The Genus Chlorosplenium (Helotiales, Leotiomycetes) from China with Notes on C. chlora Complex

The small fruitbodies of Chlorosplenium are greenish yellow and mainly grow on woody substrates. The species diversity of the genus in China was investigated based on specimens formerly deposited in the Herbarium Mycologicum Academiae Sinicae as well as new collections gained in recent years. Our phylogenetic results revealed the species diversity of the genus is underestimated and the commonly known Chlorosplenium chlora is a species complex. Based on morphology studies and sequence analyses of three regions (ITS, LSU and RPB1), the Chinese collections represent two new species which are described and illustrated here as C. sinicum and C. sinochlora. Chlorosplenium fusisporum is quite possibly a species of the genus Chlorociboria, and C. hyperici-maculati should be excluded from the genus.

Many saprotrophic inoperculate cup-fungi have been placed in Chlorosplenium on the basis of the phenotypic character grayish-to greenish-yellow apothecia, and 57 names were published under the genus (http://www.indexfungorum.org/Names/Names.asp (accessed on 15 August 2021)). However, many of them are obviously not congeneric with the type species C. chlora, and they were even scattered in 11 different families according to the recent taxonomic treatments [3]. Chlorosplenium was once treated as a member of Helotiaceae or Dermateaceae [1,4]. According to the results of multi-locus phylogenetic analysis, the genus appeared as a clade distinct from any other fungal groups, thus a new family Chlorospleniaceae was proposed [5].
Five species are currently known in Chlorosplenium [4], i.e., C. chlora, C. hypochlorum (Berk. & M.A. Curtis ex W. Phillips) J.R. Dixon [1,2], C. cenangium (De Not.) Korf [6], C. fusisporum Liou & Z.C. Chen [7], and C. hyperici-maculati Svrček [8], among which two were previously recorded from China [9]. Species of the genus are somewhat easily recognized in the field, and are characterized by discoid to shallowly cupulate, grayish to greenish yellow apothecia with enrolled margin, usually sessile, reaching a size of 0.5-4 mm in diameter; hyphal extensions of different lengths present on receptacle surface or having a glabrous surface; ectal excipulum of textura angularis with the outer cell layer brown and the inner cells subhyaline to hyaline, medullary excipulum of textura intricata with hyaline hyphae and non-gelatinous tissues, subhymenium usually present and appearing as a densely interwoven hyphal layer; asci cylindric-clavate and containing eight ascospores; Life 2021, 11, 1167 2 of 12 ascospores subcylindrical, hyaline and commonly unicellular; and paraphyses filiform and not or slightly exceeding the asci. Members of the genus are wood-inhabiting.
During our work on species diversity of Dermateaceae and its allies in China, herbarium specimens and recent collections of Chlorosplenium were studied based on morphological features and DNA sequence data. As a result, the occurrence of C. chlora in China should be re-considered, and two new species are discovered. The taxonomic position of the previously recorded C. fusisporum Liou & Z.C. Chen is doubtful based on the original description and illustration [7] and is tentatively excluded from the genus.

