Taxonomy, Phylogenetic and Ancestral Area Reconstruction in Phyllachora, with Four Novel Species from Northwestern China

The members of Phyllachora are biotrophic, obligate plant parasitic fungi featuring a high degree of host specificity. This genus also features a high degree of species richness and worldwide distribution. In this study, four species occurring on leaf and stem of two different species of grass were collected from Shanxi and Shaanxi Provinces, China. Based on morphological analysis, multigene (combined data set of LSU, SSU, and ITS) phylogenetic analyses (maximum likelihood and Bayesian analysis), and host relationship, we introduce herein four new taxa of Phyllachora. Ancestral area reconstruction analysis showed that the ancestral area of Phyllachora occurred in Latin America about 194 Mya. Novel taxa are compared with the related Phyllachora species. Detailed descriptions, illustrations, and notes are provided for each species.

Currently, Phyllachorales has four families, including Phaeochoraceae, Phaeochorellaceae, Phyllachoraceae, and Telimenaceae [12,13]. They are morphologically characterized by black stromata of various shapes in the host plant; having paraphyses; unitunicate asci cylindrical to clavate in shape, with an inconspicuous apical ring, usually 8-spored; and aseptate ascospores, which in most species are hyaline and 1-celled, appearing as brown in a few species (e.g., Phyllachora stenostoma) [1,4,6,8,14]. The asexual morph of Phyllachorales has been reported as a coelomycetous morph [15]. Large-scale phylogenetic studies comprising many representative species have confirmed the position of Phyllachorales in the subclass Sordariomycetidae with high support (100% MLBP) as well as the monophyly of the order [4,8,16]. Mardones et al. [8] used three morphological characteristics and one ecological characteristic to reconstruct the ancestral state of genera in Phyllachorales based on the Likelihood Ancestral States method, reasoning that these characteristics had evolved independently numerous times. The ancestral state of members of Phyllachorales were monocotyledonous host plants with immersed perithecia, which was lost in the family Phaeochoraceae and evolved into erumpent or superficial perithecia in some species of Phyllachoraceae. The presence of clypeus as a morphological characteristic was lost only once in Phaeochoraceae. Therefore, it is thought that the presence of clypeus in these fungi is an evolutionarily stable characteristic. The ancestor of the Phyllachorales species had a black stroma, and the presence of bright black stromata may have evolved at least twice.
The family Phyllachoraceae was introduced by Theissen and Sydow [17] with Phyllachora as the type genus [3,18]. It is the largest family in Phyllachorales and currently comprises 54 genera [13]. Members of the family are characterized by forming leaf spots on the host that are abundant but scattered, raised, mostly rounded to oblong or elongated, sometimes parallel with leaf venation, surrounded by a light-brown necrotic region; lacking periphyses; having numerous paraphyses, branched or unbranched; 8-spored asci, persistent, cylindrical to fusiform, often present with an apical ring; ascospores fusiform to narrowly oval, hyaline, often with a mucilaginous sheath [4]. The type genus, Phyllachora, was introduced based on P. agrostis, which is a single species on the herbarium label in Fuckels exsiccate series 'Fungi Rhenani' [5]. Phyllachoraceae is similar to Phaeochoraceae, but Phaeochoraceae species are characterized by 6-8-spored asci, usually without apical structure, yellow to olivaceous ascospores or in various shades of brown, thick-walled; conversely, Phyllachoraceae species are characterized by 8-spored asci, an often-present an apical ring, usually hyaline ascospores, rarely pale brown, thin and smooth-walled [4,8]. These morphological characteristics can be used to distinguish the two families, and they form two independent branches in the phylogenetic tree [8].
Phyllachora is the type genus of Phyllachoraceae. Clements [19] designated the lectotype as Phyllachora graminis. Currently, Phyllachora is the largest genus within Phyllachoraceae, and about 1513 epithets are listed in the Index Fungorum (Index Fungorum 2022; accession date: 28.03.2022). Nevertheless, only 1382 species are accepted in the Species Fungorum (accession date: 28.03.2022). Species of the genus are morphologically characterized by clypeate pseudostroma in leaf tissues; generalized infection of the entire section of the mesophyll forming leaf spots on the host, mostly rounded to oblong or elongated, surrounded by a light-brown necrotic region; perithecium globose; numerous paraphyses, branched, slightly longer than asci; asci 8-spored, persistent, cylindrical to fusiform, short pedicellate, an apical ring often present; and ascospores 1-3 seriate, fusiform to narrowly oval, hyaline, sometimes with a gelatinous sheath [4,18,20]. Some members of the genus can inflict crop diseases, leading to yield loss. Phyllachora maydis is an example occurring in the United States, which can seriously impact quality and corn yield [21][22][23][24]. Owing to its biotrophic habit and high degree of host specificity, most Phyllachora species are given names based on host association and coevolution with the host [5,8,20,25]. Phyllachora species cannot grow on agar media since they are biotrophic [8]. Phyllachora species have been reported as pathogenic species on more than 1000 plant species (belonging to 121 families, including Cyperaceae, Fabaceae, Lauraceae, Moraceae, Myrtaceae, Poaceae, Proteaceae, and Rosaceae), and they are commonly found with Poaceae [20,26,27].
In this study, several specimens with tar spot diseases were collected. Based on polyphasic approaches (e.g., morphological analyses, information of host plant, and phylogenetic analyses), four novel species of Phyllachora are introduced herein. Based on paleontological evidence and paleoclimate records, we also reconstructed the ancestral area of Phyllachora. The analysis was restricted to members of Phyllachora, considering the history of their biogeographic diversity and dispersal route as well as estimating the divergence time and ancestral location of this genus.

