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Phytotaxa 558 (2): 185–202
https://www.mapress.com/pt/
Copyright © 2022 Magnolia Press Article PHYTOTAXA
ISSN 1179-3155 (print edition)
ISSN 1179-3163 (online edition)
Accepted by Sinang Hongsanan: 25 Jul. 2022; published: 15 Aug. 2022
https://doi.org/10.11646/phytotaxa.558.2.3
185
Morpho-phylogenetic insights reveal Bisporella montana as Calycina montana
comb. nov. (Pezizellaceae, Helotiales)
ANIS S. LESTARI1,2,3,4 & K. W. THILINI CHETHANA1,2,5*
1 School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand
2 Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand
3 Research Organization for Life Sciences, National Research and Innovation Agency (BRIN), Cibinong 16911, Indonesia
4
�
anislestari1@gmail.com; https://orcid.org/0000-0002-1606-9884
5
�
tchethi@yahoo.com; https://orcid.org/0000-0002-5816-9269
*Corresponding author:
�
tcheti@yahoo.com;
�
kandawatte.thi@mfu.ac.th
Abstract
Calycina-like specimens were collected in Doi Inthanon National Park, Thailand. The combined phylogeny of ITS and LSU
based on maximum likelihood and bayesian inference analyses confirmed the taxonomic placement of our specimen in
Pezizellaceae. Based on morphology and multi-gene phylogeny, the new discomycete specimen was identified as Calycina
montana comb. nov., and was documented for the first time in Thailand. Calycina montana is characterized by the amber
color, stipitate apothecia, gelatinized ectal excipulum, cylindrical asci and ellipsoidal ascospores. The morphology was
observed together with the type strain, Bisporella montana. Our phylogenetic analyses and observation of the morphology
of the type strain also confirmed the new combination of Bisporella shangrilana as Calycina shangrilana comb. nov., which
resolves the confusion of Bisporella placement in Pezizellaceae.
Keywords: 2 new combination species, Ascomycota, discomycetes, Leotiomycetes, saprobic
Introduction
Pezizellaceae was introduced with Pezizella as the type genus (Velenovský 1934), while Peziza sordida (type
species of Pezizella) was transferred to Calycina (Baral 1989). Pezizella species were also re-observed and re-
assigned to different genera, such as Allophylaria, Arachnopeziza, Calloria, Calycellina, Ceratophacidium, Cistella,
Clavidisculum, Conchatium, Crocicreas, Cyathicula, Dasyscyphus, Gemmina, Helotium, Hyalinia, Hyalopezia,
Hyalorbilia, Hyaloscypha, Hymenoscyphus, Ijuhya, Incrupila, Laetinaevia, Leptodontidium, Mniaecia, Mollisia,
Mollisina, Naeviopsis, Niptera, Neomensularia, Ombrophila, Orbilia, Rodwayella, Pachydisca, Pithyella, Psilachnum,
Pseudohelotium, Pyrenopeziza, Thelebolus, Trichopeziza and Unguiculella (Baral 1989, Baral 1993, Baral & Quijada
2020, Index Fungorum 2022). To date, the delimitation of genera in Pezizellaceae remains unresolved (Suija &
MotieJűnaitë 2017).
Calycina was proposed by Nees von Esenbeck (1817), which was raised to generic rank by Gray (1821), and
later Calycina herbarum (basionym: Peziza herbarum) was designated as its type species (Dumont 1972). Calycina,
previously placed in Hyaloscyphaceae, was then transferred to Pezizellaceae (Jaklitsch et al. 2016, Han et al. 2014).
Calycina is a controversial genus with its complicated taxonomic relationships with Bisporella and Hymenoscyphus
(Helotiaceae), both of which are usually characterized by calycina-like specimens (Korf & Carpenter 1974, Korf &
Bujakiewicz 1985, Zhuang et al. 2017, Ekanayaka et al. 2019). Several studies conducted on the morphological and
phylogenetic placement of Calycina, Bisporella and Hymenoscyphus resulted in synonymizing Bisporella citrina,
B. discendens, B. drosodes and Hymenoscyphus elisii under Calycina citrina, C. claroflava, Calycina drosodes and
Calycina lactea, respectively (Gray 1821, Baral et al. 2013).
Approximately 30 species of Calycina have been discovered worldwide (Baral 2016), and the hypomycetous
fungus, Chalara, was reported as the asexual morph of Calycina (Baral & Rämä 2015, Baral 2016). The lifestyles of
Calycina species are mostly saprobic on various lignocellulosic substrates found on land (Dennis 1956, Quijada &
Beltrán-Tejera 2017) and marine splash zone habitats (Baral & Rämä 2015). Calycina-like specimens were found in
LESTARI & CHETHANA
186 • Phytotaxa 558 (2) © 2022 Magnolia Press
subtropical and tropical regions in Asia (Zhuang et al. 2017, Han et al. 2014, Ekanayaka et al. 2019), Europe (Baral &
Rämä 2015, Suija & Motiejűnaitë 2017, Quijada & Beltrán-Tejera 2017, Crous et al. 2019), New Zealand (Fukami et
al. 2010), North America (Korf & Bujakiewicz 1985), Russia (Milekhin & Prokhorov 2007), South Africa (Saccardo
1889), South America (Carpenter et al. 1975, Gamundi et al. 2004), and UK (Baral & Rämä 2015). The only calycina-
like specimens found in Southeast Asia were Calycina claroflava (synonym: B. discedens) from the Philippines and
Thailand (Lizon & Korf 1995, Ekanayaka et al. 2019). Thus, more fungal exploration in Southeast Asia is vital for this
genus.
