Morphological and Phylogenetic Analyses Reveal Four New Species of Acrodictys (Acrodictyaceae) in China

During our ongoing survey of dematiaceous hyphomycetes associated with dead branches in tropical forests, eight Acrodictys isolates were collected from Hainan, China. Morphology from the cultures and phylogeny based on partial small subunit (SSU), entire internal transcribed spacer regions with intervening 5.8S (ITS), partial large subunit (LSU) of rRNA gene, partial beta-tubulin (tub2), and partial RNA polymerase II second largest subunit (rpb2) genes were employed to identify these isolates. As a result, four new species, namely Acrodictys bawanglingensis sp. nov., A. diaoluoshanensis sp. nov., A. ellisii sp. nov., and A. pigmentosa sp. nov., are introduced. Illustrations and descriptions of the four taxa are provided, along with comparisons with closely related taxa in the genus. For facilitating relative studies, an updated key to all accepted species of this genus is also compiled.


Introduction
Acrodictys M.B. Ellis was erected by Ellis and typified by A. bambusicola M.B. Ellis [1]. In the protologue, this genus was characterized by globose to subglobose, uniformly pigmented, angularly, or obliquely septate conidia. This broad, generic circumscription of Acrodictys has been followed for nearly 40 years until recent segregations starting at the beginning of this century [2][3][4].
Species in Acrodictys sensu stricto are saprobic on dead branches. They are thought to be wood-decaying fungi, promoting the carbon cycle in the ecosystem by converting cellulose, hemicellulose, and lignin into inorganic substances [7]. Although having a worldwide distribution, they are mainly recorded from tropical areas, such as Brazil, Thailand, and Mexico (https://www.gbif.org, accessed on 25 June 2022). Hainan Province (18 • 10 -20 • 10 N,108 • 37 -111 • 05 E) is an island in southern China. Its annual mean 2 of 14 temperature is 22-27 • C, and its annual precipitation is 1000-2600 mm. It is a typical tropical rainforest climate, suitable for the growth and reproduction of wood-decaying fungi.
In China, 12 isolates representing 9 species (A. bambusicola, A. elaeidicola, A. fluminicola, A. globulosa, A. hainanensis, A. liputii, A. malabarica, A. peruamazonensis, and A. porosiseptata) have been documented from tropical areas in Hainan and Yunnan Provinces [6,7]. The samples in this study were collected from the Bawangling National Nature Reserve and Diaoluoshan National Nature Reserve in Hainan Province. Dead branches of unidentified trees were collected in tropical rain forests dominated by Lauraceae and Fagaceae trees, and the fungi in pure culture were isolated from these dead branches. Fungi from these axenic cultures were identified based on classic morphological and modern molecular approaches. As a result, four new species are described and illustrated herein in the genus Acrodistys sensu stricto.

Isolation and Morphological Observation
Samples were collected from the Bawangling National Nature Reserve and Diaoluoshan National Nature Reserve, Hainan Province, China (108 • 37 -117 • 50 E, 3 • 58 -20 • 20 N), and taken to the laboratory in plastic bags. The samples were placed in Petri dishes with moist filter paper and cultured in an incubator at 25 • C for 1-2 weeks. The samples were examined using a stereomicroscope (SZX10 model, Olympus, Tokyo, Japan, https: //www.olympus-lifescience.com.cn/zh/microscopes/, accessed on 25 June 2022) to locate sporulating structures. Isolations were made with single spore methods according to the following steps: Spores were suspended with sterilized water and spread onto PDA (PDA: 200 g potato, 20 g dextrose, 20 g agar, 1000 mL distilled water, pH 7.0) plate and incubated for one day under a biochemical incubator. After germination, colonies were transferred to a new PDA plate to obtain a pure culture. After 30 days of incubation, morphological characters were recorded. Photographs of the colonies were taken on the 30th day using a digital camera (G7X model, Canon, Tokyo, Japan, https://www.canon.com.cn/, accessed on 25 June 2022). The micromorphological characteristics of colonies were observed using two stereomicroscopes (SZX10 model and BX53 model, Olympus, Tokyo, Japan, https: //www.olympus-lifescience.com.cn/zh/microscopes/, accessed on 25 June 2022), both fitted with a digital camera (DP80 model, Olympus, Tokyo, Japan, https://www.olympuslifescience.com.cn/zh/microscopes/, accessed on 25 June 2022). Sporulating structures were mounted in water for microscopic observation and photomicrography. At least 50 mature conidia and conidiophores were measured using the cellSens software (Olympus, Tokyo, Japan), and images were used for further processing with Photoshop ver. CS5 (Adobe Systems, San Jose, CA, USA, https://www.adobe.com/cn/, accessed on 25 June 2022). The cultures were stored in 10% sterilized glycerin and sterile water at 4 • C for further studies. Specimens were deposited in the Herbarium Mycologicum Academiae Sinicae, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China (HMAS), and the Herbarium of the Department of Plant Pathology, Shandong Agricultural University (HSAUP). Living cultures were deposited in the Shandong Agricultural University Culture Collection (SAUCC). New taxa were submitted to the taxonomic database My-coBank (http://www.mycobank.org, accessed on 25 June 2022) and obtained relative deposition numbers.

