Taxonomic Reappraisal of Periconiaceae with the Description of Three New Periconia Species from China

As a result of an ongoing research survey of microfungi in Yunnan, China, several saprobic ascomycetes were collected from various host substrates. Preliminary morphological analyses identified a few of these taxa as Periconia species. We obtained DNA sequence data of the Periconia species from pure cultures and investigated their phylogenetic affinities. Phylogenetic analyses of a combined LSU, ITS, SSU and tef1-α sequence dataset demonstrated that five isolates of Periconia formed well-resolved subclades within Periconiaceae. Accordingly, three new Periconia species are introduced viz. P. artemisiae, P. chimonanthi and P. thysanolaenae, and new host and geographical records of P. byssoides and P. pseudobyssoides, are also reported from dead branches of Prunus armeniaca and Scrophularia ningpoensis. Periconia celtidis formed a monophyletic clade with P. byssoides in the present phylogenetic analyses. Results of the pairwise homoplasy index (PHI) test indicated significant recombination between P. byssoides and P. celtidis. Therefore, P. celtidis has been synonymized under P. byssoides. In addition, we re-illustrated and studied the type specimen of the sexual genus Bambusistroma. As a type species of Bambusistroma, B. didymosporum features similar morphology to the sexual morph of Periconia homothallica and P. pseudodigitata. We therefore synonymize Bambusistroma under Periconia based on morphological and phylogenetic evidence. Furthermore, our new isolates produced brown conidia of asexual morph in agar media typical of the genus Noosia. Based on morphological comparison with Periconia in vitro and phylogenetic status of Noosia, we also treat Noosia as a synonym of Periconia. Detailed descriptions and illustrations of three novel taxa and two new records of Periconia byssoides and P. pseudobyssoides as well as the illustration of P. didymosporum comb. nov. are provided. An updated phylogenetic tree of Periconiaceae using maximum likelihood and Bayesian inference analyses is constructed. Generic circumscription of Periconia is amended.

Fungal specimens were collected from Chuxiong, Kunming and Xishuangbanna in Yunnan Province, China, and stored in disposable plastic Zip-loc bags. The specimens were observed and examined after 1-2 days incubation at room temperature in the laboratory. The microscopic morphological features (e.g., conidiophores, conidiogenous cells, conidia) were examined with an OLYMPUS SZ61 stereomicroscope and Nikon ECLIPSE Ni compound microscope equipped with a Canon DS126311 digital camera. In addition, Bambusistroma didymosporum and Noosia banksiae were re-illustrated. Morphological features viz. ascomata, peridium, pseudoparaphyses, asci, ascospores (sexual morph); conidiophores, conidiogenous cells and conidia (asexual morph) were measured using Tarosoft (R) Image Frame Work (IFW) version 0.9.7, and the photographic plates were processed with Adobe Photoshop CS6 Extend version 10.0 software (Adobe systems, San Jose, CA, USA).
Pure cultures of the new isolates were obtained by single spore isolation [44]. The conidial masses on the surface of fungal hosts were picked up using a sterilized surgical needle and soaked in sterilized water droplets for spore suspension. The spore suspension was spread onto the surface of potato dextrose agar plates (PDA) and incubated overnight at room temperature. Germinated conidia were singularly transferred to two new PDA plates (five conidia in each plate) and incubated at room temperature in normal day/nightlight cycle. The colony developed of single conidium was transferred to new PDA plates and incubated at room temperature for one week. Culture characteristics and growth were recorded at one week and after four weeks. The in vitro sporulation was observed  (Table 1) was generated for further phylogenetic analyses based on recent publications [6,43,[50][51][52]. Individual sequence datasets were aligned separately via MAFFT online version 7.475 using default settings (http://mafft.cbrc.jp/alignment/server; accessed on 25 June 2021) [53] and improved where necessary using BioEdit v. 7.0.9.0 [49]. Individual sequence datasets were prior analyzed by maximum likelihood (ML) criterion for checking the congruence of tree topologies and further phylogenetic analyses of the concatenated LSU, SSU, ITS and tef1-α sequence dataset were performed by maximum likelihood (ML) and Bayesian inference (BI) criteria.
