Ecophysiological performance of terrestrial diatoms isolated from biocrusts of coastal sand dunes

Abstract

Terrestrial diatoms are widespread in a large variety of habitats and are regularly recorded in biocrusts. Although diatoms have long been known to live in terrestrial habitats, only a few studies have focused on their diversity of ecophysiology. Here we present a study on the ecophysiological performance of five terrestrial diatom cultures from biocrusts, which were collected in sand dunes of the German coast of the Baltic Sea. The sampling sites were selected along a gradient of human impacts on the dunes. The richness of diatom species, roughly estimated from permanent slides, was around 30 species per sampling site. The species abundance was calculated in the same way revealing a high proportion of broken diatom frustules. All diatom cultures established in the laboratory showed no photoinhibition and high oxygen production along a light gradient. The desiccation tolerance differed among the strains, with high recovery observed for Hantzschia abundans and Achnanthes coarctata and low to no recovery for Pinnularia borealis and Pinnularia intermedia. The maximum growth rate for most strains was between 25 and 30°C. These temperatures can be easily reached in their natural environments. Nevertheless, during short-term exposure to elevated temperatures, oxygen production was recorded up to 35°C. Interestingly, two of five diatom cultures (Hantzschia abundans and Pinnularia borealis) produced mycosporine-like amino acids. These UV-protective substances are known from marine diatoms but not previously reported in terrestrial diatoms.

Full text

Frontiers in Microbiology 01 frontiersin.org
Ecophysiological performance of
terrestrial diatoms isolated from
biocrusts of coastal sand dunes
KarinGlaser 1, SandraKammann 2*, NiklasPlag 2 and
MirkoDressler
3
1 Institute for Biosciences, Biology/Ecology, TU Bergakademie Freiberg, Freiberg, Germany, 2 Institute of
Biological Sciences, Applied Ecology and Phycology, University of Rostock, Rostock, Germany,
3 Department of Physical Geography, Institute for Geography and Geology, University of Greifswald,
Greifswald, Germany
Terrestrial diatoms are widespread in a large variety of habitats and are regularly
recorded in biocrusts. Although diatoms have long been known to live in terrestrial
habitats, only a few studies have focused on their diversity of ecophysiology. Here
wepresent a study on the ecophysiological performance of five terrestrial diatom
cultures from biocrusts, which were collected in sand dunes of the German
coast of the Baltic Sea. The sampling sites were selected along a gradient of
human impacts on the dunes. The richness of diatom species, roughly estimated
from permanent slides, was around 30 species per sampling site. The species
abundance was calculated in the same way revealing a high proportion of
broken diatom frustules. All diatom cultures established in the laboratory showed
no photoinhibition and high oxygen production along a light gradient. The
desiccation tolerance diered among the strains, with high recovery observed
for Hantzschia abundans and Achnanthes coarctata and low to no recovery for
Pinnularia borealis and Pinnularia intermedia. The maximum growth rate for most
strains was between 25 and 30°C. These temperatures can beeasily reached in
their natural environments. Nevertheless, during short-term exposure to elevated
temperatures, oxygen production was recorded up to 35°C. Interestingly, two
of five diatom cultures (Hantzschia abundans and Pinnularia borealis) produced
mycosporine-like amino acids. These UV-protective substances are known from
marine diatoms but not previously reported in terrestrial diatoms.
KEYWORDS
light-irradiance curve, growth rate, desiccation, temperature extremes, stress tolerance,
biocrust, terrestrial, sand dune
1 Introduction
Sand dunes are the rst geological formation along the sea on many natural shores. Coastal
dunes are unique ecosystems in the transition zone between terrestrial and marine environments,
where interactions between geomorphology, climate, and vegetation create highly dynamic
environments (Martínez et al., 2004; Miller etal., 2010). However, dune ecosystems are
threatened worldwide by human inuences, such as land use and erosion (Hernández-Cordero
etal., 2017). Coastal dune growth depends on sediment supply and stability, which are inuenced
by biotic (vegetation cover) and abiotic factors (wind, waves, and precipitation). erefore, this
ecosystem is vulnerable to declining sediment replenishment or surface degradation. Coastal
dunes along public beaches are highly disturbed by trampling, mechanical cleaning, or
OPEN ACCESS
EDITED BY
Liang Peng,
Hunan Agricultural University, China
REVIEWED BY
Leena Virta,
University of Helsinki, Finland
Gang Li,
Chinese Academy of Sciences (CAS), China
Linda Nedbalová,
Charles University, Czechia
*CORRESPONDENCE
Sandra Kammann
sandra.kammann@uni-rostock.de
RECEIVED 22 August 2023
ACCEPTED 04 December 2023
PUBLISHED 19 December 2023
CITATION
Glaser K, Kammann S, Plag N and
Dressler M (2023) Ecophysiological
performance of terrestrial diatoms isolated
from biocrusts of coastal sand dunes.
Front. Microbiol. 14:1279151.
doi: 10.3389/fmicb.2023.1279151
COPYRIGHT
© 2023 Glaser, Kammann, Plag and Dressler.
This is an open-access article distributed under
the terms of the Creative Commons Attribution
License (CC BY). The use, distribution or
reproduction in other forums is permitted,
provided the original author(s) and the
copyright owner(s) are credited and that the
original publication in this journal is cited, in
accordance with accepted academic practice.
No use, distribution or reproduction is
permitted which does not comply with these
terms.
TYPE Original Research
PUBLISHED 19 December 2023
DOI 10.3389/fmicb.2023.1279151

