Exploring the Diversity and Systematics of Phaeosphaeriaceae: Taxonomic Novelties from Ecologically Diverse Habitats and Their Phylogenetic Resolution

Simple Summary Our study describes several species of Phaeosphaeriaceae found in terrestrial habitats in Sichuan Province, China. We used morphological and molecular data to accurately delimit these species and determine their taxonomic relationships within the family. Our findings contribute to understanding fungal diversity in China and provide a basis for future studies exploring the ecological roles and biotechnological potential of these fungi. Additionally, our multi-gene phylogenetic approach provides increased accuracy and resolution in the delimitation of species boundaries within Phaeosphaeriaceae. Abstract The family Phaeosphaeriaceae is a diverse group of ascomycetous fungi that are commonly found in terrestrial habitats worldwide. In recent years, there has been increasing interest in the biodiversity of Phaeosphaeriaceae in China, particularly in Sichuan Province, which has not been fully explored for its high fungal diversity. In our study, we conducted extensive surveys in Sichuan Province to identify and describe new species of Ascomycota with diverse habitats. Here, we present a taxonomic revision of Phaeosphaeriaceae with taxonomic novelties from terrestrial habitats in Sichuan Province based on a multi-gene phylogenetic approach. Our study focuses on the description of four new species of Phaeosphaeriaceae, representing novel additions to the diversity of this fungal family. Using a combination of morphological and molecular data, we provide detailed descriptions of the new taxa and their placement within the family. Additionally, we discuss the phylogenetic relationships of these new taxa with other members of Phaeosphaeriaceae, providing insights into the correct taxonomic classification of the family. Our taxonomic revision contributes to understanding fungal diversity in China and provides a foundation for future studies investigating the taxonomy and ecological roles of Phaeosphaeriaceae fungi. Furthermore, our multi-gene phylogenetic approach provides increased resolution and accuracy in the delimitation of species boundaries within the family. Our study highlights the importance of continued exploration and taxonomic revision in order to fully understand the diversity and distribution of fungal species in China and beyond. New species: Paraloratospora sichuanensis, Phaeosphaeria chengduensis, P. sichuanensis, and Septoriella shoemakeri. New combinations: Paraloratospora breonadiae, P. fructigena, Septoriella ammophilicola, S. asparagicola, S. festucae, S. luzulae, and S. verrucispora. New names: Septoriella paradactylidis, and S. neomuriformis.


Introduction
The family Phaeosphaeriaceae comprises a diverse group of ascomycetous fungi that are commonly found in terrestrial habitats worldwide [1].Despite their abundance, the taxonomy of Phaeosphaeriaceae remains obscure, with many species remaining undescribed or poorly characterized.In recent years, there has been an increasing interest in the biodiversity of Phaeosphaeriaceae.Researchers have conducted extensive surveys of the mycobiota, resulting in the discovery of over 135 new taxa in Phaeosphaeriaceae within the last five years: 2019-2023 [2].In particular, several new species of Phaeosphaeria have been identified, representing novel additions to the diversity of this fungal family.However, the taxonomy of Phaeosphaeriaceae is still challenging due to the high degree of morphological variability within the family.Many species have been described based on only a single specimen, and the use of molecular data to aid in the delimitation of species boundaries for the species published prior to the 1990s is still inadequate.Currently, the family consists of 84 genera [3], but their exact classification is debatable.The phylogenetic relationships within the family are also not well resolved.Twenty-one of the genera are represented by a single strain in the family (viz., Acericola, Alloneottiosporina, Arezzomyces, Banksiophoma, Bhagirathimyces, Bhatiellae, Camarosporioides, Equiseticola, Hydeopsis, Jeremyomyces, Kwanghwaensis, Longispora, Melnikia, Neosphaerellopsis, Ophiosimulans, Pseudophaeosphaeria, Scolicosporium, Vittaliana, Vrystaatia, Xenophaeosphaeria, and Xenophoma), whereas thirty of the genera are represented by a single species (monotypic).Additionally, some of the species are not monophyletic with their type (i.e., Muriphaeosphaeria, Loratospora, and Phaeosphaeria).Since the last revision [4], there has been no proper attempt to resolve the inter-and intra-generic classification of Phaeosphaeriaceae, indicating the need for a new taxonomic revision of the family.