Maximum likelihood (ML) was analyzed by RAxML-HPC2 on XSEDE (8.2.10) [54,55] via the CIPRES science Gateway V.3.3 web server [56] using default settings, but with adjustments by setting up the substitution evolution model as GTRGAMMAI and 1000 replicates of rapid bootstrap. The evolutionary model of nucleotide substitution was selected independently for each locus (LSU, SSU, ITS and tef1-α) using MrModeltest 2.3 [57]. The best-fit model is the GTR + I + G substitution model for each locus under the Akaike Information Criterion (AIC).
Bayesian inference (BI) was performed by MrBayes on XSEDE (3.2.7a) via the CIPRES science Gateway V.3.3 web server [56]. Bayesian posterior probabilities (BYPP) [58,59] were evaluated by Markov Chain Monte Carlo sampling (MCMC). Parameters were set as defaults, but with the adjustments as two parallel runs of six simultaneous Markov chains. Starting random tree topology was run with 3,000,000 generations for a combined dataset, and trees were sampled every 100 generations (resulting in 30,000 total trees). The effective sampling sizes (ESS) and the stable likelihood plateaus and burn-in value were determined by Tracer version 1.7.1 software [60]. The first 6000 sampled trees (20%) representing the burn-in were discarded and the remaining trees were used to calculate the Bayesian posterior probabilities (BYPP) in a 50% majority rule consensus tree.

Genealogical Concordance Phylogenetic Species Recognition (GCPSR) Analysis
The recombination level within phylogenetically closely related species of newly generated strains   [61] performed in SplitsTree4 [62,63], using the GCPSR model [64]. The combined ITS, LSU, SSU and tef1-α sequence dataset of these phylogenetically closely related species was applied for the test to refine incompatibility between pairs of sites regarding a genealogical history does not involve recurrent or convergent mutations that can be parsimoniously inferred [61]. The split graph showing relationships between newly generated strains KUMCC 20-0263, KUMCC 20-0264 and other closely related species was visualized and constructed in LogDet transformation and splits decomposition options. The pairwise homoplasy index resulted in a threshold lower than 0.05 (Φw < 0.05), indicating significant recombination in the dataset.

Phylogenetic Analyses
The concatenated ITS, LSU, SSU and tef1-α sequence dataset consisted of 69 strains of representative taxa in Periconiaceae, and the other two related families, Didymosphaeri-  Figure 1. The matrix had 1095 distinct alignment patterns, with 43.72% undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.238246, C = 0.255371, G = 0.267517, T = 0.238865; substitution rates AC = 1.513277, AG = 2.455316, AT = 1.810928, CG = 1.226908, CT = 9.347925, GT = 1.000000; gamma distribution shape parameter α = 0.201661. The final average standard deviation of split frequencies at the end of total MCMC generations was calculated as 0.009307 in BI analysis.   Phylogenetic analyses of the concatenated ITS, LSU, SSU and tef1-α sequence dataset based on ML and BI were similar in overall tree topologies. Periconia artemisiae sp. nov. (KUMCC 20-0265) formed a distinct clade with Periconia sp. (strains Otu0123, G1782 and C75) with high support (100% ML, 1.00 PP; Figure 1). Periconia artemisiae also formed a wellresolved clade with P. alishanica Tennakoon, C.H. Kuo and K.D. Hyde, P. pseudobyssoides, P. salina, P. byssoides and P. celtidis with high support (100% ML, 1.00 PP). Periconia chimonanthi sp. nov. shared the same branch length with Periconia sp. CY137 (isolated from the nest of Cyphomyrmex wheeleri in Texas, USA) and is sister to P. cortaderiae Thambug. and K.D. Hyde with high support in BI analysis but shows insignificant support in ML analysis (62% ML, 0.99 PP; Figure 1). While P. thysanolaenae sp. nov. formed a separate branch sister to P. minutissima Corda strain MUT 2887 with insignificant support in ML and BI analyses (67%, 0.80 PP) and clustered with P. homothallica and Noosia banksiae.