During our collection trip to Doi Inthanon National Park, Chiang Mai, Thailand, calycina-like specimens were
found on unidentified rotten bark. The current study aims to characterize the newly found calycina-like specimens
based on polyphasic approaches and provide complete descriptions, sequence data, and illustrations.
Materials and methods
Sample collection and morphological studies
Discomycetous specimens were collected in Doi Inthanon National Park, Chiang Mai Province, Thailand in October
2021. Specimens enclosed in paper boxes were brought to the laboratory. The samples were examined following
the methodology described in Senanayake et al. (2020). The macromorphology was examined and photographed
using a Leica EZ4 stereomicroscope (Leica Microsystems Company, Germany) connected to an Olympus SC 180
digital camera. The apothecial sections were mounted in distilled water or 5% KOH and stained in Congo red and
Melzer reagent for the amyloid test. Microscopic characters were observed and photographed using the Nikon Eclipse
Ni-U compound microscope fitted to the Nikon DS-Ri2 digital camera. Microscopic characters, such as excipulum
layers, paraphyses, asci, and ascospores, were measured using the Tarosoft (R) Image Frame Work program. The
photoplates were prepared with Adobe Photoshop CS3 Extended version 10.0 software (Adobe Systems, USA). The
dried herbarium specimens were deposited in the Mae Fah Luang University Herbarium (MFLU). The type strains,
Bisporella montana (HMAS 275566) and B. shangrilana (HMAS 275568), were loaned from Herbarium Mycologicum
Academiae Sinicae Beijing, China (HMAS). Faces of fungi numbers and Index Fungorum numbers were obtained as
in Jayasiri et al. (2015) and Index Fungorum (2022). The data is added to the details of GMS microfungi database
(Chaiwan et al. 2021).
DNA extraction, amplification and sequencing
Apothecia were collected into 2 ml microtubes and ground using the TissueRuptor homogenizer (Qiagen, USA),
followed by genomic DNA extraction with the E.Z.N.A Fungal DNA Mini Kit D3390-02 (Omega Bio-Tek, USA)
using the manufacturer’s instructions with slight modifications.The modification was conducted in the elution step
with the total volume of extracted DNA was 50 μl. Extracted DNA were stored subsequently at -20 °C. The Internal
Transcribed Spacer (ITS) and Large Sub-Unit of 28 S rRNA (LSU) gene regions were amplified using primers ITS5/
ITS4 (White et al. 1990) and LROR/LR5 (Vilgalys & Hester 1990), respectively. The PCR reactions were carried out
in 50 μl volumes, containing 25 µl of 2 × Go Taq@ Green Master Mix (Promega. com, USA), one μl of each primer
(20 μM), one μl genomic DNA and 22 μl of de-ionized water. PCR amplification conditions for ITS and LSU are as
follows: initial denaturation at 94 oC for 3 minutes, followed by 35 cycles of denaturation at 94 oC for 40 seconds,
annealing at 55 oC for 50 seconds, extension at 72 oC for 90 seconds and a final extension at 72 oC for 10 minutes. The
PCR products were stained with DL5000-DNA Fluorescent loading dye (Smobio Technology Inc, Taiwan), visualized
on 1.5% agarose gels and sequenced at Biogenomed Co. Ltd Bangkok, Thailand. The newly produced sequences were
deposited in the GenBank (Table 1).
Sequence alignments and phylogenetic analyses
Sequences of ITS and LSU were checked for their quality and assembled with SeqMan V. 7.0.0 (DNASTAR, Madison,
WI), and then aligned in BioEdit v 7.0.9.0 (Hall 1999). The newly generated sequences were confirmed their identity
via NCBI Blastn while reference sequences from related studies (Cantrell & Hanlin 1997, Fukami et al. 2010, Koukol
2011, Baral et al. 2013, Han et al. 2014, Baral & Rämä 2015, Guatimosim et al. 2016, Hosoya & Zhao 2016, Suija &
MotieJűnaitë 2017, Zhuang et al. 2017, Crous et al. 2019, Ekanayaka et al. 2019, Vu et al. 2019, Mitchell 2021, Senn-
Irlett et al. 2021) were retrieved from the GenBank (Table 1). Each data set was aligned using MAFFT v. 7, edited
manually using BioEdit v. 7.2 (Hall 1999) then trimmed using trimAl v1.2 software with parameter -gappyout (Capella-
REVEAL BISPORELLA MONTANA AS CALYCINA MONTANA Phytotaxa 558 (2) © 2022 Magnolia Press • 187
Gutiérrez et al. 2009). Phylogenetic analyses of single-gene alignments and the combined genes (ITS and LSU) were
performed based on Maximum Likelihood (ML) and Bayesian Inference (BI) analyses. Maximum likelihood analysis
was performed for 1000 bootstrap replicates using RaxML-HPC2 on XSEDE (8.1.12) in the CIPRES Science Gateway
(Miller et al. 2010, Stamatakis 2014). The GTR+G model was selected as the substitution model for ML analysis.
TABLE 1. Accession numbers and origin of fungal taxa used in the phylogenetic analysis.
