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The Genus Otidea (Pezizales, Pyronemataceae) from Pakistan

AROOJ NASEER1*, HAFIZA SIMAB ASGHAR1, RUBAB KHURSHID1, ABDUL REHMAN


NIAZI1 & ABDUL NASIR KHALID1
1
Department of Botany, University of the Punjab, Lahore.
Corresponding author*: arooj.hons@pu.edu.pk

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

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were designated with reference to mColorMeter application (Yanmei He, Mac App
Store). Specimens were deposited in the Herbarium, Department of Botany, University of
the Punjab, Lahore, Pakistan (LAH) and University of Florida Fungal Herbarium,
Gainesville FL, USA (FLAS). These were rehydrated in distilled water for two hours and
then sectioned in Melzer’s reagent (MLZ) and 8% KOH. Measurements of structures and
photographs were made with or better contrast and examined using a Meiji Techno
MX4300H compound microscope.

DNA extraction, amplification and sequencing

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).

Molecular phylogenetic analyses

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

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online MUSCLE by EMBL-EBI (http://www.ebi.ac.uk/Tools/msa/muscle/). A maximum
likelihood tree was inferred for each alignment using RAxML-HPC2 v8.1.11 (Stamatakis
2014) with a GTR + gamma model of nucleotide substitution. One thousand bootstrap
iterations were performed with rapid bootstrapping. Significant support was considered to
be ≥70 %. All phylogenetic analyses were performed on the CIPRES Portal v. 3.1.
(Miller et al. 2010). The phylogeny from ML analysis was displayed with FigTree 1.4.2
(Rambaut 2014) and exported to Adobe Illustrator.

Otidea alutacea (Pers.) Massee, Brit. Fung.-Fl. (London) 4: 446 (1895)

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).

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Figure 1. Macromorphology of Otidea alutacea. A. FLAS-F59409. B. LAH35220. Scale
bar = 1 cm.

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Figure 2. Scanning electron microphotographs of Otidea alutacea (LAH35563)

Figure 3. Light micrographs of Otidea alutacea.

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Molecular phylogenetic characterization

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).

[SYLWAN., 158(X)]. ISI Indexed 6


MN495933 O alutacea PAKISTAN
MN495934 O alutacea PAKISTAN
MN495935 O alutacea PAKISTAN
100 MN495936 O alutacea PAKISTAN
MN495937 O alutacea PAKISTAN
7 KM010073_O_alutacea_France
KM010074_O_alutacea_Denmark
100
KM010072_O_alutacea_Spain
KM010071_O_alutacea_Norway
KT818925_O_alutacea_England
99 KM010075_O_alutacea_Italy
AF072069_O_alutacea_Italy
AF072071_O_alutacea_Norway
100
AF072070_O_alutacea_USA
AF072073_O_alutacea_France
KM010065_O_alutacea_Sweden
KM010064_O_alutacea_Norway
1 0 0 KM010076_O_alutacea_Sweden
KM010063_O_alutacea_Sweden
JF908507_O_leporine_USA
KM010066_O_alutacea_Sweden
KT818924-O_alutacea_England
1 0 0 KM010067_O_alutacea_Denmark
93 KU987013_Otidea_alutacea_China
KU987014_0_alutacea_China
KM010068_O_alutacea_Sweden
KM010061_O_alutacea_Denmark
KM010060_O_alutacea_Swedan
EU784380_O_aff._alutacea
100
KM010059_O_alutacea_Swedan
KM010062_O_alutacea_Sweden
KT818926_O_alutacea_England
98
KM010069_O_alutacea_Italy
96
EU784381_O_aff._alutacea
KM010070_O_alutacea_USA
1 0 0 KM010117_O_tuomikoskii
JN942776_O_tuomikoskii
JN942777_O_tuomikoskii
96 KM010116_O_tuomikoskii
NR_120293_O_tuomikoskii
100 100
KF717584_O_tuomikoskii
KM010114_O_tuomikoskii
100 100 KM010089_O_leporine
KM010091_Otidea_leporine
100 KM010105_O_papillata
NR_120291_O_papillata
100 NR_121353_O_subterranea
90 FJ404767_O_subterranea
71 KM010086_O_daliensis
1 0 0 EU784382_O_apophysata
76 KM010077_O_apophysata
9 5 KM010107_O_platyspora
9 9 KM010106_O_platyspora
EU784393_O_platyspora
KM010108_O_platyspora
JF908568_Peziza_vesiculosa

0.2

Figure 4. Maximum likelihood phylogram of Otidea alutacea based on ITS ribosomal


DNA as generated with RAxML with 1000 bootstrap iterations . Bolded lettering refers
to sequences generated in this study.

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KM823250_O_tuomikoskii

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

MN493146 O alutacea PAKISTAN

MN493147 O alutacea PAKISTAN

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

Figure 5. Maximum likelihood phylogram of Otidea alutacea based on LSU ribosomal


DNA as generated with RAxML with 1000 bootstrap iterations . Bolded lettering refers
to sequences generated in this study.

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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).
O. alutacea s.l. is considered to encompass a species composite. It is documented by the
standard brown, cup-shaped, fragmented apothecia, an ectal excipulum with only thin
resinous exudates, mainly oblong spores. Although sometimes treated as a well-delimited
species (Harmaja 2009), spore sizes of O. alutacea provided by different authors vary
considerably, e.g. 14–16 × 7–9 µm (Dissing 2000) or 12.5–14.5 × 6.2–7.3 µm (Harmaja
2009). Actually different members of O. cochleata has been detached from O.
alutacea interpretation of bigger spores ranges16–18 × 7–8 µm also differ due to darker
apothecia (Mornand & Courtecuisse 2005,Liu & Zhuang 2006, Zhuang 2006).
Temporarily, two taxa belongs to O. alutacea making species composite have been
detached in North America due to apothecia distinct color, varying spore size and shape
(Peterson 1998). In present study LSU phylogeny fixed numerous clades within O.
alutacea, which are strongly reinforced in the multigene phylogeny (Hansen & Olariaga
2015). The different clusters seem that the spore sizes within individually clade have an
equally narrow range in which overlap occurs among the clades. Outlines of interior
speciation are suggested that two clades have Sweden specimens, and the rest contain
samples from Norway, France, England, Italy and Denmark. The specimens have little
difference in spore size range but clusters together phylogenetically. Specifically,
Morphoanatomical features suggested that samples from Pakistan similar to O.
alutacea of North America and Europe specimens and new record from Pakistan.
Our Otidea alutacea collections represent an addition to the mycobiota of Pakistan and a
first report of the species from South Asia.
Acknowledgments

We thank the two anonymous reviewers for their corrections and suggestions to improve

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our work. Thanks to the University of the Punjab, Lahore, Pakistan for providing
financial support (Research Grant 2014-2015) to Arooj Naseer for this Research work.

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