Genealogical Concordance Phylogenetic Species Recognition (GCPSR)
Twelve representative strains of Periconia byssoides, P. celtidis, P. pseudobyssoides and P. salina were implied in a pairwise homoplasy index (PHI) test for determining the recombination level of these four species. The results of PHI test indicated no significant recombination among P. byssoides, P. pseudobyssoides and P. salina in ITS (Φw = 0.2398), LSU (Φw = 0.6534), tef1-α (Φw = 0.4152), and a combined ITS, LSU, SSU and tef1-α sequence dataset (Φw = 0.654) while SSU showed that there are too few informative characters to implemented; however, it is significant recombination between P. byssoides and P. pseudobyssoides in LSU region. PHI results of single gene ITS, LSU, tef1-α and a combined ITS, LSU, SSU, tef1-α sequence dataset indicated significant recombination between P. byssoides and P. celtidis. Therefore, these two species are shown to be conspecific and thus, P. celtidis is treated as a synonym of P. byssoides ( Figure 2). Sexual morph: Ascomata solitary to gregarious, sometimes with pseudostromatic or clypeus-like basal structure on host, scattered to clustered, immersed to erumpent, dark brown to black, globose to subglobose, or conical, ostiolate, papillate; ostiolar canal filled with hyaline periphyses. Peridium composed of several layers of thin-or thick-walled, brown to dark brown pseudoparenchymatous cells of textura angularis to textura prismatica. Hamathecium composed of numerous, cellular, hyaline, septate, branched, anastomosed pseudoparaphyses, embedded in a gelatinous matrix. Asci 8-spored, bitunicate, fissitunicate, cylindrical, with short, rounded or furcate pedicel, apically rounded with a shallow ocular chamber. Ascospores overlapping 1-2-seriate, hyaline, fusiform, 1-septate, with guttules, smooth-walled, with an entire sheath. Asexual morph: Conidiophores macronematous, mononematous, sometimes lacking, erect, straight or slightly flexuous, branched or unbranched, pale brown to dark brown, septate, smooth-walled, or slightly echinulate, sometimes swollen near the base, with spherical conidial heads on main stipe and/or apical branches. Conidiogenous cells monoblastic or polyblastic, terminal, integrated or discrete, ovoid to subglobose, or subspherical, lightly pigmented, branched, smooth to slightly echinulate, sometimes with small, pimple-like pores. Conidia solitary or catenate, in acropetal chains, globose to subglobose, aseptate, occasionally ellipsoidal to cylindrical, pale brown to dark brown, smooth-walled or verruculose. In vitro: Mycelium hyaline to brown, branched, smooth to verruculose with age, frequently aggregating into hyphal strands. Conidiophores sometimes reduced to conidiogenous cells; when present, macronematous, erect, single, sometimes in pairs, pigmented, septate, thick-walled. Conidiogenous cells mono-to polyblastic, solitary, discrete or integrated, determinate, or inconspicuous, lateral and terminal, pigmented, ellipsoidal, ovoid to clavate, aseptate, sometimes with small, pimple-like pores, or with percurrent proliferations. Conidia dimorphic; primary conidia globose to subglobose, or fusoid-ellipsoidal, subhyaline to brown or dark brown, smooth to verruculose, solitary or in short, branched chains, sometimes with minute, unthickened basal pores; secondary conidia phragmoconidia, brown, verruculose, arising from disarticulating hyphal cells (adopted from Crous et al. [16], Calvillo-Medina et al. [51]).  Type species: Periconia lichenoides Tode. Notes: Periconia was recognized as a polyphyletic genus in Periconiaceae due to the other three monotypic genera that always clade with other Periconia species [12,[18][19][20][21][22]. However, morphological study of Bambusistroma and Noosia demonstrated that these two genera resemble the sexual morph of Periconia homothallica and P. pseudodigitata as well as the sporulation in the culture of P. artemisiae, P. chimonanthi and P. pseudobyssoides. Bambusistroma always clustered with Flavomyces in previous studies [12,[20][21][22]43]. In this study, we include more taxon sampling in Periconiaceae, and the phylogenetic analyses indicated that B. didymosporum is sister to Sporidesmium tengii (HKUCC 10837), clustering with other Periconia species with insignificant support. There is only one LSU sequence available for the non-type strain HKUCC 10837 of S. tengii, and therefore, the phylogenetic status of S. tengii remains unclarified and is pending further study. Crous et al. [16] introduced the genus Noosia due to the lack of conspicuous conidiophores, which is different from other Periconia in nature. However, we were able to induce the sporulation of P. artemisiae, P. chimonanthi and P. pseudobyssoides on PDA after three months. Morphologically, sporulation in the cultures of P. artemisiae, P. chimonanthi and P. pseudobyssoides resembles Noosia banksiae. Hence, in the present study, we treated Bambusistroma and Noosia as synonyms of Periconia based on morphological characteristics and phylogenetic analyses.
