Fungi from Admiralty Bay (King George Island, Antarctica) Soils and Marine Sediments

Abstract

Extreme environments such as the Antarctic can lead to the discovery of new microbial taxa, as well as to new microbial-derived natural products. Considering that little is known yet about the diversity and the genetic resources present in these habitats, the main objective of this study was to evaluate the fungal communities from extreme environments collected at Aldmiralty Bay (Antarctica). A total of 891 and 226 isolates was obtained from soil and marine sediment samples, respectively. The most abundant isolates from soil samples were representatives of the genera Leucosporidium, Pseudogymnoascus, and a non-identified Ascomycota NIA6. Metschnikowia sp. was the most abundant taxon from marine samples, followed by isolates from the genera Penicillium and Pseudogymnoascus. Many of the genera were exclusive in marine sediment or terrestrial samples. However, representatives of eight genera were found in both types of samples. Data from non-metric multidimensional scaling showed that each sampling site is unique in their physical-chemical composition and fungal community. Biotechnological potential in relation to enzymatic production at low/moderate temperatures was also investigated. Ligninolytic enzymes were produced by few isolates from root-associated soil. Among the fungi isolated from marine sediments, 16 yeasts and nine fungi showed lipase activity and three yeasts and six filamentous fungi protease activity. The present study permitted increasing our knowledge on the diversity of fungi that inhabit the Antarctic, finding genera that have never been reported in this environment before and discovering putative new species of fungi.

Full text

FUNGAL MICROBIOLOGY
Fungi from Admiralty Bay (King George Island, Antarctica) Soils
and Marine Sediments
Lia Costa Pinto Wentzel
1
&Fábio José Inforsato
1
&Quimi Vidaurre Montoya
1
&Bruna Gomes Rossin
2
&
Nadia Regina Nascimento
2
&André Rodrigues
1
&Lara Durães Sette
1
Received: 3 December 2017 / Accepted: 31 May 2018 /Published online: 19 June 2018
#Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
Extreme environments such as the Antarctic can lead to the discovery of new microbial taxa, as well as to new microbial-derived
natural products. Considering that little is known yet about the diversity and the genetic resources present in these habitats, the
main objective of this study was to evaluate the fungal communities from extreme environments collected at Aldmiralty Bay
(Antarctica). A total of 891 and 226 isolates was obtained from soil and marine sediment samples, respectively. The most
abundant isolates from soil samples were representatives of the genera Leucosporidium,Pseudogymnoascus, and a non-
identified Ascomycota NIA6. Metschnikowia sp. was the most abundant taxon from marine samples, followed by isolates from
the genera Penicillium and Pseudogymnoascus. Many of the genera were exclusive in marine sediment or terrestrial samples.
However, representatives of eight genera were found in both types of samples. Data from non-metric multidimensional scaling
showed that each sampling site is unique in their physical-chemical composition and fungal community. Biotechnological
potential in relation to enzymatic production at low/moderate temperatures was also investigated. Ligninolytic enzymes were
produced by few isolates from root-associated soil. Among the fungi isolated from marine sediments, 16 yeasts and nine fungi
showed lipase activity and three yeasts and six filamentous fungi protease activity. The present study permitted increasing our
knowledge on the diversity of fungi that inhabit the Antarctic, finding genera that have never been reported in this environment
before and discovering putative new species of fungi.
Keywords Extremophiles .Fungal diversity .Marine mycology .Maritime Antarctica .Cold-adapted enzymes
Introduction
The Antarctic environment is characterized by low tempera-
tures, high UVincidence, dryness, freeze and thaw cycles, low
nutrient content, and high salinity, in addition to long periods
of darkness [1–3]. Despite these extreme conditions, many
microorganisms can thrive in this environment. Fungi and
bacteria play great ecological roles in the Antarctic environ-
ment, but their diversity information is still poorly explored
[4]. Fungi that inhabit marine sediments and soil work as
decomposers and are dominant components of the biomass
in Antarctic ecosystems [5,6].
Antarctic microorganisms, considered extremophiles, de-
veloped adaptation mechanisms, including high concentra-
tions of unsaturated membrane lipids, antifreeze proteins,
and enzymes that are active at low temperatures known as
cold-adapted enzymes [7–10]. Cold-adapted enzymes are ac-
tive at low and moderate temperatures, require lower activa-
tion energy, and are stable at higher temperatures (the inacti-
vation temperature usually is higher than the catalysis optimal
temperature, unlike the mesophilic analogs). All these advan-
tages are attractive for industrial processes because they can
decrease energy costs. Besides this, in processes conducted
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00248-018-1217-x) contains supplementary
material, which is available to authorized users.
*Lara Durães Sette
larasette@rc.unesp.br
1
Instituto de Biociências, Departamento de Bioquímica e
Microbiologia, São Paulo State University (UNESP), Av 24A, 1515,
Rio Claro 13506-900, SP, Brazil
2
Instituto de Geociências e Ciências Exatas, Departamento de
Planejamento Territorial e Geoprocessamento, São Paulo State
University (UNESP), Avenida 24A, 1515, Rio Claro 13506-900, SP,
Brazil
Microbial Ecology (2019) 77:12–24
https://doi.org/10.1007/s00248-018-1217-x

with enzymes that are extremely efficient at low temperatures,
the contamination by other microorganisms can be avoided
[11,12].
Oxidative enzymes can be applied in a wide range of in-
dustrial processes in the food and textile industries. Besides,
they have pharmaceutical and nanotechnological applications
and can also be used for effluent treatment [13–15].
Hydrolytic enzymes such as proteases account for approxi-
mately 60% of the world enzyme trade and are used for
treating industrial rejects and in the production of pharmaceu-
ticals [16,17].
