A new geographical record of Lichenostigma chlaroterae from China

Lichenostigma was introduced by Hafellner in 1982 to accommodate the lichenicolous fungus L. maureri and was initially placed in Arthoniales based on ascocarp morphology^[1]. The species grows on Usnea spp. and Pseudevernia furfuracea and has been reported from the Eastern Alps as well as other parts of Eurasia. Ertz et al.^[2] provided the first phylogenetic study of Lichenostigma and synonymized two lichenicolous genera, Diederimyces and Phaeosporobolus, under Lichenostigma^[2]. Based on the combined phylogenetic analyses of LSU and mtSSU sequence data, together with detailed morphological evidence, Lichenostigmatales was established within Arthoniomycetes to accommodate the family Phaeococcomycetaceae, with Lichenostigma designated as the type genus^[2]. Phaeococcomycetaceae presently includes four genera, namely Antarctolichenia, Etayoa, Lichenostigma, and Phaeococcomyces^[3].

Presently, Lichenostigma comprises approximately 34 species^[3,4], with Lichenostigma maureri as the type species. The genus is polyphyletic, with its taxa distributed across both Lichenotheliaceae (Dothideomycetes) and Arthoniomycetes^[2]. Ertz et al.^[2] proposed that the genus comprises two subgenera: subgenus Lichenostigma, placed within Arthoniales, and corresponding to Lichenostigma sensu stricto, subgenus Lichenogramma, which is phylogenetically clustered within Dothideomycetes; these subgenera are primarily distinguished by differences in cell division. Lichenostigma sensu stricto divides by budding, whereas the latter produces cells through septation. The sexual morph lacks hamathecial filaments, with asci developing directly among stromatic cells; the ascus tholus may be I+ reddish and K/I+ blue, while ascospores are 1-septate, with the outer wall occasionally K/I+ blue. The asexual morph is characterized by conidiomata composed of similar globose stromatic cells, from which conidiogenous cells arise, producing multicellular, subspherical to ellipsoid conidia^[2].

The first checklist of lichenicolous fungi in China was published in 2003, reporting the occurrence of Lichenostigma cosmopolites on Xanthoparmelia scabrosa from Hong Kong^[5]. In the present study, a specimen collected in China, growing on Lecanora sp., was examined. Based on morphological characters and phylogenetic analyses of LSU and mtSSU sequence data, the specimen was identified as Lichenostigma chlaroterae, representing the first geographic record of this species from China.

A specimen of Lichenostigma was collected in 2018 from the bark of an unidentified tree hosting a Lecanora species in Lijiang (22°36′38.8′′ N, 100°10′33.5′′ E), Yunnan Province, China. The specimen was observed and examined using a stereo-microscope (SteREO Discovery V12, Carl Zeiss Microscopy GmbH, Germany) and a compound microscope (Nikon ECLIPSE 80i, Nikon, Japan), equipped with a Nikon DS-Ri2 digital camera (Nikon, Japan). The measurements were done with Tarosoft® Image Frame Work v0.9.7, and the morphological analysis was performed following Senanayake et al.^[6]. The symbol '$\overline {\rm x} $' indicates the mean value. Illustrations were prepared and finalized using Adobe Photoshop 2020 (Adobe Systems, San Jose, CA, USA). The specimen was deposited in the Herbarium of Lichen, Kunming Institute of Botany, Chinese Academy of Sciences (KUN-L). The Faces of Fungi number was obtained following Jayasiri et al.^[7].

DNA extraction, polymerase chain reaction amplification, and sequencing

Genomic DNA was extracted directly from fungal fruiting bodies using the Omega Bio-Tek Kit (Anoren [Beijing] Biotechnology Co., Ltd), following the manufacturer's protocol. PCR amplification of the LSU region was carried out with primers LR0R/LR5 following the conditions recommended by Vilgalys & Hester^[8]. The mtSSU region was amplified using primers mrSSU1/mrSSU3R^[9]. The PCR reaction mixture contained a total volume of 25 μL, consisting of 12.5 μL of 2 × PCR G013 Taq MasterMix with Dye (ABM, Canada), 1 μL of each primer (10 μM), 2 μL of genomic DNA, and 8.5 μL of sterile distilled water. The parameters of PCR amplifications are displayed in Table 1. The PCR products were visualized through electrophoresis on a 1% TAE gel and detected using TSJ003 GoldView nucleic acid dye (Qingke, Kunming, China). Sequencing was performed at Qingke Company, Kunming, China.

