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Research Article
Three new Pyrenula species with 3-septate ascospores with red or orange oil when over-mature (Ascomycota, Pyrenulales, Pyrenulaceae) from China
expand article infoMingzhu Dou‡, Shengnan Liu‡, Jiechen Li‡, André Aptroot§, Zefeng Jia‡
‡ Liaocheng University, Liaocheng, China
§ Universidade Federal de Mato Grosso do Sul, Mato Grosso do Sul, Brazil

Corresponding author: Zefeng Jia ( zfjia2008@163.com )

Academic editor: Thorsten Lumbsch
© 2024 Mingzhu Dou, Shengnan Liu, Jiechen Li, André Aptroot, Zefeng Jia.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Dou M, Liu S, Li J, Aptroot A, Jia Z (2024) Three new Pyrenula species with 3-septate ascospores with red or orange oil when over-mature (Ascomycota, Pyrenulales, Pyrenulaceae) from China. MycoKeys 102: 107-125. https://doi.org/10.3897/mycokeys.102.113619
Open Access

Abstract

The lichenised fungal genus Pyrenula is a very common crustose lichen element in tropical to subtropical forests, but little research has been done on this genus in China. During our study on Pyrenula in China, based on morphological characteristics, chemical traits and molecular phylogenetic analysis (ITS and nuLSU), three new 3-septate species with red or orange oil in over-mature ascospores were found: Pyrenula inspersa sp. nov., P. thailandicoides sp. nov. and P. apiculata sp. nov. Compared to the known 3-septate species of Pyrenula with red or orange oil, P. inspersa is characterised by the inspersed hamathecium; P. thailandicoides is characterised by the IKI+ red hamathecium and the existence of an unknown lichen substance; and P. apiculata is characterised by the absence of endospore layers in the spore tips and the absence of pseudocyphellae. It is reported for the first time that the presence of a gelatinous halo around the ascospores of Pyrenula is common. A word key for the Pyrenula species with red or orange oil in over-mature ascospores is provided.

Key words

morphology, new species, phylogeny, Pyrenulaceae, taxonomy

Introduction

The lichen genus Pyrenula Ach. (Pyrenulaceae) was first established by Acharius, with Pyrenula nitida (Weigel) Ach. as the type species (Acharius 1814). Pyrenula is mainly a tropical and subtropical genus (Mendonça et al. 2016) and the Neotropics are the centre of diversity of the genus, which typically grow on bark (Aptroot 2012). The genus is characterised by UV– or UV+ yellow thallus, with or without pseudocyphellae, with or without lichexanthone or anthraquinones, perithecioid ascomata, occasionally inspersed hamathecia, unbranched filaments and distoseptate, transversely septate or (sub)muriform ascospores (Aptroot 2012; Mendonça et al. 2016).

In a world key of Pyrenula species, Aptroot (2012) accepted 169 species out of the ca. 745 named taxa in the genus. Since then, many new species of Pyrenula have been described and the genus now comprises ca. 238 species (Aptroot 2012, 2021; Aptroot et al. 2018; Ingle et al. 2018; Miranda-González et al. 2022; Mishra et al. 2022; Lücking et al. 2023; Soto-Medina et al. 2023), of which 41 species have so far been found in China (Aptroot 2012, 2021; Wang et al. 2018; Fu et al. 2018, 2019; Wei 2020; Xie et al. 2021).

Harris (1989) was the first to recognise the presence of red or orange oily granules in over-mature ascospores of some Pyrenula species and to point out the significance of the degradation stage of spores for the taxonomy of Pyrenula. Aptroot et al (2013) described the degradation process in detail: in a few species, the old spores assume a reddish tinge, the wall becomes red-brown and the remains of the lumina develop into red or orange granules. Now, a total of seven species with red or orange oil in over-mature ascospores have been reported, of which four have transverse distoseptate ascospores, viz. P. concastroma R.C. Harris, P. bahiana Malme, P. sexlocularis (Nyl.) Müll. Arg. and P. thailandica Aptroot; three have (sub)muriform ascospores, viz. P. seminuda (Müll. Arg.) Sipman & Aptroot, P. breutelii (Müll. Arg.) Aptroot and P. macularis (Zahlbr.) R.C. Harris. Our study adds three septate Pyrenula species with red or orange oily granules in over-mature ascospores.

