diversity

Article
A New Species and Two New Records of the Lichen Genus
Fissurina from China
Kaijie Shi, Zefeng Jia and Xin Zhao *

College of Life Sciences, Liaocheng University, Liaocheng 252059, China

* Correspondence: [email protected]

Abstract: The lichenized fungal genus Fissurina with mostly slit-like lirellae, belongs to Graphidaceae
and is mainly distributed in tropical and subtropical regions. A total of 17 Fissurina species have
been reported from China. During a survey of the lichen diversity of southern China, a new species
Fissurina wuyinensis K.J. Shi, Z.F. Jia and X. Zhao, sp. nov. was found, which is characterized by a
corticolous thallus without detected secondary substances, uncarbonized lirellae, and an exposed disc
with pruina, muriform and amyloid ascospores. Furthermore, two new records of F. pseudostromatica,
F. subcomparimuralis have been identified by morphological, anatomical, chemical and molecular
studies. Phylogenetic analyses of three loci (ITS, nuLSU and mtSSU) supported the position of these
species within Fissurina. Detailed morphological descriptions as well as high-resolution photographs
of the morphology and anatomy of the three species are provided, as well as a comparison and
discussion of the characteristics of similar species. The studied specimens were deposited in the
Fungarium of the College of Life Sciences, Liaocheng University (LCUF).

Keywords: lichenized fungi; Ascomycota; Ostropales; taxonomy

1. Introduction
Graphidaceae is the largest family of tropical and subtropical crustose lichens. It
is a symbiotic ecosystem composed of lichenized fungi of various genera and species
of Chlorophyta Chlorococcaceae Trebouxia spp. and Trentepohliaceae Trentepohlia spp.
Citation: Shi, K.; Jia, Z.; Zhao, X. A
from the plant kingdom. It belongs to Ascomycota, Lecanoromycetes, Ostropomycetidae
New Species and Two New Records
and Ostropales. The currently accepted Graphidaceae includes 4 subfamilies, more than
of the Lichen Genus Fissurina from
China. Diversity 2023, 15, 959.
80 genera, and a total of more than 2000 species [1–4].
https://doi.org/10.3390/d15090959
The lichen genus Fissurina Fée belongs to Graphidaceae, and is characterized by a pale
yellow-brown to olive green (rarely whitish), mostly smooth and glossy thallus, mostly
Academic Editor: Gothamie slit-like lirellae and ovoid ascospores with a halo, transversely septate or muriform, amy-
Weerakoon
loid to non-amyloid [5–7]. Fée established the genus with Fissurina dumastii Fée as the
Received: 22 July 2023 type species [8], it was subsumed within Graphis Adans. And Graphina Müll. Arg. [9–11].
Revised: 15 August 2023 In 1992, with the description of Fissurina quadrispora by Kalb and Hafellner, Fissurina was
Accepted: 24 August 2023 resurrected to the generic level [12], and in 1999, Staiger and Kalb redescribed Fissurina [13].
Published: 25 August 2023 Staiger studied the combination of the phenotypic characteristics (ascomata characteristics,
asci structure, ascospores characteristics, etc.) and genotype of Graphidaceae. She pro-
posed a taxonomy of the family based on features other than spores combined with existing
molecular data. Enter the embryonic phase of the combination of phenotype and genotype;
Copyright: © 2023 by the authors. a more natural classification of the family Graphidaceae was defined and generally ac-
Licensee MDPI, Basel, Switzerland. cepted [14]. After that, Kalb et al., Archer, Lücking et al., etc., revised Graphidaceae [15–18],
This article is an open access article
and so far the lirellate-morph Graphidaceae classification system of 24 genera has been
distributed under the terms and
generally recognized, and the genus level of Fissurina status tends to be more scientifically
conditions of the Creative Commons
defined. Subsequent phylogenetic studies confirmed its phylogenetic position in the family
Attribution (CC BY) license (https://
Graphidaceae [19]. Phylogenetic studies indicate that Fissurina as currently defined is
creativecommons.org/licenses/by/
polyphyletic, and together with some taxa of Dyplolabia A. Massal., Ocellularia G. Mey. and
4.0/).

Diversity 2023, 15, 959. https://doi.org/10.3390/d15090959 https://www.mdpi.com/journal/diversity

Diversity 2023, 15, 959 2 of 13

Myriotrema Fée (the only common characteristic is the astrothelioid ascospores) constitute a
monophyletic group, which is clearly distinct from other taxa of the family Graphidaceae
and included in Fissurinoideae [1,20–22]. Kraichak and Lücking used a temporal banding
approach to conduct in-depth studies on several orders of the two main subclasses of
Lecanoromycetes O.E. Erikss. & Winka., the results split Graphidaceae into four families:
Diploschistaceae Zahlbr, Fissurinaceae B.P. Hodk, Graphidaceae s.str. and Thelotremat-
aceae [22,23]. This classification system was adopted in the latest “Outline of Fungi and
fungus-like taxa”, a classification system for eukaryotic microorganisms [24].
There are more than 150 species of Fissurina in the world, and this genus is mainly
distributed in tropical and subtropical regions [25–31]. The subtropical zone of China is
south of the Qinling Mountains and the Huaihe River, north of the Leizhou Peninsula and
east of the Hengduan Mountains; the tropical zone generally refers to the area between the
Tropic of Cancer, such as the entire Hainan Province, the Nansha Islands, and southern
Guangxi, Guangdong, Yunnan and Taiwan. The species resources are very rich in these
regions and are therefore the key to revealing the biodiversity of lichens [32,33]. Until now,
17 species of Fissurina have been described and reported in China, all distributed in tropical
and subtropical zones [7,34,35]. During a survey of the lichen diversity of southern China,
a new species of Fissurina collected from Fujian Province, and two newly recorded species
from Yunnan and Guangdong provinces were discovered and they are described in this
paper; phylogenetic trees were also constructed for these species.

