10.6092/issn.2531-7342/18172
We report the finding of three recently described species in late autumn and winter (October to December) in the Bracciano-Martignano Regional Natural Park in Lazio (Italy). The species are Amanita calida, Lepiota elseae and Xerocomellus sarnarii. This is the first record of Lepiota elseae for Italy. The collection site, a Quercus thicket, is located on the slopes of San Bernardino del Malpasso in Trevignano Romano, between Monte Rocca Romana (Sabatini mountains) and the nearby Bracciano Lake. A description of the specimens is given, and a comparison is made with the few descriptions of the ambient available for previous records of the respective species.
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Italian Journal of Mycology
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Three new species found in the Bracciano-Martignano Regional Natural Park in Lazio, Italy
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Ital J Mycol
Irja Saar
2024/02/05
10.6092/issn.2531-7342/18172
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Institute of Ecology and Earth Sciences, University of Tartu, J. Liivi 2, 50409 Tartu, Estonia
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Weber RWS, Davoli P (2002) Autophagocytosis of carotenoid-rich lipid droplets into vacuoles during aeciospore ageing in Puccinia distincta. New Phytologist 154:471–479 https://doi.org/10.1046/j.1469-8137.2002.00377.x
Three new species found in the Bracciano-Martignano Regional Natural Park in Lazio, Italy
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                     IJM - Italian Journal of Mycology
                     ISSN 2531-7342 - Vol. 53 (2024): 1-12
                     Journal homepage: https://italianmycology.unibo.it/

Research article


          Three new species found in the Bracciano-Martignano
                  Regional Natural Park in Lazio, Italy
                                   Amalia Ferretti1, Arnold Knijn1, Irja Saar2

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 29/09/23; accepted 23/12/23
https://doi.org/10.6092/issn.2531-7342/18172

Abstract
We report the finding of three recently described species in late autumn and winter (October to December) in the
Bracciano-Martignano Regional Natural Park in Lazio (Italy). The species are Amanita calida, Lepiota elseae and
Xerocomellus sarnarii. This is the first record of Lepiota elseae for Italy. The collection site, a Quercus thicket, is located
on the slopes of San Bernardino del Malpasso in Trevignano Romano, between Monte Rocca Romana (Sabatini
mountains) and the nearby Bracciano Lake. A description of the specimens is given, and a comparison is made with the
few descriptions of the ambient available for previous records of the respective species.

Keywords
Amanita calida, Lepiota elseae, Xerocomellus sarnarii, Quercus forest, Natural Parks

________________________________________________________________________________

Introduction

Three species, described as new during the last decade, belonging to the genus Amanita, Lepiota
(Agaricales) and Xerocomellus (Boletales), were found in the same area near Rome in Lazio. The
species are Amanita calida Plaza & Illescas (2022), Lepiota elseae A. Caball., Vizzini, G. Muñoz &
Contu (2015) and Xerocomellus sarnarii Simonini, Vizzini & U. Eberh. (2015) respectively.
      Amanita calida is inserted in the section Vaginatae (Tulloss 1994; Tulloss and Yang, 2022) and
was recently described from Spain (Illescas and Plaza, 2022) based on collections obtained in
Andalusia and Catalonia, associated with Quercus, in spring-summer and in October.
Macroscopically, this species is characterized by a whitish, beige to grayish cuticle, with a prominent
darker brown umbo, a widely striate margin of the pileus, and a very elongated stipe. This Amanita
occurs in Spain (OK316924, ITS of holotype) and in Italy with two sequences present in the
International Nucleotide Sequence Database (INSD) with temporary codes, later confirmed as A.