As far as we can tell, there have been no reports of a gelatinous halo around the ascospores in Pyrenula. This could mislead lichen taxonomists into believing that ascospore gelatinous haloes are absent in this genus. However, during our study of Pyrenula in southern China, we found that gelatinous haloes are common in this genus and present in all the three new species described here.

In term of molecular data, the attempts to infer relationships within Pyrenulaceae presented two well-supported groups that do not seem to differ based on their morphology, apart from the presence/absence of pseudocyphellae; meanwhile, delimitation problems in few taxa, for instance, P. quassiicola and P. mamillana, were highlighted (Weerakoon et al. 2012; Gueidan et al. 2016). Our phylogenetic analysis using ribosomal genes (nuLSU and ITS) confirmed the above conclusions and supported the description of three new species.

Materials and methods

Morphological and chemical analyses

The specimens were collected in southern China and deposited in the Fungarium, College of Life Sciences, Liaocheng University, China (LCUF). Morphological and anatomical characters of thalli and apothecia were examined and photographed under an Olympus SZX16 dissecting microscope and an Olympus BX53 compound microscope. The lichen secondary metabolites were detected and identified by thin-layer chromatography using solvent C and B (Orange et al. 2010; Jia and Wei 2016).

DNA extraction, PCR sequencing and phylogenetic analysis

Genomic DNA was extracted from ascomata using the Hi-DNA-secure Plant Kit (Tiangen, Beijing, China) according to the manufacturer’s protocol. The nuLSU and ITS regions were amplified using the primer pair AL2R/LR6 (Vilgalys and Hester 1990; Mangold et al. 2008) and ITS1F/ITS4 (White et al. 1990; Gardes and Bruns 1993). The PCR amplification progress followed Dou et al. (2018) and the PCR products were sequenced by Biosune Inc. (Shanghai). The newly-generated sequences were submitted to GenBank (Table 1).

Table 1.
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Information for the sequences used in this study. Newly-generated sequences are shown in bold.

