Content uploaded by Muhammad Shahid Iqbal
Author content
All content in this area was uploaded by Muhammad Shahid Iqbal on Oct 04, 2023
Content may be subject to copyright.
1
ISSN 1062-3590, Biology Bulletin, 2023. © Pleiades Publishing, Inc., 2023.
A New Species and a New Record of Family Ramalinaceae
(Lichenized Ascomycetes) from Pakistan
Muhammad Shahid Iqbala, * (ORCID: 0000-0002-6244-3267), Hafiza Simab Asghara, and Abdul Nasir Khalida
a Fungal Biology and Systematics Research Laboratory, Institute of Botany, University of the Punjab,
Qaid-e-Azam Campus, Lahore, 54590 Pakistan
*e-mail: mshahidiqbal012@gmail.com
Received April 19, 2023; revised April 24, 2023; accepted April 27, 2023
Abstract—Toninia pakistanica sp. nov. is described from Darel Valley, Gilgit Baltistan, Pakistan. Its place
within the genus Ton i nia A. Massal. was validated by a comparative morpho-anatomical investigation and
ITS based molecular studies. The taxon is characterized by dark brown lobate squamules, epruinose to weakly
pruinose with dark olive to black thallus, densely pruinose upper surface, more thicker epinecral layer 60 μm,
25–45 μm thick grey to blackish grey lower cortex and with the absence of apothecia and secondary metab-
olites. Its positioning in a separate branch in the phylogenetic tree also makes it distinct from the other known
species. Phylogenetic analysis based on ITS-nrDNA sequencing placed our species relative to Toninia cinereo-
virens (Schaer.) A. Massal. Kiliasia nordlandica (Th. Fr.) Kistenich, Timdal, Bendiksby & S. Ekman is included
and reported as new record to Pakistan.
Keywords: Toninia, Kiliasia, Ramalinaceae, molecular analysis, taxonomy
DOI: 10.1134/S1062359023602203
INTRODUCTION
The Ramalinaceae C. Agardh is the fourth-largest
family of lichen-forming ascomycetes, with 42 genera
and 913 species (Lücking et al., 2017a, 2017b). This
family contains members with a chlorococcoid photo-
biont, mostly biatorine or lecideine apothecia (when
present) and hyaline and often transversely septate
ascospores. Ton i n i a, widely present in dry and arid
regions, is a crucial part of desert lichen ecosystems
(Timdal, 1991, 2002). Molecular genetic characteris-
tics (Sun et al., 2019), the asci and paraphyses are the
primary factors used in the present delimitation of the
genus (Kistenich et al., 2018; Ekman 2001, Ekman
et al., 2008). Toninia A. Massal is characterized by a
crustose to squamulose and epruinose to densely pru-
inose thallus; lecideine, epruinose to densely pruinose
apothecia; dark brown to colourless hypothecium;
usually colourless, grey, green, or brown epihymenium
and exciple that changes colour in K and N; clavate,
Biatora-type, 8-spored asci; and elliptical to spindle-
shaped, colourless, 1–8-celled ascospores. 357 species
worldwide belong to this genus Toninia (Indexfungo-
rum November 10, 2022). Kiliasia Hafellner is genus
of the Ramalinaceae, which was included in Toninia
by Timdal (1991), includes species from his groups 1,
3, and 7. Morphologically, it is characterized by a
more or less crustose thallus, although both non-
lichenized and squamulose species occur. The asco-
spores vary from ellipsoid, 1-septate to shortly bacilli-
form, 3-septate (Kistenich et al., 2018). Eleven species
worldwide belong to this genus Kiliasia (Indexfungo-
rum November 10, 2022).
During our exploration of the lichen diversity of
Pakistan, collections were made from Gilgit–Baltis-
tan and Khyber Pakhtunkhwa, Pakistan. We were able
to confirm the existence of one new species and one
new record of the genera Toninia and Kiliasia respec-
tively, from Pakistan.
