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Environmental Microbiology (2015) 17(10), 3882–3897 doi:10.1111/1462-2920.12872

Melting glacier impacts community structure of

Bacteria, Archaea and Fungi in a Chilean
Patagonia fjord

Marcelo H. Gutiérrez,1,2* Pierre E. Galand,3 Introduction

Carlos Moffat1,2,4 and Silvio Pantoja1,2
1
Most glaciers in Patagonian Ice Fields have retreated
Department of Oceanography,
2
over the past half-century (Rignot et al., 2003; Willis et al.,
COPAS Sur-Austral Program, Universidad de
2012), with annual ice mass losses of 24.4 ± 1.4 Gt, which
Concepción, Concepción, Chile.
3
are equivalent to a sea level rise of 0.067 ± 0.004 mm
Sorbonne Universités, UPMC Univ Paris 06, CNRS,
year−1 (Willis et al., 2012). Jorge Montt glacier (Fig. 1), the
Laboratoire d’Ecogéochimie des Environnements
northern-most glacier in the Southern Patagonian Ice
Benthiques (LECOB), Observatoire Océanologique,
Field, has experienced a rapid retreat of c. 19.5 km
F-66650, Banyuls sur Mer, France.
4
between 1898 and 2011, and an accelerated rate of ter-
Institute of Marine Sciences, University of California
minal ice loss in recent years (Rivera et al., 2012). Calving
Santa Cruz, Santa Cruz, CA, USA.
of the glacier front as well as submarine melting and
run-off result in an input of freshwater that impacts the
Summary hydrographic structure and water circulation in the
proglacial fjord (Moffat, 2014).
Jorge Montt glacier, located in the Patagonian Ice
The hydrographic structure of Patagonian fjords and
Fields, has undergone an unprecedented retreat
channels in southern Chile is characterized by two or
during the past century. To study the impact of the
more layers originating from high freshwater discharge
meltwater discharge on the microbial community of
from terrestrial drainage, rivers, precipitation and ice
the downstream fjord, we targeted Bacteria, Archaea
melting, and by the deep input of Subantarctic waters
and Fungi communities during austral autumn and
from the adjacent Pacific Ocean (Silva et al., 1998; Dávila
winter. Our results showed a singular microbial com-
et al., 2002). The freshwater flux supplies allochthonous
munity present in cold and low salinity surface waters
organic and inorganic matter to these fjords (Sepúlveda
during autumn, when a thicker meltwater layer was
et al., 2011; Silva et al., 2011; Vargas et al., 2011;
observed. Meltwater bacterial sequences were related
González et al., 2013), which may influence the plank-
to Cyanobacteria, Proteobacteria, Actinobacteria and
tonic trophic web (Vargas et al., 2011), and autotrophic
Bacteriodetes previously identified in freshwater and
and heterotrophic microbial activity (Montero et al., 2011).
cold ecosystems, suggesting the occurrence of
In particular, meltwater represents a sizable source of
microorganisms adapted to live in the extreme con-
freshwater (Rignot et al., 2003; Willis et al., 2012) charac-
ditions of meltwater. For Fungi, representative
terized by high load of suspended sediment (Boldt et al.,
sequences related to terrestrial and airborne fungal
2013), high concentration of silicic acid and low nitrate
taxa indicated transport of allochthonous Fungi by
and phosphate content (González et al., 2011; 2013) that
the meltwater discharge. In contrast, bottom fjord
can impact primary productivity and the structure of
waters from autumn and winter showed representa-
pelagic community in the fjord system adjacent to the ice
tive Operational Taxonomic Units (OTUs) related to
fields (González et al., 2013).
sequences of marine microorganisms, which is con-
The microbial community in cold aquatic environments
sistent with current models of fjord circulation. We
includes representative members of the domains Bacte-
conclude that meltwater can significantly modify the
ria, Archaea and Eukarya (Margesin and Miteva, 2011).
structure of microbial communities and support the
Among Bacteria, Alphaproteobacteria, Betaproteo-
development of a major fraction of microorganisms in
bacteria, Cyanobacteria, Bacteriodetes and Actinoba-
surface waters of Patagonian fjords.
cteria frequently predominate in cryoconites holes
(Edwards et al., 2011; Cameron et al., 2012), snow
Received 20 October, 2014; revised 2 April, 2015; accepted 2 April,
2015. *For correspondence. E-mail magutier@udec.cl; Tel. (+56) 41 and glacial meltwater (Møller et al., 2013), glacier fed
266 1287; Fax (+56) 41 222 5400. streams (Wilhelm et al., 2013), high mountain glaciers
© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd
 Microbial community in meltwaters of Patagonian fjords 3883

Fig. 1. Study area and sampling sites during autumn (red circles) and winter (yellow triangles) in the fjord adjacent to Jorge Montt glacier with
a colour scale of bathymetry (A). Salinity sections (coloured scale) and temperature isolines (black trace) for the April (B) and August (C) 2012
field campaigns.

(Liu et al., 2011) and glacial fjords (Zeng et al., 2009).
Among Archaea, Thaumarchaeota has been detected in
Antarctic cryoconite holes (Cameron et al., 2012) and
sea ice (Cowie et al., 2011). Although the composition of
the fungal community remains less known in cold envi-
ronments, yeast, melanized Fungi and filamentous
Ascomycota have been found in supraglacial systems
(Uetake et al., 2012; Edwards et al., 2013) and in Arctic
ice (Gunde-Cimerman et al., 2003). In fjords and high
latitude waters influenced by glacial meltwater, members
of classes Gammaproteobacteria and Alphaproteo-
bacteria and the phylum Cytophaga–Flavobacterium–
Bacteroides frequently dominate bacterial communities
(Zeng et al., 2009; 2013; Piquet et al., 2010; 2011).
Moreover, changes in hydrographic properties as a result
of meltwater input can induce temporal and spatial
changes in the structure of the microbial community
(Piquet et al., 2010; Zeng et al., 2013). Differences in
bacterial composition have been observed associated to
varying meltwater inflow (Piquet et al., 2010) and between
fjord waters and the adjacent ocean (Zeng et al., 2009).
Among photosynthetic microorganisms, meltwater inflow
seems to favour the occurrence of small size nano- and
picophytoplankton (Piquet et al., 2014).
Patagonian glaciers are retreating (Rignot et al., 2003;
Rivera et al., 2007; Willis et al., 2012) and since meltwater
has the potential to change the physicochemical environ-
ment of the downstream ecosystems, we hypothesize that
melting impacts the microbial community composition in
Patagonian proglacial fjords by favouring the develop-
ment of meltwater-adapted microorganisms. In order to
test our hypothesis, we analysed the spatial (vertical and
horizontal) and seasonal variability of Bacteria, Archaea
and Fungi communities in the water column and sedi-
ments of the fjord adjacent to Jorge Montt glacier.
Results
Environmental variability
During autumn (April), cold (<4°C) and fresh (<20 psu)
surface water occupied the top 10 m of the water column
near the glacier terminus (Fig. 1B). The layer thinned and
its temperature and salinity increased from the terminus
towards the mouth of the fjord connecting to Baker
Channel (c. 20 km, Fig. 1B). Below this layer, temperature
increased gradually from 6°C to c. 10°C in bottom waters
of the fjord basin and c. 9°C in Baker Channel (Fig. 1B).
Salinity also showed a gradual increase from c. 24 to

© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 3882–3897
3884 M. H. Gutiérrez, P. E. Galand, C. Moffat and S. Pantoja
Table 1. Sample ID and chemical and biological characteristics of the waters sampled during April and August 2012 in the fjord adjacent to Jorge
Montt glacier.

Fungal
Sample Depth Nitrate Phosphate Silicic acid Chlorophyll-α Prokaryote filaments
Date ID Station Location (m) (μM) (μM) (μM) (mg m−3) (×108 cell l−1) (ng C l−1)

April 2012 A2-3m A2 48°15.8’S 3 n.d. 0.01 ± 0.01 16.1 ± 0.4 0.14 3.2 ± 0.2 224.7 ± 219.0
73°26.7’W
A3-3m A3 48°14.8 S 3 5.3 ± 1.0 n.d. 18.2 ± 0.7 0.29 1.6 ± 0.0 38.8 ± 40.7
73°26.7 W
A4-3m A4 48°13.9′ S 3 n.d. n.d. 24.5 ± 0.4 0.32 4.0 ± 0.0 28.9 ± 33.6
73°28.2′ W
A5-3m A5 48°06,6′ S 3 2.5 ± 0.6 0.2 ± 0.01 9.5 ± 1.8 0.10 3.0 ± 0.2 10.3 ± 13.8
73°29,7′ W
A1-210m A1 48°15.9’S 210 7.6 ± 0.4 0.8 ± 0.01 3.3 ± 0.1 n.d. 1.1 ± 0.2 n.d.
773°26.9′ W
A2-290m A2 48°15.8’S 290 8.0 ± 0.1 1.0 ± 0.02 6.2 ± 0.2 n.d. 1.6 ± 0.0 n.d.
73°26.7’W
August 2012 W1-0m W1 48°17,8′ S 0 0.8 ± 0.0 0.08 ± 0.00 20.6 ± 0.0 0.02 2.9 ± 0.0 95.2 ± 4.1
73°27,3′ W
W2-0m W2 48°14,2′ S 0 1.0 ± 0.0 0.09 ± 0.00 25.8 ± 0.0 0.6 2.6 ± 0.1 71.8 ± 17.0
73°28,2′ W
W5-3m W5 48°08,8′ S 3 1.4 ± 0.0 0.12 ± 0.00 29.2 ± 0.0 0.34 2.4 ± 0.0 253.8 ± 228.3
73°26,1′ W
W1-300m W1 48°17,8′ S 300 17.7 ± 0.0 0.94 ± 0.00 6.21 ± 0.0 n.d. 1.2 ± 0.0 194.1 ± 115.3
73°27,3′ W
W2-50m W2 48°14,2′ S 50 11.0 ± 0.0 1.05 ± 0.00 4.6 ± 0.0 n.d. 1.2 ± 0.0 6.1 ± 4.1
73°28,2′ W
W5-100m W5 48°08,8′ S 100 24.7 ± 0.0 1.40 ± 0.00 12.5 ± 0.0 n.d. 1.0 ± 0.0 71.8 ± 17.0
73°26,1′ W
W3-sed W3 48°11,6′ S Surf. sed.
73°30,2′ W
W4-sed W4 48°11,5′ S Surf. sed.
73°29,3′ W

Concentrations of nutrients and abundance of prokaryote and biomass of fungal filaments are average ± standard deviation of duplicate samples.
n.d., not detected.

30 psu in waters in the fjord basin and maximum values
(c. 34 psu) in the deeper waters of Baker channel
(Fig. 1B). The hydrographic structure within the fjord is
strongly influenced by the presence of a sill located at the
mouth of the fjord that blocks the inflow of water below
60 m deep from Baker Channel (Fig. 1A). On the distal
side of the sill, water column was characterized by a
subsurface temperature maximum of c. 11.5°C centered
at 30 m, while from the sill and inward, water was colder
and fresher, a result of mixing with the fresh and cold
meltwater. A tongue of warm and salty water was
observed intruding at depth from the sill, which is located
around 35 m along this section and reaches a maximum
depth of 45 m (Fig. 1B).
During winter (August), the fresh surface layer was
thinner than in autumn, with the halocline located at c. 5 m
deep (Fig. 1C). Temperature and salinity in the surface
layer was not homogenous, with colder (<3°C) and fresher
(<15 psu) water located close to the glacier front and at the
mouth of the fjord, the latter suggesting an intrusion of
freshwater from Baker Channel (Fig. 1C). Both tempera-
ture and salinity increased gradually with depth from c. 5°C
to 10°C and 29 to over 32 psu in the inner fjord and in Baker
Channel (Fig. 1C). In this period, the salinity below 15 m in
the fjord basin and in Baker Channel was higher than
30 psu. A subsurface temperature maximum was found
deeper in Baker Channel (at around 45 m depth) and had
a maximum value roughly one degree lower (c. 10.5°C)
than in autumn (Fig. 1B and C).
Surface waters had lower nitrate and phosphate con-
centrations than bottom waters during autumn and winter
(Table 1). In contrast, higher concentrations of silicic acid
were observed in surface relative to bottom waters
(Table 1). Chlorophyll-α in the surface layer showed an
average concentration of 0.17 ± 0.09 mg m−3 in autumn
and 0.30 ± 0.22 mg m−3 in winter (Table 1), and no major
differences were observed from the head to the mouth of
the fjord. In addition, prokaryote abundance and fungal
biomass were higher in surface than bottom waters during
both seasons (Table 1). There were significant differences
in silicic acid concentration at different depths and during
both seasons as well as in nitrate and phosphate concen-
trations and prokaryote abundance between surface and
bottom waters (Table 2).

© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 3882–3897
 Microbial community in meltwaters of Patagonian fjords 3885
Table 2. Two-way ANOVA to test statistical differences on the rank- Fungi (Fig. S1) indicated that all samples had reached the
transformed data of environmental variables and bacterial and fungal
curvilinear phase of sampling effort, and that for Archaea,
richness between seasons and depths.
most of the curves had started to plateau.
Variable Factor df F P-value OTU richness estimated by Chao1 after resampling
ranged from 57 to 367 for Bacteria, 1 to 97 for Archaea
Nitrogen Period 1 0.00 1.000
Depth 1 58.3 0.000 and 5 to 146 for Fungi (Table 3). In 12 out of 14 samples,
Period×Depth 1 18.0 0.005 the observed number of bacterial and fungal OTUs rep-
Phosphate Period 1 0.6 0.467 resented over 80% of the OTU richness estimated by
Depth 1 20.2 0.004
Period×Depth 1 0.6 0.468 Chao1 (Table 3). For Archaea, the abundance detected in
Silicic acid Period 1 5.45 0.048 samples with a relative high number of OTUs (>20) rep-
Depth 1 30.21 0.000 resented over 70% of the richness estimated by Chao1.
Period×Depth 1 0.28 0.612
Prokaryote Period 1 1.12 0.321 Bacterial diversity (Chao1, Table 3) was higher in the
Depth 1 19.18 0.002 bottom (saline) than the surface waters (low salinity) of the
Period×Depth 1 0.04 0.837 fjord basin in autumn and winter (Stations A1-A4 and
Chlorophyll – – – –
Fungal filaments – – – – W1-W2; Table 3). In contrast, fungal richness in the fjord
Chao1 Bacteria Period 1 3.31 0.106 basin was higher in the surface than the bottom layer in
Depth 1 0.81 0.395 both periods (Table 3). Archaeal diversity was highest in
Period×Depth 1 0.18 0.686
Chao1 Archaea Period 1 1.09 0.328 sediments collected during winter close to the fjord mouth
Depth 1 1.46 0.262 and in surface waters from Baker channel (stations W3
Period×Depth 1 0.92 0.365 and W4, Table 3). There were no significant differences
Chao1 Fungi Period 1 0.43 0.530
Depth 1 0.61 0.456 (at 95% confidence level) in bacterial, fungal and archaeal
Period×Depth 1 4.58 0.065 diversity between seasons and depths (Table 2).
Bacterial diversity in surface waters in both autumn and
–, insufficient data for analysis. The values highlighted in bold are
statistically significant at 95% confidence level.
winter was higher in the Baker Channel (sites A5 and W5)
than in the fjord basin (sites A2-A4 and W1 and W2;
Table 3). Fungal communities were more diverse in
Bacteria, Archaea and Fungi community diversity
surface waters in the fjord basin (sites A2-A4) than in
Microbial diversity was analysed by comparing the distri- Baker Channel (site A5) in autumn. In winter, Baker
bution of 34 263 and 21 692 16S rRNA gene sequences of Channel waters had the highest fungal diversity (Table 3).
Bacteria and Archaea respectively, and 38 891 ITS rDNA OTU richness for Bacteria and Fungi showed higher
sequences of Fungi (Table 3). A total of 1443 Operational values during austral autumn (April) compared with winter
Taxonomic Units (OTUs) of Bacteria, 164 of Archaea and (August) in surface waters (Table 3). In bottom waters in
298 of Fungi were identified. Differences in OTU abun- the fjord basin (sites A1, A2, W1 and W2), diversity was
dances among depths, sampling sites and seasons were higher in April for bacteria and in August for Fungi
observed (Table 3). Rarefaction curves for Bacteria and (Table 3). Additionally, in August the highest Bacteria,

Table 3. Number of sequences, OTU abundance and richness after resampling (Chao1) estimated for microbial assemblages from waters and
sediments of the fjord adjacent to Jorge Montt glacier.

Bacteria Archaea Fungi

Sample ID Reads OTUs Chao1 Reads OTUs Chao1 Reads OTUs Chao1

A2-3m 1999 184 167 1402 136 146

A3-3m 2790 199 186 60 3 1894 73 70
A4-3m 5836 285 104 2767 8 1 1085 97 99
A5-3m 3038 342 252 1369 22 17
A1-210m 1865 324 300 18 7 1827 20 18
A2-290m 1106 251 188 378 21 23 6361 26 18
W1-0m 627 47 57 1277 8 4 3549 33 25
W2-0m 347 54 68 1998 9 10 6709 56 23
W5-3m 1872 310 211 2340 82 42 1951 36 41
W1-300m 2835 138 92 8 3 760 42 53
W2-50m 1825 124 90 1855 33 47 4765 47 35
W5-100m 5559 376 201 7031 60 25 1465 83 82
W3-sed 4564 589 367 2264 122 97 1645 54 53
W4-sed 1696 3 2 4109 10 5

© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 3882–3897
3886 M. H. Gutiérrez, P. E. Galand, C. Moffat and S. Pantoja

Archaea and Fungi diversity was observed in surface
sediment collected near the fjord mouth (site W3, c. 38 m
depth, Table 3).
Composition of bacterial communities
Bacterial communities were separated according to their
composition at the OTU level into three major clusters with
a similarity level (based on a Bray–Curtis distance matrix)
higher than 70%. Cluster B1 grouped the surface water
community from autumn, B2 included the bottom water
community from winter and B3 the bottom water commu-
nity from autumn (Fig. 2A). There were two exceptions to
this grouping: sites W2-0m in cluster B2 and W5-3m in
cluster B3. The clustering was consistent with the pattern
observed in the ordination plots derived from principal
coordinate analysis (PCoA; Fig. S2).
Cluster B1 was mainly characterized by OTUs matching
clones of uncultured Synechococcus, Comamonadaceae
Betaproteobacteria, Alphaproteobacteria and Actino-
bacteria of the group Actinomycetales (Fig. 2B, Table 4).
In this group (surface waters in autumn), Cyanobacteria
dominated in waters in the fjord basin (more than 40% of
the total Bacteria sequences), followed by Proteobacteria,
Actinobacteria and Bacteroidetes (Fig. 2C). In contrast,
in surface waters of Baker Channel (sample A5-3m)
sequences of Proteobacteria were the most abundant
(50%) followed by Cyanobacteria (c. 25%; Fig. 2C).
Among Proteobacteria (Fig. 2D), sequences of Alphapro-
teobacteria predominated in surface waters in the fjord
basin (36–44% of total Proteobacteria sequences) and
Gammaproteobacteria in surface waters from Baker
Channel (site A5, 41% of Proteobacteria sequences;
Fig. 2D).

