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Acta Botanica Hungarica 63(3–4), pp. 335–342, 2021
DOI: 10.1556/034.63.2021.3-4.6
POLYCOCCUM, VARIOSPORA, AND ZEROVIELLA,
THREE NEW GENERA FOR PAKISTAN
*, M. and A. N.
Fungal Biology and Systematics Research Lab, Department of Botany, University of the Punjab
Quaid-e-Azam Campus-54590, Lahore, Punjab, Pakistan
*E-mail: kamranhabiib@gmail.com; corresponding author
(Received: 24 March 2021; Accepted: 10 August 2021)
Two lichen species, Variospora kudratovii and Zeroviella laxa, and a lichenicolous species,
Polycoccum clauderouxii are reported for the first time from Pakistan and represent first ge-
neric records as well. Detailed macro- and micromorphological descriptions and spot test
results are provided along with ITS-based molecular phylogenetic analysis.
Key words: Khaplu, nrDNA, Teloschistaceae, xanthorioid lichen, Ziarat
INTRODUCTION
During lichen exploration, authors collected specimens of xanthorioid
-
mens from all other known members of Pakistan’s xanthorioid and calopla-
coid lichens (Ahmad 1965, Aptroot and Iqbal 2012). Identity of the species
-
mens with published description (Kondratyuk et al. 2013, 2015, 2016). As
these represent new generic records for Pakistan, detailed descriptions and
The Teloschistaceae subfamilies Xanthorioideae and Caloplacoideae contain
42 and 31 genera (Kondratyuk et al. 2014a, b, Mishra et al. 2020). Previous re-
cords from Pakistan include only one genus of xanthorioid lichens, i.e. Xan-
thoria (Fr.) Th. Fr. (with six species reported) and two genera of caloplacoid
lichens, i.e. Caloplaca Th. Fr. (with 67 species reported) and Seirophora Poelt
(with one species reported) (Ahmad 1965, Aptroot and Iqbal 2012, Habib et
al. 2017).
This work will bring some more insight in the lichens of Pakistan as most
of the lichens of the country are very poorly known with very little informa-
tion and will be helpful in compilation of taxonomic diversity of lichens of
Pakistan.
Acta Bot. Hung. 63, 2021
336 HABIB, K., ARSHAD, M., BANO, A. and KHALID, A. N.
MATERIAL AND METHODS
Morphological characters of the thalli were observed under the Meiji
Techno, EMZ-5TR, Japan stereomicroscope. Colours were designated ac-
cording to Munsell (1975). The morphology of fruiting bodies was studied
separately. For micromorphological analysis, sections of the apothecia were
cut and mounted on glass slides using water and 5% KOH (5/100 aqueous)
as mounting media and examined under compound microscope MX4300H,
Meiji Techno Co., Ltd., Japan. The spot tests were performed by applying
10% potassium hydroxide (K-test), calcium hypochloride (C-test) and KC-test
to the lichen fragment and the changes were observed under a stereomicro-
scope. Voucher specimens were deposited in the LAH Herbarium, University
of the Punjab, Quaid-e-Azam Campus, Lahore, Pakistan.
-
plified using ITS1F/ITS4 primer pair following amplification protocol of Khan
et al. (2018). The amplified DNA fragments were visualised in 1.2% agarose gel
(Sambrook and Russell 2001) and PCR products were sequenced. The nucleo-
-
et al.
for phylogenetic analysis. Online MUSCLE was used to align the sequences
alignments were trimmed to nearly equal number of sites for all sequences.
Phylogenetic analysis was performed at 1,000 bootstraps in MEGA 6.0 (Tamu-
ra et al. 2013) by constructing maximum likelihood (ML) trees.
RESULTS AND DISCUSSION
Zeroviella laxa
(Fig.1)
Thallus foliose, saxicolous, thick, centrally squeezed, warty, rough, rusty
patches on the surface; upper surface cylindrical to subcylindrical, flattened
toward lobes, with up to 1.5 mm black galls of lichenicolous fungus, yellow
(7.5YR 8/4) to light orange (7.5YR8/3,8/4); lower surface centrally hollow, at-
tached by margins. Lobes 0.5–1.5 mm wide, semiconvex to flattened. Pseudo-
cyphellae well developed, numerous in the centre, up to 1 mm in diam. Apo-
thecia not found. Spot tests: cortex K+ (violet red), C–; medulla K–, C–, KC–.