Culture characteristics: Conidia germinated on PDA within 24 h. Colonies on PDA reaching 20 mm diam in one week at room temperature (10-25 • C), dense, circular, flattened, dull, cottony, surface smooth with entire edge, hairy at the margin, pale grey at the margin and pale brown to white at the middle from the above; greenish-brown at the margin and dark brown to black at the middle in reverse; not producing pigmentation on agar medium. as synonyms of Periconia based on morphological characteristics and phylogenetic analyses.
Notes: Based on ITS nucleotide BLAST search, the isolate KUMCC 20-0264 is most similar to Periconia byssoides H4853 (GenBank no. MW444854) with 99% similarity (535/536 bp). A nucleotide base comparison of the ITS and tef1-α sequences between the new isolate KUMCC 20-0264 and other representative strains of P. byssoides also revealed nucleotide differences less than 1.5% (Table 3), indicating that the new isolate is conspecific with P. byssoides [65]. Phylogenetic analyses demonstrated that the new isolate KUMCC 20-0264 is basal to other P. byssoides and P. celtidis, with 68% ML and 0.71 PP support ( Figure 1) and most closely related with P. byssoides strains MFLUCC 18-1548 and MFLUCC 18-1553, which were isolated from decaying pods of Peltophorum sp. (Fabaceae) in Thailand [21]. Based on Farr and Rossman [3], P. byssoides is reported as a saprobe on Prunus armeniaca in Yunnan, China for the first time.
Tennakoon et al. [40] introduced Periconia celtidis as a saprobe on dead leaves of Celtis formosana and Macaranga tanarius from Taiwan. Their phylogenetic analyses showed that P. celtidis form a distinct lineage and clustered with P. byssoides, P. pseudobyssoides and P. alishanica with 90% ML and 1.00 PP support (Tennakoon et al. [40] (p. 37)). In the present phylogenetic analyses, P. celtidis grouped with other P. byssoides, and this result was also supported by PHI results of ITS, LSU, and a combined ITS, LSU, SSU and tef1-α sequence dataset ( Figure 2). Therefore, we treat P. celtidis as a synonym of P. byssoides in this study. Morphological characteristics of the new isolate KUMCC 20-0264 are slightly different from other representative isolates of P. byssoides and P. celtidis in size of conidiophores and conidia which is detailed in Table 4.
Index Fungorum number: IF559497. Etymology: The specific epithet "chimonanthi" refers to the host genus, Chimonanthi, from which the fungus was collected.
Culture characteristics: Conidia germinated within 18-20 h on PDA. Colonies reaching 15 mm diam in one week at 20-25 • C in normal light, dense, circular, flattened to slightly raised, surface slightly rough, with entire edge, floccose to cottony, radially furrowed at the margin, pale greenish grey at the margin, dark greenish towards the center from above and below; not producing pigmentation on medium. Periconia byssoides Periconia chimonanthi E.F. Yang, H.B. Jiang and Phookamsak, sp. nov. Figure 5. Index Fungorum number: IF559497 Etymology: The specific epithet "chimonanthi" refers to the host genus, Chimonanthi, from which the fungus was collected.
Notes: In the ITS and tef1-a nucleotide BLAST search, the newly generated strain KUMCC 20-0263 is identical to Periconia pseudobyssoides (strain DUCC 0850) with 99.60% and 98.73% similarities and is also closely related to P. pseudobyssoides (strains H4151/MAFF 243868, and H 4790/MAFF 243874). Phylogenetic analyses indicated that the generated strain KUMCC 20-0263 clustered with P. pseudobyssoides strains DUCC 0850, MAFF 243868 and MAFF 243874 with 58% ML, 0.86 PP support (Figure 1). A nucleotide base comparison of the ITS and tef1-α sequences between the new isolate KUMCC 20-0263 and other representative strains of P. pseudobyssoides also revealed nucleotide differences of less than 1.5% (Table 5). Moreover, the ITS nucleotide base comparison between KUMCC 20-0263 and the ex-type strain of P. pseudobyssoides (BILAS 50334) also revealed the conspecific status of KUMCC 20-0263 with P. pseudobyssoides (3/562 bp of ITS (0.53%, <1.5%)). Thus, we identified our newly generated strain KUMCC 20-0263 as P. pseudobyssoides, which is reported from Scrophularia ningpoensis in Yunnan, China for the first time.
