<em>Punctularia atropurpurascens</em> in the Villa Ada urban Park in Rome, Italy
10.6092/issn.2531-7342/8349
Il fungo lignicolo saprofita Punctularia atropurpurascens, specie che cresce in zone tropicali e subtropicali, è stato rinvenuto su un ceppo marcescente di quercia da sughero, in un bosco misto di sughere, pini e lecci, all’interno del Parco cittadino di Villa Ada a Roma. P. atropurpurascens, con la sua morfologia appariscente e la colorazione violacea, viene considerata una specie rara, interessante anche per le proprietà biologiche delle molecole chimiche che produce. L’identità del fungo è stata confermata attraverso l’analisi del DNA.
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Amalia Ferretti
Italian Journal of Mycology
Villa Ada urban Park
<em>Punctularia atropurpurascens</em> in the Villa Ada urban Park in Rome, Italy
Text.Serial.Journal
https://italianmycology.unibo.it/
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Articles
2018-07-02
2018-06-29
2018-01-29
2020-01-20
29-39
Ital J Mycol
Amalia Ferretti
2018/07/02
10.6092/issn.2531-7342/8349
29
2531-7342
Villa Ada urban Park
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<em>Punctularia atropurpurascens</em> in the Villa Ada urban Park in Rome, Italy
https://italianmycology.unibo.it/article/download/8349/7994
47
A. Knijn, A. Ferretti                                     Italian Journal of Mycology vol. 47 (2018) ISSN 2531-7342
DOI: https://doi.org/10.6092/issn.2531-7342/8349




  Punctularia atropurpurascens in the Villa Ada
  urban Park in Rome, Italy
  _______________________________________________________________________________________

                                  Arnold Knijn1, Amalia Ferretti2

  1M.Sc.  Physics
  2Ph.D.  Biology
  1,2Via Arno 36, 00198 Rome, Italy


  Corresponding Author e-mail: knijn_roma@hotmail.com

  Abstract

     The wood-decay saprophytic fungus Punctularia atropurpurascens, a species normally growing in
     tropical and subtropical zones, has been observed on a marcescent cork oak stump, in a mixed wood
     composed of cork oaks, pines and holm oaks, in the Villa Ada urban Park in Rome, Italy.
     P. atropurpurascens, with its striking morphology and purplish colouration, is considered a rare species,
     also interesting for the biological properties of the chemical molecules it produces. Fungus identity was
     confirmed by DNA-based analysis.

     Keywords: Punctularia atropurpurascens; Punctulariaceae; phylogeny; Villa Ada urban Park


  Riassunto

     Il fungo lignicolo saprofita Punctularia atropurpurascens, specie che cresce in zone tropicali e
     subtropicali, è stato rinvenuto su un ceppo marcescente di quercia da sughero, in un bosco misto di
     sughere, pini e lecci, all’interno del Parco cittadino di Villa Ada a Roma. P. atropurpurascens, con la sua
     morfologia appariscente e la colorazione violacea, viene considerata una specie rara, interessante anche
     per le proprietà biologiche delle molecole chimiche che produce. L’identità del fungo è stata confermata
     attraverso l’analisi del DNA.

     Parole chiave: Punctularia atropurpurascens; Punctulariaceae; filogenesi; Parco urbano di Villa Ada

  Introduction

  The genus Punctularia, corticioid Basidiomycota in the family Punctulariaceae, contains two species:
  Punctularia atropurpurascens (Berkeley & Broome) Petch (Petch, 1916), synonym P. subhepatica, and
  P. strigosozonata (Schweinitz) P.H.B. Talbot. The family Punctulariaceae was created in 1964 by the
  mycologist Marinus Anton Donk in order to accommodate the genus Punctularia, and included in the
  Aphyllophorales group based upon microscopic characteristics (Donk, 1964). Successively, its
  taxonomic positioning was modified and the fungus was collocated into the Corticiales (Larsson,


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DOI: https://doi.org/10.6092/issn.2531-7342/8349



