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Deadly mushrooms of the genus Galerina found in Antarctica
colonized the continent as early as the Pleistocene
ISAAC GARRIDO-BENAVENT 1, ROBERT A. BLANCHETTE 2and ASUNCIÓN DE LOS RÍOS 3
1
Departament de Botànica i Geologia, Facultat de Ciències Biològiques, Universitat de València, C/Doctor Moliner 50, E-46100 Burjassot,
Valencia, Spain
2
Department of Plant Pathology, 1991 Upper Buford Circle, 495 Borlaug Hall, University of Minnesota, St. Paul, MN 55108-6030, USA
3
Department of Biogeochemistry and Microbial Ecology, National Museum of Natural Sciences (MNCN), CSIC, E-28006 Madrid, Spain
Isaac.Garrido@uv.es
Abstract: Fungi are probably the most diverse group of eukaryotic organisms in the Antarctic continent
and nearby archipelagos, and they dominate communities in either mild or harsh habitats. However, our
knowledge of their global distribution ranges and the temporal origins of their Antarctic populations is
rather limited or almost absent, especially for species that do not lichenize. We focused for the first time on
elucidating the taxonomic identity and phylogenetic relationships of several Antarctic collections of the
deadly fungal Basidiomycota genus Galerina. By using molecular sequence data from the universal
fungal barcode and a dataset encompassing 178 specimens, the inferred phylogeny showed that
the Antarctic specimens corresponded with the sub-cosmopolitan species Galerina marginata,
Galerina badipes and Galerina fallax, and their most closely related intraspecific genetic lineages were
from northern Europe and North America. We found that these species probably host Antarctic-
endemic intraspecific lineages. Furthermore, our dating analyses indicated that their Antarctic
populations originated in the Pleistocene, a temporal frame that agrees with that proposed for the
Antarctic colonization of plants such as the grass Deschampsia antarctica, mosses and some
amphitropical lichens. Altogether, these findings converge on the same temporal scenario for the
assembly of the most conspicuous terrestrial Antarctic plant and fungal communities.
Received 8 November 2022, accepted 29 April 2023
Key words: biogeography, dating analysis, fungal endemism, Galerina marginata, long-distance dispersal,
non-lichenized fungi
Introduction
Fungi are probably the most widespread and diverse group
of eukaryotic organisms inhabiting Antarctica, with a
known fossil record dating back to the Permian period
(White Jr & Taylor 1991, Harper et al. 2016). They are
involved in key processes in terrestrial ecosystems, such
as decomposition and symbiotic mutualism (Treseder &
Lennon 2015, Asplund & Wardle 2017), and therefore
they contribute greatly to biogeochemical cycles in
otherwise low-nutrient habitats. The number of known
species in territories south of 60°S and archipelagos at
lower latitudes, such as South Georgia, is ∼1500
(Øvstedal & Lewis Smith 2001,2011, Onofri et al. 2005,
Bridge et al. 2008, Bridge & Spooner 2012). Almost a
third of them associate symbiotically with eukaryotic
algae (chlorophytes) or cyanobacteria, forming
macroscopic lichen thalli. Lichens are in fact one of the
most conspicuous elements of Antarctic terrestrial
habitats, and their communities develop profusely in
maritime areas (Søchting et al. 2004, Peat et al. 2007),
and even in rocky outcrops at harsher locations in the
continent (Kappen et al. 1981, Broady & Weinstein
1998, Pérez-Ortega et al. 2012). On the other hand,
non-lichenized fungi generally remain unnoticeable
because either they are unicellular (e.g. yeasts) or they
form unseen mycelia and small reproductive structures.
Microfungi are the most abundant in Antarctic soils
(Vishniac & Hempfling 1979, Ruisi et al. 2007, Arenz &
Blanchette 2011, Arenz et al. 2014), and many
Ascomycota and even some Basidiomycota have been
reported on wood brought to Antarctica (Arenz &
Blanchette 2009, Blanchette et al. 2010, Arenz et al.
2011, Held & Blanchette 2017). Additional research
using next-generation sequencing techniques has also
revealed a high diversity of fungi even in the most
unexpected habitats (Coleine et al. 2018,
Garrido-Benavent et al. 2020, Rosa et al. 2021), with
similarities in species diversity and community
composition to the Arctic (Cox et al. 2016).
Antarctic Science page 1 of 14 (2023) © The Author(s), 2023. Published by Cambridge
University Press on behalf of Antarctic Science Ltd. This is an Open Access article,
distributed under the terms of the Creative Commons Attribution licence (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution
and reproduction, provided the original article is properly cited. doi:10.1017/S0954102023000196
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https://doi.org/10.1017/S0954102023000196 Published online by Cambridge University Press
Macrofungi (i.e. non-lichenized species that form
relatively large fruiting bodies or 'mushrooms') are
infrequently reported in Antarctica, with a few dozen
species growing in the climatically milder sub-Antarctic,
Maritime Antarctica and occasionally in the western
Antarctic Peninsula, often occurring on large carpets of
mosses and vascular plants (Pegler et al. 1980,
Gumin
ska et al. 1994, Bridge et al. 2008, Putzke et al.
2012, Held & Blanchette 2017, Newsham et al. 2021).
Bridge & Spooner (2012) and Newsham et al. (2021)
suggested that the general absence of large land animals
and higher plants with woody components in Antarctica
is a limiting factor for the development of these fungi.