Periconia pseudobyssoides was introduced by Markovskaja and Kacergius [24]. The species was found to be a saprobe on dead stalks of Heracleum sosnowskyi in Lithuania. Periconia pseudobyssoides (KUMCC 20-0263) is morphologically similar to the type of P. pseudobyssoides (BILAS 50334) and P. pseudobyssoides (DUCC 0850) in producing macronematous, brown, septate, unbranched, verruculose conidiophores, mono-to polyblastic, light brown to brown, ovoid to subglobose, discrete, determinate conidiogenous cells, and globose, brown, aseptate, verruculose conidia, which are borne singly or catenate in short chains. However, these collections vary slightly in size of conidiophores and conidia, likely depending on environmental factors and host associations [20,24]. Table 5. Polymorphic nucleotides from the ITS, LSU, and tef1-α sequence data of Periconia pseudobyssoides. The newly generated strain is indicated in bold black and the type strain is indicated as superscript "T".     Phylogenetic analyses demonstrated that KUMCC 20-0262 is sister to P. minutissima with 67% ML, 0.80 PP support and clustered with P. homothallica (KT 916) and P. banksiae (CBS 129526) with low support, distancing from P. macrospinosa (Figure 1). Based on a nucleotide pairwise comparison of ITS and tef1-α sequences, P. thysanolaenae sp. nov. (KUMCC 20-0262) differs from P. minutissima strain MUT 2887 in 5/458 bp of ITS (1.09%), differs from P. homothallica strain KT 916 (type strain) in 25/530 bp of ITS (4.72%), and differs from P. banksiae strain CBS 129526 (type strain) in 48/602 bp of ITS (7.97%). Periconia minutissima strain MUT 2887 formed a distinct clade with P. minutissima strain MFLUCC 15-0245 in this study. Bovio et al. [70] isolated P. minutissima strain MUT 2887 from Sycon ciliatum in the Atlantic Ocean using a culture method and identified the strain as P. minutissima based on a high percentage of homologies, with sequences available in GenBank. However, P. minutissima strain MFLUCC 15-0245 remains unpublished. Our new isolate KUMCC 20-0262 is not significantly different from P. minutissima strain MUT 2887 based on the ITS nucleotide base comparison (1.09%); however, P. minutissima strain MUT 2887 has no morphological support for species identification. Unfortunately, the ex-type strain of P. minutissima is unavailable. We, therefore, introduced our new collection as P. thysanolaenae sp. nov.

Species
Morphological characteristics of Periconia thysanolaenae align with described Periconia species in having erect or flexuous, faint to reddish brown, or dark brown, conidiophores, with spherical conidial head at the apex, polyblastic, ovoid to subglobose, light brown to brown, terminal conidiogenous cells, and catenate, globose, and brown to dark brown aseptate conidia. However, P. thysanolaenae can be distinguished from other phylogenetically related species in having proliferating, 2-3 secondary conidiophores. Periconia minutissima is characterized by effuse, brown, colonies, with dark brown, loose, very slender, few septate, translucent conidiophores, divided above into very short branches, with botrytislike apex and globose, colorless conidia [71], while P. homothallica is represented by its sexual morph, and P. banksiae formed an asexual morph in vitro that lack conspicuous conidiophores [12,16].