DNA Extraction and Amplification
Genomic DNA was extracted from fungal mycelia grown on PDA, using a modified cetyltrimethylammonium bromide (CTAB) protocol as described in Guo et al. [8]. Five loci, namely the internal transcribed spacer regions with intervening 5.8S rRNA gene (ITS), partial large subunit (LSU) rRNA gene, the partial small subunit (SSU) rRNA gene, the partial RNA polymerase II second largest subunit (rpb2), and part of the beta-tubulin gene region (tub2) were amplified with the primer pairs and polymerase chain reaction (PCR) processes listed in Table 1. PCR was performed using a thermocycler (Mastercycler X50, Eppendorf, Hamburg, Germany, https://www.eppendorf.com/CN-zh/, accessed on 25 June 2022). Amplifications were performed in a 25 µL reaction volume, which contained 12.5 µL 2 × Taq Plus Master Mix II (Vazyme, Nanjing, China), 1 µL of each forward and reverse primer (10 µM) (Tsingke Company Limited, Qingdao, China), 1 µL template genomic DNA (approximately 1 ng/µL), and 10 µL distilled deionized water. PCR products were visualized through 1% agarose gel electrophoresis. Sequencing was bidirectionally conducted by a company (Tsingke Company Limited, Qingdao, China), and a consensus was obtained using MEGA 7.0 [9]. All sequences generated in this study were deposited in GenBank under the accession numbers listed in Table 2.

Phylogenetic Analyses
Novel sequences were generated from the eight strains in this study, and all available reference sequences of Acrodictys species were downloaded from GenBank [7,10]. No rpb2 sequences are available in GenBank, and only two sequences are available for tub2. Multiple sequences for ITS, LSU, and SSU were aligned and concatenated using an online program (MAFFT v.7.11, http://mafft.cbrc.jp/alignment/server/, accessed on 25 June 2022) [11], with default settings and manually corrected where necessary (Supplementary File S1). To establish the identity of the isolates at a species level, phylogenetic analyses were conducted for each locus and for a combination of all three loci (ITS, LSU, and SSU), based on maximum likelihood (ML) and Bayesian inference (BI). The best evolutionary model for each partition was determined using MrModeltest v. 2.3 [12] and then incorporated into the BI analyses. ML and BI were run through the CIPRES Science Gateway portal (https://www.phylo.org, accessed on 25 June 2022) using RaxML-HPC2 on XSEDE 8.2.12 (Heidelberg, Germany) [13,14] and MrBayes on XSEDE 3.2.7a (Stockholm, Sweden), respectively [15][16][17]. ML was performed using the default parameters, and BI was carried out using the rapid bootstrapping algorithm with the automatic halt option. Bayesian analyses included four parallel runs of 5,000,000 generations and a sampling frequency of 500 generations. The burn-in fraction was set to 0.25, and posterior probabilities (PP) were determined from the remaining trees. The resulting trees were plotted using MF370954 -Notes: New species established in this study are in bold. Ex-type strains are marked with "*".
The Bayesian analysis lasted for 65,000 generations, resulting in 1301 total trees, of which 976 trees were used to calculate the posterior probabilities. The ML tree topology is consistent with that from the BI analyses, and therefore, the ML tree is presented with BI posterior probabilities being plotted together ( Figure 1      Notes-Strains SAUCC 1342 and SAUCC 1343 have similar morphological features and identical DNA sequences and form a monophyletic group with a long branch and robust support values (MLBV = 100% and BIPP = 1.00, Figure 1)   Culture characteristics-Colonies on PDA are flat with an entire margin, attain 27.0-32.0 mm in diameter after 14 days at 25 °C, with a growth rate of 1.5-2.5 mm/d they have white-to-pale-brown aerial mycelia that are floccose cottony; the reverse black. Colonies on MEA are flat with an entire margin, with white-to-gray aerial myc that are floccose cottony; the reverse is black with a pale brown margin.