Species Name Specimen No. Locality GenBank accession number
ITS nuLSU
P. thailandicoides M.Z. Dou & Z.F. Jia FJ220208 China Fujian OR578593 —
YN18212 China Yunnan OR578589 OR578570
YN18015 China Yunnan OR578590 OR578571
P. inspersa M.Z. Dou & Z.F. Jia HN17058 China Hainan OR578591 OR578572
P. apiculata M.Z. Dou & Z.F. Jia YN18172 China Yunnan OR578592 OR578573
P. cf. acutalis R.C. Harris F_19092_b Australia — DQ329026
P. aff. aggregataspistea Aptroot & M. Cáceres AA11618 Brazil — KT808561
P. aggregataspistea Aptroot & M. Cáceres AA11216 Brazil KT820112 KT808557
P. anomala (Ach.) A. Massal. AA11222 Brazil KT820168 KT808607
AA11607 Brazil KT820116 —
AA15591 Brazil KT820113 —
P. arthoniotheca Upreti AA11887 Brazil KT820120 —
P. aspistea (Ach.) Ach AA11263 Brazil KT820121 KT808560
AA13547 Brazil KT820123 —
CBS_109078 Hong Kong — EF411063
CG3030 Vietnam KT820124 KT808562
CG3060 Vietnam KT820125 KT808564
CG3070 Vietnam KT820126 —
CG3071 Vietnam KT820127 —
GW1042 Sri Lanka JQ927450 JQ927469
GW1044 Sri Lanka JQ927451 JQ927470
RAMK17271 Thailand KT820128 —
RAMK17277 Thailand KT820129 KT808563
P. astroidea (Fée) R.C. Harris RAMK17281 Thailand KT820088 —
P. bahiana Malme RVH1 Laos KT820090 —
RVH2 Laos KT820091 KT808614
RVH3 Laos KT820092 KT808605
P. balia (Kremp.) R.C. Harris CG3063 Vietnam KT820130 KT808566
P. brunnea Fée CG3023 Vietnam KT820093 —
P. cf. subglabrata (Nyl.) Müll. Arg CG3028 Vietnam KT820140 KT808574
P. chlorospila (Nyl.) Arnol CG1520b England JQ927452 JQ927471
P. cornutispora Aptroot & M. Cáceres AA11938 Brazil KT820131 KT808618
ISE_AA11938 Brazil NR_158911 NG_060160
P. corticata (Müll. Arg.) R.C. Harris AA11443 Brazil KT820132 KT808568
AA11466 Brazil KT820133 KT808569
P. confinis (Nyl.) R.C. Harris AA13575 Brazil — KT808567
P. cruenta (Mont.) Vain Green_PYCR12 USA KC592268 —
Green_PYCR16 USA KC592269 —
Green_PYCR4 USA KC592267 —
Lutzoni_9806174 Puerto Rico — AF279407
P. fetivica (Kremp.) Müll. Arg CG1963 Vietnam KT820134 —
P. fetivica (Kremp.) Müll. Arg GW307A Sri Lanka JQ927453 JQ927472
GW835 Sri Lanka JQ927454 —
P. infraleucotrypa Aptroot & M. Cáceres AA11105 Brazil KT820114 KT808558
AA11468 Brazi KT820136 —
AA11499 Brazi KT820115 —
AA15450 Brazi KT820142 KT808575
AA15451 Brazi KT820117 KT808559
P. inframamillana Aptroot & M. Cáceres AA11220 Brazi KT820137 KT808572
AA11272 Brazi KT820138 KT808571
AA11897 Brazi KT820139 KT808573
P. laevigata (Pers.) Arnold OL_206758 Norway MK812685 —
OL_206773 Norway MK812185 —
Palice 5608 Slovakia — AY607736
P. cf. leucostoma Ach. F_19082 Australia — DQ329024
P. macrospora (Degel.) Coppins & P. James CG1520a England JQ927455 JQ927473
P. mamillana (Ach.) Trevis. AA11342 Brazil KT820143 KT808576
AA11610 Brazil KT820144 KT808615
AA11846 Brazil KT820145 KT808617
AA15465 Brazil KT820146 KT808579
CG3014 Vietnam KT820147 KT808580
CG3034 Vietnam KT820149 KT808582
CG3058 Vietnam KT820150 KT808583
CG3059 Vietnam KT820151 KT808584
P. aff. mamillana (Ach.) Trevis. GW818A Sri Lank JQ927456 JQ927474
P. massariospora (Starbäck) R.C. Harris CG3061 Vietnam KT820153 KT808585
CG3062 Vietnam KT820154 KT808586
GW1028 Sri Lanka JQ927457 JQ927475
P. minor Fée AA11505 Brazil KT820155 KT808620
AA13516 Brazil — KT808587
P. minutispora Aptroot & M. Cáceres AA11877 Brazil KT820119 —
ABL_AA11877 Brazil NR_136140 —
P. nitida (Weigel) Ach. 17076 Poland MN387114 —
17081 Poland MN387115 —
17146 Poland MN387116 —
17189 Poland MN387117 —
F_5929 Czech Republic JQ927458 DQ329023
s. n. Germany — AY607737
P. nitidella (Flörke) Müll. Arg. 17082 Poland MN387139 —
CG3027 Vietnam KT820156 —
P. occidentalis (R.C. Harris) R.C. Harris OL_206777 Norway MK811633 —
P. ochraceoflava (Nyl.) R.C. Harris Gaya_160308_EGB11 USA KC592275 —
P. punctella (Nyl.) Trevis. Tripp4522 — KT232213 —
P. pyrenuloides (Mont.) R.C. Harris CG1545 Vietnam KT820094 —
P. quassiicola Fée CG3001 Vietnam KT820098 KT808588
CG3019 Vietnam KT820101 KT808591
CG3032 Vietnam KT820104 KT808592
CG3033 Vietnam KT820105 KT808593
RVH6 Laos KT820107 KT808595
P. sanguinea Aptroot, M. Cáceres & Lücking 15707F Brazil — KF697129
P. leucostoma Aptroot & Gueidan AFTOL_ID387 USA DQ782845 —
DUKE_0047599 — NR_119610 NG_068722
Reeb VR 14 VI 025 USA — AY640962
P. reginae E.L. Lima, Aptroot & M. Cáceres ELL0010 Brazil — KT808596
P. rubronitidula Aptroot & M. Cáceres AA11332 Brazil KT820157 KT808597
AA15603 Brazil KT820158 —
AA11697 Brazil KT820159 KT808616
ISE_AA11697 Brazil NR_158913 NG_06015
P. scutata (Stirt.) Zahlbr CG1635 Vietnam KT820160 KT808598
P. septicollaris (Eschw.) R.C. Harris AA13534 Brazil KT820166 KT808610
AA13546 Brazil KT820161 —
AA13555 Brazil KT820167 —
AA15009 Brazil — KT808599
AA15012 Brazil KT820162 KT808600
AA15021 Brazil KT820163 KT808601
AA15023 Brazil KT820164 KT808602
AA15038 Brazil — KT808603
AA15042 Brazil KT820165 KT808604
P. sexlocularis (Eschw.) R.C. Harris RAMK17261 Thailand KT820108 KT808606
P. sp. F19113n Australia — DQ329027
CG3009 Vietnam KT820110 KT808611
F19082r Australia JQ927461 DQ329025
LHD210 Vietnam AB935436 —
P. subelliptica (Tuck.) R.C. Harris RVH5 Laos KT820106 KT808594
P. subglabrata (Nyl.) Müll. Arg. CG3069 Vietnam KT820169 KT808608
P. subpraelucida Müll. Arg. F_17550_f Costa Rica — DQ329015
P. thelemorpha Tuck. F_19082 Australia JQ927460 —
P. viridipyrgilla Aptroot & M. Cáceres AA11864 Brazil KT820170 KT808619
ISE_AA11864 Brazil NR_158914 —
Cyphellophora europaea (de Hoog, Mayser & Haase) Réblová & Unter. CBS129_96 — EF551553 FJ358248
Endocarpon pusillum Hedw. CG470 — JQ927447 EF643754

