Download Microbial eukaryotic distribution in a dynamic Beaufort Sea and the

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Microbial eukaryotic distribution in a
dynamic Beaufort Sea and the
Arctic Ocean
CONNIE LOVEJOY * AND MARIANNE POTVIN
DÉPARTEMENT DE BIOLOGIE, INSTITUT DE BIOLOGIE INTÉGRATIVE ET DES SYSTÈMES (IBIS) AND QUÉBEC-OCÉAN, UNIVERSITÉ LAVAL,
1045 AVE
DE LA MÉDECINE, QUÉBEC, CANADA G1V
0A6
*CORRESPONDING AUTHOR: [email protected]
Received July 20, 2010; accepted in principle August 20, 2010; accepted for publication August 31, 2010
Corresponding editor: John Dolan
When Pacific Waters enter the Arctic Ocean, there is an abrupt change from
temperature to salinity stratification of the upper water column. This change
coincides with a faunal change as Pacific and Bering Sea zooplankton and fish
species are replaced by Arctic species. The clear changes in distributions of larger
organisms suggest that the Arctic is an ideal environment to test hypothesis of
endemism in single-celled planktonic groups. Here, we investigate the distribution
of phylotypes of small protists identified by their 18S rRNA gene. We constructed
nine new clone libraries from three different water masses from samples collected
along the continental shelf and offshore of Beaufort Sea, Western Canadian
Arctic. The new data combined with all other available sequences from the Arctic
were used to identify possible phylotypes with restricted Arctic distributions.
Among those only reported to date from the Arctic were an oligotrichous ciliate, a
chlorarachniophyte and a rhizarian. In the near-surface shelf sample, we also
retrieved sequences from Pacific species that had not been previously reported in
the Arctic. The occurrences of those phylotypes were best explained by incursions
of Pacific Water as coastal currents in combination with elevated temperatures in
2005 that would have been favourable to the non-Arctic phylotypes. Overall, we
found support for the notion of microbial biogeography and our results suggest
that the Arctic may be vulnerable to microbial community changes.
KEYWORDS: Beaufort Sea; Arctic; Chlorarachniophyta; phytoplankton; protists;
Radiolaria; Ciliophora; Micromonas
I N T RO D U C T I O N
Although the debate continues on the extent of
microbial biogeography (Caron, 2009), there is little
doubt that the environment can be a strong filter and
that common or more abundant groups are favoured by
local conditions, such as irradiance, nutrients and temperature and salinity (TS; Bouman et al., 2006; Lozupone
and Knight, 2007). These variables in the ocean are
ultimately determined by global circulation patterns
(Di Lorenzo et al., 2008). One consequence of present
trends in global warming and climate change is that
these patterns are shifting (Bersch et al., 2007; Hakkinen
and Rhines, 2009) and many larger marine species
track these shifts (Hatun et al., 2009). The oceans are
neither vertically nor horizontally uniform, but made
up of regionally formed water masses, with distinct TS
characteristics, that move around the globe over
doi:10.1093/plankt/fbq124, available online at www.plankt.oxfordjournals.org. Advance Access publication September 29, 2010
# The Author 2010. Published by Oxford University Press. All rights reserved. For permissions, please email: [email protected]
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different spatial and temporal scales (Stommel, 1958;
Broecker, 1991; Lozier, 2010). Although larger organisms may actively seek different water masses for whatever reason, small planktonic organisms are at the
whim of advective forces (Durham et al., 2009) and may
become confined to their water mass of origin
(Hamilton et al., 2008) resulting in distinct spatially separated microbial communities (Varela et al., 2008;
Galand et al., 2009b, c). Water masses and associated
currents then are a tool for predicting distribution of
both larger and smaller species. One consequence of
global climate change, which influences water mass distribution, could be changed distributions of microbes as
well as larger animals. The result may well be community changes and perhaps extinctions of local ecotypes
and loss of their distinct genetic heritage.
Small protists are key components of pelagic marine
ecosystems. They are responsible for significant global
photosynthetic production and as bacterial grazers
much of the global heterotrophic production, and therefore, they have a major impact on marine carbon and
energy budgets (Sherr et al., 2007). In general, the identity and biogeography of most small fragile marine flagellates and other protists is poorly known.
Environmental surveys of marine planktonic communities using molecular techniques have resulted in an
increased appreciation of the diversity of these protists
(López-Garcı́a et al., 2001; Moon-van der Staay et al.,
2001), and the 18S rRNA gene has proved to be a
means of classifying these uncultivated organisms
(Guillou et al., 2008; Vaulot et al., 2008). Unseen and
uncultured organisms can be hierarchically ranked to
resemble taxonomic levels. The 18S rRNA gene also
provides a valuable tool for tracking distributions of
organisms from phylum to ecotypes (Massana et al.,
2006a; Lovejoy et al., 2007). Environmental gene surveys
over the World Ocean from diverse geographic regions,
water masses and environmental conditions offer an
opportunity to test theories of biogeography and endemism in the open ocean (Martiny et al., 2006; Massana
et al., 2006a).
