10.6092/issn.2531-7342/13464
We report the finding of an Agaricus specimen in the section Sanguinolenti and describe it as a new species Agaricus hortusdamarum. Based on ITS data, the species is distinct and well separated from all known sequenced species within this section. The type specimen grew in late autumn, under Cupressus sempervirens, near a Pinus pinea alley in the Villa Borghese Gardens in Rome, in Italy. The new species is phylogenetically close to Agaricus iranicus, a rare species of temperate climate, which is so far described only in the Guilan province in Iran. Its morphological characters and phylogenetic pattern are described and divergence from A. iranicus is discussed.
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Italian Journal of Mycology
Villa Borghese
A new species of <em>Agaricus</em> (section <em>Sanguinolenti</em>) from Rome, Italy
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2024-01-22
2021-08-23
2022-02-21
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Ital J Mycol
Arnold Knijn
2022
10.6092/issn.2531-7342/13464
1
Via Arno 36, 00198 Roma, Italy
2531-7342
Villa Borghese
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Zhao R-L, Zhou J-L, Chen J, Margaritescu S, Sánchez-Ramírez S, Hyde KD, Callac P, Parra LA, Li G-J, Moncalvo J-M (2016) Towards standardizing taxonomic ranks using divergence times – a case study for reconstruction of the Agaricus taxonomic system. Fungal Diversity 78:239–292. https://doi.org/10.1007/s13225-016-0357-x
A new species of <em>Agaricus</em> (section <em>Sanguinolenti</em>) from Rome, Italy
https://italianmycology.unibo.it/article/download/13464/17472
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                     IJM - Italian Journal of Mycology
                     ISSN 2531-7342 - Vol. 51 (2022): 1-10
                     Journal homepage: https://italianmycology.unibo.it/

Research article


       A new species of Agaricus (section Sanguinolenti)
                      from Rome, Italy
                                     Amalia Ferretti1, Irja Saar2, Arnold Knijn1
1
    Via Arno 36, 00198 Roma, Italy
2
    Institute of Ecology and Earth Sciences, University of Tartu, J. Liivi 2, 50409 Tartu, Estonia

Corresponding author e-mail:knijn_roma@hotmail.com
______________________________________________________________________________
ARTICLE INFO
Received 22/11/2021; accepted 30/01/2022
https://doi.org/10.6092/issn.2531-7342/13464
The contribution was amended on 22/01/2024; see https://doi.org/10.6092/issn.2531-7342/18530

Abstract
We report the finding of an Agaricus specimen in the section Sanguinolenti and describe it as a new species Agaricus
hortusdamarum. Based on ITS data, the species is distinct and well separated from all known sequenced species within
this section. The type specimen grew in late autumn, under Cupressus sempervirens, near a Pinus pinea alley in the Villa
Borghese Gardens in Rome, in Italy. The new species is phylogenetically close to Agaricus iranicus, a rare species of
temperate climate, which is so far described only in the Guilan province in Iran. Its morphological characters and
phylogenetic pattern are described and divergence from A. iranicus is discussed

