Six Additions to the Genus Periconia (Dothideomycetes: Periconiaceae) from Graminaceous Plants in China

Periconia is a polyphyletic and asexual morphic genus within the family Periconiaceae (Pleosporales). The genus is characterized by a pale to dark brown stipe with an apical conidial head and ellipsoidal to oblong conidia. Species of Periconia are widely distributed throughout the world in various hosts, while most species are isolated from graminaceous plants. During our investigations of microfungal in Sichuan Province, China, 26 Periconia isolates were collected from a wide variety of graminaceous plants. These isolates corresponded to 11 species based on the examination of morphology and multi-locus phylogenetic analysis (SSU, ITS, LSU, TEF1, RPB2). This includes six new species (P. chengduensis, P. cynodontis, P. festucae, P. imperatae, P. penniseti, and P. spodiopogonis) and five new records (P. byssoides, P. chimonanthi, P. cookie, P. pseudobyssoides, and P. verrucosa). A comprehensive description and illustrations of the new species are provided and discussed with comparable taxa. These discoveries expand our knowledge of the species diversity of Periconia taxa in graminaceous plants in China.

Periconia is a polyphyletic genus in the family Periconiaceae [21]. The members of this genus were previously classified under Massarinaceae. Based on phylogenetic analysis, Tanaka et al. [7] showed that Periconiaceae is a sister clade distinct from Massarinaceae [22]. Phukhamsakda et al. [23] showed that Periconiaceae and Massarinaceae diverged in the

Phylogenetic Analyses
The raw sequencing fragments of corresponding Sanger sequencing chromatograms were manually edited, trimmed, and assembled into consensus sequences using SeqMan Pro version 11.1.0 (DNASTAR, Inc. Madison, WI, USA). Barcode sequences of Periconia species currently available in GenBank and the outgroup taxon Massarina cisti (CBS 266. 62) were downloaded from the NCBI nucleotide database using an in-house python script. Table 1. Loci used in this study with the corresponding PCR primers and conditions. (72 • C: 5 min) × 1 cycles [44,45] The multiple sequence alignment was conducted using MAFFT version 7.310 [46] with options "-maxiterate 1000 -genafpair -adjustdirectionaccurately", and the alignment results were further trimmed using trimAl version 1.4 [47] with the option "-gapthreshold 0.5", which only allows 50% of taxa with a gap in each site. The best-fit nucleotide substitution models for each alignment dataset were selected using PartitionFinder version 2.1.1 [48] under the Akaike Information Criterion (AIC).

ITS
Maximum likelihood (ML) and Bayesian analysis (BI) were conducted based on the individual and combined datasets. ML phylogenetic trees were obtained using the IQ-TREE version 2.0.3 [49], and the topology was evaluated using 1000 ultrafast bootstrap replicates. The BI was conducted using parallel MrBayes version 3.2.7a [50]. Two different runs with 20 million generations and four chains were executed, and the initial 25% of sample trees were treated as burn-in. Tracer version 1.7.1 [51] was used to confirm that the MCMC runs reached convergence with all ESS values above 200. Then, the ML tree was annotated by TreeAnnotator version 2.6.6 implemented in BEAST version 2.6.6 [52] based on MrBayes MCMC trees with no discard of burn-in and no posterior probability limit. The tree was visualized using ggtree [53] and edited in Adobe Illustrator version 20.0.0.
The best-scoring ML consensus tree (lnL = −20,240.300) with ultrafast bootstrap values from ML analyses and posterior probabilities from MrBayes analysis at the node is shown in Figure 1. Phylogenetic analyses showed that our newly collected 26 isolates clustered into 11 clades and can be recognized as 5 known species (P. byssoides, P. chimonanthi, P. cookie, P. pseudobyssoides, and P. verrucosa) and six new species (P. chengduensis, P. cynodontis, P. festucae, P. imperatae, P. penniseti, and P. spodiopogonis).  The newly generated sequences are indicated in red, and the ex-type strains are in bold. Missing sequences are indicated by "-".