MATERIALS AND METHODS
Morphological and Chemical Studies
The collections were made during a lichen survey
of different sites of Gilgit–Baltistan and Khyber Pakh-
tunkhwa, Pakistan in 2020-22. Morphological charac-
ters were observed under a stereomicroscope (Meiji
Techno, EMZ-5TR, Japan). Standard microscopy
and spot tests (Hale, 1979) were used for further iden-
tification. The sections were observed with a com-
pound microscope (MX4300H, Meiji Techno,
Japan). Free hand sections of thallus and Apothecia
were mounted in water and observed at different mag-
nifications for anatomical characterization and mea-
BOTANY
2
BIOLOGY BULLETIN 2023
MUHAMMAD SHAHID IQBAL et al.
surements. Minimum twenty measurements were
made for each diagnostic feature.
Molecular Characterization
We extracted fungal DNA from the thallus using a
2% CTAB protocol (Gardes and Bruns, 1993). Molec-
ular data were generated for the internal transcribed
spacer (ITS) region. The primer pair ITS1F (Gardes
and Bruns, 1993) and ITS4 (White et al., 1990) was
used to amplify the ITS region under the PCR condi-
tions used by Khan et al. (2018). PCR products were
visualized in a 1% agarose gel (Sambrook and Russell,
2001) and sent to BGI Hong Kong, for sequenc-
ing.The ITS locus was amplified and sequenced for all
specimens yielded fragments of about 600–700 bp
each. The BioEdit sequence alignment editor was used
to reassemble forward and reverse sequences (Hall,
2011). The nucleotide sequence comparison was per-
formed using the Basic Local Alignment Search Tool
(BLAST) of the National Centre for Biotechnology
Information (NCBI) (Altschul et al., 1990). The mul-
tiple sequence alignment was performed using
MAFFT v.7 with all parameters set to default values
(Katoh and Standley, 2013). The phylogenetic tree was
executed using software MEGA 6.0 (Tamura et al.,
2013) with ML method based on General Time
Reversible model. Nodal support was evaluated using
the “rapid bootstrapping” option with 1000 replicates.
For rooting purpose of the tree, Phyllopsora borbonica
Timdal and Krog (MG925988) and Sporacestra per-
texta (Nyl.) Stapnes and Timdal (MG926000) were
selected as an out-group.
RESULTS
Phylogenetic Analysis
The new ITS nrDNA sequences are nested within
the phylogenetic branch of the genus Toninia, repre-
senting the species unknown yet, described here as
Toninia pakistanica. Altogether 47 ITS rDNA
sequences were analyzed, including sequences
obtained from the GenBank. There were 566 charac-
ters in the alignment file of which 224 were conserved,
325 variables, 266 parsimony informative and 56 were
singleton variants. In the ITS phylogram Toninia paki-
Fig. 1. (a) Habitus of Toninia pakistanica sp. nov. (Holotype, LAH 37604); (b) Squamules; (c) Section of thallus.
(c)
80 Pm
(a)
0.5 cm 1.5 mm
(b)
BIOLOGY BULLETIN 2023
A NEW SPECIES AND A NEW RECORD OF FAMILY RAMALINACEAE 3
stanica sp. nov. formed a sister clade with T. cin e r eo-
virens with strong support (100% BS). The analysis
represents the independent position of the Pakistani
taxon. Similarly our another collection GC-60C
nested within specimens of the genus Kiliasia from
Spain (MG838188), from Norway (AF282113). Kilia-
sia nordlandica (Th.Fr. Kistenich, Timdal, Bendiksby
and S. Ekman) is first time reported from Pakistan
based on ITS data.
TAXONOMIC TREATMENT
Toninia pakistanica M.S. Iqbal & Khalid sp. nov.
MYCOBANK # MB 846607.
Etymology: The epithet “pakistanica” refers to the
type locality of the country Pakistan.
Diagnosis: Toninia pakistanica sp. nov. differs from
its closely related species T. cinere ovirens by dark
brown lobate squamules, epruinose to weakly pru-
inose with dark olive to black thallus, densely pruinose
upper surface, more thicker epinecral layer 60 µm, 25–
45 µm thick grey to blackish grey lower cortex and with
the absence of apothecia and secondary metabolites.
