Ecological Indicators 126 (2021) 107704

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Ecological Indicators
journal homepage: www.elsevier.com/locate/ecolind

Bacterial diversity and community structure along the glacier foreland of

Midtre Lovénbreen, Svalbard, Arctic
Siddarthan Venkatachalam a, Vatharamattathil Mohanan Kannan b,
Vadakke Neelamanakesavan Saritha b, Dinesh Sanka Loganathachetti a, Mahesh Mohan b,
Kottekkatu Padinchati Krishnan a, *
a
Arctic Ecology and Biogeochemistry Division, National Centre for Polar and Ocean Research (NCPOR), Ministry of Earth Sciences (Govt. of India), Vasco-da-Gama,
Goa, India
b
School of Environmental Sciences, Mahatma Gandhi University, Kottayam, Kerala, India

A R T I C L E I N F O A B S T R A C T

Keywords: Polar regions provide an ideal environment to investigate the succession of bacterial communities. In the present
Glacier foreland study, we investigated the bacterial diversity and community structure of the Midtre Lovénbreen glacier foreland
Soil development ecosystem using a chronosequence approach. The alpha diversity indices of the samples collected from recently
Bacterial succession
deglaciated sites comprised of less diverse, yet abundant bacterial groups compared to the samples from
Amplicon sequencing
comparatively older sites, where the bacterial diversity was very rich and evenly distributed. Bacterial phyla viz.
(a) Proteobacteria (14–44.58%) comprising classes of alpha- and gamma Proteobacteria along with (b) Acti­
nobacteriota (8.8–33.8%) were predominantly distributed across the samples, while phyla Bacteroidota (up to
21%) was mainly distributed in the recently deglaciated samples and phyla Acidobacteriota (up to 24%) in
deglaciated samples which were older. Bacterial families (Sulfurovaceae and Sulfurimonadaceae) affiliated with
bio weathering of rocks for their energy metabolism was also detected in the present study from the recently
deglaciated region. Bacterial genera belonging to Luteolibacter (up to 10.25%), Polaribacter (up to 12.32%),
Acidimicrobium (6%) and Sulfitobacter (13.6%) were highly abundant in the recently deglaciated samples, while
Candidatus_Udaeobacter (up to 17%) and RB41 (up to 10.5%) were found to be abundant in the older stage
samples. Linear discriminant analysis revealed 121 Operational Taxonomic Units that could be attributed to the
differences in the community diversity between the two groups. Among the analyzed environmental variables,
pH, Cr, Cd and Ca significantly contributed to the differences in the bacterial community structure.

1. Introduction rhizosphere, which in turn participate in biogeochemical processes in

Arctic terrestrial ecosystems are constantly undergoing rapid
changes due to global warming, resulting in the increase of average
Arctic temperatures twice the global average for the past 50 years
(Koenigk et al., 2020). These rapid changes have proven to affect Arctic
ecosystems, including a decline in sea-ice, thawing of permafrost and
retreat of glaciers. When a glacier is retreating, new landscapes are
exposed that have been otherwise locked under the ice for thousands of
years. This provided an opportunity for the colonization and succession
process of various life forms in time and space (Pendleton et al., 2019).
In glacier foreland ecosystems, microbes play an essential role in
modulating the vegetation structure during colonization, while plants
influence and recruit their own symbiotic communities through the
the high latitude regions (Bai et al., 2019; Brown and Jumpponen,
2014). Thus, microbes play a vital role in nutrient cycling, contributing
to soil development, its fertility and also support the colonization of
diverse vegetation at higher trophic levels (Kim et al., 2017; Schuette
et al., 2010; Schütte et al., 2009).
The concept of ecological succession has a long history and has been
investigated over the centuries in many different geographical regions.
These processes vary from different geographical locations based on
their local environmental conditions and other abiotic factors like
extreme temperatures, altitude, nutrient availability and seasonality (Li
et al., 2020). Glacier foreland ecosystems in the Arctic are highly pro­
ductive despite their extreme environmental conditions with substantial
seasonal changes in ice and snow cover. However, the peak of primary

* Corresponding author.
E-mail addresses: venkatachalam@ncpor.res.in (S. Venkatachalam), krishnan@ncpor.res.in (K.P. Krishnan).