2. Materials and Methods

2.1. Morphological and Chemical Analyses
The Chinese materials for this study were collected from Hunan, Yunnan, Guizhou, Fu-
jian, Guangdong and Hainan provinces, and were deposited in the Fungarium of the College
of Life Sciences, Liaocheng University (LCUF) after drying and low-temperature treatment.
Morphological and anatomical features were observed and photographed using a
stereo microscope (OLYMPUS SZX16) and an optical microscope (OLYMPUS BX53 with
digital camera OLYMPUS DP74).
Morphological observations were made, including the growth type, color, surface
state and accessory structure of the thallus, the disc and labia, and mode of initiation of the
ascomata. The specific methods of anatomical research are as follows:
First, remove the well-developed ascomata, thallus and a small part of substrate as
one with a blade, gently flatten the sample on white cardboard, and slice the ascomata
with part of the substrate by hand with a blade. Next, select a complete, uniform, and
thin slice, place it in the sterile water droplet on the glass slide, add a coverslip, absorb
excess water from one side with absorbent paper, and remove air bubbles. According to
the characteristics of the genus, observe the characteristics of the epithecium, hymenium,
paraphyses and ascospores in detail, and take pictures and make records. This process
requires a light press of the coverslip to disperse the paraphyses so that the branches at the
ends of the paraphyses can be easily observed. After the ascus is exposed, the number, size
and type of the ascospores in the ascus can be clearly observed. Finally, add Lugol’s iodine
solution dropwise on one side of the coverslip, and absorb it with absorbent paper on the
other side, and test the amyloidity of the hymenium and ascospores using Lugol’s solution.
Spot tests were performed on the thallus surface and thin thallus sections by adding K
(10% KOH solution), C (saturated aqueous NaClO solution) and P (dissolving p-phenylenediamine
in anhydrous ethanol to prepare a 5% ethanol solution) reagents. The secondary metabolites
of the lichens were analyzed and identified by thin layer chromatography (TLC) with the C
solvent system [36–38], specific steps are as follows:
In this study, Lethariella cladonioides (Nyl.) Krog. containing atranorin and norstictic
acid was used as the partition standard sample.
Firstly, do the preparatory work: Prepare the solvent according to the formula (toluene:
acetic acid = 200:30 mL). Then, use a 2B pencil to lightly draw a straight line about
1.5 cm from the bottom edge of the long side of the glass silicone board, (take the size
Diversity 2023, 15, 959 3 of 13

of 100 mm × 200 mm as an example), and draw a parallel straight line 0.2 cm above and
below the straight line, take a point every 1 cm between the two ends of the first straight
line, except the 1st, 10th and 19th, these three points are numbered as standard samples,
the rest are coded as 1–16 in the sequence for sampling.
Next, sample preparation, extraction and spotting: use a blade (75% alcohol disinfec-
tion) to scrape a small amount of thallus cortex and medulla onto white paper, transfer
them to sterilized small centrifuge tubes and number them; add a small amount of acetone
to the small centrifuge tube until the sample is buried. After soaking for about 1–2 h, the
capillary can be used to spot the sample according to the number.
Exposure layer: Pour an appropriate amount of C system solvent into the chromatogra-
phy cylinder, put the glass silicone board with sample placed on it into the chromatography
cylinder, and the straight line at the bottom of the glass silicone board should be above
the chromatography solution. After the glass silicone board has been saturated in the
chromatographic solution for 15–20 min, and before the solvent reaches 1 cm from the end
of the chromatography plate, the glass silicone board can be taken out, and the surface of
the plate can be dried with a hair dryer.
Color development: Observe the position and color of the chromatographic spots
under sunlight; observe the fluorescence of the chromatographic spots under 365 nm and
254 nm ultraviolet light. Then, spray the silica gel plate evenly with 10% sulfuric acid
solution, observe whether there are fat spots while wet, and record results. Then, put it in
an oven at 85 ◦ C for 10–15 min, so that the chromatographic color develops well.
Partition and component research: the three straight lines at the beginning of the
chromatography on the silica gel plate are zone 1; draw the tangent lines on the upper
and lower borders of the norstictic acid and atranorin acid spots, respectively, and define
them as zone 4 and zone 7; draw a line between zone 1 and zone 4, and divide them into
zone 2 and zone 3 in the middle; draw a line between zones 4 and 7 to divide them evenly
into zones 5 and 6; above zone 7 is zone 8. Record the color and position of each spot, and
record the Rf value if necessary.

