Taxonomy, Phylogeny, Divergence Time Estimation, and Biogeography of the Family Pseudoplagiostomataceae (Ascomycota, Diaporthales)

Species of Pseudoplagiostomataceae were mainly introduced as endophytes, plant pathogens, or saprobes from various hosts. Based on multi-locus phylogenies from the internal transcribed spacers (ITS), the large subunit of nuclear ribosomal RNA gene (LSU), partial DNA-directed RNA polymerase II subunit two gene (rpb2), the partial translation elongation factor 1-alpha gene (tef1α), and the partial beta-tubulin gene (tub2), in conjunction with morphological characteristics, we describe three new species, viz. Pseudoplagiostoma alsophilae sp. nov., P. bambusae sp. nov., and P. machili sp. nov. Molecular clock analyses on the divergence times of Pseudoplagiostomataceae indicated that the conjoint ancestor of Pseudoplagiostomataceae and Apoharknessiaceae occurred in the Cretaceous period. and had a mean stem age of 104.1 Mya (95% HPD of 86.0–129.0 Mya, 1.0 PP), and most species emerged in the Paleogene and Neogene period. Historical biogeography was reconstructed for Pseudoplagiostomataceae by the RASP software with a S–DEC model, and suggested that Asia, specifically Southeast Asia, was probably the ancestral area.

The classifications were initially based on phenotype, and with the development of molecular technology, phylogenetic analysis of multi-gene provided reliable evidence for the classifications of phenotype [11][12][13]. However, this has led to significant changes in many lineages, and many unsuitable introductions of secondary ranking. Recently, Hyde et al. [13] used 'temporal banding' to revalued the position of higher taxa in the Ascomycota Caval.-Sm. They believed that the taxa of higher hierarchical levels should be older than lower levels. Thus, 'temporal banding' was regarded as a novel approach, using molecular clock analyses to standardize taxonomic ranking [11,[13][14][15][16][17]. The concept of molecular clock studies is evaluating divergence times of lineages based on the assumption that mutations occur at balanced rate over time, and gradually become a reliable tool to calculate evolutionary events and explore new insights into genetic evolution [18][19][20]. Moreover, Hyde et al. [13] proposed a series of evolutionary periods including, families: 50-150 Mya, orders: 150-250 Mya, subclasses: 250-300 Mya, classes: 300-400 Mya, subphyla: 400-550 Mya, phyla > 550 Mya, and provided recommendations for ranking taxa with evidence for divergence times. The key to draw conclusions from divergence data was stabilize the phylogenetic trees.
In this article, three new species were described by combining phylogeny and morphology, viz. Pseudoplagiostoma alsophilae sp. nov., P. bambusae sp. nov., and P. machili sp. nov. At the same time, a hypothesis for specific divergence time and origin of Pseudoplagiostomataceae was proposed.

Isolation and Morphology
Diseased leaves of Alsophila spinulosa (Wall. ex Hook.) R. M. Tryon, Bambusoideae sp., Machilus nanmu (Oliver) Hemsley were collected from Fujian and Hainan Province during 2021 and 2022 in China. The cultures of Pseudoplagiostomataceae were isolated from diseased and non-diseased tissues of sample leaves using tissue isolation methods [21]. The diseased leaves with obvious disease spots were selected as experimental materials, and the surfaces of the materials were cleaned with sterile deionized water. The leaf samples with typical spot symptoms were first surface sterilized for 30 s in 75% ethanol, then rinsed in sterile deionized water for 45 s, in 2.5% sodium hypochlorite solution for 2 min, then rinsed four times in sterile deionized water for 45 s [22]. The pieces were blotted on sterile filter paper to dry, then transferred onto the PDA flats (PDA medium: potato 200 g, agar 15-20 g, dextrose 15-20 g, deionized water 1 L, pH~7.0, available after sterilization), and incubated at 23 • C for 3-5 days. Hyphal tips were then removed to new PDA flats to gain pure cultures Simultaneously, inoculate on Petri dishes containing pine needle agar (PNA) [23], and incubated at 23 • C under continuous near ultraviolet light to promote sporulation.
After 10-14 days of incubation, morphological characters should be recorded, including graphs of the colonies were taken at the 10th and 14th day using a digital camera (Canon G7X), morphological characters of conidiomata using a stereomicroscope (Olympus SZX10), and micromorphological structures were observed using a microscope (Olympus BX53). All cultures were deposited in 10% sterilized glycerin and sterile water at 4 • C for future studies. Micromorphological structural measurements were taken using the Digimizer software (https://www.digimizer.com/, accessed on 6 December 2022), with 25 measurements taken for each structure [22]. Voucher specimens were deposited in the Herbarium Mycologicum Academiae Sinicae, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China (HMAS), and Herbarium of the Department of Plant Pathology, Shandong Agricultural University, Taian, China (HSAUP). Ex-holotype living cultures were deposited in the Shandong Agricultural University Culture Collection (SAUCC). Taxonomic information of the new taxa was submitted to MycoBank (http://www.mycobank.org, accessed on 6 December 2022).