Comments: In our phylogram (Fig. 4), the sequences of Z. laxa formed
a sister clade with East Asian Z. mandschurica (Zahlbr.) S. Y. Kondr. et Hur.
Morphologically, Zeroviella laxa differs from Z. mandschurica in having wider
Acta Bot. Hung. 63, 2021
337
POLYCOCCUM, VARIOSPORA, AND ZEROVIELLA, NEW GENERA FOR PAKISTAN
lobes (Kondratyuk et al. 2015). Zeroviella laxa is also similar to Z. esfahanensis
S. Y. Kondr., Zarei-Darki et Hur but differs by having squeezed, thick, cy-
lindrical to subcylindrical and warty thallus. The morphological comparison
and the ITS-based phylogeny demonstrated the identity of our collection with
Chinese Z. laxa, reported (KU056854), having exactly the same ITS sequence.
Previously, Zeroviella laxa was reported from China in Asia. This study added
this taxon as a new record to the lichen biota of Pakistan.
2018 (LAH35905).
Fig. 1. Zeroviella laxa
galls of lichenicolous fungus on thallus
Acta Bot. Hung. 63, 2021
338 HABIB, K., ARSHAD, M., BANO, A. and KHALID, A. N.
Variospora kudratovii S. Y. Kondr., Kärnefelt, Elix, A. Thell, Jung Kim,
(Fig. 2)
Thallus crustose, saxicolous, areolate, rugose, slightly pruinose; areoles
scattered or distant, more or less aggregated, angular to irregular, determi-
nate, 0.5–1.5 mm wide; upper surface rough, yellow (7.5YR8/4) to orange
(7.5YR8/6), distinctly wrinkled, not fissured. Photobiont Myrmecia (Chloro-
phyceae), globose to subglobose, 12–20.2 µm in diam. Ascomata apothecial,
initially 1–3 per areole, usually 1 adult per areole, slightly glossy, 0.2–1 mm
diam. Disc orange, rounded to subrounded. Hymenium hyaline, 50–80 µm
tall. Asci clavate, Teloschistes-type, 8-spored, 41–60 × 10–18 µm. Ascospores
bilocular, ellipsoidal, 10–16 × 4.5–6.5 µm. Hamathecial elements: paraphysis,
septate, apex swollen up to 4.5 µm wide, capitate, branched. Spot tests: thallus
and apothecia K+ purple, C–, KC–.
Comments: The Pakistani specimen of Variospora kudratovii (Z18-3A) is
morphologically similar to the type specimen of V. kudratovii except for hav-
ing paraphyses with narrower apices (4.5 µm). Variospora kudratovii is also
Fig. 2. Variospora kudratovii -
cospores; E = paraphyses
Acta Bot. Hung. 63, 2021
339
POLYCOCCUM, VARIOSPORA, AND ZEROVIELLA, NEW GENERA FOR PAKISTAN
similar to V. dolomiticola, but differs in having larger thalline areoles, un-
cracked distant areoles and larger ascospores. There is a difference of four
nucleotide between the Pakistani collection of V. kudratovii (Z18-3A) and the
Iranian specimens of V. kudratovii (KJ021245-KJ021247) at site 91, 92, 406 and
489, but similar at the same positions with the type specimen sequence of
V. kudratovii (KJ021242-KJ021244). The combination of morphological studies
and ITS-based molecular phylogenetic analysis confirmed the identity of the
Pakistani material with V. kudratovii. In Asia, it has been reported earlier only
from Iran (Kondratyuk et al. 2013). This study added Variospora kudratovii as a
new record to the lichen biota of Pakistan.
dry temperate area, on rock; coll.: A. N. Khalid (Z18-3A), 2 July 2018 (LAH36070).