  2007), having molecular analysis pointed out the distance between families Punctulariaceae and
  Corticiaceae in its narrowest sense (Hjortstam, 1995).
  P. atropurpurascens is a purplish coloured, easily observable saprophytic fungus that grows on
  marcescent wood, in prevalence deciduous. Its fruiting body spreads on the growth substrate, its
  superior surface is fluffy, membranous, waxy, with a purplish-brownish, coloured, central zone. The
  fungus can also appear in the form of brown violet coloured crusts, with a whitish margin. An aqueous
  exudate in the form of reddish coloured droplets is often present. The lower surface is adherent to the
  growth substrate. The hymenium is found on the upper surface and it is distributed in a not uniform
  manner, in spots. At a microscopic level, spores (6.5-9×3.5-5µm) are ellipsoid, hyaline or brownish,
  generally described as smooth. Lipid bodies are observed within some spores, that are reported to
  have various functional roles in fungal cells (Wang et al., 2002; Narvaes da Rocha Campos et al., 2008;
  Brondz et al., 2004) as well as in mammalian cells (Ferretti et al., 1999). The hyphal system is
  monomitic, made up of only generative hyphae, which appear branched, septate and with clamp
  connections. Basidia produce four sterigma. Violet crystal encrustations are sometimes present within
  the hymenium, the conidia, produced through sporogenesis, are chained, subglobose or ellipsoid, with
  a violet brown colour (Martini, 2016).
  The species is used for the isolation of several chemical compounds like ‘Phlebiarubrone’ and its
  hydroxylated derivatives, as well as the ‘Phlebiakauranol’ aldehyde, a diterpene with a kaurene core
  that is intermediate in the synthesis of gibberellins, molecules acting as hormones in fungi and plants
  (Dawson, 1968). All these components hold a range of biological activities, such as antifungal,
  antimicrobial and antitumoral (Anke et al., 1986).
  The main role of shikimic acid, an important metabolic intermediate in plants, fungi and
  microorganisms, is to supply aromatic amino acids essential to the organisms. Furthermore, the
  shikimic acid pathway provides intermediate molecules for the biosynthesis of other aromatic
  compounds, that can be divided into simple benzene derivatives, diphenyl benzoquinones and related
  molecules specific to fungi, together with other particular compounds synthesised by fungi and
  superior plants (Turner, 1971). ‘Phlebiarubrone’ (4,7-diphenyl-1,3-benzodioxole-5,6-dione) (Fig. 1) is
  a molecule belonging to the diphenyl benzoquinones or terphenyl quinones and a red pigment initially
  isolated from the fruiting bodies of basidiomycetes, in particular from the mycelium of Punctularia
  strigosozonata (formerly Phlebia strigosozonata) (McMorris et al., 1963). This diphenyl benzoquinone
  molecule and its hydroxylated derivatives, that are violet pigments, have been observed also in P.
  atropurpurascens (Gill et al., 1987; Anke et al., 1984). The importance of these intermediate polycyclic
  terphenyls, isolated from fungi of the genus Punctularia, has been highlighted by other studies that
  noted their capacity to act as bioactive molecules, possessing cytotoxic, antibacterial, anti-
  inflammatory and antioxidant properties (Calì et al., 2003).
  Interest in P. atropurpurascens, a species reported as subtropical, derives from the fact that, while
  being rather rare in Italy (there have been only a few reports from some zones of Sicily; Mondello,
  2017), a specimen has been observed in one of the greater urban parks of Rome: Villa Ada Savoia.