From a taxonomic and biogeographical viewpoint, the
scarcity of available collections of mushroom-forming
fungi from the Antarctic has so far impeded detailed
comparisons with species that are known from elsewhere.
More specifically, the lack of genetic data has prevented
the deciphering of the most probable temporal and
spatial origins of the Antarctic populations of certain
species. In fact, despite being one of the most diverse
groups in the whole continent, there is still a general lack
of knowledge as to whether non-lichenized fungi, and
particularly the Antarctic macrofungi, form specific
populations of cosmopolitan species or constitute true
endemic species (Bridge & Spooner 2012, Arenz et al.
2014).
To provide an answer to this question, the present work
uses recent collections of fruiting bodies of Galerina
species to assess their taxonomic identity and estimate a
date for their Antarctic origin within a phylogenetic
framework. This genus of basidiomycetous fungi
encompasses ∼300 species worldwide (Horak 1994,
Gulden et al. 2005), which form relatively small,
yellowish to reddish-brown fruiting bodies with
campanulate, convex to flat pilei and slender stipes.
Several Galerina species are well known for posing a
poisoning risk due to the production of deadly
amatoxins (Landry et al. 2021). The genus shows a
broad distribution in Mediterranean, temperate and
boreal regions in the Northern Hemisphere (GBIF
2022), where saprotrophic species generally grow on
dead parts of bryophytes in peat bogs or are associated
with woody remnants or other plant debris in forests, on
which this genus degrades wood cell wall components
(Gulden et al. 2005, Grzesiak & Wolski 2015, Kohler
et al. 2015). In the Antarctic continent and nearby
archipelagos, the number of Galerina species reported is
∼11, with Galerina antarctica Singer, Galerina glebarum
(Berk.) Singer and Galerina perrara Singer originally
being described, and these are known only from these
regions (Fig. 1; Berkeley 1847, Singer & Corte 1962,
Pegler et al. 1980, Bridge et al. 2008). Based on the
inferred phylogeny, we aim to ascertain whether the
sequenced Antarctic specimens belong to geographically
restricted, species-level lineages (i.e. putative endemic
species) or conform to particular intraspecific lineages of
cosmopolitan Galerina (non-endemic species). In
lichenized fungi, Antarctic endemic species have been
shown to have a relictual, pre-Pleistocene origin, whereas
Antarctic populations of amphitropical lichens are
generally much younger, dating back from the
Pleistocene onwards (Fernández-Mendoza & Printzen
2013, Garrido-Benavent et al. 2016,2018,2021). The
temporal frame estimated with the time-calibrated
Galerina phylogeny will further help us to discern
whether their evolution in Antarctica conforms to either
of these two scenarios.
Material and methods
Fieldwork and morpho-anatomical study of fruiting bodies
Several fruiting bodies of Galerina growing in a localized
area, and therefore probably corresponding to a single
mycelium, were collected in March 2018 from
Livingston Island (South Shetland Islands) and more
specifically in Punta Hannah (62°39'15.37" S,
60°36'27.44" W, 377 m above sea level), which is the
second largest island in the South Shetland Islands, a
mountainous archipelago located in Maritime
Antarctica. These fruiting bodies grew abundantly on
soil, with a profuse development of cryptogams (mosses
and the chlorophyte macroalgae Prasiola) and the
Antarctic hair grass Deschampsia antarctica Desv.
(Fig. 2). Sampling permit no. CPE-2017-3 was obtained
through the Spanish Polar Committee. Specimens were
frozen until further processing at the laboratory, where
they were observed under a Leica S8APO dissecting
microscope equipped with a Leica EC3 image capture
system. Handmade sections of lamellae were rehydrated
in distilled H
2
O to describe anatomical characteristics.
Microscopic observations were made using a Zeiss
Axioplan 2 microscope fitted with 'Nomarski'
differential interference contrast, and photographs were
taken with a Zeiss AxioCam digital camera.
Microscopic measurements were made by means of the
Zeiss Axiovision 4.8 imaging system. Reported data are
averages followed by standard deviations, and the
maximum and minimum values are given in parentheses.
DNA extraction and polymerase chain reaction
amplification
The isolation of genomic DNA from a single Galerina
basidioma (pl. basidiomata; i.e. basidiomycete fruiting
bodies) was done from a piece of lamellae and using the
Speed Tools DNA Extraction Kit (Biotools, Madrid,
Spain), following the manufacturer's recommendations.
The extracted DNA was eluted in a final volume of 60 μl
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with sterile purified water (SIGMA). Sequence data of the
internal transcribed spacer of the nuclear ribosomal DNA
(the so-called fungal barcode marker; Schoch et al. 2012)
was amplified using the primer pair ITS1F-KYO2 and
ITS4-KYO2 (Toju et al. 2012). Polymerase chain
reaction (PCR) experiments were performed in a total
volume of 10 μl, containing 1 μl of reaction buffer
(Biotools
®
), 2 μl of dNTPs (1 mM), 0.5 μlofeach
primer (10 μM), 0.2 U of DNA polymerase (Biotools
®
)
and 1.5 μl of the genomic DNA elution; the final
volume was reached by adding distilled water (SIGMA).
The following PCR temperature profile was employed:
5 min at 95°C, then 30 cycles of 30 s at 95°C, 1 min at
52°C and 1.5 min at 72°C, with a final extension of
10 min at 72°C. The PCR experiments were visualized
on 1% agarose gel stained with PRONASAFE nucleic
acid stain solution (CONDA Laboratories). The PCR
products were purified and cleaned using the UltraClean
PCR Clean-Up Kit (MOBIO Laboratories, Inc.). Both
complementary DNA strands were sequenced at
Macrogen Europe (Spain) using the same primer set as
for the initial amplification. Electropherograms were
checked and assembled using SeqManII v.5.07
©
(DNASTAR, Inc.).
