Rinodina Moziana (Lichenized Ascomycetes Physciaceae), a New Record From Pakistan

doi:10.21203/rs.3.rs-4870877/v1

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Rinodina Moziana (Lichenized Ascomycetes Physciaceae), a New Record From Pakistan

https://doi.org/10.21203/rs.3.rs-4870877/v1

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published 02 Sep, 2024

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During the field survey conducted in Malakand district, Khyber Pakhtunkhwa, Pakistan, Rinodina species was discovered. The taxonomic and chemical characters along with an ITS-based phylogenetic analysis and notes on ecology and distribution considered it as Rinodina moziana (lichenized Ascomycota). A comprehensive study of the available literature corroborates this discovery, and marking it as the first documented occurrence of Rinodina moziana in Pakistan.

Biodiversity

physciaceae

saxicolous

taxonomy

A cosmopolitan genus of Lichens, Rinodina (Ach.) Gray, is distributed in both Hemispheres from polar to tropical latitudes (Nadyeina et al., 2010). The estimated number of worldwide species of Rinodina are 300 (Sheardet al., 2017). The genus has been studied extensively during in last few decades in Australia and New Zealand (Mayrhofer 1983; Mayrhoferet al., 1999; Kaschik 2006; Elix 2011), in Europe (Mayrhofer and Poelt 1979; Mayrhofer 1984; Giralt 2001; Mayrhofer and Moberg 2002) and in North America (Sheard 2004, 2010). So far 43 species of this genus have been reported from North-eastern Asia (Sheardet al., 2017). Mayrhofer (1984) has dealt with species from Africa and Asia in addition to Europe. Due to deforestation, landscape fragmentation, habitat degradation, pollution and climatic changes it is very important to focus on documentation of lichens communities from unexplored area (Essen and Coxon, 2015). From Pakistan, nine species of Rinodina, R. olea Bagl., R. bohilinii H. Magn. R. bischoffii Körb.,R. cana (Arnold) Arnold, R. malangica (Norman) Arnold, R. roboris (Duf. ex Nyl.) Arnol R. cinnamomea (Th. Fr.) Rasanen, R. mniaraea (Ach.)Korb and R. mniaraeiza (Nyl.) Arnold have been reported (Aptroot and Iqbal, 2012 and Resl et al., 2016). Among these, only R. cana has been reported on the basis of morphological characters from Malakand division (Aptroot and Iqbal, 2012).

During the field survey, Rinodina moziana is collected from Malakand division, Khyber Pakhtunkhwa province, Pakistan. Its morphological characters, along with chemical and molecular phylogenetic analysis proved it as a new record from Pakistan.

Sampling site

The sample was collected from Barang, Malakand division, Khyber Pakhtunkhwa province, Pakistan (Fig. 1).

Map of Malakand

Morpho-anatomical studies

The sample was observed under stereomicroscope (MEIJI TECHNO EMZ–5TR) to study the type of thallus, upper and lower surfaces, margins. For slide preparation, dried sample of Rinodina were placed in water for 15 minutes. And mounted in 10% KOH.Lugol's solution was used for staining of the asci. The slides were examined under microscope LABOMED (CXRII). These slides were micro-photographed using HDCE–X5 camera attached to the compound microscope. Illustrations of hymenium and its components were made. Length and width of asci, ascospores, paraphysis, algal cells and thickness of hymenium was taken by ScopeImage 9.0 (X5)

Chemical analysis

Thin-layer chromatography was carried out according to the methods of Culberson and Kristinsson (1970) and Culberson (1972) with later modifications.

DNA extraction, amplification, sequencing, and molecular phylogenetic analysis

DNA Extraction AND PCR Amplification

DNA was extracted by 2% CTAB method (Grades and Bruns, 1996). Polymerase chain reaction (PCR) was performed to amplify Internal Transcribed Spacers (ITS) using primer pair of ITS1F and ITS4 (Grades and Bruns, 1993; White et al., 1990).

The PCR products were separated on 1% agarose gel, using ethidium bromide. The PCR products are visualized through Gel Documentation System. Sequencing was carried out in both directions using the same pair of amplification primers at sequence service BGI China.

Phylogenetic Analysis

A consensus sequence was obtained from both the sequences generated from forward and reverse primer in BioEdit software (7.2 version) (Hall, 1990). This sequence was BLAST searched at NCBI (https://www.ncbi.nlm.nih.gov)

The ITS region of Rinodina moziana was successfully amplified and produced throughout this work. The ITS sequences were aligned using Mafft v7's 'automatic' option, with all other parameters set to default. Each one-locus alignment's ambiguous positions were deleted. Maximum Likelihood was used to do the phylogenetic analysis (ML). ML tree was generated using RAxML 8.2.11 on GENEIOUS (Stamatakis 2014), with 1000 bootstrap pseudoreplicates and the GTR + G + I substitution model. FigTree 1.4.2 was used to visualise the tree file (Rambaut 2014). The analysis have 548 conserved, 317 variable, 187 Prisomy informative and 129 singleton sites. A total of 23 nucleotide sequences were used in the analysis. The sequences were obtained from Genbank, as well as from the literature, Our sequence is nested with R. moziana (DQ849306) from Austria. Mycoblastus sanguinarius (JF744910) used as an outgroup (Resl et al., 2016).