                                                      ______________
    Department of Agricultural and Food Science in partnership with the Italian Mycological Society (UMI).
    The journal is hosted and maintained by AlmaDL. © 2024 The Authors. This work is licensed under CC BY 4.0
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calida (MT073006, MW013162) from collections made in Central Italy (Terni, Umbria), in woods
of Quercus ilex L. and Fagus, between the beginning of July and the beginning of August of 2018.
(Tulloss and Yang, 2022). One sequence obtained from a soil sample (UDB0153760) in Estonia also
coincided with that of the holotype.
       Lepiota elseae was found and described in Spain in 2015 (Caballero et al., 2015). This species
belongs to the section Lepiota and is known from Mediterranean areas in Spain in association with
Q. ilex and determined as a new species on both morphological and molecular characteristics (analysis
of the ITS region of the ribosomal RNA gene sequences). No evidence of previous findings of
fruitbodies in Italy has been reported. A soil sample from Italy (UDB02883342) and two samples
from Bulgaria (UDB02886835, UDB02886840) contained DNA from L. elseae.
       Regarding X. sarnarii, findings in Sardinia and Tuscany have been recorded. Based on Simonini
et al. (2016) some specimens of this species have also been found in France. It is described as a rare
Boletales species, so far known from Italy, France, some middle eastern region of Europe and in
Cyprus (Ariyawansa et al., 2015; Loizides et al., 2019). Its habitat are the Mediterranean areas,
especially under Quercus suber L. and Q. ilex trees. The main fruiting period is late autumn
(November). It is reported that this taxon seems to prefer climatically moderate zones and undisturbed
habitats. For this species, eDNA from soil samples have been recorded in Georgia (UDB07503546),
Greece (UDB07503362) and Italy (UDB07502840).
       Our initial tentative attributions of the three species found in the Quercus wood zone in the
Bracciano-Martignano Regional Natural Park, based on the morphological characteristics, indicated
an Amanita of the section Vaginatae with characteristics between Amanita vaginata (Bull.) Lam. and
Amanita battarrae (Boud.) Bon, the other two species as Lepiota clypeolaria (Bull.) P. Kumm., and
Xerocomellus chrysenteron (Bull.) Šutara or Xerocomellus porosporus (Imler ex Watling) Šutara.
The correct identifications of specimens were obtained by molecular analysis of the rDNA ITS
barcode.


Materials and Methods

Area of study and sample
The small wood area is part of the Bracciano-Martignano Regional Natural Park in Lazio and located
on the volcanic slopes of San Bernardino del Malpasso. The total area measures somewhat less than
5,000 m² with a maximum length of 160 m, a maximum width of 45 m at about 200 amsl (Fig. 1), in
Trevignano Romano between Monte Rocca Romana (Sabatini mountains) and the nearby shores of
the Bracciano Lake at 42°09'19" N 12°16'17" E. This thicket is crossed by an almost permanently dry
ditch and is located close to uncultivated fields and vineyards and inhabitations too (Via delle
Fossette). It displays the peculiar morphology of fertile volcanic soil. In the Sabatino volcanic
system, mainly pyroclastic soils, andosols and acid brown soils born on beds of volcanic scoria and
ash are found. The volcanic soils examined in the territory have a low content of calcium carbonate
and a subacid to neutral reaction (pH = 5.3–7.8) and in general are fertile but fragile
pedoenvironments (Various authors, 2009). The soils in the area facing the lake tend to take on the
typical characteristics of arid environments which adapt well to the coenoses of the xerophilous
Mediterranean vegetation. The Bracciano-Martignano area figures as a transition zone from a more
distinctly Mediterranean climate to a temperate climate, typical of the inland areas of the Lazio


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Apennines (Various authors, 2009). Differently from the other side of the Monte Rocca Romana
(Knijn et al., 2021), this wooded Quercus area overlooking the lake does not show a humid climate.
The rainfall in the autumn and early winter period is given by light rains, the month with the highest
rainfall is November (30%) while it drops to 10% in June. As for the temperature, in this part of the
Bracciano Lake in the autumn months it varies from 23 °C, perceived as 25 °C, to 15 °C during the
day and in December it averages around 13 °C, while in June the maximum temperatures reach an
average of 26 °C. The thicket vegetation, characteristic of the Mediterranean woods, mainly consists
of various types of oaks, such as downy oaks (Quercus pubescens Willd.) and Turkey oaks (Quercus
cerris L.) as deciduous plants and holm oaks (Q. ilex) as evergreens. The undergrowth is made up of
butcher’s broom (Ruscus aculeatus L.), Asparagus acutifolius L. for which the area is very popular,
ivy (Hedera helix L.) and cyclamen.
      The presence of the various fungal species was noted in several specimens for A. calida in
autumn-winter period from 2018 to 2022, as well as for X. sarnarii, the discovery of L. elseae instead
took place only in October 2020. The specimens of A. calida and X. sarnarii morphologically and
molecularly characterized in this study were harvested in October 2022. The analysed specimens are
conserved as a part of The Mycological Collections of the Natural History Museum and Botanical
Garden of The University of Tartu, Estonia.