Fig. 2. Dendrogram based on Bray–Curtis similarity between bacterial assemblages in surface and bottom waters from autumn and winter in
the fjord adjacent to Jorge Montt glacier (A). Heatmap of the main bacterial OTUs (B), relative abundance (percentage of total sequences) of
the main bacterial groups (C) and detail for Proteobacteria (D). B1, B2 and B3 represent the bacterial communities identified with more than
70% of similarity.

© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 3882–3897
 Microbial community in meltwaters of Patagonian fjords 3887
Table 4. Best taxonomic match for the main OTUs of Bacteria, Fungi and Archaea communities in the water column and sediment of the fjord
adjacent to Jorge Montt glacier.

Representative Accession
OTU ID cluster/sample Best taxonomic match number % identity Environment Reference

BacJM1 B1 Synechococcus AY151250 99 Subalpine and high altitude lakes Crosbie et al., 2003a, b; Ernst
EU703201 et al., 2003; Xing et al., 2009
EU703437
EU703201
BacJM2 B1, B2 Comamonadaceae DQ234234 100 Estuarine waters Liao et al., 2007
Unclassified Bacteria JF429247 River waters Liu et al., 2012
BacJM3 B1, B2 Alphaproteobacteria DQ063213 98 Coastal environment influenced by Riemann et al., 2008
freshwater discharge
BacJM4 B2 Unclassified Bacteria HQ730061 99 Bacteria associated to Antarctic Murray et al., 2011
icebergs
BacJM5 B1 Actinomycetales HM856562 97 Yellowstone lake Clingenpeel et al., 2011
BacJM8 B2 Unclassified Bacteria EU287086 100 Surface sediments from the Pacific Li et al., 2009
FR685473 Arctic Ocean Newbold et al., 2012
Fjord waters
BacJM9 B3 Unclassified Bacteria EU491101 97–98 Ocean crust and deep sea mud Santelli et al., 2008;
HQ588368 96 volcanoes Pachiadaki et al., 2011
FJ640811 95 Hydrothermal vents Wang et al., 2009
JF344181 Subtidal sediments Acosta-Gonzalez et al., 2013
BacJM10 B2 Alphaproteobacteria of the RSU63934 99 Waters of the Baltic Sea Pinhassi et al., 1997
genus Rhodobacter
BacJM11 B2 Unclassified Bacteria DQ015848 97 Antarctic lakes Glatz et al., 2006; Tang et al.,
Bacteriodetes JX948651 95 Subantarctic waters 2013
(Flavobacteriales) AY794159 Prabagaran et al., 2007
BacJM13 B3 Unclassified Bacteria EU035849 100 Deep-water coral reefs Jensen et al., 2008
HQ673544 99 Subarctic waters Allers et al., 2013
HQ729998 99 Associated to Antarctic icebergs Murray et al., 2011
BacJM19 B2 Unclassified bacteria EU919855 99 Glacial Arctic fjord Zeng et al., 2009
BacJM37 B3, W3-sed Alphaproteobacteria KC492638 100 Sponge-associated bacterial Croué et al., 2013
Unclassified bacteria KC197685 100 community Sjöstedt et al., 2014
Marine environments
BacJM51 W1-0m Gammaproteobacteria (Vibrio) KF942293 100 Upwelling region, Monterey Bay Mansergh and Zehr, 2014
BacJM70 W1-0m, B2 Unclassified bacteria FJ628262 100 Anoxic tidal fjord Schmidtova et al., 2009
EF379591 98 Estuarine environment Zhang et al., 2007
BacJM182 W1-0m Gammaproteobacteria (Vibrio) KF941811 100 Upwelling region, Monterey Bay Mansergh and Zehr, 2014
FunJM1 F1, F2, F3 Basidiomycota GU062301 100 Indoor dust and associated to Arhipova et al., 2011, Wirsel
Unclassified Fungi clone AJ279465 100 terrestrial plants et al., 2001
FJ820495 Air particulate matter Fröhlich-Nowoisky et al., 2009
FunJM2 F4, F1 Cladosporium (Ascomycota) KF225878 100 Soil Hu et al., 2013
KF367474 Freshwater Oliveira et al., 2013
HE608784 Coastal environment Scopel et al., 2013
FunJM3 F2, F3 Basidiomycota, Agaricales KC176337 95–97 Soils Thorn et al., 1996;
KC669316 Ramírez-Cruz et al., 2013
FunJM4 W4-sed Cryptococcus (Basidiomycota) DQ643977 100 In association with mycorrhizal Alonso et al., 2008
DQ317387 fungi Arenz et al., 2006
Antarctic soils
FunJM5 W1-0m Unclassified fungi JF945133 <90 Forest environment Cordier et al., 2012
FunJM6 F1, F2, F3 Cryptococcus (Basidiomycota) FR717837 99 Arbuscular mycorrhizal roots and Yurkov et al., 2012
AJ581048 spores Renker et al., 2004
FunJM7 F3, F4 Unclassified fungi FJ235861 99 Soils Hong et al., 2010
FunJM8 W4-sed Unclassified fungi JF497138 99 Deep-Sea sediments Singh et al., 2012
FunJM9 F1 Unclassified Fungi KF296942 <90 Arctic soils Timling et al., 2014
FunJM10 A5-3m Chytridiomycete HQ191313 95 Lake ecosystem Monchy et al., 2011
FunJM14 F3 Unclassified fungi AB615542 99 Subsurface marine sediments Y. Nagano, T. Nagahama, M.
Konishi, T. Kubota, F. Abe,
K. Takahashi, Y. Hatada,
unpublished
FunJM18 F1, F2, F4 Unclassified Fungi KC965831 100 Arctic soils Timling et al., 2014
Cladosporium (Ascomycota) HM148149 Cosmopolitan fungi Bensch et al., 2010
KF156274 Root associated fungi Stenström et al., 2013
FunJM21 F3 Penicillium (Ascomycota) GU062216 100 Associated with plants Arhipova et al., 2011
FunJM23 F1, A5-3m Hypocreales (Ascomycota) 99 Root associated fungi in saline Macia-Vicente et al., 2012
gradient
FunJM57 F1 Unclassified Fungi JX974768 <90 Estuarine sediments B. Xing, X. Zhang, J. Gong,
unpublished
ArcJM1 A4-3m, W2-50m, W5-100m Unclassified Archaea HE796570 99 Freshwater environments including Auguet and Casamayor, 2012;
KC604531 high mountain lakes Flynn et al., 2013; Kato
AB722183
et al., 2013
ArcJM2 W2-50m, W5-100m, W3-sed, Thaumarchaeota EF069371 100 Antarctic sediments Gillan and Danis, 2007
W2-0m, W5-3m, W4-sed
ArcJM4 W3-sed, W5-3m, W1-0m Thaumarchaeota, Marine JX015356 100 Meromitic saline lake La Cono et al., 2013
group I GQ387725 Mediterranean surface waters Galand et al., 2010

© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 3882–3897
3888 M. H. Gutiérrez, P. E. Galand, C. Moffat and S. Pantoja
Waters from August (cluster B2, Fig. 2) shared main
bacterial OTUs with the microbial community from the
surface meltwater in April (cluster B1; Fig. 2, Table 4). In
addition, OTUs matching clones of unclassified Bacteria,
Alphaproteobacteria of the genus Rhodobacter and
Bacteriodetes, were also representative of this community
(Table 4, Fig. 2). Cluster B2 (mostly bottom waters from
August) was also characterized by a predominance of
Proteobacteria reaching c. 50% of the total bacterial
sequences, followed by Cyanobacteria and Bacteriodetes
(Fig. 2C). Among Proteobacteria, sequences of Alphapro-
teobacteria predominated (40–54% of total Proteoba-
cteria sequences) followed by Betaproteobacteria and
Gammaproteobacteria (Fig. 2D). The bacterial community
in surface waters from site W2 was also included in cluster
B2.
Cluster B3 was mainly composed of two OTUs match-
ing clones of an unclassified Bacteria and an Alpha-
proteobacteria from the marine environment (Fig. 2B,
Table 4). The bacterial community grouped in B3 (bottom
waters from April) was dominated by sequences of
Proteobacteria representing more than 70% of bacterial
sequences (Fig. 2C), with Gammaproteobacteria accoun-
ting for over 65% of total Proteobacteria sequences, fol-
lowed by Alphaproteobacteria, Deltaproteobacteria and
Betaproteobacteria (Fig. 2D). The bacterial community in
surface waters collected in Baker Channel in August (W5-
3m) showed a similar pattern to those observed in bottom
waters grouped in B3.
Sediments from the area close to the fjord mouth
(W3-sed) and surface waters from the area close to the
glacier (W1-0m) collected in August were not included in
any main cluster (Fig. 2A). In these communities, OTUs
matching clones of Alphaproteobacteria, unclassified
Bacteria and Gammaproteobacteria were representatives
(Fig. 2B, Table 4) and a predominance of sequences of
Alphaproteobacteria and Gammaproteobacteria was
observed (Fig. 2D).
Composition of fungal communities
Cluster analysis of fungal community at the OTU level
showed four main groups with more than 60% similarity
(Fig. 3A). Group F1 was composed of surface water com-
munity in autumn, F2 included a mix of surface and
bottom waters in winter, F3 includes shallow sediments
and 100 m waters from Baker Channel collected in winter,
and F4 was composed of bottom waters collected in
autumn (Fig. 3A). The clustering was consistent with the
pattern observed in the ordination plots derived from
PCoA (Fig. S3).
Main OTUs in cluster F1 (surface waters from autumn in
the fjord basin) were assigned to Basidiomycota, Asco-
mycota and uncharacterized Fungi (Fig. 3B; Table 4).
© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 3882–3897
Ascomycota sequences accounted for between 36% and
66% and Basidiomycota from 26% to 50% of the fungal
sequences (Fig. 3C). In the same cluster, sequences of
unclassified fungal OTUs accounted for 7–18% of the
total sequences (Fig. 3C).
In F2 (waters from August), the community was
characterized by OTUs related to clones of Basidio-
mycota of the order Agaricales, uncharacterized Fungi
and Cladosporium spp. (Fig. 3; Table 4). Basidiomycota
sequences represented more than 87% and 50% of the
sequences in surface and bottom waters respectively
(Fig. 3C).
Cluster F3 shared main OTUs with F2 and included
OTUs matching Ascomycota of the genus Penicillium and
uncharacterized Fungi (Fig. 3B; Table 4). This community
(water from 100 m depth in Baker Channel and sediment
close to the fjord mouth in winter) was characterized by a
predominance of Ascomycota Fungi sequences (46–
53%; Fig. 3C). In addition, sequences of Chytridiomycota
represented c. 8% in waters from Baker Channel (100 m)
and unclassified fungal sequences accounted for 24% in
surface sediments (Fig. 3C).
Group F4 was mainly composed of OTUs assigned to
Ascomycota of the genus Cladosporium from different
environments (Fig. 3B; Table 4). In this community,
Ascomycota represented more than 93% of the total
fungal sequences (Fig. 3C).
Samples collected in surface waters of the area close
to the front of the glacier and in surface sediments from
the outside basin in August and surface waters of Baker
Channel in April were not included in any cluster
(Fig. 3A). These fungal communities were mainly com-
posed of OTUs related to unclassified Fungi (W1-0m),
OTUs matching Chitridiomycota and Ascomycota (A5-
3m), and OTUs assigned to Basidiomycota (W4-sed;
Fig. 3C).
Composition of archaeal communities
With the exception of sample A4-3m, no sequences or
only low numbers (0–378 reads) were obtained for
Archaea during autumn (Table 3). During winter,
although we recovered a higher number of sequences in
most of the sampling sites (8–7031 reads), they repre-
sented a small fraction of all OTUs (Table 3). We
attributed this result to the set of primers used, which
were probably not able to detect typical Archaea from
glacial fjords waters. Among the sequences recovered,
more than 90% were related to sequences of
Thaumarchaeota, with main OTUs matching clones pre-
viously detected in fresh and marine waters, sediments
and cold environments (Table 4). Most of the character-
istic archaeal OTUs were found in samples collected in
winter (Table 4).
 Microbial community in meltwaters of Patagonian fjords 3889

Fig. 3. Dendrogram based on Bray–Curtis similarity of the fungal assemblages in surface and bottom waters from autumn and winter in the
fjord adjacent to Jorge Montt glacier (A). Heatmap of the main fungal OTUs (B), and relative abundance (percentage of total sequences) of
main fungal groups (C). F1, F2, F3 and F4 represent the fungal communities identified with more than 60% of similarity.

Discussion retreat in the Patagonian Ice fields (Rivera et al., 2012).