Polycoccum clauderouxii
(Fig. 3)
Lichenicolous, dispersed on thalline lobes when young, in mature state
globose to subglobose, black, up to 1.2 mm, slightly pruinose, up to 2 mm
inside thalline lobes. Pycnidia not
found. Ascomata perithecioid, im-
mersed in the galls, globose to pyri-
form, black, ostiolate, glossy. Para-
physis frequently branched. Asci
elongate, hyaline, clavate, bilayered,
8-spored, 100–115 × 12–20 µm. As-
cospores brown to blackish brown,
septate, 2-celled, obclavate to cylin-
drical, 20–25 × 5.5–8 µm, granules
3–4.5 µm. Host: Zeroviella laxa, grow-
ing on rock in cold deserts.
Comments: The lichenicolous
fungus Polycoccum clauderouxii on
Pakistani material of Zeroviella laxa is
morphologically similar to the Chi-
nese type specimen of P. clauderouxii,
except for having larger asci (100–115
Fig. 3. Polycoccum clauderouxii: A = galls on
-
cospores
Acta Bot. Hung. 63, 2021
340 HABIB, K., ARSHAD, M., BANO, A. and KHALID, A. N.
Fig. 4. Molecular phylogenetic analysis of Zeroviella laxa. The evolutionary history was in-
ferred by using the maximum likelihood method based on the Tamura 3-parameter model.
A discrete Gamma distribution was used to model evolutionary rate differences among
sites. The analysis involved 17 nucleotide sequences. Sequence generated from local col-
Fig. 5. Molecular
phylogenetic anal-
ysis by maximum
likelihood meth-
od. The evolution-
ary history was
in ferred by using
the maximum
like lihood meth-
od based on the
Kimura 2-param-
eter model. The
analysis involved
34 nucleotide se-
quences. All posi-
tions containing
gaps and missing
data were elimi-
nated. Sequence
generated from
local collection is
marked with t
Acta Bot. Hung. 63, 2021
341
POLYCOCCUM, VARIOSPORA, AND ZEROVIELLA, NEW GENERA FOR PAKISTAN
vs. 65–70 µm) and ascospores (22–25 vs. 16–21 µm). Our Pakistani specimen
differs from Polycoccum clauzadei Nav.-Ros. et Cl. Roux (described from France
colonising on a Xanthoria host; Navarro-Rosinés and Roux 1998) by its larger
galls on a Zeroviella host thalli, 8-spored asci, and longer ascospores.
Previously Polycoccum clauderouxii was known from the type locality,
China, on Zeroviella laxa growing on soil in mountain deserts. This study de-
scribes Polycoccum clauderouxii found on Zeroviella laxa, growing on rock in
cold desert. This fungus is reported here for the first time outside the type
locality and is an addition to the fungi of Pakistan.
E; 2,600 m a.s.l.; dry temperate area, cold desert, on Zeroviella laxa
20 July 2018, (LAH35905).
PHYLOGENETIC ANALYSIS
The aligned data set of Zeroviella laxa comprised of 538 characters con-
taining gaps, of which 422 sites were conserved, 113 variable, 95 parsimo-
ny informative and 18 were singleton. Verrucoplaca verruculifera (Vain.) S. Y.
Kondr., Kärnefelt, Elix, A. Thell, Jung Kim, M. H. Jeong, N. N. Yu, A. S. Kondr.
et Hur (EU639624 & JQ301669) was chosen as an outgroup. The Pakistani col-
lection, Zeroviella laxa (KPL-01) clustered with the Chinese Z. laxa (KU056854)
with 94% bootstrap support (Fig. 4) and formed a sister clade with the East
Asian Z. mandschurica (Zahlbr.) S. Y. Kondr. et Hur.
The aligned data set of Variospora kudratovii included 542 characters in-
cluding gaps, of which 372 sites were conserved, 163 variable, 109 parsimony
informative and 53 singletons. Ioplaca pindarensis (Räsänen) Poelt et Hinter
(JQ301672) was chosen as an outgroup. The Pakistani collection of V. kudratovii
(Z18-3A) clustered with Iranian specimens of V. kudratovii (KJ021245-KJ021247)
with 71% bootstrap support (Fig. 5) and formed a sister clade with V. dolomiti-
colaCaloplaca sororicida Steiner et Poelt.
*
Acknowledgements – We are sincerely thankful to Prof., Dr Sergey Y. Kondratyuk, Kholodny
-
hore, for pre-submission review of the manuscript.
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