Materials and Methods
Specimens examined were all deposited in the Herbarium Mycologicum Academiae Sinicae (HMAS) and collected during 1933−2020 from Anhui, Chongqing, Guangdong, Guangxi, Henan, Hubei, Hunan, Jiangxi, Shaanxi, and Yunnan provinces of China. Photographs of fresh apothecia were taken by a Canon PowerShot G16 digital camera. Dried apothecia were rehydrated with distilled water and sectioned at a thickness of 15−20 µm with a Yidi YD-1508A freezing microtome (Jinhua, China). Mounting media was lactophenol cotton blue. Iodine reactions of ascus apical apparatus were tested in Melzer's reagent and Lugol's solution with or without 3% KOH solution pretreatment. An Olympus BH-2 microscope (Tokyo, Japan) was used for microscopic examination and measurements. Microscopic photographs were taken using a ZEISS Axiocam 305 color microscope camera (Göttingen, Germany) attached to a Zeiss Axioskop 2 Plus microscope (Göttingen, Germany).
Newly generated sequences were assembled using SeqMan (DNASTAR, Lasergene 7.1.0) and BioEdit 7.0.5.3 [15]. The new sequences were deposited at GenBank and additional sequences used for phylogenetic analyses were downloaded from GenBank ( Table 1). The sequence matrix was aligned by MUSCLE [16] and manually edited using BioEdit 7.0.5.3 [15]. Introns and ambiguous sites were removed and excluded from the analysis process. Dermea acerina and Pezicula carpinea were chosen as outgroup taxa.  Note: Type specimens are indicated with superscript "T" and newly generated sequence data are indicated in black bold.
Maximum likelihood (ML) and Bayesian inference (BI) analyses were conducted with RAxML 8.0 [17] and MrBayes 3.1.2 [18], respectively. Parameters of the ML analysis were set as default, and statistical support values were obtained from 1000 replicates of bootstrap (BP) tests. The BI analysis was performed using the best-fit evolutionary model estimated by MrModeltest 2.3 [19] with the Akaike information criterion. Two parallel runs of four simultaneous chains of Markov Chain Monte Carlo were performed for 2,000,000 generations with the trees sampled every 100 generations. The first quarter of sampled trees were discarded as the burn-in phase, and the remaining trees were used for calculating posterior probabilities (PP) in the majority rule consensus tree. The phylogenetic trees were visualized in FigTree 1.4.4 [20].

Phylogenetic Analyses
The final ITS matrix included 31 sequences of Chlorosplenium and eight of related taxa, and the alignment contained 595 nucleotide sites. GTR+I+G was selected as the best-fit model for BI analysis. The phylogenetic trees yielded by ML and BI analyses are similar in topology, and the ML tree is presented in Figure 1 with bootstrap values at nodes. All the Chlorosplenium sequences clustered together (MLBP/BIPP = 88%/1.00) that confirmed the Life 2021, 11, 1167 4 of 12 monophyly of the genus. Seven well-supported clades/lineages (A-G) were recognized, including the two proposed new taxa from China.

Phylogenetic Analyses
The final ITS matrix included 31 sequences of Chlorosplenium and eight of related taxa, and the alignment contained 595 nucleotide sites. GTR+I+G was selected as the bestfit model for BI analysis. The phylogenetic trees yielded by ML and BI analyses are similar in topology, and the ML tree is presented in Figure 1 with bootstrap values at nodes. All the Chlorosplenium sequences clustered together (MLBP/BIPP = 88%/1.00) that confirmed the monophyly of the genus. Seven well-supported clades/lineages (A-G) were recognized, including the two proposed new taxa from China. Phylogenetically, all collections of C. sinicum were strongly supported in clade A which were further divided into a few subclades. Chlorosplenium sinochlora appeared as an isolated lineage F not closely related to the others. Chlorosplenium chlora from the USA Phylogenetically, all collections of C. sinicum were strongly supported in clade A which were further divided into a few subclades. Chlorosplenium sinochlora appeared as an isolated lineage F not closely related to the others. Chlorosplenium chlora from the USA materials clustered in clade B, and they were typical representatives of the species. Clades C, D, E and G consisted of unidentified samples of Chlorosplenium from New Zealand, Australia, Mexico, and Honduras, which were shown as Chlorosplenium spp. We suspect they might represent either un-named species or the existing species which have not been connected with any DNA sequences.
Due to the lack of LSU and RPB1 sequence data in most Chlorosplenium collections, the combined ITS-LSU-RPB1 dataset only included 13 sequences of the Chinese material, as well as four related inoperculate discomycete taxa. The final alignment comprised 2234 sites including gaps, of which 655 bp for ITS, 825 bp for LSU and 754 bp for RPB1. The best-fitting model for BI analysis was GTR+I for the multi-locus sequence data. Figure 2 showed the ML tree resulted from ITS-LSU-RPB1 sequence analysis with bootstrap values higher than 50%. Similar to the ITS analysis, C. sinicum is divided into several subgroups. materials clustered in clade B, and they were typical representatives of the species. Clades C, D, E and G consisted of unidentified samples of Chlorosplenium from New Zealand, Australia, Mexico, and Honduras, which were shown as Chlorosplenium spp. We suspect they might represent either un-named species or the existing species which have not been connected with any DNA sequences.
Due to the lack of LSU and RPB1 sequence data in most Chlorosplenium collections, the combined ITS-LSU-RPB1 dataset only included 13 sequences of the Chinese material, as well as four related inoperculate discomycete taxa. The final alignment comprised 2234 sites including gaps, of which 655 bp for ITS, 825 bp for LSU and 754 bp for RPB1. The best-fitting model for BI analysis was GTR+I for the multi-locus sequence data. Figure 2 showed the ML tree resulted from ITS-LSU-RPB1 sequence analysis with bootstrap values higher than 50%. Similar to the ITS analysis, C. sinicum is divided into several subgroups.