Multi-locus (ITS and nuLSU) phylogenetic analysis was performed. The combined analysis included 187 sequences (Table 1), of which nine sequences were newly generated and 178 were downloaded in GenBank (Lutzoni et al. 2001; Geiser et al. 2006; Weerakoon et al. 2012; Gueidan et al. 2016). The dataset represented 121 taxa, amongst which two out-group species, Endocarpon pusillum and Cyphellophora europaea, were chosen, based on previous studies (Weerakoon et al. 2012; Gueidan et al. 2016). All Pyrenula taxa that could be found in GenBank were included in our data matrix.

The alignment of sequences for each marker (ITS and nuLSU) was undertaken independently by applying MAFFT 7 (Katoh and Standley 2013). We used the “maskSegment” function in the R package AlignmentFilter (Zhang et al. 2023) to mask ambiguously-aligned or overly-divergent segments (stringency-controlling parameter prob set to 0.05) and then used the “degap” function to remove sites with more than 50% gaps. The congruence of the two datasets was tested using a 70% reciprocal bootstrap criterion (Mason-Gamer and Kellogg 1996): the two matrices (nuLSU, ITS) were analysed separately with RAxML v.8.2.12 (Stamatakis 2014) using 100 bootstrap pseudoreplicates and implementing a GTRGAMMA model on the CIPRES Web Portal (http://www.phylo.org). The resulting trees were compared and any hard conflicts detected were eliminated by pruning sequences or taxa out of the datasets. The two single-locus alignments were concatenated in PhyloSuite v.1.2.2 (Zhang et al. 2020). The concatenated data matrix comprised 1581 characters (674 for ITS and 907 for nuLSU). For BI (Bayesian Inference) analysis, PartitionFinder 2 (Lanfear et al. 2017) was used to determine the best-fit model for each partition. The dataset was partitioned into gene groups, with the GTR+I+G and SYM+I+G substitution models applied to ITS gene and nuLSU gene, respectively. BI analysis was performed with MrBayes 3.2.7 (Ronquist et al 2012). Two runs of four chains were carried out for 10,000,000 generations and trees were sampled every 1000 generations. The convergence of parameters was checked with the programme Tracer v.1.6 (Rambaut et al. 2014). The first 25% of the convergence runs were discarded as burn-in. Construction of the ML (Maximum Likelihood) tree was undertaken by applying RAxML v.8.2.12 (Stamatakis 2014), using 100 bootstrap pseudoreplicates and a GTRGAMMA model on the CIPRES Web Portal (http://www.phylo.org). ML bootstrap values (BS) ≥ 70% and Bayesian posterior probabilities (PP) ≥ 0.95 were considered as significantly supported. The datasets/alignments were deposited in TreeBase (http://purl.org/phylo/treebase/phylows/study/TB2:S31046).