  Fig. 1. ‘Phlebiarubrone’ chemical structure
  Fig. 1. ‘Phlebiarubrone’ struttura chimica

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A. Knijn, A. Ferretti                                        Italian Journal of Mycology vol. 47 (2018) ISSN 2531-7342
DOI: https://doi.org/10.6092/issn.2531-7342/8349



  Materials and methods

  Area of study
  The Villa Ada urban Park (Fig. 2) consists of tree grooves for about 80% of its 160 hectares and the
  specific plant diversity, provided by both native and exotic species, makes it a mix of plots, consisting
  of monospecific or polyspecific stands. The dominant tree species is pine (Pinus nigra J.F. Arnold, P.
  pinaster W. Aiton, P. pinea L.), but groups of locusts (Robinia pseudoacacia L.), cedars (Cedrus atlantica
  (Endl.) Carrière, C. deodara (Roxb.) G. Don, C. libani A. Rich.), olive trees (Olea europaea L.), chestnut
  trees (Castanea sativa Mill.), walnut trees (Juglans californica S. Watson, J. regia L.) and yews (Taxus
  baccata L.) also grow in the meadows or on the borders. Monospecific woods are composed of holm
  oak (Quercus ilex L.) laurel (Laurus nobilis L.) or maple (Acer sp. L.), whereas polyspecific ones by oak
  species like pubescent oak (Quercus pubescens Willd.), cork oak (Quercus suber L.) and holm oak
  (Quercus ilex L.), or mixed conifer plots composed of redwood (Sequoia sempervirens (D. Don) Endl.),
  spruce (Picea abies (L.) H. Karst.) and cypresses (Cupressus sempervirens L.).




  Fig. 2. A glimpse of the Villa Ada urban Park, an area of holm oaks and cork oaks.
  Fig. 2. Uno scorcio del Parco cittadino di Villa Ada, una zona di lecci e sughere.



  Morphological analysis
  Microscope images of a fragment of the fungus were obtained by means of a digital camera AmScope
  MU500 mounted on an AmScope B490 microscope, operating at 800× and 2000× magnifications.
  Samples were analysed dry mount and wet mount in H2O without dye.
  Molecular analysis
  DNA-based analysis was performed on a sample of the fungus at the University of Tartu in Estonia by
  Prof. U. Kõljalg and Dr. I. Saar, in the frame of the “Submit specimens for sequencing” service of the

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A. Knijn, A. Ferretti                                  Italian Journal of Mycology vol. 47 (2018) ISSN 2531-7342
DOI: https://doi.org/10.6092/issn.2531-7342/8349



  UNITE project. DNA extraction, PCR amplification and sequencing were as in Lindahl et al., 2013. In
  particular, a sequence of 915 base pairs was obtained that included the nuclear ribosomal internal
  transcribed spacer (ITS1) region, the 5.8S internal gene, the nuclear ribosomal internal transcribed
  spacer (ITS2) region, and a portion of the 28S Large Subunit LSU. This sequence was subjected to Basic
  Local Alignment Search Tool (BLAST 2.7.1+, Camacho et al., 2009) on the GenBank (Benson et al.,
  2018) and UNITE (Kõljalg et al., 2013) databases.
  To create a dataset for phylogenesis, sequences from various fungi belonging to the Punctulariaceae
  were gathered from UNITE and GenBank databases and one sequence of P. atropurpurascens
  published on the MushroomObserver website. Three species having an increasing phylogenetic
  distance were chosen as outgroups to polarise the phylogenetic tree: Vuilleminia comedens (Nees)
  Maire from the Corticiales (same order), Mycoacia fuscoatra (Fr.) Donk and Lepista sordida
  (Schumach.) Singer from the Agaricomycetes (same class). Their sequences were obtained from
  GenBank (Tab. 1).
  During further bioinformatic analysis, sequences were aligned with the MUSCLE algorithm (Edgar,
  2004), utilising MEGA 7.0.26 software (Kumar et al., 2016) and then fine-tuned manually. For
  phylogenetic analyses, two methods were adopted: Maximum Likelihood and Bayesian inference. For
  Maximum Likelihood in PhyML 3.1 (Guindon et al., 2010), Nearest Neighbour Interchange (NNI) has
  been set up as topology research, initial trees were obtained applying BIONJ and Neighbour-Joining to
  a matrix of pairwise distances and HKY85 was used as the nucleotide substitution model. Otherwise
  for Bayesian inference, MrModeltest 2.3 (Nylander, 2004) suggested SYM+G as the best model for
  nucleotide evolution for all three regions (ITS1, 5.8S and ITS2). This model was applied in MrBayes
  3.2.6 software (Ronquist et al., 2012): the Markow Chain Monte Carlo (MCMC) algorithm was run
  using four chains (three hot and one cold), setting a temperature of 0.2 and iterations for 2×106
  generations on two trees with a diagnose frequency every 5,000 generations and a 25% burn-in. The
  final mean standard deviation of the separated frequencies was 0.004, indicating good convergence.
  The phylogram reporting the posterior probabilities that two branches belonged to the same clade is
  visualised with FigTree v1.4.3.