Taxon name Strain codesaGenBank accession no Origin
ITS LSU
Bisporella pallescens DMS-10078235T MW203182 - Denmark
Bisporella pallescens DMS-10075832T MW203180 - Denmark
Bisporella subpallida GM20160214 KY462818 KY462818 Luxembourg
Calycellina fagina SBRH 840 OL744073 - Netherlands
Calycellina fagina SBRH925 OL752703 OM218631 Netherlands
Calycellina punctata Cantrell GA18 U57494 - USA
Calycellina lachnobrachyoides KUS-F52576 JN033424 JN086727 Korea
Calycellina lachnobrachyoides KUS-F52183 JN033412 JN086715 Korea
Calycellina ulmarie K(M):200003 MZ159550 - UK
Calycina alstrupii (T) BILAS Motiejunaite 10761 NR154846 NG068538 Norway
Calycina alstrupii Pz167 KY305096 KY305098 Norway
Calycina citrina G.M. 2014-12-14-4 KY462815 KY462815 Luxembourg
Calycina citrina ICMP 16997 GQ411507 - New Zealand
Calycina citrina F14046 AF335454 AF335454 Canada
Calycina citrina HMAS 275570 KX781361 - China
Calycina citrina F115889 KC412004 - Spain
Calycina citrina JAC16028 OL653028 - New Zealand
Calycina citrina FH LQH-9a MW203183 MW115642 USA
Calycina claroflava F132983 KC412006 - Spain
Calycina claroflava MFLU 18-2673 MK584952 MK591982 Thailand
Calycina claroflava TNS F37025 LC169492 - Japan
Calycina cortegadensis MSS906 MN017444 MN017503 Spain
Calycina herbarum KUS-F52362 JN033407 JN086710 Korea
Calycina herbarum KUS-F51458 JN033390 JN086693 Korea
Calycina lactea iNAT:18000156 MZ209003 - USA
Calycina lactea HB7224 KC412007 - Germany
Calycina languida F116599 KC412002 - Spain
Calycina languida F116600 KC412003 - Spain
Calycina marina TROM F26093 KT185677 KT185670 Noway
Calycina marina TROM:F26101 KT185674 Norway
Calycina montana MFLU 22-0055 ON156524 ON156523 Thailand
Calycina montana MFLU 22-0056 ON176689 ON325438 Thailand
Calycina montana comb. nov. (T) HMAS 275566 NR 153627 - China
Calycina shangrilana comb. nov. (T) HMAS 275568 NR153628 - China
Calycina shangrilana HMAS 275569 KX781360 - China
Calycina shangrilana HKAS 90655a MK584972 MK591998 China
Calycina shangrilana HKAS 90655b MK584971 MK591997 China
Chalara pseudoaffinis (T) CCF 3979 NR154761 NG067520 Czech Republic
Chalara pseudoaffinis CBS 261.75 FR667225 MH872651 France
......continued on the next page
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188 • Phytotaxa 558 (2) © 2022 Magnolia Press
TABLE 1. (Continued)
Taxon name Strain codesaGenBank accession no Origin
ITS LSU
Chlorencoelia torta KUS-F52256 JN033400 JN086703 Korea
Chlorencoelia torta ICMP 21732 MH682234 - New Zealand
Chlorencoelia torta JAC14068 MK432798 MK431490 New Zealand
Gemmina gemmarum H.B. 6883 OM456209 - Germany
Gemmina gemmarum SBRH 862 KX501127 OM218628 Netherlands
Gemmina sp. H.B. 6910 OM456210 OM456211 Netherlands
Micropeziza umbrinella K(M):168087 MZ159422 - UK
Micropeziza umbrinella ZT-Myc-64170 MW489557 - Switzerland
Microscypha ellisii KUS-F52489 JN033418 JN086721 Korea
Microscypha ellisii KUS-F52663 JN033428 JN086731 Korea
Microscypha sp. TNS-F18016 JN033444 JN086745 Japan
Mollisina uncinata TNS-F38901 JN033457 JN086757 Japan
Mollisina uncinata KUS-F52307 JN033404 JN086707 Korea
Psilachnum staphyleae KUS-F52105 JN033396 JN086699 Korea
Rodwayella citrinula KUSF52443 JN033414 JN086717 Korea
Rubropezicula thailandica (T) MFLU 16-0592 NR163781 - Thailand
Scleropezicula alnicola CBS 200.46 MH856161 MH867686 Canada
Scleropezicula alnicola CBS 119905 MH863066 - Lithuania
Zymochalara lygodii (T) CPC 24699 NR154510 NG059653 Brazil
Zymochalara cyatheae (T) CPC 24665 NR154509 NG059652 Brazil
a Cantrell: S.A. Cantrell collection, Georgia, USA; CBS: Central Bureau voor Schimmelcultures, Utrecht, The Netherlands;
CPC: Collection of Pedro Crous housed at CBS; F: Fundación Medina’s Fungal Culture collection, Spain; FH: Farlow
Herbarium of Cryptogamic Botany, Cambridge, USA; GM: G. Marson collection, Luxembourg; H.B: Hans Otto Baral
collection, Germany; HMAS: Herbarium Mycologicum Academiae Sinicae Beijing, China; ICMP: International Collection
of Microorganisms from Plants, New Zealand; iNAT: inaturalist.org collection, California, USA; K(M): Kew Mycology
collection, Royal Botanic Garden, London, UK; JAC: J.A. Cooper collection, New Zealand; The Kew Mycology collection,
Richmond, UK; KUS: Korea University Herbarium, Seoul, Korea; MFLU: Mae Fah Luang University Herbarium, Chiang
Rai, Thailand; SBRH: S.B. Helleman collection, Netherlands; TNS: National Museum of Nature and Science, Tsukuba,
Japan; TROM: Tromso University Museum, Norway; ZT: Zuercherstrasse, Zurich, Switzerland. Ex-type strains are labeled
by (T), newly generated sequences are in bold, “–” indicates unavailable sequences.
