Abstract
The apothecia of Otidea were collected from different forests of Pakistan. Based on morphology
and molecular phylogeny, the specimens were identified as Otidea alutacea. This is first report of its
occurrence in Pakistan.
Key words: Ascomycota, Biodiversity, ITS, 28S.
Introduction
The genus Otidea was established by Fuckel in 1870 and is easily distinguished by its
peculiar shapes of the apothecia and paraphyses. This genus is distinct from other
members of Pezizales due to cupulate fruit bodies (split or ear shaped), filiform
paraphyses (curved or capitate at apex), subcylinderical, nonamyloid asci and elliptical,
biguttulate ascospores. (Kanouse 1949; Dennis, 1968; Hansen and Knudsen, 2000).
Multilocus phylogenetic analyses have proved that genus is monophyletic (Hansen et al.
2013). The genus is ectomycorrhizal with diverse host plants of gymnosperms and
angiosperms, also reported as ECM symbionts of Quercus (Smith et al 2007, Moser et al
2009).
From Pakistan, Otidea leprina has been reported previously from Murree and Kaghan
valley (Ahmed 1955a, 1978; Khalid et al., 1992). Thorough and systematic macrofungal
explorations of Swat, Ayubia National Park and Bagh, Kashmir, different fruiting bodies
of Otidea alutacea have been collected and anaylzed. Nuclear ribosomal DNA sequence
analyses of the ITS and 28S regions from fruit bodies were combined with detailed
morphological examination to confirm our Pakistani collections were Otidea alutacea.
Materials & Methods
Collection, morphological and anatomical study
Apothecia were photographed in the field using Nikon D70S digital camera. Colours
DNA was extracted from apothecia by modified CTAB method (Bruns 1995). ITS and
LSU regions were amplified by the primer pairs ITS1F/ITS4 and LR0R/LR5,
respectively (Vilgalys and Hester 1990, White et al. 1990, Gardes and Bruns 1993).
Polymerase chain reactions (PCR) were performed in 25 µl volume reactions. All PCR
products were evaluated for successful amplification using SYBR Green and 1.5%
agarose gels with TAE buffer for gel electrophoresis. Amplicons were prepared for
sequencing via enzymatic purification using Exonuclease I and Shrimp Alkaline
Phosphatase enzymes (Werle et al. 1994). Purified products were sequenced by the
University of Florida’s Interdisciplinary Center for Biotechnology Research
(http://www.biotech.ufl.edu/). Sequence chromatograms were trimmed, edited, and
assembled using Sequencher 4.1 (GeneCodes, Ann Arbor, MI). DNA sequences
generated for this study were deposited in GenBank under accession
numbers (MN493146, MN493147, MN495933, MN495934, MN495935, MN495936,
MN495937).
Consensus sequences for ITS and LSU generated in BioEdit software were used to query
GenBank database using BLAST searches. Sequences with closest match were selected
from GenBank to reconstruct phylogeny. Published sequences of the most closely related
species were also included in the final data set. Multiple sequences were aligned using
APOTHECIA 2–5 cm tall, 2–4 cm broad, yellow to light brown (2.5Y 5.5/3.8), initially
ear shaped, then expand to capulate, sploit, erect, in clusters, subsessile or short stipe,
asymmetrical, truncate at apex, margin wavy.
SPORES 14–15.5 × 7–8 µm, hyaline to yellowish green in 5% KOH, ellipsoid to
elliptical, smooth, guttulated, usually two. ASCI 146–178 × 11–12 µm, cylindrical, non-
amyloid. PARAPHYSIS 3–4 µm wide, hyaline in 5% KOH, slender, hooked, branched
below. EXCIPULUM Textura globosa, cells compactly arrange, parenchymatous cells,
11–14 µm wide cells, terminal cells larger than medullary excipulum, hyaline,
interconnected, septate hyphae.
Material examined: PAKISTAN, Khyber Pakhtunkhwa Province, Swat district, Toa,
2800 m a.s.l., on soil under Quercus spp., 15th July 2015, Arooj Naseer & Abdul Nasir
Khalid, AST18 (FLAS-F59409); AST36 (LAH35220); Abottabad District, Ayubia
National Park, Khanspur, 2575 m a.s.l., on moist soil, in the vicinity of broad leaved trees
of conifers, 13th August 2017, Simab Asghar & A. R. Niazi, KH454 (LAH35563); Azad
Jammu and Kashmir, Bagh, at 2,625 m a.s.l., under Conifers, solitary, 09th September
2017, Rubab Khurshid, RK50 (LAH35631).
Consensus sequences for the ITS region of Otidea alutacea yielded 580–631 base
pairs sequences. BLAST searches in NCBI revealed it as 99% identical with Otidea
alutacea (KM010072, KM010071 & KM010075) from Norway and Spain with 100%
query cover and 0.0 E value and also showed 97% identity with Otidea alutacea
(EU846245) from Portland, USA, with 100% query cover, 0.0 E value.
The consensus sequence for the LSU region of Otidea alutacea yielded 840–860
base pairs. Initial BLAST analysis revealed it as 99% identical with Otidea alutacea
(KM823186, KM823185 & KM823164) with 100% query and 0.0 E value.
The sequences were aligned with ITS and LSU sequences of the other related
taxa. The final data set included 52 and 35 nucleotide sequences for ITS and LSU
respectively to construct phylogeny. Peziza vesiculosa (JF908568) was chosen as
outgroup to construct phylogenetic tree. The analysis revealed that sequences clustered
with sequences of Otidea alutacea from different parts of world in same clade.