  Results

  Collection and morphological identification
  The “Sughereta” is an area of the Villa Ada Savoia park of about 7 hectares composed of cork oaks with
  pine trees and holm oaks, in the centre of which a creek flows. Here, in October 2016, on a chopped
  cork oak stump, a P. atropurpurascens specimen proliferated, a showy wood-decay fungus, woolly,
  violet, more or less brightly coloured (Fig. 3). The spotted specimen was located laterally on the moss
  of the bark, on the top surface and, observed from above, on the bark and on areas of the bast, the
  cambium and the growth rings. The stump was photographed again in the autumn (Fig. 3a-b) of the
  following year, when P. atropurpurascens had a cottony morphology, feathery, with variable lilac tones,
  almost white in the most external parts and, in this period of maximum growth, presented reddish
  aqueous droplets, presumably containing the red pigment ‘Phlebiarubrone’. During the winter months,
  on the contrary, the fungus (Fig. 3c) spread out on the bark of the stump as a compact layer, was less
  woolly, with several zones dried out, and had an intense violet colour. During spring it appeared dry,
  dark-brown, adherent to the bark but partly powdered, and probably occurred on the inside of the
  stump (Fig. 3d). These observations indicate that the fungus undergoes strong morphological seasonal
  variations and, in any case, is persistent throughout the year on the colonised host.

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DOI: https://doi.org/10.6092/issn.2531-7342/8349




  Fig. 3. Morphological characteristics of P. atropurpurascens in different seasonal periods (a, b: autumn; c: winter;
  d: spring, summer).
  Fig. 3. Variazioni nella morfologia di P. atropurpurascens nei diversi periodi stagionali (a, b: autunno; c: inverno;
  d: primavera, estate).

  Optical microscopic characteristics, performed on violet filaments, are consistent with what reported
  in the literature (Martini, 2016; Mondello, 2017). Hymenial structures with short branches, a
  monomitic hyphal system and chained conidia. The spores show an ellipsoid form and their walls
  appear frequently brownish; on the inside of some spores both lipid bodies and pinkish droplets are
  found (Fig. 4).
  Identification of the fungus was not immediate. This because another species exists, very similar
  macroscopically, differing little from the morphological description of P. atropurpurascens:
  Hypochnella violacea Auersw. ex J. Schröt.. This latter species is also not very widespread, considered
  from rare to very rare. In the Czech Republic (Holec et al., 2006) and in The Netherlands (Arnolds et
  al., 2008) it is included in a list of threatened species, while in Italy it has been proposed for inclusion
  in the European Red List (Saitta et al., 2011).
  The discrimination among the two species is not clear even at microscopic level. Differences between
  P. atropurpurascens and H. violacea have been described regarding spores (thin walls rather than
  thick, hyaline or brownish, no-amyloid in the first species, brownish violet and weakly amyloid in the
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DOI: https://doi.org/10.6092/issn.2531-7342/8349



  other species); hyphae, with clamp connections occurring in the first and absent in the second; length
  of basidia (40-50 [-80] μm against less than 30 μm) (Hjortstam et al., 1987). In the various samples
  examined (Fig. 4), microscopy of the mycelium did not seem to be dissimilar from what is reported for
  both species and hyphae presented sporadic clamp connections. The spores showed an ellipsoid form,
  with a smooth wall, neither thin nor thick, with a prevalently brownish-violet colour.




