Diorygma tiantaiense sp. nov. and a Checklist and Key to Diorygma Species from China

Abstract

A new species Diorygma tiantaiense Z.F. Jia, sp. nov. was found, which is characterized by a corticolous thallus with norstictic acid, oval or oblong apothecia, immersed to ± raised discs with white pruina, and large muriform ascospores (120–210 × 35–60 µm). Detailed morphological descriptions, photographs, and a comparison and discussion of similar species are provided. A checklist and key to the species of Diorygma known from China is presented.

1. Introduction

The lichenized fungi genus Diorygma was established by Eschweiler (1824) [1], and D. hieroglyphicum (Pers.) Staiger & Kalb was chosen as the lectotype for the genus by Staiger (2002) [2]. Kalb et al. (2004) described the monograph of 24 species known in the world of the genus [3]. Feuerstein et al. (2014) described three new species and a revised world key which contained 52 species [4]. Subsequently, another species was described from tropical to subtropical distribution [5,6,7,8,9]. Wimalasena et al. (2022) described the genus to include 77 species [10]. The total has now increased to 85 [11,12,13]. The genus Diorygma belongs to Graphidaceae, Graphidales, Ostropomycetidae, and Lecanoromycetes, usually on bark [3,10,14,15]. Diorygma is characterized by a crustose, off-white to pale olive-green thallus; lirelliform to irregularly rounded ascomata with a pruinose disc; exciple uncarbonized or sometimes carbonized; hymenium hyaline, not inspersed, branched or anastomosing paraphyses with a thick gelatinous wall; asci clavate, 1–8-spored; ascospores hyaline (rarely brownish), transversely septate to mostly muriform; substances including norstictic or stictic acid or the protocetraric acid complex [3,6,11].

Ten species of the genus Diorygma from China were reported: Diorygma erythrellum [≡ Graphina erythrellum], D. fuscum, D. hieroglyphicum, D. hololeucum, D. isabellinum [≡ Graphina isabellina], D. junghuhnii [= Graphina mendax], D. macgregorii, D. megasporum, D. pruinosum, and D. soozanum [≡ Graphina soozana] [3,6,7,16,17,18,19,20,21,22,23]. Here, we describe a new species to science collected in Zhejiang Province, China, for which we propose the name Diorygma tiantaiens. We provide a working key and the descriptions and known distribution of the Chinese species.

2. Materials and Methods

2.1. Specimens and Morphology

The specimens were collected from Zhejiang, Hunan, Sichuan, Guizhou, Guangdong, Guangxi, Yunnan, Fujian and Hainan provinces, China, and deposited in the Fungarium of College of Life Sciences, Liaocheng University (LCUF), the Herbarium Mycologicum Academiae Sinicae-Lichenes (HMAS-L), and the Botanical Herbarium, Shandong Normal University (SDNU). The collected time and location of the specimens are described for each species in the part of taxonomy. A dissecting microscope (Olympus SZX16) was used to observe the structure of apothecia, and compound microscope (Olympus BX53) for microscopic characters. Measurements of apothecia, exciples, paraphysis, asci, and ascospores were obtained from mature vertical sections of fruit bodies mounted in water.

2.2. Chemistry

Ascospores were tested by using Lugol’s solution (1% iodine solution). Spot tests were performed on the thallus surface (10% KOH, saturated aqueous NaOCl, and saturated p-phenylenediamine in ethanol). The lichen substances were detected and identified by thin-layer chromatography (TLC), using solvent C [24,25,26].

2.3. DNA Extraction and PCR Sequencing

Genomic DNA was extracted from ascomata of the specimens by using the Hi-DNA-secure Plant Kit (Tiangen, Beijing, China) according to the manufacturer’s protocol. The nuLSU region was amplified using the primer pair AL2R/LR6 [27]. Reactions were carried out in a 50 µL reaction system containing 2 µL of each primer solution, 2 µL of genomic DNA, 19 µL of ddH₂O, and 25 µL of 2×Taq PCR Master Mix (Tiangen, Beijing, China). PCR conditions were as follows: initial denaturation for 5 min at 94 °C and 35 cycles of 94 °C for 30 s, 52 °C for 30 s, followed by an extension at 72 °C for 90 s, and a final extension at 72 °C for 10 min. The target products of PCR were purified and sequenced at Biosune Biotechnology Company (Jinan, China).

