Morpho-molecular characterization of rock-inhabiting lichen Dermatocarpon miniatum (Verrucariaceae, Ascomycota) and its symbiont in Indian Himalayas

Abstract

The genus Dermatocarpon (Verrucariaceae) is a rock-inhabiting lichen, mostly grows along the edges of lakes, rivers, streams, and watercourses. Dermatocarpon species are widely distributed from the tropics to the polar regions. In present study, D. miniatum samples were collected from the Indian Himalayas; the mycobiont and their photobionts are identified using morphological and molecular methods. The ITS rDNA markers was amplified for the DNA extracted from cultured photobiont isolates and mycobiont. The light and confocal laser scanning microscope were used for morphological evaluation of the photobionts. The nuclear ITS rDNA gene of the mycobionts and photobionts were sequenced to confirm identity. The phylogenetic trees of mycobionts and photobionts were constructed using the Maximum likelihood method that revealed an evolutionary affinity of lichen D. miniatum and photobiont Diplosphaera chodatii with similar taxa. The D. chodatii (Trebouxiophyceae) was associated with all samples of D. miniatum. This study concludes that Diplosphaera chodatii is the primary photobiont associated with D. miniatum. To the best of our knowledge this is the first study of diversity for the photobiont associated with D. miniatum from India.

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https://doi.org/10.1007/s13237-021-00349-0
ORIGINAL ARTICLE
Morpho‑molecular characterization ofrock‑inhabiting lichen
Dermatocarpon miniatum (Verrucariaceae, Ascomycota) andits
symbiont inIndian Himalayas
KhemChandSaini1 · FelixBast1 · SanjeevaNayaka2 · AjayKumarGautam1
Received: 27 September 2020 / Accepted: 23 February 2021
© Archana Sharma Foundation of Calcutta 2021
Abstract
The genus Dermatocarpon (Verrucariaceae) is a rock-inhabiting lichen, mostly grows along the edges of lakes, rivers,
streams, and watercourses. Dermatocarpon species are widely distributed from the tropics to the polar regions. In present
study, D. miniatum samples were collected from the Indian Himalayas; the mycobiont and their photobionts are identified
using morphological and molecular methods. The ITS rDNA markers was amplified for the DNA extracted from cultured
photobiont isolates and mycobiont. The light and confocal laser scanning microscope were used for morphological evaluation
of the photobionts. The nuclear ITS rDNA gene of the mycobionts and photobionts were sequenced to confirm identity. The
phylogenetic trees of mycobionts and photobionts were constructed using the Maximum likelihood method that revealed
an evolutionary affinity of lichen D. miniatum and photobiont Diplosphaera chodatii with similar taxa. The D. chodatii
(Trebouxiophyceae) was associated with all samples of D. miniatum. This study concludes that Diplosphaera chodatii is
the primary photobiont associated with D. miniatum. To the best of our knowledge this is the first study of diversity for the
photobiont associated with D. miniatum from India.
Keywords Diplosphaera· ITS rDNA· Mycobionts· Phylogeny· Symbionts
Introduction
Verrucariaceae is a large family of lichenized fungi com-
prising more than 50 genera [19, 25] and about 750 species
[16]. Within the Verrucariaceae, the genus Dermatocarpon
Eschw. forms a well-supported monophyletic clade [15, 16,
39]. It is a widely distributed genus found growing at dif-
ferent latitudes from tropics to the polar regions [16, 21]. In
index fungorum database (http:// www. inde x fungo rum. org/),
a total of 426 entries are available for this genus, which actu-
ally comprises 20 currently accepted species worldwide
[29]. From India, three species are reported which includes
D. meiophyllizum Vain., D. miniatum (L.) Mann, and D.
vellereum Zschacke [36, 40]. They are mainly distributed in
Himachal Pradesh, Uttarakhand, Jammu and Kashmir, Tamil
Nadu, Madhya Pradesh, Rajasthan and Maharashtra [36].
Among these, D. miniatum is the most common species,
which includes five varieties in India [40].
The genus Dermatocarpon is characterized by saxicolous,
foliose, umbilicate thallus, green algal photobiont, presence
of epinecral layer, perithecioid ascomata, colourless, sim-
ple ascospores [1, 36]. It is usually found growing on rocks
along the margins of waterfalls, streams, lakes, and rivers [9,
10]. Although it is considered obligately saxicolous, a few
species are also able to grow on soil [1]. Since it regularly
experiences water level fluctuations, it is also considered a
“subaquatic” lichen [2, 9]. Dermatocarpon species remained
poorly studied due to the lack of uniform, discrete charac-
ters, and morphological variability [1, 18]. The recent mor-
phological and molecular studies have addressed the various
fundamental questions about evolution, diversity, and phylo-
genetic position of the genera [1, 7, 9, 16, 20, 43].
Corresponding Editor: Amita Pal; Reviewers: TAM Jagdeesh
Ram, Krishna Pal Singh.
* Felix Bast
felix.bast@gmail.com
1 Department ofBotany, Central University ofPunjab,
Ghudda, Bathinda, Punjab151401, India
2 Lichenology Laboratory, CSIR-National Botanical Research
Institute, Rana Pratap Marg, Lucknow, UttarPradesh226001,
India