                     Fig. 1. – Aerial photograph of the ticket where the three species were found.
                     The site measures about 160 × 45 m (area < 5,000 m2) at 200 amsl and is
                     located alongside Via delle Fossette at 42°09'19" N 12°16'17" E. Maxar
                     (Vivid) imagery captured on 2 Sep 2022. Esri Community Maps Contributors,
                     Esri, HERE, Garmin, Foursquare, GeoTechnologies, Inc, METI/NASA,
                     USGS | Maxar, Microsoft.




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Microscopic analyses
Photographs and notes were taken of fresh basidiomata. Microscopic investigations of dried
basidiomata were carried out using a light microscope Leica DM750 with Leica Application Suite
v4.13.0 at ×1000. Measurements of basidiospores were made in 3% KOH and stained in Congo Red.
Melzer's reagent was used to test amyloid or dextrinoid reaction.

Molecular analyses
Ribosomal DNA-based analysis was performed on the specimens in the frame of the UNITE project
(Kõljalg et al., 2013). DNA extraction, PCR amplification of SSU partial, ITS1, 5.8S, ITS2, LSU
partial regions and sequencing were performed as in Voitk et al. (2018). Initial molecular analyses
consisted in BLAST 2.13.0+ (Camacho et al., 2009) alignment of the obtained sequences against the
Full UNITE+INSD v9.0 dataset for Fungi (Abarenkov et al., 2022).
      In the case of the Xerocomellus specimen, the result of this analysis was used to select six
closely related sequences from X. sarnarii for phylogenetic analyses together with two sequences
from the sister species X. chrysenteron as outgroup. These sequences were aligned using the L-INS-
I method of the MAFFT v7.520 algorithm (Katoh and Standley, 2013) in Aliview 1.27 (Larsson,
2014) and trimmed to include only nucleotides from the ITS1, the 5.8S ribosomal RNA gene and the
ITS2 as determined by ITSx v1.1.3 software (Bengtsson-Palme et al., 2013) resulting in an alignment
of 642 nucleotide sites.
      Maximum Likelihood and Bayesian inference were engaged with HKY+G4 as the model for
nucleotide evolution as suggested by AIC, BIC and AICc criteria applied using ModelTest-NG v0.1.7
(Darriba et al., 2020; Flouri et al., 2014). 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 and 100 replicates for non-parametric bootstrap analysis. 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 500 generations and a 25% burn-in. The final mean standard
deviation of the separated frequencies was 0.002. The phylogenetic tree of the sequences combined
with their Single-Nucleotide Variants (SNVs) were visualised using FigTree v1.4.4 (Rambaut et al.,
2018) and snipit v1.1 softwares (O’Toole and Tomkins-Tinch, 2021) and annotated manually. The
sequences of the three specimens are available in the UNITE database at https://unite.ut.ee/ (Amanita
calida: UDB07673016; Lepiota elseae: UDB0799853; Xerocomellus sarnarii: UDB07673020).