For BI analysis, nucleotide substitution models were selected for ITS and LSU sequence data using JModeltest
2.3 in the CIPRES platform (Nylander 2004). The GTR+I+G model was selected for both data sets. The BI analysis
was conducted in MrBayes v. 3.2.2 with six simultaneous Markov Chain Monte Carlo (MCMC) chain, run for 1
million generations, and sampled the trees at every 1000th generation (Ronquist & Huelsenbeck 2003). The first 25%
of the trees were discarded as ‘burn-in’, and the remaining were used to calculate posterior probabilities of the majority
rule consensus tree. Phylogenetic trees were viewed in FigTree v. 1.4.4 and edited in Adobe Illustrator CS v. 6 (Adobe
Systems, USA; Rambaut 2012).
Results
Phylogenetic analyses
The combined alignment of ITS and LSU contains 59 fungal taxa including the new geographical record taxon. The
final optimization likelihood value for ML is -7905.704513 and was generated from nucleotide alignments of 5840
and 5020 bp to 466 and 854 bp of ITS and LSU (respectively). The matrix comprises 391 distinct alignment patterns
with 32.33 % of gaps and undetermined characters. Base frequencies were estimated as follows; A = 0.231368, C =
0.245017, G = 0.276913, T = 0.246701 with substitution rates AC = 1.606335, AG = 2.7108, AT = 2.390752, CG
REVEAL BISPORELLA MONTANA AS CALYCINA MONTANA Phytotaxa 558 (2) © 2022 Magnolia Press • 189
= 0.527032, CT = 7.140609, GT = 1.00; gamma distribution shape parameter α = 0.222331. BI analysis generated
1000 trees after 1,000,000 generations. The burn-in phase in the analysis discarded the first 250 trees and retained the
remaining results to determine the posterior probabilities distribution. The final average standard of split frequencies
of BI posterior probability was 0.008.
Multigene phylogenetic analyses based on the ITS and LSU gene regions of Pezizellaceae taxa resulted in well-
supported clades as depicted in the phylogram (Figure 1). The Calycina clade encompasses 10 species with available
molecular data, including our geographical records species, C. montana (MFLU 22-0055 and MFLU 22-0056). In the
phylogram, C. montana (MFLU 22-0055 and MFLU 22-0056) form a sister relationship with B. montana (HMAS
275566) with statistical support of 99% ML and 0.99 BI. Additionally, the type strain of B. shangrilana (HMAS
275568) forms a distinct lineage from the C. citrina-cluster with statistical support (78% ML/0.99 BI) (Figure 1). Both
type strains of B. montana (HMAS 275566) and B. shangrilana (HMAS 275568) are positioned in the Calycina clade
instead of Bisporella, Helotiaceae clade (Figure 1).
FIGURE 1. The phylogram of combined ITS and LSU sequence data for genera in Pezizellaceae. Maximum likelihood bootstrap values
greater than 70% and posterior probability values greater than 0.95 given near the nodes. The new geographical record highlighted in red
and type strains are in bold. The tree is rooted to Chlorencoelia torta (JAC 14068, KUSF 52256 and ICMP 21732).
LESTARI & CHETHANA
190 • Phytotaxa 558 (2) © 2022 Magnolia Press
Taxonomy
Calycina montana (W.Y. Zhuang & H.D. Zheng) Lestari & Chethana, comb. nov. (FIGURE 2–4)
Basionym: Bisporella montana W.Y. Zhuang & H.D. Zheng, Zhuang, Zheng & Ren, Mycosystema 36(4): 407 (2017)
Index Fungorum number: IF 570309, Facesoffungi number: FoF 10812
Holotype: HMAS 275566
FIGURE 2. Calycina montana (MFLU 22-0055, new geographical record). a Rotten wood substrates covered with mosses. b–c Apothecia
on the substrate. d Close-up of an apothecium sectioning. e Close-up of hymenium at the margin. f Phialide cells on lower flank surface.
g–h Conidiogenous cells bearing conidiospore i–j A conidiogenous cell (j Mounted in Congo red). Scale bars: b = 950 μm, c = 470 μm, d
= 315 μm, e = 141 μm, f = 40 μm, g = 17 μm, h–j = 13 μm.
Saprobic on decayed woods covered with mosses. Sexual morph: Apothecia 1.1–1.6 × 0.6–1.4 mm, gregarious, arising
singly or in clusters, amber colored, stipitate. Stipe 0.6–1.2 x 0.2–0.4 mm in dried condition. Receptacle discoid, bright
amber color to hyaline. Margin hyaline with brown outer layer at the flank. Disc concave. Ectal excipulum 55–68 µm (
= 59.8 µm, n = 10) at the flank near the stipe, composed of gelatinized thick-walled, hyaline cells of textura porrecta.