Keywords
Pseudochitonia, single-nucleotide variants, urban park, Villa Borghese
________________________________________________________________________________
Introduction
Agaricus (Basidiomycota, Agaricales, Agaricaceae) specimens are well-defined and
straightforwardly assigned to its genus but the internal taxonomy of the genus in toto has been
revolutionised during the last 25 years with the advent of molecular phylogenetic studies (Bunyard et
al., 1996; Geml et al., 2004; Kerrigan et al., 2005; Zhao et al., 2011; Chen et al. 2015; Parra et al.,
2015; Zhao et al., 2016; Chen et al. 2017). Currently, a revised taxonomic system has been proposed,
identifying 6 subgenera and 24 sections in the genus, based on a combination of morphological
characters with phylogenetic analyses and estimation of divergence time (Zhao et al., 2016; He et al.,
2018; Jaichaliaw et al.; 2021). Currently, a total of over 500 species are recognised (Wang et al.,
2015; Callac and Chen, 2018) and new species of this genus are still found frequently, as for instance
Agaricus iranicus Mahdizadeh, Safaie, Goltapeh, L.A. Parra & Callac, the epithet meaning “from
Iran”, the country where the holotype specimen was collected (Mahdizadeh et al., 2018). This species
belongs to the section Sanguinolenti within the subgenus Pseudochitonia (Zhao et al., 2016).
       The section Sanguinolenti has been redimensioned since several species have been shifted to
other sections according to the new phylogenetic analyses (Zhao et al., 2011; Zhao et al., 2016). It is
considered together with sections Bohusia and Nigrobrunnescentes among those that contain fungal
species that grow in a temperate environment (Mahdizadeh et al., 2018). The section now includes
just six species: the best-known A. sylvaticus Schaeff. and A. benesii (Pilát) Pilát together with A.
                                                      ______________
    Department of Agricultural and Food Science in partnership with the Italian Mycological Society (UMI).
    The journal is hosted and maintained by ABIS-AlmaDL. © 2022 The Authors. This work is licensed under CC BY 4.0
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hupohanae Kerrigan, A. cordillerensis Kerrigan, A. thujae Kerrigan and the before mentioned A.
iranicus (Mahdizadeh et al., 2018). Agaricus iranicus is therefore inserted among the species that
prefer a temperate climate, and its presence has been reported only in the Guilan province, in the
South of the Caspian Sea, in Iran (Mahdizadeh et al., 2018; Ghobad-Nejad et al., 2020). The species
found in Rome is described here by morphological characters as a new species belonging to Agaricus
section Sanguinolenti through its phylogenetic pattern inferred from a combined dataset of internal
transcribed spacer (ITS) and partial nuclear 28S rDNA (LSU) sequence data. Divergence of this new
species from A. iranicus is discussed.

Materials and Methods
Area of study and sample
A specimen was found in November 2020 in the area called ‘Parco dei Daini’ (Fallow Deer Park) of
the Villa Borghese Gardens, the most popular urban park in Rome, Italy. The basidiomata grew
terricolous on litter of pine and cypress needles and grass, under Cupressus sempervirens L. trees, in
an alley formed by Cupressus and Pinus pinea L. and close to a Cedrus libani A. Rich. tree, at 50 m
a.s.l. (Fig. 1). The collected specimens (Fig. 2) are conserved as part of the Mycological Collections
of the Natural History Museum and Botanical Garden of the University of Tartu, Estonia.




                              Fig. 1 - “Parco dei Daini” (Fallow Deer Park) in the Villa Borghese
                              Gardens: occurrence site of the new Agaricus species.



Morphological analyses
Microscopic images were obtained with a digital camera AmScope (Irvine CA, USA) MU 500
mounted on an AmScope B490 microscope, operating at ×800 magnification. Fresh lamella
fragments were mounted in distilled water to determine the presence of cheilocystidia. The
observation of these elements must be carried out on fresh material because in most Agaricus species
they are no longer observable after drying (Basso, 2012). Microscopic images on the dried specimen
were obtained using a light microscope Leica DM750 with Leica Application Suite v4.13.0 [Leica




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Microsystems (Schweiz) AG] at magnification ×1000. Dried lamella fragments were mounted in 3%
KOH.

Molecular analyses
Ribosomal DNA-based analysis was performed on the holotype specimen in the frame of the UNITE
project (Kõljalg et al., 2013; https://unite.ut.ee/). DNA extraction, PCR amplification of SSU partial,
ITS1, 5.8S, ITS2, LSU partial regions and sequencing were performed as in Voitk et al. (2018).
BLAST 2.8.1+ (Camacho et al., 2009) was used to select 17 sequences for phylogenetic analyses.
These sequences were aligned using the MUSCLE v3.8.425 algorithm (Edgar, 2004) in Aliview 1.26
(Larsson, 2014) and adjusted manually. Maximum Likelihood and Bayesian inference were applied.
In PhyML 3.1 (Guindon et al., 2010), Nearest Neighbour Interchange was set up as topology research,
initial trees obtained applying BIONJ and Neighbour-Joining to a matrix of pairwise distances. In
MrBayes 3.2.6 (Ronquist et al., 2012), the Markow Chain Monte Carlo algorithm was run using four
chains (three hot, one cold), a temperature of 0.1 and iterations for 2×106 generations on two trees
with a diagnose frequency every 5×103 generations and a 25% burn-in. The phylogenetic tree was
visualised using FigTree v1.4.3. A further alignment was performed on 657 overlapping bases of the
8 sequences of A. iranicus clade species only and their Single-Nucleotide Variants were highlighted
by the snipit software (O’Toole and Tomkins-Tinch, 2021). The ITS sequence (UDB0799866) is
available in the UNITE database.