Results

In the autumn-winter periods of the various years, the following fungi typically grew in this quercous
wooded area of the park, among others: Amanita pantherina (DC.) Krombh., Amanita rubescens
Pers., Cantharellus alborufescens (Malençon) Papetti & S. Alberti, Helvella crispa (Scop.) Fr., while
Helvella panormitana Inzenga (UDB0799868) under downy oaks further ahead. Hydnum ibericum
Olariaga, Liimat. & Niskanen and Hydnum ovoideisporum Olariaga, Grebenc, Salcedo & M.P.
Martín, such as the basidiomata described from the chestnut forest of Monte Rocca Romana (Knijn
et al., 2021), Psathyrella bipellis (Quél.) A.H. Sm., Russula rubroalba (Singer) Romagn., sometimes
in proximity with Xerocomellus sarnarii, Russula globispora (J. Blum.) Bon (UDB0799051) and


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Russula violeipes Quél. (UDB07673018), various Tricholoma species. Boletaceae fungi such as
Hemileccinum impolitum (Fr.) Šutara (UDB0799854), Leccinellum crocipodium (Letell.) Della
Magg. & Trassin., Suillellus queletii (Schulzer) Vizzini, Simonini & Gelardi (UDB07673017).
Lignicolous fungi: Lentinellus castoreus (Fr.) Kühner & Maire (UDB0754099) and Lentinus
arcularius (Batsch) Zmitr. (UDB07672111).




         a                                           b                                             c




          d                                           e                                           f
      Fig. 2 – Amanita calida (a) Specimen photographed in December 2018; (b,c) Specimens found in
      October 2022 and environment; (d,e,f) Specimen of October 2022 corresponding to TUF137142.

      Morphological and molecular characteristics of the three species found in Lazio:
Amanita calida (Fig. 2): pileus at first conical to convex, then plane, with a prominent umbo; light
beige, light greyish towards brown, usually obviously marked with concentric colour bands, umbo
darker as well as the band towards the edge of the cap; margin broadly striated to sulcate; whitish
patches of veil remnants rarely present, mostly disappeared. Lamellae rather crowded, free, white;
lamellulae of different lengths which are truncated at ninety degrees. Stipe cylindrical, very long
(much longer than pileus diameter), smooth; ring absent, almost rooting when buried in the ground;
the colour varies from whitish to light ochre, especially in the specimen with the beige-brown cap, in
this case the colour is darker in the centre of the stem and lightens towards the portion under the cap
and towards the base; sac-like, membranous, fragmented, whitish volva brown-spotted. Pileus and
stipe surface may stain darker when handled. Context whitish. Smell indistinct. Associated with
Quercus trees.



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      Microscopy (Fig. 3): Basidiospores subglobose to broadly ellipsoid, rarely globose or ellipsoid
with an apiculum, hyaline, inamyloid, with large lipid droplets inside; (n = 20) 10.4–16.5 × 7.9–12.2
μm, average 11.6 × 9.8 μm, Q = 1.1–1.4, Qav = 1.2. Claviform basidia, with small and large lipid
droplets inside, 4-spored, 43–55 × 13–14 μm. Molecular analysis of a specimen resulted in a 100%
match of the ITS barcode with that of the A. calida type specimen JA-CUSSTA 9324 (OK316924).




                           Fig. 3 – Amanita calida basidia and basidiospores in Congo Red.

Lepiota elseae (Fig. 4): pileus convex, about 6 cm in diameter, whitish to brownish, covered by dark
brown, appressed felty squamules or scales, particularly in the centre of the cap; margin appendiculate
for velar remnants. Lamellae crowded, free, white. Stipe cylindrical, widens a little at the base, fragile,
white, felted, smooth above ring zone; ring silky-floccose, evanescent, white; below ring zone whitish
or brownish floccose-scaly. Context white, thin. Smell indistinct. Saprobic on Quercus litter.
      Microscopy (Fig. 4): basidiospores (n = 20) 11.5–16.3 × 5.2–6.3 μm, average 14.2 × 5.6 μm, Q
= 2.1–3.0, Qav = 2.6, fusiform, amygdaliform, hyaline, dextrinoid. Claviform basidia, 4-spored, 32–
35 × 8.5–9.6 μm. Molecular analysis of a specimen gave a 100% match with the ITS barcode of the
L. elseae type specimen AH-40487 (KP640556).