DNA Extraction and Amplification
Genomic DNA was extracted from fungal mycelia grown on PDA, using a kit (OGPLF-400, GeneOnBio Corporation, Changchun, China) according to the manufacturer's protocol [24]. Gene sequences were obtained from five loci including the internal transcribed spacer regions with the intervening 5.8S nrRNA gene (ITS), the partial large subunit nrRNA gene (LSU), the partial DNA-directed RNA polymerase II subunit two gene (rpb2), the partial translation elongation factor 1-alpha gene (tef1α), and the partial beta-tubulin gene (tub2) were amplified by the primer pairs and polymerase chain reaction (PCR) programs listed in Table 1. Amplification reactions were performed in a 20 µL reaction volume, which contained 10 µL 2 × Hieff Canace ® Plus PCR Master Mix (With Dye) (Yeasen Biotechnology, Cat No. 10154ES03), 0.5 µL of each forward and reverse primer (10 µM) (TsingKe, Qingdao, China), and 1 µL template genomic DNA, adjusted with distilled deionized water to a total volume of 20 µL. PCR amplification products were visualized on 2% agarose electrophoresis gel. DNA Sequencing was performed using an Eppendorf Master Thermocycler (Hamburg, Germany) at the Tsingke Company Limited (Qingdao, China) bi-directionally. Consensus sequences were obtained using MEGA 7.0 [25]. All sequences generated in this study were deposited in GenBank (Table 2).  Lasmenia sp.

Phylogenetic Analyses
Novel sequences obtained in this study and related sets of sequences from Mu et al. [2] were aligned with MAFFT v. 7 and corrected manually using MEGA 7 [33]. Multilocus phylogenetic analyses were based on the algorithms maximum likelihood (ML) and Bayesian inference (BI) methods. The ML was run on the CIPRES Science Gateway portal (https://www.phylo.org, accessed on 6 December 2022) [34] using RaxML-HPC2 on XSEDE v. 8.2.12 [35] and employed a GTRGAMMA substitution model with 1000 bootstrap replicates. Other parameters were default. For Bayesian inference analyses, the best model of evolution for each partition was determined using Modeltest v. 2.3 [36] and included the analyses. The BI was performed in MrBayes on XSEDE v. 3.2.7a [37][38][39], and two Markov chain Monte Carlo (MCMC) chains were run, starting from random trees, for 2,000,000 generations. Additionally, sampling frequency of 100th generation. The first 25% of trees were discarded as burn-in, and BI posterior probabilities (PP) were conducted from the remaining trees. The consensus trees were optimized using

Divergence Time Estimation
An ITS + LSU + rpb2 + tef1α + tub2 sequence dataset with 54 strains was used to infer the divergence times of species in the family Pseudoplagiostomataceae (Figure 2). An XML file was conduct with BEAUti v. 2 and run with BEAST v. 2.6.5. The rates of evolutionary changes at nuclear acids were estimated using MrModeltest v. 2.3 with the GTR substitution model [36,40]. Divergence time and corresponding CIs were taken with a Relaxed Clock Log Normal and the Yule speciation prior. Three fossil time points, i.e., Protocolletotrichum deccanense [41], Spataporthe taylorii [42], and Paleopyrenomycites devonicus [43,44], representing the divergence time at Capnodiales, Diaporthales, and Pezizomycotina were selected for calibration, respectively. The offset age with a gamma distributed prior (scale = 20 and shape = 1) was set as 65, 136, and 400 Mya for Colletotrichum, Diaporthales, and Pezizomycotina, respectively. After 100,000,000 generations, the first 20% were removed as burn in. Convergence of the log file was checked for with Tracer v. 1.7.2 (ESS > 200 was considered convergence). Afterwards, a maximum clade credibility (MCC) tree was integrated with TreeAnnotator v. 2.6.5, and annotating clades with posterior probability (PP) > 0.7.

Inferring Historical Biogeography
The Reconstruct Ancestral State in Phylogenies (RASP) v. 4.2 was used to reconstruct historical biogeography for the family Pseudoplagiostomataceae [45,46]. Maximum clade credibility (MCC) tree, consensus tree, and states were checked with RASP before analysis. Based on the results, we select the Statistical Dispersal-Extinction-Cladogenesis (S-DEC) model. The geographic distributions for Pseudoplagiostomataceae were identified in four areas: (A) Asia, (B) Oceania, (C) South America, and (D) North America.