Collecting species in Phaeosphaeriaceae is crucial because it enables more accurate and precise identification of its species, which is particularly important for linking their holomorphs and finding DNA-based sequence data for old herbarium specimens.Moreover, this helps fill the gap in our understanding of their diversity and evolution [5].Since Phaeosphaeriaceae are cosmopolitan and ecologically important, studying their ecological relationships is essential to understand them in a broader sense [1,4].In addition, some genera (i.e., Polydomus and Setophoma) have significant practical applications, including as sources of biological activities and biotechnological products [6][7][8].Therefore, conducting research on Phaeosphaeriaceae is an important step towards unlocking their potential applications.Accurate identification of Phaeosphaeriaceae species is also necessary for assessing their conservation status and identifying areas of high biodiversity that require protection.
This paper presents a taxonomic revision of Phaeosphaeriaceae, focusing on the genera Paraloratospora, Phaeosphaeria, and Septoriella.Four new species, seven new combinations, and two new names are proposed, supported by detailed morphological and molecular analyses to establish their placement within this family.Furthermore, we investigate the phylogenetic relationships of these novel taxa with other members of Phaeosphaeriaceae and their current taxonomic classification.Additionally, we confirm the species list that includes DNA data in Phaeosphaeria sensu stricto.This study highlights the significance of ongoing exploration and taxonomic revision to comprehensively understand fungal species diversity and distribution in China and beyond.It contributes to our knowledge of the diversity and evolution of Phaeosphaeriaceae in terrestrial habitats and establishes a foundation for future taxonomic and ecological investigations within this important fungal family.

Isolates and Specimens
During our fieldwork in Sichuan Province, China, we collected typical black ascomata/conidiomata from dead twigs across various regions including Chenghua District, Qingyang District, and Dayi County during both wet (August 2021) and dry (January 2023) seasons.The collected samples were placed in Ziploc bags and transported to the mycology laboratory of the Kunming Institute of Botany, where they were stored in paper envelopes.Single spore isolation was conducted according to the methods described in Wanasinghe et al. [9], and germinated spores were handled appropriately.Dried specimens were preserved in the fungarium of the Cryptogams Kunming Institute of Botany, Academia Sinica (KUN-HKAS), and representative cultures were deposited in the Kunming Institute of Botany Culture Collection (KUMCC) and the University of Electronic Science and Technology Culture Collection (UESTCC), Chengdu, China.Nomenclatural data for fungal novelties were deposited in MycoBank [10].

Morphological Observations
To examine the ascomata, conidiophores, and conidia from natural substrates, we rehydrated them with tap water and viewed them using a Motic SMZ 168 series stereomicroscope (Motic Asia, Kowloon, Hong Kong).Morphological characteristics were evaluated by hand-sectioning sporocarps placed on water-mounted glass slides.We assessed various characteristics such as the diameter, height, color, and shape of the ascomata/conidiomata, as well as the width of the peridium and the height and diameter of the ostioles.Microscopic photography was carried out using a Nikon ECLIPSE Ni (Nikon Corporation, Konan, Minato-ku, Tokyo, Japan) compound microscope with differential interference contrast (DIC) and phase contrast (PC) illumination.Images of microscopic structures were captured using a Canon EOS 600D (Canon Inc., Ota, Tokyo, Japan) camera.Additionally, macroscopic images of colonies were documented using an iPhone XS Max (Apple Inc., Cupertino, CA, USA) in daylight.We used the Tarosoft (R) Image Frame Work program to measure dimensions, and images used for figures were processed using Adobe Photoshop CS6 (Adobe Systems, San Jose, CA, USA).