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The diversity of photobionts associated with lichenized
fungi of Verrucariaceae is believed to be greater among all
lichenized fungi [23]. According to Thüs etal. [43], Proto-
coccus, Stichococcus and Diplosphaera are the most com-
mon photobionts associated with Verrucariaceae while
additional taxa includes Auxenochlorella, Chlorella, Coc-
cobotrys, Dilabifilum, Myrmecia, and Trochiscia [33, 44,
45]. Whereas the primary photobiont associated with genus
Dermatocarpon belongs to genus Diplosphaera [9, 11, 43].
Stocker-Wôrgôtter and TüRk [41] identified Hyalococcus
dermatocarponis as a photobiont in D. miniatum. In many
lichen species, the co-existence of multiple photobionts in a
single thallus was observed [8, 30, 31].
The present study is aimed to reveal the diversity of pho-
tobionts associated with rock-inhabiting lichen Dermato-
carpon in Indian Himalayas using culture-dependent and
independent methods. Morphological and molecular studies
were performed to resolve the better taxonomic and phy-
logenetic position of Indian isolates and their symbionts.
DNA sequence data were obtained using the nuclear Inter-
nal Transcribed Spacer (ITS) rDNA gene regions for both
fungal and associated algal symbionts. As indicated through
the literature survey, there are no previous studies available
regarding the diversity of photobionts in Dermatocarpon
species in Indian Himalayas.
Materials andmethods
Taxon sampling andphenotypic characterization
The Dermatocarpon samples were collected by scraping
the thallus from the rock surfaces from various localities
of Himachal Pradesh and Jammu & Kashmir, India. Five
distinct populations of Dermatocarpon are included for
analyses in the present study (Table1). The samples were
dried and stored at 4°C until processing. All vouchers are
deposited in the Herbarium LWG of CSIR-National Botani-
cal Research Institute, Lucknow, India, and duplicates
were deposited in the Herbarium of Department of Botany,
Central University of Punjab, Bathinda, India. The overall
methodology used in this study is diagrammatised in Sup-
plementary Fig.1.
Morphological and anatomical features of lichen thalli
were observed using stereo zoom Leica S8APO and light
DM500 microscopes attached with a camera following the
methods explained by Nayaka [32]. Thin sections of thallus
were mounted in distilled water and observed under the light
microscope. The spot test was carried out using aqueous
solution of 10% potassium hydroxide (K), calcium hypochlo-
rite (C) and alcoholic solution of p-phenylendiamine (P).
Thin layer chromatography (TLC) was performed to identify
the secondary metabolites, if any. A small part of the lichen
thallus is used for extracting lichen compounds in acetone.
The solvent system C (toluene:acetic acid 170:30ml) was
used to run the TLC pate; 10% sulphuric acid was sprayed
on to the plate and then charred in hot air oven pre-heated to
110° C and secondary metabolite spots are identified follow-
ing Orange etal. [34]. The specimens were identified with
the help of macrolichen key of Awasthi [3].
Photobiont isolation, culturing andmicroscopy
Lichen thalli were examined under a stereomicroscope and
washed with distilled water to remove surface contamina-
tions, epiphytes, or infections. The photobionts were isolated
from the lichen thalli (HP-28 and HP-56) by following the
micro method of Gasulla etal. [14]. The isolated photo-
bionts were streaked on sterile 1.2% agar with 3N-BBM
media [5]. The Petri plates were kept on the culture shelves
at an irradiance of 40µmol m−2 s−1 with 12:12h light: dark
cycle at 20–25°C. After 40–45days, algal colonies grown
Table 1 Collection information of different specimens of Dermatocarpon miniatum var. miniatum (L.) Mann
a Lichen specimens used for culture experiments
b H.P. = Himachal Pradesh
c J&K = Jammu and Kashmir
Sample Id CUP voucher no LWG herbaria no Collection date Coordinates Collection site
HP-28aCAL-CUPVOUCHER-HP-
28L-2018-1
LWG 38451 19-06-2018 32° 02′ 48.68″ N; 77° 10′
38.19″ E
Jana water fall, Manali, H.P.b
HP-56aCAL-CUPVOUCHER-HP-
56L-2018-1
LWG 39412 27-10-2018 32° 31′ 45.92″ N; 76° 2′ 4.36″
E
Lakkar Mandi, Dalhousie, H.P
JK-13 CAL-CUPVOUCHER-JK-
13L-2018-1
LWG 38452 10-07-2018 33° 33′ 29.3″ N; 75° 24′ 45.6″
E
Cheerward, Anantnag, J&Kc
JK-14 CAL-CUPVOUCHER- JK-
14L-2018-1
LWG 38453 09-07-2018 33° 42′34.4″N; 75° 26′ 27.3″E Gawran, Anantnag, J&K
JK-15 CAL-CUPVOUCHER- JK-
15L-2018-1
LWG 38454 10-07-2018 33° 36′35.83″ N; 75° 26′
18.190″ E
Dksum, Anantnag, J&K