Holotype: PAKISTAN: Gilgit Baltistan, Darel Val-
ley 35°37′ N, 73°27′ E, elev. 2000 m, on rocks,
August 10, 2022, Muhammad Shahid Iqbal DR-170
(LAH37604), (ITS GenBank accession number
OP879646).
Fig. 2. Kiliasia nordlandica (Th.Fr.) Kistenich, Timdal, Bendiksby and S. Ekman (a–d): (a, b) Showing crustose-Squamulose
thallus with apothecia, (c) Areoles, (d) Cross section of apothecium, (e) Cross section of Thallus, (f) Ascus with Ascospores,
(g) Paraphyses.
(e)
100 Pm
(f)
8 Pm
(g)
5 Pm
0.5 mm
2 mm
(b)
0.1 cm
(a)
(c)
100
P
m
(d)
4
BIOLOGY BULLETIN 2023
MUHAMMAD SHAHID IQBAL et al.
Description: Thallus: Squamulose, determinate,
dark brown squamules upto 3 mm in diameter, epru-
inose to weakly pruinose, occasionally dispersed when
young, later randomly imbricated, not countinous,
sometime scattered lobed when young, later deeply
lobed, weakly concave to weakly convex. Upper Sur-
face: dark brown to greyish brown, densely pruinose,
dull, somewhat shiny, Lower Surface: pale brown to
grey. Color: dark olive to black when dry, unchanged
green when wet. Upper cortex: 70–100 µm thick, light
paler brown, paraplectenchymatous, texture rounded
to globular, cell 12–17 µm diam., overlain by a distinct
60 µm thick epinecral layer. Medulla: white, well
developed, hyphae 3–4.5 µm thick. Algal layer: 60–
100 µm thick, chlorococcoid, cells broadly ellipsoid to
globose, 10–20 µm diam. Lower cortex: grey to black-
ish gray, 25–45 µm thick, Apothecia: not found.
Chemistry: Cortex: K-, C-, KC- ; Medulla: K-, C-,
KC-.
TLC: No substance detected.
Ecology: The collections of new species in an open
situation exposed to Sun and rain, found at the hilly
topography of Darel valley with an altitude of 2000 m.
In area, summers are warm and clear and the winters
are freezing, snowy, and partly cloudy. The specimens
were on calcareous sedimentary rocks. Common flo-
ral species are Cedrus deodara (Roxb. ex Lambert)
G. Don, Pinus wallichiana A.B. Jacks. The average
annual precipitation in the valley is 100–300 mm,
mostly occurring during winter and early spring in the
form of snow. Mean temperatures range from –10°C
in winter to +35°C in summer.
Additional Specimen: PAKISTAN: Gilgit Baltistan,
Darel Valley 34°36′ N, 72°26′ E, elev. 1900 m, on
rocks, October 21, 2022, Muhammad Shahid Iqbal
DR-355 (LAH37605), (ITS GenBank accession
number OP879682).
Kiliasia nordlandica (Th.Fr.) Kistenich,
Timdal, Bendiksby and S. Ekman
Thallus saxicolous, sub-squamulose, scattered
about 1cm wide bunches of minute squamules up to
2.8 mm in diam., irregularly lobed, becomes rounded
globules, raised, convex, whitish grey, often with a
brownish base, weakly to slightly pruinose, rough sur-
face, smooth or narrowly fissured from below light
brown. Upper cortex 25–60 µm tall, distinct thick
epinecral layer dark brown; algal layer 100–120 µm
continuous, Medulla indistinct, Photobiont chloro-
coccoid, algal cell globose to sub globose up to 15 µm
in diameter lower cortex absent. Apothecia lecideine,
connate prominent black, up to 1mm across, epru-
inose, convex and without distinct margins. Proper
exciple 40–50 colourless to slightly grey Epihymenium
15–20 µm grey, Hymenium colourless slightly brown
40–60 µm high Hypothecium 50–60 µm colourless
Asci 8-spored, clavate, well-developed ocular cham-
ber, bacidia-type. Paraphyses straight, septate,
unbranched almost 1.8–2.5 µm thick, the apical cells
clavate, up to 7 µm wide, Ascospores hyaline, oblong
to ellipsoid, 11–14 × 5–7 µm, 1-septate.