Sequencing and Sequence Alignment
To analyze the sequences generated from different primers of the five genes, we conducted a BLAST search to identify sequences with high similarity indices and find the closest matches with taxa in Phaeosphaeriaceae, following recent publications [19][20][21][22].Later, we analyzed them with other sequences retrieved from GenBank (Table 1).We used MAFFT v. 7 [23] to automatically generate multiple alignments of all consensus sequences as well as the reference sequences.We manually corrected the alignments using BioEdit v. 7.0.5.2 [24] where necessary.
Table 1.Taxa used in the phylogenetic analyses and their corresponding GenBank numbers.Isolates/sequences in bold were isolated/sequenced in the present study.The superscripted "T" in the strain column denotes ex-type, ex-isotype, ex-paratype, or ex-epitype strains.

Phylogenetic Analyses
We examined the single-locus datasets for any topological incongruence among the loci for members of the analyses.The resulting alignments were then concatenated into a multi-locus alignment and analyzed using maximum likelihood (ML) and Bayesian (BI) phylogenetic methods in the CIPRES Science Gateway [25].We obtained an ML tree using RAxML-HPC2 on XSEDE v. 8.2.10 [26] with a GTR + G + I model and calculated support values with 1000 bp replicates [27].Nodes with ML bootstrap values equal to or greater than 75% are indicated above each node.
The best-fit model was selected based on Bayesian Information Criterion (BIC) scores using the IQ-TREE web application at http://iqtree.cibiv.univie.ac.at (accessed on 27 June 2023) [28].We restricted the pool of available models to JC, F81, HKY, SYM, and GTR for model selection.We performed BI with two parallel runs of 50 M generations, using four chains in each, and retaining one tree every 100 generations.The dataset was partitioned by gene, and HKY + I + G (SSU), SYM + I + G (LSU), and GTR + I + G (ITS, tef 1, and rpb2) models were applied to each partition, ending the run automatically when the standard deviation of split frequencies dropped below 0.01 with a burn-in fraction of 0.25.A 50% majority rule consensus tree was obtained after discarding the first 25% of trees, and posterior probabilities were used as a measure of nodal support.Nodes with posterior probabilities in BI (BYPP) greater than 0.95 are indicated above each node.Finally, we visualized the phylograms using the FigTree v1.4.0 program [29] and reorganized them in Microsoft PowerPoint (2019).

Phylogenetic Analyses
To examine the evolutionary relationships of our new strains within Phaeosphaeriaceae, we conducted phylogenetic analyses based on the combined SSU, LSU, ITS, tef 1, and rpb2 DNA sequences of 258 representatives of the family.We used two strains from Quixadomyces hongheensis (Parapyrenochaetaceae) as the outgroup taxon.The full dataset consisted of 4413 characters, including gaps (SSU = 999 characters, LSU = 846, ITS1 + 5.8S + ITS2 = 663, tef 1 = 871, and rpb2 = 1034).The RAxML analysis of the combined dataset yielded a best-scoring tree with a final ML optimization likelihood value of −60,293.418721.The matrix had 2211 distinct alignment patterns, with 39.5% undetermined characters or gaps.We used the GTR + I + G model of the combined amplicons for the analysis, with the following parameters: estimated base frequencies of A = 0.245652, C = 0.237255, G = 0.265783, and T = 0.251310; substitution rates of AC = 1.259144,AG = 3.770291, AT = 2.003190, CG = 0.806828, CT = 7.047351, and GT = 1.000; proportion of invariable sites I = 0.575954; and gamma distribution shape parameter α = 0.641179.The Bayesian analysis ran 40,280,000 generations before the average standard deviation for split frequencies reached below 0.01 (0.009998).The analyses generated 40,281 trees, from which we sampled 30,211 trees after discarding the first 25% as burn-in.The alignment contained a total of 2217 (SSU: 340, LSU: 325, ITS: 461, tef 1: 415, and rpb2: 676) unique site patterns.Where applicable, the phylogenetic results obtained (Figure 1) are discussed in the descriptive notes below.Notes: This study presents an updated and comprehensive phylogenetic classification of the genus Paraloratospora, incorporating SSU, LSU, ITS, tef 1, and