Studio di due specie a rischio (<em>Myriostoma coliforme</em> e <em>Battarrea</em> sp.) che condividono lo stesso microhabitat
10.6092/issn.2531-7342/10039
Basidiomi di Myriostoma coliforme e Battarrea sp. sono stati trovati all’interno di uno stradello fiancheggiato da filari di cipressi, nel borgo di Trevignano Romano, in provincia di Roma. Inoltre, altri esemplari di Myriostoma coliforme sono stati raccolti nel parco urbano di Villa Ada, nella città di Roma. L’attribuzione di specie attraverso l’analisi morfologica dei basidiomi del genere Myriostoma è difficoltosa e per il genere Battarrea ancora in fase di indagine, sono state quindi effettuate analisi del genoma sui campioni esaminati in questo studio. Invero, è risultato che il basidioma Battarrea non appartiene alla specie coincidente phalloides/stevenii. Sebbene entrambi i generi siano ampiamenti diffusi, le loro popolazioni sono sporadiche e di piccole dimensioni perché condividono un habitat molto particolare. Tanto è vero che entrambe le specie oggetto di studio sono idonee per la valutazione ai fini dell’inserimento nelle future Red List europee dei funghi minacciati di estinzione. Il luogo di raccolta potrebbe essere utile per lo studio degli aspetti ambientali e microclimatici del loro habitat di sviluppo e, di conseguenza, per la conservazione di queste due rare specie
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Arnold Knijn
Italian Journal of Mycology
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On two endangered species (<em>Myriostoma coliforme</em> and <em>Battarrea</em> sp.) sharing the same microhabitat
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Ital J Mycol
Arnold Knijn
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Voitk A, Saar I, Trudell S, Spirin V, Beug M, Kõljalg U (2018) Polyozellus multiplex (Thelephorales) is a species complex containing four new species. Mycologia 109(6):975-992. https://doi.org/10.1080/00275514.2017.1416246
On two endangered species (<em>Myriostoma coliforme</em> and <em>Battarrea</em> sp.) sharing the same microhabitat
https://italianmycology.unibo.it/article/download/10039/10485
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Ferretti et al.                                                                       Italian Journal of Mycology 49:1-7 (2020)

DOI: https://doi.org/10.6092/issn.2531-7342/10039

Short Note

         On two endangered species (Myriostoma coliforme and
             Battarrea sp.) sharing the same microhabitat
                                     Amalia Ferretti1, Irja Saar2, Arnold Knijn1
1
Via Arno 36, 00198 Roma, Italy
2
Institute of Ecology and Earth Sciences, University of Tartu, Ravila 14A, 50411 Tartu, Estonia

Corresponding author e-mail: knijn_roma@hotmail.com
Received 20/11/2019; accepted 26/01/2020


Abstract
Specimens of Myriostoma coliforme and Battarrea sp. were found in the same habitat in a cypress-aligned alleyway in
the town of Trevignano Romano in the province of Rome, Italy. Furthermore, other specimens of M. coliforme were
found in the Villa Ada Urban Park in the city of Rome. Species attribution by morphological features is difficult in
Myriostoma and still under investigation in Battarrea and was therefore also assessed by DNA-based analysis.
Specifically, the Battarrea sample was found to not belong to the phalloides/stevenii complex. Although both genera are
found widely spread, only small and scattered populations exist because their habitats are quite peculiar and in some terms
coinciding. In fact, both species are presently relevant for assessment for the future European Red list of endangered
macrofungi. The places of discovery could be useful for the study of the environmental and microclimatic aspects for the
conservation of these two rare species.