https://doi.org/10.1016/j.ecolind.2021.107704
Received 21 September 2020; Received in revised form 12 February 2021; Accepted 7 April 2021
Available online 25 April 2021
1470-160X/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
S. Venkatachalam et al.
production is mostly restricted to the short summer season (Kosek et al.,
2019). Studies on ecological succession in the glacier forelands are
mainly carried out by chronosequence method by investigating soil’s
structure and development at a linear distance from the glacier edge
(Walker et al., 2010). Most of the studies on succession mainly focused
on plant traits based on vegetation structure in the glacier forelands of
polar and alpine environments (Hodkinson et al., 2003; Matthews, 1992;
Reiners et al., 1971; Sitzia et al., 2016). Studies focusing on microbes,
the primary colonizers of glacier forelands in the polar regions, have
been undertaken only since the last one decade (Mapelli et al., 2018) by
using culture-independent amplicon sequencing based approaches to
assess bacterial community dynamics. Over the years, our understand­
ing of the microbial community structure and its functions has signifi­
cantly increased with the advancement in genomics.
The landscape of Svalbard has been undergoing rapid changes due to
deglaciation. It has been reported that about 7% of the glaciers in
Svalbard have disappeared over the last 30 years (Hanna et al., 2020;
Nuth et al., 2013). Midtre Lovénbreen glacier is a polythermal valley
glacier located on the Brøggerhalvøya peninsula of the northwest
Fig. 1. Map showing the location of sampling coordinates along with Midtre Lovénbreen glacier foreland ecosystem. The map was generated in ArcGIS (v10.3)
software using source files obtained from (https://www.pgc.umn.edu/data/arcticdem/).
2
Ecological Indicators 126 (2021) 107704
Spitsbergen Island (78◦ 55′ N, 12◦ 10′ E). The glacier is about 5 km long,
located 50–650 m above sea level, and has been retreating since the end
of the Little Ice Age (Seok et al., 2016). However, in the past few de­
cades, the glacier’s retreating ratio has accelerated rapidly, and current
numerical modeling studies predict that the glacier could disappear in
the next 100 years (Ai et al., 2019; Schuler et al., 2020). Recently
deglaciated sites in this glacier foreland are composed of different
bedrocks belonging to the Lower, Middle and Upper Proterozoic era
(Wynn et al., 2007), which are being weathered by microbes continu­
ously, thereby providing optimal substrates for soil development and
plant colonization (Mapelli et al., 2011). The gradually exposed soil is
subject to freeze-thaw cycles throughout the year (Wietrzyk et al., 2018;
Yoshitake et al., 2011), providing an excellent environmental setting for
microbial ecologists to investigate changes in microbial communities in
connection with soil exposure and environmental parameters that can
help to predict the response of bacterial communities to changing
environmental conditions during the process of deglaciation. With this
background, the present study was undertaken to assess the bacterial
community structure of the Midtre Lovénbreen glacier foreland
S. Venkatachalam et al. Ecological Indicators 126 (2021) 107704