The Arctic Basin has long been considered an
Oceanic Province with a distinct macrofauna
(Longhurst, 1998). Many larger diatoms and thecate
dinoflagellates are considered either endemic to the
Arctic or bipolar (Okolodkov and Dodge, 1996; von
Quillfeldt, 2000, 2001, 2004) as are several foraminifera
(Darling et al., 2004, 2007) and the small naked dinoflagellate Polarella glacialis (Montresor et al., 2003). One
ecotype of the otherwise cosmopolitan polyphyletic
green alga, Micromonas pusilla, has only ever been recovered in the Arctic (Lovejoy et al., 2007), and the 18S
rRNA gene from several other groups of uncultivated
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protists has only been reported from the Arctic (Lovejoy
et al., 2006; Terrado et al., 2009). However, the Arctic
Ocean is not completely isolated; both Pacific and
Atlantic Waters flow into the Arctic Basin. The inflow
from these other oceans is modified by local conditions
and sinks to predictable depths determined by density.
Following such transformations, Pacific or Atlantic
macrofauna are rapidly replaced by Arctic species
(Berline et al., 2008). On the Pacific side of the Arctic,
some surface Pacific/Bering Sea water persists in the
Beaufort Sea as a coastal current along the coast of
Alaska, but mostly is incorporated into the colder
fresher surface mixed layer, mixing with melting sea ice
and river inflow in summer or cooling and sinking
deeper in winter. Deeper Pacific Waters also intrude
into the Canada Basin of the Arctic Ocean and eventually form the Arctic Cold Halocline (ACH), which
persists between the surface mixed layers and deep
Atlantic Water entering from the Barents Sea region
(Shimada et al., 2001; Macdonald et al., 2002;
McLaughlin et al., 2005).
Although Arctic species should be favoured by the
colder fresher conditions in the surface mixed layer of
the Arctic Ocean, longer ice-free open water periods
may lead to increased thermal stratification and warmer
surface waters. The resulting warmer mixed layer conditions would be predicted to favour the emergence and
persistence of Pacific – Bering Sea species. To test this
notion, we compared the communities from selected
water masses from the Beaufort Shelf and from a site at
the edge of the Canada Basin in the offshore Beaufort
Sea. Our working hypothesis was that community composition at the shallower and warmer shelf site would
be more likely to contain Pacific – Bering Sea taxa,
whereas other colder waters at the deeper offshore site
would continue to favour the Arctic taxa.
METHOD
Sample collection and preparation
Samples were collected onboard CCGS Amundsen on
2 and 5 September 2005 in the Beaufort Sea. Stations
selected for this study were from either end of a transect
from shallow coastal water in Amundsen Gulf [Station
(Stn) 204; 71808.860 N, 128812.040 W] with a maximum
depth (Zmax) of 64 m to a deep station at the edge of the
Canada Basin (Stn 10, 71833.650 N, 140806.710 W; Zmax
22500 m; Fig. 1). The region and the distribution of
photosynthetic nanoplankton (2–20 mm diameter) and
picoplankton (2–0.2 mm) in the euphotic zone have been
described in detail elsewhere (Tremblay et al., 2009).
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Fig. 1. Map of study region indicating location of Stn 10 (offshore) and Stn 204 (shelf).
Samples were collected with 12-L Niskin-type bottles
(Ocean Test Equipment Inc., Fort Lauderdale, FL, USA)
mounted on a rosette system equipped with a conductivity temperature and depth (CTD) profiler (SBE-911
CTD; Sea-Bird Inc., Bellevue, WA, USA). Additional
probes fitted onto the Rosette system included transmissivity (C-Star Transmissometer, WET Labs Inc.,
Philomath, OR, USA), photosynthetically available radiation (Biospherical Instruments Inc., San Diego, CA,
USA) and relative nitrate (MBARI-ISUS, Satlantic Inc.,
Halifax, NS, Canada), fluorescence (Seapoint Sensors
Inc., Exeter, NH, USA) and dissolved oxygen (SBE 43
oxygen sensor, Sea-Bird Inc.). Oxygen data from the
sensor were calibrated on board using a micro-Winkler
technique following the recommendations of the manufacturer (Sea-Bird Application note 64-2). Water samples
were collected from six depths near surface (3 m), the
subsurface chlorophyll maximum layer, the nitracline
and at three deeper depths as described in Hamilton
et al. (2008). The fluorometer and nitrate profilers were
used to identify the chlorophyll maximum layer and
nitracline, respectively, and bottles were tripped specifically at these depths for this study.
Seawater for environmental DNA was collected into
clean carboys directly from the Niskin bottles. Six litres
were immediately sequentially filtered through a 50-mm
nylon mesh, a 47-mm diameter 3-mm polycarbonate
filter and finally through a 0.2-mm Sterivex unit
(Millipore Canada Ltd, Mississauga, ON, Canada).
Fractionation with 3 mm filters was chosen to be consistent with a previous work (Massana et al., 2004a;
Lovejoy et al., 2006; Medlin et al., 2006). The 0.2– 3-mm
size fraction was used for this study. Buffer was added
to the Sterivex units (1.8 mL of 50 mM Tris – HCl,
0.75 M sucrose and 40 mM EDTA; pH 8.3) and the
samples were stored at 2808C until nucleic acid extraction as in Potvin and Lovejoy (2009).
Size-fractionated chlorophyll a (Chl a) samples were
collected at each target depth by filtering 500 mL of
water onto Whatman GF/F filters (Whatman, GE
Healthcare, Piscataway, NJ, USA) before (total Chl a)
and after pre-filtration through 3 mm pore-size polycarbonate membranes (AMD Manufacturing, Mississauga,
ON, Canada) and stored at 2808C until analysis on
shore. Pigments were extracted from the filters in 95%
ethanol at 708C for 5 min (Nusch, 1980), and concentrations were determined by spectrofluorometry (Varian
Cary Eclipse, Palo Alto, CA, USA). The larger or
3 mm Chl a concentrations were estimated by subtracting the ,3 mm fraction from the total fraction.
