<em>Schizophyllum amplum</em> (Agaricales, Schizophyllaceae): un raro basidiomicete da Malta e dall’Estonia
10.6092/issn.2531-7342/9426
Schizophyllum amplum (Agaricales, Schizophyllaceae) è stato trovato per la prima volta a Malta ed in Estonia. I campioni maltesi sono stati esaminati morfologicamente e confrontati con quelli precedentemente pubblicati. Le regioni ITS del DNA ribosomiale di tutti i campioni analizzati sono stati sequenziati dopo amplificazione PCR con i primer ITS1F e ITS4. Le sequenze sono state confrontate con quelle presenti nei repositori pubblici, tramite il software BLASTn. Le sequenze ITS dei campioni maltesi ed estoni avevano una similarità del 99% ma mostravano alcune differenze dalle altre sequenze di S. amplum (L43381 e L43382) depositate in GenBank. Un approccio multigenico con un maggior numero di campioni dovrebbe essere applicato per definire i limiti genetici delle specie di Schizophyllum attualmente accettate.
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2531-7342
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Irja Saar
Italian Journal of Mycology
phylogeny
<em>Schizophyllum amplum</em> (Agaricales, Schizophyllaceae): A rare Basidiomycete from Malta and Estonia
Text.Serial.Journal
https://italianmycology.unibo.it/
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Articles
2019-08-08
2019-05-10
2019-03-01
2020-01-20
50-56
Ital J Mycol
Irja Saar
2019/08/08
10.6092/issn.2531-7342/9426
50
University of Tartu
2531-7342
phylogeny
en
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Wojewoda W (2006) Auriculariopsis albomellea (Agaricales, Schizophyllaceae) new for Poland, Acta Mycologica 41(1): 49–54. https://doi.org/10.5586/am.2006.008
<em>Schizophyllum amplum</em> (Agaricales, Schizophyllaceae): A rare Basidiomycete from Malta and Estonia
https://italianmycology.unibo.it/article/download/9426/9590
48
Sammut et al.                                                             Italian Journal of Mycology 48: 50-56 (2019)

DOI: https://doi.org/10.6092/issn.2531-7342/9426

Short note

       Schizophyllum amplum (Agaricales, Schizophyllaceae):
           a rare Basidiomycete from Malta and Estonia
                              Carmel Sammut1, Pablo Alvarado2, Irja Saar3
1
 216/1 Joseph Flats St., Rue d’Argens, Gzira, GZR1367, Malta
2
 ALVALAB, La Rochela 47, 39012 Santander, Spain
3
 Institute of Ecology and Earth Sciences, University of Tartu, 14A Ravila St., 50411 Tartu, Estonia

Corresponding author email: stizzy2499@yahoo.com
Received 10/05/2019; accepted 22/07/2019


Abstract
Schizophyllum amplum (Agaricales, Schizophyllaceae) is reported for the first time in Malta and Estonia. The collections
from Malta were examined morphologically and compared with previously published accounts of this species. The
specimens from Estonia and Malta were sequenced after PCR amplification with ITS1F and ITS4 for ITS region. The
sequences were then compared to other sequences deposited in public databases using BLASTn. Genetically the ITS
rDNA data obtained from the Maltese and Estonian specimens are 99% similar but show some differences from other
sequences of S. amplum (L43381 and L43382) deposited in GenBank. A multigenetic approach with a larger number of
specimens should be employed to define the currently accepted genetic limits for Schizophyllum species.

Key words: Auriculariopsis ampla, ITS rDNA, mycobiota, Mediterranean, morphology, phylogeny


Introduction

Schizophyllum Fr. is a fungal genus with lamellar hymenophore classified within family
Schizophyllaceae, order Agaricales, along with the sister genera Fistulina Bull. and Porodisculus
Murrill (Singer, 1949; Binder et al., 2002; Moncalvo et al., 2002; Bodensteiner et al., 2004; Matheny
et al., 2006; Binder et al., 2010). Schizophyllum was erected by Fries (1815) for Schizophyllum
commune Fr., one of the most widespread basidiomycete species, and a model taxon for research
whose complete genome has been already sequenced (Ohm et al., 2010). Most sequences of
Schizophyllum in public databases come from S. commune (99.4% in July 2019), and very few other
species of this genus are represented. Schizophyllum radiatum Fr., S. umbrinum Berk., S. fasciatum
Pat., S. leprieurii Linder, and S. amplum (Lév.) Nakasone are the only other species present in
GenBank. Schizophyllum radiatum was sometimes considered a synonym of S. commune because of
the morphological and ITS rDNA genetic similarity of both species (Cooke, 1961), but recent
multigenic analyses concluded that both represent closely related but independent taxa (Siqueira et
al., 2016). Schizophyllum umbrinum has a brownish surface and striking orange-brown lamellae
(Robledo et al., 2014). Schizophyllum leprieurii has been rarely collected, and was always reported
to resemble S. umbrinum (Linder, 1933; Martin, 1941; Cooke, 1961; Robledo et al., 2014), both
species displaying brownish basidiomata with a whitish attachment disc. Schizophyllum leprieurii
and S. umbrinum have a similar structure of the context and basidia but differ by the slightly larger
and yellowish-brown colored basidiospores (S. leprieurii). Robledo et al. (2014) found also some
genetic differences between two sequences of S. umbrinum and one of S. leprieurii which support
their recognition as distinct taxa. Schizophyllum fasciatum was proposed by Patouillard (1887) to
accommodate specimens collected in Mexico and displaying a zonate pileus and fuliginous lamellae.
All sequences available in public databases come from a single strain (CBS267.60) obtained from a
Mexican specimen.
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Sammut et al.                                                       Italian Journal of Mycology 48 (2019)