   a                                                                                                           c
Fig. 2 - Agaricus hortusdamarum a) holotype on litter of conifers needles; b) holotype ex situ; c) holotype scraped stem
slowly coloured reddish brown.

Results
Molecular analyses
Phylogenetic analysis of the 17 most similar Agaricus ITS sequences from the GenBank databases
(Fig. 3) revealed the genetic similarity of the new specimen with the A. iranicus clade. Because the
specimen did not fit perfectly within the clade (PP=100%), a Single-Nucleotide Variants (SNV)
analysis was performed on the 8 sequences of this clade only to further investigate intraspecies
differences (Fig. 4). As could already be seen from the phylogenetic tree, SNV analysis confirmed
that the sequences from the specimens collected by V. Mahdizadeh (accessions KY474551-
KY474556) in December 2013 in Iran are identical between themselves.




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Fig. 3 - Phylogenetic tree generated from Bayesian analysis of ITS sequences of Agaricus section Sanguinolenti,
identified by their UNITE/GenBank accession numbers and species. The sequences KM657927 from A. campestris and
KJ877774 from Agaricus rubribrunnescens Murrill were used as outgroup. The sequence from the new sample described
in the article (accession UDB0799866) is indicated by an asterisk and the sequences from the Agaricus holotypes by a
hash symbol. Bayesian posterior probabilities are reported on the main branches. The final mean standard deviation of
the separated frequencies was < 0.007.




Fig. 4 - Alignment of the overlapping 657 nucleotides of 8 most similar ITS sequences, indicated by their
UNITE/GenBank accession. Accession KY474551 is a sequence from the A. iranicus holotype specimen VM035. Single-
Nucleotide Variants are indicated with respect to the UDB0799866 sequence. The nucleotides at positions 153 and 259
are considered species-specific ITS markers for A. iranicus. The barcode regions are annotated at the bottom.


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      A single sequence from a specimen found on the Xianshan Mountain in the Beijing region by
Xu et al. (accession JN907016) is very similar to the A. iranicus holotype sequence with only two
nucleotide variations (position 163 and 603). The sequence of the new Italian specimen (accession
UDB0799866) on the other hand, has seven nucleotide variants between the 13th and 279th nucleotide
positions (ITS1 region) and one at the 543rd nucleotide position (ITS2 region) with respect to the A.
iranicus holotype.


Taxonomy
Agaricus hortusdamarum A. Ferretti, I. Saar & A. Knijn, sp. nov.

Index Fungorum IF559484
Typification – Holotypus: Italy, Rome, Parco dei Daini, urban park, under Cupressus sempervirens,
41.916111°N, 12.493055°E, 14 Nov 2020, leg. Arnold Knijn, Amalia Ferretti (TUF105944, ITS
sequence UDB0799866).
      Etymology – hortusdamarum is a compound noun created from the Latin words for park/garden
(hortus) of red deer (damarum, genitive plural), a translation of Parco dei Daini, where the holotype
was collected.