Glaser et al. 10.3389/fmicb.2023.1279151
Frontiers in Microbiology 02 frontiersin.org
permanent structures (e.g., groins for coastal protection) that impede
wind-induced natural sand replenishment (Stancheva et al., 2011;
Santoro etal., 2012).
Besides these human impacts on the coastal dune system, harsh
environmental conditions with a variety of challenging environmental
stressors such as strong winds, substrate mobility, nutrient, and soil
water scarcity, occasionally extremely high near-surface temperatures,
intense radiation, ooding, and salt spray (Maun, 2009; Miller etal.,
2010) make the establishment of vascular plants quite challenging.
Under these conditions, the growth and development of a closed
vascular plant cover is restricted, except for anthropogenically planted
marram grass (Ammophila arenaria (L.) Link). Under natural
conditions, only specialized/stress-tolerant groups of organisms can
establish on the dune surface; this includes cryptogamous communities
such as biological soil crusts (biocrusts) (Schulz etal., 2016).
Biocrusts are formed by living organisms and their by-products,
creating a topsoil layer of inorganic particles bound together by
extrapolymeric organic compounds. Biocrusts are found on all
continents, in arid, semiarid, and other habitats where soil moisture is
limiting and cover of higher plants is sparse (Belnap etal., 2001). In
temperate zones, these habitats include, for example, sandy coastal and
inland dunes, disturbed areas (windbreaks, burned areas, etc.), or
barren soils; biocrusts usually cover all soil areas not occupied by
vascular plants and thus comprise up to 70% of the living cover (Belnap
etal., 2001). Biocrusts form the most productive microbial biomass
worldwide in the so-called ‘Earth’s Critical Zone’, which is the upper
approx. 10 mm of soil in most dry areas. In ‘new’ vegetation-free or
disturbed landscapes such as volcanic areas, glacier forelands, etc.,
biocrusts form the basis for further ecosystem development and
succession (Eldridge and Tozer, 1996; Cutler etal., 2008; Yoshitake etal.,
2010). Together with other microorganisms such as heterotrophic
bacteria, archaea, and fungi, as well as macroscopic lichens and mosses,
cyanobacteria and algae comprise the most important phototrophic
components of biocrusts (Elbert etal., 2012). Because of their impact
on various ecosystem functions, biocrusts can be characterized as
‘ecosystem engineers’. e cryptogam community forms water-stable
aggregates that play important, multifunctional ecological roles in
primary production, nutrient cycling, mineralization, water retention,
soil stabilization, and dust binding (Evans and Johansen, 1999; Reynolds
etal., 2001; Lewis, 2007; Castillo-Monroy etal., 2010). An overview of
these microbiotic crusts clearly shows the important ecological role of
these communities for global carbon (C) xation (about 7% of terrestrial
vegetation) and nitrogen (N) xation (about 50% of terrestrial biological
N xation; Elbert etal., 2012).
Biocrusts in the temperate zone support a diverse algal community
(Glaser etal., 2018; Mikhailyuk etal., 2019). Hundreds of dierent
phototrophic species of cyanobacteria and algae (including diatoms)
live in association with biocrusts. Terrestrial diatoms are widespread
in many terrestrial habitats such as biocrusts, mosses, soil, caves, or
articial environments (Norbäck Ivarsson etal., 2013; Falasco etal.,
2014; Kopalová etal., 2014; Schulz etal., 2016; Zhang etal., 2020).
ey contribute to soil stability by producing extrapolymeric
substances, which function as glue for sand particles (Kidron, 2021).
Furthermore, terrestrial diatoms are oen reported even in high cell
counts in biocrusts (Meadow and Zabinski, 2012; Schulz etal., 2016).
However, their biodiversity, ecophysiology, and taxonomy are still
inadequately characterized (Barragán etal., 2018). Marine diatoms are
known to produce mycosporine-like amino acids (MAA) as
UV-protective substances in response to increased solar radiation
(Helbling etal., 1996; Jerey etal., 1999). In detail, there are solid
indications that those MAAs are embedded in the silica frustule
(Ingalls etal., 2010). MAAs were also recorded from terrestrial algae
isolated from biocrusts, like for example Mesotaenium and
Klebsormidium (Remias etal., 2012; Hartmann etal., 2020). It seems
likely that terrestrial diatoms also follow this strategy and accumulate
MAAs as UV-protective substances. Nevertheless, this fact has not
been experimentally proven up to now. Only a few experimental
ecophysiological studies on terrestrial diatoms have been undertaken
under controlled conditions (Soureau etal., 2010, 2013; Hejduková
et al., 2019). ese authors found similar response patterns for
dierent terrestrial diatom species, as reected in higher tolerances to
extreme freezing and desiccation events that were lethal to isolates of
freshwater diatom species. All other publications have provided
comprehensive species lists and have attempted to nd correlations
between the occurrence or lack of terrestrial diatom taxa with key
environmental factors (Foets etal., 2021). By applying an array of
statistical methods (e.g., redundancy analysis), the preferential
occurrence or lack of taxa can beexplained by specic autecological
requirements, such as pH or soil organic-matter content (Antonelli
etal., 2017; Foets etal., 2021), yet experimental evidence is mostly
missing. Nevertheless, taxa-specic indicator values for undisturbed
and disturbed soil habitats, as well as tolerance ranges can at least
be estimated from such correlations, and terrestrial diatoms are
sensitive to multiple environmental factors such as pH, anthropogenic
disturbance caused by farming practices (land-use intensity), and soil
moisture and nitrogen contents (Foets etal., 2021). Farming practices
play a key role in structuring soil diatom communities (Antonelli
etal., 2017; Foets etal., 2021; and references therein), as disturbed
areas were found to beless diverse and land uses with dierent
disturbance levels could be dierentiated based solely on the
community composition. ese authors also found that the
composition of soil diatom species remained stable throughout the
year and that diatoms were always present, in contrast to higher plants.
Consequently, terrestrial diatoms could serve as indicator species in
soils, similarly to the freshwater-diatom biotic indices used extensively
to assess water quality. Recent studies have proven the suitability of
diatom species assemblages as bioindicators for human disturbances,
heavy-metal contamination, and soil environmental assessment
(Wanner etal., 2020; Zhang etal., 2020; Minaoui etal., 2021).
In this study, we present a comprehensive ecophysiological
characterization of ve diatom cultures originating from biocrusts in
coastal sand dunes, accompanied by an insight into the biodiversity of
terrestrial diatoms in biocrusts, based on morphological identication.
Our study aimed (1) to enhance our understanding of terrestrial
diatoms by revealing unknown community structures of a
yet-unstudied diatom habitat, the biocrusts in coastal sand dunes; and
(2) to obtain insights into the ecophysiological performance of
cultured terrestrial diatoms.
2 Materials and methods
2.1 Site description
Biocrusts were collected on October 05, 2021 at three sampling
sites along the coast of the Darss-Zingst-Peninsula, Germany