2.4. Phylogenetic Analysis

The newly generated sequence of the new species, Diorygma tiantaiense, was submitted to GenBank. Nineteen related sequences for phylogenetic tree construction were downloaded from GenBank and Trapelia placodioides Spribille Bjoerk 09 was selected as the outgroup (

Table 1. Specimen and sequences used in the phylogenetic analysis. The newly generated sequence is shown in bold.

Species	Specimen	nuLSU
Diorygma antillarum	MPN322	JX046465
Diorygma hieroglyphicum	Wirth 26647	AY640015
Diorygma junghuhnii	Lumbsch 20539l	JX421474
Diorygma junghuhnii	Kalb 33937	AY640018
Diorygma karnatakense	CRG668RATO14	OP235520
Diorygma karnatakense	CRG668RATO12	OP235518
Diorygma karnatakense	CRG668RATO10	OP235517
Diorygma karnatakense	CRG668RATM05	OP235516
Diorygma karnatakense	CRG668RATO13	OP235519
Diorygma poitaei	DNA3210	HQ639627
Diorygma pruinosum	Kalb 26578	AY640014
Diorygma pruinosum	Mangold 28g	JX421476
Diorygma sipmanii	Berger 14011	AY640020
Diorygma tiantaiense	Jia ZJ19123	MW750692
Diorygma tibellii	Luecking 28533	JX421475
Ocellularia erodens	UGDA19568	MK542902
Ocellularia erodens	UGDA19754	MK542900
Ocellularia microstoma	Lumbsch 19108m	JX421575
Ocellularia microstoma	RivasPlata 1085B	JX421577
Trapelia placodioides	Spribille Bjoerk 09	MH627046

). The alignment was undertaken by applying Geneious 9.0.2 with the option of MUSCLE Phylogenetic relationships to be inferred using maximum likelihood (ML) and Bayesian inference (BI) on the CIPRES Scientific gateway portal (http://www.phylo.org/portal2/ (accessed on 3 February 2024) [28]. ML was performed using RAxML-HPC BlackBox v. 8.2.12 [29], with a GTRGAMMA model and bootstrap statistics calculated from 1000 bootstrap replicates. For BI analysis, jModelTest 2.1.6 [30] was used to determine the best-fit model. Based on the result, we used the GTR+I+G model for nuLSU. BI analysis was performed using MrBayes on XSEDE (3.2.7a) [31] on CIPRES with 2 independent runs, searching for 10,000,000 generations. Each run included four independent chains and sampling every 1000 generations. After discarding the burn-in, the remaining 75% were used to calculate the consensus tree [29,31,32,33,34]. Bootstrap support above 70% and posterior probabilities above 0.95 were considered significant support values. The generated phylogenetic tree was visualized under FigTree v.1.4.3.

3. Results and Discussion

3.1. Phylogenetic Results

The phylogenetic trees obtained from maximum likelihood (ML) and Bayesian Inference (BI) exhibited similar topologies; we therefore present only the BI tree (Figure 1). The phylogenetic analyses strongly supported the two clades of Diorygma and Ocellularia (PP = 1). The nuLSU sequences supports that our specimen belongs to the Diorygma genus, and D. tiantaienser is shown as sister to the clade consisting of D. karnatakense B. O. Sharma & Khadilkar with strong support (PP = 0.99; ML = 95%).

3.2. Taxonomy

Eleven species of Diorygma from China are reported in this paper, and one of them is new to science. D. pachygraphum(Nyl.) Kalb, Staiger & Elix [≡ Graphis pachygrapha Nyl., Acta Soc. Sci. Fenn. 7: 472, 1863.], reported from China [21,22,23], is excluded in the present paper because of the synonym, the current name is Allographa pachygrapha (Nyl.) Lücking & Kalb in Mycobank. D. poitaei (Fée) Kalb et al. was reported in China as Graphina cf. virginea (Eschw.) Müll. Arg. [35]. However, after consulting the literature and examining photos, we found that its appearance is not like D. poitaei, which is different from the previously reported of Kalb et al. in 2004. In addition, the number and size of its ascospores are also different from the latter, which have (4–)6–8/ascus, ascospores 40–65 × 10–18 μm [3]. Therefore, it is not found in China now, and we correct it here. A working key to the Chinese species is provided. The descriptions, microphotographs, and discussions for the species new to China are also given.