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Table I: Primer pairs used in this study combination NSF4/18 and EukR yeilded ca. 1750 bp
fragments clones, NSF4/18 and NLR204/21 (reverse primer) yeilded fragments ca. 2000 bp
(including the ITS and 5S regions since the reverse primere targets the Large Subunit of the rRNA gene)
Sequence
NSF4/18
528f
EukR
NLR204/21
0
0
5 -CTGGTTGATYCTGCCAGT-3
50 -GCGGTAATTCCAGCTCCAA-30
50 -TGATCCTTCTGCAGGTTCACCTAC-30
50 -ATATGCTTAARTTCAGCGGGT-30
S. cerevisiae
Reference
SS 4 –22
SS 573 –592
SS 1772 –1794
LS 1184 – 204
Hendriks et al. (1989)
Elwood et al. (1985)
Medlin et al. (1988)
Van der Auwera et al. (1994)
Target postions of the primers used including the internal sequenining primer (528f) on the Saccharomyces cerevisiae ribosomal gene are indicated,
small subunit (SS) and large subunit (LS).
Samples for nutrients were collected directly from the
Niskin bottles and immediately analysed on board with
an Autoanalyzer 3 (Bran þ Luebbe, Norderstedt,
Germany) following the colorimetric methods adapted
from Grasshoff et al. (1999). Bacteria and viral size particle abundance were estimated by filtering 1 mL of
fresh sample onto a 22-mm diameter 0.02 mm pore-size
Anodisc filter (Whatman, GE Healthcare) and staining
with a 1/10 (v/v_dilution) of SybrGold (GE
Healthcare), essentially as described in Noble and
Fuhrman (1998). The slides were examined on board
within 18 h of collection at 1000 using an Olympus
51 fluorescence microscope fitted with a broad blue
band filter block (Olympus Canada Inc., Markham,
ON, Canada). A minimum of 400 cells for bacteria and
800 virus-size particles were counted for each sample.
This method does not distinguish between Archaea and
Bacteria, and the term bacteria is used for convenience.
Clone libraries and phylogenetic analysis
With the aim of detecting maximum differences among
the communities, the two stations at the ends of the
transect were selected for clone library analysis. In
addition, since microbial biomass is typically low in the
Beaufort Sea region (Tremblay et al., 2008), we selected
samples from depths with maximum microbial biomass
deduced from Chl a and bacterial concentrations.
These two depths corresponded to the subsurface chlorophyll maximum (15 m for Stn 204 and 48 m for Stn
10) and within the nitracline, which is the depth where
nitrate concentrations rapidly increase with depth (30 m
for Stn 204 and 72 m for Stn 10).
Following the suggestion of (Stoeck et al., 2006, 2010),
combinations of commonly used forward and reverse
primer pairs NSF4/18 – EukR and NSF4/18 –
NLR204/21 (Table I) were employed to construct
libraries (Medlin et al., 1988; Diez et al., 2001; Vaulot
et al., 2008). To check if there were obvious differences
between the results of the primer pairs, one additional
library was constructed for the Stn 10_72 m sample
using the NSF4/18 – EukR pair, making a total of nine
libraries for the four samples. Additional information
for these primers and detailed protocols are given in
Potvin and Lovejoy (2009). Amplified polymerase chain
reaction (PCR) products of the clones were digested
with the restriction enzyme HaeIII (Invitrogen,
Carlsbad, CA, USA) and run on a 2.5% low melting
point agarose gel for restriction fragment length polymorphism (RFLP) analysis (Diez et al., 2001). For each
clone library, 1 – 10 clones of each RFLP pattern were
sequenced with the internal 528f primer (Diez et al.,
2001) at Service de séquençage et génotypage du
Centre Hospitalier de l’Université Laval (CHUL) with
ABI 3730xl system (Applied BioSystems, Foster City,
CA, USA). Selected clones whose partial 18S rRNA
gene matched sequences previously reported only from
the Arctic were sequenced with internal forward and
reverse primers as in Lovejoy et al. (2006) to obtain a ca.
1750 bp 18S rRNA gene sequence. All sequences were
manually checked, trimmed and edited using Chromas
software version 2.3 (Technelysium, Holland Park,
Australia) and submitted to NCBI BLAST (Altschul
et al., 1990). Sequences with their closest GenBank
match under 97% were checked for chimeras with
additional BLASTs of several sections of the sequence.
Chimeras and sequences ,650 bp were excluded from
further analysis. The protist sequences were aligned
using the CLUSTAL W package (Thompson et al.,
1994) and checked manually, with GeneDoc version
2.6.003 (Nicholas and Nicholas, 1997) and Bioedit
(Hall, 1999). Rarefaction curves were calculated with
DOTUR (Schloss and Handelsman, 2005). Multivariate
correspondence analyses were carried out using PAST
software. Because of inherent PCR biases and variable
gene copy numbers in different protists, the groupings
only provide a basis for comparing communities when
similar protocols are employed (Potvin and Lovejoy,
2009) as was done in this case. All sequences reported
in this study have been deposited in GenBank under
accession numbers EU664587– EU664590 and
EU682529– EU682664. Clone names ending in r were
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Table II: Biological, nutrient and physical properties at the time of sampling
Station
Depth (m)
T (8C)
sal
NO3 (mM)
PO4 (mM)
SiO4 (mM)
O2 mL L21
Chl a S
(mg L21)
Chl a L
(mg L21)
Bacteria
(108 L21)
VLP
(109 L21)
Stn 204
15
30
48
72
3.76
20.51
20.46
21.06
28.45
31.31
30.96
32.04
1.74
4.08
6.83
14.45
0.63
0.92
0.95
1.43
2.70
6.29
6.74
17.75
8.73
8.54
8.80
8.50
0.20
0.18
0.18
0.04
0.25
0.04
0.02
0.02
5.58
2.91
2.59
1.63
5.74
6.92
2.75
2.39
Stn 10
Temperature (T, 8C), practical salinity units (sal), Chlorophyll a with Chl a S , 3 mm and Chl a L . 3 mm. Bacteria (cells L-1) including Archaea , VLP
are virus-like particles.