Glaser et al. 10.3389/fmicb.2023.1279151
Frontiers in Microbiology 03 frontiersin.org
(Figure1). On that day, a temperature of 12°C and precipitation of
30.5 L m
−2
were measured at the weather station in Zingst. e three
sampling sites were selected according to their degree of disturbance
by human activity: the highly disturbed site is located in a camping
area (N54.45551, E12.54914); the moderately disturbed site is
located at a public beach distant from the next village (N54.44103,
E12.77921) in the care and development zone of the ‘Vorpommersche
Boddenlandscha’ National Park; and the little-disturbed site is
located in the core zone of the national park (N54.44327, E12.90223).
At the highly disturbed site, the dunes were clearly inuenced by
human trampling. Most of the area was bare sand and biocrust grew
as thin biolms between trampled paths. At the moderately
disturbed site, trampled paths within the dunes were also visible,
although it is forbidden to walk in the dunes. Biocrusts were visible
as thin biolm and were more frequently observed than at the highly
disturbed site. At the little-disturbed site, no trampling was visible
because a wooden boardwalk leads through the dunes. Here, the
biocrusts were thicker, and even moss and lichen thalli could
develop. A randomly chosen plot of 1 m2 was established at each of
the three sampling sites for further material collection. Biocrusts
were sampled by gently pushing a Petri dish into the surface and
liing it with a spatula. Five Petri dish samples were collected per
site. ese were sealed using laboratory sealing lm and transported
to the laboratory for further analysis.
e biocrust stability was measured in the eld, using a
penetrometer (fruit hardness tester FHT-15, tip width 3.5 mm
diameter). e penetrometer was pushed at a 90° angle onto the
biocrust surface until the biocrust broke. e measurement was
repeated ve times per sampling site.
In the laboratory, the chlorophyll a (Chl a) content of the biocrusts
was measured as an estimator for photosynthetic biomass. A dened
area of 0.5 cm2 biocrust was picked from the Petri dishes using a cork
borer pushed approximately 1 cm into the sample. e biocrust
material was incubated in 3 mL 96% ethanol at 78°C for 30 min. e
solution was centrifuged and measured spectrophotometrically
(Shimadzu UV-2401 PC, Kyoto, Japan) at 632, 649, 665, 696, and
750 nm absorbance. e last served as a control for turbidity (Ritchie,
2008). e extraction with ethanol was repeated until no chlorophyll
a could bedetected in the biocrust samples. ree replicates were
measured for each sampling site. Total carbon and nitrogen (C
t
and
N
t
) contents were determined by dry combustion of about 100 mg
ground biocrust sample material in an element analyzer (UNICUBE
®
Elementar Analysensysteme GmbH, Langenselbold, Germany). Total
phosphorus (P
t
) was extracted from 100 mg of ground material by
sub-boiling digestion using an acidic persulfate solution in Teon®
tubes for 24 h at 90°C (Berthold et al., 2015). All samples were
neutralized using NaOH aer digestion. e neutralized extract was
further analyzed spectrometrically for phosphate, using the
molybdenum blue method at 885 nm wavelength (Murphy and Riley,
1962). All total element contents were measured in ve replicates per
sampling site.
2.2 Preparation of permanent slides and
morphological identification
Preparation of permanent slides followed the procedure
described by Schulz etal. (2016). Briey, approximately 0.5 g biocrust
FIGURE1
(A) The Darss-Zingst Peninsula in northern Germany. (B) Overview of sampling sites along the coastline of the Darss-Zingst-Peninsula, including
impressions of biocrusts at these sites. Blue: camping area (highly disturbed); yellow: public beach (moderately disturbed); green: core zone of the
National Park (little disturbed).

Glaser et al. 10.3389/fmicb.2023.1279151
Frontiers in Microbiology 04 frontiersin.org
material was mixed with 4 mL distilled water and shaken.
Immediately aer shaking, 100 μL of overlying water was gently
dripped onto glass coverslips, which were rst air-dried and then
combusted in a mue oven (Elektra M26) at 550°C for 35 min. Aer
cooling, the glass coverslips were mounted on glass microscope
slides using Naphrax®. Diatom species were morphologically
identied with the aid of a light microscope (Zeiss Axioplan,
oil-immersion Plan-Apochromat objective, aperture 1.4) with 1,000-
fold magnication. In total, over 400 valves that were at least 50%
intact were counted per sampling site. e proportion of valves at
least 90% and 100% intact was also recorded.
2.3 Establishment of diatom cultures
e establishment of terrestrial diatom cultures was challenging.
e diatoms stuck tightly to the glass surface of the slides, making it
impossible to pick single cells using a micromanipulator under a light
microscope. erefore, the pure cultures had to bedeveloped by
repeating transfer steps. Dierent culture media, both liquid and
agarized, were tested to maximize the cultivation success: WC,
Diat+Vit.mix (Bacillariophycean medium + vitamin mixture), BG11
(medium for cyanobacteria) and f/2 (enriched seawater medium)
(recipes according to SAG). Initially, cultivation in agarized Petri
dishes was more successful. Aer several transfer steps, only ve
diatom strains remained. ey were transferred to liquid media where
they could beestablished as unialgal cultures. ese unialgal diatom
cultures were cultivated in a Diat+Vit.mix medium at 20°C under low
light (50 μmol photons m−2s−1).
2.4 PCR and sequencing for identification
DNA was extracted from the ve diatom cultures, following the
instructions of the NucleoSpin® Plant II Mini Kit (Macherey and
Nagel, Düren, Germany). For identication, the rbcL gene (RuBisCO
large subunit) was amplied in a PCR using a commercial
PCR-Mastermix (Bioline). e primers Diat-rbcl-iR and Diat-rbcl-F
were applied with the respective PCR protocol (Abarca etal., 2014).
Sequencing was carried out by a commercial company (Eurons,
Luxembourg), using the same forward primer as for PCR. e
sequences were uploaded to NCBI under the accession numbers
OR387857–OR387860.
2.5 Desiccation experiment
e experiment followed the procedure described by Karsten etal.
(2016). Briey, the diatom cultures were grown for 1 week on glass-
ber lters (ve replicates per strain), which were transferred for the
experiment to a desiccation chamber lled with 100 mL silica gel.
ese were kept at a room temperature of ~25°C. e yield of
photosystem II (YII) was recorded during desiccation every 10 min
for 4 h as a proxy for the tness of the cells, using non-invasive pulse
amplitude modulation uorometry (PAM2500, Walz, Germany).
Aer a signal could no longer bemeasured, the lters were rewetted
with 250 μL medium and transferred to another water-saturated
chamber. e relative humidity in the chamber was continuously
recorded in each second, using a multifunctional data logger (MSR
145 W; MSR Electronics GmbH, Switzerland).
2.6 Photosynthesis-irradiance curves
Photosynthesis-irradiance (PI)-curves of the ve diatom
strains (four replicates per strain) were measured according to
Prelle etal. (2019). Briey, 3.1 mL of thin log phase algal suspension
of each strain and 31 μL sodium bicarbonate (NaHCO
3
, 2 mM nal
concentration) were added to four airtight water-tempered (20°C)
oxygen electrode chambers (DW1, Hansatech Instruments, King’s
Lynn, UnitedKingdom). e oxygen concentration was measured
at ten increasing photon ux density levels ranging from 0 to
~1.500 μmol photons m−2s−1 of photosynthetically active radiation
(PAR), using a non-invasive oxygen dipping probe (DP sensors
PreSens Precision Sensing GmbH, Regensburg, Germany).
Measurements consisted of a 30 min respiration (dark) phase,
followed by a 10 min photosynthesis (light) phase for each light
level. e rst and last minutes of each phase were excluded from
the calculation. Aer the last measurement, Chl a was extracted
from the 3.1 mL algal suspension (10 mL, 96% ethanol at 70°C for
10 min) and quantied spectrophotometrically (Ritchie, 2008). e
mathematical photosynthesis model of Walsby (1997) was used for
tting and calculation of the maximum rates of net primary
production (NPP
max
), respiration (R), light utilization coecient
(α), photoinhibition coecient (β), light saturation point (Ik), and
the light compensation point (Ic).
2.7 Temperature curve
e photosynthetic and respiratory response of each strain (four
replicates per strain) at temperatures between 5°C and 40°C was
measured using the same oxygen optode system as for the PI-curves
(Karsten et al., 2010). Aer 20 min incubation in the dark, the
respiratory oxygen consumption (10 min in the dark), followed by the
photosynthetic oxygen production (10 min under light-saturated
conditions at 335 μmol photons m
−2
s
−1
PAR) were determined.
Measurements were normalized to the total Chl a concentration (see
procedure above). e model of Yan and Hunt (1999) was used to t
the temperature values, including optimum and
maximum temperature.
2.8 Growth rate
e uorescence of Chl a was used as a proxy for biomass to
calculate the growth rates of the ve diatom strains according to the
temperature. e in-vivo Chl a uorescence measurements were
performed with a self-constructed growth uorimeter based on the
basic electronic unit of an MFMS uorimeter (Hansatech Instruments,
King’s Lynn, UnitedKingdom) according to the protocol of Karsten
et al. (1996). Bright-blue light LED emission (Nichia, Nürnberg,
Germany) with a peak emission wavelength of 470 nm was selected
for excitation of the Chl a uorescence and pulsed with a modulation
frequency of 870 Hz. Chl a uorescence was detected as relative units
by an amplied photodiode and was separated from scattered