• Key to the species of Diorygma known from China

• Diorygma tiantaiense Z.F. Jia, sp. nov. (Figure 2).
  MycoBank: MB 852800
  Diagnosis: Differs from Diorygma karnatakense by its ascocarps ± raised when mature, whitish to greyish disc, divergent exciple, ascospores 1/ascus, I–, and the absence of salazinic acid.
  Type: China, Zhejiang Province, Tiantai County, Huading National Forest Park, on bark of Rhododendron simsii Planch., 29°15′16″ N, 121°05′30″ E, alt. 950 m, 27/IV/2019, Z.F. Jia ZJ19123 (Holotype, LCUF; GenBank MW750692 for LSU).
  Etymology: The species epithet refers to the locality of holotype.
  Description: Thallus corticolous, crustose, pale grey to greenishgrey, 100–130 µm thick, surface uneven to slightly rugose, without soralia or isidia; algal layer 40–60 µm thick; medulla poorly developed. Ascocarps numerous, oval or oblong, flexuous and branched, immersed in the thallus when young, becoming open and ± raised when mature, whitish to greyish, rounded at the ends, 0.5–3 × 0.4–2 mm; disc surrounded by entire raised thalline margins, open, rarely convex, with thick and white pruina, sometimes sparse; exciple divergent, laterally uncarbonized, basally and laterally brownish, consisting of a weakly and irregularly or brownish hyphal tissue intermingled with parts of the substrate; hymenium 150–220 µm high, not inspersed, I–; epithecium usually distinctly developed, consisting of intermingled anastomosing, hyaline or brownish paraphysis tips with short ± globular cells, hyaline granules, and dead hyphae; paraphyses 1–2 µm diam., with a gelatinous wall, often anastomosing, especially in the upper part of the hymenium and near the asci, sometimes branched at the tip. Ascospores 1/ascus, hyaline, muriform, dense spore locules of equal size, 120–210 × 35–60 µm, I–, with thin halo.
  Chemistry: K+ red, P+ yellow to red; norstictic acid.
  Ecology and distribution: This species is distributed in a subtropical forest in the southeast of China, growing on bark. The type location is in China.
  Additional specimen examined: China. Zhejiang Province, Tiantai County, Huading National Forest Park, on bark of Rhododendron simsii, 29°15′16″ N, 121°05′30″ E, alt. 950 m, 27/IV/2019, Z.F. Jia ZJ19124, ZJ19125 (LCUF).
  Discussions: Diorygma tiantaiense is characterized by the oval or oblong apothecia, the large muriform ascospores (20–210 × 35–60 µm) and the presence of norstictic acid only. The species is shown as sister to the clade consisting of D. karnatakense, but different in the latter having concealed and brownish black disc, convergent exciple, I+ in hymenium, ascospores 1–8/ascus, longer ascospores (75–220 × 18.5–51.5 µm), I+ blue violet, and the presence of salazinic acids [36]. Morphologically, it resembles D. africanum Kalb, Staiger & Elix, D. reniforme (Fée) Kalb, Staiger & Elix, D. salvadoriense Kalb, Staiger & Elix, and D. soozanum (Zahlbr.) M. Nakan. & Kashiw. in ascocarps, but is distinguished from those species by D. africanum having protocetraric acid and the absence of norstictic acid, D. reniforme having larger ascospores (110–230 × 35–80 µm) and the presence of norstictic, protocetraric, and salazinic acids, D. salvadoriense having wider ascospores (150–200 × 50–75µm) and the presence of norstictic and salazinic acids, D. soozanum having thick white pruinate discs and the presence of norstictic and connorstictic acids [3].