ecosystem using deep amplicon sequencing approach to determine the
distribution of bacterial communities across different deglaciation
stages and to delineate further the influence of environmental parame­
ters in shaping the community composition.
2. Materials and methods
2.1. Sample collection
Samples were collected from the glacier foreland associated with
Midtre Lovénbreen glacier, Svalbard, during the Indian Arctic expedi­
tion (June 2019). A total of 8 soil samples (200 g) were collected
(Fig. S1) from a transect using chronosequence approach from glacier
snout to the fjord (Fig. 1, Table 1), which was about 1.8 km covering a
deglaciation period of up to 1900 years (Hodkinson et al., 2003). Sam­
ples (ML1–ML3) from recently de-glaciated sites (snout area) containing
barren soil will be henceforth referred to as ’recent stage’ samples.
Samples (ML4–ML8) from older deglaciated sites where the soil is well
developed to support vegetation structure will be referred to as ’older
stage’ samples. The present classification was adapted based on the
previous literature from this region (Mapelli et al., 2011). Larger gravel
and other plant debris were removed before samples were taken. About
200 g of soil per sample was collected from the top layer (15 cm) using
an ethanol-cleaned shovel and transferred to Whirl-Pak bags (Nasco, WI,
USA). The samples were brought to the National Centre for Polar and
Ocean Research, India, at − 20 ◦ C and further stored at the same tem­
perature till further analysis.
2.2. Trace metals chemical analysis
Soil samples were dried, pulverized and then sieved (mesh size of
1.70 mm – ASTM No .230) prior to analysis as described by Link et al.
(1998) and Yuan et al. (2004). The samples were digested with suprapur
nitric acid (Sigma-Aldrich, USA), followed by filtration (0.2 μm nylon
filter) and dilution of samples (25 parts). Trace metals were then
quantified using an ICP-MS (iCAP Q, Thermo Fisher Scientific, USA).
Continuous calibration was performed with standard and blank solu­
tions during measurements. Standard solutions were prepared in 1%
HNO3 using ICP-multielement (30 elements) standard solution VI
(Merck Millipore, Germany). The soil pH was measured in a 1:2 (w/v)
ratio of soil: deionized water using a pH meter (Orion, Thermo scientific,
USA).
2.3. Genomic DNA extraction and 16S rRNA gene sequencing
According to the manufacturer’s protocol, the total genomic DNA
was isolated from the soil samples using PureLink™ microbiome DNA
purification Kit (Invitrogen, USA). The isolated DNA was visually
checked for quality assessment by agarose gel electrophoresis (1%
agarose) and quantified using Biospectrometer (Eppendorf, Germany).
The V3–V4 hyper-variable region of the bacterial 16S rRNA gene was
amplified using Pro341F/Pro805R primer sets (Takahashi et al., 2014).
The reaction mixture (25 µL) contained 10 ng genomic DNA, 12.5 µL of
AmpliTaq Gold® 360 PCR master mix (Invitrogen, USA) and 0.25 mM of
each primer. The PCR conditions for amplification of DNA were as fol­
lows: initial denaturation at 98 ◦ C for 2 min, followed by 25 cycles of
95 ◦ C for 30 s, annealing at 55 ◦ C for 1 min and extension at 72 ◦ C for 30
s. The amplified PCR products were purified by using gel extraction kits.
The amplicon sequencing libraries were prepared using NEBNext®
Ultra™ II DNA Library Prep Kit (New England Biolabs, USA) according
to the manufacturer’s protocol and sequenced on Illumina HiSeq plat­
form 2500. The sequence datasets were submitted to the NCBI Sequence
Read Archive (SRA) library under the accession number: PRJNA658121.
2.4. Bioinformatics and statistical analysis
The raw sequence datasets were quality trimmed to remove primer
and adapter sequences using Cutadapt (Martin, 2011). The trimmed
sequence datasets were further analyzed using Mothur (v.1.44.0) to
characterize the bacterial community structure (Schloss et al., 2009).
The sequence analysis method includes, the removal of short sequences
of <300 nts, reads that contained ambiguous nucleotides and homo­
polymeric runs of >7 from the dataset. Mitochondrial, chloroplast and
archaeal sequences were also excluded from the analysis. The chimeric
sequences were removed subsequent to identification using the
VSEARCH algorithm (Rognes et al., 2016). Classification of the reads
was done to the genus level using the Naïve Bayesian classifier algorithm
(Cole et al., 2014; Wang et al., 2007) against the SILVA database version
138 (Quast et al., 2012) with a confidence threshold of 80% to assign
taxonomic identity. A pairwise distance matrix was created from the
curated aligned datasets to group sequences into Operational Taxo­
nomic Units (OTUs) at a confidence level of 97%. The alpha diversity
indices (observed species, Simpson, Shannon, and Chao1) were calcu­
lated using the phyloseq package (McMurdie and Holmes, 2013) in R v-
4.0.2. The Linear discriminant analysis Effect Size (LEfSe) to identify the
differentially abundant features (Segata et al., 2011) among the bacte­
rial taxa was calculated using the microbiome Analyst R server (Chong
et al., 2020) with described default parameters. Statistically significant
groups were reported with nonparametric factorial Kruskal Wallis (KW)
rank-sum test (p < 0.05) and high LDA (Linear Discriminant Analysis)
threshold score values >2.0 (Segata et al., 2011). The OTU reads con­
taining less than two numbers were discarded and the remaining OTUs
within each sample were converted into relative percentages, square-
root transformed and then Bray-Curtis resemblance matrix was

Table 1
Sampling locations along with details of the data obtained by high throughput sequencing from the Midtre Lovénbreen glacier foreland ecosystem.
Sample name GPS Coordinates Soil type Raw sequence reads Sub sampled reads Total number of OTUs Goods coverage (%)