Glaser et al. 10.3389/fmicb.2023.1279151
Frontiers in Microbiology 05 frontiersin.org
excitation light through a long-pass glass lter (RG 665; Schott, Mainz,
Germany) and a bright-red gelatin lter (Lee, Brussels, Belgium).
In-vivo Chl a uorescence units correlate well to the cell number and
the concentrations of organic carbon and Chl a in diatoms, as shown
by Karsten etal. (1996) and Gustavs etal. (2009). e cultures were
grown in disposable plastic Petri dishes with cover lids, in a volume of
20 mL culture medium, and measured every 24 h for 10 days, following
the procedure of Gustavs etal. (2009). e light was kept constant at
45–105 μmol photons m
−2
s
−1
, following a 16:8 light:dark cycle
(Lumilux Deluxe Daylight L15W/950; OSRAM). e cultures were
kept in water baths or air-conditioned rooms to ensure constant
temperature conditions at ve tested temperatures (5, 15, 20, 25, and
30°C), and all diatom strains were measured in triplicate. To ensure
constant temperature over the course of the experiment, the light
intensities were a bit below saturated conditions, because the light
bulbs would otherwise have caused unwanted temperature
uctuations. Growth rates were calculated separately for each replicate,
using the phase where the uorescence signal increased exponentially
(Gustavs etal., 2009). For 30°C, no growth rate could becalculated,
because decreasing signal intensities were measured during the
duration of the experiment. e model of Yan and Hunt (1999) was
used to t the temperature values, including optimum and
maximum temperatures.
2.9 Detection of biochemical
UV-protective substances
e biocrust biomass was extracted and further processed for
HPLC (High-performance liquid chromatography) analysis as
described by Karsten etal. (2009). Samples were analyzed with an
Agilent HPLC system (Agilent, Waldbronn, Germany), and
mycosporine-like amino acids (MAAs) were separated on a
Phenomenex Synergi Fusion RP-18 column (Reversed Phase, Polar
embedded C18 with TMS endcapping; 4 μm, 250 × 3.0 mm I.D.)
protected with an RP-18 guard cartridge (20 × 4 mm I.D., Phenomenex,
Aschaenburg, Germany). e mobile phase contained 2.5%
methanol (v/v) in 0.1% acetic acid (v/v) in HPLC water (0.055 μS
cm−1) and was run isocratically at a ow rate of 0.5 mL min−1 at 30°C
column temperature for 20 min. MAAs were detected with a
photodiode array detector at 330 nm wavelength, and absorption
spectra (290–400 nm) were recorded in each second, directly on the
HPLC-separated peaks. MAA standards were run within the sample
sequence. ose included Asterina-330 (retention time 3.33 min,
absorbance at 330 nm), Shinorine (4.71 min, 334 nm), Prasiolin
(9.6 min, 323 nm), Porphyra-334 (5.89 min, 334 nm), Mycosporine-
glycine (3.8 min, 310 nm), Klebsormidin A (5.35 min; 323 nm), and
Klebsormidin B (5.74 min; 323 nm).
2.10 Statistical analyses
All statistical analyses were done in R, version 4.2.1 (R Development
Core Team, 2022) or Microso Excel. Signicant dierences between
measured soil parameters for the biocrusts of dierent sites were
calculated in R, using one-way ANOVA. Photosynthetic irradiance
curves were tted using the Walsby model in Excel, based on least-
square methods (Walsby, 1997). Temperature curves (both growth rate
and oxygen production) were tted using the Yan and Hunt (1999)
model in R, also based on the least-square model. e model by Yan and
Hunt is a simplied exponential and polynomial model, which
represents the plant growth below and above the temperature optimum
better than bi-or multilinear models. It has proven to t the experimental
growth data of microalgae well (Losa etal., 2020). Signicant dierences
between oxygen production and consumption along the temperature
gradient were calculated using one-way ANOVA followed by a post-hoc
Tukey test (p < 0.05). Condence intervals for maximum oxygen
production, maximum growth rate, and optimum and maximum
temperatures were calculated using the command ‘connt2’ (package
nlstools; Baty etal., 2015).
3 Results
3.1 Analyses of biocrust characteristics
e Chl a content of the little-disturbed site was much higher
(189.17 mg m
−2
) than for the moderately and highly disturbed sites (72.32
and 76.62 mg m
−2
respectively; Table1). Further, the biocrust at the little-
disturbed site was signicantly more stable (1.12 MPa) than at the other
two sites (0.5 MPa at the moderately and 0.4 MPa at the highly disturbed
site). e stability of the biocrusts diered signicantly between the little-
disturbed and the other two sites (p < 0.05). e concentrations of total
N (3.68 g kg
−1
) and total C (78.76 g kg
−1
) were signicantly higher
(p < 0.05) at the little-disturbed site. In contrast, signicantly higher
(p < 0.05) total P concentrations were measured in the biocrust samples
from the moderately disturbed sampling site (50.7 mg kg−1).
3.2 Diatom species and relative abundance
in biocrusts
In total, weobserved 47 diatom species in biocrusts from three sand
dunes on permanent slides (Supplementary Table S1). Each site showed
a similar richness (28–30 species), all diatom taxa were pennate species.
Six of the 47 diatom species could beassigned to a pure terrestrial life
cycle, according to the literature. ese were Pinnularia borealis,
Pinnularia. intermedia, Hantzschia amphioxys, Luticola terrestris,
TABLE1 Biocrust characterization of all three sampling sites.
Study area Degree of
disturbance
Ct (g kg−1) Nt (g kg−1) Pt (mg kg−1) Chl a (mg m−2) Stability (MPa)
Camping area High 9.5 ± 3.6 0.5 ± 0.2 6.8 ± 4.4 76.6 ± 29.0 0.4 ± 0.2
Public beach Moderate 1.5 ± 0.3 0.2 ± 0 50.7 ± 19.8 72.3 ± 49.9 0.5 ± 0.1
National Park Low 78.8 ± 23.7 3.7 ± 1.1 19.4 ± 9.4 189.2 ± 24.3 1.1 ± 0.1
Means ± standard deviations (stability, Ct = total carbon, Nt = total nitrogen, Pt = total phosphorus, n = 5; Chl a n = 3).