Considering the ecological relevance, the potential of fungi
to produce oxidases and hydrolases and the properties of cold-
adapted enzymes, fungi from the Antarctic environment could
be considered as interesting microbial resources to be
exploited and biotechnologically applied [18]. In this sense,
the aim of the present study was to characterize and compare
the fungal diversity in marine sediment and in different
Antarctic soil samples and also to evaluate the capability of
these fungi to produce oxidative (terrestrial fungi) and hydro-
lytic (marine fungi) enzymes.
Methods
Sampling Site
Soil, plants, and marine sediment samples were collect-
ed in triplicate at eight different points in the Admiralty
Bay region (King George Island, South Shetlands
Archipelago, Maritime Antarctica) (Fig. 1) in January
2015. At Yellow Point, two different types of soil (yel-
low and dark brown soils) were collected (5 cm depth).
At Punta Hennequin and Punta Plaza, the vascular
plants Deschampsia antarctica and Colobanthus
quitensis were collected with root-aggregated soil.
Marine sediment samples were collected with a Van
Veen grab. All samples were stored in sterile plastic
bags and kept under refrigeration for up to two months
until isolation took place. The triplicates were merged to
form composite samples, yielding the final samples
listed in Table 1.
Soil and Marine Sediment Physical and Chemical
Characterization
Soil and sediment granulometry was measured by the method
adapted from Camargo et al. [19]. To determine iron (Fe) and
aluminum (Al) content, three different measurements were done:
the dithionite-citrate-bicarbonate procedure as performed by
Mehra and Jackson [20], the ammonium oxalate procedure ac-
cordingtoMcKeagueandDay[21], and sodium pyrophosphate
after McKeague et al. [22].
Isolation of Soil Microorganisms
Soil samples (12.5 g) and root-associated soil (6.25 g) were
added to Erlenmeyer flasks containing 112.5 mL of Savitha’s
minimal medium [23]. As inducers for the production of
ligninolytic enzymes, 0.2% of sterilized sugarcane bagasse and
rice straw were added to the samples, separately. The
Erlenmeyer flasks were agitated at 140 rpm and incubated at
5 °C and 15 °C for 7 days. After the incubation period, 200 μl
of each sample were taken and serially diluted in a 0.085% NaCl
solution. The dilutions of 10
−1
and 10
−3
of each sample were
plated on Petri dishes. Four different culture media were used (in
gL
−1
): MA2%: 20 malt extract, 15 agar; MA2% 10× diluted;
BSA: 15 malt extract, 2 yeast extract, 15 agar, 2 lactic acid
(added after autoclaving), and B&K: 10 glucose, 2 peptone, 1
yeast extract, 20 agar, 4 mM guaiacol (added after autoclaving).
Isolation of Marine Sediment Microorganisms
Isolation was carried out in two steps, with and without sample
enrichment. For the enrichment step, 25 g of each sample were
added to Erlenmeyer flasks containing 225 mL of artificial
seawater (ASW) and enzymatic inductors for lipase or protease.
Olive oil 1.5% (v/v) and Tween 80 1.5% (v/v)wereusedas
lipase inductors and Skim Milk 2% (m/v) as protease inductor.
Each sample was agitated at 150 rpm and incubated at 5 °C and
15 °C for 10 days. After this period, 200 μL of the dilutions
10
−1
and 10
−3
were inoculated in Petri dishes on four different
culture media (in g L
−1
): PDA: 200 potato, 20 glucose, 15 agar;
PDA 10× diluted; BSA, and MA - Marine Agar: 5 peptone, 1
yeast extract, 0.1 ferric citrate, 19.45 NaCl, 8.8 MgCl
2
,3.24
Na
2
SO
4
,1.8CaCl
2
,0.55KCl,0.16NaHCO
3
,0.08KBr,
34.0 mg L
−1
SrCl
2
,22.0mgL
−1
HBr, 4.0 mg L
−1
Na
2
SiO
3
,
1.6 mg L
−1
NH
4
NO
3
,8.0mgL
−1
Na
2
HPO
4
, 15 agar. Plates
were incubated at 5 and 15 °C. Isolation without the enrichment
followed the same principle: 25 g of each sample in 225 mL of
artificial seawater (ASW), but without any inductor. Each sam-
ple was homogenized for 60 min at 150 rpm, at 5 and 15 °C.
Then, 200 μL of the dilutions 10
−1
and 10
−3
were inoculated in
Petri dishes in the same culture media listed above.
For both isolation processes, streptomycin (0.01 g L
−1
)and
chloramphenicol (0.1 g L
−1
) were added to all culture media
after their autoclavation. Plates were incubated up to 2 months
at 5 and 15 °C to retrieve both psychrophilic and
psychrotrophic fungi. Individual colonies of fungi were puri-
fied in their isolation media. Long- and medium-term preser-
vation were carried out at −80 and 4 °C using cryotubes with
sterile 10% glycerol and water, respectively. All of the fungal
isolates obtained in this study were deposited in the Microbial
Resource Center Culture Collection of the São Paulo State
University, Rio Claro, Brazil, under the codes CRM and
LAMAI included in the sequences deposited in Genbank
(see the topic Accession numbers).
Fungi from Admiralty Bay (King George Island, Antarctica) Soils and Marine Sediments 13

Fungal Identification
Soil filamentous fungi were grouped in morphotypes. Fungal
macromorphology was used to separate the morphotypes, and
one representative of each morphotype was randomly chosen
for DNA sequencing. For marine sediment fungi, all isolates
had their DNA sequenced. DNA extraction of the filamentous
fungi followed the method of Lacerda et al. [24]. The ITS
region was amplified and sequenced with the primer pair
ITS4 and ITS5 [25].