Taxonomy
Periconia byssoides Pers., Syn. meth. fung. (Göttingen) 2: 686 (1801) Figure 2.  HUEST 22.0140) share similar morphological characteristics in shape and color of conidiophores and conidia with the type of P. byssoides. Therefore, we identified our new isolates as P. byssoides based on the overlapping morphological characteristics and the multi-locus phylogenetic tree, and one collection (HUEST 22.0133) is a new host record from Imperata cylindrica.

MycoBank: MB 847458
Etymology: Name refers to Chengdu, the city where the fungus was collected.
Notes: The phylogenetic tree shows the isolates UESTCC 22.0126, UESTCC 22.0140, UESTCC 22.0141, UESTCC 22.0142, and UESTCC 22.0143 form a clade sister to the isolates P. chimonanthi including the ex-type KUMCC 20-0266 (100% ML, 1.00 PP; Figure 1). Periconia chimonanthi was introduced based on the collection from decaying branches of Chimonanthi praecox (Calycanthaceae) in China [8]. Our collections have similar morphological characteristics in the shape of conidiophores and conidia with the P. chimonanthi on natural substrate. However, P. chengduensis differs from P. chimonanthi in having shorter conidiophores (240-370 µm vs. 410-635 µm) [8]. Furthermore, the culture of P. chimonanthi is black on the reverse side of PDA media; however, P. chengduensis (UESTCC 22.0126) is generally pale yellow on the reverse [8]. Thus, considering the difference in morphological characteristics and phylogenetic analysis, we describe the isolates (   (c,d) conidiophores with spherical conidial heads; (e,f) apically branch conidiophores with conidial head; (g-l) conidia; (m) germinating conidium; (n,o) colony on PDA from above and below. Scale bars: (c) = 100 µm, (e,g,m) = 10 µm. Scale bar of (c) applies to (d). Scale bar of (e) applies to (f). Scale bar of (g) applies to (h-l).  .0144) clustered with other P. chimonanthi isolates, including type isolate (KUMCC 20-0266), that were introduced from decaying branches of Chimonanthi praecox (Calycanthaceae) in China [8]. Our two isolates display similar and overlapping morphological characteristics with the holotype of P. chimonanthi [8]. We identified our two collections (UESTCC 22.0133, UESTCC 22.0144) as P. chimonanthi, and this is the first report of P. chimonanthi isolated from Arundo donax and Imperata cylindrica.  (c,d) conidiophores with spherical conidial heads; (e,f) conidial heads bearing conidiogenous cells and conidia; (g-k) conidia; (l) germinating conidium; (m,n) colony on PDA from above and below. Scale bars: (c) = 100 µm, (e,g,i) = 10 µm. Scale bar of (c) applies to (d). Scale bar of (e) applies to (f). Scale bar of (g) applies to (h-k). Culture characteristics: Colony on PDA reaching 55 mm diam after 19 days in an incubator under dark conditions at 20 • C, oval almost circular, cottony, hairy at the margin, white at the margin, and gray-green at the middle; reverse: white at the margin and mud yellow to black toward the center.
Notes: Periconia cookei was introduced by Mason and Ellis [33] based on the morphology, characterized by conidiophores that are unbranched, septate, pale brown to dark brown, polyblastic and 13-16 µm diameter, catenate, verrucose, brown, mostly spherical conidia. The phylogenetic tree showed that our isolate UESTCC 22.0134 from the dead culms of Digitaria sanguinalis clustered with the isolate of P. cookie MFLUCC 17-1679 [54]. Thus, we identified the isolate UESTCC 22.0134 as a P. cookie. This is the first report of P. cookie occurring on Digitaria sanguinalis in Sichuan Province, China.  (k) germinating conidium; (l,m) colony on PDA from above and below. Scale bars: (c) = 100 µm, (e,g,k) = 10 µm. Scale bar of (c) applies to (d) Scale bar of (e) applies to (f). Scale bar of (g) applies to (h-j).
Notes: Periconia cookei was introduced by Mason and Ellis [33] based on the morphology, characterized by conidiophores that are unbranched, septate, pale brown to dark brown, polyblastic and 13-16 µm diameter, catenate, verrucose, brown, mostly spherical conidia. The phylogenetic tree showed that our isolate UESTCC 22.0134 from the dead culms of Digitaria sanguinalis clustered with the isolate of P. cookie MFLUCC 17-1679 [54]. Thus, we identified the isolate UESTCC 22.0134 as a P. cookie. This is the first report of P. cookie occurring on Digitaria sanguinalis in Sichuan Province, China.