Excluded and Doubtful Species of Chlorosplenium Chlorosplenium fusisporum Liou & Z.C. Chen
Notes-Chlorosplenium fusisporum was originally described from Taiwan, China [7]. It is characterized by green apothecia and with a centrally placed stipe, ectal excipulum with coiling or undulate hairs encrusted by green granules, acerose ascospores with 1-3 septa, 20-35 × 2.5-3.5 µm ( Figure 5). Although we did not access the voucher specimen of C. fusisporum which lacks sequence data, the combination of apothecial color, ectal excipulum structure and short and granulate hairs on the receptacle surface strongly suggests its affinity to Chlorociboria. The type material of the fungus has not been examined, and therefore formal nomenclatural treatment is not proposed for the time being.

Excluded and Doubtful Species of Chlorosplenium
Chlorosplenium fusisporum Liou & Z.C. Chen Notes-Chlorosplenium fusisporum was originally described from Taiwan, China [7]. It is characterized by green apothecia and with a centrally placed stipe, ectal excipulum with coiling or undulate hairs encrusted by green granules, acerose ascospores with 1-3 septa, 20-35 × 2.5-3.5 μm ( Figure 5). Although we did not access the voucher specimen of C. fusisporum which lacks sequence data, the combination of apothecial color, ectal excipulum structure and short and granulate hairs on the receptacle surface strongly suggests its affinity to Chlorociboria. The type material of the fungus has not been examined, and therefore formal nomenclatural treatment is not proposed for the time being.

Chlorosplenium hyperici-maculati Svrček
Notes-Chlorosplenium hyperici-maculati was originally described from Slovakia on stem of Hypericum maculatum [8]. In his original description, Svrček indicated that "the beautiful color of this discomycete is conspicuously deep green in all outer parts of apothecia, and the disc has an almost velvet appearance." As to its similar or closely related fungus, he compared C. hyperici-maculati with C. aeruginellum (P. Karst.) P. Karst., which is now being placed in the genus Dasyscyphus Nees ex Gray [≡ Dasyscyphus aeruginellus (P. Karst.) Korf & J.R. Dixon] belonging to a different family [1]. Judged by the original description and illustration of the fungus, we conclude that C. hyperici-maculati should be excluded from Chlorosplenium in a modern sense. Morphological details provided in the literature are not enough for accurate species identification of Chlorosplenium because macro-and microscopic features are usually overlapping. Molecular study was seldom carried out in the genus and it is necessary to use molecular approaches to facilitate species recognition. Concerning the taxonomic characters, structure of excipulum and morphology of asci and ascospores are useful in species identification. According to the results of our phylogenetic analyses, inter-specific variations of ITS rDNA, the universal barcode for fungi, are higher than those of the protein-coding gene RPB1 in Chlorosplenium, which is suitable for species distinction. Based on the results of the detailed morphological comparison and phylogenetic analyses, the Chinese collections previously identified as C. chlora were proved to represent two novel species. Thus, C. chlora should be excluded from the fungal species catalogue of China [9]. Its occurrences in other Asian countries are also doubtful.
Based only upon specimens collected from China, this study has shown that the worldwide species diversity of Chlorosplenium has not been investigated using modern methods, and many hidden species are expected to be discovered. Future studies based on large-scale samplings combining with ecological, morphological, and molecular data will lead to a better understanding of the species diversity and phylogeny of the genus. Data Availability Statement: Names of the new species were formally registered in the database Fungal Names (http://www.fungalinfo.net/fungalname/fungalname.html (accessed on 26 August 2021)). Specimens were deposited in the Herbarium Mycologicum Academiae Sinicae (HMAS). The newly generated sequences were deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank (accessed on 28 August 2021)).