Collecting, Morphological Study, and Depositing Specimens
Phyllachora-like fungi were collected from living leaves of Cenchrus flaccidus (Poaceae) and Chloris virgata (Poaceae) during field surveys in 2019 in Shanxi and Shaanxi Provinces, China. Specimens were taken to the laboratory in paper envelopes. Specimens were processed and examined with microscopes, and photos of ascomata and host were taken using a compound stereomicroscope (KEYENCE CORPORATION V.1.10 with camera VH-Z20R) following Wu et al. [28]. Hand sections were made under a stereomicroscope (OLYMPUS SZ61) and mounted in water and blue cotton, and photomicrographs of fungal structures were taken with a compound microscope (Nikon ECLIPSE 80i).
Images For our specimens, no culture was obtained by multiple single-spore isolation or tissue isolation.

DNA Isolation, Amplification, and Sequencing
In accordance with the manufacturer's instructions, genomic DNA was extracted from ascomata at room temperature using the Forensic DNA Kit (OMEGA, New York, NY, USA). The primers LR0R and LR5 were used to amplify the large subunit (LSU) rDNA [29]. The internal transcribed spacer (ITS) rDNA was amplified and sequenced with the primers ITS5 and ITS4 [30]. The partial small subunit (SSU) rDNA was amplified using primers NS1 and NS4 [30]. PCR reactions were in accordance with instructions from Golden Mix, Beijing TsingKe Biotech Co. Ltd, Beijing, China: initial denaturation at 98 • C for 2 min, followed by 30 cycles of 98 • C denaturation for 10 s, 56 • C annealing for 10 s and 72 • C extensions for 10 s (ITS and SSU) or 20 s (LSU), and a final extension at 72 • C for 1 min. All PCR products were sequenced by Biomed (Beijing Biomed Gen Technology Co., Ltd., Beijing, China). PCR products were sequenced by Biomed using the same primers as before.
Maximum Likelihood (ML) analysis using the aligned sequences as input was conducted with the help of RAxNLGUI v. 2.0 [35]. Telimena bicincta (MM-108) and T. bicincta (MM-133) were selected as an outgroup. One thousand nonparametric bootstrap iterations were employed with the "ML + rapid bootstrap" tools and "GTRGAMMA" arithmetic.
For Bayesian analysis, MrModeltest 2.3 [36] was used to estimate the best-fitting model for the combined LSU, SSU, and ITS loci, and model GTR+G was the best fit. In MrBayes v.3.2 [37], six simultaneous Markov chains were run for 2,000,000 generations; trees were sampled and printed every 100 generations. The first 5000 trees were submitted to the burn-in phase and discarded, while the remaining trees were used for calculating posterior probabilities in the majority rule consensus tree [38][39][40][41].