ML1 78 53.906
◦ ′
N Barren 526,533 166,487 2482 99.86
12◦ 03.221′ E
ML2 78◦ 54.173′ N Barren 1,031,559 166,487 1760 99.82
12◦ 02.792′ E
ML3 78◦ 54.314′ N Barren 1,005,450 166,487 2276 99.37
12◦ 02.489′ E
ML4 78◦ 54.395′ N Developing 1,179,332 166,487 4603 99.04
12◦ 02.449′ E
ML5 78◦ 54.543′ N Mature 753,585 166,487 3163 99.37
12◦ 02.428′ E
ML6 78◦ 54.627′ N Mature 1,091,119 166,487 4086 99.22
12◦ 02.337′ E
ML7 78◦ 54.755′ N Mature 1,128,477 166,487 4513 99.01
12◦ 02.222′ E
ML8 78◦ 54.833′ N Mature 951,736 166,487 2630 99.49
12◦ 02.122′ E

3
S. Venkatachalam et al. Ecological Indicators 126 (2021) 107704

calculated. A comparative nonmetric multidimensional scaling (nMDS)
analysis at the OTU level of 97% was performed among the sample
datasets to find the differences in the bacterial community composition
by using the multivariate statistical software Primer (PRIMER E) version
7 (Clarke and Gorley, 2015). Distanced-based multiple linear regression
tests (Dist-LM) and distance-based constrained redundancy analysis (db-
RDA) were performed to analyze the contribution of environmental
physicochemical parameters attributed to the variability in bacterial
community structure.
3. Results
Species coverage indices (Goods coverage) of all the samples were above
99%, which indicated an adequate level of sequencing had been carried
out to identify most of the diversity within the samples.
The alpha diversity indices (Observed, Chao-1, Shannon and Simp­
son) revealed that richness and evenness of bacterial taxa in the samples
belonging to the recent stage samples were comparatively less than
those of older stage samples from well-developed foreland ecosystem
(Fig. 2). Furthermore, the observed indices and Chao-1 richness indices,
which estimate the number of unobserved species in the given samples,
almost fell within the same range suggesting the present study described
near to complete diversity among the analyzed sequence datasets.

3.1. Environmental characteristics of Midtre Lovénbreen glacier foreland 3.3. Distribution of bacterial community structure

The samples were collected along the glacier foreland of Midtre
Lovénbreen glacier from glacier snout to the fjord mouth of Kongsfjor­
den. The vegetation structure was commonly observed among the older
deglaciated regions of Midtre Lovénbreen glacier foreland, where the
soils are fully developed, whilst samples collected from the glacier snout
area was classified as barren soil type (Table 1). Along the transect, soil
pH decreased in a linear scale ranging from pH 8.5 in the vicinity glacier
snout to pH 6.7 near Kongsfjorden. At the time of sample collection, the
mean air temperature was around 6.3–6.8 ◦ C (Norwegian Meteorolog­
ical Institute, www.met.no). Elemental analysis revealed that except for
Na and P, all of the remaining analyzed elements were found to be
highest at ML2 (Table 2), which was collected from a typical moraine
environment. Other elements like Ca (817.8–2352.8 µg g− 1) and Zn
(1040–1099.7 µg g− 1) were found to have the highest concentrations in
recent stage samples (ML1–3) in comparison to those of older stage
samples (ML4–8). We could not observe any specific patterns among the
remaining elements between recent and older stage sample groups. In
general, we observed that concentrations of metal elements varied
among collected samples across the studied transect (Table 2).
3.2. Alpha diversity of glacier foreland samples
A total of 7,667,791 good quality sequence reads were obtained from
eight samples with an average length of 430 nucleotides that belonged
to the 16S rRNA V3–V4 region. The sequence data that was normalized
to 1,66,487 reads per sample yielded a range of 1760–4513 OTUs per
sample (Table 1). At this defined sequencing depth, rarefaction curve
analysis revealed a reasonable coverage of bacterial richness among the
datasets (Supplementary Fig. S2). In general, samples collected from
recent stage sites contain fewer OTUs compared to the older stages.
Based on the phylogenetic classification of sequence datasets, a total
of 33 bacterial phyla were identified, of which Acidobacteriota
(4.1–23.8%), Actinobacteriota (8.8–33.8%), Proteobacteria
(14–44.58%), Chloroflexi (4.1–19%) and Verrucomicrobiota
(1.5–21.56%) were predominantly distributed among the samples with
different relative abundances (Fig. 3A). The phylum Proteobacteria,
comprising mainly of alpha- and gamma Proteobacteria were dominant
among samples. Phyla Bacteroidota (2–22%) was mainly distributed in
the recent stage samples, whereas Acidobacteriota (18–24%) was
commonly distributed in older stage samples. Likewise, a minor fraction
of reads representing phyla Campilobacterota and superphylum Pates­
cibacteria were also identified in recent and older late stage samples,
respectively. We also observed two other phyla, namely Myxococcota
and Gemmatimonadota, accounting for a small fraction (1–7%) of reads.
Further taxonomic classification of sequence reads at 97% similarity
level showed about 93 different classes, 224 orders, 332 families and
454 different genera among the samples. Class Bacteroidia
(15.83–21.92%), Gammaproteobacteria (6.56–20.57%) and Actino­
bacteria (12.17–15.98%) were predominantly distributed in the recent
stage samples. However, older stage samples were equally dominated
(6–10% of reads) by class Acidimicrobiia, Vicinamibacteria, and Blas­
tocatellia (Fig. 3B). The families, Rubritaleaceae (12.36%), Fla­
vobacteriaceae (9.82%), Microbacteriaceae (13.06%)
Comamonadaceae (12.84%) were identified as dominant families in the
recent stage group. Further, the families Sulfurovaceae (7.29%) and
Sulfurimonadaceae (1.4%) were unique to sample ML1, which is located
near the glacier snout area (Supplementary Table 1). Similarly, Chtho­
niobacteraceae (18.7%), Pyrinomonadaceae (10.28%) were identified
as dominant families in the late stage samples, respectively. At the genus
level, Luteolibacter, Polaribacter, Acidimicrobium, Candidatus_Aquiluna,