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on Petri plates were observed and subcultured in axenic con-
ditions. The algal biomass was harvested by scraping the
cultures. All the isolated photobionts were maintained on
the 3N-BBM agar media at 4°C for future work.
The morphology of the photobiont was investigated by
both light microscope (LM) and confocal laser scanning
microscope (CLSM). The color, shape, and size of pho-
tobionts were observed under the light microscope (Leica
DM500), while CLSM (Olympus FV1200, Tokyo) was used
for observation of the internal structures such as chloroplast
of the photobionts. Alexa Fluor 647, with 665 emission
wavelengths, was used for chloroplast autofluorescence visu-
alization. The fluorescence images were captured using the
60X and 100X oil objectives. Olympus FluoView Ver.4.2a
viewer was used for the final processing of the images.
Molecular analysis
Total genomic DNA from lichen thalli and cultivated pho-
tobionts were separately extracted using HiPurA™ Algal
Genomic DNA Extraction Kit (HiMedia Laboratories Pvt.
Ltd., Mumbai) according to the manufacturer’s protocol.
Polymerase chain reaction (PCR) amplifications were per-
formed using specific primers that amplify the internal tran-
scribed spacer (ITS-5.8S) regions. For fungus, fungal-spe-
cific primers ITS1F [12], ITS4 [47], ITS4A [28] were used,
and for photobionts algal-specific primers ITS1T, ITS2T,
and ITS4T [26] (Fig.1 and Table2) were used. The 20μl
PCR reactions containing 10μl One Taq® Hot Start Quick-
Load® 2X Master Mix (New England BioLabs (NEB),
Ipswich, MA, USA), 2μl of each primer, 2μl of template
DNA, and the rest sterile water. After an initial denaturation
at 95°C for 5min, 35 cycles of amplification consisting of
95°C for 1min, 52–54°C for 1min, and 72°C for 1.3min;
final extension at 72°C for 7min, were performed in a
programmable thermal cycler (Veriti, ABI, USA). Ampli-
cons were evaluated by agarose gel electrophoresis (1.2%),
visualized under Bio-Rad Gel Doc (Bio-Rad Laboratories,
California, USA), and purified using the ExoSAP-IT® PCR
clean-up kit (USB Corporation, Cleveland, OH, USA).
The purified amplicons were sequenced using dideoxy
chain termination protocol with ABI BigDye Terminator
Cycle Sequencing Ready® Reaction Kit v3.1 (Applied
Biosystems, Foster City, CA, USA) following the manufac-
turer’s instructions. Precipitation and clean-up of the cycle
sequenced product to remove excess fluorescent dyes was
carried out by the ethanol/EDTA precipitation method [22].
The dried samples were suspended in 15 μL of Hi-Di™
Formamide (Applied Biosystems, Foster City, CA, USA)
and loaded to a 96-well plate for sequencing (Applied Bio-
systems 3730xl Genetic Analyzer, Foster City, CA, USA).
Sequence analysis and contig assembly were performed
using licensed software Geneious® prime v2020.0.4 (Bio-
matters Limited, New Zealand available at https:// www.
genei ous. com). For the sequence homology, the BLASTn
search (Basic Locus Search Alignment Tool, www. blast.
ncbi. nlm. nih. gov) was used. The newly generated consensus
sequences were deposited into the NCBI (National Center
for Biotechnology Information) GenBank database.
Phylogenetic analysis
The newly generated ITS rDNA sequences were aligned
with related taxa retrieved from the NCBI GenBank data-
base (https:// www. ncbi. nlm. nih. gov/ nucle otide/) (Supple-
mentary Tables1 and 2) using the MUSCLE algorithm
[6] in Geneious® prime v2020.0.4. The alignments of ITS
rDNA sequences were improved by trimming of ends and
refined by the eyes. The most appropriate substitution model
was estimated by the Bayesian information criterion (BIC)
using the ML Model test in MEGA X (www. megas oftwa
re. net/) [27]. The phylogenetic trees were inferred by the
Maximum Likelihood (ML) method using PhyML 3.3 [17]
in Geneious® prime for both ITS rDNA datasets of mycobi-
onts and photobionts.
Mycobionts ITS rDNA dataset consists of 26 sequences
inferred by the ML method, and substitution bias was
modelled by the Tamura-Nei model [42] with Gamma
Fig. 1 Diagrammatic representation of the relative positions of nrITS
regions; arrows indicate the positions of the primers used in this
study for amplification and sequencing
Table 2 Primers used for amplification and sequencing in the present
study
Primer Primer sequence (5′–3′) References
Forward primers
ITS1-F CTT GGT CAT TTA GAG GAA GTAA [12]
ITS1-T GGA AGG ATC ATT GAA TCT
ATC GT
[26]
Reverse primers
ITS4-A CGC CGT TAC TGG GGC AAT
CCCTG
[28]
ITS4 TCC TCC GCT TAT TGA TAT GC [47]
ITS2-T TTC GCT GCG TTC TTC ATC GTT [26]
ITS4-T GGT TCG CTC GCC GCT ACT A [26]