Xerocomellus sarnarii (Fig. 5): Pileus convex to plane, felty/tomentose, pruinose cuticle cracking,
revealing the whitish or very pale yellowish context with pinkish outlines, reddish spots can be seen
when dry; bronze, brown or reddish brown. Tubes long, lemon yellowish, yellowish-ochre, bluing
when bruised, sometimes decurrent on the stem. Pores same colour as tubes, at first narrow then
become wide, angular, staining slowly dark blue when touched. Stipe cylindrical, wider at the base,
fistulous, ribbed to form a very large gride (lattice), fibrillose, whitish, beige background, sometimes
with intense yellow colours upper part and even dark purple red at the base. Context whitish,
yellowish, soft in pileus, where we have not noticed a change to dark blue even after a certain time
but possibly a slight change to light blue, at the base of the stipe the darkening of the red. Smell
indistinct. Sometimes clusters of three specimens were observed. Associated with Quercus trees.



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         a                                               b                                                                               c
      Fig. 4 – Lepiota elseae: (a,b) Basidiomata; (c) Basidiospores (TUF105926) in Melzer's reagent.




         Fig. 5 – Xerocomellus sarnarii: (a) specimen photographed in December 2020; (b) specimen
         photographed in November 2021; (c,d) specimen photographed in October 2022 corresponding to
         Accession TUF137146; (e) basidiospores in 3% KOH.




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Fig. 6 – On the left, a Bayesian MCMC inferred phylogenetic tree of the ITS1-5.8S-ITS2 sequences of six specimens of
Xerocomellus sarnarii indicated by their INSD accession together with the UNITE sequence UDB07673020 of the
specimen under study. Accession KT271749 is a sequence from the species’ holotype MCVE 28577. AF402139 and
HQ207692 are sequences from the sister species X. chrysenteron used as outgroup. Posterior probabilities (left) and
maximum likelihood bootstrap (right) values of the nodes are indicated. On the right, Single-Nucleotide Variants of the
same sequences without the two sequences from the outgroup are shown with respect to the holotype sequence
(highlighted in the red box). The barcode regions are annotated at the bottom.

        Microscopy (Fig. 5): basidiospores (n = 20) 11.4–14.0 × 4.8–6.8 μm, average 12.7 × 5.7 μm,
Q = 2.0–2.6, Qav = 2.2, fusiform, olive brown with lipid droplets inside. Molecular alignment of the
ITS barcode of a specimen resulted in a close 98.9% match with that of the holotype of X. sarnarii
(MCVE28577; KT271749). Including six sequences from the same species, a monophyletic tree was
obtained (Fig. 6). Observing the Single-Nucleotide Variants (SNVs) associated with these clades,
grouped SNVs seem to exist as a basis of these clades. The specimen found in this study displays an
SNV at locus position 234 in the 5.8S rRNA region, together with four mutations in the ITS1 region
and three in the ITS2 region (Fig. 6). Inclusion of 34 sequences from global soil samples (Tedersoo
et al., 2014; Supplementary Table S1) revealed the existence of various clades in this region but did
not allow to assign the sequence UDB07673020 to a specific phylospecies. UDB07673020 had all
seven SNVs in the ITS1 and ITS2 regions in common with most of these soil sequences with respect
to the sequences included in Fig. 6 but the SNV at locus gatAaag@234 was unique, preventing
UDB07673020 to cluster with other sequences (Supplementary Fig. S1).

Discussion

Several of our findings in the zones surrounding the Bracciano Lake within the Bracciano-Martignano
Regional Natural Park in Lazio (Italy), show similarities with findings in Spain (Knijn et al., 2021).
The small wood area under investigation in this study is located on the slopes of San Bernardino del
Malpasso in Trevignano Romano, between Monte Rocca Romana (Monti Sabatini) and the nearby
Bracciano Lake. The Mediterranean vegetation mainly consists in downy oaks (Q. pubescens),
Turkey oaks (Q. cerris) as deciduous plants and holm oaks (Q. ilex). The three different fungal species
discussed here, were found in this area growing in close proximity to each other, in the same
environment with homogeneous ecology.

