10.6092/issn.2531-7342/12445
Coprinellus andreorum sp. nov. is described for the first time from Malta. A full description with illustrations of the macro- and micromorphological characters, as well as its phylogenetic position is provided. This species differs from Coprinellus aureogranulatus by the large pleurocystidia, the narrower spores and multidigitate caulocystidia. Some species from sect. Domestici are discussed and compared.
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Italian Journal of Mycology
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<em>Coprinellus andreorum</em>: a new species from Malta and South America
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Articles
2021-04-26
2021-02-22
2021-02-08
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Ital J Mycol
Alexander Karich
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10.6092/issn.2531-7342/12445
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White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. - In: Innis M., Gelfand D., Sninsky J. & White T. (eds.): PCR protocols: a guide to methods and applications, pp. 315-322. Academic Press, New York. https://doi.org/10.1016/B978-0-12-372180-8.50042-1
<em>Coprinellus andreorum</em>: a new species from Malta and South America
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                 IJM - Italian Journal of Mycology
                 ISSN 2531-7342 - Vol. 50 (2021): 21-29
                 Journal homepage: https://italianmycology.unibo.it/

Short note

                          Coprinellus andreorum: a new species
                             from Malta and South America

                                    Carmel Sammut1, Alexander Karich2

1 - 216 Flat 1 St. Joseph Flts., Rue d´Argens, Gzira, GZR 1367, Malta
2 - Technische Universität Dresden - International Institut Zittau, Markt 23, 02763 Zittau, Germany

Corresponding author e-mail: stizzy2499@yahoo.com


Article info
Received 22/2/2021; accepted 14/4/2021
https://doi.org/10.6092/issn.2531-7342/12445

Abstract
Coprinellus andreorum sp. nov. is described for the first time from Malta. A full description with illustrations of the macro-
and micromorphological characters, as well as its phylogenetic position is provided. This species differs from Coprinellus
aureogranulatus by the large pleurocystidia, the narrower spores and multidigitate caulocystidia. Some species from sect.
Domestici are discussed and compared.

Keywords
Agaricales, Psathyrellaceae, Aureogranulati, morphology, taxonomy



Introduction
Following a recent clearing of a small area in Buskett (Siggiewi, Malta) several large dead branches
of Ceratonia siliqua L. were torn down to smaller pieces and dispersed above the soil together with
other dead branches from Quercus ilex L. The sudden abundance, on the soil, of degraded lignicolous
material coupled with abundant rain in the early weeks of October 2020 has resulted in a number of fast
growing coprinii appearing over a few days towards the end of October. The list of lignicolous fungi
noted in the area were both previously encountered as well as new records for the area and include
Parasola conopilea (Fr.) Örstadius & E. Larss., Coprinopsis melanthina (Fr.) Örstadius & E. Larss.,
Coprinellus radians (Desm.) Vilgalys, Hopple & Jacq. Johnson, Coprinellus subdisseminatus (M.
Lange) Redhead, Vilgalys & Moncalvo, Coprinellus xanthothrix (Romagn.) Vilgalys, Hopple & Jacq.
Johnson as well as an unidentified species of Coprinellus with yellow-brown coloured velar remnants.
The macromorphology of the collected specimens appeared similar to Coprinellus aureogranulatus
(Uljé & Aptroot) Redhead, Vilgalys & Moncalvo (A. Melzer, pers. comm.) but micromorphology was
different (Uljé et al., 1998; Melzer et al., 2016).
   The genus Coprinellus in the family Psathyrellaceae consists of small to large, dark spored,
saprotrophic basidiomycete fungi growing on soil, wood and herbivore dung (Nagy et al., 2012;
Hussain et al., 2018). The genus has a worldwide distribution with more than 80 species listed in Index
fungorum (http://www.indexfungorum.org/). Coprinellus has recently been re-evaluated and split into
nine different sections based on morphological characters supported by phylogenetic data (Wächter
and Melzer, 2020). Coprinellus aureogranulatus was originally placed in Coprinus subsect. Setulosi
J.E. Lange by Uljé et al. (1998) based on the presence of pileocystidia, and later in Coprinellus sect.