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distribution. Tamura-Nei model was the best model in our
ML model test to find the best fitting substitution models
with a BIC score of 4915.737. While the photobionts ITS
rDNA dataset consists of 22 sequences inferred by the
ML method, and substitution bias was modelled by the
Kimura‐2‐parameter model [24] with Gamma distribution.
The Kimura-2 model was the best model in our ML model
test to find the best fitting substitution models with a BIC
score of 4963.474. We performed 1000 bootstrap replicates
under the ML criterion to estimate interior branch support
for both the datasets. The phylogeny of the mycobionts was
out group rooted with Endocarpon pulvinatum (KY697127)
and Verrucaria muralis (KY697147); the algal phylogeny
was out group rooted with Trebouxia jamesii (MN397116
and MN397121) [4].
Results
Morphological observations
Morphological investigation ofthelichen thallus
Morphological investigation revealed that the specimens
belong toDermatocarpon miniatum var. miniatum (L.)
Mann. It is characterized by saxicolous and foliose thallus
(Figs.2, 3). The thallus is monophyllous or polyphyllous,
umbilicate, up to 5cm across, the upper surface of the thal-
lus is grey, lower surface light brown to brown, lacunose,
or wrinkled and lacking rhizines. The lower cortex of the
thallus is Dermatocarpon-type (sensu Harada [18]). Peri-
thecia numerous, immersed, ostiole brown to dark brown;
ascospores hyaline, simple, sometimes with one pseudo
septa, broadly ellipsoidal,10.2–13.5 × 6.1–7.9µm.
Chemistry No chemical substances were detected in the
thallus of studied specimens. The spot test results are K‐, C‐,
KC‐ and P− and no secondary metabolites were observed
in TLC.
Fig. 2 Habitat and thallus of the lichens. a Habitat of HP-28 specimen; b upper surface of the thallus (HP-28); c habitat of HP-56 specimen; d
upper surface of the thallus (HP-56). Scale bars represents (b, d)