Table 2
Physico-chemical analysis of Midtre Lovénbreen glacier foreland samples. Given Data values are average of three technical replicates.
Element concentration (µg g− 1) ML 1 ML 2 ML 3 ML 4 ML 5 ML 6 ML 7 ML 8

Sodium (Na) 110.7 127.2 292.9 147 55.8 114.1 ND 210.9

Phosphorus (P) 611.3 713.9 725.4 652.1 617.0 726.7 613.2 682.0
Potassium (K) 2776.0 8548.8 3370.7 2842.1 3237.3 3393.8 4185.1 3224.4
Calcium (Ca) 2036.3 2352.8 817.8 197.5 659.4 649.2 540.7 537.0
Lithium (Li) 11.3 25.4 13.5 10.7 15.1 14.0 19.5 15.7
Beryllium (Be) 0.691 1.4 0.822 0.684 0.884 0.742 1.1 0.825
Aluminium (Al) 13744.3 39008.0 15503.5 12561.2 16229.2 15145.2 29259.1 17395.9
Chromium (Cr) 24.6 35.1 19.8 13.1 27.6 20.7 26.6 29.9
Cadmium (Cd) 0.342 0.446 0.425 0.219 0.108 0.107 0.125 0.149
Manganese (Mn) 369.1 1079.5 527.1 389.3 627.7 550.4 609.5 323.4
Iron (Fe) 30427.7 53774.2 29499.0 25547.4 35347.3 30201.2 39295.4 31499.5
Cobalt (Co) 11.6 21.0 11.9 9.9 14.3 11.6 13.8 9.4
Copper (Cu) 18.1 36.4 24.8 27.0 25.9 17.2 19.9 31.1
Zinc (Zn) 1040.0 1099.7 1054.2 43.8 983.0 642.3 556.1 692.4
Arsenic (As) 7.5 9.9 6.5 7.6 7.2 6.8 5.6 6.3
Strontium (Sr) 25.9 35.2 18.9 9.2 14.6 15.6 17.4 22.2
Lead (Pb) 10.1 16.2 10.9 19.6 4.6 2.0 5.6 1.0
pH 8.5 8.3 8.3 7.5 7.8 7.4 6.7 6.9
Mean Air Temperature 6.3 ◦ C 6.8 ◦ C 6.7 ◦ C 6.7 ◦ C 6.7 ◦ C 6.7 ◦ C 6.7 ◦ C 6.7 ◦ C

ND: Not detected.

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S. Venkatachalam et al. Ecological Indicators 126 (2021) 107704

Fig. 2. Alpha diversity indices from the soil samples of Midtre Lovénbreen glacier foreland. The samples ML1-ML3 is grouped as recent stage and samples ML4-ML8
is grouped as older stage based on their deglaciation time and phase.