A mini satellite-primed PCR (MSP-PCR) was done to sep-
arate the soil yeasts in different fingerprints. Yeast DNA
Table 1 Data of the terrestrial and
marine samples collected at
Admiralty Bay
Site Sample Geographic
coordenate
Average T
(°C)
Ave r ag e
depth (m)
Yellow Point (S1) Yellowish soil 62° 04.479′S; 58°
23.726′W
3.6 Superficial
(5 cm)
Yellow Point (S2) Dark brown soil 62° 04.479′S; 58°
23.726′W
2.5 Superficial
(5 cm)
Punta Hennequin (S3) C. quitensis
root-associated soil
62° 07.216′S; 58°
23.677′W
3.6 Superficial
(5 cm)
Punta Hennequin (S4) D. antarctica
root-associated soil
62° 07.216′S; 58°
23.677′W
3.5 Superficial
(5 cm)
Punta Plaza (S5) D. antarctica
root-associated soil
62° 05.363′S; 58°
24.691′W
6.4 Superficial
(5 cm)
Punta Plaza (S6) C. quitensis
root-associated soil
62° 05.363′S; 58°
24.691′W
6.3 Superficial
(5 cm)
Botany Point (Sed1) Marine sediment 62° 05.734′S58°
19.919′W
0.6 24.7
EACF
a
(Sed2) Marine sediment 62° 05.130′S58°
23.356′W
0.4 22.9
Punta Ullman (Sed3) Marine sediment 62° 05.015′S58°
20.987′W
0.3 20.3
Refugio 2 (Sed4) Marine sediment 62° 04.373′S58°
25.335′W
0.1 20.0
Refugio 2 intertidal
zone (Sed5)
Marine sediment 62° 04.341′S58°
25.233′W
2.4 Superficial
(5 cm)
a
EACF in front of the Brazillian Station
14 Wentzel L. C. P. et al.
Fig. 1 Sampling sites in Aldmiralty Bay (King George Island, South Shetlands Archipelago, Maritime Antarctica)

extraction followed the method adapted from Sampaio et al.
[26]andDeAlmeida[27]. MSP-PCR was performed using
(GTG)
5
primer according to Duarte et al. [28]. One represen-
tative of each different fingerprint was randomly selected for
DNA sequencing. The LSU (D1/D2) region was amplified
and sequenced with the primer pair NL1 and NL4 [29]. PCR
for LSU was performed according to Duarte et al. [28].
Amplicons from filamentous fungi and yeasts were puri-
fied using the enzymes Exonuclease I and Alkaline phospha-
tase (Thermo Scientific, Massachusetts, USA) according to
the manufacturer’s protocol. Samples were quantified in
NanoDrop® (Thermo Scientific) and sequenced using the
BigDye Terminator® v.3.1 kit (Applied Biosystems,
California, USA) after the manufacturer’sinstructionsinan
ABI 3500 sequencer (Applied Biosystems). The sequencing
conditions for all molecular markers were 95 °C/min followed
by 28 cycles at 95 °C/15 s, 50 °C/45 s, and 60 °C/4 min. The
generated sequences were assembled into contigs in BioEdit
v.7 . 2.5 [30] and compared to homologous sequences deposit-
ed in the NCBI-GenBank database using BLAST. Data sets
comprising the assembled sequences (those generated in this
study and the sequences obtained from the database) were
aligned in MAFFT v 7 [31]. The alignments were prepared
separately for each fungus genus. Phylogenetic trees were
inferred in MEGA v.7.1 [32], using the Neighbor-joining
method and with Kimura 2-parameters nucleotide substitution
model [33]. The robustness of the trees was calculated using
the bootstrap method, with 1000 generations.
Accession Numbers
Sequences generated in this study were deposited in Genbank
under accession numbers MG735773-MG736057 (yeasts)
and MH128164-MH128317 (filamentous fungi).
Soil/Marine Sediment-Taxonomy Correlation, Species
Diversity, Richness, and Distribution
Soil and marine sediment physical-chemical characteristics
were related to the species composition profile of each
sample by non-metric multidimensional scaling (NMDS).
An analysis of similarities (ANOSIM) using a Bray-Curtis
dissimilaritymatrixwasperformedtoverifyiftherewere
differences between soil sample groups (C. quitensis root-
associated soil and D. antarctica root-associated soil) and
between the two isolation temperatures. All analyses were
performed using PAST v. 2.17c [34]. To quantify species
diversity, the Shannon and the inverse Simpson indices
were calculated and for species richness, the Chao-1 esti-
mator was used. The similarities among fungal taxa from
different samples were estimated using the Bray-Curtis,
Sorensen, and Jaccard indices. All indices and estimators
were calculated in EstimateS v.9.1.0 [35].
Screening of Cold-Adapted Enzymes
Soil isolates were screened for ligninolytic enzymes laccase,
manganese peroxidase, and lignin peroxidase. The first screen-
ing was done on B&K medium with guaiacol 4 mM (during the
isolation step) accordingtoVermaetal.[36]. The presence of
an intense brown color under the mycelium and surrounding it
indicated the probable presence of laccase activity. Isolates that
showed this characteristic were selected for the quantitative
tests. Marine sediment isolates were screened for lipase and
protease. Screening for lipase was done according to Kouker
and Jaeger [37], with modification on Tubaki medium (in g
L
−1
): 1 peptone, 0.5 yeast extract, 15 agar, 31.25 mL of olive
oil and Rhodamine B solution 0.01% (v/v). Lipase production
was detected by the presence of a degradation halo around the
colonies. For protease screening, the fungi were inoculated on
skim milk medium (in g L
−1
): 20 skim milk,20 agar, and 0.2 M
pH 7 phosphate buffer. The presence of a clear degradation halo
around the colonies indicated protease production.
Cold-Adapted Enzymes Activity Quantification
Soil fungi screened positive for ligninolytic enzymes were
cultivated in MA2% medium. After their growth, two 5-mm
cylinders from the margin of the isolates’colonies were trans-
ferred to 150-mL flasks containing 50 mL of malt broth 2%.