Phylogenetic Analyses
Alignment contained 25 strains representing Pseudoplagiostomataceae and Apoharknessiaceae, and the strain CBS 243.76 of Nakataea oryzae was used as outgroup. The dataset had an aligned length of 3343 characters including gaps were obtained, viz. LSU: 1-842, ITS: 843-1544, rpb2: 1545-2215, tef1α: 2216-2813, tub2: 2814-3343 (Supplementary File S1). Of these, 2059 were constant, 303 were parsimony-uninformative, and 981 were parsimonyinformative. The ModelTest suggested that the BI used the Dirichlet base frequencies, and the GTR + I + G evolutionary mode for LSU, ITS, and tub2, GTR + I for rpb2, and HKY + G for tef1α. The topology of the ML tree was consistent with that of the Bayesian tree, and, therefore, only shown the topology of the ML tree as a representative for recapitulating evolutionary relationship within the family Pseudoplagiostomataceae. The final ML optimization likelihood was −14,845.00184. The 25 strains were assigned to 18 species clades on the phylogram (Figure 1). Based on the phylogenetic resolution and morphological analyses, the present study introduced three novel species of the Pseudoplagiostomataceae, viz. Pseudoplagiostoma alsophilae sp. nov., P. bambusae sp. nov., and P. machili sp. nov.

Divergence Time Estimation for Pseudoplagiostomataceae
Divergence time estimation (Figure 2) showed that Pseudoplagiostomataceae occurred early with a mean stem age of 104.1 Mya [95% highest posterior density (HPD) of 86.0-129.0 Mya, 1.0 PP], and a mean crown age of 91.6 Mya (95% HPD of 73.4-117.6 Mya, 0.9 PP), which was consistent with a previous study [13]. The clade of Pseudoplagiostoma eucalypti and P. oldii with a mean stem age of 10.7 Mya (95% HPD of 4.9-20.9 Mya), and a mean crown age of 4.6 Mya (95% HPD of 1.5-9.7 Mya), which was consistent with previous studies [47]. While the clade of Pseudoplagiostoma eucalypti and P. oldii evolved most recently, the clade of P. myracrodruonis and P. castaneae diverged the earliest in the genus with a stem age of 68.1 Mya (95% HPD of 39.7-98.8 Mya). The stem/crown age of other species are shown in Table 3. Table 3. Inferred divergence time of species in the genus Pseudoplagiostoma.
Currently, the divergence and ranking of taxa across the kingdom Fungi, especially the phylum Ascomycota, have significant theoretical and practical significance, and gradually become a reliable and referential evidence before introducing new higher taxa [11,[13][14][15][16][17]. Our analysis of molecular clock indicates that Pseudoplagiostomataceae was closely related to Apoharknessiaceae, which was most deeply diverged during the Paleogene, with a mean stem age of 104.1 Mya (95% HPD of 86.0-129.0 Mya), and full supports (1.0 PP, Figure 2 and Table 3). Even though Hyde et al. [13] only included two species of the Pseudoplagiostomataceae, its divergence time was coincided with this study. In the present study, a mean stem age of Diaporthales reached 188.2 Mya and was fully supported earlier than in the previous study [13,47]. Therefore, both new fossil findings and new species findings have an impact on the divergence time of the orders. Of course, the impact was controllable, and it must be in certain evolutionary periods.
Macrofungi have been widely applied for biogeographical analyses [24,[48][49][50][51]. Our study suggested that the species distribution and speciation of Pseudoplagiostomataceae had a particular biogeographical pattern, and these species appeared to originate in Asia, particularly in Southeast Asia. Previous studies suggested that the Indian continent collided with the Eurasian continent at~60 Mya, which was consistent with some speciation of the Pseudoplagiostomataceae, and formed the Hengduan-Himalayan area which was a global biodiversity hotspot [52][53][54][55][56][57]. Based on the discovered specimens and biogeographical information, this study is more inclined to explain that Pseudoplagiostomataceae species originated in Asia and spread to Hawaii and South America through Malaysia, Australia, New Zealand, and more than 20,000 independent islands in the South Pacific, and frequent hurricanes and circulating ocean currents in the South Pacific are the best spore carriers. The humid climate in the southern hemisphere and the rich tropical host plants, such as Quercus sp. and Eucalyptus sp., are also suitable for the reproduction and evolution of Pseudoplagiostomataceae species [58,59]. Dispersal, vicariance, and extinction of species may be related to the Indian continent collided with the Eurasian; however, this claim needs more species and fossil evidence to support it.

Institutional Review Board Statement:
Not applicable for studies involving humans or animals.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The sequences from the present study were submitted to the NCBI database (https://www.ncbi.nlm.nih.gov/, accessed on 6 December 2022) and the accession numbers were listed in Table 2.