Results and discussion

Phylogenetic analyses

The dataset includes 105 ITS sequences and 82 LSU sequences, of which five ITS sequences and four LSU sequences are newly generated in this study. The BI and ML trees showed similar topologies, so only the BI tree is provided here as Fig. 1. Compared with the dataset of Gueidan et al. (2016), our phylogenetic analysis includes nine additional species (Pyrenula punctella, P. nitidella, P. cf. acutalis, P. cf. leucostoma, P. sanguinea, P. occidentalis and three new species) and confirms the presence of two main well-supported monophyletic groups in accord with the presence/absence of pseudocyphellae as shown in Weerakoon et al. (2012) and Gueidan et al. (2016). Our phylogenetic results also indicate that delimitation problems affect several taxa, for example, P. mamillana, P. quassiicola and P. rubrostigma, which is consistent with Gueidan et al. (2016).

Figure 1. 

Phylogeny of the family Pyrenulaceae, based on a two-gene dataset (ITS and nuLSU) and 121 taxa a overview of the entire tree and details of Group 1 b details of Group 2. Most likely tree obtained using MrBayes. Support values are reported above the branches [posterior probability (PP)/bootstrap value (BS)]. Only significant values (higher than 95% PP and higher than 70% BS) are shown. Cyphellophora europaea and Endocarpon pusillum are the out-group taxa.

The three specimens of Pyrenula thailandicoides form a well-supported monophyletic group (1/100 and 0.99/83). Pyrenula thailandicoides is sister to P. sexlocularis, but with very low support (0.52/-, Suppl. material 1). Pyrenula inspersa is sister to P. quassiicola clade 1 with low support (0.79/-) and P. apiculata forms the first diverging lineage in Group 1 with strong support (1/100). The three new species all belong to Group 1.

Taxonomy

Pyrenula inspersa M.Z. Dou & Z.F. Jia, sp. nov.

Fungal Names: FN 571675
Fig. 2

Diagnosis

The new species can be distinguished from the most similar species Pyrenula thailandica Aptroot by the hamathecium densely inspersed with minute granules and colourless oil droplets.

Type

China. Hainan Province: Changjiang County, Bawangling Nature Reserve, Yajia, 19°05′07′′N, 109°07′25′′E, alt. 444 m, on bark, 10 December 2017, X.H. Wu HN17058 (LCUF:holotype: HN17058; GenBank OR578591 for ITS and OR578572 for LSU).

Description

Thallus corticolous, crustose, brown, surface dull, uneven, corticate with pseudocyphellae, UV-. Ascomata perithecioid, emergent, dispersed, aggregated occasionally when crowded, hemispherical, 1–1.5 mm diam., with crystals, KOH-. Ostioles apical. Hamathecium heavily inspersed with minute granules and colourless oil droplets (close-up in Suppl. material 2), IKI-. Ascospores 8 per ascus, irregularly biseriate, with gelatinous halo before becoming old, 3-septate, 28.5–50 × 10–20 μm; middle lumina diamond-shaped, end lumina triangular, with a thick layer of endospore in the spore tips; hyaline when young, brown when mature, over-mature ascospores with orange oil.

Figure 2. 

Pyrenula inspersa (LCUF HN17058) A thallus with apothecia B apothecia and pseudocyphellae C, F–H ascospores at different developmental stages, over-mature ascospores with orange-oil can be seen in C, F and H D section of apothecium E section visualised with polarised light showing cortex of apothecium with crystals, red stars in D–H show the inspersion in hamathecium I–K young ascospores, red arrows show gelatinous halo. Scale bars: 2 mm (A); 1 mm (B); 10 μm (C, I); 200 μm (D, E); 50 μm (F, H); 35 μm (G); 20 μm (J, K).

Chemistry

Thallus K-, C-, KC-, UV-, hamathecium IKI-.

Ecology and distribution

The new species is currently only known from the tropical regions of southern China on bark.

Etymology

The specific epithet inspersa refers to the inspersed hamathecium.