Spot tests: thallus K-, C-, KC-.
Chemistry: no substance detected.
Material Examined: PAKISTAN. Khyber Pakh-
tunkhwa: Chitral, Garam Chashma; 35°59′50′′ N and
71°33′50′′ E, 2550 m a.s.l.; on rocks; November 14,
2020, M. Usman GC-60C Holotype: LAH (LAH-
37606); (GenBank no. OP872703).
Ecology: Saxicolous in dry temperate climate at an
altitude of 2100–2550 m a.s.l in open situations
exposed to sun and rain, in hilly topography having
maximum and minimum ranging from 20 to 36°C in
the summer to –4°C in the winter respectively. The
area receives an average rainfall of about 603 mm.
Comments: Toninia pakistanica sp. nov. is morpho-
logically similar to T. ci n e r eovi r e n s as both have upto
3 mm wide squamules, weakly concave to weakly con-
vex lobate thallus. Phylogenetically, T. pakistanica sp.
nov. and T. ci n e reov i r e ns are found to be strongly sup-
ported relative taxa (100% of BS) (Fig. 3). Our taxon
can be differ from T. c i n e reov i r ens by having dark
brown lobate squamules, densely pruinose upper sur-
face, more thicker epinecral layer 60 µm, epruinose to
weakly pruinose thallus and 25–45 µm thick grey to
blackish grey lower cortex vs (dark deeply lobed squa-
mules, medium brown to dark brown often with oliva-
ceous tinge upper surface, less thicker epinecral layer
40 µm, epruinose thallus, poorly developed lower cor-
tex) (Upreti et al., 2010). Morphologically, the new
species can be easily distinguished from the other spe-
cies of genus Toninia tristis subsp. asiae centralis and
T. squal i d a with dark olive to black thallus, dark brown
lobate squamules, epruinose to weakly pruinose with
dark olive to black thallus, densely pruinose upper sur-
face, more thicker epinecral layer 60 µm, and 25–
45 µm thick grey to blackish grey lower cortex (Upreti
et al., 2010).
In the present study Kiliasia nordlandica (Th.Fr.) is
morpho-anatomically and molecularly characterized.
The phylogenetic analyses clearly separated K. nord-
landica and K. sculpturata. Our results supported inde-
pendent phylogenetic position of K. nordlandica and
clearly got separated from K. sculpturata (Fig. 3).
K. nordlandica is differs from K. sculpturata by pale
grey to white thallus, grey epithecium vs (thallus pale
brown, epithecium dark brown). (Miao et al., 2020).
This study reports K. nordlandica as new record to the
lichen biota of Pakistan.
BIOLOGY BULLETIN 2023
A NEW SPECIES AND A NEW RECORD OF FAMILY RAMALINACEAE 5
Fig. 3. Phylogenetic relationships of Toninia pakistanica sp. nov. and Kiliasia nordlandica (Th. Fr.) Kistenich, Timdal, Bendiksby &
S. Ekman, based on a Maximum Likelihood analysis of the ITS region. The tree was rooted using Phyllopsora borbonica Timdal
and Krog (MG925988) and Sporacestra pertexta (Nyl.) Stapnes and Timdal (MG926000).
MN103178.1 Toninia squalida
KP314433.1 Toninia sp.