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Mayamaea fossalis, and Nitzschia cf. pusilla. ese six diatom species
were abundant in all the biocrust samples: 26% in biocrusts from the
national park, 50% in biocrusts from the moderately disturbed area, and
18% in the highly disturbed dune area (Supplementary Table S1). e
Simpson diversity index for the moderately disturbed site was lower
(0.77) than at the other two sites (0.91 and 0.92). is was mainly
because one species, P. intermedia, dominated the community at the
moderately disturbed site, with an abundance of 45%.
Notably, many diatom frustules were partly disrupted. At the
little-and moderately disturbed sites, approximately 48% were at least
90% or 100% intact. At the high disturbed site, only 25% were at least
90% or 100% intact (more details on the exact numbers of totally/
mostly intact frustules can befound in Supplementary Table S1).
Most of the diatom species were found at all three sites (Figure2)
but some (~38%) were found exclusively at one site. ese unique
species were in most cases scarce, whereas the common species for all
three sites were highly abundant.
3.3 Identification of diatom cultures
We successfully developed ve unialgal cultures of terrestrial
diatoms from the sand dune biocrusts. Unfortunately, one culture
collapsed during the experiments. is culture (P. intermedia) had
relatively small cells (smaller than average according to the literature)
and those cells became smaller and smaller over time until the culture
was no longer viable. is phenomenon is known to occur in some
diatom cultures. With each cycle of asexual reproduction, the average
size of diatoms in a culture decreases due to the formation of silica
frustules, until the species’ physical limit is reached. In the case of sexual
reproduction, the daughter cells can become larger than the parent cells.
However, not all diatoms reproduce sexually under culture conditions.
Most likely, our culture collapsed because it reached its lower cell size
limit and could not reproduce sexually under our culture conditions.
Diatoms in the other cultures did not decrease in average size.
We successfully cultivated Hantzschia amphioxys-aggr. (#1),
Achnanthes coarctata (#2), Hantzschia abundans (#3), Pinnularia
intermedia (#4), and Pinnularia borealis-aggr. (#6) (Figure 3).
H. amphioxys and P. b o re al is originated from the highly disturbed site,
whereas the other three cultures originated from the moderately
disturbed site. e cultures were identied morphologically and also
at the molecular level, based on the rbcL gene. e rbcL sequence of
P. bo re al i s was very similar (99.8% similarity) to the strain TAS17-48-
10, which was assigned to clade 3 within the Pinnularia borealis-
complex (Pinseel et al., 2019). e closest hit for the cultured
P. intermedia was P. acrosphaeria, with 97.7% identity. ere are no
records of P. intermedia in the NCBI databases.
3.4 Desiccation tolerance
e diatom cultures were subjected to desiccating conditions to
estimate their desiccation tolerance. Aer around 4 h, the lters with
diatom cultures dried completely in the desiccation chamber
(Figure4). e drop in relative humidity from 40% to ~25% was
accompanied by a drop in the yield of photosystem II (YII) to 0%. Few
minutes later, the lters were transferred to a water-saturated chamber.
P. intermedia and P. b or ea li s showed only minor recovery to the initial
Y(II) aer rewetting. H. amphioxys recovered to around 40% of its
initial Y(II). H. abundans and A. coarctata performed best, ending
with 80% of the initial Y(II) aer 24 h (Figure4).
3.5 Light-dependent photosynthesis
e photosynthetic performance of the diatom cultures was
measured as oxygen production along an increasing light gradient.
H. abundans had a lower NPPmax, lower alpha, and lower respiration
rate compared to the other four cultures (Figure 5; Table 2). All
cultures showed no to only slight photoinhibition up to an irradiance
of 1,500 μmol photons m2 s−1.
3.6 Temperature-dependent
photosynthesis
e photosynthetic performance of the cultures showed a broad
temperature tolerance (Figure6; Table3). e optimal temperature
range, where at least 80% of the maximum oxygen production
occurred, was about 13°C (Figure7A). P. intermedia had a slightly
higher range of about 15°C. In addition, the temperature range
(between 10°C and 25°C) of optimal oxygen production for this
species was below the range of the other four cultures, which was
around 13–18°C to 27–30°C. e respiration of all diatom cultures had
a higher optimum temperature (30°C to 35°C) than photosynthesis.
3.7 Temperature-dependent growth rate
e dependence of the growth rate on temperature was similar for
H. amphioxys, A. coarctata, and P. b or ea l i s . ese three cultures
FIGURE2
Venn diagram displaying the overlapping and unique diatom species
present in biocrusts from dunes with high, moderate, or low degrees
of human disturbance. Results based on morphological
determinations from permanent slides.

