, these
rates would be expected to deplete the standing stock of
DOC within 1–2 yr. The total DOC pool is likely an overesti-
mate of the bioavailable fraction, as the labile pool is con-
sumed quickly, concurrent with the phytoplankton bloom
(Ducklow 2003). Residence times in the MIS/RIS cavity may
vary between water masses and are not well constrained, but
estimates range from  2 yr to 8 yr (Smethie and Jacobs
2005; Reddy et al. 2010). Based on this, our data imply that
another organic carbon source would be required to main-
tain heterotrophic potential beneath the ice shelf during the
water residence time.
The production of organic matter via fixation of CO2 into
biomass (chemoautotrophy) may also be important under
the ice. Our microbial diversity, in concert with results from
other studies (e.g., Gryzmski et al. 2012; Williams et al.
2012; Christner et al. 2014) show that aerobic ammonia-
oxidizing microorganisms are widespread in Antarctic
aquatic environments, and may be key sources of new
organic carbon to ecosystems shielded from sunlight. Iso-
lated representatives of the proposed phylum Thaumarch-
aeota, a major group identified in our mHSSW sample, are
aerobic ammonia oxidizers that fix CO2 (K€onneke et al.
2014). We measured dark CO2 fixation of  3 nmol C L21
d21 in the 0.2–3 lm size fraction in both the shallow AASW
and deep mHSSW samples. We attribute this CO2 fixation to
bacteria and/or archaea, rather than phytoplankton-
mediated anapleurotic reactions because our flow cytometry
data showed that the 0.2–3 lm size fraction was chiefly com-
prised of bacterioplankton. Still, dark CO2 fixation was an
order of magnitude less than heterotrophic bacterial carbon
production at our study site, implying that heterotrophic
demand in this region of the MIS must also rely on some
combination of the previously discussed carbon sources
(advected organic matter and biomass or the DOC pool).
Dissolved organic matter quality and microbial growth
Dissolved organic carbon concentrations, while higher in
the surface waters (42.1 lmol L21) than at depth (31.8 lM)
were similar to Ross Sea wintertime background values (40–
42 lmol L21; Ducklow 2003). With terrestrial sources limited
by a paucity of land plants, autochthonous production dom-
inates the DOM pool in Antarctic waters (e.g., McKnight
et al. 2001). Fluorescence indices derived from our EEMS
datasets were consistent with those of microbially produced
organic matter, where a high index ( 1.8) corresponds to
DOM of microbial origin, and a low index ( 1.2) corre-
sponds to DOM of terrestrial origin (McKnight et al. 2001;
Cory and McKnight 2005). Humification indices increase
with the presence of more humified and/or older DOM (up
to >10 for fulvic acids; Zsolnay et al. 1999), while a Fresh-
ness index >1 corresponds to recently produced microbial
DOM (Huguet et al. 2009). Both the humification and fresh-
ness indices calculated for our samples are consistent with
DOM of recent microbial origin. The 55% higher humifica-
tion and 22% lower freshness indices in the AASW water
mass relative to the deeper mHSSW water mass reveal that
the near-surface DOM was more degraded, perhaps due to
high rates of heterotrophic activity associated with phyto-
plankton production in the photic zone of McMurdo Sound
before being advected into the sub-ice cavity.
Ducklow (2003) showed that labile DOC produced during
the summer Ross Sea phytoplankton bloom is quickly con-
sumed, leaving a mainly recalcitrant pool available for con-
sumption during the winter. This is consistent with our
findings of more degraded near-surface DOM. All three indi-
ces of biological activity (3H-TdR, 3H-Leu, and 14C-bicarbon-
ate uptake) revealed increased metabolic rates in the deeper
mHSSW water mass. Increased biological activity may
explain the stronger signal for freshly produced DOM,
including the elevated maximum tryptophan-like fluores-
cence in the mHSSW relative to the AASW.
Although the ability to take up leucine and thymidine
can differ among bacterial groups (Perez et al. 2010), the
ratio of Leu (protein production or biomass maintenance) to
TdR (DNA synthesis or cell division) incorporation rates can
be used as a metric for understanding changes in rates of
biomass maintenance relative to reproduction (Chin-Leo and
Kirchman 1988; Shiah and Ducklow 1997; Vick and Priscu
2012). The lower Leu: TdR we detected in the mHSSW sam-
ple may indicate faster heterotrophic bacterioplankton
growth rates at depth relative to the surface, possibly a prod-
uct of the availability of higher quality DOM.