Medullary excipulum 36–80 µm ( = 57.7 µm, n = 10) at the flank near the stipe, distinctively separate from ectal
excipulum, composed of thin-walled, hyaline cells of textura oblita, hypha with refractive guttules. Hymenium 141–
180 µm ( = 168.9 µm, n = 15), amber colored. Paraphyses 1.8–2.5 µm in diam. ( = 2.1 µm, n = 15) at the terminal,
filiform, septate, branched at the 1/3 length from the base, guttules. Asci 101–118 × 4.7–6.3 µm ( = 108.6 × 5.5 µm, n
= 15), unitunicate, 8-spored, cylindrical and subclavate at the top, rounded (in alive state) to conical apex (dead state),
J+ (eu-amyloid, in Melzer’s with and without KOH treatment), long substipitate base, arising from croziers. Ascospores
7.1–9.2 × 4.1–5 µm ( = 8 × 4.4 µm, n = 20), ellipsoid, 0-1 septate, partially biseriate to uniseriate, hyaline, rounded at
both ends with guttules. Asexual morph: Hypomycetous, on the lower flank and receptacle of the apothecia’s surface.
Conidiogenous cells 12–19 × 3.7–5.1 ( = 14.3 × 4.3 µm, n = 10) µm, phialidic, black to dark brown. Conidiophore
1.5–3.7 × 1.6–2.4 ( = 3 × 1.9, n = 5) µm, black to dark brown. Conidia 3.2–3.8 × 1.7–2 ( = 3.5 × 1.9 µm, n = 2) µm
ellipsoid, aseptate, hyaline, smooth.
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FIGURE 3. Calycina montana (MFLU 22-0055, new geographical record). a Close up of excipulum cells. b Ectal excipulum cells (b
mounted in Congo Red). c Medullary excipulum cells. d–e Filiform paraphyses with refractive guttules. f–g Asci (mounted in Congo red).
h Asci (alive state). i Close up of crozier. j Tip of the ascus (J+, in Melzerr’s agent with KOH treatment). k–m Ascospores (m mounted in
Congo red). Scale bars: a = 40 μm, b–c = 25 μm, d–h = 14 μm, i = 10 μm, j–m = 4 μm.
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192 • Phytotaxa 558 (2) © 2022 Magnolia Press
FIGURE 4. Calycina montana (HMAS 275566, holotype). a A herbarium package. b An apothecium on the woody substrate. c–d Cross
sections of an apothecium (d mounted in Congo red). e Close up of hymenium at the margin. f Close up of ectal excipulum cells. g–h
Filiform paraphyses (dead state). i–l Asci (k mounted in Melzer’s agent, l mounted in Congo red). m Tip of the ascus (J+, in Melzer’s agent
with KOH treatment). n Close up of crozier at the base of an immature ascus. o–p Ascospores (o mounted in Congo red). Scale bars: b =
500 μm, c–d = 380 μm, e = 43 μm, f = 25 μm, g–h = 23 μm, i–l = 19 μm, m = 4 μm, n = 8 μm, m, o–p = 4 μm.
REVEAL BISPORELLA MONTANA AS CALYCINA MONTANA Phytotaxa 558 (2) © 2022 Magnolia Press • 193
Material examined: Thailand, Chiang Mai Province, Chomthong District, Doi Inthanon, Kew Mae Pan nature
trail, 20 October 2021, Anis S. Lestari, KMP3 21 (MFLU 22-0055), KMP3 41 (MFLU 22-0056) (Figure 2 & 3);
CHINA, Yunnan, Shangrila, Pingbian, Daweishan on rotten hard wood, 5 September 1999, X.Q. Zhang & D.Z. Ren,
(HMAS 275566, holotype) (Figure 4).
GenBank numbers: MFLU 22-0055: ITS–ON156524, LSU–ON156523; MFLU 22-0056: ITS–ON176689, LSU–
ON325438
Notes: In the current study, Bisporella montana (HMAS 275566, holotype) and our specimens clustered under
Calycina, as Calycina montana. Calycina montana (HMAS 275566), previously known as Bisporella montana, was
discovered in a mountainous area in China as a saprobe on woody substrates (Zhuang et al. 2017). The characters of
both specimens, especially the amber-colored apothecia and gelatinized textura porrecta cells of ectal excipulum,
fit the generic concept of Calycina (Baral 1993). Another character which further confirms the combination is the
appearance of a calycina-like apical ring shown clearly in our collection (Figure 3j) and the type specimen (Figure
4m). Based on phylogenetic analyses using ITS and LSU sequence data, our collection clusters with the type strain
with high statistical support of 99% ML and 0.99 BI (Figure 1). Across the 466 base pairs of ITS (+5.8S) gene of our
Calycina montana strains (MFLU 22-0055 and MFLU 22-0056) and the ex-type HMAS 275566, only one base pair
difference (0.22 %) was observed.