Fig. 3. Distribution of bacterial community structure of the foreland of Midtre Lovénbreen glacier. Phylogenetic classification at (A) Phylum and (B) Class level. The
datasets were classified using Silva reference database v138 using the Naïve Bayesian classifier algorithm.

5
S. Venkatachalam et al.
Sphingomonas and Sulfitobacter were predominantly found recent stage
samples, whereas Candidatus_Udaeobacter and RB41 were found to be
abundant in the older stage samples. Genera, KD4_96_Chloroflexus and
Vicinamibacteraceae_ge were equally distributed throughout the samples
(Fig. 4A).
Lefse analysis was carried out to determine the effect size of various
differentially abundant taxa among the samples. A total of 121 bacterial
OTUs can be attributed to the differences in the community diversity
between the recent and older stage groups, of which top 15 bacterial
OTUs are represented in the Fig. 4B. Three bacterial OTUs belonging to
members of genera Chloroflexi_CM66, Candidatus Actinomarinales, and
Luteolibacter contributed significantly to the variations in recent stage
samples, whereas OTUs that belonged to Candidatus Udaeobacter, KD4-
96_ge, Pyrinomonadaceae_RB41 and Vicinamibacterales significantly
contributed to the variations observed in the older stage samples
(Fig. 4B). Further, NMDS and ANOSIM based multivariate analysis
revealed that bacterial community structure between recent stage (n =
3) and older stage (n = 5) were found to be significantly different
(Fig. 5A) among the sample datasets (R = 0.713, P < 0.01).
3.4. Correlation of physicochemical parameters with bacterial community
structure
Although NMDS and ANOSIM showed significant differences in
bacterial community structure between two groups of glacier foreland
ecosystem, it does not specify which environmental variables explain
those differences in community structure. To elucidate the environ­
mental factors that best explain bacterial community structure, stepwise
distance-based linear models (DistLM) and distance-based redundancy
analysis (db-RDA) were carried out. The analysis showed that the
examined variables accounted for about 62.3% of the bacterial com­
munities total variability, suggesting that metals were among the major
factors in shaping the community diversity of the glacier foreland eco­
systems (Fig. 5B). The DistLM analysis showed top six environmental
variables viz., Sr, Mn, Cr, Cd and Ca, along with pH mainly contributed
to the variations in the bacterial community composition (Table 3).
Among them, pH, Cd, Ca and Cr were shown to be significantly
contributing to the variation, whereas that of Sr and Mn were not sta­
tistically significant.
Fig. 4. Identification of dominant bacterial genera (A) from the glacier foreland samples and (B) their size effect by differential abundance analysis (LEfSe).
6
Ecological Indicators 126 (2021) 107704
4. Discussion
Global warming has accelerated the retreat of glaciers immensely in
the last decade resulting in the exposure of new terrains that were once
covered under the ice. These recently deglaciated sites are composed
mainly of rocks and barren soils characterized by unvegetated and
nutrient-poor regions (Mapelli et al., 2018, 2011; Schuette et al., 2010;
Walker et al., 2010; Wietrzyk et al., 2018). These deglaciated terrains
undergo various stages of soil development processes that depend upon
many factors like barren substrate, topography, climate, biota, time and
their interactions among each other (Wietrzyk et al., 2018). Autotrophic
microorganisms are the initial colonizers in these deglaciated environ­
ments playing a significant role in developing barren lands into fertile
vegetative ecosystems through nutrient recycling and remineralization
of biological substrates (Mapelli et al., 2011; Schütte et al., 2009).
The alpha diversity measurements in the present study suggested
that the bacterial community composition of recently deglaciated
(recent stage) samples were less diverse than those of older stage sam­
ples (Fig. 2). Phylogenetic analysis showed that specific phyla like
Proteobacteria, Bacteroidota and Actinobacteriota solely contributed
(55%–85%) to the recent stage samples, while most of the phyla were
evenly distributed among older stage samples (Fig. 3A). The differences
observed in the bacterial community composition along glacier foreland
regions was in accordance with previously reported glacier foreland
studies from alpine and polar regions (Bai et al., 2019; Brown and
Jumpponen, 2014; Hodkinson et al., 2003; Kim et al., 2017; Mapelli
et al., 2018; Schuette et al., 2010). In brief, recent stage samples were
less diverse and abundant, while the trajectory shifted more evenly in
the older stage samples with a more complex bacterial community
structure. Nutrient availability, well-developed soil conditions, and
vegetation structure play a key role in establishing highly diverse and
complex bacterial community structure in the older stage samples
(Mapelli et al., 2018). NMDS and db-RDA analysis showed that the
sample ML1 sample has a different bacterial community structure
compare to the other samples. Among the analyzed environmental
variables, pH was found to be the closest that may have influenced the
bacterial community composition at ML1. Novel phylum Campilo­
bacterota, comprising of families Sulfurovaceae (7.29%) and Sulfur­
imonadaceae (1.4%), were identified in the sample ML1 which is located
S. Venkatachalam et al. Ecological Indicators 126 (2021) 107704