Taxonomy

Rinodina moziana (Nyl.) Zahlbr., Cat. Lich. Univers. 7: 544 (1931)

Macro and Micro Morphological Characters

Thallus crustose, thin, rimose, or areolate, surface is dark grey to dark brown, dull, smooth or slightly granular surface. the areoles up to 0.5–0.8 mm wide, flat, with a slightly verrucose surface. Margins length 5 cm. Prothallus is absent. Apothecia at first lecanorine and more or less immersed in thallus, becoming pseudolecanorine and adnate, 0.3–0.6 mm diam,, with a black, usually flat disc, an entire, finally often excluded thalline margin, and a dark brown to black, persistent proper margin. Disc is flat, dark brown to black. Thalline exciple corticate; proper exciple colourless or more often blue-green to black-brown in upper and outer parts; Algal cells are globose. Hymenium hyaline, and lacks pigmentation, 59.44–56.51 µm tall. Epihymenium 19.93 µm length. Paraphyses are septate branched, apices expanded with a brown cap, 1–2 µm wide. Hypothecium colourless, up to 200 µm high. Asci 8-spored, clavate, Lecanora-type, 48–20 µm. Ascosporess are simple, brown, ellipsoid to broadly ellipsoid in shape, brown, septate, 17.09–25.14 × 9.17–13.40 µm, Mischoblastia-type, often swollen at septum at maturity, the torus poorly developed and the walls lightly pigmented. Pycnidia immersed. Conidia bacilliform, 2–4 µm. Spot tests: thallus K + yellow, C-, KC-, P + faintly yellow. Chemistry: cortex with atranorin.

Spot tests: thallus K + yellow, C-, KC-, P + faintly yellow. Secondary metabolites: atranorin detected.

Habitat

On calciferous rock in subtropical broadleaf forest, an altitude of 1058 m.a.s.l., Dalbergia sissoo Roxb. ex DC., Acacia modesta (Linn.) Wall., Ficus spp., and Terminalia spp. Western Himalayan ranges, maximum and minimum temperature of 35°C and 4°C respectively, annual rainfall varying between 1000–1200 mm.

Material examined: PAKISTAN. Khyber Pakhtunkhwa: District Malakand, Barang; at 1058 m a.s.l., December 16, 2016, H. Wahab, T. Wahab. H-01 (OR526811).

Remarks

It is characterized by dark grey to dark brown thallus, dull, smooth or slightly granular surface, smaller ascospores 17.09–25.14 × 9.17–13.40 µm.

The worldwide genus Rinodina (Ach.) Gray is widely spread in both Hemispheres from polar to tropical latitudes, with over 265 species (Kirk et al. 2008). Spore size and structure are very important characters in distinguishing among Rinodina species (Nadyeina et al., 2010).

Previously ten species of Rinodina, R. olea Bagl., R. bohilinii H. Magn. R. bischoffii Körb.,R. cana (Arnold) Arnold, R. malangica (Norman) Arnold, R. roboris (Duf. ex Nyl.) Arnol R. cinnamomea (Th. Fr.) Rasanen, R. mniaraea (Ach.)Korb, R. mniaraeiza (Nyl.) Arnold and R. iqbalii have been reported (Aptroot and Iqbal, 2012 and Resl et al., 2016, Zulfiqar et al., 2022). Among these, only R. cana has been reported on the basis of morphological characters from Malakand division (Aptroot and Iqbal, 2012).

Rinodina moziana discussed extensively in the present study, It is characterized by dark grey to dark brown thallus, dull, smooth or slightly granular surface, smaller ascospores 17.09–25.14 × 9.17–13.40 µm. ITS-based molecular research revealed genetic resemblance to a taxon from Austria (DQ849306). Their similarities are supported by morpho-anatomical and chemical characters. (Resl & Mayrhofer 2016). It is a new record from Pakistan.

Author Contribution

HiraWahab*1 has written the complete manuscript and did experimental work.Siraj uddin1 helped in the field survey and collected the specimen.Afshan Wahab1 completed the phylogeny.Abdul Nasir Khalid2 has provided the resources for the study and examined the morphological characters.Helmut Mayrhofer3 did the chemistry to detect secondary metabolites.and all authors reviewed the manuscript.

Acknowledgements

Authors are grateful to Asst.Prof. Mustafa YAVUZ (PhD.) Istanbul Medeniyet University, Faculty of Arts and Humanities, History of Science Department, A202 South Campus, 34720 Kadikoy, ISTANBUL / TURKIYEfor help in literature survey and anatomical comparisions. Sincere thanks to Dr. Sana Jabeen (University of Education, Lahore) for help in finalizing this work. I am also grateful to Ms. AfshanWahab for help in molecular work. Thanks is also due to Mr. Zia Ullah for help in phylogenetic analysis and Mr. Taimoor Wahab to have nice company during field work.

Data Availability

the Accession number is available on GenBank National Institutes of Health (NIH) (.gov)

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No competing interests reported.

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You are reading this latest preprint version

Rinodina Moziana (Lichenized Ascomycetes Physciaceae), a New Record From Pakistan

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials and Methods

Sampling site

Map of Malakand

Morpho-anatomical studies

Chemical analysis

DNA extraction, amplification, sequencing, and molecular phylogenetic analysis

DNA Extraction AND PCR Amplification

Results

Phylogenetic Analysis

Taxonomy

Macro and Micro Morphological Characters

Discussion

Declarations

Author Contribution

Acknowledgements

Data Availability

References

Additional Declarations

Status:

Journal Publication

Version 1