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showed a similar maximum growth rate of ~0.5 μ day
−1
(Figure8;
Table4). H. abundans had a higher growth rate than the others. e
maximum temperature for growth (Table 4) was highest in
H. amphioxys (34°C), followed by A. coarctata (~31°C), whereas
H. abundans and P. b or e al is reached their maximum temperature for
growth already at 26°C and 28°C, respectively (Figure7B).
3.8 Biochemical UV protection
In two of ve cultures, H. abundans and P. b o re a l i s , the same
mycosporine-like amino acid (MAA) was detected (Figure9). e
absorbance of this MAA was 333.5 nm wavelength and had a retention
time of 6 min. is was a longer retention time than for our test
standards, and therefore the MAA in these diatoms could not
beidentied.
4 Discussion
Depending on environmental conditions and external inuences,
biocrusts can reach dierent successional stages, from a thin algal
crust to moss-dominated or lichen crusts (Langhans et al., 2009;
Garcia-Pichel, 2023). e biocrusts observed at the little-disturbed site
FIGURE3
Microphotographs of five unialgal living diatom cultures and their frustules, isolated from biocrusts in coastal sand dunes, Mecklenburg-Vorpommern,
Germany. Species identification is based on morphological and molecular features. (A) Hantzschia abundans, (B) Hantzschia amphioxys, (C) Pinnularia
borealis, (D) Achnanthes coarctata, (E) Pinnularia intermedia; scale bar = 20 μm.

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TABLE2 Parameters of photosynthesis-irradiance (PI)-curves (Figure4) of the five terrestrial diatom cultures (n = 4 ± standard deviation) measured at
20°C in Diat+Vit.mix medium.
Achnanthes
coarctata
Hantzschia
abundans
Hantzschia
amphioxys
Pinnularia
borealis
Pinnularia
intermedia
NPPmax 71.27 ± 1.4 19.6 ± 4 64.93 ± 5.1 56.42 ± 3.7 82.84 ± 10.8
alpha 2.3 ± 0.5 1.11 ± 0.9 2.5 ± 0.1 1.72 ± 0.1 1.85 ± 0.2
beta −0.01 ± 0.01 −0.01 ± 0.004 −0.02 ± 0 0 ± 0.004 −0.01 ± 0.01
Respiration −35.42 ± 2.5 −22.62 ± 1.2 −42.3 ± 1.6 −43.24 ± 4 −54.56 ± 4.5
Ik46.29 ± 12.8 38.17 ± 8.9 42.91 ± 8.3 57.96 ± 12.3 74.32 ± 12.1
Ic18.68 ± 2.2 29.13 ± 4.3 21.51 ± 1.1 32.91 ± 1.6 37.56 ± 2.5
NPPmax, maximum rates of net primary production in μmol O2 mg−1 Chl a h−1; alpha, light utilization coecient; beta, photoinhibition coecient; Ik, light saturation point; Ic, light
compensation point.
FIGURE4
Eect of controlled desiccation and rehydration on the eective quantum yield (Y(II)) of PSII to five diatom cultures isolated from biocrusts in sand
dunes (n = 4, only negative standard deviation is displayed for a better overview). Eective quantum yield values were standardized to the starting Y(II)
to 100% for better comparison. The dashed line represents the measurement of relative humidity in the desiccation chamber.
FIGURE5
Photosynthetic-irradiance curve of five diatom cultures isolated from biocrusts in sand dunes. The points represent the mean of the measured values
(n = 4 ± standard deviation), and the line is the fitting curve after Walsby (1997).

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were mostly dominated by mosses, representing a later stage in
succession (Lan etal., 2012; Garcia-Pichel, 2023). One reason for this
may bethe high protection status of the dunes within the national
park. e dunes are protected against human trampling and biocrust
can form stable layers on the dune sediment, as shown in this study.
ese stable biocrusts accumulated more organic material, as
evidenced by the signicantly higher total carbon and Chl a contents
compared to the other two sites. Phosphorus is transported into the
ecosystem mostly by wet and dry deposition (Berthold etal., 2019).
Further weathering of phosphorus-containing parent rock material is
an important mechanism through which phosphorus becomes
available to the biocrust community in the sand dunes (Filippelli,
2002). e signicantly higher phosphorus concentration at the
moderately disturbed site might beexplainable by a selective input of
an external P source such as bird droppings, which could have
inuenced the measurement.
Terrestrial diatoms have long been known, but have received only
slight interest from biologists. Recent studies have pointed toward a
FIGURE6
Temperature-dependent oxygen production of five diatom cultures isolated from biocrusts in sand dunes. The points represent the mean of the
measured values (n = 4 ± standard deviation), and the dotted line is the fitting curve after Yan and Hunt (1999). Dierent letters indicate significant
dierences for photosynthesis (blue) or respiration (red), respectively (ANOVA with post-hoc Tukey test). (A) Achnanthes coarctata, (B) Hantzschia
abundans, (C) Hantzschia amphioxys, (D) Pinnularia intermedia, (E) Pinnularia borealis.