2.2. DNA Extraction, Amplification and Sequencing

Genomic DNA was extracted from the ascomata and thallus of the specimens using the
REDExtract-N-Amp Plant PCR Kits (Sigma-Aldrich, Saint Louis, MO, USA) according to the
manufacturer’s protocol. The internal transcribed spacer region (ITS), nuclear large subunit
rDNA (nuLSU) and mitochondrial small subunit rDNA (mtSSU) regions were amplified
using the primer pair ITS1F/ITS4 [39,40], AL2R/LR6 [41] and mrSSU1/mrSSU3R [42],
respectively. The 50 µL PCR reaction system consisted of 2.5 µL each primer solution,
4 µL genomic DNA, 16 µL ddH2O and 25 µL 2×Taq PCR MasterMix (Tiangen, Beijing,
China). Thermocycling conditions for ITS comprised initial denaturation at 94 ◦ C (3 min),
35 denaturation cycles at 94 ◦ C (30 s), annealing at 52 ◦ C (30 s), extension at 72 ◦ C (1.5 min)
and a final extension at 72 ◦ C (10 min); for nuLSU conditions comprised initial denaturation
at 94 ◦ C (5 min), 35 denaturation cycles at 95 ◦ C (30 s), annealing at 58 ◦ C (30 s), extension
at 72 ◦ C (1 min) and a final extension at 72 ◦ C (10 min); for mtSSU conditions comprised
initial denaturation at 95 ◦ C (5 min), 35 denaturation cycles at 94 ◦ C (45 s), annealing at
50 ◦ C (1 min), extension at 72 ◦ C (1.5 min) and a final extension at 72 ◦ C (10 min). The target
product of PCR was affirmed by electrophoresis on 1% agarose gels and sequenced by
Biosune Inc. (Shanghai, China) and Tsingke Biotech Co., Ltd. (Beijing, China). The newly
generated sequences were submitted to GenBank (Table 1). Porina aenea (Körb.) Zahlbr.
and P. leptalea (Durieu & Mont.) A.L. Sm. were selected as the outgroup [21].
Diversity 2023, 15, 959 4 of 13

Table 1. Specimens and sequences used for phylogenetic analyses. Newly generated sequences are
shown in bold.

Voucher
Species Country ITS mtSSU nuLSU
Specimens
Clandestinotrema
Costa Rica Sipman 44327 — JX421014 —
clandestinum
C. leucomelaenum Ecuador Luecking 26216 — JX421016 —
C. leucomelaenum Costa Rica Luecking LCB6b — JX421015 —
C. stylothecium Nicaragua Luecking 28636 — HQ639597 HQ639662
Cruentotrema cruentatum Brazil Luecking 263 — HQ639587 HQ639660
Cr. thailandicum Myanmar TNS:YO12327 LC573996 — —
Lumbsch
Cr. thailandicum Thailand — JF828960 JF828975
19955d1
Lumbsch
Cr. thailandicum Thailand — JX421020 —
19955d2
Lumbsch
Cr. thailandicum Thailand — JX421021 —
19955d3
Dyplolabia afzelii Australia MEL:2382721 KP012902 — —
D. afzelii / RMG246 MK503256 — —
D. afzelii USA Luecking 26509 — HQ639594 HQ639628
D. afzelii Australia Kalb 33915 — DQ431950 AY640013
Fissurina adscribens China HNX18044 OR264094 OR264108 OR264070
F. adscribens China FJ220250 OR264085 OR264107 OR264068
F. adscribens China FJ211195 OR264084 — —
F. adscribens Thailand Lumbsch 20200c — JX421032 —
F. aff. dumastii Thailand Kalb 38899 — JX421034 JX421487
A. Frisch No.
F. aff. humilis Tanzania — DQ431948 DQ431921
99/Tz2002
F. aggregatula Peru RivasPlata 107C — JX421036 JX421490
F. aggregatula Thailand Luecking 24019 — JX421038 —
F. aggregatula Thailand Kalb 38890 — JX421037 —
F. amazonica Brazil Caceres 11030 — KJ608636 KJ608633
Luecking
F. astroisidiata Mexico — JX421040 JX421491
RLD057
F. bullata Australia Mangold 6f — JX421041 KF875537
F. comparimuralis El Salvador Luecking 28103 — JX421042 JX421492
F. crassilabra China FJ211425 OR264081 OR264114 OR264065
F. crystallifera USA Mercado 4462 — — KF875538
Frisch &
F. dumastii Cameroon Tamnjong Idi — DQ384926 —
99/Ka3855
F. illiterata USA Common 9115B — JX421045 —
F. inabensis China GZ18113 OR264087 OR264105 OR264071
F. inabensis China YN221499 OR264086 OR264109 OR264067
F. inabensis China FJ211545 OR264088 OR264104 OR264066
F. inabensis Russia LE L-18645 OP901516 — —
F. insidiosa China HN19093 OR264080 OR264111 —
F. insidiosa / AFTOL-ID 1662 — DQ972995 DQ973045
F. insidiosa USA Spribille 39035 KR017123 KR017325 KR017185
F. inspersa USA Common 9113G — JX421047 —
F. marginata Australia Kalb 33944 — DQ431951 AY640012
F. marginata Thailand Luecking 24122 — HQ639613 JX421493
F. monilifera Puerto Rico Mercado 838 — KJ435167 KJ440941
Rivas Plata
F. nigrolabiata Philippines — JF828961 JF828976
1198B
Diversity 2023, 15, 959 5 of 13

Table 1. Cont.