from the libraries constructed using the EukR reverse
primer, whereas names ending in n were from libraries
using the NLR204/21 reverse primer.
The taxonomy of all partial sequences, initially
checked using KeyDnaTools (http://keydnatools.com/;
Viprey et al., 2008), was confirmed following the construction of phylogenetic dendrograms, which incorporated related sequences from GenBank. Phylogenetic
relatedness was estimated by the maximum likelihood
(ML) model HKY with rate heterogeneity and a proportion of invariable sites (PAUP 4.0b10, Sinauer
Associates, Inc., Sunderland, USA). Libraries from the
same sample but different primer pairs clustered
together, as reported in Potvin and Lovejoy (2009) using
the Bray– Curtis algorithm and PAST software
(Hammer et al., 2001), and libraries from the same
sample were combined. Sequences with 99% similarity to other Arctic sequences were selected for further
analysis. Related sequences previously reported from
the Arctic (Lovejoy et al., 2006; Scarcella, 2009; Terrado
et al., 2009; this study), their closest non-Arctic matches
and their nearest neighbours identified using the Tree
View Widget from NCBI BLAST (1 July 2009) were
included in a neighbour-joining analysis. All Arctic
sequences from this and other studies .650 bp were
aligned with their nearest BLAST matches and reference sequences to construct neighbour-joining trees
within their higher level groups. To test for the evidence
of Arctic phylotypes or clades, sequences ,900 bp and
those that were distant from our Arctic sequences were
then removed to build subsequent ML dendrograms as
above. Finally, the ML topologies were bootstrapped
(100 runs) to test the robustness of the resulting
branches. Individual putative Arctic phylotypes were
defined as those that were .99% similar to each other
over at least 900 nucleotides of the 18S rRNA gene and
contained a minimum of one distinct nucleotide motif
with .80% boot-strap support in the ML trees. We
further restricted our definition of Arctic phylotypes to
those where sequences had been recovered from at least
two different studies and where a minimum of six
sequences were available for alignment. We then performed a BLAST search (Altschul et al., 1990) of the
NCBI database 16 June 2010 to verify that no new
sequences matching the Arctic phylotypes from other
regions had been released by GenBank since the original analysis.
R E S U LT S
Environmental characteristics
Temperatures were typically near the freezing point
except at Stn 204_15 m, which was nearly 48C. Salinity
was also lower (28.45) in that sample. Salinities for the
remaining three samples were ca. 31– 32. All oxygen
values were high, ranging from 7.5 mL L21 for the
deepest sample to ca. 8.8 mL L21 in the 15-m sample
(Table II). Nutrient concentrations, especially silicate
and nitrate, increased with sample depth (Table II).
Chl a concentrations were generally low; ,0.22 mg Chl
a L21 at the three colder sites and 0.45 mg Chl a L21
for Stn 204_15 m. This was also the only sample with a
large proportion (45%) of the Chl a in the .3 mm size
fraction (Table II). Temperature, salinity, Chl a, nutrients, bacteria and viral-like particles concentrations
were similar for Stn 204_30 m and Stn 10_48 m
(Table II). Multivariate correspondence analysis of the
physical and nutrient data in Table II grouped Stn
204_30 m and Stn 10_48 m together, whereas the deep
sample from Stn 10 and shallowest sample from Stn
204 were separated (Fig. 2a).
Clone libraries
Clones from one 96-well plate per library were
re-amplified with the vector M13 primers. Resulting
amplicons of the correct size were then screened using
RFLP analysis to identify distinct patterns. A total of
354 clones were sequenced. When there were more
than two representatives of a single RFLP pattern, we
sequenced at least two, and often more. Clones with the
same RFLP banding pattern yielded the same sequence
as has been reported in other studies (Diez et al., 2001;
Lovejoy et al., 2006). Chimeras made up fewer than 2%
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Fig. 2. Correspondence analysis based on physical and nutrient characteristics (a) and on community composition (b) from sequence
frequencies.
of sequences and were discarded. Metazoans were
common in three of the combined libraries, in particular the appendicularian Oikopleura, from both depths at
the Stn 10 offshore (82 clones) and copepods from the
deeper sample of Stn 204 inshore (19 clones). These
metazoans were not included in subsequent analysis.
The four communities were compared with each other
by grouping the remaining sequences into operational
taxonomic units (OTUs) defined at a .98 similarity
level, yielding a total of 69 OTUs. For this analysis, the
relative abundance of clones belonging to the OTUs
was based on the frequency of their matching patterns
in the RFLP screen. Multivariate correspondence analysis of the proportions of OTUs in the samples grouped
Stn 204_30 m and Stn 10_48 m together with the other
two samples separating out (Fig. 2b).