  Fig. 4. P. atropurpurascens mycelium and spores. Bar length = 10 µm.
  Fig. 4. Morfologia di micelio e spore di P. atropurpurascens. Lunghezza della barra = 10 µm.
  Molecular identification
  Molecular methods were applied to obtain a definitive identification. Ribosomal DNA sequencing is
  largely used for identification and systematics purposes. The target region was a fragment of the rRNA
  operon located between 18S and 28S rRNA genes, including the internal 5.8S rRNA gene and the
  hypervariable, flanking ITS1 and ITS2 spacers. This region provides superior taxon resolution
  compared to other popular DNA markers and it has been therefore proposed as the consensus primary
  fungal barcode (Schoch et al., 2012). The genomic sequence of P. atropurpurascens (UNITE acc. no.
  UDB034596) was subjected to a BLAST search for the highest homologies in the GenBank and UNITE
  databases, providing a positive identification of the fungus (similarity=99%; bit-score=1026; E-
  value=0; gaps=0%).
  Based on this outcome, a dataset was created for further analysis, combining the sequences of various
  fungi belonging to the Punctulariaceae. Unfortunately, a sequence of H. violacea could not be
  introduced in this dataset because no sequence of this species has been deposited in neither database.

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DOI: https://doi.org/10.6092/issn.2531-7342/8349



  The final dataset with the gaps added during the alignment process, resulted in a selection of 26
  sequences with a length of 1334 base pairs (Tab. 1). For phylogenetic analysis, two methods were
  adopted: Maximum Likelihood and Bayesian inference. Both methods produced substantially the same
  tree (only the phylogram obtained through Bayesian inference is shown here) (Fig. 5). The sequence
  from the newly found fungus in the urban park in Rome finely fitted into the P. atropurpurascens clade.
  In detail, the sequence is identical to the sequence of a finding in California in a contiguous way along
  the latter’s length of 432 base pairs, but dissimilar by 2 and 4 substitutions and 4 gaps from the
  sequences of the findings in Italy, which in turn are one substitution without gaps diverse from the
  American sequence.




  Fig. 5. Phylogenetic tree based upon alignment of the nuclear ribosomal ITS sequences reported in Table 1. In
  bold the newly sequenced sample.
  Fig. 5. Albero filogenetico basato sull'allineamento delle sequenze del gene nucleare ITS indicate nella Tabella 1.
  In grassetto il nuovo esemplare sequenziato.

  Discussion and conclusions

  Before the present report, Punctularia atropurpurascens had been found only three times in Italy, all
  reports being from Sicily (Mazzaforno and Monte Pellegrino in the province of Palermo, Birribbaida in
  the province of Trapani; metadata from the UNITE database, acc. nos. UDB028311, UDB028313 and
  UDB028312 respectively). The occurrence of this fungus in an urban park in Rome is remarkable,
  considering it is regarded a tropical or subtropical species. This finding is thus the fourth observation
  of P. atropurpurascens in Italy and the first in the Latium region. What's important to notice, is that all
  reports were made in a ten-year time frame. This fungus importance lies not only in its rareness
  (Mondello, 2017), but also in the potential of its molecules and pigments such as ‘Phlebiarubrone’ and
  ‘Phlebiakauranol’ aldehyde, a characteristic common to other members of the of the genus Punctularia,
  holding different biological activities and great prospects for biomedical applications (Anke et al.,
  1986; Calì et al., 2003; Järvinen et al., 2016). For instance, in E. coli the antibacterial activity of
  ‘Phlebiarubrone’ has been correlated with its capacity to inhibit DNA transcription, translation and
  replication, thus it was supposed that its mechanism of action could be related to protein synthesis