Voucher
Species Country ITS mtSSU nuLSU
Specimens
Rivas Plata
F. nigrolabiata Philippines — — JX421494
1016E
F. nitidescens USA Common 9113C — JX421048 —
F. pseudostromatica China YN222451 OR264089 OR264098 OR264074
F. pseudostromatica China YN222474 OR264090 OR264099 OR264072
F. pseudostromatica USA Common 9126B — JX421051 —
F. pseudostromatica USA Common 9113A — JX421050 —
F. pseudostromatica Bolivia Luecking 29014 — JX421049 —
F. pseudostromatica Thailand Kalb 38827 — — JX421495
F. pseudostromatica China YN210691 OR264091 OR264100 OR264075
F. rufula Fiji Lumbsch 20521l — JX421053 JX421497
F. rufula Fiji Lumbsch 20500l — JX421052 —
F. subcomparimuralis China GD19219 OR264092 OR264112 OR264079
F. subcomparimuralis China GD19359 OR264093 OR264113 OR264078
F. subfurfuracea Brazil Caceres 11981 — KJ608635 KJ608632
F. subundulata China FJ220370 OR264082 OR264110 —
F. subundulata China GZ18148 OR264083 OR264106 OR264069
A. Frisch No.
F. triticea Tanzania — DQ431952 AY640011
99/Tz1855
F. wuyinensis China FJ230605 OR264095 OR264101 OR264076
F. wuyinensis China FJ230610 OR264097 OR264103 OR264073
F. wuyinensis China FJ230464 OR264096 OR264102 OR264077
PRA-
Porina aenea Czech Republic OQ718026 OQ646398 —
Vondrak25464
PRA-
P. leptalea Czech Republic OQ718029 OQ646400 —
Vondrak24457
Pycnotrema pycnoporellum USA Luecking 26546 — JX421295 JX421615

2.3. Phylogenetic Analysis

The contigs were assembled and edited using the program Geneious v. 9.0.2 (Biomat-
ters Ltd., Auckland, New Zealand), and subjected to BLAST searches for an initial verifi-
cation of their identities. The sequences were aligned using MAFFT v 7.308 with settings
appropriate for the variability of each locus [43]. For ITS sequences, we used the L-ING-i
alignment algorithm with the remaining parameters set to default values. For nuLSU, the
G-ING-i algorithm and “leave gappy regions” were selected. Then, we used the E-ING-i
algorithm for mtSSU with the remaining parameters set to default values. A concatenated,
3-locus matrix was generated using Geneious v. 9.0.2. This matrix contained both Fissurina
species and species belonging to related genera in the Fissurinoideae of the Graphidaceae
family [4,44]. In addition to our newly generated sequences, other related sequences were
downloaded from GenBank and added to the matrix (Table 1). Maximum likelihood (ML)
and Bayesian inference (BI) were performed using the CIPRES Scientific gateway portal
(http://www.phylo.org/portal2/) (accessed on 12 July 2023) [45]. Maximum likelihood
bootstrapping analysis was performed with RAxML HPC v. 8, using the locus-specific
model partitions with the default parameters and the GTRGAMMA model as implemented
on the CIPRES, NSF XSEDE resource with bootstrap statistics calculated from 1000 boot-
strap replicates [46]. For the Bayesian analysis, the best substitution models of the three loci
were estimated using the Akaike information criterion in jModelTest 2.1.6 [47]. Based on the
results, we used the GTR+G model for ITS, SYM+I+G for nuLSU and GTR+I+G for mtSSU.
Bayesian analysis was performed using MrBayes v. 3.2.2 on CIPRES with 2 independent
runs, searching for 10 000 000 generations with four independent chains and sampling
every 1000th tree [48]. After discarding the burn-in, the remaining 7500 trees of each run
were pooled to calculate a 50% majority-rule consensus tree. Clades that received bootstrap
Diversity 2023, 15, 959 6 of 13

support ≥70% under ML and posterior probabilities ≥0.95 were considered significant.
Generated phylogenetic trees were visualized under Figtree v. 1.4.2 [49].