The impact of glacier meltwater on fjord microbes was
assessed by analysing Bacteria, Archaea and Fungi com-
munities in the water column and sediments of the fjord
adjacent to Jorge Montt, a glacier experiencing the fastest
During austral autumn and winter, we found lower con-
centrations of nitrate and phosphate and higher concen-
trations of silicic acid in surface compared with bottom
waters. These chemical characteristics are considered
indicative of the influence of glacial-derived waters in

© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 3882–3897
3890 M. H. Gutiérrez, P. E. Galand, C. Moffat and S. Pantoja
Patagonian fjords (González et al., 2013). Moreover, the
thicker and more extensive surface meltwater layer,
overall lower salinities, and higher stratification of the
water column in autumn compared with winter indicate
that the fjord is subjected to a higher freshwater discharge
from the glacier in autumn. This seasonal variability in
meltwater content sets the framework to evaluate poten-
tial changes in microbial communities exposed to increas-
ing glacial melting. The autumn conditions, reflecting a
higher contribution of meltwater, favoured the occurrence
of a microbial community adapted to cold and freshwater
in the surface layer of the fjord. It indicates that a sus-
tained input of glacial-derived waters have the potential to
change microbial community composition in the
Patagonian fjords, displacing autochthonous microorgan-
isms and supporting the development of others, better
adapted to live under the extreme conditions observed
under an ice melting scenario.
Bacterial richness was higher in autumn than winter in
the surface layer of the fjord, suggesting both that melt-
water supports the development of different bacterial taxa
and that glacial-derived waters carry microorganisms to
the proglacial fjord. Although we lack direct measure-
ments of cell transport by meltwater, this would be
consistent with recent reports of substantial cellular
flux to downstream ecosystems from glacier surfaces
(Irvine-Fynn and Edwards, 2014). Cyanobacteria, identi-
fied previously as Synechococcus in cold and freshwater
environments, were the predominant bacterial OTUs of
surface meltwater during austral autumn. Cyanobacteria
are considered the dominant phototrophic microorgan-
isms in cold freshwater environments (Vincent, 2000;
Zakhia et al., 2008; Vincent and Quesada, 2012) and
important contributors to nitrogen fixation in polar waters
(Vincent, 2000). In particular, the group Synechococcus is
a dominant part of picophytoplanktonic prokaryotes in
freshwater environments (Callieri, 2007), and a conspicu-
ous microorganism of Arctic and Antarctic water bodies
(Vincent, 2000; Vincent et al., 2000), where potential
adaptations have been proposed for distinctive lineages
(Huang et al., 2012). Cyanobacteria can play a main role
in photosynthetic production of organic matter under low
nutrient (Callieri and Stockner, 2002; Callieri, 2007) and
oligotrophic conditions (Bell and Kalff, 2001; Callieri,
2007), in high latitude freshwaters bodies (Vincent and
Quesada, 2012), and in glacial fjords (Piquet et al., 2014).
Considering the low nitrate and phosphate concentrations
observed in the surface oligotrophic meltwater of the fjord,
Cyanobacteria could contribute significantly to photosyn-
thetic production in the waters adjacent to Jorge Montt
glacier during autumn. In agreement with that, Piquet
et al. (2014) found that meltwater inflow favoured the
occurrence of nano- and picophytoplankton cells, includ-
ing cyanophytes in waters of the Arctic fjords.
© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 3882–3897
Sequences of Proteobacteria, Actinobacteria and
Bacteriodetes were also abundant in surface meltwater
during autumn (Fig. 2), with most of main OTUs related to
sequences previously identified in cold and freshwater
environments (Table 4). These Bacteria phyla are fre-
quently identified in glacial environments such as
cryoconite holes (Edwards et al., 2011; Cameron et al.,
2012), glacier-fed streams (Wilhelm et al., 2013), and
fjords and polar waters influenced by glacial meltwater
(Zeng et al., 2009; 2013; Piquet et al., 2010; 2011).
Among Proteobacteria, Alphaproteobacteria, Betapro-
teobacteria and Gammaproteobacteria showed similar
relative abundance. This is consistent with previous
findings showing high abundance of clones of Alphapro-
teobacteria, Betaproteobacteria and Gammaproteo-
bacteria in waters of Arctic fjords (Piquet et al., 2010)
and with a predominance of Alphaproteobacteria and
Gammaproteobacteria in sea-ice environments (Brown
and Bowman, 2001) and in surface waters of high latitude
and fjords ecosystems influenced by glacial meltwaters
(Zeng et al., 2009; Piquet et al., 2011). In addition,
Alphaproteobacteria and Betaproteobacteria are common
in cryoconite holes (Edwards et al., 2011, 2013, 2014;
Cameron et al., 2012) and Betaproteobacteria in
subglacial Antarctic lakes (Christner et al., 2014). In par-
ticular, the occurrence of abundant OTUs identified as
Comamonadaceae is consistent with previous observa-
tions of this group of Betaproteobacteria in cryoconites
holes (Cameron et al., 2012), glacial-fed streams
(Wilhelm et al., 2013) and subglacial waters (Chen and
Foght, 2007).
Among Fungi, a predominance of Ascomycota over
Basidiomycota-related sequences was observed in melt-
water in the fjord basin in autumn. Consistently, a sig-
nificant proportion of Ascomycota have been isolated
from cryoconite sediments (Edwards et al., 2013), and in
soils influenced by alpine glaciers, they dominate the
fungal community in recently retreated soils (Zumsteg
et al., 2012). A different pattern was observed in surface
waters of Baker Channel where a predominance of
sequences related to Chytridiomycetes was observed
(Fig. 3C). Chytrid Fungi are ubiquitous in aquatic envi-
ronments, where they are saprotrophs of plant material
and parasites of algae (James et al., 2006). This group
has been identified previously in periglacial soils
(Freeman et al., 2009) as well as in cryoconites holes
(Edwards et al., 2013). Due to their role as saprotrophs,
we suggest that chytrids can play an important function
in plant litter degradation in waters of Baker Channel,
where detritus from the adjacent forest may represent a
significant source of organic matter. Our results show a
recurrent fraction of unclassified fungal sequences in
surface waters during autumn (up to 18% of total fungal
sequences), suggesting that glacial waters, among
 Microbial community in meltwaters of Patagonian fjords
others, can be a source of unknown Fungi in Patagonian
fjords. Consistently, we observed higher abundance of
fungal OTUs in surface compared with bottom waters
and inside the fjord compared with Baker Channel in
autumn, as well as higher biomass of filamentous Fungi
in surface compared with bottom water and in meltwater