Conclusions
The sub-McMurdo Ice Shelf cavity provides an important
conduit for organisms and nutrients between the Ross Sea
and the sub-Ross Ice Shelf cavity. Our results show that the
decomposition of phytoplankton coupled with chemoauto-
trophic production of new carbon may be important in sup-
porting ecosystems beneath the MIS. While inputs from
phytoplankton blooms are available only during the austral
summer, chemoautotrophic production is available year-
round. Our sequence-based community analysis corroborates
our contention and those of previous reports suggesting that
new carbon produced by chemoautotrophic ammonia-
oxidizing organisms can supply organic carbon to partially
sustain ecosystem processes beneath Antarctic ice shelves
such as the MIS and RIS. Other abundant phylotypes in our
deep-water sample are related to a chemoautotrophic sulfide
Vick-Majors et al. Biogeochemistry under the MCM ice shelf
582
oxidizer, suggesting that reduced sulfur compounds may also
be an important energy source to fuel chemoautotrophic
production beneath the MIS.
Given that many of the ice shelves surrounding Antarcti-
ca’s coastline are thinning at increasing rates (Paolo et al.
2015) and are susceptible to collapse (Joughin et al. 2014),
their losses should lead to significant changes in the biogeo-
chemistry of Antarctic coastal systems supported by sub-ice
shelf processes. Changes in Southern Ocean ice cover can
impact carbon biogeochemistry at local as well as global (see
review by Sigman et al. 2010) scales. In the case of the RIS/
MIS, the relative biogeochemical importance of chemoauto-
trophic production and material advected beneath the ice
from the Ross Sea would decrease if photosynthetic primary
production began to occur in newly open water. Assuming
that the rates we measured are relatively constant over the
year, the sum of heterotrophic and autotrophic C produc-
tion under the MIS is currently<1% of the photosynthetic
C production in the open water of the Ross Sea (per km2 per
year in the top 100 m of water column; Ross Sea estimates in
Arrigo et al. 2008). Considering such disparity in productiv-
ity between ice shelf covered and open water, the loss of ice
shelves can be expected to have cascading effects on elemen-
tal cycling in the region via shifts in dominant biological
processes. In a less dramatic scenario than massive ice-shelf
loss, climate change induced changes to winds and tempera-
tures that modulate incursions of Circumpolar Deep Water
onto the continental shelf could severely disrupt the Ross
Sea food web (Smith et al. 2014), leading to major effects on
sub-MIS biogeochemistry. Baseline data from existing sub ice
shelf environments, such as the MIS, inform the prediction
of the biogeochemical impacts of ice shelf collapse and cli-
mate change in the Southern Ocean.
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Acknowledgments
The authors would like to thank two anonymous reviewers for helpful
comments, Robert Edwards for project management, Andrew T. Fisher
and Kenneth D. Mankoff for providing sediment samples, the WISSARD
Science Team, the individuals working as part of the Antarctic Support
Contractor managed by Lockheed-Martin, for logistical support, as well
as K. Welch and A. Chiuchiolo for analytical and laboratory assistance
and I. Alekhina for field support. The Whillans Ice Stream Subglacial
Access Research Drilling (WISSARD) project was funded by National Sci-
ence Foundation grants (0838933, 0838896, 0838941, 0839142,
0839059, 0838885, 0838855, 0838763, 0839107, 0838947, 0838854,
0838764 and 1142123) from the Office of Polar Programs. Partial sup-
port was also provided by funds from NSF award 1023233 (B.C.C.), NSF
award 1115245 (J.C.P.), the American Association of University Women
Dissertation Fellowship (T.J.V.), the NSF’s Graduate Research Fellowship
Program (1247192; A.M.A.), the Chilean Fulbright-CONICYT Scholarship
(P.S), the Italian National Antarctic Program (C.B.), and fellowships from
the NSF’s IGERT Program (0654336) and the Montana Space Grant
Consortium (A.B.M.). The drilling was directed by F.Rack and imple-
mented by D.Blythe, J.Burnett, C.Carpenter, D.Duling (chief driller),
D.Gibson, J. Lemery, A. Melby and G. Roberts.
Submitted 15 May 2015
Revised 13 August 2015; 11 October 2015
Accepted 30 October 2015
Associate editor: Ian Hewson
Vick-Majors et al. Biogeochemistry under the MCM ice shelf
586