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large hidden diversity within morphological species. For example, the
species complexes of P. b o re al is and H. amphioxys have been divided
into several species (Pinseel etal., 2019; Maltsev etal., 2021), which
share morphological similarities but can beclearly distinguished by
molecular methods. Such studies provide evidence of the gap in our
current understanding of terrestrial diatoms.
In our study, weobserved 28–30 diatom taxa per sampling site (a
total of 47 species at the three sites combined), which is within the
common range of species per sample compared to other studies (Van
de Vijver etal., 2008; Norbäck Ivarsson etal., 2013; Kopalová etal.,
2014; Schulz etal., 2016; Zhang etal., 2020; Fazlutdinova etal., 2021;
Foets etal., 2021). However, weobserved a large number of marine
diatoms (for example Achnanthes lemmermannii, Catenula adhaerens,
Cocconeis peltoides, Navicula germanopolonica, and Navicula
viminoides; for ecological preferences of these diatoms see Witkowski
et al., 2000; Plinski and Witkowski, 2020). ese marine species
probably did not live in the biocrusts, and their valves may have been
blown onto the beach or transported via sea spray. is idea is
supported by our observation of many partly destroyed valves. is
would beuncommon if the diatoms were living and reproducing in
this habitat. erefore, it appears that only 4 to 6 species were actually
abundant and alive in the biocrust samples. Among these, wefound
typical terrestrial diatoms such as P. b o r e a l i s and H. amphioxys.
P. b o r e a l i s is reported worldwide in a variety of terrestrial habitats
(Pinseel etal., 2019), even in less-favorable environments such as
volcanic soils a few months aer an eruption (Fazlutdinova etal.,
2021). is species has outperformed other diatom species in
surviving frequent and extreme freezing (−180°C) conditions
(Hejduková etal., 2019). Nevertheless, it is important to keep in mind
that our current understanding of P. b o r e a l i s as a ubiquitous and
highly stress-tolerant species might change. Pinseel etal. (2019) found
that P. b o r e a l i s is a complex consisting of around eight species.
Similarly, H. amphioxys, also reported worldwide from a variety of
habitats, also seems to bea complex of approximately six species.
Single species within a species complex may diverge to dierent
habitats. A. coarctata is also occasionally reported from terrestrial
habitats, especially since it seems to live in mosses. For example,
wecultured A. coarctata, which was reported from moss samples in
Antarctica (Kopalová etal., 2014). However, wedid not observe this
species on our combusted slides, which may indicate that it was
present in low abundance in our samples. e dominance of
P. intermedia in the moderately disturbed area was remarkable.
Wealso cultured this species, although wedid not succeed in keeping
it in culture for long. Notably, P. intermedia is only rarely reported and
is also missing in the sequence databank. is species was observed
from sand dunes (Round, 1957; Schulz etal., 2016) and mosses
(Beyens, 1989), but since then has only rarely been reported in
terrestrial habitats.
TABLE3 Parameters of oxygen production along a temperature gradient after fitting with Yan and Hunt (1999) model including 5% confidence interval
for four diatom cultures (n = 4).
Achnanthes
coarctata
Hantzschia
abundans
Hantzschia
amphioxys
Pinnularia
borealis
Pinnularia
intermedia
NPPmax 62.8 (53–72.7) 56.5 (46.8–66.2) 60 (46.6–73.4) 35.6 (29.4–41.9) 64.2 (53.2–75.2)
optimum temperature [°C] 20.3 (17.8–22.7) 21.6 (19.2–24) 22.1 (18.9–25.3) 24.5 (22.3–26.6) 17.1 (13.8–20.5)
maximum temperature [°C] 34.8 (33.6–36) 34.5 (33.3–35.6) 35 (33.5–36.5) 35.5 (34.5–36.5) 35.1 (33.4–36.8)
NPPmax, maximumrates of net primary production in μmol O2 mg−1 Chl a h−1.
FIGURE7
Comparison between short-term; [(A) few hours, measured as oxygen production] and long-term; [(B) few days, measured as growth rate] eects of
temperature treatment on five (or four) diatom cultures isolated from biocrusts in sand dunes. Values were calculated based on the fitting results
presented in Figures6, 8. A – Achnanthes coarctata, B – Hantzschia abundans, C – Hantzschia amphioxys, D – Pinnularia borealis, E – Pinnularia
intermedia.

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4.1 Indicator value
Freshwater diatoms have long been used as bioindicators in
aquatic environments because some species are sensitive to pollution
(e.g., high nutrient concentrations) and thus provide valuable
information concerning water quality. e idea to use terrestrial
diatoms as bioindicators is relatively new, and the criteria for
assessment are not yet dened (Antonelli etal., 2017; Barragán etal.,
2018). However, this approach seems promising (Wanner etal., 2020;
Zhang etal., 2020; Minaoui etal., 2021). For example, a study in
suburban soils around Marrakesh, Morocco, supported the idea of
using diatoms for indication of high nutrient content, pH, and
conductivity (Minaoui et al., 2021). Although our study showed
dierent community patterns, possibly depending on the degree of
human disturbance, there was no clear indicator species for low or
high disturbance levels. In our case, maybe the high proportion of
broken diatom frustules might serve as an indicator for high
disturbance levels. Broken frustules indicate that these diatoms had
been dead for some time and therefore the frustules became disrupted
due to physical forces. ese forces can bestronger under regular
trampling because in such cases the sand grains would grind the silica
frustules. In coastal dunes, such broken frustules could also becarried
in from the sea via sea spray. We derive this assumption from our
results, but further research is needed to conrm our hypothesis and
to establish the proportion of broken frustules as a valid indicator
value in the future.
4.2 Ecophysiological performance of soil
diatom cultures
Most studies on terrestrial diatoms rely on estimation of
morphological diversity based on valves, a well-developed method
(Nagy, 2011). Nevertheless, it remains unclear if the valves represent
living and reproducing diatom species or if those diatoms were dead,
or if the valves were merely blown into place because valves can
remain stable for several centuries and are used in paleobiology
(Dreßler etal., 2011). Only a few studies on terrestrial diatoms rely on
culture techniques. Culturing of terrestrial diatoms is oen quite
challenging due to unknown requirements and because the diatoms
tend to live closely attached to soil particles. Consequently, our study
is one of only a few that have examined the ecophysiological tolerance
of soil diatoms. Our study showed that the diatoms can use and
tolerate a wide range of irradiance intensities. Also, short-term
FIGURE8
Temperature-dependent growth rate of four diatom cultures isolated from biocrusts in sand dunes. Points represent the measured values (n = 3), and
the line is the fitting curve after Yan and Hunt (1999). (A) Achnanthes coarctata, (B) Hantzschia abundans, (C) Hantzschia amphioxys, (D) Pinnularia
borealis.
TABLE4 Parameters of growth rate along a temperature gradient after fitting with Yan and Hunt model including 5% confidence interval for four
diatom cultures (no results for Pinnularia intermedia; n = 3).
Achnanthes coarctata Hantzschia abundans Hantzschia amphioxys Pinnularia borealis
maximum growth rate [μ day−1]0.44 (0.31–0.57) 0.96 (0.66–1.26) 0.53 (0.51–0.55) 0.42 (0.39–0.46)
optimum temperature [°C] 15.4 (11.4–19.4) 22.2 (21.3–23) 17.2 (16.6–17.9) 13.9 (12.9–14.8)
maximum temperature [°C] 30.6 (22.2–39.0) 26.2 (25.1–27.4) 34.1 (32.1–36.2) 28 (26.7–29.2)