close to the glacier front. We also found unclassified
fungal OTUs in surface sediments, as well as in the
surface waters closest to the glacier front in winter,
where represented more than 80% of the total
sequences. Our findings, together with the report by
Edwards and colleagues (2013) of abundant uncultured
Fungi in Arctic cryoconite holes, suggest that glacial
environments can be a significant reservoir of fungal
diversity and biomass.
A higher richness of bacterial OTUs was observed in
bottom than in surface waters of the fjord basin during
autumn. Physicochemical characteristics of bottom
waters are representative of marine conditions, suggest-
ing that the bacterial community is more diverse in marine
than meltwaters in the fjord. In waters deeper than 200 m
of the fjord basin during autumn, a distinctive microbial
community was detected. This community was character-
ized by a dominance of Ascomycota Fungi and
Proteobacteria sequences, with Gammaproteobacteria
accounting for more than 65% of total Proteobacteria. The
main bacterial OTUs in these bottom waters were related
to sequences previously identified in deep ocean environ-
ments, such as ocean crust, deep-sea mud volcanoes
and hydrothermal vents, as well as subtidal sediments,
deep-water coral reefs, and Subarctic and Antarctic
waters (Table 4), suggesting the transport of microorgan-
isms in deep waters from the open ocean. That is con-
sistent with the model of circulation proposed for
Patagonian fjords, which describes the inflow of Subant-
arctic waters to the deepest strata of the fjords (Silva
et al., 1998). This deep inflow of waters of oceanic origin
was also observed in the proglacial fjord studied here
(Moffat, 2014).
The composition of major groups of Fungi detected in
bottom waters during autumn (Fig. 3C) is consistent with
the global spatial distribution of major phyla of Fungi
observed in coastal and marine airborne environments
(Fröhlich-Nowoisky et al., 2012), which agrees with the
model of water circulation of these fjords and suggests
that Subantarctic waters carry microbes to deep waters. A
strong dominance of sequences related to Ascomycota
was observed in the community of bottom waters during
autumn. This dominance appears to be a general charac-
teristic of fungal community in aquatic environments and
sediments (Shearer et al., 2007; Mohamed and Martiny,
2011). Main fungal OTUs in this community included
sequences related to Cladosporium spp. and unchar-
acterized Fungi detected previously in freshwater and
© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 3882–3897
3891
coastal waters, as well as in Arctic soil (Table 4). Although
the genus Cladosporium is a cosmopolitan Ascomycota
Fungi in terrestrial and air environments (Bensch et al.,
2012), it is also found in isolates from coastal and oceanic
waters (Wang et al., 2012), from endosymbiotic commu-
nities of seaweed (Suryanarayanan, 2012) and from
extreme environments such as the Dead Sea (Oren
and Gunde-Cimerman, 2012) and salterns (Zajc et al.,
2012).
A different microbial assemblage was observed during
austral winter. The composition of this community was
similar to those generally describing oceanic prokaryotes
(Biers et al., 2009; Zinger et al., 2011). The main OTUs
included sequences related to Alphaproteobacteria,
Betaproteobacteria and Bacteriodetes identified previ-
ously in coastal and estuarine environments, Antarctic
and Subantarctic waters, Pacific Arctic waters and sea-
water from the Baltic Sea (Table 4). During the same
period, sequences of Archaea were also recurrent in
waters and sediments, with main OTUs related to
sequences of Thaumarchaeota previously detected in
marine environments, including sediments of the Antarctic
region (Table 4). We suggest that a lower dilution of
marine conditions, produced by a decrease in the melt-
water discharge from the glacier, results in the predomi-
nance of marine prokaryotes during winter. In addition, the
occurrence of mixing events breaking water column strati-
fication could explain the presence of common microbial
taxa in surface and bottom waters. Similarly, Piquet and
colleagues (2011) found that in coastal Antarctic waters
wind-forced mixing events breaking meltwater stratifica-
tion can produce changes in the composition of bacterial
community.
The Fungi community during winter was dominated by
Basidiomycota-related sequences and was consistent
with the global pattern described for continental airborne
Fungi (Fröhlich-Nowoisky et al., 2012). Since Basidio-
mycota are predominant in terrestrial environments
(Buée et al., 2009), we suggest that air transport of
allochthonous Fungi can play an important role in deter-
mining fungal composition in Patagonian glaciers and
fjords.
Overall, the proglacial fjord surface meltwater commu-
nity during autumn was characterized by a predominance
of Cyanobacteria and Ascomycota over Basidiomycota-
related sequences. In contrast, waters from winter were
characterized by a higher proportion of Proteobacteria, a
predominance of Basidiomycota sequences (over 80% of
total fungal sequences in surface waters) and the occur-
rence of archaeal OTUs. In bottom waters from autumn,
Proteobacteria dominated bacterial community (more
than 70%), with a significant proportion of Gamma-
proteobacteria, and the fungal community was dominated
by Ascomycota.
3892 M. H. Gutiérrez, P. E. Galand, C. Moffat and S. Pantoja
In conclusion, our results suggest that meltwater from
Jorge Montt glacier sets favourable conditions for the
development of microbes adapted to cold and low salinity
surface waters of the proglacial fjord during a high dis-
charge scenario. During these periods, Cyanobacteria
dominate the bacterial community and are likely involved
in the photosynthetic synthesis of organic matter. Among
Fungi, the presence of a significant fraction of airborne
and terrestrial taxa suggests the transport of
allochthonous Fungi by meltwater to the fjord, including
unknown taxa.
We also found that the deep-water circulation supplies
marine microorganisms to the bottom waters of the fjord.
During autumn, this marine signal is diluted by meltwater,
but in winter a reduction in the thickness of the meltwater
layer and probably the occurrence of mixing events pro-
motes a dominance of marine microbes and sustain more
homogenous microbial communities in the water column.
In the relatively higher meltwater scenario, a fresh and
cold water signal was detected c. 20 km away from the
front of the glacier, suggesting that microbes associated
with meltwater can represent a significant proportion of
the microbial community in Patagonian fjords.
Experimental procedures
Study area and sampling
Sample collection was carried out in the proglacial fjord adja-
cent to Jorge Montt glacier, located in the northern section of
the Southern Patagonian Ice Field (48°20’S; 73°30’W;