Key words Red List; conservation; habitat; Cupressus sempervirens; microclimate; ITS



Introduction

The European Council for the Conservation of Fungi (ECCF) promotes the protection of fungi. Its
activities comprise the redaction of a European Red List of endangered macrofungi. In the most recent
evaluation of candidates (ECCF, 2019), two fungal species are present with the status of “relevant for
assessment for the future European Red list”: Myriostoma coliforme (Dicks.) Corda (Agaricomycetes,
Geastrales) and Battarrea phalloides (Dicks.) Pers. (Agaricomycetes, Agaricales). Both species are
saprotrophic fungi with a wide global distribution but with small and scattered populations restricted
to a few sites, mostly sandy soils. Although their final status could be “Least Concern” globally, at
the local level both species are present in national Red Lists with a status ranging from “Vulnerable”,
“Endangered”, “Critically Endangered” up to “Extinct” and are generally considered rare (Dahlberg
and Croneborg, 2003; Amalfi, 2011; Fraiture and Otto, 2015).
       Myriostoma Desv. was long considered a monotypic genus, however recently five species have
been defined (Sousa et al., 2017; Sousa et al., 2019). We have found specimens of M. coliforme for
the first time in 2016 in Rome, inside the Villa Ada Urban Park. They grew under olive trees nearby
a secular cypress and were present in this same location every subsequent year. Furthermore, in
spring/summer 2019 we found other specimens of M. coliforme in a cypress-aligned alleyway in
Trevignano Romano, a village inside the Bracciano-Martignano Regional Natural Park.
       The taxonomy of the genus Battarrea has undergone significant changes in time, especially
upon molecular analyses (Ivančević et al., 2016). Most interestingly, by DNA barcoding the
synonymy of B. stevenii with B. phalloides has been ascertained (Martín and Johannesson, 2000). At
present, four distinct clades are included in the genus, one of which coincident with the type species
B. phalloides (Martín et al., 2013).
       Concomitantly with the finding of the M. coliforme specimens in Trevignano Romano, we
noted specimens of Battarrea in the same cypress-aligned alleyway leading towards the Bracciano
Lake.
ISSN 2531-7342 - Department of Agricultural and Food Sciences in partnership with the Italian Mycological Society (UMI)
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Ferretti et al.                                                         Italian Journal of Mycology 49:1-7 (2020)




      These findings, together with the description of the two sites, represent an addition to the
knowledge of the microambients of these two endangered species, useful for their preservation and
in the specific case of the genus Battarrea also for the research of distinctive markers between the
four clades recognised by DNA sequence data. With respect to the Battarrea, our intent is to study a
major number of specimens, also in collaboration with interested researchers/mycologists, from a
genetic and biochemical point of view in relation to their habitat because it does not seem a
coincidence that other specimens found in Italy, also associated with cypresses in proximity of a body
of water, belong to the same clade.

Materials and Methods

Collection of samples
Trevignano Romano is a small town, municipality of the Metropolitan City of Rome Capital and part
of the Bracciano-Martignano Regional Natural Park. It is located about 35 km north-west of Rome
on the north coast of the volcanic Bracciano lake and south at the toe of the slope of Monte Rocca
Romana (612 m asl). The site of collection (Fig. 1, 42°09'36" N 12°13'44" E, 173 m amsl) is a 140
meters long cypress-aligned alleyway perpendicular to the lake’s coast connecting the lake front with
an interior road. The alleyway is private but freely accessible; it is a dirt road, covered by cypresses’
debris. The collections examined are indicated in Table 1.

                                          road
                         N




                                     road
                                        beach

                        a               lake      b
                       Fig. 1 – Satellite (a) and land (b) images from the site collection of the
                       specimens in Trevignano Romano. The yellow circle (42°09'36" N
                       12°13'44" E) indicates the site of Battarrea sp., red circles of M.
                       coliforme. (Source of the satellite image: Esri, i-cubed, USDA, USGS,
                       AEX, GeoEye, Getmapping, Aerogrid, IGN, IGP, UPR-EGP and the
                       GIS User Community)



Morphological analysis
Microscope images of the fungi were obtained with a digital camera AmScope (Irvine CA, USA)
MU500 mounted on an AmScope B490 microscope, operating at ×800 and ×2000 magnifications.
Samples were analysed dry mount.