Sequence alignment and phylogenetic analyses

All newly generated sequences were checked for quality using BioEdit v7.0.9^[10]. Forward and reverse sequences were assembled using DNASTAR Lasergene SeqMan Pro v7.1.0^[11]. The assembled sequences were then queried against the NCBI database^[12] to identify close relatives. Preliminary identification of the newly generated sequences was performed through standard BLAST searches of GenBank. For phylogenetic analyses, LSU and mtSSU gene regions from representative species of Lichenostigmatales, as well as the outgroup taxa from Arthoniales, were retrieved from GenBank (Table 2). Each gene dataset was aligned using MAFFT v7 (https://mafft.cbrc.jp/alignment/server/index.html), subsequently trimmed with TrimAl v1.3 software, with a gap threshold set to 0.6 for both datasets^[13]. The aligned LSU and mtSSU datasets were combined into a combined matrix using SequenceMatrix v1.8, and file formats were converted using AliView v1.19^[14] for further analyses. The phylogenetic tree for maximum likelihood analysis was generated using the Phylip format, while the Nexus format was used for Bayesian inference analysis^[15]. The ML analysis was performed using IQ-TREE with the ML+ rapid bootstrap setting and 1,000 replicates^[16,17]. Bayesian inference was performed with MrBayes v3.1.2 to estimate Bayesian posterior probabilities (BYPP)^[18]. The resulting trees were visualized using FigTree v1.4.0 (https://tree.bio.ed.ac.uk/software/figtree). The identification of potential species followed previously proposed species delineation approaches^[19,20].

The combined LSU and mtSSU dataset comprised 2,867 base pairs in total, with positions 1–1,826 corresponding to the LSU region and positions 1,827–2,867 representing the mtSSU region. The dataset consists of 1,231 distinct alignment patterns, with 39.97% of undetermined characters or gaps with Arthonia didyma, A. dispersa, Cryptothecia candida, and Briancoppinsia cytospora as outgroup taxa (Fig. 1). A total of 32 strains were included in the combined analyses. The best RAxML tree with a final likelihood value of −14109.293201 is presented in Fig. 1. Estimated base frequencies were as follows: A = 0.275194, C = 0.200804, G = 0.275049, T = 0.248953, substitution rates AC = 0.791749, AG = 2.569367, AT = 1.900859, CG = 1.275594, CT = 4.854511, and GT = 1.000000. Single-gene analyses were also performed to evaluate topological congruence and clade stability relative to the combined-gene analyses. The single-gene analyses produced tree topologies that were consistent with those inferred from the combined gene analyses. The ML analysis generated tree topologies consistent with those from the Bayesian inference (BI) analysis, supporting the reliability of our phylogenies generated herein.

Figure 1. 

Phylogram of RA × ML analysis based on the combined LSU and mtSSU sequence datasets. Bootstrap support values for Maximum Likelihood equal to or greater than 70%, and Bayesian posterior probabilities equal to or greater than 0.90 BYPP are indicated at the nodes as ML/BYPP. The newly generated strain in this study is indicated in blue bold text. The tree is rooted with Arthonia didyma (Ertz 7587 BR), A. dispersa (UPSC2583), Cryptothecia candida (Ertz 9260 BR), and Briancoppinsia cytospora (Ertz 15244 BR).

Taxonomy

Lichenostigma chlaroterae (F. Berger & Brackel) Ertz & Diederich, Fungal Diversity 66: 128 (2014) [MB#804672]. Fig. 2

Figure 2. 

Lichenostigma chlaroterae (HKAS148603). (a)–(h) Conidiomata on Lecanora sp. host. (i) Vertical section through conidioma. (j)–(r) Conidia. (s) Development of conidia. Scale bar: (i) = 20 μm, (j)–(r) = 10 μm.

=Phaeosporobolus chlaroterae F. Berger & Brackel, Herzogia 24(2): 351 (2011)

Index Fungorum: IF561182; Faces of Fungi Number: FoF 19647

The mycelium is extremely sparse, brown, and superficial. Asexual morph: Conidiomata macroscopically indistinguishable, 40–45 µm diam. × 60–70 µm high. ($\overline x $ = 43 × 65 µm, n = 10), scattered, dark brown to black, subspherical to ellipsoid, with an indistinctly truncated base. Conidiophores indistinct. Conidiogenous cells pale to medium brown, developing from spherical stromatic cells, shortly subcylindrical to almost ellipsoid, with an indistinctly truncate base, polyblastic. Conidia 4.8–6.6 × 4.4–7.1 µm ($\overline x $ = 5.9 × 5.8 µm, n = 30), multicellular, composed of 3–6 cells, ellipsoid, brown, smooth, but sometimes with a verrucose to echinulate ornamentation when over-mature. Conidial cells 2.2–4.8 × 2.1–4.5 µm ($\overline x $ = 3.3 × 3.2 µm, n = 30), subspherical to ellipsoid. Sexual morph: Unknown.

Material examined: China, Yunnan Province, Lijiang City, on a disc of Lecanora sp. on unidentified bark, 22°36' 38.8' N, 100°10' 33.5' E, elevation 3,351 m, 3 September 2018, V. Thiyagaraja, D1L18 (HKAS148603).

Distribution: China (This study). Europe, Macaronesia, Mongolia^[21], North America^[2], Russia^[21], and South America (Bolivia).