Macromorphology – Pileus convex then plane and depressed, diameter ~9 cm, reddish-brown,
squamulose, white trama exposed under the scales. The pileus margin slightly exceeded the lamellae,
appendiculate with remains of the universal veil (Figs. 2a,b). Lamellae free, crowded, intercalated
with lamellule, ventricular, at first pale, with age brown to dark brown. Stipe about 8.5 cm,
cylindrical, central, fistulose, greyish-white, with an evanescent disappearing greyish-white
membranous annulus, with a bulbous base covered with white mycelium. Stipe trama whitish, turning
very slowly reddish brown when handled or cut (Fig. 2c). Odour: slight fungal smell.
      Micromorphology – Basidiospores (one basidioma, n = 32) smooth, ellipsoid, the major part
with a brown colouration, few of them with apiculum, lipid droplets and some granules were evident
inside (Fig. 5a). Their average size was 6.1  4.6 µm [(7.1)–6.1–(5.2)  (5.2)–4.6–(3.9) µm], Qm =
1.3 Q = [(1.5)–1.3–(1.2)]. Basidia bisporic and trisporic (Fig. 5b), 18–25  7–8 µm, with sterigmata
up to 3.8 µm long (average 3.4 µm). Cheilocystidia abundant, hyaline, septate, cylindrical and clavate
(Fig. 5a), single septated elements measuring 24–48 µm  11–17 µm. Such cheilocystidia origin from
the lamellar trama, as was described for Agaricus iranicus Mahdizadeh, Safaie, Goltapeh, L.A. Parra
& Callac.

Habitat – On needle debris of pine and cypress, in urban park, autumn.

Distribution – So far only known from Italy.

Comments – Most similar to the closely related species A. iranicus. Agaricus hortusdamarum differs
in an evident although slow reddish-brown discoloration when touched, scraped and cutted the stem
surface and in cap with darker brown squamules. Microscopic evidence of larger cheilocystidia
supports the distinction between the two species. Both species grow in temperate environments but
A. iranicus presence is reported only in Asia and A. hortusdamarum is found in southern Europe. ITS
barcode: eight single-nucleotide variations occur between both species, two of which identified as
species-specific markers for A. iranicus.



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  a                                                                                         b




Fig. 5 - Microcharacters of Agaricus hortusdamarum. a) fresh lamella fragments showing the large cheilocystidia and
the basidia (in the insert) for comparison. b) dried lamella fragments mounted in KOH, basidia and basidiospores; Bar
= 10 μm.



Discussion
We report the finding of an Agaricus fungus growing in autumn in the Villa Borghese Gardens in
Rome (Italy), that we describe as a new species belonging to the section Sanguinolenti. This Agaricus
species is well defined from all known sequenced species within the section Sanguinolenti and has a
phylogenetically definite position. In fact, the new species is phylogenetically very close to but
distinct from A. iranicus, another species included in section Sanguinolenti. Agaricus section
Sanguinolenti, with the other two groups Bohusia and Nigrobrunnescentes are considered among
those containing fungal species growing in a temperate environment, 75% of the species in the first
two groups show a preferential coniferous habitat and it was suggested that it might reflect an ancient
association (Mahdizadeh, 2018).
      The genus Agaricus from Iran has been well studied from a taxonomical and phylogenetical
point of view (Mahdizadeh et al., 2016; Mahdizadeh et al., 2018; Ghobad-Nejad et al., 2020), and A.
iranicus is reported to grow in some sites of the Guilan province, in particular under Cupressus trees.
The new Italian species confirms the preferential association of the species in section Sanguinolenti
with conifers, in this case with C. sempervirens also in the presence of P. pinea and C. libani.
      Fungi of the genus Agaricus present a saprophytic behaviour, but some studies (La Chiusa,
2013; AMINT, 2015), report that many species of Agaricus, after a first phase of development of the
mycelium in which they are saprophytic, tend to establish a relationship with arboreal such as with
herbaceous plants.

       The Villa Borghese Gardens in Rome and the ‘Parco dei Daini’ were created in the 17th century,
at the time of the construction of the Casino Nobile. The back of the Villa overlooked a large square,
from which one could enter in an area called second enclosure, surrounded by walls that separated it
from the surrounding countryside. The zone today corresponds to Parco dei Daini (Hortus Damarum
in Latin), so called because there were fallow deer and gazelles that were left free in the holm oak
grove. Afterwards, the gardens underwent changes until the first years of 1900, but the present P.
pinea and Cupressus trees in the Gardens are centenary. During the second world war, the area was
used for the cultivation of horticultural crops (“orti di guerra”, so-called war gardens) as a response


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to the food crisis (Campitelli, 2005). If the hypothesis of a symbiotic-like association between
Agaricus and coniferous plants is possible, the presence of the new species in this part of the Villa
Borghese Gardens would be due to a non-recent interaction.
      The macroscopic morphology of the sample from Rome does not show strong differences from
A. iranicus, the most relevant is the presence of squamules on the cap that seems to be darker brown.
Microscopic analyses showed a similar size of basidiospores and basidia within biological variability,
however, in this new studied specimen the abundant presence of very large cheilocystidia is evident,
with about twice the size of those found in lamellae of A. iranicus. This notable difference probably
reflects the divergences found in the molecular phylogeny between the aforementioned different
species.