AF282127.1 Toninia coelestina
MZ244014.1 Toninia cf. mesoidea
MG838183.1 Toninia squalida
AF282103.1 Toninia squalida
MG838182.1 Toninia squalida
MG838181.1 Toninia squalida
MG838200.1 Toninia squalida
Toninia pakistanica sp. nov. DR-170
Toninia pakistanica sp. nov. DR-355
AF282104.1 Toninia cinereovirens
MN103171.1 Toninia cinereovirens
OM065442.1 Toninia tristis
OM521966.1 Toninia tristis subsp. asiae centralis
MN103177.1 Toninia tristis subsp. tristis
KT232214.1 Thalloidima sedifolium
HQ650689.1 Toninia sedifolia
MG925988.1 Phyllopsora borbonica
MG926000.1 Sporacestra pertexta
AF282119.1 Toninia opuntioides
AF282121.1 Toninia rosulata
MG838191.1 Toninia albilabra
MG926131.1 Toninia albilabra
AF282118.1 Toninia taurica
AF282117.1 Toninia candida
AF282116.1 Toninia alutacea
MG838188 Kiliasia nordiandica
AF282113.1 Toninia nordiandica
Kiliasia nordiardica GC60C
MG838198.1 Kiliasia nordiandica
MG838159.1 Kiliasia nordiandica
AF282112.1 Toninia philippea
AF282110.1 Kiliasia scuipturata
MG926034.1 Kiliasia scuipturata
KF024737.1 Toninia ruginosa
KF024735.1 Toninia ruginosa
KF024736.1 Toninia ruginosa
KF024738.1 Toninia ruginosa
MN103173.1 Toninia lutosa
MN103172.1 Toninia lutosa
AF282114.1 Toninia lutosa
AF282107.1 Toninia plumbina
AF282108.1 Toninia talparum
OK333025.1 Toninia aromatic
KX132986.1 Toninia populorum
MG925950.1 Toninia populorum
0.1
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
89
98
98
98
70
73
81
54
90
91 96
74
99
53
79
75
76 96
98
56
95
6
BIOLOGY BULLETIN 2023
MUHAMMAD SHAHID IQBAL et al.
Table 1. Voucher specimens and NCBI GenBank accession numbers of the sequences used in the phylogenetic analysis
Species names ITS GenBank Accession
numbers Voucher name Origin
Phyllopsora borbonica MG925988 O:L 797 Reunion
Sporacestra pertexta MG926000 hb Perez-Ortega Cuba
Toninia albilabra MG838191 O-L-144850 Peru
Kiliasia nordlandica OP872703 LAH 37606 Pakistan
Kiliasia nordlandica AF282113 Haugan and Timdal 8129 (O) Norway
Kiliasia nordlandica MG838188 O-L-200953 Spain
Kiliasia nordlandica MG838198 O-L-195601 Norway
Kiliasia nordlandica MG838159 O-L-184351 Norway
Kiliasia sculpturata AF282110 Haugan and Timdal 7829 (O) Norway
Kiliasia sculpturata MG926034 O:L 19092 Russia
Toninia albilabra MG92 6131 O:L 19 88 96 Spa in
Toninia aromatica OK333025 – Czech Republic
Toninia alutacea AF282116 Haugan and Timdal 4824 (O) Norway
Toninia cinereovirens AF282104 Haugan and Timdal 7953 (O) Norway
Toninia cinereovirens M N10 3171 17-0 4 25 –
Toninia coelestina AF282127 Haugan 5985 (O) Norway
Toni n ia cf. me s oid e a MZ244014 Leavitt 18-574ton BRY-C USA
Toninia candida AF282117 Bratli and Timdal 8733 (O) Norway
Toni n ia lu t osa MN103173 16-0003 –
Toni n ia lu t osa MN103172 10-0014 –
Toni n ia lu t osa AF282114 Timdal SON28/08 (O) Mexico
Toninia opuntioides AF282119 Haugan and Timdal 8057 (O) Norway
Toninia pakistanica sp. nov OP879646 LAH 37604 Pakistan
Toninia pakistanica sp. nov OP879682 LAH 37605 Pakistan
Toninia philippea AF282112 Haugan and Timdal H3750 (O) Norway
Toninia plumbina AF282107 Haugan 4352 (O) Norway
Toninia populorum KX132986 – Switzerland
Toninia populorum MG925950 BG Austria
Toninia ruginosa KF024736 DS8341 USA
