The Phylogeny and Taxonomy of Cryptothecia (Arthoniaceae, Ascomycota) and Myriostigma (Arthoniaceae, Ascomycota), including Three New Species and Two New Records from China

Cryptothecia and Myriostigma are important elements of crustose lichen communities in tropical to subtropical forests, but little research has been done on these two genera in China. Morphological and molecular phylogenetic approaches to investigate species diversity of Cryptothecia and Myriostigma from Southern China were carried out in this study. We find five species of Cryptothecia and Myriostigma in our study, including three new species (M. flavescens, M. hainana and M. laxipunctata) and two new records (C. bartlettii and C. inexspectata). In addition, a phylogenetic tree based on mtSSU, RPB2 and nLSU illustrates the placement of the five species and supports the delimitation of the three new taxa. Detailed descriptions of morphological, ecological and chemical characteristics and illustrations are provided for every species. A key to all known Chinese Cryptothecia and Myriostigma species is also provided.


Morphology and Anatomy
The specimens examined were collected in Fujian, Guangxi, Hainan and Yunnan Provinces, China, and preserved in the Lichen Section of Botanical Herbarium (SDNU, Shandong Normal University).External morphological features were studied with a dissecting microscope (COIC XTL7045B2) (Olympus, Toyko, Japan), and photos were taken under an Olympus SZX16 (Olympus, Toyko, Japan) with DP72.Internal anatomical features were observed and measured by a microscope (Olympus CX41) (Olympus, Toyko, Japan), and images were taken with an Olympus BX61 (Olympus, Toyko, Japan) with DP 72.

Colour Reaction
The thallus and medulla colouring reactions were carried out by P (a saturated solution of p-phenylenediamine in 95% ethyl alcohol) (Tianjin Fuyu Fine Chemical Co., Tianjin, China), C (a saturated solution of aqueous sodium hypochlorite) (Tianjin Fuyu Fine Chemical Co., Tianjin, China), K (a 10% aqueous solution of potassium hydroxide) (Tianjin Damao Chemical FTY., Tianjin, China), I (a 3% solution of Lugol's iodine) (Tianjin Fuyu Fine Chemical Co., Tianjin, China), and long-wavelength UV light.Polarized light microscopy (pol) was used to locate crystals in the thallus sections.Then, H 2 SO 4 (a 25% solution of sulfuric acid) (Yantai Yuandong Fine Chemical Co., Yantai, China), was used to test whether the characteristic needle-shaped crystals formed when calcium oxalate was present in the thallus.

Chemical Analysis
The lichen substances were identified using standardized thin-layer chromatography (TLC).The particularly stable and reliable solvent C (toluene/acetic acid 170:30) was used in this study as it often provides the best discrimination of lichen substances.In this study, Lethariella cladonioides (Nyl.)Krog.containing atranorin and norstictic acid was used as the partition standard sample.
We used a pencil, carefully drawing a straight line 1.5 cm from the bottom of the glass silicone board.A point was marked every 1 cm on the straight line, which was the sample point.We soaked the lichen fragments in c. 1 mL of acetone for 30 min in a small test tube.Then, we used microcapillary tubes to sample them separately according to the position of the sampling points on the glass silicone board.After sampling, we placed the silicone board in a chromatography cylinder and placed it 1 cm below the solvent level.When the leading edge of the solvent moved from the origin to about 1 cm from the top of the silicone board, we removed the silicone board and dried the solvent on the board surface with a hair dryer.The silicone board was dried and then examined under short wavelength (254 nm) ultraviolet light for pigments.Later, it was sprayed with 10% sulfuric acid and heated at 100 • C in an oven for 10 min to develop the spots.The Rf values and color of each lichen substance were recorded and immediately examined under long wavelength (365 nm) ultraviolet light.The Rf values, as well as the fluorescent properties, were compared and analyzed to confirm the identity of the substance [19,20].