MycoBank: MB 847465
Etymology: Name reflects the host genus, Cynodon, from which the fungus was collected.
Notes: The phylogenetic tree shows that the isolate Periconia cynodontis UESTCC 22.0127 formed a distinct clade within Periconia. Periconia cynodontis resembles P. lateralis in having curved conidiophores, conidiogenous cells directly formed in the middle part of the conidiophores, and solitary or catenate, globose, brown to dark brown conidia [55,56]. However, P. cynodontis differs from P. lateralis by having smaller conidia (7.5-12.5 µm vs. 10.5-15 µm) [55]. Periconia cynodontis differs from P. penniseti and P. neobrittanica by conidiogenous cells that are directly formed in the middle part of the conidiophores [57]. Thus, considering the difference in morphological characteristics and phylogenetic analysis, we describe the isolate UESTCC 22.0127 as Periconia cynodontis sp. nov. conidiophores; (f,g) conidiophores bearing conidia laterally; (h-j) conidia; (k) germinating conid ium; (l,m) colony on PDA from above and below. Scale bars: (d) = 20 µm, (f,h,k) = 10 µm. Scale bar of (d) applies to (e). Scale bar of (f) applies to (g). Scale bar of (h) applies to (i,j).  (d,e) conidiophores; (f,g) conidiophores bearing conidia laterally; (h-j) conidia; (k) germinating conidium; (l,m) colony on PDA from above and below. Scale bars: (d) = 20 µm, (f,h,k) = 10 µm. Scale bar of (d) applies to (e). Scale bar of (f) applies to (g). Scale bar of (h) applies to (i,j). Notes: The isolate UESTCC 22.0128 is grouped in a well-supported clade and appears to be phylogenetically distinct (98% ML, 0.95 PP; Figure 1) from other sister species. Our collection HKAS 126516 shares similar morphological characteristics in the shape and color of conidiophores and conidia with the P. cookie and P. verrucosa on the natural substrate [54]. However, it differs from P. cookie and P. verrucosa by having significantly wider conidiophores (10.5-20 µm vs. 8.5-14.5 µm and 10.5-15 µm) [34]. The culture of P. cookie is dark green to black on the reverse side of PDA media. However, P. festucae is yellow on the reverse. Periconia festucae and P. verrucosa differ in the number of septa on conidiophores (4-8-septate vs. 2-4-septate). Considering the significant differences in morphology and molecular data, we introduce the isolate UESTCC 22.0128 as a new species.  (d,e) conidiophores with spherical conidial heads; (f,g) apical branch conidiophores with conidial head; (h-l) conidia; (m) germinating conidium; (n,o) colony on PDA from above and below. Scale bars: (d) = 100 µm, (f,h,m) = 10 µm. Scale bar of (d) applies to (e). Scale bar of (f) applies to (g). Scale bar of (h) applies to (i-l). Culture characteristics: Colony on PDA reaching 47 mm diam after 19 days in an incubator under dark conditions at 20 • C, oval to almost circular, cottony, hairy and white at the margin, and pale yellow to white at the middle; in reverse yellow at the margin and mud yellow at the middle.

Periconia festucae
Notes: The isolate UESTCC 22.0128 is grouped in a well-supported clade and appears to be phylogenetically distinct (98% ML, 0.95 PP; Figure 1) from other sister species. Our collection HKAS 126516 shares similar morphological characteristics in the shape and color of conidiophores and conidia with the P. cookie and P. verrucosa on the natural substrate [54]. However, it differs from P. cookie and P. verrucosa by having significantly wider conidiophores (10.5-20 µm vs. 8.5-14.5 µm and 10.5-15 µm) [34]. The culture of P. cookie is dark green to black on the reverse side of PDA media. However, P. festucae is yellow on the reverse. Periconia festucae and P. verrucosa differ in the number of septa on conidiophores (4-8-septate vs. 2-4-septate). Considering the significant differences in morphology and molecular data, we introduce the isolate UESTCC 22.0128 as a new species.