Out of the total of 69 OTUs defined using Dotur at
98% similarity, 26 were only recovered from offshore
Stn 10, whereas 34 OTUs were retrieved only from Stn
204 on the Beaufort Shelf. There were nine OTUs in
common between the two stations; these included the
OTU exclusively made up of Arctic Micromonas, recovered from both Stn 10 depths and Stn 204_30 m. One
marine stramenopiles (MAST) 7, one MAST 1a, one
cryptophyte, one dinoflagellate and four oligotrich ciliates were also common between the two stations
(Supplementary Table S1).
The libraries from Stn 204_15 m were dominated by
marine alveolate group I (MALV I) and those from offshore Stn 10_72 m were dominated by Radiolaria,
which accounted for ca. 40% of retrieved clones. We
also recovered sequences matching “picobiliphytes”,
diplonemids, haptophytes and choanoflagellates only
from the offshore Stn 10 libraries. Sequences with
closest matches to the marine phagotroph Telonema (incertae sedis class Telonemia) were recovered from Stn
204_15 m and Stn 10_48 m (Supplementary Table S1).
Rarefaction curves of the libraries did not approach the
asymptote, indicating undersampling, which is usual
using this approach (Vaulot et al., 2008). As with all such
surveys, only the presence of a specific phylotype can be
considered a conclusive result since the absence cannot
be proven (Epstein and López-Garcı́a, 2008).
Arctic phylotypes
Following the criteria above (the “Method” section), five
putative Arctic phylotypes from different protist groups
were recovered from the libraries (Table III). These
included the previously identified Arctic Micromonas
(CCMP2099) and a diatom, (Chaetoceros neogracile
ArM0004) both with cultured representatives (Lovejoy
et al., 2007; Choi et al., 2008). In addition, we propose
three additional provisionally Arctic phylotypes: a ciliate
(Alveolata), a chlorarachniophyte (Cercozoa) and a nassellarian (Radiolaria). The Arctic ciliate was represented
by 12 clones that were 99% similar to each other and
matched the members of the Strom A group originally
suggested by Lovejoy et al. (2006). The nearest matches
of the Strom A were ,97% similar to other environmental sequences and the recently described
Pseudotontonia simplicidens (Shan et al., 2009; corrigium,
Gao et al., 2009). The 18 Strom A sequences from the
Arctic were distinguished by a GAAGT nucleotide
motif beginning at position 664 and a CTAT nucleotide
motif beginning at position 1406 from the 50 -end of the
Saccharomyces cerevisiae RDN18 – 1. We have designated
clone CD8.16 as a reference sequence for this phylotype. Among Radiolaria, we recovered a Nassellaria
(Burki and Pawlowski, 2006; Kunitomo et al., 2006) that
fell within an environmental group with nearest known
species in the family Plagoniidae. The Arctic sequences
differed from the other sequences in this group by seven
specific transitions and transversions over an 86 nucleotide region beginning at position 651 from the 50 -end of
the S. cerevisiae RDN18-1 gene (Fig. 3). The closest
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Table III: Distribution of groups is identified as either Arctic, phylotypes exclusively retrieved in Arctic
waters (see text), or widespread clades (Wide), for those retrieved from other oceanic regions in addition to
the Arctic
Taxon
Distribution
Stn 204_15 m
SML
Telonema
Dinoflagellates
Ciliate Strom A
Cilates Other
Marine Alveolates I
Marine Alveolates II
Stramenopiles
Dictyochophyta
Pelagophyta
Bacillariophyta
MAST 1a
MAST 1c
MAST 2
MAST 4
MAST 7
Haptophyceae
Cryptophyceae
Picobiliphytes
Choanoflagellata
Fungi
Rhizaria
Acantharea
Collodaria/Nassellaria
Cercozoa
Chlorarachniophyte
Cryothecomonas
Prasinophyceae
Micromonas clade Ea
Micromonas clade C
Bathycoccus
Diplonemida
Total
% Widespread
% Arctic
Wide
Wide
Arctic
Wide
Wide
Wide
4
8
1
6
26
4
Wide
Wide
Arctic
Wide
Wide
Wide
Wide
Wide
Wide
Wide
Wide
Wide
Wide
Stn 204_30 m
WML
Stn 10_48 m
WML
13
7
2
2
1
4
12
1
4
Stn 10_72 m
ACH
10
0
5
4
4
1
1
1
1
1
1
9
2
3
2
2
1
1
1
1
4
2
3
2
3
1
Wide
Arctic
16
7
Arctic
Wide
1
1
Arctic
Wide
Wide
Wide
3
2
74
96
4
16
42
2
51
55
45
66
30
70
1
61
85
15
Water masses follow the designation given in the text, SML, WML, and ACH. Sequence information and nearest matches are given in Supplementary
Table S1.
cultured match, Lithomelissa sp8012 (AB246694.1) as
well as the nearest non-Arctic environmental clone
(IND72.23 - EU562100.1) were 95% similar to this
group. We have designated clone NW617.13 as a reference sequence for this phylotype. The majority of previously reported representatives of this phylotype were
from samples collected from deeper than 50 m
(Supplementary Table S2). Finally, we identified an
Arctic cercozoan phylotype that also grouped with
other environmental sequences (Fig. 4). The nearest cultured representatives to this group belong to the enigmatic Chl b containing Chlorarachniophyceae. The
closest match to the Arctic phylotype were other environmental sequences represented by DQ369016.1, clone
UEPACAHp3 from the Pacific, which was 98% similar
over 1700 bp to the Arctic phylotype. The Arctic phylotype differed from other environmental chlorarachniophytes, by six transitions over 91 nucleotide beginning at
position 647 from the 50 -end of the S. cerevisiae RDN18-1
gene. We have designated clone NW617.36 as the reference sequence for this phylotype. The environmental
cluster is distant from all cultivated chlorarachniophytes
(Fig. 4), and although the presence of a chloroplast has
not been confirmed, we note that all Arctic sequences
were retrieved near the surface (5 – 15 m) supporting the
notion that they are photosynthetic.