Toninia ruginosa KF024738 DS8349 USA
Toninia ruginosa KF024737 DS83 42.1 USA
Toninia ruginosa KF024735 DS8343.1 USA
Toni n ia ro s u lat a AF282121 Timdal 8640 (O) Norway
Toni n ia sq u alid a MG838183 O-L-169774 Norway
Toni n ia sq u alid a AF282103 Haugan 4970 (O) Norway
Toni n ia sq u alid a MG838182 O-L-182301 Norway
Toni n ia sq u alid a MG838181 O-L-184426 Norway
Toni n ia sq u alid a MG838200 O-L-184539 Norway
Toni n ia sq u alid a MN103178 10-000 9 –
Toni n ia sp. KP3144 33 ZT2013191 Svalbard
Toni n ia se difo l ium KT232214 D. Ertz 7601 (DUKE0047686) –
Toni n ia se difo l ia HQ650689 – –
Toninia tristis OM065442 Han LF201308047-1 ITS4 –
Toninia tristis subsp. asiae centralis OM521966 201899728 –
Toninia tristis subsp. tristis MN103177 10-0276 –
Toni n ia ta l paru m AF282108 Timdal SON120/01 (O) USA
Toni n ia ta u rica AF282118 Haugan and Timdal 8060 (O) Norway
BIOLOGY BULLETIN 2023
A NEW SPECIES AND A NEW RECORD OF FAMILY RAMALINACEAE 7
FUNDING
No funding was received for conducting this study.
COMPLIANCE WITH ETHICAL STANDARDS
The authors declare that they have no conflicts of interest.
This article does not contain any studies involving animals
or human participants performed by any of the authors.
AUTHOR CONTRIBUTIONS
All authors contributed to the study conception and
design. Samples and morpho-anatomical data was collected
by Muhammad Shahid Iqbal. Material preparation and
analysis were performed by MSI, HSA and ANK. The first
draft of the manuscript was written by Muhammad Shahid
Iqbal and all authors commented on previous versions of
the manuscript. All authors read and approved the final
manuscript.
REFERENCES
Al tschul, S.F., Gis h, W., Miller, W. , Myers, E.W., a nd Lip -
man, D.J., Basic local alignment search tool, J. Mol. Biol.,
1990, vol. 215, no. 3, pp. 403–410.
Ekman, S., Andersen, H.L., and Wedin, M., The limita-
tions of ancestral state reconstruction and the evolution of
the ascus in the Lecanorales (lichenized Ascomycota), Syst.
Biol., 2008, vol. 57, no. 1, pp. 141–156.
https://doi.org/10.1080/10635150 801910 451
Ekman, S., Molecular phylogeny of the Bacidiaceae (Le-
canorales, lichenized Ascomycota), Mycol. Res., 2001,
vol. 105, no. 7, pp. 783–797.
https://doi.org/10.1017/s095375 620100 4269
Gardes, M. and Bruns, T.D., ITS primers with enhanced
specificity for basidiomycetes-application to the identifica-
tion of mycorrhizae and rusts, Mol. Ecol., 1993, vol. 2,
no. 2, pp. 113–118.
h tt p s :/ / d oi . or g / 10 .1111 /j .1 36 5 - 29 4 X .19 9 3 .t b 00 0 0 5. x
Hale, M.E., How to Know the Lichens, WC Brown Co.,
1979.
Hall, T., Biosciences, I., and Carlsbad, C.J.G.B.B.,
BioEdit: an important software for molecular biology,
GERF Bull. Biosci., 2011, vol. 2, no. 1, pp. 60–61.
Indexfungorum, 2022. http://www.indexfungorum.org/
names/Names.asp. Accessed November 10, 2022.
Katoh, K. and Standley, D.M., MAFFT multiple sequence
alignment software version 7: improvements in perfor-
mance and usability, Mol. Biol. Evol., 2013, vol. 30, no. 4,
pp. 772–780.
Khan, M., Khalid, A.N., and Lumbsch, H.T., A new spe-
cies of Lecidea (Lecanorales, Ascomycota) from Pakistan,
MycoKeys, 2018, vol. 38, pp. 25–34.