MycoBank: MB 847467
Etymology: Name after the host genus from which the fungus was isolated, Imperata. Saprobic dead leaves and culms of Poaceae. Asexual morph: Colonies on the natural substrate numerous, effuse, dark brown to black, floccose. Conidiophores 280-520 µm long (x= 378, n = 15), 10-16.5 µm wide (x = 12.5 µm, n = 15), macronematous, mononematous, straight or slightly flexuous, branched, solitary, rarely 1-2 together on stroma, brown to dark brown, 3-7-septate, smooth to minutely verruculose, thick-walled. Conidiogenous cells polyblastic, pale brown to brown, terminal, integrated or discrete, oval to subglobose, smooth to verruculose. Culture characteristics: Colony on PDA reaching 58 mm diam after 11 days in an incubator under dark conditions at 20 • C, circular, cottony, hairy and white at the margin and yellow toward the middle; yellow at the margin and yellow to brown at the middle in reverse; producing yellow pigments on PDA.
MycoBank: MB 804763 Scale bar of (e) applies to (f). Scale bar of (g) applies to (h,i). Scale bar of (j) applies to (k,l). Culture characteristics: Colony on PDA 23 mm diam after 2 weeks in an incubator under dark conditions at 20 • C, irregular circular, cottony, hairy at the margin, colonies from above white grayish to creamy white; in reverse, white at the margin and pale yellow to khaki at the middle.
Notes: The phylogenetic tree shows that the isolate UESTCC 22.0130 clustered with the ex-type strain of P. neobrittanica (CBS 146062), which was isolated from leaves of Melaleuca styphelioides (Myrtaceae) in California, USA [57]. Periconia penniseti can be distinguished from P. neobrittanica in having longer conidiophores (285-540 µm vs. 100-300 µm) and that are more septate (2-5 vs. 0-1) [57]. Periconia penniseti differs from P. cynodontis in the position of conidiogenous cells. In P. penniseti, the conidiogenous cells arise from the apical part of the conidiophores, while in P. cynodontis, the conidiogenous cells arise from the middle part of the conidiophores. Thus, considering the difference in morphological characteristics and phylogenetic analysis, we describe the isolate UESTCC 22.0130 as a new species.
Periconia pseudobyssoides Markovsk. and A. Kačergius, Mycol. Progr. 13 (2): 293 (2014) Figure 10. Culture characteristics: Colony on PDA reaching 46 mm diam after 10 days in an incubator under dark conditions at 20 °C, irregular circular, cottony, hairy at the margin, white; reverse: pale yellow at the margin and brown to black at the middle.
Notes: The phylogenetic tree showed that our two isolates (UESTCC 22.0135, UESTCC 22.0147) clustered with other P. pseudobyssoides isolates. Morphologically, our two collections are similar to the description of P. pseudobyssoides. Thus, we identified our collections as P. pseudobyssoides. This is the first record of Periconia species occurring on Digitaria sanguinalis. MycoBank: MB 847469 Etymology: Name reflects the host genus, Spodiopogon, from which the fungus was (d,e) conidiophores with spherical conidial heads; (f,g) conidial heads bearing conidiogenous cells and conidia; (h-j) conidia; (k) germinating conidium; (l,m) colony on PDA from above and below. Scale bars: (d) = 100 µm, (f,h,k) = 10 µm. Scale bar of (d) applies to (e). Scale bar of (f) applies to (g). Scale bar of (h) applies to (i,j). Figure 11. Periconia spodiopogonis (HKAS 126519, holotype). (a-c) Colonies on the natural substrate (d,e) conidiophores; (f,g) apically branch conidiophores with conidial head; (h-k) conidia; (l) germinating conidium; (m,n) colony on PDA from above and below. Scale bars: (d) = 100 µm, (f,g,h,j,l) = 20 µm. Scale bar of (d) applies to (e). Scale bar of (h) applies to (i). Scale bar of (j) applies to (k).  (l) germinating conidium; (m,n) colony on PDA from above and below. Scale bars: (d) = 100 µm, (f,g,h,j,l) = 20 µm. Scale bar of (d) applies to (e). Scale bar of (h) applies to (i). Scale bar of (j) applies to (k).  (MFLUCC 17-2158). The type species of P. verrucosa was isolated from decaying stems of Clematis viticella in Belgium [34]. Morphologically, there are no significant differences between our collections and the type species of P. verrucosa. Therefore, our five collections are identified as P. verrucosa, and this is the first report of P. verrucosa from terrestrial habitats in China. (e,f) apically branch conidiophores with conidial head; (g-i) conidia; (j) germinating conidium; (k,l) colony on PDA from above and below. Scale bars: (c) = 100 µm, (e,g,j) = 10 µm. Scale bar of (c) applies to (d). Scale bar of (e) applies to (f). Scale bar of (g) applies to (h-i). (e,f) apically branch conidiophores with conidial head; (g-i) conidia; (j) germinating conidium; (k,l) colony on PDA from above and below. Scale bars: (c) = 100 µm, (e,g,j) = 10 µm. Scale bar of (c) applies to (d). Scale bar of (e) applies to (f). Scale bar of (g) applies to (h-i).  (MFLUCC 17-2158). The type species of P. verrucosa was isolated from decaying stems of Clematis viticella in Belgium [34]. Morphologically, there are no significant differences between our collections and the type species of P. verrucosa. Therefore, our five collections are identified as P. verrucosa, and this is the first report of P. verrucosa from terrestrial habitats in China.