Assays were incubated at 15 °C for 7 days at 150 rpm in
duplicates. After this step, the liquid media were centrifuged
at 10,000 rpm for 30 min at 4 °C. Enzymatic activity was
measured in triplicate using the broth obtained.
Quantification of lignin peroxidase followed the method
adapted from Arora and Gill [38]. Manganese peroxidase
was quantified following the method from Wariishi et al.
[39], and the determination of laccase activity was measured
by the method reported by Buswell et al. [40]. Lipase quanti-
fication was determined following the method reported by
Yang et al. [41]. The protease activity was determined as de-
scribed by Charney and Tomarelli [42]. For ligninolytic en-
zymes, one enzymatic unit (U) was defined as the amount of
enzyme needed to oxidize 1 μmol of substrate per minute.
One unit of lipase activity (U) was defined as the amount of
enzyme capable of releasing 1 μmol of p-NPP per mL per min
of reaction. One unit of protease (U) was defined as the
amount of enzyme required to increase the absorbance by
0.01 under the conditions used.
Results
Fungi from Soil Samples
A total of 891 isolates was obtained comprehending 399
yeasts and 492 filamentous fungi. The yeasts were recovered
Fungi from Admiralty Bay (King George Island, Antarctica) Soils and Marine Sediments 15

on MA2% (n= 117), B&K (n= 107), BSA (n= 96), and
diluted MA2% (n= 79). For filamentous fungi, BSA (n=
172), B&K (n= 142), MA2% (n= 90), and diluted MA2%
(n= 88). From the two soil samples, 141 isolates were re-
covered and from the four root-associated soil samples,
750. The temperature of 15 °C resulted in a higher number
of isolates (58.5%), whereas 41.5% were recovered at 5 °C.
The ANOSIM for the temperatures confirmed that there
was a significant difference between both employed tem-
peratures (R= 0.479; p=0.002).
The morphotyping step resulted in 66 morphotypes at
15 °C, and 85 morphotypes at 5 °C. For the yeasts, 114
different fingerprints were obtained. Thus, from the total
891 fungal isolates, 667 were sequenced. They were iden-
tified in 35 different taxa within the phyla Ascomycota,
Basidiomycota, and Mucoromycota (Table 2). Most of the
filamentous fungi belonged to the Ascomycota, and most
of the yeasts belonged to the Basidiomycota. Six fungal
taxa (Ascomycota) presented low molecular similarities in
comparison to sequences in the NCBI-GenBank database.
Even after phylogenetic trees were built, their identification
remained inconclusive and therefore they were classified as
NIA (non-identified ascomycetes; their phylogenetic trees
are available in the Supplementary Material, Figs. S1-S5).
Among the yeasts, the most represented taxa were
Leucosporidium sp. (44.4%), the NIA6 (21.4%),
Goffeauzyma sp.(11.2%),andHoltermanniella sp.
(10.8%). Among the filamentous fungi,
Pseudogymnoascus was the dominant taxon (31.7%) after
NIA2 (5.1%) and Mortierella sp. 1 (4.5%). In contrast, the
taxa Vishniacozyma sp., Cadophora sp., Cosmospora sp.3,
Laetinaevia sp., Thelebolus sp., Mortierella sp.2, and NIA3
were the least dominant, with an abundance of ≤1.2%
(Table 2). The most dominant genera, Leucosporidium
and Pseudogymnoascus, were also dominant in all types
of samples. Mortierella sp.2 was found exclusively in the
soil sample S2. Cadophora sp., Oidiodendron sp., and
Thelebolus sp. occurred exclusively in the D. antarctica
root-associated soil sample S5. On the other hand,
Cosmospora sp.3, Laetinaevia sp., and Purpureocillium
sp. occurred exclusively in the C. quitensis root-
associated soil sample S6. There were no species that oc-
curred exclusively according to the plant species (C.
quitensis or D. antarctica). Some taxa were isolated only
at 5 °C, such as NIA3, NIA5, Cadophora sp., and
Thelebolus sp. By contrast, the taxa NIA1, NIA2,
Cosmospora sp.2 and Cosmospora sp.3, Laetinaevia sp.,
Penicillium sp., Pochonia sp., Purpureocillium sp.,
Trich oderm a sp., and Mortierella sp.2 were isolated only
at 15 °C. According to Table 2, 22.9% of total identified
isolates were recovered from sample S6, 21.4% from sam-
ple S5, 19% from sample S4, 18.4% from sample S3,
10.8% from sample S2, and 7.3% from sample S1.
Fungi from Marine Sediment Samples
A total of 226 isolates was obtained. In the first phase (with
enrichment), 146 yeasts and 24 filamentous fungi were recov-
ered from the five samples. The yeast isolates were recovered
Table 2 Fungal taxa from soil samples
Taxon S1S2S3 S4 S5 S6 Total
Ascomycota
ANI1 0230218
ANI2 0 0 4 4 6 11 25
ANI3 0010001
ANI4 00224311
ANI5 0020114
ANI6 321223181169
Antarctomyces sp. 1 0 11 2 2 3 19
Cadophora sp. 00 00101
Cladosporium sp. 0100618
Cosmospora sp1 00 11169
Cosmospora sp2 00 00358
Cosmospora sp3 00 00011
Fusarium sp. 0 0 1 2 10 5 18
Laetinaevia sp.00 00011
Leptosphaeria sp. 0 1 0 1 0 12 14
Microdochium sp. 00161311
Oidiodendron sp. 0000202
Penicillium sp. 10 21105
Pochonia sp. 0000112
Pseudogymnoascus sp1 4 9 25 7 15 6 66
Pseudogymnoascus sp2 0 0 1 4 2 3 10
Pseudogymnoascus sp3 1 13 17 19 13 17 80
Purpureocillium sp. 0000033
Thelebolus sp. 00 00101
Trichoderma sp. 00007310
Varicosporium sp. 00 22004
Basidiomycota
Cystobasidium sp. 0 0 3 0 3 4 10
Goffeauzyma sp. 1 4 6 21 1 3 36
Holtermanniella sp. 1 5 5 5 12 7 35
Leucosporidium sp. 292320231731143
Mrakia sp.00232613
Naganishia sp.74 110114
Vishniacozyma sp. 00 10102
Mucoromycota
Mortierella sp.1 1 7 0 0 10 4 22
Mortierella sp.2 0100001
Total 49 72 123 127 143 153 667
S1 Yellow Point (yellowish soil), S2 Yellow Point (dark brown soil), S3
Punta Hennequin (C. quitensis root-associated soil), S4 Punta Hennequin
(D. antarctica root-associated soil), S5 Punta Plaza (D. antarctica root-
associated soil), S6 Punta Plaza (C. quitensis root-associated soil)