Kistenich, S., Timdal, E., Bendiksby, M., and Ekman, S.,
Molecular systematics and character evolution in the lichen
family Ramalinaceae (Ascomycota: Lecanorales), Tax on,
2018, vol. 67, no. 5, pp. 871–904.
https://doi.org/10.12705/675.1
Lücking, R., Hodkinson, B.P., and Leavitt, S.D., The 2016
classification of lichenized fungi in the Ascomycota and Ba-
sidiomycota—approaching one thousand genera, Bryolo-
gist, 2017a, vol. 119, pp. 361–416.
https://doi.org/10.1639/0007-2745-119.4.361
Lücking, R., Hodkinson, B.P., and Leavitt, S.D., Correc-
tions and amendments to the 2016 classification of liche-
nized fungi in the Ascomycota and Basidiomycota, Bryolo-
gist, 2017b, vol. 120, pp. 58–69.
https://doi.org/10.1639/0007-2745-120.1.058
Table 2. Comparison of Toninia pakistanica sp. nov. to closely related species
Characters Toninia
pakistanica sp. nov. Toninia cinereovirens Toninia tristis subsp.
asiae centralis Toninia squalida
Thallus morphology Epruinose to densely
pruinose
Epruinose Entirely pruinose –
Squamules (Type) Lobate squamules Deeply lobed squamules Bullate squamules –
Squamules (Color) Dark brown Dark Brown Pure brown
Thallus Color Dark olive to black Brown to dark brown Brown Pure brown
Upper Surface (Color) Dark brown to greyish
brown
Medium brown to dark
brown often with oliva-
ceous tinge
Castaneous brown to
dark Brown with green
tinge
–
Upper surface Densely pruinose Epruinose – –
Lower Surface (Color) Pale brown to grey Pale brown to medium
brown
Pale brown to black –
Epinecral layer (Thickness) 60 µm 40 µm 100 µm –
Lower cortex (Thickness) 25–45 µm Poorly developed – –
Chemistry No chemical
compounds
No chemical Triterpenes –
References In this paper (Upreti et al., 2010) (Upreti et al., 2010) (Upreti et al., 2010)
8
BIOLOGY BULLETIN 2023
MUHAMMAD SHAHID IQBAL et al.
Miao, C., Sun, M., Zhang, X., Ren, Z., and Hu, L., New
records of Toninia from China, Mycotaxon, 2020, vol. 135,
no. 3, pp. 569–578.
Sambrook, J. and Russell, D.W., Detection of DNA in aga-
rose gels, in Molecular Cloning, A Laboratory Manual, New
York: Cold Spring Harbor Laboratory Press, 2001, 3rd ed.,
pp. 5–14.
Sun, M.J., Yan, S.K., Tang, R., Wang, C., and Zhang, L.,
New records of Bilimbia and Toninia from China,
Mycotaxon, 2019, vol. 134, no. 1, pp. 139–146.
Tamura, K., Stecher, G., Peterson, D., Filipski, A., and
Kumar, S., MEGA6: molecular evolutionary genetics anal-
ysis version 6.0, Mol. Biol. Evol., 2013, vol. 30, no. 12,
pp. 2725–2729.
Timdal, E., A monograph of the genus Toninia (Lecideaceae,
Ascomycetes), Opera Bot., 1991, vol. 110.
Timdal, E., Toninia, in Lichen Flora of the Greater Sonoran
Desert Region, Nash, T.H. et al., Eds., Tempe, Arizona State
University: Lichens Unlimited, 2002, vol. 1, pp. 488–501.
Upreti, D.K., Joshi, Y., Nayaka, S., and Joshi, S., New re-
cords of squamulose lichens from Western Himalaya,
Geophytology, 2010, vol. 38, nos. 1–2, pp. 85–91.
Whit e, T.J.T., Bru ns., S .J., L ee, W.T., a nd Taylor, J., Am -
plification and direct sequencing of fungal ribosomal RNA
genes for phylogenetics, PCR Protocols: a Guide to Methods
and Applications, 1990, vol. 18, no. 1, pp. 315–322.
https://doi.org/ 10.1016/B978-0-12-372180-8.500 42-1