      More recently, Nakasone (1996) combined Cyphella ampla Lév., a species with a smooth
hymenophore, into Schizophyllum as S. amplum (Lév.) Nakasone, because of its dimitic hyphal
structure, ellipsoid basidiospores, and also genetic data, in agreement with previous observations of
similarities in the development of the hymenophore by Stalpers (1988). Schizophyllum amplum is
generally found on dead twigs or branches of Populus and Salix, mainly in Europe, but also in North
America, central Asia, Australia and New Zealand. The purpose of the present work is to cite S.
amplum from Malta and Estonia providing a brief morphological and genetic study to support the
identification.

Materials and Methods

Specimens
Two collections of S. amplum from Malta and one from Estonia were examined. The Maltese
specimens are preserved in the herbarium of one of the authors (CS) and the Estonian specimen is
preserved in the fungarium of the University of Tartu (TU).

Morphological studies
Fresh samples were examined with a light microscope. Thin sections and squashed tissue were stained
using Congo Red/Phloxine (1% each in 5% ammonia), Melzer reagent and/or Toluidine blue (1%).
All measurements were taken on fresh material mounted in water with the aid of Piximètre
(downloadable from http://ach.log.free.fr/Piximetre/).

DNA extraction, amplification and sequencing
Total DNA was extracted from dry specimens employing a modified protocol based on Murray &
Thompson (1980). A portion of each sample was blended with the aid of a micropestle in 600 µL
CTAB buffer (CTAB 2%, NaCl 1.4 M, EDTA pH 8.0 20 mM, Tris-HCl pH 8.0 100 mM). The
resulting mixture was incubated for 15 min at 65 °C. A similar volume of chloroform: isoamyl alcohol
(24:1) was added and carefully mixed with the samples until their emulsion. It was then centrifuged
for 10 min at 13,000 g, and the DNA in the supernatant was precipitated with a volume of isopropanol.
After a new centrifugation of 15 min at the same speed, the pellet was washed in cold ethanol 70%,
centrifuged again for 2 min and dried. It was finally resuspended in 200 µL ddH2O. PCR amplification
was performed with the primers ITS1F and ITS4 (White et al., 1990; Gardes and Bruns, 1993) for
ITS region. PCR reactions were performed under a program consisting of a hot start at 95 °C for 5
min, followed by 35 cycles at 94 °C, 54 °C and 72 °C (45, 30 and 45 s respectively) and a final 72 °C
step 10 min. PCR products were checked in 1% agarose gels, and positive reactions were sequenced
with one or both PCR primers. Chromatograms were checked searching for putative reading errors,
and these were corrected.

Phylogenetic analyses
BLASTn (Altschul et al., 1990) was used to select the most closely related sequences from INSD
public databases. Sequences came mainly from Nakasone (1996), Robledo et al. (2014), and Siqueira
et al. (2016). Sequences first were aligned in MEGA 5.0 (Tamura et al., 2011) software with its
Clustal W application and then corrected manually. The final alignment included 233/560 variable
and 59/560 parsimony-informative sites. The alignment was loaded in PAUP* 4.0b10 (Swofford,
2001) and subjected to MrModeltest 2.3 (Nylander, 2004) in PAUP* 4.0b10. Model GTR+G was
selected and implemented in MrBayes 3.1 (Ronquist and Huelsenbeck, 2003), where a Bayesian
analysis was performed (data partitioned, two simultaneous runs, six chains, temperature set to 0.2,
sampling every 100th generation) until convergence parameters were met after about 0.51 M
generations, standard deviation having fell below 0.01. Finally, a full search for the best-scoring
maximum likelihood tree was performed in RAxML (Stamatakis, 2006) using the standard search


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algorithm (data partitioned, GTRMIX model, 2000 bootstrap replications). Significance threshold
was set above 0.95 for posterior probability (PP) and 70% bootstrap proportions (BP).

Results and Discussion

      ITS rDNA data obtained from the specimen found at Wied il-Qlejgha (MH013190) was found
to be 100% similar to two S. amplum records in GenBank (DQ097353 and AF141873), and 99%
close to one in UNITE (UDB031960; Kõljalg et al., 2013). Other sequences of S. amplum in GenBank
(L43381 and L43382) showed a 50 bp deletion and 8 differences at other positions, resulting in a
87.5% similarity with the lineage of the samples analyzed in the present work. However, phylogenetic
inferences grouped them all together in a significantly supported clade different from that of S.
commune, S. umbrinum and S. fasciatum (Fig. 1).