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Morphological investigation ofthephotobionts
The photobiont Diplosphaera chodatii is characterized
by unicellular, spherical, and rarely oval celled, solitary
or group of 2–8 cells (Figs.4, 5). The cells were usually
3.5–7.5 × 2–6.5μm in size (Fig.5a,c), chloroplasts are pari-
etal or plate-like, without a pyrenoid (Fig.5b,d).
Phylogenetic analyses
Mycobiont phylogeny
The NCBI BLAST searches of ITS rDNA consensus
sequences of mycobionts generated in the present study
showed the best match (based on percentage identity) > 94%
with Dermatocarpon miniatum (Supplementary Table3).
Maximum-likelihood (ML) analysis of the ITS rDNA
dataset showed Dermatocarpon species used in this study
were clustered together with sequences retrieved from
GenBank (Fig.6). The ITS rDNA phylogenetic analysis of
mycobiont represents 26 sequences, five sequences from this
study, and 21 GenBank accessions, two of which were used
to out-group rooted following the recommendations of Ver-
bruggen and Theriot [46]. The phylogenetic analysis resulted
in well-resolved phylograms, with three clades.
Clade 1 is supported by 91% bootstrap, comprised of D.
miniatum samples, and form a monophyletic clade. Indian
samples HP-28 (MT873515), JK-13 (MT873517), and JK-15
(MT873519) grouped together with 100% bootstrap. Inter-
estingly, these isolates are positioned with D. miniatum
(AF333161 and AF333162) specimens of Indian Himala-
yas. Similarly, two Indian isolates HP-56 (MT873516), and
JK-14 (MT873518), grouped between the GenBank acces-
sions MF680078 and MF521951 from the Himalayas of
Pakistan and AF333165 from Indian Himalayas. Clade 2 is
supported by 94% bootstrap comprises of D. luridum species
of Austria, Canada, Sweden, and USA. Clade 3 represent D.
meiophyllizum (AF333171 and AF333172) with 99% boot-
strap support. The result from the ML phylogenetic analysis
showed that Indian isolates identified as D. miniatum in this
study were related to other ITS rDNA GenBank accessions
of D. miniatum with some degree of intraspecific distance.
Fig. 3 Thallus structure of the lichen specimens. a Upper surface of the thallus (JK-13); b upper surface of the thallus (JK-14); c upper surface
of the thallus (JK-15)
Fig. 4 Light photomicrographs of photobiont Diplosphaera chodatii isolated from D. miniatum. a Isolate from sample HP-28, and b isolate
HP-56. Scale bars 10µm

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Photobiont phylogeny
The NCBI BLAST searches of ITS rDNA consensus
sequences of photobiont generated in the present study
showed the best match (based on percentage identity) > 94%
with Diplosphaera species (Supplementary Table3).
Maximum-likelihood (ML) analysis of the ITS rDNA
dataset of photobionts showed Diplosphaera chodatii
species used in this study were clustered with sequences
retrieved from GenBank (Fig.7). The ITS rDNA phyloge-
netic analysis of photobiont represents 22 sequences, five
sequences from this study, and 16 GenBank accessions, two
of which were used to out-group rooted following the rec-
ommendations of Verbruggen and Theriot [46]. The phylo-
genetic analysis resulted in well-resolved phylograms, with
two clades.
Clade 1 is strongly supported by 100% bootstrap, com-
prised of Diplosphaera chodatii samples, and form a
monophyletic clade. Indian isolates HP-28 (MT872222),
HP-56 (MT872312), and JK-13 (MT872351) grouped with
79% bootstrap. These isolates were grouped between the
GenBank accessions of Austria (KF317619) and Switzer-
land (MT078182). Similarly, two Indian isolates JK-14
(MT873598) and JK-15 (MT872371), were grouped with
the GenBank accessions of Canada and Austria. Clade 2 is
supported by 99% bootstrap comprises of Trebouxia species.
Fig. 5 Confocal photomicro-
graphs of photobiont Diplo-
sphaera chodatii isolated
from D. miniatum (HP-28 and
HP-56). a, c Normal cells; b, d
autofluorescence signals of the
chloroplast. Arrow indicates the
cell wall (CW) and chloroplast
(CP). Scale bars 10µm