Fig. 1. Diversity and distribution of Galerina species in Antarctica based on collection data provided by Bridge et al. (2008), Arenz et al.
(2014), Krishnan et al. (2016) and Canini et al. (2020). Species for which samples have been included in the present work are in bold.
3PLEISTOCENE ANTARCTIC COLONIZATION OF THE MUSHROOM GENUS GALERINA
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Compilation of the specimen-based nrITS dataset and
sequence alignment
The newly produced sequence was submitted to the
BLAST online tool (Altschul et al. 1990) to check for
possible PCR product contamination and to identify
and retrieve available, highly similar nrITS sequences. To
this purpose, the GenBank (http://www.ncbi.nlm.nih.gov/),
UNITE (Nilsson et al. 2019) and BOLD (Ratnasingham
& Hebert 2007) nucleotide databases were used as
references. A total of 118 sequences (97 GenBank,
13 UNITE and 8 BOLD) spanning a 97–100%
similarity range were downloaded. Most were accessions
labelled with the species name Galerina marginata
(Batsch) Kühner. A closely related nrITS sequence of a
Galerina collection from Amsler Island (Antarctic
Peninsula) was included as well. This was also collected
from an area where mosses were growing under permit
ACA-2012-013. DNA extraction and sequencing were
done using methods previously described (Blanchette
et al. 2016). An additional search in public databases
was conducted to select and retrieve any available
nrITS data for other Antarctic Galerina collections. Two
sequences obtained from soil isolates were found:
MK537266, which was generated in a study by Canini
et al. (2020) from Victoria Land; and MF692967, from
King George Island (Krishnan et al. 2018). BLAST
searches against the GenBank database revealed a close
Fig. 2. Galerina marginata:a. fruiting bodies growing on a carpet of Deschampsia antarctica and Prasiola sp. in Punta Hannah
(Livingston Island), b. a detail of the fruiting body pileus, c. a basidium (i.e. basidiomycete sporangium) with developing spores and
d. spores. Scale bars: 10 μm.
4ISAAC GARRIDO‐BENAVENT et al.
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match of these accessions to nrITS sequences labelled
with the species names Galerina badipes (Pers.) Kühner
and Galerina fallax A.H. Sm. & Singer. Twenty-five
sequences hosted in GenBank belonging to these two
species were downloaded and incorporated into the
dataset as well. Finally, the dataset was completed by
including additional Galerina species based on works
by Gulden et al. (2005)andLathaet al. (2015), which
provided two of the most comprehensive Galerina
phylogenies published to date. The final nrITS
consisted of 178 sequences. We followed Gulden et al.
(2005) in selecting adequate outgroup taxa for our
phylogenetic analyses. Although the genus Galerina
was revealed to be polyphyletic by these authors,
they considered a group of taxa referred to 'tubariopsis'
to be a suitable outgroup. In our dataset, this is
represented by the following species: Galerina arctica
(Singer) Nezdojm., Galerina clavata (Velen.) Kühner,
Galerina discreta E. Horak, Senn-Irlet, M. Curti
& Musumeci, Galerina laevis Singer, Galerina
pseudocerina A.H. Sm. & Singer and Galerina stordalii
A.H. Sm.
The program MAFFT v.7.308 (Katoh & Standley
2013) was used to generate a multiple-sequence
alignment with the following parameters: the FFT-NS-I
x1000 algorithm, the 200PAM/k= 2 scoring matrix, a
gap open penalty of 1.5 and an offset value of 0.123. The
resulting alignment was manually optimized in Geneious
v.9.0.2 to 1) trim alignment ends of longer sequences that
included part of the 18S–28S ribosomal subunits, 2) replace
gaps at the ends of shorter sequences with an International
Union of Pure and Applied Chemistry (IUPAC) base
representing any base ('N') and 3) replace doubtful base
calls at the extremes with 'N'. The software GBlocks 0.91b
(Castresana 2000) was subsequently used to automatically
deal with ambiguously aligned regions, implementing the
least stringent parameters but allowing gaps in 50% of the
sequences. Alignments were deposited in FigShare
(DOI: 10.6084/m9.figshare.22219546).
Maximum-likelihood phylogenetic analyses
The online version of RAxML-HPC2 hosted at the
CIPRES Science Gateway (Stamatakis 2006, Stamatakis
et al. 2008, Miller et al. 2010) was used to estimate two
maximum-likelihood (ML) phylogenies based on the
GBlocks-trimmed (GB) and untrimmed (ORG) alignments.
This approach would allow us to evaluate the effect of
alignment uncertainty on the inferred nodal support. The
analyses used the GTRGAMMA nucleotide substitution
model for the two delimited partitions within the nrITS
(ITS1+2, 5.8S), and nodal support was evaluated by
conducting 1000 rapid bootstrap pseudoreplicates.
The resulting phylogenetic trees were visualized in FigTree
v.1. 4 ( http://tree.bio.ed.ac.uk/software/tracer/), and
Adobe Illustrator CS5 was used for artwork. Tree nodes
with bootstrap support (BS) values ≥70% were regarded
as significantly supported.