Department of Agricultural and Food Science in partnership with the Italian Mycological Society (UMI).
The journal is hosted and mantained by ABIS-AlmaDL. © 2021 The Authors. This work is licensed under CC BY 4.0
Sammut, Karich                                               Italian Journal of Mycology 50 (2021): 21-29

https://doi.org/10.6092/issn.2531-7342/12445



Micacei (Fr.) D.J. Schaf. by Huang and Bau (2018) but in a recent revision of the Psathyrellaceae
by Wächter and Melzer (2020) a separate section was created in Coprinellus to accommodate C.
aureogranulatus in sect. Aureogranulati Wächt. & A. Melzer. In the present study morphological and
phylogenetic studies were conducted to characterize Coprinellus andreorum sp. nov.

Materials and Methods
Specimens
Fresh specimens were collected from Buskett (Siggiewi, Malta) on dead branches in a humid
environment at 194 m asl in an open area between broadleaf trees. The specimens were dried at 40°C
for several hours, over 2 days. A small branch, from which the original specimen was collected, was
transferred to a moist chamber (100% relative humidity) at room temperature and a further 3 batches
of the same species were collected. The exsiccate were deposited in the Herbarium Senckenbergianum
Görlitz (GLM F127896).

Morphological studies
Fresh samples were examined with a light microscope, a few hours after collection. Specimens in
the moisture chamber were examined over several hours during growth. Hand cut thin sections were
examined microscopically by squash mounts. Pleurocystidia were observed in living specimens,
during growth, with a hand lens. Fresh material was examined in water, Congo Red/Phloxine stain
(1%) in 5% ammonia solution and toluidine blue (1%).
   Dried material was re-examined after wetting with 10% ammonia and treated similarly to
fresh material. All measurements were taken on fresh material mounted in water with the aid of
Piximètre (downloadable from http://ach.log.free.fr/Piximetre/). Measurements are given on the basis
of N number of spores as indicated in each entry using free spores in the mounts. The following
abbreviations are used in text L – spore length, W – spore width, B – spore width in side view, Q1 =
L/W, Q2 = L/B, Me - mean of L & W.

DNA extraction, amplification and sequencing
DNA extraction, amplification and sequencing of the fungus were performed by Alvalab (Oviedo,
Spain) using protocols previously reported (Alvarado et al., 2018; Sammut et al., 2019). The genomic
DNA was extracted from dried fruiting bodies and amplification was carried out with the ITS1F-
ITS4 primer pair (White et al., 1990). The following molecular phylogenetic markers were used for
the phylogenetic analysis: ITS1, 5.8S, ITS2. The nucleotide sequences for the tree inference were
taken from NCBI (https://www.ncbi.nlm.nih.gov) (Supplementary Table S1). As outgroup, a set of
Narcissea patouillardii s.l. sequences were selected.
   The initial alignment of the ITS region was performed with MAFFT v7.450 (Katoh and Standley,
2013) using the L-INS-i method and corrected manually. The final maximum likelihood analysis was
done with PhyML v3.3.20180621 (Guindon et al., 2010), applying the GTR substitution model and
1000 ML bootstrap inferences were calculated. Of these, 1000 trees were sampled and the best tree
was labeled with the ML bootstrap support values over 70%.




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  Fig 1. Maximum Likelihood tree based on selected ITS-sequences from specimen of section Aureogranulati and
  Domestici applying the GTR-substitution model in PhyML (Guindon et al., 2010). The tree is drawn to scale, with branch
  lengths measured in the number of substitutions per site. Only bootstrap support values over 70% are depicted in the tree.


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Taxonomy
Coprinellus andreorum Sammut & Karich sp. nov. - Mycobank - MB 838887. Figs. 1, 2, 3;
Supplementary Table S2.

   Diagnosis: Differs from C. aureogranulatus, the closest species, by the voluminous pleurocystidia,
narrower spores and multi-furcated caulosystidia.
   Typification: MALTA: Siggiewi, Buskett, 35° 51.375N, 14° 23.939E, 194 m asl, on a dead
corticated branch of Ceratonia siliqua L. on the ground, 29 October 2020, leg. C. Sammut, CS1247
(GLM-F127896); GenBank ITS sequence MW621497, GenBank LSU sequence MW621007,
GenBank Tef-1α sequence MW633114.
   Etymology: The specific epithet is derived from the name Andreas/André and is dedicated to
Andreas Melzer whose collaboration over several years is much appreciated and to André Sammut
who accompanied, collected and encouraged the first author on a number of forays.