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Fig. 6 Molecular phylogeny of the mycobiont D. miniatum. Values at
branches refer to bootstrap values—branches with high statistical sup-
port (100%) indicated by bold lines. Newly obtained sequences in this
study are in bold. Scale bar represents the probable number of substi-
tutions per site

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Discussion
According to Gärtner and Ingolic [13], the accurate iden-
tification of photobiont at species level is possible using
photobiont cultures. However, we identified Diplosphaera
chodatii using both culture-dependent and culture-independ-
ent approaches. In photobiont phylogeny, our sequences
were clustered with sequences of Diplosphaera chodatii
SAG 11.88 (MT078177) and SAG 49.86 (MT078182) and
those generated by Fontaine etal. [9] from Dermatocar-
pon luridum. The placement of our sequences with standard
culture collection sequences strongly supports the accurate
identification and placement of our isolates in the photo-
biont phylogeny. However, previous literature has shown
various photobionts associated with Dermatocarpon spe-
cies are Diplosphaera sp. Protococcus, Stichococcus [9, 43];
Hyalococcus [37, 41]; Myrmecia [37]; and Pleurococcus
[3, 35, 38].
The mycobiont phylogeny constructed from the ITS
rDNA sequences data reveals the monophyletic nature of the
D. miniatum. Our sequences were clustered with sequences
of D. miniatum from the Himalayas of Pakistan and those
generated by Heidmarsson [20] from Indian Himalayas. The
geographical affinities are interesting as the Asian speci-
mens, including five specimens used in the present study
group together, while the USA and Canadian specimens
group with the North American ones. Moreover, all speci-
mens generated in the present study were best hit with D.
miniatum sequences.
In conclusion, to the best of our knowledge this is the
first study of diversity for the photobiont associated with
D. miniatum from Indian Himalayas. The present and pre-
vious studies validates Diplosphaera chodatii is a primary
photobiont associated with Dermatocarpon species. Fur-
thermore, extensive studies on Dermatocarpon species
may explore the multiple photobionts associated with this
genus from Indian Himalayas.
Electronic supplementary material The online version of this article
(https:// doi. org/ 10. 1007/ s13237- 021- 00349-0) contains supplementary
material, which is available to authorized users.
Acknowledgements This study was supported by grant-in-aid from
CSIR-Sponsored Research Scheme (60(0114)/17/EMR-II) awarded
to FB.We are grateful to Dr. Kiran Toppo, Senior Technical Officer,
Fig. 7 Molecular phylogeny
of the photobiont Diplo-
sphaera chodatii. Numbers at
branches indicate bootstrap
values. Significantly supported
(bootstrap ≥ 99%) internodes are
indicated by bold lines. Newly
obtained sequences in this study
are in bold. Scale bar represents
the probable number of substi-
tutions per site

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Algology Laboratory, CSIR-National Botanical Research Institute,
Lucknow, for providing us light microscopic images of photobionts.
Figures were created withBioRender.com.
Author contributions FB conceived the idea of the manuscript. KCS
collected the samples, performed experiments, analyzed the data,
and drafted the manuscript. AKG helped in culturing and sequenc-
ing experiments. SN helped in the morphological analysis of lichen
samples and reviewed the manuscript. FB proof checked and finalized
the manuscript.
Funding This work was supported by the Council of Scientific and
Industrial Research (CSIR), New Delhi (No. 60(0114)/17/EMR-II).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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