In this study, we re-evaluate the taxonomic status of Bambusistroma and Noosia based on morphological and multigene phylogenetic approaches. Morphological comparison of Bambusistroma and the sexual morphs of Periconia species (viz. P. homothallica and P. pseudodigitata) showed that Bambusistroma has similar morphology with the sexual morph of Periconia species (see notes of P. didymosporum), while P. artemisiae, P. chimonanthi and P. pseudobyssoides sporulated in vitro are not significantly different from Noosia (see notes of P. banksiae). Therefore, these two monotypic genera are treated as synonyms of Periconia in this study. Knapp et al. [4] introduced a monotypic genus Flavomyces to accommodate the root endophytic species, F. fueloephazae (as F. fulophazii), isolated from the root of Festuca vaginata in Hungary based on culturable and phylogenetic approaches. Morphological characteristics of Flavomyces are undescribed. In this study, F. fueloephazae formed an independent clade basal to Periconia prolifica Anastasiou, P. citlaltepetlensis Calvillo, Cobos-Villagrán and Raymundo, P. igniaria, P. epilithographicola Coronado-Ruiz et al., P. caespitosa Cantillo, Gusmão and Madrid, P. variicolor S.A. Cantrell, Hanlin and E. Silva, P. minutissima and P. macrospinosa with significant support in ML analyses (73% ML; Figure 1). Since morphology is poorly known, the generic status of Flavomyces may need to be clarified.
Phylogenetic relationships of many Periconia species are not well-resolved, such as the relationships of P. cookei E.W. Mason and M.B. Ellis, P. delonicis Jayasiri, E.B.G. Jones and K.D. Hyde, P. elaeidis T. Sunpapao and K.D. Hyde, P. palmicola J.F. Li and Phookamsak and P. verrucosa Phukhams. et al. These species are grouped together with significant support in ML analyses (98%; Figure 1); however, the interspecific relationships of these species could not be resolved. These species may be conspecific and contain a high degree of genetic variation. The conspecific status of these ambiguous species should be re-evaluated. Moreover, phylogenetic relationships of P. byssoides, P. celtidis, P. pseudobyssoides and P. salina are not well-resolved in this study and it is in agreement with previous studies [18,51,72]. We re-evaluated the conspecific status of P. byssoides, P. celtidis, P. pseudobyssoides and P. salina based on the pairwise homoplasy index (PHI) test. The PHI test of the individual gene (ITS, LSU, and tef1-α) and the combined ITS, LSU, SSU and tef1-α sequence dataset resulted in no significant recombination among P. byssoides, P. pseudobyssoides and P. salina (Figure 2), indicating that P. byssoides, P. pseudobyssoides and P. salina are not conspecific. In contrast, the PHI test resulted in significant recombination between P. byssoides and P. celtidis, and polymorphic nucleotide comparison between P. byssoides and P. celtidis (Table 2) also supported that P. celtidis is conspecific with P. byssoides. Hence, P. celtidis is synonymized under P. byssoides in this study.
Out of 121 morphologically accepted species, only 28 species (including three new species and two novel combinations provided in this study) have molecular data to clarify their phylogenetic placements in Periconiaceae [2,40]. Most available sequences of Periconia species are limited to ITS and LSU gene regions. Sixteen species have sequence data of a protein-coding gene (tef1-α), but most species do not have useful phylogenetic markers (i.e., mtSSU, rpb1, rpb2 and tub2) to identify them to the species level in Periconiaceae. Moreover, some Periconia species show an intraspecific variation, such as P. byssoides, P. cortaderiae, P. prolifica and P. pseudobyssoides. More reliable phylogenetic markers may be required to support their conspecific status. In addition, two strains of P. minutissima (MUT 2887 and MFLUCC 15-0245) and two strains of P. igniaria (CBS 379.86 and CBS 485.96) form a distinct clade separated from each other within Periconiaceae. These strains are not represented by the ex-type strains. Therefore, the phylogenetic status of P. minutissima and P. igniaria remain in doubt. The epitypification of P. minutissima and P. igniaria are in urgent need of clarification regarding their phylogenetic status within Periconiaceae.
Nevertheless, the type specimens of Periconia lichenoides have been lost while, the DNA sequence data of this type species are also unavailable in GenBank. Tanaka et al. [12] judged Periconia to be a member of Dothideomycetes due to the morphological resemblance of Periconia sensu stricto (e.g., P. byssoides, P. cookei, P. igniaria, and P. digitata) with P. lichenoides. The taxonomic treatment proposed by Tanaka et al. [12] was followed by various subsequent authors [2,6,[17][18][19][20][21][22]43,51,52,72]. However, this taxonomic status is somewhat questionable due to the lack of information from the type species of Periconia. Neotypification or designation of reference specimens of P. lichenoides is urgently needed to clarify the taxonomic status of Periconia in Dothideomycetes.