Reconstruction of Ancestral State
Members of Phyllachora were coded based on their collection locality according to field notes and references. Six areas were delimited based on the distribution data of Phyllachora: A = East Asia, B = Southeast Asia, C = North America, D = South America, E = Latin America, F = Central Europe, G = Unknown, using species from Asia, Europe, North America, South America, Latin America, and Central Europe. In MrBayes v.3.2, chains were run for 1 00000 generations; trees were sampled and printed every 100 generations. RASP 4.2 (Reconstruct Ancestral State in Phylogenies, http://mnh.scu.edu.cn/soft/ blog/RASP, accession date: 10 April 2022) was used to reconstruct the ancestral state, and the most-optimal model was DEC [42].

Calibration Procedure
The second calibration time referenced the results of Dayarathne et al. [33] and Hongsanan et al. [43]. We followed the conclusion that the family Phyllachoraceae divergence time was about 217 Mya as a calibration point (root) for ancestral distribution reconstruction.

Molecular Phylogenetic Results
We analyzed a three-loci (LSU, SSU, ITS) data set of Phyllachora. Based on the combined data of LSU, SSU, and ITS sequences. It was found that the two topological trees obtained by maximum likelihood (ML) and Bayesian were similar, and the best scoring RAxML tree was used as the representative tree ( Figure 1). We generated a total of 161 sequences from 74 taxa of Phyllachorales, 57 sequences of LSU, 34 sequences of SSU, 70 sequences of ITS, and concatenated sequences of three genes, with 3341 characters including gaps. Bootstrap values of ML higher than 50% are shown on the phylogenetic tree, while values of Bayesian posterior probabilities above 0.5 are shown on the tree (Figure 1). Phylogenetic analysis showed that all four new taxa belonging to Phyllachora cluster together with Phyllachora panicicola with bootstrap values of 68% (in ML analysis) and Bayesian posterior probability of 0.92. Phyllachora panicicola and four new taxa form two clades independent from each other with bootstrap values of 100% and Bayesian posterior probabilities of 1.00.

Ancestral Area Reconstruction Analysis for Phyllachora
Ancestral area reconstruction analysis revealed that Phyllachora species originated from Latin America about 194 Mya (Figure 2, node 145). Dispersal, vicariance, extinction, and other historical events affected the biogeographical distribution of the species. The evolutionary history of ancestors from the genus Phyllachora reveals that the species of this genus underwent 20 dispersals, 13 vicariances, and 1 extinction (Figure 2, blue coils represent dispersal, green coils represent vicariance, and orange coils represent extinction).