16 Wentzel L. C. P. et al.

with PDA (n= 52), diluted PDA (n= 49), BSA (n=23),and
MA (n= 22). For filamentous fungi, PDA (n= 12), PDA di-
luted (n=4), BSA (n= 8), and no fungi were isolated from
MA medium. From the second isolation phase (without en-
richment), 56 isolates were recovered. A total of 20 yeasts was
recovered from the four media: PDA (n= 5), PDA diluted
(n= 8), MA (n= 7), and no isolate from BSA. Thirty-six fila-
mentous fungi were recovered: PDA (n= 4), PDA diluted
(n=5),BSA (n=8), and MA(n= 19). From the total of iso-
lates, 68% were isolated at 15 °C. The ANOSIM for the tem-
peratures confirmed that there was a significant difference
betweenbothemployedtemperatures(R=0.33;p=0.03).
According to sequencing data and phylogenetic analyses,
representatives of nine yeast genera were recovered from the
sediment samples (Table 3). The genus Metschnikowia was
the most abundant (45.1%), followed by isolates from the
genera Mrakia (21.6%), Cryptococcus and Glaciozyma
(7.8%), Meyerozyma and Holtermanniella (5.4%),
Rhodotorula (4.2%), Cystobasidium (1.8%), and
Phenoliferia (0.6%). For filamentous fungi, representatives
of eight genera were isolated from the marine sediment sam-
ples (Table 3). The genera Penicillium and
Pseudogymnoascus were the most abundant (40.0%), follow-
ed by the genera Cadophora (6.6%), Cladosporium (5.0%),
Toxicocladosporium,Pseudocercosporella,Pestalotiopsis,
and Paraconiothyrium (1.6%). Some of the fungi identified
in the present study have never been reported in Antarctic
environments before (Toxicocladosporium,
Pseudocercosporella,andParaconiothyrium) and one isolate
was not identified. According to Table 3, 35.8% of total iso-
lates were recovered from sample Sed5, 24.7% from sample
Sed4, 17.2% from sample Sed3, 11.5% from sample Sed1,
and 10.6% from sample Sed2.
Although different culture conditions were applied to iso-
late fungi from terrestrial and marine samples, they shared the
taxa Pseudogymnoascus,Penicillium,Cladosporium,
Cadophora,Mrakia,Goffeauzyma,Cystobasidium,and
Holtermanniella (Fig. 2).
Soil–Taxonomy Correlation, Species Diversity,
Richness, and Distribution
The NMDS analysis (Fig. 3a) revealed that the samples were
separated by sampling location by coordinate 1 (samples S3
and S4 –Punta Hennequin, samples S5 and S6 –Punta Plaza).
Samples S1 and S2, despite collected at the same place, they
presented distinct physical and chemical characteristics, ex-
cept for the carbon content, and appear distant from each other
on the graph (Fig. 3a). The pH and the Fe and Al amounts are
the characteristics that contribute to this distance. Data from
soil physical and chemical characterizations are available in
the Supplementary Information (Tables S1 and S2). The main
factors that grouped samples S5 and S6 were the carbon
content and the amounts of Fe and Al (Piro). There was no
separation of samples by type (e.g., samples S3 and S6, C.
quitensis root-associated soil, did not form a group) but by
sampling location. The ANOSIM for the groups of samples
C. quitensis root-associated soil and D. antarctica root-
associated soil showed that there was no significant difference
between the two groups (R=−1; p= 1). This means that the
type of plant is not significant for the composition of the
fungal communities.
The indices and richness estimator are listed in Table 4.
Estimator Chao 1 showed that sample S6 has the major esti-
mated richness. Shannon and Inverse Simpson indices were
higher in samples S5 and S6. Species sharing analysis showed
that the highest similarity occurred between samples S3 and
S4 (68%) followed by samples S3 and S5 (63.9%) and S5 and
S6 (63.5%). Samples that shared the least species were sam-
ples S1 and S5, with 31.3% of shared species, and samples S1
and S3, with 38.4% of species similarity.
Table 3 Fungal taxa from sediment samples
Taxon Sed1Sed2Sed3Sed4Sed5Total
Ascomycota
Pseudogymnoascus sp.110104 6
Pseudogymnoascus sp.200010 1
Pseudogymnoascus sp.36221415
Pseudogymnoascus sp.400020 2
Penicillium sp. 5395224
Cladosporium sp.1 01000 1
Cladosporium sp.2 00100 1
Cladosporium sp.3 00100 1
Pestalotiopsis sp. 00001 1
Pseudocercosporella sp.00010 1
Paraconiothyrium sp.00010 1
Toxicocladosporium sp.00010 1
Cadophora sp. 04000 4
Metschnikowia sp. 1 6 2 22 44 75
Meyerozyma sp. 70101 9
Nonidentified 00100 1
Basidiomycota
Rhodotorula sp. 00016 7
Mrakia sp. 3 6 10 16 1 36
Cryptococcus sp. 00111113
Glaciozyma sp.1 02013 6
Glaciozyma sp.2 10000 1
Glaciozyma sp.3 20004 6
Cystobasidium sp. 00030 3
Holtermanniella sp. 00009 9
Phenoliferia sp. 00001 1
Total 2624395681226
Sed1 Botany Point, Sed2 EACF, Sed3 Punta Ullman, Sed4 Refúgio 2
(Sediment), Sed5 Refugio 2 (intertidal zone)
Fungi from Admiralty Bay (King George Island, Antarctica) Soils and Marine Sediments 17

Marine Sediment–Taxonomy Correlation, Species
Diversity, Richness, and Distribution
The NMDS analysis revealed that samples do not form a co-
hesive group since each sample is in a different place in the
graph (Fig. 3b). All samples were collected at different places
in Antarctica and showed unique characteristics. Data from
sediment physical and chemical characterizations are avail-
able in the Supplementary Information (Tables S1 and S2).