Fig. 5. Multivariate analysis of the glacier foreland samples. (A) Non-metric Multidimensional Scaling (NMDS) plot showing the relationship between bacterial
groups. (B) Distance-based Redundancy analysis (db-RDA) shows the best explained environmental variables in the glacier foreland’s bacterial community structure.
The samples ML1-ML 3 belong to recent stage, while ML 4-ML 8 are from older stage.

remineralization in sediments (Bradley et al., 2016; Mapelli et al.,

Table 3
2011). Similarly, members of genera Luteolibacter and Sulfitobacter are
The distance-based linear model (DistLM - sequential test) for the most signifi­
shown to be involved in the degradation of polycyclic aromatic hydro­
cant environmental parameters contributing to the observed microbial com­
munity variation in the glacier foreland ecosystem.
carbon (PAHs) and sulfur cycling, respectively (Zhang et al., 2014). In
the present study, we observed that the abundance ratio of these bac­
Variable Pseudo- P- Proportion of variation Cumulative variation
terial genera gradually declined along the transect. Hence, these bac­
F value (%) (%)
terial groups could be among the keystone species in the recently
pH 2.9398 0.001* 32.88% 32.88% deglaciated environments (recent stage), involved in recruiting highly
Cd 3.2234 0.005* 26.31% 59.19%
Ca 2.1083 0.042* 14.09% 73.28%
complex bacterial community structure.
Cr 1.9188 0.047* 10.43% 83.70% In well-developed soils (older stage), the abundance of phyla Acid­
Mn 1.4777 0.315 6.93% 90.63% obacteriota, Actinobacteriota, Chloroflexi and Verrucomicrobiota were
Sr 1.1982 0.478 5.11% 95.74% relatively uniform, suggesting that soil community pattern has stabilized
*P < 0.05 = significant. over time (Schuette et al., 2010; Schütte et al., 2009). The pH for the
older stage soil samples was slightly acidic compared to the recent stage
near to the glacier snout area. This phylum was created by consolidating samples. This could be attributed to the abundance of high organic
class Epsilonproteobacteria and order Desulfurellales (Deltaproteobac­ content in the matured soil samples due to the well-developed vegeta­
teria) (Waite et al., 2015). Bacterial groups of Sulfurovaceae and Sul­ tion structure. Also, the presence of bacterial taxa belonging to members
furimonadaceae are known to be involved in the process of sulfur of Rhizobiales (7–10%), which are known for their nitrogen fixation and
oxidization (D’Angeli et al., 2019). They have been reported previously symbiotic relations with plants, further provide evidence for a presence
for its crucial role in bio weathering of rocks in the moraine systems, of a well-developed ecosystem. The vegetation structure in this region
thus assisting in establishing vegetation structure in the barren lands was mainly dominated by vascular plants, dwarf-shrubs and crypto­
(Mapelli et al., 2011). The study also revealed the prevalence of the gamic species. In contrast, vegetation was only observed in patches at a
bacterial families, Actinomarinales_uncultured (15.88%), Micro­ few places in the recent stage samples. A study by Mapelli et al. (2018)
bacteriaceae (13.06%), Rubritaleaceae (12.36%), Comamonadaceae revealed, plants recruit their own microbiome community from bulk soil
(12.84%) in the recent stage samples. Members of Microbacteriaceae through their rhizosphere and participate in symbiotic biogeochemical
have been frequently retrieved among the glacier habitats, and their processes in glacier foreland ecosystems.
presence in the recently deglaciated site suggested it may be originated Predominant distribution of phylum Acidobacteriota was observed
from the glacier ecosystem (Mapelli et al., 2018). in all samples except ML1, which further substantiates that glacier
The relationship between microbial community structure and soil forelands may be oligotrophic with low nutrient availability for sup­
organic carbon is relatively well established in the alpine and polar porting organism growth (Hodkinson et al., 2003; Kim et al., 2017). The
glacier foreland regions. However, the significance of elemental sequences belonging to members of RB41 was predominant (7–11%) in
composition, especially in the recently deglaciated areas, is relatively the older stage samples. RB41 belonged to the Blastocatellaceae family,
still poorly represented (Hodkinson et al., 2003; Kim et al., 2017; widely known to be playing an essential role in maintaining the meta­
Mapelli et al., 2018; Schuette et al., 2010; Schütte et al., 2009). In the bolic and biogeochemical functions of soil under long-term low-nutrient
present study, redundancy analysis revealed that elements Cr, Cd, and stress conditions (Sun et al., 2020). About 12–27% of sequences
Ca along with pH could be attributed to the total variations in the bac­ retrieved among the samples belonged to the members of uncultured
terial community structure among the sample groups. The concentration genera, along with few dominant candidatus genera, including Aquiluna
of metals varied based on soil types, vegetation structure and microcli­ and Udaeobacter, which have been reported for the first time from a
matic conditions in the glacier foreland ecosystem (Wietrzyk et al., glacier foreland ecosystem (Fig. 4). The predominance of candidatus
2018, 2020). Elevated levels of metal concentrations in the recent stage Udaeobacter, an ubiquitous oligotrophic soil colonizing group in the
samples, especially ML2, were found to correlate with highly abundant older stage samples, indicates its ability to survive and replicate by
bacterial taxa belonging to Luteolibacter, Polaribacter, Acidimicrobium, acquiring amino acids and vitamins from the environment (Brewer
Candidatus_Aquiluna, Sphingomonas and Sulfitobacter (Fig. 4A). Members et al., 2016). Overall, at the genera level, bacterial community structure
of Acidimicrobium are represented by chemolithotrophic groups was dominated by several uncultured and candidatus groups in the
commonly known to be involved in iron oxidation and organic glacier foreland ecosystem, signifying the need to step up culture-based