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DOI: https://doi.org/10.6092/issn.2531-7342/8349



  inhibition or DNA replication (Järvinen et al., 2016). Furthermore, some proteins that bind
  carbohydrates and glycoconjugates such as lectins, have been purified from the mycelium of P.
  atropurpurascens, in particular a new lectin with specificity to N-acetylglucosamine (Alborés et al.,
  2014). Lectins are implicated in several biological processes. For instance, in fungi they induced the
  formation of mycelium structures and fruiting bodies and facilitate the adherence of parasitic fungi to
  the host substrate. These fungal lectins have biological activity also in animals and humans, being
  involved in inflammation and in mechanisms of immune defence. On the other hand, articles studying
  the exploitation of basidiomycetes for their capacity to degrade xenobiotics like insecticides and
  pesticides, report that P. atropurpurascens is susceptible to the endosulfan insecticide and does not
  grow in presence of this compound (Rivero Machado et al., 2017).
  DNA analysis finally identified the specimen unequivocally as P. atropurpurascens. It’s striking that the
  macro- and microscopic differences with H. violacea are smaller than the morphological variations of
  the same specimen during its life cycle. Since in systematics they are placed in different genera,
  families and orders of the Agaricomycetes, we can hypothesise that the strong resemblance of these
  two species could be a case of evolutionary convergence. The morphological variations of P.
  atropurpurascens seem related with different biochemical mechanisms at various growth stages as
  well as with different environmental and climatic conditions. Moreover, the fruiting body’s persistence
  on the oak stump in any period of the year made it possible to observe these variations with the
  seasonal changes. A certain seasonality seems to exist, probably as a function of humidity since the
  specimen anticipated the transformation of the fluffy stage to the crustose stage in the extremely dry
  spring of 2017 with respect to the more rainy spring of 2018.
  The Villa Ada Park, due to its extent and variety of environments, turned out to be rich in mycological
  diversity. Indeed, many fungal genera and species were observed there, some of which are infrequent
  and therefore not much spotted in wild natural environments, like Myriostoma coliforme (Dicks.)
  Corda and Podoscypha multizonata (Berk. & Broome) Pat. (Knijn et al., 2017). The question arises, how
  these fungal species ended up growing in a city park; probably through the planting of trees
  originating from remote localities, or through the supply of soil and bark chips, as happened for
  Leratiomyces ceres (Cooke & Massee) Spooner & Bridge, an Australian species that has been conveyed
  in this way into England and hence in other European green spaces (Noordeloos, 2011), including the
  Villa Ada Park (Knijn et al., 2017). In this connection, it turns interesting that the sequence of the
  fungus is identical to an example from California and slightly different from findings of the same
  species in Sicily. The occurrence of this uncommon and important species in a green urban space, in
  areas to some extent neglected and degraded, poses a nontrivial aspect of the problem of the cure,
  conservation and requalification of Parks and their vegetation in large urban areas. Certainly, it is of
  the utmost importance that fungal biodiversity is included in whatsoever Urban Green Space
  Maintenance Plan.

  Acknowledgements

  We would like to thank Prof. U. Kõljalg and Dr. I. Saar of the University of Tartu in Estonia for
  sequencing a portion of the rDNA of the fungus. Furthermore, we thank both of them and Prof. T.W.M.
  Kuyper, of the University of Wageningen, The Netherlands, for valuable advice, and the reviewer of the
  manuscript for precious suggestions.




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DOI: https://doi.org/10.6092/issn.2531-7342/8349



  Tab. 1. Sequences used in the phylogenetic analysis. In bold the newly sequenced sample. (*) The sequences are
  misidentified as P. strigosozonata in the UNITE database (Irja Saar, personal communication, 23/02/2018).
  Tab. 1. Sequenze utilizzate per le analisi filogenetiche. In grassetto il nuovo esemplare sequenziato. (*) Le
  sequenze sono state erroneamente identificate come P. strigosozonata nel database UNITE (Irja Saar,
  comunicazione personale, 23/02/2018).