Samples Sed1, Sed2, and Sed3 are separated from Sed4 and
Sed5. Besides this, coordinate 2 separated samples Sed3 and
Sed4 from the others. Estimator Chao 1 showed that sample
Sed4 has the highest estimated richness (Table 5); this sample
has more rare species (singletons and doubletons) than the
others. Shannon index was higher in sample Sed1, while
Inverse Simpson was higher in sample Sed2 (Table 5).
Species sharing (β-Diversity) showed that the major similarity
occurred between samples Sed4 and Sed5 (42.3%), followed
by samples Sed3 and Sed4 (40.0%). The least similarity oc-
curred between samples Sed3 and Sed5 (only 15%).
Screening of Cold-Adapted Enzymes and Activity
Quantification
From the 249 soil isolates grown on B&K, 35 showed prob-
able laccase activity. Only one was from a soil sample, the
others were from root-associated soil samples. Two isolates
were recovered at 5 °C and the rest at 15 °C. Three isolates
showed enzymatic activity higher than or equal to 1 U L
−1
for
lignin peroxidase: isolate 9P-1.9 (5.41 U L
−1
), isolate 12P-
3.10B (1.7 U L
−1
), and 9P-3.19 (1 U L
−1
). There was no
detectable activity for manganese peroxidase and values for
laccase were under 0.2 U L
−1
. The 170 isolates (marine sedi-
ment) from the first isolation phase were tested for lipase
production. In the qualitative step, 60 yeasts and 19 fungi
showed a positive result. After this, the positive ones were
tested in quantification phase, and 16 yeasts and nine fungi
showed measurable lipase activity. Nevertheless, only nine
yeast isolates were able to produce lipase above 0.5 U mL
−1
.
The best result (0.88 U mL
−1
) was achieved by
Metschnikowia sp. CRM1589. After the experimental design,
this isolate increased its lipase production to 1.05 U mL
−1
(data not shown). All the isolates from marine sediment were
also tested for protease production. In the qualitative step,
three yeasts and six filamentous fungi showed a positive re-
sult. After this, the positive isolates were tested in quantifica-
tion phase, and all of them showed measurable protease activ-
ity. In this case, six filamentous fungi were able to produce
protease above 2.0 U mL
−1
. The best result(6.21 U mL
−1
)was
from Pseudogymnoascus sp. CRM1533. After the experimen-
tal design, the isolate Pseudogymnoascus sp. CRM1533 in-
creased its protease production to 11.47 U mL
−1
(data not
shown).
Discussion
Fungal Isolation and Identification
The number of isolates and the different genera iden-
tified in this study revealed that yeasts and filamentous fungi
could be recovered from extreme-condition samples of the
Antarctic terrestrial and marine environments. As expected,
there were more isolates retrieved from soil samples than
from marine sediment, and more from root-associated soil
than from bulk soil. As the roots release organic carbon, it is
reasonable to believe that more isolates can be found in this
environment [43]. Moreover, according to Berríos et al. [44],
Fig. 2 Diagram showing the
genera isolated from each type of
sample
18 Wentzel L. C. P. et al.

some microorganisms from the rhizosphere of D. antarctica
can have a significant role in the survival and adaptation of
this plant in maritime Antarctica.
Ascomycota is the most common fungal phylum found in
Antarctica [45]. In our study, we also had most of the filamen-
tous fungi as representatives of this phylum. On the other
hand, most of the yeasts were Basidiomycota.
Metschnikowia was the most abundant yeast genus found in
marine sediment samples, while Leucosporidium was the
most abundant in soil samples. Representatives of the genus
Metschnikowia are very common in Antarctica and also re-
ported from water, ice, algae, sediments, and soil [28,46–48].
On the other hand, Leucosporidium is widespread in alpine
and polar environments [49] and was isolated from different
substrates. The genera Penicillium and Pseudogymnoascus
were the most abundant in marine sediments. In the soil sam-
ples, the most abundant ones were Mortierella and
Pseudogymnoascus. According to Hayes [50], species from
the genus Pseudogymnoascus are distributed globally and are
common in cold environments. Moreover, Arenz et al. [51]
suggest that this genus takes part in the decomposition and
nutrient cycling in Antarctica. The genera Penicillium and
Pseudogymnoascus are spread worldwide, and several species
can develop in different and extreme conditions [50,52].
An interesting result is thepresence ofToxicocladosporium,
Pseudocercosporella,andParaconiothyrium in marine sedi-
ment samples. These genera have not been reported in the
Antarctic environment yet. Toxicocladosporium is related to
Cladosporium but now is recognized as a different genus
[53]. The isolates in this study showed high sequence
Fig. 3 Two non-metric multidi-
mensional scaling (NMDS)
biplots based on Bray-Curtis dis-
tances displaying the total fungal
community distribution. aSoil
samples and bmarine sediment
samples
Fungi from Admiralty Bay (King George Island, Antarctica) Soils and Marine Sediments 19

similarity with Toxicocladosporium strelitziae; however, the
phylogenetic tree did not confirm this identification. This ge-
nus is commonly associated with plants, and a large number of
species are phytopathogens. Also, some species were
recovered from clinic samples [53–57]. The genus
Pseudocercosporella has several species that are associated
with plants and also as phytopathogens [58]. The isolates
showed 99% identity similarity with Pseudocercosporella
fraxini, but the phylogenetic tree did not confirm this data.