Ancestral Area Reconstruction Analysis for Phyllachora
Ancestral area reconstruction analysis revealed that Phyllachora species originated from Latin America about 194 Mya (Figure 2, node 145). Dispersal, vicariance, extinction, and other historical events affected the biogeographical distribution of the species. The evolutionary history of ancestors from the genus Phyllachora reveals that the species of this genus underwent 20 dispersals, 13 vicariances, and 1 extinction (Figure 2, blue coils represent dispersal, green coils represent vicariance, and orange coils represent extinction). Species of Phyllachora migrated from Latin America to Southeast Asia during the Jurassic period, with two dispersal events noted (Figure 2, node 127). In approximately 60-155 Mya, there were frequent dispersal and vicariance events, and moreover, vicariances were always accompanied by dispersal events. There is only low support suggesting that species belonging to Phyllachora may have migrated from Latin America to Southeast Asia 119 Mya ( Figure 2, node 108). About 100 Mya, species migrated from East Asia or North America to central Europe, with one dispersal and one vicariance (Figure 2, node 77).
Species of Phyllachora migrated from Latin America to Southeast Asia during the Jurassic period, with two dispersal events noted (Figure 2, node 127). In approximately 60-155 Mya, there were frequent dispersal and vicariance events, and moreover, vicariances were always accompanied by dispersal events. There is only low support suggesting that species belonging to Phyllachora may have migrated from Latin America to Southeast Asia 119 Mya (Figure 2, node 108). About 100 Mya, species migrated from East Asia or North America to central Europe, with one dispersal and one vicariance (Figure 2, node 77).
The hosts of Phyllachora sphaerosperma (= Phyllachora cenchricola), P. flaccidudis and P. sandiensis belong to species of Cenchrus. However, the host of P. cenchricola is Cenchrus echinatus, which has been found in Brazil, the southern United States, South America, and the West Indies. The ascospores are nearly spherical, wider than both P. flaccidudis and P. sandiensis ( Table 2).
Phyllachora flaccidudis and P. chloridis [33] have similar morphological characteristics, but their host plants are different. The host of P. flaccidudis and P. sandiensis was reported from Cenchrus flaccidus (Poaceae), while the host of P. chloridis is Chloris sp. Morphologically, the asci of P. flaccidudis and P. sandiensis are significantly longer than those of P. chloridis ( Table 2) and feature pedicels, but they are absent in P. chloridis. Ascospores of P. flaccidudis and P. sandiensis have 1-2 or more guttules, while P. chloridis has only one central guttule, which can serve as an important characteristic for species delimitation.

Discussion
In this study, we introduced four new taxa of Phyllachora (P. flaccidudis, P. sandiensis, P. virgatae, and P. jiaensis) that have morphological characteristics typical of Phyllachora: black leaf spots, peridium clypeate, multilocular, asci cylindrical, an unobvious apical ring, shortly pedicellate, numerous paraphyses and slightly longer than asci, and aseptate ascospores with guttules [4,33,50]. All novel taxa were introduced based on morphological characteristics and novel phylogenetic lineages in Phyllachora (Figure 1). We compared the morphological characteristics of the four new species and similar Phyllachora taxa ( Table  2).
Phylogenetically, P. virgatae and P. jiaensis clustered together with high bootstrap support and probability value (100/1.0), with P. jiaensis forming a long branch. The LSU, SSU, and ITS loci differ by 8 bases, 109 bases, and 3 bases, respectively. Phylogenetically, P. virgatae grouped with P. jiaensis to form one clade, and P. flaccidudis with P. sandiensis to form another clade, with high bootstrap and probability values (100/1.0), but they occur on different hosts. P. virgatae and P. jiaensis both occur on Chloris virgata, and the host of P. flaccidudis and P. sandiensis is Cenchrus flaccidus. The ascospores of P. flaccidudis and P. sandiensis are acute at one end and blunt at the opposite end, while the ascospores of P. virgatae and P. jiaensis are blunt at both ends.
The four new species described herein have ascospores with gelatinous sheaths that differ in black ink (Figure 4). The gelatinous sheaths of P. flaccidudis and P. sandiensis are larger than in P. virgatae and P. jiaensis. Hence, based on both morphological and phylogenetic evidence, we introduce the novel species, P. jiaensis.