              Species                          Origin           DataBase        Accession              Declared sequence                  Length
  Dendrocorticium polygonioides      Finland                GenBank             HM046877     18S partial, ITS1, 5.8S, ITS2 partial         662 bp

  Dendrocorticium polygonioides      Finland, Kainuu        UNITE               UDB031607 18S, ITS1, 5.8S, ITS2                            596 bp

  Dendrocorticium sp                 USA, Wisconsin         GenBank             KJ140586     18S partial, ITS1, 5.8S, ITS2 partial         564 bp

  Dendrocorticium sp                 USA, Wisconsin         GenBank             KJ140606     18S partial, ITS1, 5.8S, ITS2 partial         509 bp

  Dendrocorticium sp                 USA, Wisconsin         GenBank             KJ140694     18S partial, ITS1, 5.8S, ITS2 partial         494 bp

  Punctularia atropurpurascens       Italy, Rome            UNITE               UDB034596 18S partial, ITS1, 5.8S, ITS2, 28S partial       915 bp

  Punctularia atropurpurascens       USA, California        Mushroom Observer   198256       ITS                                           432 bp

  Punctularia atropurpurascens (*)   Italy, Palermo         UNITE               UDB028311 ITS1, 5.8S, ITS2                                 865 bp

  Punctularia atropurpurascens (*)   Italy, Trapani         UNITE               UDB028312 ITS1, 5.8S, ITS2, LSU D1, LSU D2                1115 bp

  Punctularia atropurpurascens (*)   Italy, Palermo         UNITE               UDB028313 ITS1, 5.8S, ITS2, LSU D1, LSU D2                1115 bp

  Punctularia atropurpurascens       USA                    GenBank             KP814559     18S partial, ITS1, 5.8S, ITS2, 28S partial    668 bp

  Punctularia atropurpurascens       Costa Rica, San José   UNITE               UDB024827 ITS1, 5.8S, ITS2                                 653 bp

  Punctularia strigosozonata         Japan, Iwate           GenBank             AB374288     18S, ITS1, 5.8S, ITS2, 28S                    595 bp

  Punctularia strigosozonata         ?                      GenBank             DQ398958     ITS1 partial, 5.8S, ITS2 partial              551 bp

  Punctularia strigosozonata         China                  GenBank             EF488381     18S partial, ITS1, 5.8S, ITS2, 28S partial    607 bp

  Punctularia strigosozonata         USA, Wisconsin         GenBank             KU668972     18S partial, ITS1, 5.8S, ITS2, 28S partial    621 bp

  Punctularia strigosozonata         Gabon                  GenBank             KY449385     ITS1 partial, 5.8S, ITS2, 28S partial         599 bp

  Punctularia strigosozonata         Gabon                  GenBank             KY449386     ITS1 partial, 5.8S, ITS2, 28S partial         607 bp

  Punctularia strigosozonata         Gabon                  GenBank             KY449387     ITS1 partial, 5.8S, ITS2, 28S partial         607 bp

  Punctularia strigosozonata         Estonia, Valga maakond UNITE               UDB016433 ITS1, 5.8S, ITS2                                 564 bp

  Punctulariopsis cremeoalbida       USA                    GenBank             KR494275     18S partial, ITS1, 5.8S, ITS2, 28S partial    692 bp

  Punctulariopsis efibulata          USA                    GenBank             KR494276     18S partial, ITS1, 5.8S, ITS2 partial         599 bp

  Punctulariopsis subglobispora      Argentina              GenBank             HM046917     18S partial, ITS1, 5.8S, ITS2, 28S partial    600 bp

  Vuilleminia comedens               Russia                 GenBank             MG609033     18S partial, ITS1, 5.8S, ITS2, 28S partial    658 bp

  Mycoacia fuscoatra                 USA, Massachusetts     GenBank             KY948754     18S partial, ITS1, 5.8S, ITS2, 28S partial    639 bp

  Lepista sordida                    India                  GenBank             MF384327     ITS1, 5.8S, ITS2, 28S partial                 691 bp




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A. Knijn, A. Ferretti                                   Italian Journal of Mycology vol. 47 (2018) ISSN 2531-7342
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