Note

This new species is similar to Pyrenula thailandica, P. bahiana and P. concastroma in having 3-septate ascospores with red or orange oil when over-mature. It differs from P. thailandica by an inspersed hamathecium and larger ascomata, which are in the latter species 0.6–1.1 mm wide (Aptroot 2012; Aptroot et al. 2012, 2013; Ingle et al. 2018). This new species differs from P. bahiana by larger ascospores, which are in the latter species 26–33(–35) × 10–13(–15) μm (Malme 1929; Aptroot 2012; Aptroot et al. 2013; Ingle et al. 2018). Pyrenula concastroma differs from the new species by the mostly aggregated ascomata with fused walls, but separate ostioles (Aptroot 2012; Schumm and Aptroot 2021). Although P. quassiicola and P. pyrenuloides are phylogenetically close to this new species, they can be distinguished easily by the morphology. P. quassiicola has smaller ascomata (0.3–0.7 mm), smaller ascospores (28–35 (–40) × 12–16 μm) containing colourless oil when over-mature and not inspersed, IKI+ (orange) hamathecium (Harris 1989). P. pyrenuloides has smaller ascomata (0.5–1.0 mm), larger ascospores (50–62 × 18–24 μm) containing no oil when over-mature and not inspersed, IKI+ (orange) hamathecium (Harris 1989).

Pyrenula thailandicoides M.Z. Dou & Z.F. Jia, sp. nov.

Fungal Names: FN 571676
Fig. 3

Diagnosis

The new species can be distinguished from the most closely-related species Pyrenula thailandica by the IKI+ red hamathecium and an unidentified lichen substance.

Type

China. Yunnan Province: Mengla County, Xishuangbanna Tropic Botanical Garden, Chinese Academy of Sciences, Rainforest Valley, 21°54′51′′N, 101°11′28′′E, alt. 626 m, on bark, 26 January 2018, X.H. Wu YN18212 (LCUF: holotype: YN18212; GenBank OR578589 for ITS and OR578570 for LSU).

Description

Thallus corticolous, crustose, olive-green, corticate with few pseudocyphellae, UV-. Ascomata perithecioid, emergent, dispersed, conical, 0.8–1.6 mm diam., with crystals, KOH-. Ostioles apical, white, 0.25–0.45 mm. Hamathecium not inspersed (close-up in Suppl. material 3), IKI+/I+ red (Fig. 2 and Suppl. material 4). Ascospores 8 per ascus, irregularly biseriate, with gelatinous halo before becoming old, 3-septate, (30–)35–55 × (12–)15–23 μm; middle lumina diamond-shaped, end lumina triangular, with a thick layer of endospores in the spore tips; hyaline when young, reddish-brown when mature, over-mature ascospores with red oil.

Figure 3. 

Pyrenula thailandicoides (LCUF YN18212) A, B thallus with apothecia C, D ascospores at different developmental stages, over-mature ascospores with red-oil can be seen in C, red arrows in D show gelatinous halo E IKI+ red hamathecium. Scale bars: 2 mm (A); 1 mm (B); 30 μm (C); 20 μm (D); 50 μm (E).

Chemistry

Thallus K+ orange–brown, C-, KC+ yellow, UV-, hamathecium IKI+ red, TLC showed an unidentified substance at Rf four of solvent C (Suppl. material 5).

Ecology and distribution

The new species is currently only known from the tropical and subtropical regions of southern China on bark.

Etymology

The specific epithet thailandicoides refers to the similarity to Pyrenula thailandica.

Additional specimens examined

China. Yunnan Province: Mengla County, Xishuangbanna Tropic Botanical Garden, Chinese Academy of Sciences, 21°55′37′′N, 101°15′27′′E, alt. 555 m, on bark, 25 January 2018, X. Zhao YN18015 (LCUF; YN18015; GenBank OR578590 for ITS and OR578571 for LSU). China. Fujian Province: Longyan City, Dongxiao National Forest Park, Frog Stone, 24°58′07′′N, 117°01′14′′E, alt. 679 m, on bark, 12 July 2022, Z.G. Ma FJ220208 (LCUF; GenBank OR578593 for ITS).

Notes

This new species is similar to Pyrenula thailandica, P. bahiana and P. concastroma in having 3-septate ascospores with red or orange oil when over-mature. The colour reaction of hamathecium of Pyrenula species in IKI is negative (such as Pyrenula thailandica and P. bahiana) or IKI+ red/orangish (such as P. concastroma) or IKI+ blue (such as P. massariospora). This new species differs from P. thailandica by its IKI+ red hamathecium and an unidentified lichen substance (Aptroot 2012; Aptroot et al. 2012, 2013; Ingle et al. 2018). This new species differs from P. bahiana by its IKI+ red hamathecium, an unidentified lichen substance and larger ascospores, the latter 26–33(–35) × 10–13(–15) μm (Malme 1929; Aptroot 2012; Aptroot et al. 2013; Ingle et al. 2018). P. concastroma differs from the new species by the mostly aggregated ascomata with fused walls, but separate ostioles (Aptroot 2012; Schumm and Aptroot 2021).