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        The volcanic soil of these areas is subacid to neutral, probably not dissimilar to that described
for the zone of finding of A. calida in Spain (Illescas and Plaza, 2022). In fact, the authors indicate
that A. calida shows a preference for associating with Quercus in acidic or neutral soils. The
temperatures in the Bracciano Lake areas are relatively lower than those reported for the spring-
summer harvest areas in Spain. We have found A. calida in October and December of different years
from 2018 on but not in the summer (June-September), when the climate is drier.
        Based on the microscopical analyses it is possible to note the presence of numerous lipid
droplets in A. calida basidia and spores which sometimes completely occupy the internal space. Lipid
droplets act as intracellular storage organelles for neutral lipids and sterols, in most types of cells and
are principally involved in energy homeostasis and lipid metabolism. In fungi, lipid bodies are also
the storage organelle for carotenoid-type pigments which are produced by several species belonging
to all major phyla and, in this case, they assume a yellowish colour (Weber et al., 2002). The
significance of the presence of lipid bodies in fungi is yet under study. For example, some reports of
studies performed on a fungal system like Schizosaccharomyces pombe Lindner propose a novel
stress adaptation mechanism in which heat-induced triacylglycerol synthesis contributes to membrane
rigidisation and reveal that the metabolic crosstalk between membrane and storage lipids facilitates
homeostatic maintenance of the membrane physical/chemical state that resists negative effects on cell
growth and viability in response to heat stress (Péter et al., 2017). Besides, there is increasing evidence
that lipid droplets are involved in cellular detoxification. In fact, trapping of endogenous toxins and
absorbing external lipophilic toxins by lipid droplets are resistance mechanisms to reduce the
production of reactive oxygen species (Chang et al., 2015).
        Lepiota elseae was described from Mediterranean areas in Spain fund in October and associated
with Q. ilex (Caballero et al., 2015). We report the first finding of L. elseae fruitbodies for Italy, apart
from the soil sample there are no data about this finding in the Italian territory, perhaps due to the
similarities of this species with L. clypeolaria. The finding of L. elseae on litter of mixed oaks wood
only occurred once in October 2020, in five years of monitoring the area. We could suggest that
because the area is near inhabitations, rather anthropized and crowded, the area was less visited in
2020 due to the COVID pandemic constraints and remained more unaffected. There are no relevant
differences in the morphology between the A. calida and L. elseae findings of the Quercus wood zone
of the Bracciano-Martignano Regional Natural Park and the specimens described in the literature.
        On the contrary, although it has been reported that the X. sarnarii species is quite rare (Simonini
et al., 2016), we have evidenced the presence in the same site of several specimens in the autumn of
all the years we have visited the mixed Quercus thicket. Some Boletales genera contain compounds
arising from enzyme-controlled dimerization of p-hydroxyphenillpiruvic acid, derived by oxidation
of aromatic amino acids. The derived compounds that turn blue when the fungi are cut or bruised are
quinones (Bertrand, 1901; Bertrand, 1902). In 1968 Edwards demonstrated that the bluing is caused
by enzymatic oxidation of the more hydroxylated pulvinic acid derivatives: variegatic acid
(Beaumont et al., 1968) and the closely related xerocomic acid (Gill et al., 1987). However, many of
the species containing variegatic and xerocomic acid do not turn blue, probably due to loss of the
oxidase enzymes activities that produces the blue chinone methides (Nelsen, 2010). We have not
noticed, in the X. sarnarii, the described change to dark blue in the whitish, yellowish context as
occurs instead in the tubes, even after a certain time but only a slight change to light blue. This means
the specimen could be defined as a morphospecies of X. sarnarii as well as a phylospecies which


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molecular analysis seem to indicate. However, more extensive research is needed to permit the
definition of a new intermediate species between the two sister species X. sarnarii and X.
chrysenteron.

Acknowledgements
Irja Saar was supported by the Estonian Research Council (PRG1170).



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