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Ferretti et al.                                               Italian Journal of Mycology 49:1-7 (2020)




  Table 1 - The collected specimens
   Species                 Fungarium/ UNITE codes   Host                    Date       Site

                                                    Under Olea and
   Myriostoma coliforme    TU114952/UDB0754090                           2018/10/17    Rome
                                                    Cupressus
                                                                                       Trevignano
   M. coliforme            TU114971/UDB0778014      Under Cupressus      2019/04/14
                                                                                       Romano
                                                                                       Trevignano
   Battarrea sp.           TU114972/UDB0778015      Under Cupressus      2019/05/12
                                                                                       Romano


Molecular analysis
Ribosomal DNA-based analysis was performed on all three specimens of the fungi 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). Sequences were aligned with the MUSCLE v3.8.425 algorithm (Edgar, 2004) in Aliview 1.26
software (Larsson, 2014) and fine-tuned manually. The Basic Local Alignment Search Tool (BLAST
2.8.1+; Camacho et al., 2009) was used to assess sequence identity. For phylogenetic analyses,
Bayesian inference was applied as before (Knijn and Ferretti, 2018) except that in this case GTR was
used as the model for nucleotide evolution as suggested by MrModeltest 2.3 (Nylander, 2004).
Phylogenetic trees were drawn using FigTree v1.4.3 (http://tree.bio.ed.ac.uk/). The newly obtained
sequences were submitted to UNITE (accession numbers in Table 1).

Results, discussion and conclusion

Morphological characteristics
Both samples of M. coliforme basidioma display the typical morphological aspects of the genus (Fig.
2a). Basidiomata were globose/sub-globose with 7 to 9 exoperidial rays (or laciniae). The
exoperidium was composed of an ochre or light brown mycelial layer, sometimes made up of irregular
patches, a fibrous whitish-cream coloured layer and a light or dark yellow coloured
pseudoparenchymatous layer. The endoperidial globose body (1.5 × 1.0 cm) was grey-light brown
coloured, covered with evident minute warts. The number of stomata ranged from 9 to 17, and the
ochre pedicels were laterally compressed. The basidiospores (Fig. 2b) were light brown and globose
and measured 5.5 ± 0.4 µm (n = 18) in both specimens.
      The samples of Battarrea sp. (Fig. 2c) were observed from spring to autumn. Their capitulum
was initially covered by a white peridium surrounding the gleba, attached to the stem. In our
specimens, the diameter of the capitulum measured about 3.5 cm. In mature samples the upper part
of the veil was detached from the brown spore-bearing gleba. The basidiospores (Fig. 2d) were
spherical or sub-spherical, brown and decorated, 4-6 µm in diameter. Elaters were sometimes evident
at microscopic analyses (14-24 × 5 µm), cylindrical with spiral thickenings.
      Mature stems protrude from the exoperidium, a volva-like veil of whitish colour, initially
gelatinous then dry. The fibrillose and scaly stems changed colour from whitish to brownish due to
the spores, were about 11 cm tall, buried in the terrain for about 4 cm together with the 2.5 cm long
volva. The stem becomes hollow and woody with age; some specimens persisted in this dry state for
several months.

Molecular data
Myriostoma coliforme is found mainly in Europe, but also in some sites in the United States; M.
capillisporum (V.J. Staněk) Suz, A.M. Ainsw., Baseia & M.P. Martín is found in South Africa, M.
calongei Baseia, J.O. Sousa & M.P. Martín in Brazil and Argentina, M. areolatum (Calonge & M.
Mata) M.P. Martín, J.O. Sousa & Baseia in Costa Rica. These species differ from one another for
morphological and microscopical characteristics as well as biogeography (Sousa et al., 2017).
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Ferretti et al.                                                        Italian Journal of Mycology 49:1-7 (2020)




Recently a new species has been described, M. australianum J.O. Sousa, Baseia & M.P. Martín,
similar to M. capillisporum (Sousa et al., 2019).
      Both Myriostoma specimens found by us in the two sites morphologically resembled M.
calongei, but BLAST searches and phylogenetic analysis (Fig. 3) confirmed them to be M. coliforme.




     a                                                     b




      c                                                    d
    Fig. 2 - Basidioma of M. coliforme collected in Trevignano Romano under Cupressus sempervirens (a);
    basidiospores and capillitium of M. coliforme (b); basidioma of Battarrea sp. collected in Trevignano Romano
    under Cupressus sempervirens (c); basidiospores and an elater of Battarrea sp. (d). Bar = 10 μm