rpb2 DNA sequence analyses.By combining morphological and phylogenetic considerations, we have identified a new species, Paraloratospora sichuanensis, as well as proposed two new combinations, Paraloratospora breonadiae (=Phaeosphaeria breonadiae) and P. fructigena (=Phaeosphaeria fructigena), within the genus.Detailed information regarding these taxonomic changes can be found in the note sections, where an additional discussion and supporting evidence are provided.The discovery of Paraloratospora sichuanensis adds to the known diversity of Paraloratospora species and expands our knowledge of the ecological and morphological characteristics within the genus.The two newly proposed combinations, Paraloratospora breonadiae and P. fructigena, reflect the revised taxonomic understanding based on the integration of molecular and morphological data.These taxonomic changes contribute to the overall refinement and accuracy of the classification system for Paraloratospora.Descriptions and illustrations: See Crous et al. [30].Notes: The phylogenetic analysis conducted in this study yielded interesting results regarding the relationship between Phaeosphaeria breonadiae and P. fructigena with the species of Paraloratospora.The analysis revealed that type strains of these two species clustered together with Paraloratospora, forming a distinct group separate from the Phaeosphaeria sensu stricto (Figure 1).Based on these findings, it was determined that a reclassification of these two species within the genus Paraloratospora was warranted.Consequently, we propose the new combinations Paraloratospora breonadiae comb.nov.and P. fructigena comb.nov.for Phaeosphaeria breonadiae and P. fructigena, respectively.This reclassification aligns with the phylogenetic evidence and provides a more accurate taxonomic placement for these species within the Paraloratospora genus.The newly proposed combinations reflect the closer relationship and shared characteristics observed between these species and other members of Paraloratospora.

Pleosporales
Paraloratospora sichuanensis Maharachch.& Wanas.sp.nov.(Figure 2).Notes: Based on the phylogenetic analysis of multiple genes, Paraloratospora sichuanensis shares a close relationship with a putatively named strain called 'Phaeosphaeria' ), are semi-immersed to erumpent, solitary, scattered, and uniloculate, and can be globose to subglobose in shape, with a brown to dark brown color.The ostioles, which can reach up to 3-40 µm in diam., are centrally located and have a minute papilla.They are dark brown and consist of hyaline periphyses.The peridium is of unequal thickness, with a thicker portion near the apex (15-24 µm) and a thinner portion at the base (10-16 µm).It is composed of two layers, with the outer part consisting of dark brown thick-walled cells arranged in a textura angularis pattern and the inner layer made up of pale brown to hyaline thin-walled cells also arranged in a textura angularis pattern.The hamathecium is 2.5-4.5 µm wide, unbranched, septate, and composed of pseudoparaphyses that are constricted at the septum.The asci 55-90 7-14 µm (M = 62.3 × 11 µm, n = 15) are eight-spored, bitunicate, fissitunicate, cylindrical, sessile to subsessile, with a rounded apex and a thick wall.They are short pedicellate and possess a developed ocular chamber.The ascospores 20-28 × 3-5.5 µm (M = 23 × 4.5 µm, n = 25) are arranged in overlapping 1-2 seriate fashion and are 3-5 septate (mostly 5).The ascospores are hyaline to pale yellowish in color and have a fusiform shape with rounded ends.They can be straight to slightly curved and have a smooth surface.They are surrounded by a sheath.Asexual morph: the asexual form of this fungus is undetermined.
Culture characteristics: The ascospores germinated on PDA within 24 h.Following a two-week incubation period at 25 • C, the colonies on PDA medium reached a diameter of 5 cm.These colonies exhibited an undulate margin, initially appearing creamy whitish and transitioning to a creamy grey shade at the center.Towards the periphery, the color lightened further.The colonies were slightly raised in texture, with a creamy orange hue at the center and a creamy grey coloration towards the periphery when observed from the reverse side.