Calycina shangrilana (W.Y. Zhuang & H.D. Zheng) Lestari & Chethana, comb. nov. (FIGURE 5)
Basionym: Bisporella shangrilana W.Y. Zhuang & H.D. Zheng, Zhuang, Zheng & Ren, Mycosystema 36(4): 407 (2017)
Index Fungorum number: IF 570312, Facesoffungi number: FoF 05942
Holotype: HMAS 275568
Saprobic on woody substrate. Sexual morph: Apothecia 0.4–1.7 × 0.3–1.3 mm in dried state, gregarious, arising singly
or in clusters, pinkish-orange, stipitate. Stipe 0.3–0.7 x 0.1–0.4 mm in dried condition, white to cream color different
from the disc. Receptacle discoid, bright orange to yellow color. Margin cream color to hyaline. Disc concave. Ectal
excipulum 33–59 µm ( = 42.7 µm, n = 10) at flank near the stipe, composed of gelatinized-hyaline cells of textura
intricata. Medullary excipulum 24–31 µm ( = 27.1 µm, n = 10) at flank near the stipe, distinctively separate from
ectal excipulum, composed of thin-walled, hyaline cells of textura oblita. Hymenium 109–140 µm ( = 125.3 µm, n =
10). Paraphyses 1.4–1.9 µm in diam. ( = 1.8 µm, n = 10) at the terminal, filiform, septate. Asci 119–146 × 5.4–8 µm (
= 133 × 6.7 µm, n = 10), unitunicate, 8-spored, cylindrical, subclavate at the top, rounded apex, J+ (euamyloid), long
substipitate base, arising from croziers. Ascospores 7.3–11 × 3.1–4.2 µm ( = 8.3 × 3.8 µm, n = 10), ellipsoid, 0–1-
septate, partially biseriate to uniseriate, hyaline, guttulate, rounded at both ends. Asexual morph: Undetermined.
Material examined: China, Yunnan, Shangrila, Bitahai, 12 August 2008, on rotten wood, X.Q. Zhang & D.Z. Ren,
(HMAS 275568, holotype).
Notes: This specimen is the type strain of Bisporella shangrilana (HMAS 275568), synonymized under Calycina,
as C. shangrilana in the current study. Calycina shangrilana is characterized by orange-pinkish apothecia, gelatinized
textura intricata cells in the ectal excipulum, and a calycina-like apical ring, which fit with the generic concept of
Calycina (Baral & Marson 2005). Based on ITS and LSU phylogenetic analyses, the type strain (HMAS 275568)
clusters with the other C. shangrilana strains within Calycina clade (Figure 1).
Discussion
Doi Inthanon National Park in Chiang Mai Province spanned three districts (Chom Thong, Mae Wang, and Mae Chaem)
with altitudes ranging from 400 to 2562 m, and temperatures ranging from 1 oC to 23 oC (Anonimus 2001). Located in
a mountainous area and covered with rich plant diversities (Khamyong et al. 2004), many new species of bryophytes
(Akiyama et al. 2010), ferns (Iwatsuki et al. 1998), reptiles (Kunya et al. 2015) and insects (Takaoka & Choochote
2005) were uncovered. Fungal diversity in Doi Inthanon National Park has been studied, specifically aquatic fungi
(Chuaseeharonnachai et al. 2013) and Xylariaceae (Læssøe et al. 2013), however, none reported on discomycetes. In
Thailand, previous research revealed novel species and geographical records of discomycetous fungi in many areas of
northern Thailand (Ekanayaka et al. 2018, Ekanayaka et al. 2019, Zeng et al. 2019), but no studies were conducted at
the Doi Inthanon National Park; hence, more discoveries are expected, especially in Doi Inthanon National Park.
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194 • Phytotaxa 558 (2) © 2022 Magnolia Press
FIGURE 5. Calycina shangrilana (HMAS 275568, holotype). a A herbarium package b–c Apothecia on the woody substrate. d Cross
sections of an apothecium. e Close up of hymenium at the margin. f Close up of ectal excipulum cells. g Filiform paraphyses (mounted
in Congo red). h–j Asci (h, i mounted in Congo red, j mounted in Melzer’s agent). k Tip of the ascus (J+, in Melzer’s agent with KOH
treatment). l Close up of croziers at the base of an immature ascus. m–n Ascospores (mounted in Congo red). Scale bars: b–c = 475 μm, d
= 350 μm, e = 100 μm, f = 15 μm, g = 20 μm, h–j = 17 μm, k = 5 μm, l–n = 9 μm.
REVEAL BISPORELLA MONTANA AS CALYCINA MONTANA Phytotaxa 558 (2) © 2022 Magnolia Press • 195
The phylogram’s topology in our study is roughly similar to Baral & Rämä (2015) especially in the Calycina
clade, however, Calycellina populina (CBS 247.62) was not included. Calycellina populina (CBS 247.62) was placed
in the Calycina clade (Baral & Rämä 2015, Ekanayaka et al. 2019), hence its name suggested to be transferred in to
Calycina populina (Baral & Rämä 2015). Since its morphology has not been re-observed, this species was not included
in our analysis. Our phylogenetic tree’s topography is different from Ekanayaka et al. (2019) based on the gene region
and taxa selection. Our phylogenetic analysis based on ITS and LSU contrast to ITS phylogeny in Ekanayaka et al.
(2019). The taxa selection in this study is based on genera accepted under Pezizellaceae by Wijayawardene et al.
(2022). However, Bisporella citrina (ILLS 61033) and Calycina herbarum (NBRC 9520) were not included in our
analysis due to their morphology and phylogeny affinities to Helotiaceae members, Phaeohelotium monticola (Baral
et al. 2013) and Hymenoscyphus herbarum (Zhao et al. 2012), respectively.