Biogeography
The remaining clones did not meet the criteria for
belonging to a putative Arctic phylotype and were
classified as widespread taxa (Enghoff, 1996). These
included nearly all of the clones (96%) from Stn
204_15 m. In terms of numbers, two uncultivated
groups, MALV I (Guillou et al., 2008) and MAST 2
(Massana et al., 2004b), were responsible for nearly half
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Fig. 3. Naassellaria (also classified as Collodaria/Nassellaria) ML
tree based on an 811-nt alignment. Bootstrap results .70% are
indicated. The outgroup (Tetrapyle octacantha) has been removed for
clarity. Sequences from this study are indicated in boldface. The
Arctic Collodaria/Nassellaria phylotype included eight sequences in
Supplementary Table S2. A sequence from the earlier study
NW617.13 has been designated the reference sequence for this
phylotype. Isolation source of environmental sequences are
abbreviated in parenthesis. DP, Ant: Drake Passage, Antarctica;
W. Pac.: Western Pacific; Ind. Oc.: Indian Ocean; CB, Arc: Canada
Basin, Arctic; Sar. Sea, Atl: Sargasso Sea, Atlantic; CaB, Ven: Cariaco
Basin, Venezuela; HMAR: Hydrothermal Mid-Atlantic Ridge. NCBI
accession numbers for all sequences used are given in Supplementary
Table S2.
of the Stn 204_15 m clones belonging to widespread
taxa. Both of these groups were rare or absent in the
other three samples (Table III). The MALV I sequences
were diverse with closest matches to three separate
sequences from different geographical regions
(Supplementary Table S1). In contrast, all MAST 2
clones were identical, and although the best match was
to an Arctic sequence, we classified it as widespread,
since it was also 99% similar to sequences from the
Antarctic and a Norwegian fjord. Other MAST clones
in this library were also classified as widespread including representatives from MAST 1c, MAST 4 and
MAST 7 as defined by Massana et al. (2004b).
Prasinophytes recovered from Stn 204_15 m had closest
matches to a widespread clade of Micromonas (clade C;
Slapeta et al., 2006) and to Bathycoccus (Table III).
A large proportion (85%) of the clones from Stn
10_72 m were also defined as widespread (Table III).
Over 25% of the Stn 10_72 m clones belonged to one
phylotype, which was .99% similar to a previously
proposed Arctic group designated ACAN I (Terrado
et al., 2009). The ACAN I sequences were distinguished
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Fig. 4. Chlorarachniophyta-related sequences ML tree based on a
1689-bp alignment. Bootstrap results .70% are indicated. The
outgroup (Gymnophrys cometa) has been removed for clarity. Sequences
from this study are indicated in boldface. The Arctic
chlorarachniophyte phylotype includes six sequences listed in
Supplementary Table S2. A sequence from the earlier study
NW617.36 has been designated the reference sequence for this
phylotype. Isolation source of environmental sequences are
abbreviated in parenthesis. GNB, Arc: Greenland, Norwegian and
Barents Sea convergence; SP Pac.: Scripps Pier, Pacific; Svalbard, Arc:
Svalbard, Arctic. NCBI accession numbers for all sequences used are
given in Supplementary Table S2.
by the nucleotide motif TTTTATTAATTTAAA starting
at position 698 from the 50 -end of S. cerevisiae RDN18-1
gene. A single sequence .99% similar to the ACAN I
motif has recently been reported from the Deep Pacific
Ocean in San Pedrós Channel (Gilg et al., 2010), and
we therefore considered ACAN 1 to be geographically
widespread. Three ciliate sequences from Stn 10_72 m
with nearest BLAST results to other Arctic ciliates were
classified as widespread as well, since they were not
reported from the Arctic previously and did not meet
the criteria set here (Supplementary Table S1).
Over 45% of clones from Stn 204_30 m and ca.
70% of clones from Station 10_48 m were Arctic phylotypes by our strict definition (Table III). By far, the most
abundant Arctic phylotype was the previously described
Arctic Micromonas (Lovejoy et al., 2007). Strom A ciliates
were also recovered from the two libraries. The remaining clones belonged to diverse groups classified as widespread (Table III and Supplementary Table S1).