Most species of the genus Paraconiothyrium are usually asso-
ciated with plants. This genus has been previously found in
marine sponges and sea sediments [59–63].
Putative New Species
Among the total soil fungi recovered, six taxa were not identified
at the genus level and were therefore labeled as Bnon-identified
ascomycete^(NIA); NIA1 showed close relatedness to the only
two described species for the genus Rhizoscyphus,whichare
commonly associated with the roots of C. quitensis and D.
Antarctica [64]. The non-identified ascomycetes 2 and 3 had
low bootstrap values in the phylogenetic tree, and therefore their
identification remained inconclusive. NIA5 showed 93% simi-
larity with Urceolella carestiana; in the phylogenetic tree, the
isolate was placed very distantly from that species and could
represent a new genus. Isolates identified as Oidiodendron sp.
could also represent new species to science (for the phylogenetic
tree, see Supplementary Material, Fig. S6). Among the marine
isolates obtained in this study, one taxon was not identified due to
its low similarity to other sequences in Genbank (for the
phylogenetic tree, see Supplementary Material, Fig. S7). The best
match was with Phaeoacremonium santali, but the similarity
was only 86%.
Soil Diversity
There are few studies addressing fungi in root-associated soil
in the two vascular plants that inhabit Antarctica. Vaz et al.
[10] reported 12 different yeast taxa from the rhizosphere of
D. antarctica. Gonçalves et al. [65] obtained nine different
genera of filamentous fungi from the rhizosphere of D.
antarctica. Microbial communities in root-associated soils
are shaped by the plant species, and consequently by the type
of exudates that they release [66,67]. Thus, it was expected
that C. quitensis and D. antarctica would present some influ-
ence on the fungal communities on their root-associated soils.
Conversely, it was observed that the structure of the commu-
nity was similar for samples collected in the same place, re-
gardless of the plant species, and more related to the charac-
teristics of the soil. Although there was no significant differ-
ence between the microbial composition found in root-
associated soils of C. quitensis and D. antarctica, the fungal
communities varied in the presence of plants among the sam-
pling sites. The α-diversity analyses showed that samples S5
andS6(C. quitensis and D. antarctica root-associated soil –
Punta Plaza) were the richest and most diverse ones. The β-
diversity results were expected since samples collected at the
Table 4 Indices and richness estimator (αand β-diversity) for soil
samples
Sample Shannon Simpson Inv. Chao1 S
S1 (Soil1) 1.44 2.61 24.69 10
S2 (Soil2) 2.04 5.79 12.99 12
S3 (Soil3) 2.51 8.89 25.47 22
S4 (Soil4) 2.35 7.94 19.19 18
S5 (Soil5) 2.84 13.32 32.96 27
S6 (Soil6) 2.8 11.51 46.86 26
First sample Second sample Jaccard Sorensen Bray-Curtis
S1 S2 0.571 0.727 0.612
S1 S3 0.391 0.563 0.384
S1 S4 0.474 0.643 0.409
S1 S5 0.321 0.486 0.313
S1 S6 0.321 0.486 0.416
S2 S3 0.308 0.471 0.574
S2 S4 0.364 0.533 0.563
S2 S5 0.3 0.462 0.53
S2 S6 0.393 0.564 0.533
S3 S4 0.739 0.85 0.68
S3 S5 0.633 0.776 0.639
S3 S6 0.581 0.735 0.601
S4 S5 0.5 0.667 0.578
S4 S6 0.552 0.711 0.621
S5 S6 0.688 0.815 0.635
Table 5 Indices and richness estimator (αand β-diversity) for sediment
samples
Sample Shannon Simpson Inv. Chao1 S
Sed1 1.83 5.37 9.44 8
Sed2 1.8 5.43 7 7
Sed3 1.82 4.83 13.25 10
Sed4 1.79 3.99 26.75 13
Sed5 1.73 3.11 21.41 14
First sample Second sample Jaccard Sorensen Bray-Curtis
Sed1 Sed2 0.364 0.533 0.36
Sed1 Sed3 0.5 0.667 0.4
Sed1 Sed4 0.313 0.476 0.268
Sed1 Sed5 0.467 0.636 0.206
Sed2 Sed3 0.308 0.471 0.413
Sed2 Sed4 0.25 0.4 0.4
Sed2 Sed5 0.313 0.476 0.248
Sed3 Sed4 0.278 0.435 0.4
Sed3 Sed5 0.333 0.5 0.15
Sed4 Sed5 0.35 0.519 0.423
20 Wentzel L. C. P. et al.

same sampling point showed high rates of shared species.
Moreover, root-associated samples showed higher species
sharing among them.
According to Teixeira et al. [68], the presence of vascular
plants in soils of Admiralty Bay plays an important role in the
structure of bacterial communities. Possible explanations for
this are the rhizosphere effect and the soil properties that in-
fluence more the structure of fungal communities. For exam-
ple, the carbon content found in root-associated soil samples
was higher than in soil samples (except for sample S4), and as
the soil microbiota is composed mainly of heterotrophic mi-
croorganisms they rely on the carbon released by the roots as
an energy source [67]. Another environmental factor related to
the composition of the fungal communities was the tempera-
ture. There was a higher number of isolates obtained at 15 °C
than at 5 °C, and the analysis of similarities showed that there
was a significant difference between the communities’com-
position at these temperatures. Nonetheless, the test showed
that the temperature itself is not a factor that fully explains the
observed differences between the groups of isolates at 15 and
5°C(R= 0.479), and other factors need to be taken into ac-
count. Studying soil communities along a transect of many
islands in the Antarctic Peninsula, Dennis et al. [69] verified
that the fungal communities’composition was not associated
with latitude, which suggested that temperature is not a key
factor in the composition of fungal soil communities along the
Antarctic Peninsula. Comparing our results with the results
from Dennis et al., it can be inferred that temperature plays a
significant role in the local level (given that the sampling
points in our study are placed on the same island and around
the same bay), and once one analyzes soil communities on a
larger scale, this factor becomes less significant.