    Pileus 9 ‒ 20 mm diameter when closed, up to 45 mm wide when expanded, campanulate when
young to convex to applanate with uprolled margin, initially pale yellow-brown with ochraceous
centre changing to pale greyish brown with ochraceous centre as the specimen matures, sulcate
striate to almost centre; velar remnants scattered, cream coloured with distinct erect rusty brown
tufts. Lamellae L= c. 40 ‒ 60, l= 1 ‒ 3, free, narrow, initially white changing to black, deliquescent.
Stipe 30 ‒ 40 x 1 ‒ 3 mm, central, white with visible cystidia; sub-bulbous at base, often with tufts of
rust-coloured fibrils 1‒3 mm above attachment point, with rust coloured ozonium loosely attached to
substrate. Spore print red-brown.
    Spores 7.4 ‒ 10.0 × 4.3 ‒ 5.7 x 4.0 ‒ 5.2 µm, ellipsoid in front view, phaseoliform in side view,
medium red-brown with rounded base, with central germ pore, 1.6 ‒ 2.0 µm in diameter; apiculus
very small; Q1 = 1.59 ‒ 1.92; Q2 = 1.62 ‒ 1.98; N = 30; av. Q1 = 1.75, av Q2 = 1.82; av. L = 7.7 ‒
8.8 µm, av. W = 4.97 µm, av. B = 4.73 µm. Basidia 17 ‒ 28 x 6.5 ‒ 9 µm, tetrasporic. Pleurocystidia
55 ‒ 110 x 30 ‒ 45 µm, abundant in immature specimens (visible) but rapidly dissolve, ellipsoid to
ovoid to subcylindric. Cheilocystidia 40 ‒ 60 x 18 ‒ 26 (base) x 6 ‒ 8 µm (tip), lageniform. Pileipellis
composed of a layer of globose cells 20 ‒ 50 µm in diameter and lageniform pileocystidia 65 ‒ 110
x 16 ‒ 31 µm with 4 ‒ 8 µm wide cylindrical necks. Caulocystidia 75 ‒ 180 x 15 ‒ 21 µm with
cylindrical neck of 4 ‒ 9 µm at the tips, roughly lageniform but multidigitate (branched) 1‒4 digits
(2 most common). Veil 15 ‒ 60 x 5 ‒ 20 µm made up of thick walled chains of encrusted brownish
coloured subcylindrical or fusoid elements with sub-globose 25 ‒ 50 x 15 ‒ 25 µm terminal elements.
Spherical elements 20 ‒ 45 µm in diameter present. Mycelial threads are yellow brown, thick walled
(up to 1 µm) filaments, 3 ‒ 5 µm in diameter. Clamps absent.
    Habitat: on dead corticated branches of Ceratonia siliqua L., scattered, recently cleared open area
near Ceratonia siliqua L. and Quercus ilex L., Buskett, 194 m asl, CS1247, 29/10/20, leg. C. Sammut.
    Additional collections examined (cultivated from same branch in moisture chamber on subsequent
days): CS1254 (3/11/20), CS1255 (7/11/20) and CS1256 (12/11/20).




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      Fig. 2. a-m Coprinellus andreorum Sammut & Karich sp. nov. a-e – holotype (CS1247); f-g – primordia
      and ozonium; h, j – isotype (CS1254); i – isotype (CS1255); k-m – isotype (CS1256). Bar f, i - l – 10 mm;
      g, h, m – 5 mm




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       Fig. 3. a-i Coprinellus andreorum Sammut & Karich sp. nov. Microscopy: a) pileipellis, b) velar remnants,
       c) pileocystidia, d) cheilocystidia, e) basidia, f) pleurocystidia, g) caulocystidia, h) spores, i) mycelial
       hyphae. Scale bar a, b, c, d, i – 25µm; e, h – 10µm; f, g - 50µm. Staining: a, c-f, – Congo Red/Phloxine;
       b, h, i – unstained; g – 1st set Congo Red/Phloxine, 2nd set – Toluidine blue.