Haplotype networks, DNA polymorphism and neutrality
tests
The genealogical relationships among specimens included
in the G. marginata clade were calculated under a
statistical parsimony framework in PopA RT v.1.7 (Leigh
&Bryant2015) using the method of Templeton et al.
(1992). To this purpose, a sub-alignment of 120 sequences
was extracted from the GBlocks-untrimmed, original
alignment. Because the inference of haplotype networks is
sensitive to ambiguous base calls and missing data (Joly
et al. 2007), the sub-alignment was edited to remove 16
sequences with a high proportion of missing data at their
extremes and 34 sequences with ambiguous base calls
occurring at polymorphic positions. Haplotypes were
subsequently inferred with DnaSP v.5.10 (Librado &
Roza s 2009) considering sites with alignment gaps and
removing invariable sites. The network was artistically
edited in Adobe Illustrator CS5 and haplotypes were
labelled according to their geographical origin. DNA
polymorphism in the 70 remaining sequences was
evaluated with the software DnaSP v.5.10 (Librado &
Roza s 2009). The computed indices were the number of
segregating sites (s), the number of haplotypes (h),
haplotype diversity (Hd) calculated without considering
gap positions and the nucleotide diversity (π) using the
Jukes & Cantor (1969) correction. Deviations from
neutrality with Tajima's D and Fu's Fs statistics were also
assessed to infer past population size changes. The tests
were carried out in DnaSP v.5.10 using the nu mb e r o f
segregating sites, and their significance was assessed based
on 10
4
coalescent simulations. We did not infer haplotype
networks, nor do we evaluate DNA polymorphism for G.
badipes and G. fallax because of the few sequences these
species encompassed and due to the substantial amount
of missing data in sequences masking the existing
polymorphism.
Dating analyses
The inference of a time frame for the global evolutionary
history of Galerina was conducted under a Bayesian
framework with BEAST 1.8.1 (Drummond et al. 2012).
Because this Basidiomycota genus lacks a suitable fossil
record, the dating analysis used a secondary calibration
imposed on the nrITS substitution rate. Hence, the
BEAST analysis implemented the average rate of
4.61 × 10
-3
substitutions per site per million years (s/s/Ma)
inferred for the genus Phaeocollybia R. Heim in Ryberg
&Matheny(2012), because this genus and Galerina
belong into the family Hymenogastraceae (Matheny et al.
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PLEISTOCENE ANTARCTIC COLONIZATION OF THE MUSHROOM GENUS GALERINA
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2015). This analysis was referred to as Dating A. To take
into account the uncertainty associated with that rate, we
re-ran analyses using the estimates representing the
minimum (2.92 × 10
-3
s/s/Ma, Dating B) and maximum
(6.45 × 10
-3
s/s/Ma, Dating C) values of the rate's
95% credibility interval provided by Ryberg & Matheny
(2012). The analyses were conducted with the two
alignment versions (GB and ORG) to learn about the
impact on age estimates of keeping ambiguously aligned
positions in the alignment. Redundant sequences were
removed from the alignments using the FaBox v.1.41
online toolbox (Villesen 2007). PartitionFinder 1.1.1
(Lanfear et al. 2012) was used to infer optimal
substitution models for the two nrITS partitions
considering a model with linked branch lengths and the
Bayesian information criterion. This analysis favoured the
GTR+Γmodel for the ITS1+ITS2 partition and the
K80 model for the 5.8S partition. We conducted
preliminary Bayes factor comparisons (Kass & Raftery
1995) of ML estimates (MLEs) calculated with path
sampling and stepping-stone approaches (Lartillot &
Philippe 2006,Xieet al. 2011) to choose among different
BEAST tree priors and molecular clocks. The use of an
uncorrelated lognormal relaxed molecular clock over the
strict clock was strongly supported for the GB and ORG
datasets (Tables SI & SII). As for the tree priors, models
incorporating the coalescent-constant size produced
substantially higher MLE values than models using the
birth-death and Yule process priors. Runs using chain
lengths of 1.5 × 10
8
steps were implemented, and
parameters were logged every 1.5 × 10
4
steps. Resulting log
files were checked in Tracer 1.7 to ensure that all
parameters had effective sample sizes > 200 after removing
the first 20% of saved trees as burn-in. Then, the median
heights of the 1 × 10
4
post-burn-in tree samples were
annotated with TreeAnnotator 1.8.1, and the chronograms
were drawn with FigTree 1.4. Tracer 1.7 and
TreeAnnotator 1.8.1 are available at http://tree.bio.ed.ac.uk/.
We set the value of Bayesian posterior probabilities (PPs)
at a minimum of 0.97 for considering tree nodes to be well
supported.
Results
Specimen study
The G. marginata specimens studied morphologically and
phylogenetically showed relatively small pilei of up to 4 cm
in diameter, first convex and then turning flat (Fig. 2).
Basidia were tetrasporic and produced ellipsoid to
broadly amygdaloid, brownish and verrucose spores,
with a rounded apex and a visible hilar appendix and
containing one to two guttules. The size (length × width)
of 25 spores measured in water was (11.2) 13.0 ± 1.0
(15.8) × (6.2) 7.4 ± 0.8 (9.9) μm, and the length/width
ratio was (1.4) 1.8 ± 0.1 (2.0). Care must be taken when
comparing the size of pilei and microscopic
characteristics with literature data because specimens
were brought back from Antarctica frozen and were
examined after melting, a process that might have
affected the structure of these characteristics.