This paper documents a geographical record of calycina-like species found in Doi Inthanon National Park,
Thailand, based on morphology and multi-gene phylogeny. Our newly collected samples were identified as C. montana,
which was previously found in China and identified under the name Bisporella montana (Zhuang et al. 2017). The
morphological differences between Calycina and Bisporella seem to be insignificant in terms of apothecial color, asci,
paraphysis, and ascospores, except for the blueing of the apical ring visible in calycina-like fungi. In contrast to that, a
hymenoscyphus-type apical ring is visible in Bisporella species (Verkley 1993, Baral & Marson 2005).
Following observation of the holotype of Calycina montana (HMAS 275566), we confirmed the morphological
similarities between the type and our specimens. The original description of Calycina montana (HMAS 275566) is
incomplete and lacks several key characteristics, including details on asexual morphs and the calycina-like apical ring
at the ascal tips, which confirmed its relation to Calycina and its taxonomic placement in Pezizellaceae (Figure 4).
In the original description, the blue ring at the ascal tip of Calycina montana (HMAS 275566) was not confirmed by
Zhuang et al. (2017). However, we managed to re-observe and verify the appearance of a calycina-like apical ring
on the type strain (Figure 4b) and its morphological similarity with our newly collected Calycina montana (Figure
2–3). This new taxonomic placement was further supported by the ITS and LSU phylogeny, which shows different
placements for Calycina (Pezizellaceae) and Bisporella (Helotiaceae) fungi in different clades (Figure 1).
The chalara-like fungus was mentioned in Baral (2016) as the asexual morph of Calycina and also reported
in Ekanayaka et al. (2019) for C. claroclava (synonym: B. discendens). The asexual morph of the newly collected
Calycina montana (MFLU 22-0055) is revealed to be chalara-like and grows on the surface of apothecia (Figure 2).
However, the asexual morph was not described for the holotype (Zhuang et al. 2017); therefore, our findings update
information for the asexual and sexual morphs of C. montana.
Our study also synonymized Bisporella shangrilana into Calycina shangrilana comb. nov., supported by the
combined phylogeny of ITS and LSU (Figure 1) and morphology (Figure 5). Also, the morphological description of
the type specimen coincides with Calycina generic concept (Baral 1993, Baral & Marson 2005). Calycina shangrilana
(HMAS 275568) found in China was reported to have insignificant morphological differences from Calycina montana
(HMAS 275566) except in highly gelatinous and larger excipular cells (Zhuang et al. 2017). While in our study, we
only noticed that Calycina shangrilana differs from Calycina montana by having pinkish-orange apothecia and a
shorter stipe, which is not concolorous to the receptacle of Calycina shangrilana (Figure 5). However, fresh specimens
of Calycina shangrilana may reveal more differences between the two species, especially in micromorphologies.
Calycina species have been found in various regions, mainly as saprobes in plant materials (Baral & Rämä
2015, Quijada & Beltrán-Tejera 2017), living plants (Crous et al. 2019), and as lichenicolous (Suija & Motiejűnaitë
2017). More than 20 records of Calycina species are documented in the Species Fungorum (2022), and only a few
have molecular data. This study also provides a synopsis of the morphological characteristics of the Calycina species
available with molecular data, including geographical data (Table 2).
The current study resolves the taxonomic confusions related to Bisporella species placed in Pezizellaceae
(Ekanayaka et al. 2019). However, the delineation of other genera in Pezizellaceae remains unclear. Therefore, more
global explorations combined with comprehensive studies using morphology and phylogeny are vital for resolving
taxonomic confusions of other genera in Pezizellaceae or other families in Leotiomycetes.
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196 • Phytotaxa 558 (2) © 2022 Magnolia Press
Table 2. Synopsis of Calycina and calycina-like species with molecular data.
Species Origin Apothecia Paraphyses Ectal
excipulum
Medullary
excipulum
Asci Ascospore characters Notes References
Size (μm) Size (μm)
Calycina
alstrupii
Norway 0.24–0.47 mm
in diam., cream
yellowish to
orange, sessile
Filiform,
aseptate, not
exceeding
asci at length
Textura
prismatica to
angularis
Textura
prismatica to
porrecta
44–67 × 5–7;
cylindrical to
subcylindrical, J+,
arising from simple
septa, 8-spored
5–7 × 1.5–2.5; ellipsoid,
obtuse or with apices
attenuated at one or both
ends, hyaline, uniseriate
-Suija & Motiejūnaitė
(2017)
Calycina
citrina
China, Cuba,
North America,
Russia
0.5-2.5 mm in
diam., bright
yellow, very
short stipe.
Filiform,
somewhat
swollen at the
apex
Textura
prismatica to
porrecta
Textura
intricata
5–135 × 8–10;
cylindrical, rounded
apex, J- to J+
9–14 × 3–5; ellipsoidal,
hyaline, sometimes
eventually becoming 1-
septate, uniseriate
synonym:
Bisporella
citrina
Dennis (1956),
Carpenter & Dumont
(1978), Milekhin &
Prokhorov (2007)
Calycina
claroflava
Europe, New
Zealand, North
America, the
Philippines,
South America,
South Africa,
Thailand
0.7–1 mm in
diam., bright
greenish-
yellow in fresh
condition, sessile
Filiform,
septate
Textura
porrecta
Textura
epidermoidea
71–101 × 5.5–6;
cylindric-clavate,
rounded apex, J-,
arising from simple
septa
7–12.5 × 2–3;
subcylindrical to fusoid,
hyaline, obtuse to narrow
ends, 1-septate, uniseriate
to biseriate
synonym:
Bisporella
discendens
Quijada & Beltrán-
Tejera (2017),
Ekanayaka et al.
