The Genus Otidea (Pezizales, Pyronemataceae) from Pakistan

AROOJ NASEER1*, HAFIZA SIMAB ASGHAR1, RUBAB KHURSHID1, ABDUL REHMAN NIAZI1 & ABDUL NASIR KHALID1

1Department of Botany, University of the Punjab, Lahore. Corresponding author*: [email protected]

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

[SYLWAN., 163(10)]. ISI Indexed 145 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

[SYLWAN., 163(10)]. ISI Indexed 146 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).

[SYLWAN., 163(10)]. ISI Indexed 147 Figure 1. Macromorphology of Otidea alutacea. A. FLAS-F59409. B. LAH35220. Scale bar = 1 cm.

[SYLWAN., 163(10)]. ISI Indexed 148

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

Figure 3. Light micrographs of Otidea alutacea.

[SYLWAN., 163(10)]. ISI Indexed 149 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., 163(10)]. ISI Indexed 150

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 100 KM010076_O_alutacea_Sweden KM010063_O_alutacea_Sweden JF908507_O_leporine_USA KM010066_O_alutacea_Sweden KT818924-O_alutacea_England 100 KM010067_O_alutacea_Denmark 9 3 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 9 8 KM010069_O_alutacea_Italy 9 6 EU784381_O_aff._alutacea KM010070_O_alutacea_USA 100 KM010117_O_tuomikoskii JN942776_O_tuomikoskii JN942777_O_tuomikoskii 9 6 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 9 0 FJ404767_O_subterranea 7 1 KM010086_O_daliensis 100 EU784382_O_apophysata 7 6 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.

[SYLWAN., 163(10)]. ISI Indexed 151

KM823250_O_tuomikoskii

KM823235_O_tuomikoskii 9 8 KM823496_O_tuomikoskii

KM823497_O_tuomikoskii

KM823499_O_tuomikoskii 8 9 JN941092_O_tuomikoskii

KM823251_O_tuomikoskii 100

9 3 KM823216_O_leporina

100 KM823215_O_leporina 9 9 KM823213_O_leporine

KM823478_O_leporina

KM823233_O_papillata

100 FJ404767_O_subterranean

8 1 FJ404766_O_subterranean

KM823206_O_daliensis

KM823491_O_platyspora

9 2 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 1 5 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.

[SYLWAN., 163(10)]. ISI Indexed 152 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

[SYLWAN., 163(10)]. ISI Indexed 153 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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