Alignments and phylogenies under ML
The original alignment (ORG) done with MAFFT
consisted of 178 Galerina nrITS sequences and
665 positions, of which 279 were variable and
82 corresponded to singleton sites. After processing the
alignment with GBlocks (GB), 603 positions (90% of the
original alignment) were retained in 29 selected blocks;
255 positions were variable and 73 were singleton sites.
The ML analyses in RAxML estimated phylogenies
with lnL = -5168.2 (GB) and lnL = -5493.96 (ORG).
Although the topologies inferred based on the two
alignments were not identical, they showed no supported
conflicts (Supplemental Figs 1 & 2). In general, sister
relationships among the different Galerina species
included in the ingroup lacked support. Bootstrap values
> 70% were obtained for the crown nodes encompassing
all G. marginata,G. badipes and G. fallax sequences,
where the data obtained from Antarctic material are
placed in both topologies. It must be highlighted that the
G. marginata clade, hereinafter referred to as G. marginata
s.l., included sequences from specimens originally
labelled as Galerina autumnalis,G. hygroph ila,
G. pseudomycenopsis,G. unicolor and G. venenata
(including its type sequence, MH827070). Furthermore,
BS values > 78% were found for the sister relationship
of G. margi nat a s.l. and G. bad ipes,G. mi nima and
G. atkin son iana,G. pseudobadipes and G. stylifera,and
G. ce phal otri cha and G. mniophila. The sister relationship
of G. jaapii with the clade containing G. marginata s.l.
and G. badip es received a BS value of 82% (GB), whereas
the clade containing the latter three species along with
G. in dica had a BS value of 81%. Within G. margin ata s.l.,
the newly generated sequence from Livingston Island
(GenBank accession OQ569484) was located at the
bottom and close to three other sequences from
Antarctica: OP795715, which was obtained from a
basidioma collected at Norsel Point on Amsler Island
and differed by one nucleotide; KU559684, an
environmental sequence co-occurring in Antarctica and
the Arctic (Cox et al. 2016), which is shorter than the
other sequences and therefore was composed of a
number of missing nucleotides; and KT990212, labelled
as 'Arrhenia antarctica' and collected by Halina Galera
from an uncertain location within Antarctica that
showed missing and ambiguous positions together with
one diverging nucleotide. Two sequences labelled as
G. pseudomycenopsis (AJ585503 and GU234057) collected
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in the USA and Svalbard were closely related as well
and differed, in general, by fewer than five alignment
positions.
Genetic diversity in G. marginata s.l.
Forty-two haplotypes, producing a haplotype diversity
(Hd) of 0.930, were recovered from the 70 analysed
sequences of G. marginata s.l. (Fig. 3). The haplotype
network revealed a close relationship between the two
Antarctic haplotypes and others obtained from Northern
Hemisphere specimens, including either North America
(USA and Canada) or northern Europe (Scandinavia,
Baltic countries and the UK). Single mutations
segregated these haplotypes. Furthermore, the haplotype
network showed two star-like sub-networks separated by
just one mutation. The most evident sub-network was
composed of a central haplotype with a wide distribution
in North America (especially in Canada) that also
occurred in Switzerland (central Europe). Connected to
this central haplotype by just one or two mutations were
several minor haplotypes from North America and
northern Europe. In contrast, the second star-like
sub-network had a central haplotype distributed in
Europe overall and the Caucasus mountainous region,
and this was linked to minor haplotypes distributed in
Europe as well. At the bottom of the network in Fig. 3,a
number of haplotypes from Asia (Altay Republic, China,
South Korea and Japan) were connected by one or up to
four mutations to haplotypes occurring in North America
and Mexico. A couple of these haplotypes were shared by
regions on both sides of the PacificOcean.Finally,
Tajima's and Fu's neutrality tests estimated negative
values of D (-1.46358, P> 0.10; not significant) and Fs
(-24.755, P< 0.001; significant). The negative values of D
and Fs indicate an excess of low-frequency
polymorphisms relative to expectation and an excess of
the number of alleles, respectively. Collectively, the results
from these two tests suggest a population or demographic
expansion in G. marginata s. l.
BEAST phylogenies and age estimates
The average effective sample sizes were > 200 for all
parameters in the Bayesian dating analyses conducted
with BEAST based on the two alignments (GB and
ORG). No supported topological conflicts were observed
between the two BEAST topologies (Fig. 4) or between
Fig. 3. Statistical parsimony networks connecting Galerina marginata haplotypes and summary of DNA polymorphism indices.
Haplotypes were coloured according to the geographical origin of samples. The sizes of the circles in the networks are proportional to
the numbers of individuals bearing the haplotype; black-filled smaller circles indicate missing haplotypes. Mutations are shown as
hatch marks. S= segregating sites; h= number of haplotypes; Hd = haplotype diversity; π= nucleotide diversity.
7PLEISTOCENE ANTARCTIC COLONIZATION OF THE MUSHROOM GENUS GALERINA
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Fig. 4. Chronogram obtained with BEAST based on nrITS data depicting the evolutionary history of Galerina species. Dashed red
rectangles highlight the clades where the Antarctic collections are included. The mean age estimate for the divergence of selected nodes
is provided in million years ago (Ma). For each terminal in the tree, the GenBank, UNITE or BOLD nrITS accession number, the
taxonomic identity as originally deposited in these databases and the geographical origin are given. Green-filled rectangles indicate
nodal support (posterior probability (PP) ≥0.97) in analyses using the two versions of the nrITS alignment (GB and ORG). The
newly produced nrITS sequences with the corresponding GenBank codes are highlighted in bold. Numbers 1–4 in white circles
indicate phylogenetic clades where sequenced specimens of Antarctic Galerina are placed: 1–2=Galerina marginata,
3=Galerina badipes) and 4 = Galerina fallax.