Discussion
In this study, we introduced four new taxa of Phyllachora (P. flaccidudis, P. sandiensis, P. virgatae, and P. jiaensis) that have morphological characteristics typical of Phyllachora: black leaf spots, peridium clypeate, multilocular, asci cylindrical, an unobvious apical ring, shortly pedicellate, numerous paraphyses and slightly longer than asci, and aseptate ascospores with guttules [4,33,50]. All novel taxa were introduced based on morphological characteristics and novel phylogenetic lineages in Phyllachora (Figure 1). We compared the morphological characteristics of the four new species and similar Phyllachora taxa (Table 2).
Yang et al. [20] introduced Phyllachora heterocladae from Sichuan Province, and the phylogenetic tree was artificially divided into five lineages based on the host plants. Most species of Phyllachora that cluster within lineage I are graminicolous (Poaceae), but P. qualeae grows on Qualea multiflora (Vochysiaceae). They formed a distinct subclade with P. arundinellae (MHYAU:108), P. cynodontis (MHYAU:20043), and P. imperatae (MHYAU:014). Species within lineage II and lineage IV are bambusicolous fungi. Lineage III is solely composed of P. thysanolaenae (MFLU , which is an unstable species in the phylogeny. Lineage V contains only P. pomigena, associated with an unknown host plant. Li et al. [32] introduced two new species, P. dendrocalami-membranacei and P. dendrocalami-hamiltonii, and phylogenetic analysis was generated four main clades. Lineage I consisted of all Phyllachora species obtained from the subfamily Agrostidoideae of the Poaceae, except for Polystigma pusillum (MM-19), which was found growing on Fabaceae. Neophyllachora species occurred in the family Myrtaceae within Lineage II. Lineage III and Lineage IV are Phyllachora species collected from the subfamily Bambusoideae of the Poaceae. However, it is important to note that Yang et al. [20] did not include all Phyllachora species in their analysis.
In this study, the generated phylogenetic tree comprises 74 species belonging to six genera (viz., Ascovaginospora, Camarotella, Coccodiella, Neophyllachora, Phyllachora, and Polystigma). We found that the Phyllachora genus is paraphyletic. Because the host of P. pomigena remains unknown, the species Phyllachora pomigena formed a single clade [20,51]. In the phylogenetic analysis, the new species described herein are included within the Phyllachora genus and separated from other taxa with a single subclade. Their hosts are Cenchrus flaccidus and Chloris virgata, both belonging to Poaceae (graminicolous).
The study revealed that the ancestor of Phyllachora species originated from Latin America. Phyllachora species ancestors initially spread from Latin America to North America, East Asia, South America, and eventually to Central Europe. The characteristic of Phyllachora species in Latin America are consistent with the ancestral characteristics of Phyllachora genus found in Mardones et al. [8]. For example, existing species P. maydis and P. graminis still retain ancestral characteristics, such as growing on monocotyledonous hosts, immersed perithecia, black stromata, and the presence of clypeus [8]. Reconstruction analysis of ancestral location indicates that a vicariance event (i.e., the splitting of the range of a taxon or biota into two or more geographical subdivisions by the formation of natural barriers, for example, mountain building, glaciation, plate tectonics or climate change) affected speciation allowing some species to retain ancestral morphological characteristics [52]. The appearance of Polystigma could have resulted from the extinction event (Figure 2 node 143). The extinction event may have resulted in the host of Polystigma species shifting from monocotyledons to dicotyledons (Fabaceae).
During the Cretaceous geological upheaval, orogeny, continental drift as well as the emergence of the Atlantic and the Indian Ocean led to dramatic terrestrial climate changes across the earth's surface [53]. These led to the mass extinction of the dominant Mesozoic gymnosperm and ferns in the tropics, subtropical plains, and low mountains areas, which were replaced by angiosperms (the origin of Poaceae) that flourished in the Paleogene [54]. The emergence of angiosperms may have triggered the evolution and migration of the ancestors of the Phyllachora fungi.
There are few studies examining the co-evolution and ancestral state reconstruction of Phyllachora species; this is because of the scarcity of existing species with high-quality molecular data, which adds uncertainty to the process of ancestral state reconstruction. Extensive sampling and high-quality molecular data will reveal more accurate changes in the ancestral status of species in this group. Ancestor state reconstruction currently requires inferring phenotypes of ancestral species using observations from present-day species [55,56]. As new classical and molecular methods for identifying fungi continue to develop [57], ancestor state reconstruction analysis of fungal taxonomy is at the forefront of a new trend [8,[58][59][60]. Future studies on species diversity and evolution of Phyllachora species require more extensive sampling and high-quality molecular data.  Data Availability Statement: All sequence data are available in NCBI GenBank following the accession numbers in the manuscript. All species data are available in MycoBank.