Discussion
Otidea alutacea is characterized by its medium brown receptacle and light yellowish
brown basal mycelium that lacks brown resinous exudates. Morphologically it is similar
to Otidea cochleata due to very similar truncate apothecial shape. However, Otidea
alutacea can be differentiated due to woody brown apothecia from dark coloured
fruitbodies of O. cochleata.
Different species of Otidea are measured to be ectomycorrhizal, few number of species
on bases of molecular ectomycorrhizal community revisions have
documented Otidea from root samples of different hosts (Tedersoo & Smith 2013) direct
indication is deficient for most species. Recently, an additional clade from Chinese
collection in the Otidea alutacea complex has been added base on ITS and LSU regions
(Xu et al 2018).
0.2
KM823235_O_tuomikoskii
98
KM823496_O_tuomikoskii
KM823497_O_tuomikoskii
KM823499_O_tuomikoskii
89
JN941092_O_tuomikoskii
KM823251_O_tuomikoskii
100
9 3 KM823216_O_leporina
100 KM823215_O_leporina
99
KM823213_O_leporine
KM823478_O_leporina
KM823233_O_papillata
100 FJ404767_O_subterranean
81 FJ404766_O_subterranean
KM823206_O_daliensis
KM823491_O_platyspora
92 8 6 KM823238_O_platyspora
KM823237_O_platyspora
KM823196_O_apophysata
KY498605_O_alutacea_Thailand
KM823186_O_alutacea_Spain
KM823185_O_alutacea_Norway
KM823457_O_alutacea_Denmark
9 8 KC012691_O_alutacea_Sweden
KM823187_O_alutacea_Sweden
KM823188_O_alutacea_Sweden
KU987026_O_alutacea_China
3 9 KU987025_0_alutacea_China
KM823461_O_alutacea_Sweden
9 5 KM823191_O_alutacea_Sweden
0 KM823460_O_alutacea_Denmark
KM823459_O_alutacea_Sweden
15
KM823465_O_alutacea_Sweden
KM823190_O_alutacea_Norway
KM823458_O_alutacea_Sweden
AF378367_Peziza_vesiculosa
0.03
We thank the two anonymous reviewers for their corrections and suggestions to improve
References
Ahmed, S. (1955a) Pezizales of Pakistan. Biologia 1: 1-24.
Ahmed, S., (1978) Ascomycetes of Pakistan. Biological Sociey of Pakistan, Monograph 7
& 8 pp.
Bruns, T.D. (1995) Thoughts on the processes that maintain local species diversity of
ectomycorrhizal fungi. Plant and Soil 170: 63–73.
https://doi.org/10.1007/BF02183055
Dennis, R.W.G. (1968) British Ascomycetes. J. Cramer, Lehre.
Gardes, M., & Bruns, T.D. (1993) ITS primers with enhanced specificity for
basidiomycetes – application to the identification of mycorrhizae and rusts. Molecular
Ecology 2: 113–118. https://doi.org/10.1111/j.1365-294X.1993.tb00005.x
Kanouse, B.B. (1949) Studies in the genus Otidea. Mycologia 41: 660-677.
Khalid, A.N. (1992) Some operculate discomycetes (Pezizales) from Murree Hills. Sci.
Int. (Lahore) 4(1): 115-123
Harmaja, H. (2009) Studies in Otidea (Pezizales). Karstenia, 48(2), 33-48.
Miller, M.A., Pfeiffer, W., & Schwartz, T. (2010) Creating the CIPRES Science Gateway
for inference of large phylogenetic trees. Proceedings of the Gateway Computing
Environments Workshop (GCE), New Orleans, Louisiana, 1–8.
https://doi.org/10.1109/GCE.2010.5676129
Mornand, J., & Courtecuisse, R. (2005) Le genre Otidea et espèces affines en
France. Publications de la Société Linnéenne de Lyon, 74(8), 65-84.
Nouhra, E., Urcelay, C., Longo, S., & Tedersoo, L. (2013). Ectomycorrhizal fungal
communities associated to Nothofagus species in Northern
Patagonia. Mycorrhiza, 23(6), 487-496.
Olariaga, I., Van Vooren, N., Carbone, M., & Hansen, K. (2015) A monograph of Otidea
(Pyronemataceae, Pezizomycetes). Persoonia: Molecular Phylogeny and Evolution of
Fungi, 35, 166.
Rambaut, A., Suchard, M.A, Xie. D., & Drummond A.J. (2014) Tracer v1.6. Available at
http://beast.bio.ed.ac.uk/Tracer 2014]. [Verified May 2014].
Stamatakis, A. (2014) RAxML version 8: a tool for phylogenetic analysis and post-
analysis of large phylogenies. Bioinformatics, 30(9), 1312-1313.
http://beast.bio.ed.ac.uk/Tracer 2014].
Peterson, E. T. (1998) Systematics of the genus Otidea in the Pacific Northwest.
Stamatakis, A. (2014) RAxML version 8: a tool for phylogenetic analysis and post-
analysis of large phylogenies. Bioinformatics 30: 1312–1313.
https://doi.org/10.1093/bioinformatics/btu033
Smith, M. E., & Healy, R. A. (2009) Otidea subterranea sp. nov.: Otidea goes below
ground. Mycological research, 113(8), 858-866.
Hansen, K., Perry, B.A., Dranginis, A.W., & Pfister, D. H. (2013) A phylogeny of the
highly diverse cup-fungus family Pyronemataceae (Pezizomycetes, Ascomycota) clarifies