Key to the Species of Acrodictys
Together with the four new species proposed in this study, we currently accepted a worldwide total of 27 species in the genus Acrodictys. In order to facilitate identification in the future, a key to the species of Acrodictys is provided herein, updating the key compiled 11 years ago [6]. Since then, as many as eight new species have been added to this genus. The characteristics adopted in the updated key include perithecia, septa, asci, ascospores, conidiogenous cells, conidia, and chlamydospores. conidia [2][3][4][5]. Acrodictys as a single genus in Acrodictyaceae was introduced by Xia et al. [7] based on ITS, LSU, SSU, and tub2 sequence data. In previous studies, Acrodictys species have been characterized and identified based on dictyoseptate pigmented conidia seceding schizolytically from monoblastic integrated terminal determinate or lageniform to doliiform percurrently extending conidiogenous cells [1,6]. Xia et al. [7] described eight Acrodictys species, including a new species based on morphology and ITS, LSU, SSU, and tub2 sequence data. This makes it possible for us to study Acrodictys species through molecular systematics. Subsequently, Luo et al. [10] introduced a new species and a new combination for this genus. To date, 10 Acrodictys species have molecular data, viz. A. aquatica, A. bambusicola, A. elaeidicola, A. fluminicola, A. globulosa, A. hainanensis, A. liputii, A. malabarica, A. peruamazonensis, and A. porosiseptata [7,10].
Tropical forest ecosystems offer suitable habitats for microfungi, among which the anamorphic species are the most abundant and diverse. Many anamorphic species have been recorded in rainforests, forest parks, and national nature reserves of Hainan Province, China [23][24][25][26]. As decomposers in the ecosystem, saprobic hyphomycetes can decompose a large amount of litter in tropical rain forests and convert them into inorganic substances, returning them to the soil. During our studies of saprobic hyphomycetes from the national nature reserves of Hainan Province, particularly the regions of the Bawangling National Nature Reserve and Diaoluoshan National Nature Reserve, several collections were made on dead branches. Studies of their morphological characteristics and phylogenetic data revealed four new species. These four species share a feature in conidiogenous cells, namely determinate proliferations. The conidial characteristics provide useful information for species delimitation (Table 3). In this study, we also uploaded rpb2 and tub2 sequence data (Table 2), although few were deposited in GenBank by other experts. All the four new species reported herein are different from one another in these two loci. Table 3. Micromorphological comparison of phylogenetically related Acrodictys species.

Conclusions
Hainan Province has a typical tropical rainforest climate, which is very suitable for the growth and reproduction of saprotrophic fungi. In this study, we chose the Bawangling National Nature Reserve and Diaoluoshan National Nature Reserve as representative sites for sample collection. Through morphological observations and molecular date analyses, we identified four new Acrodictys species, namely A. bawanglingensis, A. diaoluoshanensis, A. ellisii, and A. pigmentosa. The morphological descriptions and molecular data of Acrodictys in this study not only enrich the world's fungal resources and diversity but also contribute materials for studies of the effects of saprobic hyphomycetes on carbon cycling in ecosystems.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/xxx/s1, Supplementary File S1: The combined ITS, LSU, and SSU multiple sequence alignment.

Data Availability Statement:
The sequences from the present study were submitted to the NCBI database (https://www.ncbi.nlm.nih.gov/, accessed on 10 June 2022), and the accession numbers are listed in Table 2.

Conflicts of Interest:
The authors declare no conflict of interest.