      Phylogenetic analysis indicates the new specimen’s affinity with the A. iranicus clade, however,
also highlights some significant distinctions. In fact, among the eight single-nucleotide variations
with respect to the holotype, two in particular seem significant (nucleotide positions 153 and 259)
since they have been identified as part of the eight species-specific ITS markers for A. iranicus
(Mahdizadeh et al., 2018), corresponding respectively to tgagAactt@153 and gtctCtaca@259
mentioned in the article.

      Another trait that could have phylogenetical significance is discoloration. The studied specimen
showed a slow but evident reddish-brown discoloration when touched, and a slow reddish-brown
discoloration scraping and cutting the stem surface. In the specimens from Iran discoloration was not
observed if not a late reddish-brown discoloration at stipe base (Mahdizadeh et al., 2018). In any case,
the behaviour is rather different from that of other species in section Sanguinolenti, because A.
sylvaticus Schaeff., A. benesii (Pilát) Pilát and A. thujae Kerrigan all turn pinkish-red when injured
and strongly turn reddish when cut.

       It is reported that the browning reactions in some Agaricus fungi are due to either mechanical
damage or bacterial activity in tissue (Naveen and Beelman, 2006). Enzymatic browning is catalysed
by the enzyme tyrosinase (polyphenol oxidase) (Weijn et al., 2013). Tyrosinase naturally occurs at
high levels in the surface tissues (Soler-Rivas et al., 2000). It has been hypothesised that tyrosinase
and its substrates are located on separate subcellular compartments in intact fungi and when the fungi
are bruised the cellular membrane is damaged and rapid browning is observed. The loss of membrane
integrity provides greater access of tyrosinase to its phenolic substrates such as γ-L-glutaminyl-4-
hydroxybenzene (GHB), allowing for the activated enzyme to oxidise the compounds into the
melanin products with associated brown discoloration of Agaricus fungi (Boekelheide et al., 1979;
Burton, 1986; Jolivet et al., 1995). It has been suggested that tyrosinase and its metabolites such as
GHB, which is also the precursor of spore wall melanin, play a role in defence systems and in the
resistance response to stress in fungi (Jolivet et al., 1998). Besides, it was reported that the variation
of bruising sensitivity observed in different A. bisporus strains correlated with the presence of
phenolic compounds (GHB) in the fungus’ skin (Weijn et al., 2013). The article suggests that the
enzymatic conversion of p-aminophenol to GHB by the enzyme γ-glutamyltransferase (GGT),
present in high levels in gills, pileipellis and stipe base (Jolivet et al., 1998), is the rate-limiting step
of the reactions in the different strains and might be caused by genetical mutations in the expression
or in altering the activity of the enzyme (Weijn et al., 2013). Therefore, variations in the production
of pigments or in the lack of discolouration in genus Agaricus could be due to alterations in the
mechanisms involved in the regulation of the enzymatic conversions also consequent to genetic
mutations.




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      This study reports further evidence of the presence of particular and/or rare fungi in the urban
Parks of Rome, such as the (sub-)tropical Punctularia atropurpurascens (Berkeley & Broome) Petch
1916 (Knijn and Ferretti, 2018). The findings and the surprising variety of fungi are probably related
with the botanical biodiversity of both autochthonous and allochthonous trees present in the various
ancient Parks of the metropolis and confirm the importance to maintain and preserve the public green
areas of the city.


Acknowledgements
Irja Saar was supported by the Estonian Research Council (PRG1170), the European Regional
Development Fund (Centre of Excellence EcolChange).



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Ferretti, Saar, Knijn                                                                        Italian Journal of Mycology 51 (2022): 1-10

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