8ISAAC GARRIDO‐BENAVENT et al.
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them and those obtained in RAxML under ML
(Supplemental Figs 1 & 2). However, the BEAST
analysis using the GB alignment found support (PP >
0.97) for a sister relationship between a Galerina sp.
collected from the Balearic Islands (MH817980) and the
bulk of G. marginata s.l. sequences. Within G. marginata
s.l., sequences from Antarctica were phylogenetically
close to the same sequences reported in the RAxML
analyses. Moreover, two highly supported clades were
revealed: one encompassing several sequences from
North America and Asia, which correspond with the
haplotypes shown at the bottom of the haplotype
network (Fig. 3); and a second clade containing several
sequences obtained from North American as well as one
from Switzerland. This clade is represented by the most
evident star-like sub-network in Fig. 3. High support
Fig. 4. Continued.
9PLEISTOCENE ANTARCTIC COLONIZATION OF THE MUSHROOM GENUS GALERINA
https://doi.org/10.1017/S0954102023000196 Published online by Cambridge University Press
was also revealed for a clade containing G. marginata s.l.
and G. badipes together with G. jaapii,G. indica,
G. triscopa f. telamonioides,G. pseudocamerina,
G. pruinatipes,G. chionophila and G. nana. Supported
sister relationships were also found for G. calyptrata,
G. sphagnicola and G. luteolosperma and for the pair
G. mniophila-cephalotricha.
The dating analysis based on the GB alignment
generated slightly lower age estimates than the analysis
that used the ORG alignment (Table I). However, the
inferred 95% highest posterior density (HPD) intervals
obtained with the two alternative analyses overlapped to
a considerable extent (Tab l e I ). For example, when the
average nrITS substitution rate of 4.61 × 10
-3
s/s/Ma was
employed (Dating A), the crown node of G. marginata
s.l. was dated back to 2.35 Ma (3.22–1.42 Ma,
95% HPD; GB alignment) and 3.09 Ma (4.44–1.74 Ma,
95% HPD; ORG alignment), a time interval at the
transition from the Pliocene to the Pleistocene. For
simplicity, the discussion below is based on the
chronogram estimated with the GB alignment (Fig. 4)
because it did not include potentially misaligned
(ambiguous) regions. This chronogram reveals that major
diversification events in Galerina took place since the
Miocene epoch (ca. 23.03 to 5.30 Ma) and extended into
the Pliocene (ca. 5.30 to 2.58 Ma). Speciation occurred
also in the Pleistocene (ca. 2.58 Ma to 11 700 years ago),
as is observed between the species G. mniophila and
G. cephalotricha and between G. minima and
G. atkin soni ana. Intraspecificdiversification in
G. marginat a s.l., G. bad ipes and G. fal lax, which include
sequences obtained from Antarctic material, occurred
mainly in the Pleistocene, with the Antarctic haplotypes
originating during the last 500 000 years on average
(Fig. 4). Finally, it should be highlighted that Dating B
and C, which used the minimum and maximum values of
the rate's 95% credibility interval provided by Ryberg &
Matheny (2012), produced older and younger age
estimates, respectively, compared with results using the
ave r ag e r ate val u e. Table I summarizes the age estimates
and corresponding 95% HPD intervals for selected nodes
(see Fig. 4) based on the three dating analyses (Dating A,
B and C).
Discussion
The present study validated by means of molecular
phylogenetics the existence in Antarctica of populations
of G. ma rg inata,G. badipes and G. fallax. The former
species (and probably G. badipes too) is well known for
producing amatoxins, which can have dramatic
consequences for human ingestion (Landry et al. 2021).
The samples of Galerina collected from Amsler Island,
Antarctica, were also found to contain alpha-amanitin
(unpublished data 2013, analyses completed by Jonathan
Walton, University of Michigan). These three Galerina
species, G. marginata,G. badipes and G. fallax, represent
relatively common macrofungi in Mediterranean and
Temperate-Arctic ecosystems in the Northern
Hemisphere (GBIF 2022). The closest genetic lineages to
sequenced Antarctic Galerina were in fact collected in
northern Europe (G. marginata), Greenland (G. badipes)
and North America (G. fallax), according to the inferred
phylogenies. Therefore, their distribution is here shown
to be potentially sub-cosmopolitan or amphitropical,
given the few occurrences in tropical regions (GBIF
2022). This biogeographical interpretation of our results
supports the opinion of Pegler et al. (1980), who used
morphological evidence to suggest a close similarity
between macrofungal species from the sub-Antarctic and
the Temperate-Arctic regions of the Northern
Hemisphere. The distribution patterns of the studied
Galerina match to a great extent with the global
geographical distribution of some non-lichenized
Antarctic microfungi (Bridge & Newsham 2009, Bridge
& Spooner 2012,Coxet al. 2016), but, most
interestingly, they also match with the amphitropical
Table I. Estimated divergence ages for the selected crown nodes in Fig. 4 representing Galerina species with Antarctic populations. The dating analyses in
BEAST used alternative nrITS alignment versions (GB vs ORG). For each of these versions, the mean age value and the corresponding 95% highest
posterior density intervals are provided in million years ago (Ma) considering the mean, minimum or maximum values of the nrITS substitution rate
inferred for the genus Phaeocollybia in Ryberg & Matheny (2012), which was used here for calibration purposes. The time frame proposed in the
'Epoch' column considered the six mean ages estimated in each row.