3. Results and Discussion

Detailed figures of the morphology and spores, together with information regarding
the chemical compositions of the three species, have been provided. Furthermore, the
phylogenetic positions of these species have been confirmed.
For this study, 50 new sequences were generated (Table 1). The concatenated, three-
locus matrix consisted of 65 individuals and 3253 aligned nucleotide position characters.
Phylogenies derived from the ML and B/MCMC analyses were generally concordant. Mi-
nor differences in the arrangement of some terminals occurred, but relationships at deeper
nodes and in well-supported clades were identical. We chose to present the ML topology,
with nodal support values from both ML bootstrap analysis and posterior probabilities
from the Bayesian inference (Figure 1).
The phylogenetic analysis showed that the subfamily Fissurinoideae exhibits a well-
supported monophyletic lineage containing the genera Clandestinotrema Rivas Plata, Lück-
ing & Lumbsch, Cruentotrema Rivas Plata, Papong, Lumbsch & Lücking, Dyplolabia A.
Massal., Fissurina and Pycnotrema Rivas Plata & Lücking. The tree shows that Fissurina is
polyphyletic in its current delimitation. The topology of our tree is similar to the results of
Rivas Plata et al. and Lumbsch et al. [2,44].
The new species Fissurina wuyinensis formed a single clade, represented by a bootstrap
support of 100, and a posterior probability of 1 for the branch (Figure 1). Its species status is
further supported by its distinctive morphological, chemical and geographic characteristics
(see below in Taxonomy).
Fissurina pseudostromatica is polyphyletic in this tree, forming a high-support clade
together with F. aggregatula, F. wuyinensis and Pycnotrema pycnoporellum (bootstrap = 99%,
posterior probability = 1). Two specimens of F. pseudostromatica from China were clustered
with the material from USA, while another specimen was clustered with F. aggregatula
collected from Peru. It appears to indicate issues with current species circumscriptions.
Similar problems also arise in the species F. adscribens, F. insidiosa and F. marginata; none of
them are monophyletic in our tree.
Fissurina subcomparimuralis from China is phylogenetically close to F. comparimuralis
and F. nitidescens; they all have fissurinoid to chroodiscoid ascomata, somewhat carbonized
lirellae and muriform spores, but F. subcomparimuralis has non-amyloid ascospores which
can be distinguished from the other two species.
 Diversity 2023, 15, x FOR PEER REVIEW 7 of 14
Diversity 2023, 15, 959 7 of 13

Figure 1. Phylogenetic tree generated from maximum likelihood (ML) analysis based on combined
Figure 1. Phylogenetic tree generated from maximum likelihood (ML) analysis based on combined
ITS, nuLSU and mtSSU sequences. ML bootstrap values and B/MCMC posterior probabilities
ITS, nuLSU and mtSSU sequences. ML bootstrap values and B/MCMC posterior probabilities (second
(second value) are displayed above each branch. New species and records from China are shown in
value) are displayed above each branch. New species and records from China are shown in bold.
bold.
 bensis have non-amyloid ascospores [7,51].
Fissurina wuyinensis is characterized by uncarbonized lirellae, exposed disc
pruina, amyloid muriform ascospores and no chemistry. The new species largely re
bles Fissurina confusa Common & Lücking, but differs in the wider lirellate (0.35–0.45
vs. 0.15–0.2 mm), thin-walled ascospores and in lacking lichen substances [52]. Fiss
Diversity 2023, 15, 959
aperta Herrera-Camp., Barcenas-Peña & Lücking and F. reticulata R.8 ofMiranda,
13
H
ra-Camp. & Lücking have more or less similar morphological features, but the forme
apically carbonized lirellae and the latter has longer ascospores about 35–45 µm [5
4. Taxonomy addition, it differs from Fissurina americana Lendemer & R.C. Harris by the prom
lirellae withK.J.
Fissurina wuyinensis exposed discJia
Shi, Z.F. [54].
and X. Zhao, sp. nov., Figure 2.