Notes: the BLAST results of the LSU sequence data revealed the highest similarity to Lichenostigma chlaroterae (Goward 12–42) and L. chlaroterae (Neuberg [h Diederich]), with 99.10% and 100.00% similarity, respectively. The mtSSU sequence data showed the highest similarity to L. chlaroterae (Neuberg [h Diederich]) with 100.00%. In the multi-gene phylogenetic analyses, the new strain L. chlaroterae (HKAS148603) clustered with extant strains of L. chlaroterae (Neuberg [h Diederich], Goward 12–42 and Goward 12–30), with 93% ML bootstrap support and 0.93 BYPP. The strain L. chlaroterae (Diederich 17329) was excluded from the present analyses due to its unstable phylogenetic placement. The base pair comparison between our strain (HKAS 148603) and L. chlaroterae (Neuberg [h Diederich]) revealed no differences in either the LSU or mtSSU regions. In comparison with L. chlaroterae (Goward 12–42), sequence divergence was 0.12% (1/822 bp) in the LSU and 0.41% (3/715 bp) in the mtSSU, with no gaps detected. Similarly, comparison with L. chlaroterae (Goward 12–30) showed a divergence of 0.29% (2/675 bp) in the mtSSU, while LSU comparison was not performed due to the lack of molecular data for that strain. The pairwise sequence comparison between our strain and L. chlaroterae (Diederich 17329) revealed 1.26% (10/792) differences in the LSU and 1.25% (9/720) differences in the mtSSU without gaps.

Lichenostigma chlaroterae was established by Ertz et al.^[2] and the species was originally introduced as Phaeosporobolus chlaroterae by Berger & von Brackel^[22] based on material collected in Austria. It has also been reported from Germany and Portugal. In Central Europe, records range from colline to montane zones, and the species is probably more widespread^[22]. The new specimen exhibits several characteristics similar to Lichenostigma chlaroterae, including superficial, smooth-walled brown hyphae, warty to granular ornamentation on the outer stromatal cells, and multicellular conidia composed of nearly spherical to ellipsoidal cells. Additionally, L. chlaroterae (Diederich 17329) occasionally produces a sexual stage with 1-septate brown ascospores, whereas no sexual structures have been observed in our sample^[2]. Our specimen slightly differs from the L. chlaroterae (Diederich 17329) in the smaller conidia (4.8–6.6 × 4.4–7.1 µm vs [5.5–]7.1–10.2[–13] × [5–]6.3–8.8[–11] µm) and a smaller number of cells (3–6 vs [3–]4–9[–16])^[2].

Since the beginning of the 21^st century, research on lichenicolous fungi in China has gradually expanded. Aptroot & Sipman^[23] reported three species in Hong Kong, including two lichenicolous fungi newly recorded from China (Lichenoconium lecanorae and Lichenostigma cosmopolites)^[23]. In 2003, four additional lichenicolous fungi were reported in Yunnan^[24]. In the same year, a checklist of 29 known lichenicolous fungi in China was systematically compiled^[5]. In 2016, Polycoccum clauderouxii was recorded in Qinghai^[25]. Subsequently, a total of 30 lichenicolous species were reported^[26]. Thiyagaraja et al.^[27] reported Sclerococcum simplex that was reported from Yunnan, China^[27]. In 2023, nine species of Cercidospora were documented in China. However, as these fungi were isolated from the medullary tissues of lichen thalli and reported as "black lichenicolous fungi", their true lichenicolous nature remains uncertain^[28]. Subsequently, a new record of Sclerococcum glaucomarioides was described from Yunnan, indicating that the diversity of lichenicolous fungi in China is far from being fully revealed, especially in Yunnan^[29]. However, research on lichenicolous fungi in China is limited, with nearly 60 species reported in the past 25 years, indicating that the documented diversity remains remarkably low given China's vast area and habitat heterogeneity^[5,23−29].

In the phylogenetic analyses, the genera within Lichenostigmales formed a monophyletic clade, with the exception of Lichenostigma, which appeared polyphyletic. This polyphyletic nature of Lichenostigma was also reported by Ertz et al.^[2] highlighting the need for further studies, particularly due to the lack of molecular data for many species within the genus. Recently, Muggia et al.^[30] introduced a new endolichenic genus, Antarctolichenia, within the order and the genus was collected from the Antarctic continent, which showed a close phylogenetic relationship with the genus Etayoa. A similar phylogenetic placement was observed in the present study. Further, our newly obtained strain of L. chlaroterae, together with previously reported strains of the same species, formed a well-supported clade with L. alpinum. This clade formed a sister clade to the type species, L. maureri, with strong statistical support (100/1.00).

Lichenostigma is a cosmopolitan genus that has been reported from a wide range of regions worldwide^[2,21,31]. This study represents the second record of the genus Lichenostigma from China. Previously, L. cosmopolites was reported on Xanthoparmelia scabrosa from Hong Kong, China^[23]. Lichenostigma cosmopolites is widely distributed in extratropical regions of both hemispheres (Africa, Asia, Australia, Europe, North America, and South America), occurring mainly in lowlands of temperate regions and at mid elevations or higher in tropical regions^[32]. Lichenostigma chlaroterae, a relatively common species in Europe, has been reported from North America^[2]. This species primarily colonizes Lecanora and other lichens, such as Buellia griseovirens, Fuscidea lightfootii, and Graphis pulverulenta^[2,33,34]. Here, we report the first occurrence of L. chlaroterae in China. This record further expands the known distribution of the species in East Asia.