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temperature shis could betolerated quite well. Both factors, light
intensity and temperature, can change rapidly in terrestrial habitats
and over a wider range than in aquatic habitats. However, our
observations of slower growth rates during several days of increased
temperatures made it obvious that soil diatoms prefer relatively low to
moderate temperatures (~15–20°C) and are sensitive to high
temperatures (>30°C). In coastal sand dunes, the surface can reach
temperatures above 40°C (Maun, 2009). It appears that the diatoms
could tolerate such a shi for a short time but need a recovery period
with moderate temperatures during the night. Also, other studies have
reported that diatoms can tolerate a wide temperature range over the
short term (Soureau etal., 2010).
Terrestrial habitats, especially the sand dunes with their low
water-holding capacity, can dry out rapidly during the day. erefore,
terrestrial organisms must deal with periodic desiccation events. ree
cultures (H. amphioxys, H. abundans, and A. coarctata) reached 50%
of their initial photosynthetic performance aer rehydration. Only the
two Pinnularia species (P. bo re al i s and P. intermedia) failed to recover
well aer the desiccation event. is is somewhat surprising because
both Pinnularia species are true terrestrial species and can live outside
a moss carpet. It might bethat our experimental setup was harsh, with
a relative humidity below 25%, which does not naturally occur in
coastal dunes in Germany. Additionally, the P. intermedia cells were
smaller than average, which indicates that the culture did not reach its
full potential. In general, pennate diatoms, such as those in our ve
cultures, are mobile and can avoid unfavorable conditions or migrate
in a positive phototactic direction (e.g., moving vertically in
sediments) under natural conditions (Consalvey etal., 2004; Poulsen
etal., 2022). Such a protective vertical movement was not possible for
the diatoms during the desiccation experiment. A study on aquatic
and terrestrial diatoms also indicated a low desiccation tolerance for
diatoms in general when only air-dried (Soureau etal., 2010). e
exceptions in this study were P. b o re al is and H. amphioxys, which were
able to grow aer drying in air for 10 min.
4.3 Mycosporine-like amino acids from
diatoms
Mycosporine-like amino acids are frequently reported from
marine or brackish water and are mostly attributed to dinophytes and
bacillariophytes (Jerey etal., 1999). Interestingly, it was suggested
that MAAs might be stored in the silica frustules, which would
substantially enhance their stability and prevent harmful UV light
from penetrating the outer cell layer (Ingalls etal., 2010). Wedid not
test for the location of the MAAs, but this seems to bean interesting
strategy, which could potentially also beused by terrestrial diatoms.
Studies on marine diatom cultures have reported seven MAAs as the
most common: Shinorine, Porpyhra-334, Mycosporine-2-glycine,
Palythine, Palythene, Mycosporine-glycine, and Mycosporine-glycine-
valine (Helbling etal., 1996; Moisan and Mitchell, 2001). Of these
frequently present diatom MAAs, we most likely detected
Mycosporine-2-glycine, with an absorbance at 334 nm. is
assumption is supported by the retention time for Mycosporine-2-
glycine in a methanol/acid mobile phase in HPLC, which is a few
seconds longer than for Porphyra-334. In our study, the MAA from
the terrestrial diatoms had a slightly longer retention time in the same
phase than the Porphyra-334 standard.
Our study contributes the rst record of MAAs in terrestrial
diatoms. Terrestrial habitats are characterized by high UV radiation
compared to aquatic habitats, especially in summer under a clear sky.
Terrestrial diatoms are typically pennate with one or two raphes,
which allow the diatoms to move, for example, in marine sediments.
erefore, wehypothesize that terrestrial diatoms could move in
FIGURE9
High-performance liquid chromatography (HPLC) results for mycosporine-like amino acid (MAA) extraction from terrestrial diatom cultures; two of five
diatom strains contained MAAs. The insert shows the absorbance of the strongest peak (maximum absorbance at 333.5 nm).

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deeper sand layers to avoid harmful radiation. is would incur a
certain cost: although the diatoms escape the harmful UV radiation,
they are less exposed to photosynthetic active radiation. As all the
cultures that wetested showed no photoinhibition, protection from
harmful UV radiation seems to bea useful strategy to exploit the high
radiation on the sand surface for ecient photosynthesis without
being harmed by UV radiation.
5 Conclusion
In this study, weevaluated the ecophysiological characteristics of
terrestrial diatoms from sand dunes. Although terrestrial diatoms have
been known for a long time, only a few studies have investigated their
functioning despite their abundance and wide distribution. Our study
is one of only a few to provide an insight into the ecology of terrestrial
diatoms based on cultures, which is a necessary basis for
understanding their ecology and distribution. Weobserved a wide
temperature tolerance of all diatom cultures concerning primary
production, but long-term exposure to dierent temperatures resulted
in a lower temperature tolerance concerning growth rates. Desiccation
tolerance diered among the cultures, with some showing a high
recovery rate aer harsh desiccating conditions and others with only
low recovery rates. Although terrestrial diatoms appear to besensitive
to warmer and drier summer conditions, the biocrust microecosystem
including a moss cushion may reduce the environmental stress for
these diatoms.
Data availability statement
e original contributions presented in the study are publicly
available. is data can befound here: NCBI, accession numbers
OR387857-OR387860.
Author contributions
KG: Formal analysis, Investigation, Conceptualization, Data
curation, Funding acquisition, Project administration, Supervision,
Visualization, Writing – original dra. SK: Formal analysis,
Investigation, Writing – review & editing. NP: Data curation, Formal
analysis, Methodology, Visualization, Writing – review & editing. MD:
Data curation, Formal analysis, Methodology, Visualization, Writing –
review & editing.
Funding
e author(s) declare nancial support was received for the
research, authorship, and/or publication of this article. is research
was nancially supported by the Rudolf-und-Helene-Glaser-Stiung.
Grant number T0083/37274/2021/kg.
Acknowledgments
e authors would like to thank the national park oce
“Vorpommersche Boddenlandscha” for granting them permission
(Aktenzeichen 32-5303.3) to sample in a strictly protected area.
Sincere thanks to the Rudolf-and-Helene-Glaser Foundation, which
nancially supported this research.
Conflict of interest
e authors declare that the research was conducted in the
absence of any commercial or nancial relationships that could
beconstrued as a potential conict of interest.
Publisher’s note
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organizations, or those of the publisher, the editors and the
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that may be made by its manufacturer, is not guaranteed or endorsed
by the publisher.
Supplementary material
e Supplementary material for this article can befound online
at: https://www.frontiersin.org/articles/10.3389/fmicb.2023.1279151/
full#supplementary-material
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