• Diorygma erythrellum (Mont. & Bosch) Kalb, Staiger & Elix, Symb. Bot. Upsal. 34(1): 150, 2004.
  ≡ Ustalia erythrella Mont. & Bosch, in Junghuhn, Pl. Jungh. 4: 478, 1856.
  ≡ Graphina erythrella (Mont. & Bosch) Zahlbr. Cat. Lich. Univers. 2: 405 1923.
  Thallus crustose, greenish grey; surface smooth; pseudocortex well developed. Ascocarps lirellae, 0.5–5 × 0.3–0.5 mm, ± raised, sometimes branched, isabelline. Disc narrow to open, with a thin white pruina; Exciple divergent, uncarbonized, brown. Hymenium clear, 120–150 µm high. Ascospores 8/ascus, hyaline, muriform, spore locules of equal size, 10–12/2–4 locular, 40–53 × 12–15 µm.
  Chemistry: Norstictic, connorstictic and stictic acids.
  Ecology and distribution: This species is a pantropical species, which is distributed in tropical to subtropical forests, growing on bark. Known from China, Java, the Philippines, Australia, New Caledonia, Thailand, and Sumatra [3].
  Specimens: China. Fujian Province: Wuyishan City, Mt. Wuyishan, alt. 540 m, 27/V/2007, Z.F. Jia FJ457 (LCUF). Hainan Province: Qiongzhong County, Mt. Wuzhishan, alt. 700 m, 21/VII/2009, J. Li HN09077 (LCUF).
  Discussions: This species is similar to Diorygma fuscum Jian Li bis & Z.F. Jia, but differs in the latter having opened discs with a thick and white pruina, and stictic acid as major chemistry, with norstictic acid absent [6]. The size of ascospores in specimens from China is smaller than the previously reported of Kalb et al. in 2004 [3]. Position in key of Feuerstein et al. (2014): couplet 22(21) Ascospores 30–65 × 12–20 µm; thallus with smooth cortex [4].

• Diorygma fuscum Jian Li bis & Z.F. Jia, Mycotaxon 131(3): 718, 2016.
  Thallus crustose, pale grey to olivegrey, surface uneven to slightly rugose or warty; pseudocortex indistinctly developed, partly lacking. Ascocarps lirellate, 1–4 × 0.3–2 mm, sometimes branched, immersed to ± raised, whitish. Disc with thick and white pruina. Exciple divergent, laterally uncarbonized, carbonization sometimes at the basal position. Hymenium clear, 100–180 µm high, I+ weakly bluish violet. Ascospores 8/ascus, hyaline to brownish, muriform, spore locules of equal size, 10–14/3–4-locular, 40–60 × 12–18 µm, I+ violet, with thin halo.
  Chemistry: Sticti, constictic, hypostictic and hypoconstictic acids.
  Ecology and distribution: This species is distributed in subtropical forests, growing on bark. Known from China [6].
  Specimens examined: Type: China. Fujian Province, Jianou City, Fangdao Town, Wanmulin, alt. 310 m, on bark, 3/VI/2007, Q.F. Meng FJ1280 (HMAS-L 137193); alt. 540 m, 2/VI/2007, J. Li FJ1066 (HMAS-L 137199).
  Discussions: This species is similar to D. pruinosum (Eschw.) Kalb et al., which differs in the latter having 1-spored asci, larger ascospores, and the presence of protocetraric acids [3]. This species is similar to D. poitaei, but differs in having opened discs, a slightly carbonized proper exciple at the base, and the presence of stictic acid (major), while D. poitaei contains hypostictic and hypoconstictic acids (major), α-acetylhypoconstictic, constictic, and stictic acids (minor, trace or absent) [3].