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Results and Discussion
Molecular and morphological studies support the assignment of this fungus to the genus Coprinellus
sect. Aureogranulati Wächt. & A. Melzer as defined in Wächter and Melzer (2020). ITS sequence
showed 99.38% and 98.82% similarity with those deposited in GenBank with the accession numbers
MK843955 and MF471294, respectively. These accessions form a well-supported clade with the
specimen analyzed in this study (Fig. 1). MK843955 corresponds to the voucher BLBS 107 from
Brazil (Schünemann, 2019), named as Coprinellus sp. MF471294 correspond to the voucher UCUE
PC M 22M23 from Ecuador, reported with a best match to AY461815 [voucher SFSU DEH1026 -
Coprinellus radians (Desm.) Vilgalys, Hopple & Jacq. Johnson] in Maldonado et al. (2020). However,
the similarity between these ITS sequences (MF471294 and AY461815) is relatively low (95%) and
the identification of the match was not based on a phylogenetic approach (see below).
   Coprinellus andreorum and C. aureogranulatus are the only known representatives of the section
Aureogranulati Wächt. & A. Melzer. Their most important characteristics are an ozonium (mycelium
mat), a well-developed veil consisting of chains of thick-walled cells as well as the presence of
pleuro- and pileocystidia (Wächter and Melzer, 2020). These are rare species, of which there are only
a few reports despite their conspicuous habit. Coprinellus aureogranulatus was found, in addition to
the isotype find in Papua New Guinea (Uljé et al., 1998), so far in Sri Lanka (Dona et al., 2019), India
(Priyamvada et al., 2015, 2017; Singh et al., 2017), China (Huang and Bau, 2018), in the Philippines
(Lopez et al., 2016), São Tomé (Desjardin and Perry, 2016), and the Seychelles (Melzer et al., 2016).
Overall, the main distribution seems to be in areas with high humidity, preferably in tropical or
subtropical climates (Fig. 4).




   Fig. 4. Distribution of Coprinellus aureogranulatus and C. andreorum.

   The two species in section Aureogranulati have many similar macroscopic and microscopic features,
so identification might prove difficult without examination of a comprehensive set of characteristics.
Coprinellus andreorum can be differentiated by the voluminous pleurocystidia, narrower spores and
multiply furcated caulocystidia, and possibly by the presence of a more robust velum. Of course, the


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constancy of these characteristics should be checked in future finds. Neither Schünemann (2019) nor
Maldonado et al. (2020) contain descriptions and hence the variability of C. andreorum cannot be
established at this point, but a reliable determination is currently guaranteed by sequencing of ITS
regions. However, there is no doubt about the status of this taxon as an independent species.

   The section Domestici (Singer) D. J. Schaf. is very closely related. This section contains some
species, which sometimes have an ozonium, like Coprinellus domesticus (Bolton) Vilgalys, Hopple
& Jacq. Johnson, Coprinellus ellisii (P. D. Orton) Redhead, Vilgalys & Moncalvo and Coprinellus
radians (Desm.) Vilgalys, Hopple & Jacq. Johnson. Furthermore, the velum is strongly developed
and brownish pigmented. From a macroscopic point of view, confusion with older specimens of C.
andreorum is quite possible.
   Overall, the section Domestici is still very incompletely studied; there are major problems with
determination and divergent species concepts, especially with C. radians (Desm.) Vilgalys, Hopple
& Jacq. Johnson and C. xanthothrix (Romagn.) Vilgalys, Hopple & Jacq. Johnson. A case in point
is highlighted above for AY461815 (Keirle et al., 2004) identified as Coprinellus radians but is in
reality Coprinellus domesticus (compare Wächter and Melzer 2020). With the help of microscopic
features, however, the delimitation of all species from the section Aureogranulati is unproblematic,
because pileocystidia are absent in the section Domestici. At the present time confusion with other
species is not possible.

Acknowledgements
We gratefully acknowledge Andreas Melzer for his helpful comments, valuable information and
suggestions as well as relevant references provided. We would like to thank Pablo Alvarado for
providing sequencing services. We would also like to thank the reviewers and the editor for their
helpful comments and suggestions.

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