Fig. 1A). Field campaigns were conducted during austral
autumn (25 April–2 May 2012) and winter (28–30 August
2012) onboard the vessel Don Tito. Water was collected with
Niskin bottles from surface (0–3 m) and bottom waters (50–
300 m) in sites along a 20 km gradient, which includes waters
from the area close to the glacier terminus and from Baker
channel (outside the fjord basin; Fig. 1, Table 1). One litre of
pre-sieved seawater (210 μm) was filtered through 0.45 μm
sterile membrane filters (Millipore) and stored at −20°C for
DNA analyses. Sediments were collected with a dredge at
two sites, inside (38 m depth) and outside (84 m depth) the
fjord basin during the winter campaign (Fig. 1A, Table 1).
Hydrographic data were recorded using a SeaBird 25
CTD in April 2012 and a RBR XRX-620 CTD in August
2012.
Samples for quantification of particulate chlorophyll and
dissolved inorganic nutrients were collected from c. 1 L sea-
water filtered throughout 0.7 µm glass fiber filters.
Chlorophyll-α was analysed by fluorometry (Parsons et al.,
1984) and nutrients by spectrophotometry (Strickland and
Parsons, 1972). Prokaryotes and fungal filaments abundance
were analysed by epifluorescence microscopy using 4,6-
diamidino-2-phenylindole (Porter and Feig, 1980) and
Calcofluor White (Gutiérrez et al., 2011) from 10 to 50 ml of
water subsamples collected directly from the Niskin bottle
and fixed with 2% and 3% formaldehyde (final concentration)
respectively.
© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 3882–3897
DNA extraction and 454 pyrosequencing
DNA from filters and sediment samples (0.2 g) were
extracted and cleaned using a PowerSoil® DNA Isolation Kit
and Power Clean® DNA Clean-up Kit (MOBIO Laboratories).
Bacteria and Archaea 16S rRNA genes were amplified using
primer sets 28F (5′-GAG TTT GAT CNT GGC TCA G-3′) and
519R (5′-GTN TTA CNG CGG CKG CTG-3′), and 341F (5′-
GYG CAS CAG KCG MGA AW-3′) and 958R (5′-GGA CTA
CVS GGG TAT CTA AT-3′) respectively. For Fungi, the inter-
nal transcribed spacer (ITS) region was amplified by using
the ITS1F (5′-CTT GGT CAT TTA GAG GAA GTA A-3′) and
ITS4R (5′-TCC TCC GCT TAT TGA TAT GC-3′) set of
primers. Amplification and sequencing on a 454 GS-FLX
system (Roche) with titanium chemistry were conducted in a
commercial laboratory (Research and Testing Laboratory,
Lubbock, TX, USA) (Dowd et al., 2008).
Sequence data processing
The full 454-pyrosequencing data set is available at the
National Center for Biotechnology Information Sequence
Read Archive under the Accession No. SRR1607287 and
SRR160331. Raw sequences were analysed using the
Pyrotagger pipeline (Kunin and Hugenholtz, 2010). The reads
were first filtered by removing low-quality reads, then trimmed
to remove reads having ≥ 3% of bases with Phred values <27
(0.2% per-base error probability). This is recommended to
ensure that when clustering at 97%, the influence of errone-
ous reads is minimized (Huse et al., 2010). The sequence
length threshold was set to 350 bp for Bacteria and Fungi and
200 bp for Archaea, and Uclust was used to cluster OTUs at
a 97% similarity threshold. Representative sequences from
each OTU were classified by comparison with the
Greengenes database (De Santis et al., 2006) for Archaea
and Bacteria and with SILVA (Quast et al., 2013) for Fungi.
Pyrotagger outputs, including OTU distribution among
samples and taxonomic affiliation, were used as input for
analyses in the qiime software package version 1.6.0
(Caporaso et al., 2010) after removal of potential chimeras.
Analysis of similarity in community composition and diversity
(Chao1) was carried out after resampling with the rarefaction
method using the minimum number of sequences per sample
for Bacteria and Fungi and a subsample of 60 sequences for
Archaea. Similarity was estimated at the OTU level based on
the Bray–Curtis distance matrix index and hierarchical cluster
analysis (UPGMA clustering). A PCoA was carried out in R
version 3.1.2 using the package vegan (Oksanen et al.,
2013) to verify the clustering pattern obtained with UPGMA
clustering.
OTU heatmaps were produced to identify the contribution
of individual OTUs to the community composition of the dif-
ferent samples. OTU heatmaps were filtered to display the
representative OTUs defined as the ones containing more
than 150 or 60 sequences per OTUs for Fungi and Bacteria
respectively. Rarefaction curves for each microbial commu-
nity were generated from the means of 100 randomized data
sets using the software EstimateS version 9 (Colwell, 2013).
Two-way analysis of variance was applied to test statistical
differences between periods and depths on the rank-
transformed data of environmental variables and community
 Microbial community in meltwaters of Patagonian fjords
diversity. This transformation allows dealing with data that do
not meet the assumptions of normality (Conover and Iman,
1981; Neter et al., 1996).
Acknowledgements
This research was funded by FONDECYT Grants Nos.
11110515 and 791220026, CONICYT, Chile. A research visit
of M. H. Gutiérrez to the Observatoire Océanologique de
Banyuls was funded by the International Associated Labora-
tory MORFUN. C. Moffat and the collection and analysis of
hydrographic data was funded by FONDECYT Grant No.
11100362. P. Galand was supported by the Agence Nationale
de la Recherche (ANR) project MICADO (ANR-11JSV7-003-
01). We acknowledge C. Córdova, A. M. Jara, R. Mansilla
and C. Iturra for their help during fieldwork in Patagonia and
laboratory analysis, R. Veas for his assistance with statistical
analysis and Waters of Patagonia and the crew of the vessel
Don Tito for their support during fieldwork. Partial funding
was obtained from Program COPAS Sur-Austral PFB-31. We
are grateful to the Hanse-Wissenschaftskolleg (HWK),
Delmenhorst, Germany, for the Fellowship awarded to S.
Pantoja.
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Supporting information
Additional Supporting Information may be found in the online
version of this article at the publisher’s web-site:
Fig. S1. Rarefaction curves for Bacteria (A), Fungi (B) and
Archaea (C) for each sampling sites and depths in the fjord
adjacent to the Jorge Montt glacier.
Fig. S2. Principal coordinate analysis (PCoA) based on
Bray–Curtis distance matrix of OTUs of Bacteria in the fjord
adjacent to the Jorge Montt glacier. Colours in three-
dimensional plots represent the groups identified by the
UPGMA clustering analysis. NC indicates samples not
grouped by the clustering method. PCo1, PCo2 and PCo3
explained 67% of the variability.
Fig. S3. Principal coordinate analysis (PCoA) on the basis of
Bray–Curtis distance matrix of OTUs of bacterial (A) and
fungal (B) communities in the fjord adjacent to the Jorge
Montt glacier. Colours in three-dimensional plots represent
the groups identified by UPGMA clustering analysis. NG indi-
cates not grouped samples. PCo1, PCo2 and PCo3
explained 49% of the variability.