• Diorygma hieroglyphicum (Pers.) Staiger & Kalb, in Kalb et al., Symb. Bot. Upsal. 34(1): 151, 2004.
  ≡ Opegrapha hieroglyphica Pers., Ann. Wetter. Gesellsch. Ges. Naturk. 2(1): 16, 1810.
  Thallus crustose, whitish grey or greenish, surface rough and matt, sometimes verrucose; pseudocortex indistinctly developed, with crystallization. Ascocarps lirellate, 0.5–3 × 0.4–0.8 mm, oblong, numerous or branched, immersed, whitish. Discs narrow to open and covered with whitish or yellowish pruina. Exciple divergent, uncarbonized, and indistinctly developed. Hymenium clear, 100–150 μm high, I+ weakly blue to violet blue. Ascospores 1/ascus, hyaline, muriform, oblong, with thick wall, spore locules of equal size, 20–30/6–10-locular, 80–150 × 20–40 µm, I+ violet blue.
  Chemistry: K+ red, P+ yellow to red; stictic, norstictic, constictic and cryptostictic acids.
  Ecology and distribution: This species is a pantropical species, which is distributed in tropical to subtropical coastal rainforests, growing on bark. Known from China, Africa, Singapore, Papua New Guinea, Philippines, Australia, and Southwest Pacific Island Countries [3,37].
  Specimens examined: China. Fujian Province: Wuyishan City, Mt. Wuyishan, alt. 500 m, 27/V/2007, Z.F. Jia FJ450 (LCUF). Hainan Province: Mangrove near the Qiongshan city, alt. 0m, 24/V/2007, Q.F. Meng M402 (HMAS-L 128726), M405 (HMAS-L 128727); Baoting contry, Mt. Qixianling, alt. 110 m, 25/VII/2009, M. Liu HN09386 (HMAS-L 115519); Qiongzhong County, Limushan National Forest Park, alt. 630 m, 24/IX/2008, Z.F. Jia HN003 (LCUF). Yunnan Province: Mengla County, Rainforest Valley Xishuangbanna National Park of Tropical Rainforests, alt. 570 m, 21/VIII/2011, Q. Ren YN-R-08 (LCUF); Pingbian County, Dawei Mountain Sand Pearl Bottom, alt. 900 m, 23/VIII/2011, Z.F. Jia 11-419, 11-421, 11-452 (LCUF).
  Discussions: This species is similar to Diorygma pruinosum, but differs in the latter having obviously opened discs, and stictic acid absent [3]. It is morphologically similar to D. megasporum Kalb, Staiger & Elix, but differs in the latter having ascospores 2–6/ascus [3]. Position in key of Feuerstein et al. (2014): couplet 36 (34) Stictic acid present in addition to norstictic acid; ascospores 95–150(–170) × 30–45 µm [4].

• Diorygma hololeucum (Mont. & Bosch) Kalb, Staiger & Elix, Symb. Bot. Upsal. 34(1): 155, 2004.
  ≡ Graphis hololeuca Mont. & Bosch, Pl. Jungh. 4: 473, 1856.
  Thallus crustose, white, creamy white to grey, surface rough and matt; pseudocortex indistinctly developed or thin, with crystallization. Ascocarps lirellate, 1–7 × 0.5–1.5 mm, scattered, elongated to oblong, numerous or branched, raised or adnate, whitish. Discs open, covered with a thick and white pruina. Exciple divergent, uncarbonized, poorly developed. Hymenium clear, 180–250 μm high, I+ weakly violet blue. Ascospores 2–4(–6 or –8)/ascus, muriform, hyaline, oblong, with thin gelatinous wall at ends, spore locules of equal size, 25–40/5–8-locular, 120–200 × 30–40 µm, I+ violet blue.
  Chemistry: K–, P–; protocetraric acid.
  Ecology and distribution: This species is a pantropical species, which is distributed in tropical to subtropical rainforests, growing on bark. Known from China, Philippines, Papua New Guinea, Malaysia, Indonesia [3]; and Australia [38].
  Specimens examined: China. Hainan Province: Ledong County, Mt. Jianfengling Core Area, alt. 950 m, 2/X/2008, J. Li HN081449 (HMAS-L 117001).
  Discussions: This species is similar to Diorygma megasporum, but differs in the latter having immersed ascocarps, with narrow to slightly opened discs, and containing stictic acid, α-acetylconstictic acid, and constictic acid [3]. Position in key of Feuerstein et al. (2014): couplet 12(11) Ascospores 125(–250) × 30–40(–50) µm [4].