Marine Sediment Diversity
The α-diversity analyses showed that sample Sed4 was the
richest one, while Sed1 was the most diverse. The triplicates
from sample Sed4 were collected at 20 m (average depth) and
at 0.1 °C (average temperature), and triplicates from sample
Sed1 were collected at 24.7 (average depth) and 0.6 °C (aver-
age temperature). These results revealed that samples collect-
ed at 20 m or deeper and at temperatures near 0 °C presented
higher richness and diversity than the sample collected in the
intertidal zone (superficial sample).
The results from β-diversity were expected; samples
Sed4 and Sed5 showed higher species sharing. Both were
collected in the same region. However, despite the higher
similarity of these samples, the sharing percentage was
low, below 50%. This hypothesis is sustained by the
NMDS graph (where the samples were totally separated).
Analyzing the distribution pattern and the isolated species,
it is possible to infer that the geographic location is the
main reason for the differences.
Samples Sed4 and Sed5 are distant from the others, prob-
ably because geographically their distance is higher. And the
difference between Sed4 and Sed5 is probably due to the
different types of the samples and their temperatures; Sed4 is
from sediment and Sed5 from the intertidal zone. By bringing
together all this information, it is possible to infer that fungal
diversity in all samples changes due to the collecting region.
Each sample is unique, and this provides the development of
distinct fungal communities.
The ANOSIM showed that there was also a significant
difference between the communities’composition at 15 and
5 °C. As for the soil samples, temperature itself is not a factor
that fully explains the observed differences between the
groups of isolates at the two temperatures (R=0.33).
Cold-Adapted Enzymes
Ligninolytic Enzymes
The majority of filamentous fungi positive for lignignolytic
enzymes in the first screening were isolated at 15 °C and come
from root-associated soil. The low amount of fungi capable of
producing ligninolytic enzymes could be related to the restric-
tion of lignocellulosic material in Antarctic environments.
Although no laccase activity was observed for yeasts,
Vishniac [70]reportedthatlineagesofCryptococcus isolated
from Antarctica could produce laccase, among other enzymes.
In a previous study from our group, with 160 filamentous
fungi isolated from different Antarctic substrates (wood, sea
stars, marine sediment, lichen, algae), it was verified that 29
had probable laccase activity using the same screening meth-
od as used here (data not publishd yet). As far as we know,
there is no study related to the production of ligninolytic en-
zymes by Antarctic filamentous fungi.
Lipase and Protease
Data from lipase in solid and liquid media revealed that yeasts
were more expressive in lipase production than the filamen-
tous fungi. Among the positive yeasts, 56.2% were recovered
from sample Sed5 (Refugio 2 - intertidal zone). This result can
be justified by the fact that this sample came from a transition
zone, where the accumulation of oil and fat (from dead ani-
mals) and pollution (oil, boat fuel) brought by tides is more
likely to happen. Duarte [71] reported lipase activity from two
genera isolated from Antartic samples: Cryptococcus and
Leucosporidium (between 0.1 and 0.23 U mL
−1
). In this study,
the highest activity (0.88 U mL
−1
) was from Metschnikowia
sp. CRM 1589. Yeasts from the phylum Ascomycota, in gen-
eral, are able to produce lipase, especially the ones from the
genera Candida,Yarrowia,andSaccharomyces [72]. Vaca et
al. [73] reported lipase activity from two isolates of M.
australis, but only the qualitative procedure was done.
Fungi from Admiralty Bay (King George Island, Antarctica) Soils and Marine Sediments 21

Protease activity was more expressive in filamentous fungi
than in yeasts. All positive isolates were recovered from sed-
iment samples (Sed1, Sed2, Sed3 e, Sed4) and none from the
intertidal zone (Sed5). Pseudogymnoascus sp. CRM1533
showed higher protease activity (6.21 U mL
−1
). The protease
production ability of the Pseudogymnoascus genus has been
reported in literature, but most of the studies only do the qual-
itative screening, and there is not much data about protease
activity quantification [74–77].
In conclusion, results from the present work revealed that
Pseudogymnoascus sp. and representatives of the genus
Penicillium were dominant in all marine sediment samples,
while Metschnikowia was the most abundant yeast genus in
this Antarctic environment. Pseudogymnoascus sp. was also
dominant in soil samples, as well as yeasts of the genus
Leucosporidium. We showed that despite being located within
the same bay, each sampling point we assessed is unique in its
physical-chemical composition and fungal community. This
shows the complexity of the Antarctic environment and that
further studies need to be performed to fully understand the
dynamics of fungal communities. Oxidative and hydrolytic
enzymes were produced by fungal isolates from soils and
marine sediments, respectively. Although few soil fungi pre-
sented the capacity to produce ligninolytic enzymes, many
fungi were able to produce protease and lipase. The fungal
collection obtained in the present study is currently being in-
vestigated in the search for plant growth promoter and anti-
cancer compounds and for biopesticides.
Funding Information This paper was supported by grants financed by
FAPESP (reference numbers: #2013/19486-0 and #2016/07957-7), and
by scholarships financed by CAPES. LDS and AR thank the National
Council for Scientific and Technological Development (CNPq) for
Productivity Fellowships 304103/2013-6 and 305341/2015-4. LDS
thanks MICROSFERA project (PROANTAR/CNPq) for the support with
sample collection.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of
interest.
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