DISCUSSION
Recently, Jungblut et al. (2010) described the distribution
of cyanobacterial mat communities from the cold biosphere where cyanobacterial 16S rRNA gene sequences,
up to 99% similar, were reported only from polar and
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C. LOVEJOY AND M. POTVIN
alpine regions. Analogous to such terrestrial systems
where altitude is an important factor in biogeographical
patterns, oceanic planktonic microbial distributions
were once thought to be primarily determined by
depth, with light and nutrient availability driving
species distributions. However, bacteria and archaea
have biogeographical distributions, even in the deep sea
where nutrient and irradiance fields vary little (Varela
et al., 2008; Galand et al., 2009a). An earlier study using
denaturing gradient gel electrophoresis, a community
fingerprinting technique, found that eukaryotic picoplankton were associated with distinct water masses in a
hydrographically complex region between Greenland
and Ellesmere Island (Hamilton et al., 2008). The
Beaufort Sea, our study region, on the other side of the
Canadian Arctic, is also hydrographically complex and
consists of water masses formed from incoming source
waters over different seasons (Carmack and
MacDonald, 2002; Shimada et al., 2005; Williams et al.,
2006). The main input of deeper Pacific Water is conditioned (cooled) in the Bering Sea to form the ACH of
the Canada Basin and Beaufort Sea. This halocline lies
over warmer, saltier Atlantic Waters (McLaughlin et al.,
2005). The ACH itself is layered with the deepest water
masses ca. 70– 180 deep originating from Bering
Sea Winter Water, which is saltier and colder than a
20– 50 m layer of Bering Sea Summer Water that lies
just above (Moore et al., 1992; Melling and Moore,
1995). Above these, ACH layers are other water mass
layers that have been modified by sea ice formation in
winter (winter mixed layer, WML) or melting sea ice
and freshwater runoff in summer (summer mixed layer,
SML). The community from Stn 204_15 m was collected from the SML. Although moderate decreases in
salinity may have little effect on biota, increasing the
temperature would be favourable for more southern
species enabling them to persist in this layer. In contrast,
the TS characteristics of Stn 240_30 m and Stn
10_48 m were typical of the WML. The presence of
WML waters at the relatively shallow depths at Stn
204_30 m is consistent with an upwelling event which
occurred under wind forcing along the eastern shelf of
the Beaufort Sea in 2005 (Williams and Carmack,
2008; Williams et al., 2008). Although the WML
samples from Stn 10 and Stn 204 were over 400 km
apart, concentrations of Chl a and bacteria were similar
and our results indicated that the two protist communities were more similar to each other than to the communities within 15 or 30 m in the vertical.
The community collected at Stn 10_72 m with TS
characteristics typical of the upper ACH was distinct,
with Radiolarians dominating. Most phylogenetic
studies tend to classify Radiolarians into two separate
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super groups, divided into Acantharians and
Polycystina. The Polycystina include the Nassellaria
(Pawlowski and Burki, 2009; Gilg et al., 2010). The polycystine Radiolaria are often more abundant deeper in
the water column (200 – 500 m) and their vertical distribution changes seasonally in responses to the presence
of bacteria and detritus exported to deeper layers
(Nimmergut and Abelmann, 2002; Okazaki et al., 2003;
Wang et al., 2006). In particular, nassellarian abundance
has been tied to food quality (Wang et al., 2006), and
their position below the deep chlorophyll maximum at
Stn 10_72 m would be consistent with sinking phytoplankton and detrital activity.
A consensus is arising that short sequences are ill
suited to resolve phylogeny of fast evolving groups
including most marine phytoplankton (Marande et al.,
2009) or for identifying ecotypes (Rocap et al., 2002). We
analysed longer sequence reads and identified potential
Arctic phylotypes by nucleotide motifs occurring within
sequences only ever recovered from the Arctic. The
absence of these sequences from other geographical
regions indicated that the phylotypes had not been
reported from elsewhere to date. Several conclusions
are possible: (i) we describe true endemicity of the taxa
recovered in our clone libraries; (ii) that the taxa are
rare elsewhere and not previously reported due to
undersampling of a “rare biosphere” (Pedròs-Aliò,
2006); (iii) the result is an artefact of poor global coverage of the protists over the planet. For example, a
recently published single sequence from a targeted
study of Radiolarians at 500 and 880 m depth in the
San Pedros Channel (California, Pacific; Gilg et al.,
2010) was identical to the ACAN I phylotype. Gilg et al.
(2010) remarked that it was puzzling to recover core
Acantharians in the deep hypoxic waters and suggested
that the deep forms could represent a single life stage,
which periodically entered the low-oxygen waters of the
Deep San Pedros Channel. The ACAN I phylotype was
very common at 200 m throughout the year in Franklin
Bay in the Beaufort Seas (Terrado et al., 2009) and was
the also most abundant sequence from Stn 10_72 m.
Interestingly, all Arctic ACAN I have been recovered
from the ACH which is a well-oxygenated
Pacific-derived water mass.
The Arctic phylotypes that we describe here may
eventually be found elsewhere with more sampling
effort. In particular, although some larger phytoplankton are reported to be bipolar (Montresor et al., 2003;
Darling et al., 2007), there has not been the same
sampling effort in the Antarctic upper water column as
the Arctic and it cannot be ruled out that some of the
small eukaryotes also have bipolar distributions.
Nonetheless, it is evident that the Arctic phylotypes
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described here are currently common in the Arctic;
they were all recovered from separate studies in different
years. On the other hand, our definition of widespread
was not stringent and additional phylotypes could be
confined to the Arctic. For example, among sequences
which were 99% similar to existing sequences in
GenBank nearly 45% of these had closest matches only
to other Arctic sequences, suggesting that a detailed
examination of variable regions in the whole rRNA
gene may also reveal that there are identifiable motifs
only occurring in Arctic sequences. In addition, we
retrieved 13 other sequences that were ,96% similar
to any GenBank entry and could represent novel phylotypes (Supplementary Table S1). Since the 18S rRNA
gene yields no information on ecological adaptation,
surveys targeting specific functional genes or using
metagenomic approaches could also uncover additional
phylotypes with restricted Arctic distributions.