• Diorygma isabellinum (Zahlbr.) Z.F. Jia & Lücking, MycoKeys 25: 24, 2017.
  ≡ Graphina isabellina Zahlbr., in Handel-Mazzetti, Symb. Sinic. 3: 58, 1930.
  Thallus crustose, creamy white, somewhat yellowish, surface rough and warty. Ascocarps lirellate, 2–4.5 × 0.2–0.35 mm, elongate, single and rarely branched. Labia obvious. Discs closed to slightly open and proper margin conspicuous; Exciple uncarbonized. Hymenium clear, 160–180 µm high, I–. Ascospores 1/ascus, muriform, hyaline, ellipsoid, 110–120 × 35–48 µm, I+ violet.
  Chemistry: Norstictic and connorstictic acids.
  Ecology and distribution: This species is a subtropical species, growing on bark. Known from China [7,39].
  Specimens examined: China. Hunan Province: Changsha City, Mt. Yuelushan, alt. 250 m, 27/I/1918, Handel-Mazzetti 11437 (W).
  Discussions: Diorygma isabellinum was reported in China as Graphina isabellina Zahlbr. [39], and then was recombined as D. isabellinum [7]. This species is similar to D. junghuhnii (Mont. & Bosch) Kalb, Staiger & Elix, but the latter differs in having I+ blue-violet hymenium and smaller ascospores sized (60–)80–125 × 21–42 µm [3].

• Diorygma junghuhnii (Mont. & Bosch) Kalb, Staiger & Elix, Sym. Bot. Upsal. 34(1): 157, 2004.
  ≡ Graphis junghuhnii Mont. & Bosch, Pl. Jungh. 4: 471, 1856.
  = Graphis mendax Nyl., Annls Sci. Nat., Bot., sér. 4 11: 244 1859.
  Thallus crustose, whitish, greyish or greenish, surface rough and matt, verrucose; pseudocortex absent or thin, with crystallization. Ascocarps lirellate, 1–6 × 0.5 mm, scattered, elongated to nearly round, single or irregular branched, immersed, whitish. Discs open, covered with greyish to brownish pruina. Exciple slightly divergent, indistinctly developed, uncarbonized. Hymenium clear, 100–130 μm high, I+ blue. Ascospores 1/ascus, muriform, hyaline, oblong, with thin gelatinous wall at ends, spore locules of equal size, 25–30/6–10-locular, 70–125 × 20–40 µm, I+ violet blue.
  Chemistry: K+ red, P+ yellow to red; norstictic and connorstictic acids.
  Ecology and distribution: This species is a pantropical species, which is distributed in tropical to subtropical rainforests, growing on bark. Known from China, Togo (Central and Southern West Africa), Tanzania (East Africa), Philippines, Australia, Fiji, Costa Rica, Guatemala, Guyana, Brazil, etc. [3]; Thailand, Australia, and South Pacific island countries [38].
  Specimens examined: China. Fujian Province: Wuyishan City, Mt. Wuyishan, alt. 500 m, 27/V/2007, Z.F. Jia FJ445, FJ446 (LCUF). Guangdong Province: Xinyi City, Mt. Datianding, alt. 1700 m, 5/XI/2010, H.Y. Wang 20107250, 20107750, 20107958 (SDNU). Guangxi Province: Longsheng County, Huaping, alt. 900 m, 6/VI/2001, J.B. Chen 20032-1-3 (HMAS-L 030808-10). Hainan Province: Ledong Country, The tropical arboretum in the Mt. Jianfengling, alt. 650 m, 30/IX/2008, Z.F. Jia HN080692 (HMAS-L 127471); Wuzhishan City, Mt. Wuzhishan, alt. 680 m–880 m, 28/IX/2008, J. Li, HN081254 (HMAS-L 127474), HN081259 (HMAS-L 117008).
  Discussion: Diorygma junghuhnii is similar to D. soozanum, but differs in the latter having narrow to open discs covered with white pruina, and I+ weakly blue in hymenium [3]. It is similar to D. macgregorii (Vain.) Kalb, Staiger & Elix in appearance and the chemical substance, but differs in the latter having larger spores (135–185 × 40–63 μm) [3]. Position in key of Feuerstein et al. (2014): couplet 41(40) Hymenium completely I+ blue-violet; ascospores (60–)80–125 × 21–42 µm [4].