Figure 2. Fissurina wuyinensis

Figure (FJ230464)
2. Fissurina (A) Thallus
wuyinensis with ascomata;
(FJ230464) (B)with
(A) Thallus Cross-section
ascomata;of(B)
apothecium;
Cross-section of ap
(C) Hymenium with asci; (D) Ascospores. Bars: A = 1.0 mm; B = 100 µm; C = 50 µm; D
cium; (C) Hymenium with asci; (D) Ascospores. Bars: A = 1.0 mm; B = 100 = 50 µm;
µm. C = 50 µm; D
µm.
Type: China, Fujian Province: Wuyishan City, Xingcun Town, Tongmu Village,
Guadun, 27◦ 430 5200 N, 117◦ 390 29
Fissurina
00 E, alt. 875 m, on bark, 9 June 2023, Jiang Shuhao FJ230464
pseudostromatica Lücking & Rivas Plata, Bull. Florida Mus. Nat.
(LCUF, holotype).49(4): 145 (2011), Figure 3.
Fungal Names:Type: FN 571620
U.S.A. Florida. Collier County, Fakahatchee Strand Preserve State Park,
Description: Thallus
Scenic Drivecorticolous
6.5 mi NNW on tree
of trunk,
rangercontinuous,
station, westepiperidermal, dark green to hardw
of old tram, Taxodium-Sabal
hammock, slough and strand, March 2009, Lücking & Rivas Plata ×
olive green, smooth, glossy. Photobiont trentepohlioid, cells angular to elongate, 6–15 5–9 µm.
26512 (F, holotype
Ascomata lirellate, Description:
prominent toThallus
sessile,corticolous,
straight, curved or continuous,
crustose, sinuous, terminally acute, olive-g
surface smooth,
single to sparsely branched,
glossy. Ascomata0.8–1.5 mm mm
0.1–0.4 long,long,
0.35–0.45
0.1–0.2mmmm wide.
wide,Disc partially
lirellae exposed
densely aggregate in p
and deeply immersed, labiawhite
dostromatic, thin but conspicuous,
clusters, short togrey, whitestraight
elongate, pruina present,
to curved, thalline
single to spa
margin remaining inclined but with the cortex splitting off and becoming erect. Excipulum
branched, immersed to slightly raised arising. Disc concealed to slightly gaping,
uncarbonized, thin,
yellowish
whitishbrown
labia,tocovered
grayishby brown, with clusters
concolorous thallineofmargin.
crystals, 28–40 µmuncarbon
Excipulum
wide; epithecium black-grey, 7–16 µm wide; hymenium colorless, clear,
brown to tawny, with clusters of crystals, 20–27 µm wide; epithecium 120–130 high;3–8 µm w
µmgrey,
hypothecium brown, 25–34 µm high; paraphyses simple, apically not or sometimes
hymenium colorless, clear, 71–89 µm high; hypothecium pale brown, 10–18 µm slightly
thickened and paraphyses
smooth, 0.5–0.8 wide;
µm1–2.5
simple, µm periphysoids absent. Asci
wide. Asci cylindrical, broadly
75–83 × 7–11clavate to
µm. Ascospores
cylindrical, 103–138
ascus,× ellipsoid,
24–42 µm.hyaline
Ascospores
to pale8 per ascus, ellipsoid,
gray-brown hyaline,
when old, muriform
4 loculars, with
15–20 × 7–11 µm
5–7 × 1–3 septa,cluding
25–35 × 16–19
1 µm µm,
thick without
wall), I–. halo, I+ deep violet-blue.
Chemistry: thallus K–, C–, thallus
Chemistry: P–; no substances
K+, C–, P–; no detected by TLC.
substances detected by TLC.
Etymology: The epithet refers to the type locality: Mount Wuyi.
Additional specimens examined: Fujian Province: Wuyishan City, Xingcun Town,
Tongmu Village, Guadun, 27◦ 430 5200 N, 117◦ 390 2900 E, alt. 875 m, on the bark of Camellia,
9 June 2023, Guo Tangli FJ230472 (LCUF); Gaoqiao, 27◦ 420 5500 N, 117◦ 430 0600 E, alt 510 m,
on the bark of Camellia, 9 June 2023, Jia Zefeng FJ230605, Jiang Shuhao FJ230610 (LCUF).
Ecology and distribution: The new species grows on bark at a comparatively low
elevation (no more than 1000 m). It is thus far known only from the type locality.
Notes: The thallus and ascomata morphology and molecular sequence data place it in
the genus Fissurina Fée [50]. In the reported Fissurina species of China, only three species
viz. Fissurina elaiocarpa (A.W. Archer) A.W. Archer, F. inabensis (Vain.) M. Nakan. & Kashiw.
 Diversity 2023, 15, x FOR PEER REVIEW 1

Diversity 2023, 15, 959 9 of 13

Selected specimens examined: China, Yunnan Province: Mengla Co
Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, 21°55′3
101°14′51″
and F. subundulata E, alt.
Kalb & Z.F. Jia530
havem,muriform
on bark, 30 June 2021,
ascospores of Zhu
similarMengli YN210691
size and (LCUF); Men
lack lichen
substances, but they all have a concealed disc, and F. subundulata and F. inabensis haveon bark, 4
Town, Linleyuan Mountain Village, 21°55′55″ N, 101°16′09″ E, alt. 540 m,
tember 2022,
non-amyloid ascospores Jia Tao YN222451, YN222474 (LCUF).
[7,51].
Ecology and distribution:
Fissurina wuyinensis is characterized Growing on tree
by uncarbonized bark exposed
lirellae, of the tropical rainforests a
disc with
pruina, amyloid muriform ascospores and no chemistry. The new species largely resembles reporte
altitudes. Previously reported from the U.S.A. and Brazil [52,55]. Newly
Fissurina confusaChina.
Common & Lücking, but differs in the wider lirellate (0.35–0.45 mm vs.
Notes:ascospores
0.15–0.2 mm), thin-walled The morphology, anatomy
and in lacking lichenand chemical
substances characteristics
[52]. Fissurina aperta of the Ch
specimens are the same as those of the type specimens,
Herrera-Camp., Barcenas-Peña & Lücking and F. reticulata R. Miranda, Herrera-Camp. & but the ascospores are w
(7–11 µm vs. 5–7 µm) [52]. Fissurina pseudostromatica is
Lücking have more or less similar morphological features, but the former has apicallydistinguished by pseudostro
clusters
carbonized lirellae and of
thelirellae,
latter hastransversely septate and
longer ascospores I– ascospores.
about 35–45 µm [53]. Fissurina mexicana and F.
In addition,
cludens have similar thallus structure, but differ in having
it differs from Fissurina americana Lendemer & R.C. Harris by the prominent lirellae with muriform ascospores
species
exposed disc [54]. is also similar to F. aggregatula Common & Lücking by its 4 locular ascospore
the absence of lichen
Fissurina pseudostromatica substances,
Lücking & Rivaswhile theBull.
Plata, latter Florida
has labiate-aggregate
Mus. Nat. Hist. lirellae and
rower ascospores
49(4): 145 (2011), Figure 3. (14–20 × 7–9 µm) [52,56].