GBlocks-trimmed alignment (GB) Original alignment (ORG)
Mean Minimum Maximum Mean Minimum Maximum Epoch
(1) Crown Galerina marginata s.l. 2.35
(3.22–1.42)
3.91
(5.03–2.22)
1.61
(2.30–1.04)
3.09
(4.44–1.74)
5.47
(7.00–2.87)
1.76
(2.38–1.01)
Pliocene-Pleistocene
(2) Crown of clade with Antarctic Galerina
marginata s.l.
0.51
(0.88–0.07)
1.1
(1.63–0.14)
0.33
(0.72–0.08)
0.57
(0.90–0.13)
2.04
(2.16–0.49)
0.55
(0.83–0.23)
Pleistocene
(3) Crown Galerina badipes 0.69
(1.31–0.17)
1.33
(2.07–0.24)
0.43
(0.92–0.12)
0.79
(1.38–0.16)
1.78
(2.14–0.26)
0.55
(1.00–0.10)
Pleistocene
(4) Crown Galerina fallax 0.84
(1.56–0.23)
1.55
(2.42–0.35)
0.54
(1.10–0.19)
0.95
(1.63–0.24)
2.04
(2.55–0.39)
0.67
(1.16–0.17)
Pleistocene
10 ISAAC GARRIDO‐BENAVENT et al.
https://doi.org/10.1017/S0954102023000196 Published online by Cambridge University Press
distribution pattern displayed by a significant proportion
of lichenized fungi, which in Antarctica account for
almost 40% of lichens (Øvstedal & Lewis Smith 2001).
The existence of nearly identical global distribution
patterns in various lichenized and non-lichenized
Antarctic fungi makes us hypothesize that, at the
geological time scale, these species overcame similar
ecological and geographical filters to acquire their
current distribution. To accumulate evidence for
supporting or rejecting this hypothesis, other Antarctic
species of Galerina and members of additional
non-lichenized macrofungi genera should be surveyed
and studied phylogenetically.
The phylogenetic and haplotype network analyses
indicated that the studied intraspecific genetic lineages of
the Antarctic Galerina might be geographically restricted
and therefore endemic to this polar region. The fact
that the considered species produced fruiting bodies in
the surveyed Antarctic localities indicates that these
macrofungi have established permanent populations, and
therefore they are not transient visitors. Basidiomata
formation represents the last step in the life cycle of
Basidiomycota fungi. Briefly, it starts with spore
germination and mycelium growth once abiotic and
biotic requirements are met, followed usually by mating
of two compatible, distinct mycelia, and, as a result,
basidiomata develop and spores are produced and
released after meiosis. We suggest that the three Galerina
species have been established in this region long enough
for mutations to accumulate in the studied genetic locus,
the nrITS, which is known to evolve at a higher rate
compared to other commonly used fungal molecular
markers (Schoch et al. 2012). Moreover, the existence of
Antarctic-endemic intraspecific lineages of these fungi is
of the utmost importance for designing conservation
policies that consider a broad spectrum of eukaryotic
organisms and not only plants and animals.
However, assessing endemicity in fungi poses some
risks, even at the intraspecific genetic level. In fact,
the existence or not of true Antarctic-endemic non-
lichenized fungal lineages has been hotly debated (e.g.
Bridge & Spooner 2012, Arenz et al. 2014) because of
the obvious difficulties in observing and/or isolating
macro- and microfungi in Antarctica, or elsewhere, due
to their complex life cycles, ecologies and/or sizes. In this
sense, Bridge & Spooner (2012) mentioned that some of
the allegedly Antarctic-endemic species reported by
Onofri et al. (2005) were found later elsewhere. Assessing
endemicity in lichenized fungi is comparatively more
straightforward because they usually form macroscopic
and enduring lichen thalli (but see Hale et al. 2019).
Hence, the proportion of Antarctic-endemic lichens has
been estimated at ∼30% (Øvstedal & Lewis Smith 2001).
In our opinion, the analysis of biogeographical patterns
in non-lichenized Antarctic fungi may be more accurate
if approached phylogenetically as long as extensive
specimen and molecular datasets are compiled. For
example, genotypes restricted to Antarctica were
revealed for widespread fungi, such as the ascomycete
Thelebolus microsporus (Berk. & Broome) Kimbr. and
other microfungi (de Hoog et al. 2005, Bridge &
Newsham 2009, Bridge & Spooner 2012, Gonçalves
et al. 2017). Even so, the lack of availability of sequence
data and collections from as yet unexplored areas in the
Southern Hemisphere makes it difficult to assess
endemicity in Antarctic fungi. Although we compiled a
large specimen dataset in the present Galerina study, it
lacked sequence data associated with reports of
G. marginata and G. badipes from Australia and New
Zealand (GBIF 2022). Because of the geographical
proximity of these austral regions to Antarctica, it would
be worth checking whether Antarctic, Australian and
New Zealand populations of Galerina share the same
genotype or are at least closely related. In this way,
endemicity or colonization routes would be judged
more correctly. Furthermore, DNA sequence length
and quality are crucial for precise interpretations
of biogeographical patterns. This is of particular
importance today due to the abundance of short
sequences from DNA metabarcoding studies. If short
and long sequences are combined in datasets, the
resulting biogeographical interpretations could be
misleading to some extent, as unrealistic phylogenetic
affinities could be inferred. For example, we used a short
nrITS sequence of G. marginata that co-occurred in
Antarctica and the Arctic (KU559684; Cox et al. 2016).