       The sample found in Rome (accession number UDB0754090) together with other three
specimens is somewhat distant from the central clade of M. coliforme sequences due to only one
single nucleotide polymorphism in the ITS2 region about 10 nucleotides away from the LSU region
(a Cytosine transformed in Adenine in these four samples).
       A relevant aspect of the Battarrea sample is that phylogenetic analysis (Fig. 4) proved that the
specimen did not belong to the phalloides/stevenii complex (clade B2 in the phylogenetic tree) but
fitted well into what has been defined clade A (Martín et al., 2013), which has not yet been assigned
a species name. No evident morphological or geographical differences, specific and reproducible,
have been found between the specimens belonging to the four clades (Garrido-Benavent, 2014),
indicating that the genetic variations could be due to cryptic characteristics. It would be an interesting
study to assert if the differences at the genetic level are correlated with a particular habitat or growth
substrate and thus to a relative different enzymatic content.




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Ferretti et al.                                                             Italian Journal of Mycology 49:1-7 (2020)




Fig. 3 - Phylogenetic tree with posterior probability values obtained through Bayesian inference based upon alignment of
the nuclear ribosomal ITS sequences of Myriostoma species, identified by their UNITE/GenBank accession number and
associated species. The sequence from M. areolatum was used to root the phylogenetic tree. The new samples described
in the article are indicated in bold. Bayesian posterior probabilities are reported on each branch. The final mean standard
deviation of the separated frequencies was 0.0076.




Fig. 4 - Phylogenetic tree with posterior probability values obtained through Bayesian inference based upon alignment of
the nuclear ribosomal ITS sequences of Battarrea specimens, identified by their UNITE/GenBank accession number.
Sequences from Chlamydopus meyenianus (AF097755) and Tulostoma beccarianum (AF097752) were chosen as
outgroups to root the tree. The new sample described in the article is indicated in bold. Clades are indicated as A, B1, B2
and C according to Martín et al. (2013). Bayesian posterior probabilities are reported on each branch. The final mean
standard deviation of the separated frequencies was 0.0053.


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Ferretti et al.                                                  Italian Journal of Mycology 49:1-7 (2020)




Conservation
An interesting result of this study is that specimens of M. coliforme and Battarrea sp. clade A were
growing contemporarily in a cypress-aligned alleyway in Trevignano Romano, probably because they
share a particular type of habitat. In Italy, both are frequently associated with cypress trees but
certainly other ambient factors like soil composition are influent since especially central Italy is very
rich in cypress groves. However Myriostoma and Battarrea specimens are not equally frequently
found. The same concomitance of these two species has previously been reported in Italy for the
Polvese island in the Trasimeno lake (Pecoraro et al., 2014; Angelini et al., 2016). Similarly, in this
case the Battarrea specimen belonged to clade A (KF780873 in Fig. 4). The Polvese island was
declared scientific and didactic park as well as a territorial laboratory for sustainable development
inside the Regional Park of the lake in 1995. Furthermore, in 2012 the island was included in the
European ecological network “Natura 2000” as a site of community interest in the form of special
conservation zones (SCZ) (Angelini et al., 2016). Thus, in the latter case the habitat is well protected
and could serve as a virtuous example of conservation.
      The situation in Trevignano Romano results more complicated, despite the area making part of
a Regional Natural Park. In fact, as a private property with public access, the habitat could be the
object of various threats. A European Red List of endangered macrofungi or at least an Italian Red
List would be important to convince local authorities to act for the preservation of such a particular
environment. An Italian Red List has not yet been redacted, although efforts in this direction are being
made based upon the Checklist of Italian fungi (Onofri et al., 2005) for instance by the Italian Institute
for Environmental Protection and Research (ISPRA; Siniscalco et al., 2019) or at a Regional level in
Tuscany (Antonini et al., 2006) and in Umbria (Angelini et al., 2017).
      The main point is that, especially in the light of the recent division in species/clades of both
genera which makes each individual species even more rare, there is a strong need to maintain and
protect current populations. A comparison between the two places of discovery could proof valuable
for the study of the particularities of their habitat in terms of environment and microclimate.

Acknowledgements

Irja Saar was supported by the Estonian Research Council (IUT20-30), the European Regional
Development Fund (Centre of Excellence EcolChange).

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