(2019)
Calycina
cortegadensis
Spain 0.2–0.5 cm in
diam., translucent
brown to
pale yellowish-
brown in fresh
condition
Filiform,
septate at
the base, not
exceeding
asci at length
Textura oblita
to intricata
Textura
porrecta
40–53 × 3.5–5;
cylindrical to
subcylindrical, a
conical or obtuse
apex, J+, arising
from simple septa
4–7.5 × 2–2.8; ellipsoid,
hyaline, obtuse apices,
aseptate, uniseriate to
biseriate
-Crous et al. (2019)
Calycina
herbarum
Russia, Turkey 2–3 mm in diam.,
whitish to cream
broad, short
stipitate
Filiform
with apex
sometime
apically
swollen
Textura
porrecta
N/A 82–86 × 4–5;
cylindric to
cylindrical clavate,
J+
12–14 × 1.5–2.5; fusoid - Milekhin &
Prokhorov (2007),
Uzun et al. (2014)
...Continued on the next page
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Table 2. (Continued)
Species Origin Apothecia Paraphyses Ectal excipulum Medullary
excipulum
Asci Ascospore characters Notes References
Size (μm) Size (μm)
Calycina
lactea
Germany 0.3–0.7 mm in
diam., milky
white, with hairs
at the margin
Filiform with
rounded apex,
not exceeding
asci at length
Textura prismatica
to oblita
N/A 120–135 × 8–9.5;
cylindric with subclavate
at the top, J+ (IKI),
arising from croziers
(not clear), 8 spored but
only with 2–4 mature
ascospores
17–19 × 22–26; fusoid with
1-3 septate and guttulate
- Baral (2013)
Calycina
languida
Germany,
Spain
Up to 0.5 mm in
diam., white to
yellowish with
robust short stipe
Filiform
with rounded
apex, slightly
exceeding asci
at length
Textura oblita to
intricata
Textura
intricata
52–68 × 4–4.3;
cylindrical with long
base, J+ (IKI), arising
from simple septa
4.8–7.8 × 2.3–3.2; fusoid
mostly with two big guttules
and small guttules, partially
biseriate to uniseriate,
aseptate
- Baral (2021)
Calycina
marina
Norway,
Scotland,
UK
0.25–0.65 mm in
diam., whitish to
bright yellowish-
orange-
ochraceous,
sessile
Slightly to
rather strongly
clavate-capitate
at the apex
Gelatinized
textura
prismatica-
globulosa
Textura
angularis-
prismatica
38–96 × 5.5–8;
cylindrical clavate with
rounded apex, arising
from simple septa
5.5–15 × 2.5–5.5;
subcylindrical to subfusoid
or ellipsoid, obtuse at both
ends, uniseriate to biseriate
- Baral &
Rämä (2015)
Calycina
montana
comb. nov.
Thailand 1.1–1.6 mm in
diam., amber
color, long
stipitate
Filiform Gelatinized
textura porrecta
Textura
oblita
101–118 × 4.7–6.3,
cylindrical with
subclavate at the top, J+
(MLZ without and with
KOH treatment), rounded
to conical apex, long
substipitate base, arising
from croziers, 8-spored
7.1–9.2 × 3.2–4.2;
ellipsoidal, uniseriate to
partially biseriate
basionym:
Bisporella
montana
This study
Calycina
shangrilana
comb. nov.
China 1–3 mm in diam.,
pinkish-orange,
stipitate
Filiform Gelatinized
textura intricata
Textura
oblita
88–105 × 4.5–5.5;
cylindric, J+ (with KOH
treatment), arising from
croziers, 8-spored
5–8 × 2.2–3.3; ellipsoidal,
uniseriate
basionym:
Bisporella
shangrilana
Zhuang et
al. (2017)
“IKI” Iodine-Potassium Iodide, “MLZ” Melzer’s agent, “N/A” Not Available
LESTARI & CHETHANA
198 • Phytotaxa 558 (2) © 2022 Magnolia Press
Acknowledgements
This study was funded by the Mae Fah Luang University (grant no. 641A01002), entitled ‘Assessing the taxonomy of
Discomycetes and plant pathogenic fungi in Doi Inthanon National Park, Thailand’, Mae Fah Luang Partial Scholarship
for doctoral degree program and Mushroom Research Foundation. The authors would like to thank the National
Research Council of Thailand for the project entitled “Comparison of diversity and biogeographical distribution of
Ascomycetous fungi from two protected areas in Turkey and Thailand” (Project no. P-19-52624) and Saranyaphat
Boonmee for their support in conducting collection visits at Doi Inthanon National Park. We are immensely grateful
to Mr. Kriangkrai Chaiphicet, the Director of the Doi Inthanon National Park, and all his staffs for their immense
help during our collecting trips to explore different areas of the national park in search of discomycetes. K. W. Thilini
Chethana would like to thank the National Research Council of Thailand for providing permission to conduct research
in the Doi Inthanon National Park, Thailand (No. 0402-2703). Anis S. Lestari would like to thank H.O. Baral for
providing English notes of Calycina lactea and C. languida.
Conflicts of Interest
The authors declare no conflict of interest.
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