                 Fig 1 - Fifty percent majority rule consensus ITS rDNA phylogram of the genus
                 Schizophyllum obtained in MrBayes from 3,825 sampled trees. Nodes supported by >
                 0.95 Bayesian PP or > 70% ML BP are shown annotated


Schizophyllum amplum (Lév.) Nakasone                                                       Figs. 2-3
MB 415948
= Cyphella ampla Lév., Ann. Sci. Nat., Bot., III 9: 126 (1848); = Chaetocypha ampla (Lév.) Kuntze,
Revis. gen. pl. (Leipzig) 2: 847 (1891); = Auriculariopsis ampla (Lév.) Maire, Bull. Soc. mycol. Fr.
18(suppl.): 102 (1902); = Merulius amplus (Lév.) Spirin & Zmitr., Nov. sist. Niz. Rast. 37: 181
(2004);
                                                                                                            52
Sammut et al.                                                             Italian Journal of Mycology 48 (2019)




= Cantharellus coemansii Rabenh., Fungi eur. exs., No 209: no. 209 (1867);
= Auricularia syringae Fuckel, in Jb. nassau. Ver. Naturk. 27-28: 9, Fung. Rhen. no 2508 (1874);
= Auricularia leveillei Cooke & Quél., Clav. Hym. Europ. (London): 213 (1878);
= Cyphella cyclas Cooke & W. Phillips, Grevillea 9(no. 51): 94 (1881); = Chaetocypha cyclas (Cooke
& W. Phillips) Kuntze, Revis. gen. pl. (Leipzig) 2: 847 (1891);
= Auricularia bresadolae Schulzer, Hedwigia 24(4): 148 (1885);
= Cytidia simulans Lloyd, Mycol. Notes (Cincinnati) 6(Letter 64): 991 (1920);
= Stereum pubescens Burt, Ann. Missouri. bot. Gdn 7(2-3): 178 (1920).

Basidiomata: 4-19 mm in diameter, disc-shaped to cupulate, pileate, sessile, lignicolous, scattered.
Abhymenial surface tomentose and white in colour; internally (hymenium) coloured a light brown to
ochre, smooth to veined, gelatinous in appearance.

Microscopy: External hyphae thick walled, straight hyphae; internal layer a filamentous mesh of
hyphae 2.3-4.0 µm in width with distinct clamps and with several anastomoses, many hyphae with
gelatinous sheaths. Basidia clavate-elongated, 4-spored, 26-33x4-4.8µm, with clamps. Basidiospores
hyaline to pale yellowish, smooth, allantoid to cylindrical, (8.1) 8.4-11.1 (11.3) × (2.3) 2.5-3.6 (4.5)
µm, Q = (2.4) 2.6-3.6 (4.5), N = 20, mean = 9.8×3.1 µm, Qe = 3.2.

Material examined: MALTA, Buskett (Wied il-Luq) on dead twigs under Populus alba, 35.85887 °N
14.40157 °E, 29.09.2010, (CS95); Wied il Qlejgha, on dead broken P. alba branch, 35.89395 °N
14.3922 °E, 22.03.2017, (CS1112, GenBank MH013190); ESTONIA, Lääne-Viru county, Vinni, on
fallen branch of Populus tremula, 59.2941667 °N 26.4408333 °E, 30.09.2015, leg. Urmas Ojango
(TU117324, UNITE UDB031960).




          Fig 2 - Schizophyllum amplum. a-c. collection CS1112; a) in habitus; b) younger specimen; c)
          abhymenial surface. Scale Bar = 10mm




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Sammut et al.                                                              Italian Journal of Mycology 48 (2019)




      This is the first published record of S. amplum in the Maltese Islands and Estonia.
Schizophyllum amplum is a very rare species in Malta, always associated with Populus alba in areas
near small streams. The single Estonian collection was growing on Populus tremula. The species is
also rare in other parts of Europe (Wojewoda, 2006; Robinson et al., 2011). The Maltese specimens
are morphologically similar to other published records (Eriksson and Ryvarden, 1975; Nakasone,
1996).




                      Fig 3 - Schizophyllum amplum. Microscopic characters of collection
                      CS1112. a) Tramal hyphae; b) Tramal hyphae stained in toluidine blue;
                      c) Basidiospores; d) Basidia, lateral view

      Genetically, the clade of S. amplum shows variability in ITS rDNA (8/496 bp variable sites),
but most remarkably, sequences L43381 and L43382 present important indels (up to 56 bp in 5 sites).
This could mean that two closely related species are hidden within the concept of S. amplum. The
limited phylogenetic signal provided by ITS rDNA alone does not support the existence of
homogeneous and genetically isolated groups within any of the clades of Schizophyllum, and so all
variability found here is considered intraspecific by now. However, multigenic analyses have already
shown that different species could have been misidentified within these clades, e.g. S. radiatum within
the S. commune complex (Siqueira et al., 2016). Therefore, a multigene approach including a high
number of specimens analyzed from a broad geographical range could change the currently accepted
genetic limits of Schizophyllum species.

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