• Diorygma macgregorii (Vain.) Kalb, Staiger & Elix, Sym. Bot. Upsal. 34(1): 159, 2004.
  ≡ Helminthocarpon pervarians var. macgregorii Vain., Ann. Acad. Sci. Fenn., Ser. A 15(6): 266, 1921.
  Thallus crustose, creamy white, grey or greenish grey, surface rough and matt with small granula; pseudocortex absent or thin, with crystallization. Ascocarps lirellate, 1–5 × 0.5–2 mm, scattered, oval to elongated, branched, distinctly raised. Discs distinctly open, covered with white or greyish pruina. Exciple divergent, indistinctly developed, uncarbonized. Hymenium clear, 140–200 μm high, I+ weakly violet blue near exciple. Ascospores 1/ascus, muriform, hyaline, oblong, with the central cells larger than the peripheral ones, 135–180 × 35–50 µm, I+ violet blue.
  Chemistry: K+ red, P+ yellow to red; norstictic, connorstictic, cryptostictic and stictic acids.
  Ecology and distribution: This species is a pantropical species, which is distributed in tropical to subtropical rainforests, growing on bark. Known from China, Southeast Asia, and Papua New Guinea [3].
  Specimens examined: China. Hunan Province: Sangzhi County, Mt. Badagongshan, alt. 1400 m, 19/VIII/1997, J.B. Chen, D.P. Wang, S.L. Wang 9633 (HMAS-L 121023). Hainan Province: Changjiang County, Mt. Bawangling, alt. 980 m, 18/V/2007, Q.F. Meng M488 (HMAS-L 128742); Ledong Coutry, Mt. Jianfengling, alt. 180 m, 1/X/2008, J. Li, HN081360 (HMAS-L 117074). Guizhou province: Tongren City, Mt. Fanjingshan, alt. 1570 m, 1/IX/1963, J.C. Wei 621 (HMAS-L 047731).
  Discussion: This species is similar to Diorygma hieroglyphicum, but differs in the latter having immersed lirellae with narrow to open discs and smaller spores sized 95–150(–170) × 30–45 μm [3]. It is similar to D. junghuhnii, but differs in the latter having smaller spores sized (60–)80–125 × 21–42 µm [3].

• Diorygma megasporum Kalb, Staiger & Elix, Symb. Bot. Upsal. 34(1): 160, 2004.
  Thallus crustose, whitish, creamy white, yellowish, or weakly grayish green, surface rough and matt, with small granula; pseudocortex indistinctly developed or thin, with crystallization. Ascocarps lirellate, 0.5–5 × 0.1–0.7 mm, scattered, sub round to elongated, branched, immersed. Discs narrow to slightly open, and covered with a thick greyish pruina. Exciple divergent, uncarbonized, poorly developed. Hymenium clear, 170–200 μm high, I+ violet blue on upper parts and exciple. Ascospores 2–6/ascus, muriform, hyaline, irregular round, with thick wall, spore locules of equal size, 30–60/7–12-locular, 80–200 × 20–50 µm, I– or I+ violet blue.
  Chemistry: K+ yellow, P+ yellow to orange; stictic, α-acetylconstictic and constictic acids.
  Ecology and distribution: This species is a pantropical species, growing on bark. Known from China and India [3].
  Specimens examined: China. Guizhou Province: Tongren City, Mt. Fanjing Mountain, Scissor Gorge, alt. 1570 m, 6/IX/1963, J.C. Wei 0675 (HMAS-L 047752).
  Discussions: This species is similar to Diorygma hololeucum, but differs in the latter having protocetraric acid, and adnate ascocarps with open discs [3]. This species is recorded containing chemical components such as norstictic and hypostictic acids by Kalb et al. (2004); however, we did not detect these components in Chinese specimens. Position in key of Feuerstein et al. (2014): couplet 49(48) Ascospores 231–244 × 59–76 µm [4].