Pyrenula apiculata M.Z. Dou & Z.F. Jia, sp. nov.

Fungal Names: FN 571678
Fig. 4

Diagnosis

The new species can be distinguished from the most similar species Pyrenula bahiana by the absence of endospore layers in the spore tips and the absence of pseudocyphellae.

Type

China. Yunnan Province: Mengla County, Xishuangbanna Tropic Botanical Garden, Chinese Academy of Sciences, Green Stone Forest, Buttress Roots, 21°54′39′′N, 101°17′05′′E, alt. 672 m, on bark, 26 January 2018, X. Zhao YN18172 (LCUF: holotype: YN18172; GenBank OR578592 for ITS and OR578573 for LSU).

Description

Thallus corticolous, crustose, olive-green, corticate without pseudocyphellae, UV-. Ascomata perithecioid, emergent, dispersed, conical, flattened, 0.3–0.5 mm diam., with crystals, the sides partly covered by the thallus, KOH-. Ostioles apical, black. Hamathecium not inspersed, IKI-. Ascospores 8 per ascus, uniseriate, with gelatinous halo before becoming old, 3-septate, 18–34 × 10–15 μm; middle lumina triangular to round, end lumina triangular, without layer of endospore in the spore tips; hyaline when young, reddish-brown when mature, over-mature ascospores with red oil.

Figure 4. 

Pyrenula apiculata (LCUF YN18172) A, B thallus with apothecia C–E over-mature ascospores with red oil F–I ascospores at different developmental stages, red arrow in F shows gelatinous sheath. Scale bars: 2 mm (A); 1 mm (B); 5 μm (C, E, I); 10 μm (D, F, H); 20 μm (G).

Chemistry

Thallus K-, C-, KC-, UV-, hamathecium IKI-.

Ecology and distribution

The new species is currently only known from the tropical region of southern China on bark.

Etymology

The specific epithet apiculata refers to the pointed bulge of the end locules of ascospores.

Additional specimens examined

China. Yunnan Province: Mengla County, Xishuangbanna Tropic Botanical Garden, Chinese Academy of Sciences, Green Stone Forest, Buttress Roots, 21°54′39′′N, 101°17′05′′E, alt. 672 m, on bark, 26 January 2018, X. Zhao YN18173 (LCUF, GenBank for ITS and for LSU), same locality, YN18174; China.

Notes

This new species is similar to Pyrenula thailandica, P. bahiana and P. concastroma in having 3-septate ascospores with red or orange oil when over-mature. It differs from P. thailandica by the absence of pseudocyphellae, the absence of endospore layers in the spore tips and reddish-brown and smaller ascospores, which measure in the latter (30–)35–51 × (10–)14–20 μm (Aptroot 2012; Aptroot et al. 2012, 2013; Ingle et al. 2018). This new species differs from P. bahiana by the reddish-brown ascospores when mature, absence of endospore layers in the spore tips and absence of pseudocyphellae (Aptroot 2012; Aptroot et al. 2013; Ingle et al. 2018). P. concastroma differs from the new species by the mostly aggregated ascomata with fused walls, but separate ostioles (Aptroot 2012; Schumm and Aptroot 2021).