New records
In contrast to identifying limited distributions in a confined region, defining a new record, which could
become an invader, requires finding a species originating
elsewhere and not reported previously in the new geographical region. We found two new records of phylotypes that were 99% similar to sequences reported
from warmer latitudes and had never been previously
recorded in the Arctic including from surveys along the
Beaufort Shelf (Lovejoy et al., 2006; Scarcella, 2009;
Terrado et al., 2009; this study). The first was a new
record of a Micromonas belonging to a widespread clade
(Clade C; Slapeta et al., 2006). In addition, the
Micromonas belonging to Clade C appeared to replace
the Arctic Micromonas phylotype, which was absent in the
Stn 204_15 m library. The cultured representative of the
Arctic Micromonas (CCMP2099) is adapted to cold
waters with fluctuating light regimes (Lovejoy et al.,
2007) and the phylotype persists under ice throughout
winter (Terrado et al., 2009). In the WML, Arctic
Micromonas were abundant, representing one-third of
clones in the deeper shelf sample (Stn 204_30 m). The
presence of Micromonas Clade C only in the warmer
SML waters suggests either a recent intrusion of surface
Pacific Water into the SML or conditions that favoured
the more southern clade to persist and replace the more
typical Arctic ecotype. A second potential invading
group belonged to an uncultivated marine stramenopile
belonging to the MAST 4 clade. No MAST 4 sequences
have been previously reported from the Arctic (Lovejoy
et al., 2006; Massana, 2006b; Hamilton et al., 2008;
Scarcella, 2009; Terrado et al., 2009) and they were not
detected using fluorescence in situ hybridisation probes
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targeting MAST 4 in samples collected over 2003 – 2004
near the Beaufort Shelf (Massana et al., 2006a). As with
the Micromonas Clade C, the MAST 4 sequences were
found only in the SML of Stn 204.
There are several mechanisms whereby Pacific Water
may be transported relatively quickly to the far side of
the Beaufort Shelf (Okkonen et al., 2009). The most
direct would be a continuation of the shallow coastal
current that is thought to transport Pacific Euphausiids
to Barrow, Alaska (Berline et al., 2008). Bering/Chukchi
waters may also be pumped onto the Beaufort Shelf,
when winds are weak or from the southwest (Williams
et al., 2008), these waters are considerably conditioned
(modified) once they enter the Arctic. It is not surprising
then that microorganisms may arrive in the Eastern
Beaufort Shelf region from the Pacific, but the relative
importance of Pacific taxa has been difficult to quantify
given the sporadic sampling effort and poor taxonomic
resolution for small flagellates using traditional methodologies. If conditions are unfavourable for survival of
Pacific taxa, their numbers would remain very low as
source waters are mixed with colder fresher Arctic
Waters. An analogous situation occurs when freshwater
picocyanobacteria enter the Beaufort Sea from the
Mackenzie River and rapidly disappear as waters mix
with cold saline Arctic Waters (Waleron et al., 2007).
Year 2005 was the first of several exceptionally warm
years with record high surface temperatures and low ice
extent in the Western Arctic (Li et al., 2009). The longer
ice-free conditions and warmer surface waters likely led
to conditions favourable for widespread and previously
“absent” groups. Such “invaders” were able to increase
population levels sufficiently for detection in clone
libraries (Pedròs-Aliò, 2006). The new records of temperate pan-oceanic Micromonas and MAST 4 in the Arctic
indicates that, as with larger animals, microbes from far
afield may penetrate deep into Arctic, similar to the
range expansion of Gray Whales (Moore, 2008) and
reports of Pacific salmon along the Canadian Beaufort
Shelf, far from their historical range (Stephenson, 2006).
Just as the eventual success of the salmon and Gray
Whales in their new habitat is uncertain (Babaluk et al.,
2000; Moore, 2008), a key question is whether microbial
“invaders” will persist and if so whether they would
increase diversity or replace existing species
CONCLUSIONS
The goal of describing and predicting the distribution
and ecology of marine protists (including phytoplankton)
has a long tradition among microbial ecologists and
oceanographers. Patterns of distribution and diversity of
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C. LOVEJOY AND M. POTVIN
microbes provide baseline information needed to identify mechanisms that determine what specific species will
colonise and persist within a given environment. We
found that similar to Northern Baffin Bay (Hamilton
et al., 2008), geographic location or depth was less
important than water mass of origin in determining
microbial community composition. The presence of protists from temperate oceans in the Arctic reflects the
history of water masses entering the Arctic and indicates
that the protists encounter conditions that favoured their
persistence or even growth. As the Arctic experiences
longer ice-free periods and warmer summers, such invasions may represent a previously unidentified threat to
polar marine food chains. Macrozooplankton are
dependent on phytoplankton and are sensitive to species
composition (Vargas et al., 2006). In turn, zooplankton
success is tied to higher trophic levels (Hatun et al.,
2009). Frequent and widespread input of microorganisms from lower latitudes or replacement by a new flora
could have unforeseen consequences for already stressed
higher trophic levels. There is also the possibility of
global genetic loss, if environments or habitats defined
by water mass TS space disappear, threatening the
Arctic’s unique cold-adapted organisms.
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BEAUFORT SEA PROTISTS
Innovation (CFI), and the Canadian Healthty Ocean
Network and especially ArcticNet.
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