• Diorygma pruinosum (Eschw.) Kalb, Staiger & Elix, Sym. Bot. Upsal. 34(1): 166, 2004.
  ≡ Leiogramma pruinosum Eschw., in von Martius, Icon. Plant. Cryptog. 2: 12, 1828.
  ≡ Cyclographina pruinosa (Eschw.) D. D. Awasthi, Norw. J. Bot. 26: 175, 1979.
  Thallus crustose, creamy white, greenish or pale grey, surface rough and matt, partially cracked; pseudocortex distinctly developed and partially indistinctly developed, with crystallization. Ascocarps lirellate, 1–3 × 0.2–1 mm, scattered, elongated to round, single or branched, immersed to ± raised. Discs wide open and covered with a thick and white pruina. Exciple slightly divergent, indistinctly developed, uncarbonized to slightly carbonized. Hymenium clear, 110–190 μm high, I+ weakly violet blue. Ascospores 1/ascus, muriform, hyaline, oblong, spore locules of equal size, 25–40/6–10-locular, 90–170 × 20–50 µm, I+ violet blue.
  Chemistry: K–, P–; protocetraric acid.
  Ecology and distribution: This species is a pantropical species, which distributed in tropical to subtropical forests, growing on bark. Known from China, Cameroon, Nigeria (West Coast African country), Kenya (Equatorial East African country), and Tanzania Nigeria (East African countries), Indonesia, Singapore, Papua New Guinea (Western Pacific countries), Australia, Scotland, Brazil, etc. [3]; Australia, Philippines, and Solomon Islands [37].
  Specimens examined: China. Fujian Province: Jian’ou City, Fangdao Town, Wanmulin, alt. 600 m, 2/VI/2007, Q.F. Meng FJ645 (LCUF). Hainan Province: Ledong Country, Mt. Jianfengling, Rainforest Valley, alt. 670 m, 16/V/2007, Q.F. Meng M231 (HMAS-LHMAS-L128760), M333 (128737), M362 (HMAS-L128761).
  Discussion: This species is similar to Diorygma hololeucum, but differs in the latter having 2 or more spores with larger spores sized 125–230(–250) × 30–45(–50) µm [3]. It is similar to D. macgregorii in disc and the size of spores, but differs in the latter having norstictic acid [3]. Position in key of Feuerstein et al. (2014): couplet 15(13) Ascospores up to 150 µm long (or very rarely to 170 µm long), 95–150(–170) × 19–50 µm; peripheral and central ascospore locules of more or less equal size [4].

• Diorygma soozanum (Zahlbr.) M. Nakan. & Kashiw. [as ‘soozana’], in Nakanishi, Kashiwadani & Moon, Bull. Natn. Sci. Mus., Tokyo, B 29(2): 86, 2003.
  ≡ Graphina soozana Zahlbr., Feddes Repert. Spec. Nov. Regni veg. 31: 215, 1933.
  Thallus crustose, creamy white to pale grey, surface slightly rough and matt, cracked partialy; pseudocortex distinctly developed, with small crystallization. Ascocarps lirellate, 1–5 × 0.4–0.6 mm, scattered, oblong to enlarged, single or branched, prominently raised. Discs narrow to open, covered with white pruina. Exciple slightly divergent, indistinctly developed, uncarbonized. Hymenium clear, 130–160 μm high, I+ weakly violet blue. Ascospores 1/ascus, muriform, hyaline, oblong, spore locules of equal size, 20–30/7–8-locular, 110–140 × 35–45 µm, I+ violet blue.
  Chemistry: K+ red, P+ yellow to red; norstictic and connorstictic acids.
  Ecology and distribution: This species is a pantropical species, which is distributed in tropical to subtropical rainforests, growing on bark. Known from China and Japan [3,20].
  Specimens examined: China. Fujian Province: Wuyishan City, Mt. Wuyishan, alt. 500 m, 27/V/2007, Z.F. Jia FJ470, FJ453, FJ454 (LCUF); Jian’ou City, Fangdao Town, Wanmulin, alt. 320 m, 3/VI/2007, Q.F. Meng FJ942, FJ943 (LCUF). Sichuan Province: Dujiangyan City, Xiangshui Cave, alt. 1750 m, 13/VIII/1997, J.C. Wei 97141 (HMAS-L 055260). Yunnan Province: Luxi City, Mt. Santai, alt. 1340 m, 28/XI/1980, Y.M. Jiang 552-2 (HMAS-L 047681).
  Discussion: This species is similar to Diorygma tuberculosum (Stirt.) Kalb, Staiger & Elix, but differs in the latter having small and unequal spores, I– [3]. It is similar to D. junghuhnii, but differs in the latter having immersed ascocarps, with thickly pruinose discs, and I+ distinctly bluish violet in hymenium [3]. Position in key of Feuerstein et al. (2014): couplet 41(40) Hymenium weakly I+ blue-violet (mostly laterally); ascospores 110–145 × 36–45 μm [4].