Key to Pyrenula with red or orange oil in over-mature ascospores

1 Ascospores transversely septate 2
– Ascospores submuriform to muriform 8
2 Ascospores 5-septate, 22–34 × 8–14 µm Pyrenula sexlocularis (Nyl.) Müll. Arg.
– Ascospores 3-septate 3
3 Ascomata mostly aggregated, with fused walls, but with separate ostioles, ascospores 31–40 × 15–16 µm Pyrenula concastroma R.C. Harris
– Ascomata mostly simple, only aggregated by chance when crowded 4
4 Hamathecium inspersed, ascospores 28.5–50 × 10–20 μm, ascomata ca. 1–1.5 mm diam Pyrenula inspersa M.Z. Dou & Z.F. Jia
– Hamathecium not inspersed 5
5 Ascospores < 35 μm long 6
– Ascospores > 35 μm long 7
6 Terminal locules directly against the exospore wall; ascospores 18–34 × 10–15 μm; ascomata ca. 0.3–0.5 mm diam Pyrenula apiculata M.Z. Dou & Z.F. Jia
– Terminal locules separated from the exospore wall by endospore thickening; ascospores 26–33(–35) × 10–13(–15) µm; ascomata ca. 0.4–0.6 mm diam Pyrenula bahiana Malme
7 Hamathecium IKI-; no substances detected by TLC; ascospores (30–)35–51 × (10–)14–20 µm; ascomata ca. 0.6–1.1 mm diam Pyrenula thailandica Aptroot
– Hamathecium IKI+ red; TLC showed an unidentified substance at Rf four of solvent C; ascospores (30–)35–55 × (12–)15–23 µm; ascomata ca. 0.8–1.6 mm diam Pyrenula thailandicoides M.Z. Dou & Z.F. Jia
8 Ascospores submuriform, the sections usually simple, the rest bicellular, 22–40 × 10–17 µm Pyrenula seminuda (Müll. Arg.) Sipman & Aptroot
– Ascospores muriform 9
9 Ascospores 25–35 × 12–13 µm, with 8 rows of 3–4 lumina per row Pyrenula breutelii (Müll. Arg.) Aptroot
– Ascospores 35–45 × 14–16 μm, with 8 rows of 1–3 lumina per row Pyrenula macularis (Zahlbr.) R.C. Harris

Acknowledgements

We sincerely thank Fangluan Gao (Fujian Agriculture and Forestry Univercity) and Xinmei Qin (Guangxi Institute of Botany, Guangxi Zhuang Autonomous Region and Chinese Academy of Sciences) for the generous help in analysing the data.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This study was supported by the National Natural Science Foundation of China (32300005); Shandong Provincial Natural Science Foundation, China (ZR2023MC105 and ZR2023QC245); Doctoral Initiation Fund of Liaocheng University (318051813) and Research Fund of Liaocheng University (318012011).

Author contributions

Data curation: MD, JL, SL. Formal analysis: MD. Funding acquisition: MD. Methodology: JL, MD, SL. Project administration: MD. Software: SL, MD. Validation: ZJ, AA. Visualization: MD. Writing – original draft: MD. Writing – review and editing: MD, ZJ.

Author ORCIDs

Jiechen Li https://orcid.org/0009-0001-8819-8407

André Aptroot https://orcid.org/0000-0001-7949-2594

Data availability

All of the data that support the findings of this study are available in the main text or Supplementary Information.

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Supplementary materials

Supplementary material 1 

ML tree showing the internal phylogeny of the family Pyrenulaceae, based on a two-gene dataset (ITS and nuLSU) and 121 taxa

Mingzhu Dou, Shengnan Liu, Jiechen Li, André Aptroot, Zefeng Jia

Data type: pdf

Explanation note: Cyphellophora europaea and Endocarpon pusillum are the out-group taxa. Only significant values (higher than 70% BS) are shown.

This dataset is made available under the Ope.0n.0 Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (282.58 kb)
Supplementary material 2 

Section of the ascomata of Pyrenula inspersa (LCUF HN17058) showing hamathecium with inspersion

Mingzhu Dou, Shengnan Liu, Jiechen Li, André Aptroot, Zefeng Jia

Data type: jpg

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (5.44 MB)
Supplementary material 3 

Section of the ascomata of Pyrenula thailandicoides (LCUF YN18212) showing hamathecium without inspersion

Mingzhu Dou, Shengnan Liu, Jiechen Li, André Aptroot, Zefeng Jia

Data type: jpg

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (3.61 MB)
Supplementary material 4 

The colour reaction of hamathecium of Pyrenula thailandicoides (LCUF YN18212) just in I

Mingzhu Dou, Shengnan Liu, Jiechen Li, André Aptroot, Zefeng Jia

Data type: tif

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (29.35 MB)
Supplementary material 5 

TLC test of the new species Pyrenula thailandicoides using C solvent systems

Mingzhu Dou, Shengnan Liu, Jiechen Li, André Aptroot, Zefeng Jia

Data type: pdf

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (578.34 kb)
Supplementary material 6 

TLC test of the new species Pyrenula thailandicoides using B solvent systems

Mingzhu Dou, Shengnan Liu, Jiechen Li, André Aptroot, Zefeng Jia

Data type: pdf

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (566.35 kb)
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