DNA Extraction, PCR Amplification and Sequencing
DNA was extracted directly from the clean growing portions of the thalli of recently collected specimens.Genomic DNA was extracted using the Sigma-Aldrich REDExtract-N-Amp Plant PCR Kit (St. Louis, MO, USA) following the manufacturer's instructions, except for the use of only 30 µL of extraction buffer and 30 µL of dilution buffer.
In our study, we found that the ITS sequence, typically considered crucial for distinguishing between lichen species, is essentially identical in Cryptothecia and Myriostigma and cannot be used as a standard for distinguishing between species within these two genera.Therefore, three other loci were amplified: the mtSSU gene with primer pairs mtSSU1 and mtSSU3R [21], the RPB2 gene with RPB2-7cF and RPB2-11aR [22], and the nLSU gene with LIC24R and LR7 [23,24].The 50 µL PCR mixture consisted of 2 µL of DNA, 2 µL of each primer, 25 µL of 2 × Taq PCR MasterMix [Taq DNA Polymerase (0.1 unit/µL); 3 mM MgCl 2 ; 100 mM KCl; 0.5 mM dNTPs; and 20 mM Tris-HCl (pH 8. Sequencing was performed by BioSune Biological Technology (Shanghai, China).

Sequence Alignment and Phylogenetic Analysis
To ascertain that all the new sequences were reliable, the newly generated sequences were compared to the sequences available in the GenBank database (http://www.ncbi.nlm.nih.gov/BLAST/,accessed on 10 September 2023), and all the new sequences were assembled using SeqMan v.7.0 (DNAstar packages).The sequences were aligned and edited using the online version of MAFFT v.7.0.26 and MEGA v.7.0.The algorithm of MAFFT chooses automatically (FFT-NS-1, FFT-NS-2, FFT-NS-i or L-INS-i; depending on the data size).The species Chiodecton natalense Nyl. was chosen as the outgroup [12].
Phylogenetic relationships were inferred using maximum likelihood (ML) and Bayesian inference (BI).The three gene matrices were combined by Geneious Prime 2023.0.4.We used the CIPRES Science Gateway (http://www.phylo.org/portal2/,accessed on 1 March 2024) [25] and performed ML analyses using RaxML-HPC v. 8.2.12 [26] under the default parameters as implemented on CIPRES, and support values were based on 1000 nonparametric bootstrap pseudoreplicates.We used PhyloSuite [27] to infer BI phylogenies via MrBayes 3.2.6 [28] under a partition model, for which the initial 25% of the sampled data were discarded as burn-in.The stationarity of the analysis was determined by examining the standard deviation of the split frequencies (<0.01).Bootstrap support (BS) ≥ 70 and posterior probability (PP) ≥ 0.95 were considered significant support values.The phylogenetic trees generated were visualized with FigTree v. 1.4.2[29].

Phylogenetic Analyses
A total of 10 mtSSU sequences, 7 RPB2 sequences and 2 nLSU sequences were newly generated from 10 specimens.We constructed ML and BI topologies based on these mtSSU, RPB2 and nLSU sequences and 87 additional sequences downloaded from NCBI (https: //www.ncbi.nlm.nih.gov/,accessed on 25 February 2024) (Table 1).The phylogenetic trees obtained from ML and BI analyses exhibited similar topologies; therefore, we present only the ML tree, with BS ≥ 70 for the ML analysis and PP ≥ 0.95 for the Bayesian analysis (Figure 1).The combined sequence matrices revealed three new monophyletic lineages corresponding to three new species: Myriostigma flavescens J.X. Xue & Lu L. Zhang, sp.nov.; M. hainana J.X. Xue & Lu L. Zhang, sp.nov.; and M. laxipunctata J.X. Xue & Lu L. Zhang, sp.nov.All the species positions had strong support in the ML and Bayesian analyses.In our phylogenetic tree, we can see those three new species (M.flavescens, M. hainana and M. laxipunctata) are clustered with Myriostigma and Stirtonia.According to Aptroot [30], Stirtonia had transversely septate ascospores; thus, based on morphological characters and phylogenetic analysis, we propose three new species in Myriostigma.This is in addition to two new Chinese records, as both are clustered with Cryptothecia subnidulans (the type species of Cryptothecia), which supported the delimitation of two new Chinese records.
Etymology: The epithet refers to its yellow ascospores.Ecology and distribution: This species is found only in China on bamboo and trees in a humid tropical forest in Yunnan Province.
Etymology: The epithet refers to the ascigerous areas of the thallus that have loose brown dots.
Ecology and distribution: This species is found only in China on the bark of trees in a humid tropical forest in Yunnan Province.
Ecology and distribution: Cryptothecia bartlettii has previously been reported in Ne Zealand and Australia [2] and is new to China.We collected the species in Fujian Provin under similar ecological conditions as in New Zealand and Australia.Specifically, w found them on the bark of trees within the subtropical forest at an elevation of appro mately 450 to 570 m.
Notes: According to Thor [2], Cryptothecia bartlettii is characterized by a thick a