Discussion
Species of Periconia have been reported from a large number of host plants, including graminaceous plants [4,8,55]. For example, previously, P. hispidula and P. thysanolaenae were recorded from graminaceous plants in China [8,58]. It is known that this genus produces a number of bioactive secondary metabolites, including terpenes, polyketides, aromatic compounds, and carbohydrate derivatives [59]. Periconia sp. isolated from Torreya grandifolia produced taxol, known as an anticancer compound [60]. Similarly, compounds 2,4dihydroxy-6-[(1 E,3 E)-penta-1 ,3 -dienyl]-benzaldehyde isolated from P. atropurpurea had vigorous antifungal activity against Cladosporium sphaerospermum and C. cladosporioides [19]. Therefore, the genus Periconia has excellent potential for natural product exploration and the development of pharmacological agents. Thus, future genetic and secondary metabolites studies on this chemically highly diverse genus may lead to the discovery of novel biochemical properties unique to the Periconia group.
Sichuan Province along the Yangtze River is a biodiversity hot spot [61,62]. In Sichuan Province, we regularly conduct fungal diversity surveys. This survey collected samples from five sites; 26 Periconia strains were morphologically and phylogenetically characterized, which resulted in 11 species identified and six new taxa described. In the phylogenetic tree, P. salina was clustered within P. byssoides, but Yang et al. [8] showed that P. byssoides and P. salina were not conspecific by the pairwise homogeneity index (PHI) test. Periconia is an ancient and species-rich genus and among 130 morphologically accepted Periconia species [8]. However, only 43 species (including six new species in this study) have molecular data in GenBank. Even though many Periconia species only have ITS and LSU loci. As a highly diverse group, it is also difficult to distinguish species within Periconia using only ITS and LSU gene regions. Therefore, in future studies, more phylogenetic markers with high resolving power are essential to understand the species boundaries in this highly diverse genus. In addition, due to a lack of information and sequence data on many of the species, including the type of Periconia, epitype with living cultures are essential for further comprehensive studies to clarify the taxonomic status of the genus.  Data Availability Statement: All sequence data are available in NCBI GenBank following the accession numbers in the manuscript.