Figure 3. Fissurina pseudostromatica (YN210691) (A,B) Thallus with ascomata; (C) Cross-section of
Figure 3. Fissurina pseudostromatica (YN210691) (A,B) Thallus with ascomata; (C) Cross-sect
apothecium; (D) Ascospores.
apothecium; Bars: A = 2.0 mm;
(D) Ascospores. B =A
Bars: 1.0= mm; C =B100
2.0 mm; µm;
= 1.0 D =C50
mm; µm.µm; D = 50 µm.
= 100

Type: U.S.A. Florida. Collier County, Fakahatchee Strand Preserve State Park, Janes
Fissurina subcomparimuralis Common & Lücking, Phytotaxa 18: 58 (2011), Fig
Scenic Drive 6.5 mi NNW of ranger station, west of old tram, Taxodium-Sabal hardwood
Type: U.S.A. Florida. Collier County, Fakahatchee Strand Preserve State
hammock, slough and strand, March 2009, Lücking & Rivas Plata 26512 (F, holotype).
Common 7323A (holotype MSC, isotype hb. Common).
Description: Thallus corticolous, crustose, continuous, surface smooth, olive-green,
Description: Thallus corticolous, crustose, continuous, surface smooth, ye
glossy. Ascomata 0.1–0.4 mm long, 0.1–0.2 mm wide, lirellae densely aggregate in pseu-
ish-brown to olive-brown, slightly glossy. Lirellae immersed to sessile, straight to cu
dostromatic, white clusters, short to elongate, straight to curved, single to sparsely branched,
single, 0.4–1.0 mm long, 0.2–0.35 mm wide. Disc concealed to fissured; labia conspic
immersed to slightly raised arising. Disc concealed to slightly gaping, with thin, whitish
yellowish-white, covered by thalline margin; proper margin distinct, visible as thick
labia, covered by concolorous thalline margin. Excipulum uncarbonized, brown to tawny,
cipulum entire, dark brown to brown-black, 49–61 µm wide; epithecium black-
with clusters of crystals, 20–27 µm wide; epithecium grey, 3–8 µm wide; hymenium color-
11–20 µm wide; hymenium colorless, 70–85 µm high; hypothecium grayish brown, 1
less, clear, 71–89 µm high; hypothecium pale brown, 10–18 µm high; paraphyses simple,
µm high; paraphyses unbranched, glabrous, 0.5–0.8 µm wide; periphysoids short, 1
1–2.5 µm wide. Asci cylindrical, 75–83 × 7–11 µm. Ascospores 8 per ascus, ellipsoid,
µm long, indistinctly warty. Ascospores 8 per ascus, ellipsoid, colorless, muriform
hyaline to pale gray-brown when old, 4 loculars, 15–20 × 7–11 µm (including 1 µm thick
4–6 × 1–3 septa, 17–24.5 × 6–8.5 µm, weakly halonate, I–.
wall), I–.
Chemistry: thallus K+, C–, P–; no substances detected by TLC.
Selected specimens examined: China, Yunnan Province: Mengla County, Xishuang-
banna Tropical Botanical Garden, Chinese Academy of Sciences, 21◦ 550 3700 N, 101◦ 140 5100 E,
 Diversity 2023, 15, x FOR PEER REVIEW

Diversity 2023, 15, 959 10 of 13

Chemistry: thallus K–, C–, P–; no substances detected by TLC.

Selected specimens examined: China, Guangdong Province: Guangzhou, B
alt. 530 m, on bark, 30 June 2021, Zhu Mengli YN210691 (LCUF); Menglun Town, Linleyuan
Mountain, Pugu, 23°10′08″ N, 113°17′32″ E, alt. 110 m, on bark, 20 January 2019, L
Mountain Village, 21◦ 550 5500 N, 101◦ 160 0900 E, alt. 540 m, on bark, 4 September 2022, Jia Tao
GD19219 (LCUF); South China Botanical Garden, North Entrance, 23°11′19″ N, 113°
YN222451, YN222474 (LCUF).
E, alt. 22 m, on bark, 21 January 2019, Yao Zongting GD19359 (LCUF).
Ecology and distribution: Growing on tree bark of the tropical rainforests at low
Ecology and distribution: This species grows on bark in low elevations of tr
altitudes. Previously reported from the U.S.A. and Brazil [52,55]. Newly reported for China.
regions. Previously reported from Florida, U.S.A. [57]. Newly reported for China.
Notes: The morphology, anatomy and chemical characteristics of the Chinese speci-
Notes: The morphology and chemical characteristics of the specimens are the
mens are the same as those of the type specimens, but the ascospores are wider (7–11 µm
as those of the type specimens, but the disc sometimes gaping and lirellae shorte
vs. 5–7 µm) [52]. Fissurina pseudostromatica is distinguished by pseudostromatic clusters
lirellae features of GD19359 are more similar to the type specimen than GD19
of lirellae, transversely septate and I– ascospores. Fissurina mexicana and F. intercludens
having thin labia greyish-black to brown-black below corticate thalline margin.
have similar thallus structure, but differ in having muriform ascospores. The species is also
comparimuralis is similar to F. comparimuralis Staiger, but the latter differs in havin
similar to F. aggregatula Common & Lücking by its 4 locular ascospores and the absence of
yloid
lichen substances, ascospores
while and
the latter haslacking periphysoids
labiate-aggregate [14,57].
lirellae andF.narrower
subcomparimuralis
ascosporesis also sim
F. incrustans
(14–20 × 7–9 µm) [52,56]. Fée in having similar-sized, muriform ascospores and lacking liche
stances, but F. incrustans has I+ violet ascospores and exposed
Fissurina subcomparimuralis Common & Lücking, Phytotaxa 18: 58 (2011), Figure 4. disc [7,58,59].