7
S. Venkatachalam et al.
studies to unravel the potential of this untapped diversity. The occur­
rence of rapid glaciers retreat around the world in response to ongoing
climatic changes, future studies that focus on continuous monitoring
and assessment that target autotrophic and heterotrophic microbial
colonization in the glacier foreland ecosystems in conjunction with
studies that focus on dynamics and consequences of glacial retreat across
different glacier foreland systems will provide much better clarity on
relationship between glacier retreat associated microbial succession in
the glacier foreland regions.
5. Conclusion
The present study highlights the significant differences in the bac­
terial diversity and community structure along the glacier foreland of
Midtre Lovénbreen glacier. Alpha diversity indices showed that recent
de-glaciated samples exhibited less diverse but comparatively more
abundant taxa than those of the older stage samples. Taxa that belonged
to Chloroflexi_CM66, Candidatus Actinomarinales and Luteolibacter sp.
from recent stage and Candidatus Udaeobacter, KD4-96_ge, Pyr­
inomonadaceae_RB41 and Vicinamibacterales from late stage samples
contributed to the differences in the community structure and assembly.
Distance-based redundancy analysis showed that environmental vari­
ables Cr, Cd, Ca and pH, primarily influenced the variations in the
bacterial community composition.
CRediT authorship contribution statement
Siddarthan Venkatachalam: Conceptualization, Funding acquisi­
tion, Formal analysis, Writing - original draft, Writing - review & edit­
ing. Vatharamattathil Mohanan Kannan: Formal analysis. Vadakke
Neelamanakesavan Saritha: Formal analysis. Dinesh Sanka Loga­
nathachetti: Formal analysis, Writing - review & editing. Mahesh
Mohan: Formal analysis, Writing - review & editing. Kottekkatu
Padinchat i Krishnan: Conceptualization, Funding acquisition, Writing
- original draft, Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
The authors wish to express their gratitude to Director, NCPOR for
his support and encouragement. SV thanks Science and Engineering
Research Board (SERB), Department of Science and Technology, Gov­
ernment of India for his Post-Doctoral Fellowship (No.: PDF/2018/
002088) and research running grant for carrying out the present work.
This work was carried out as a part of the Indian scientific expedition to
the Arctic-2019. We also thank Dr. N.S. Magesh, Project Scientist at
NCPOR, for his assistance in the drawing sampling maps. This is NCPOR
contribution number: J- 2/2021-22.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ecolind.2021.107704.
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