Discussion
As one of the largest families of lichens, Arthoniaceae contains more than 700 lichenized species according to Lücking et al. [37].Recent phylogenetic analyses by Frisch et al. [7], Thiyagaraja et al. [38] and others have further implications for our understanding of generic limits within this family.However, it should be noted that all of these analyses were based on only a relatively low number of species described in Arthoniaceae.In previous studies, we can see that the morphological characteristics of Cryptothecia and Myriostigma are very similar, and some species of Myriostigma were described as Cryptothecia.In 2014, Frisch et al. [7] revealed through phylogenetic analysis that Cryptothecia was polyphyletic and Myriostigma Krempelhuber (1874) was resurrected because the type species Myriostigma candidum Kremp.[=Cryptothcia candida (Kremp.)R. Sant.] represents a lineage separate from Cryptothecia.They also reinstated Myriostigma for the Cryptothecia candida group, which is characterized by raised maculate ascomata-like ascigerous areas; 8-spored, thick-walled, globular and stalked asci; and bean-shaped, muriform ascospores [7].Therefore, some species of Cryptothecia, such as C. miniata and C. napoensis, were transferred to Myriostigma [10,34].However, in our study, we can see that some species of Myriostigma, such as M. hainana and M. laxipunctata, resemble Cryptothecia in morphology due to the absence of raised maculate ascomata-like ascigerous areas; thus, the distinction between the two genera based on the presence or absence of a maculate ascomata-like ascigerous areas becomes ambiguous.In addition, in our research, all species of Cryptothecia formed a cluster with the type species, C. subnidulans Stirt., on the phylogenetic trees, which exhibited a consistent presence of 1-2-spored asci, and all species of Myriostigma have 8-spored asci.The number of ascospores may thus serve as a distinguishing characteristic between Cryptothecia s. str.and Myriostigma.The confirmation of this viewpoint requires further research.
Species in Arthoniaceae often have a pantropical distribution.In southern China, there are abundant subtropical to tropical evergreen resources [39].This habitat is favourable for the lichens of Arthoniaceae.However, the family has not been sufficiently studied; thus far, only a few species have been recorded in China, including five species of Cryptothecia and one species of Myriostigma [40].In our study, we found that Arthoniaceae is highly diverse in southern China, especially in the Xishuangbanna region of Yunnan Province.Therefore, more comprehensive sampling and research are needed to determine the true diversity of Arthoniaceae in southern China.

Figure 1 .
Figure 1.Phylogenetic tree constructed by maximum likelihood (ML) analysis of Arthoniace cies based on the concatenated mtSSU-RPB2-nLSU dataset.Bootstrap support values ≥ 70 and posterior probabilities ≥ 0.95 (second value) for Bayesian methods are indicated above o the branches.Newly obtained sequences are marked in bold.

Figure 1 .
Figure 1.Phylogenetic tree constructed by maximum likelihood (ML) analysis of Arthoniaceae species based on the concatenated mtSSU-RPB2-nLSU dataset.Bootstrap support values ≥ 70 for ML and posterior probabilities ≥ 0.95 (second value) for Bayesian methods are indicated above or below the branches.Newly obtained sequences are marked in bold.
3)] (Tiangen, Beijing, China), and 19 µL of dd H 2 O.The PCR conditions for mtSSU amplification were set for initial denaturation at 94 • C for 10 min; 34 cycles of 95 • C for 45 s, 50 • C for 45 s, and 72 • C for 90 s; and a final extension at 72 • C for 10 min.The PCR conditions for RPB2 amplification were set for an initial denaturation at 94 • C for 10 min; 34 cycles of 94 • C for 45 s, 52 • C for 50 s, and 72 • C for 1 min; and a final extension at 72 • C for 5 min.The PCR conditions for nLSU amplification were set for an initial denaturation at 95 • C for 15 min; 45 cycles of 95 • C for 45 s, 53 • C for 45 s, and 72 • C for 1 min; and a final extension at 72 • C for 7 min.

Table 1 .
Specimens used for the phylogenetic analyses with the corresponding voucher information and GenBank accession numbers for the mtSSU, RPB2 and nLSU sequences.Newly obtained sequences in this study are in bold.