Figure 4. Fissurina subcomparimuralis (GD19219) (A,B) Thallus with ascomata. (C) Cross-section of
Figure 4. Fissurina subcomparimuralis (GD19219) (A,B) Thallus with ascomata. (C) Cross-sec
apothecium; (D) apothecium;
Ascospores. Bars: A = 1.0 mm;
(D) Ascospores. B=A
Bars: 1.0=mm; C =B
1.0 mm; 200
= 1.0 D =C50= µm.
µm;mm; 200 µm; D = 50 µm.
Type: U.S.A. Florida. Collier County, Fakahatchee Strand Preserve State Park, Com-
Author Contributions: Conceptualization, X.Z. and Z.J.; methodology, X.Z.; software, K
mon 7323A (holotype MSC, isotype hb. Common).
X.Z.; validation, K.S., Z.J. and X.Z.; formal analysis, K.S. and X.Z.; investigation, K.S. and
Description: Thallus
sources, Z.J.;corticolous, crustose,
data curation, continuous,
K.S. and surface smooth,
X.Z.; writing—original yellowish-
draft preparation, K.S.
brown to olive-brown, slightly glossy. Lirellae immersed to sessile, straight
ing—review and editing, X.Z.; visualization, K.S.; supervision X.Z.; project to curved,
administration, Z
single, 0.4–1.0 X.Z.;
mm funding
long, 0.2–0.35 mmZ.J.
acquisition, wide. DiscAll
and X.Z. concealed to fissured;
authors have labia conspic-
read and agreed to the published
uous, yellowish-white, covered by thalline margin; proper margin distinct, visible as
of the manuscript.
thick. Excipulum entire, dark brown to brown-black, 49–61 µm wide; epithecium black-
Funding: This research was funded by the Emergency Management Project of the National N
grey, 11–20 µm wide; hymenium colorless, 70–85 µm high; hypothecium grayish brown,
Science Foundation of China, grant number 31750001, the Young Scientists Fund of Nation
15–23 µm high;ural paraphyses unbranched,
Science Foundation glabrous,
of China, grant 0.5–0.8
numberµm wide; and
31700018 periphysoids short,
Shandong Provincial Natu
10–20 µm long,ence indistinctly warty. Ascospores
Foundation, China (ZR2023MC105). 8 per ascus, ellipsoid, colorless, muriform
with 4–6 × 1–3 septa, 17–24.5 × 6–8.5 µm, weakly halonate, I–.
Institutional Review Board Statement: Not applicable.
Chemistry: thallus K–, C–, P–; no substances detected by TLC.
Selected specimens examined:
Data Availability China,
Statement: Guangdong
Publicly availableProvince: Guangzhou,
datasets were analyzed in Baiyun
this study. This d
Mountain, Pugu, 23◦ 10from
be found 0 0800 http://www.ncbi.nlm.nih.gov/
N, 113◦ 170 3200 E, alt. 110 m,(accessed on 1420July
on bark, 2023). 2019, Li
January
Min GD19219 (LCUF); South China Botanical Garden, North Entrance, 23◦ 110 1900 N,
113◦ 210 3000 E, alt. 22 m, on bark, 21 January 2019, Yao Zongting GD19359 (LCUF).
Diversity 2023, 15, 959 11 of 13

Ecology and distribution: This species grows on bark in low elevations of tropical
regions. Previously reported from Florida, U.S.A. [57]. Newly reported for China.
Notes: The morphology and chemical characteristics of the specimens are the same as
those of the type specimens, but the disc sometimes gaping and lirellae shorter. The lirellae
features of GD19359 are more similar to the type specimen than GD19219 in having thin
labia greyish-black to brown-black below corticate thalline margin. F. subcomparimuralis
is similar to F. comparimuralis Staiger, but the latter differs in having amyloid ascospores
and lacking periphysoids [14,57]. F. subcomparimuralis is also similar to F. incrustans Fée in
having similar-sized, muriform ascospores and lacking lichen substances, but F. incrustans
has I+ violet ascospores and exposed disc [7,58,59].

Author Contributions: Conceptualization, X.Z. and Z.J.; methodology, X.Z.; software, K.S. and X.Z.;
validation, K.S., Z.J. and X.Z.; formal analysis, K.S. and X.Z.; investigation, K.S. and Z.J.; resources,
Z.J.; data curation, K.S. and X.Z.; writing—original draft preparation, K.S.; writing—review and
editing, X.Z.; visualization, K.S.; supervision X.Z.; project administration, Z.J. and X.Z.; funding
acquisition, Z.J. and X.Z. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Emergency Management Project of the National Natural
Science Foundation of China, grant number 31750001, the Young Scientists Fund of National Natural
Science Foundation of China, grant number 31700018 and Shandong Provincial Natural Science
Foundation, China (ZR2023MC105).
Institutional Review Board Statement: Not applicable.
Data Availability Statement: Publicly available datasets were analyzed in this study. This data can
be found from http://www.ncbi.nlm.nih.gov/ (accessed on 14 July 2023).
Acknowledgments: We are grateful to Jiang Shuhao and Guo Tangli for their help with field research.
Conflicts of Interest: The authors declare no conflict of interest.

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