Despite the existence of identical DNA sequences in
individuals from both poles having been observed in, for
example, the lichenized fungus Pseudephebe minuscula
(Arnold) Brodo & D. Hawksw. (Garrido-Benavent et al.
2021), it cannot be ruled out that comparison of the
Galerina sequences along their entire length would reveal
some genetic differences.
To the best of our knowledge, the present work is the
first examining the temporal origins of Antarctic
macrofungal populations. Thus, the Antarctic lineages of
Galerina probably diverged from their Northern
Hemisphere relatives during the Pleistocene, based on a
consensus estimate of divergence times inferred with the
various dating strategies implemented in the present
study. In G. ma rgi nata, the divergence was probably
linked to a demographic expansion, as revealed by the
calculated neutrality tests. Moreover, the calculated
divergence time intervals for the three Galerina agree with
the inferred Pleistocene origin of Antarctic populations of
amphitropical lichens (Fernández-Mendoza & Printzen
2013,Garrido-Benaventet al. 2021). In the lichenized
fungi Cetraria aculeata (Schreb.) Fr. and P. minuscula,a
close genetic affinity of Maritime Antarctica specimens
to South American (Chilean) collections suggested a
11
PLEISTOCENE ANTARCTIC COLONIZATION OF THE MUSHROOM GENUS GALERINA
https://doi.org/10.1017/S0954102023000196 Published online by Cambridge University Press
colonization route through the Sea of Hoces (Drake
Passage). In addition, continental specimens of the
second species were genetically close to Svalbard
(Northern Hemisphere) specimens, which indicated an
independent colonization route. The closest relatives of
G. marginata,G. badi pes and G. fallax, based on the
inferred phylogenies, grew in North America and northern
Europe, so that a direct, long-distance dispersal across the
tropics and ending in the establishment of Antarctic
Galerina populations might be assumed. However, the
possibility that these species colonized the Antarctic in a
series of stepping-stone movements from other territories
in the Southern Hemisphere, for which neither specimen
nor sequence data are yet available, must not be ruled
out. South America was in fact the region from which the
vascular plant D. antarctica is believed to have colonized
the Antarctic region, also during the Pleistocene
(Fasanella et al. 2017). It is worth recalling that the
studied Galerina species grew in tight association with
carpets of this plant as well as mosses, where these fungi
behave as saprophytes. Even some common populations
of Antarctic mosses had a Pleistocene origin (Pisa et al.
2014,Biersmaet al. 2017,2018). The overlapping
temporal frameworks for the origins of these plants and
fungi that coexist in the same Antarctic terrestrial
communities further support a relatively recent Antarctic
colonization of Galerina. The meiotic spores produced by
their basidiomata, which in general are ellipsoidal or
amygdaliform and < 15 μm in length, constitute the
expected mode of dispersal, and either wind currents or
migratory birds could be involved in such transoceanic
dispersals (Muñoz et al. 2004, Viana et al. 2016). For
example, Biersma et al. (2018) inferred aerial models that
indicated local wind patterns as the most probable
transfer mechanisms from southern South America to the
northern Maritime Antarctic. A greater research effort is
needed to corroborate these means of dispersal.
The biogeographical history of the studied Antarctic
Galerina has been interpreted on the basis of time
trees inferred using a secondary calibration (i.e. nrITS
substitution rate). Although this approach would be
expected to lead to more inaccurate dating results than
phylograms calibrated using fossil data (Schenk 2016),
the divergence times calculated for the whole Galerina
phylogeny in this study are largely coherent with
those reported for the divergence of species in other
Basidiomycota genera (e.g. Amanita,Heterobasidion,
Russula) that used different calibration strategies and
more extensive molecular sequence datasets (Chen et al.
2015, Sánchez-Ramírez et al. 2015, Looney et al. 2020).
Furthermore, the diversification events within the three
Antarctic Galerina species also agree with the inferred
colonization events of D. antarctica and mosses that,
together with these fungi, form typical terrestrial
habitats in Antarctica.
Acknowledgements
The UTM-CSIC technicians are thanked for their
assistance during Antarctic fieldwork. The authors also
acknowledge Esther Rodríguez (MNCN) for technical
assistance in the laboratory; Alexandra Isern, Director
Antarctic Earth Sciences, US National Science
Foundation, Carolyn Lipke and other personnel at
Palmer Station, Polar Programs; the US National
Science Foundation for their help collecting Galerina
samples from the Antarctic Peninsula; and Benjamin
Held, University of Minnesota, for laboratory assistance.
This research was part of POLARCSIC activities. Two
anonymous peer reviewers are thanked for their feedback.
Funding
This work was supported by grants CTM2017-84441-R
(MINECO/FEDER, UE) and PID2019-105469RB-C22
(MICINN, AEI).
Author contributions
IG-B: conceived the project, conducted the analyses and
wrote the first draft of the manuscript. RAB: contributed
to the molecular dataset and editing of the final
manuscript. AdlR: obtained funding and field resources,
conducted fieldwork (including specimen collection) and
contributed to the editing of the final manuscript.
Supplemental material
A supplemental table will be found at https://doi.org/10.
1017/S0954102023000196.
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