Notes: Phaeosphaeria fructigena, which was isolated from plant debris submerged in freshwater, is characterized by the production of clavate asci and fusiform ascospores.The sexual stage of P. fructigena has only been observed in both the original material and pure cultures by Magaña-Dueñas et al. [34].In our phylogenetic analysis, P. fructigena was grouped under Paraloratospora.Please refer to the notes under Paraloratospora breonadiae for further information.
Phaeosphaeria I. Miyake, Bot.Mag.(Tokyo) 23: 93 (1909).Notes: The genus Phaeosphaeria is renowned for its significant diversity, encompassing numerous described species and many more yet to be discovered.The presence of Phaeosphaeria species has been reported in various ecosystems, including terrestrial and freshwater environments [34,35].These fungi exhibit adaptability to a wide range of climatic conditions and substrates, allowing their distribution in both temperate and tropical regions.Within this genus, some species play a crucial role as saprophytes, participating in vital processes such as decomposition and nutrient cycling.They are commonly associated with decaying plant material, dead wood, or organic debris of monocotyledons.On the other hand, certain Phaeosphaeria species are recognized as plant pathogens, causing diseases in various hosts, including agricultural crops and forest trees [4,35].
In our study, we observed that six strains of our newly isolated species exhibit close resemblance to Phaeosphaeria strains based on BLAST similarity indices in GenBank.These findings underscore the importance of conducting further investigations and taxonomic revisions to ensure accurate classification and a better understanding of the relationships within the Phaeosphaeria genus.By utilizing a combination of morphological and molecular data from these new collections, we provide detailed descriptions of two new species within Phaeosphaeria.Additionally, we revised the species description for Phaeosphaeria poagena to update the characteristics of its sexual form.
Holotype: HKAS 129197.The fungus is saprobic, found on a dead twig of an unidentified deciduous host.Sexual state: The ascomata are 100-170 µm high, 90-150 µm in diam., and mostly scattered and immersed, with a uniloculate structure that is globose to subglobose and brown to dark brown in color.The peridium is 10-20 µm wide and consists of two layers: an outer layer composed of brown to dark brown cells arranged in a textura angularis pattern and an inner layer with loosely arranged, hyaline cells in a textura angularis to textura globulosa pattern.The hamathecium is made up of pseudoparaphyses, measuring 2-3 µm in width, which are cellular and indistinctly constricted at the septa.The asci measure 80-100 × 12-15 µm (M = 90.6 × 13.6 µm, n = 15) and are eight-spored, bitunicate, fissitunicate, broadly cylindrical, and have a pedicel.They are rounded at the apex and possess an ocular chamber.Ascospores measure 18-26 × 6.5-9 µm (M = 20.6 × 7.8 µm, n = 20) and are arranged as overlapping 1-2 seriate.They are phragmosporous, ellipsoidal with rounded ends, and initially yellowish brown but becoming pale brown as they mature.The ascospores are 4-5 septate and exhibit slight constriction at the septa, with the upper cell next to the middle septum being enlarged.Their surfaces are smooth walled.The asexual state was not observed.
Culture characteristics: Colonies on PDA reach a diameter of up to 3 cm after four weeks at 25 • C.These colonies have an irregular, flattened to slightly raised appearance, with a greenish-grey edge and a grey center.They also display various color sectors ranging from grey to dark grey.The reverse side of the colonies appears dark brown.
Culture characteristics: After four weeks at 25 • C, colonies on potato dextrose agar (PDA) attained a diameter of up to 4 cm.These colonies exhibit an irregular, flattened to slightly raised morphology and display various color sectors ranging from white to creamy orange.The reverse side of the colonies appears creamy orange, with occasional dark patches that can be observed.
Known distribution: Crous et al. [36] identified this fungus in the Netherlands (specifically in Raalte), where it was found on Poa sp.(Poaceae) in a terrestrial habitat.In this study, we found it in China (specifically in Sichuan) on deceased bamboo culms (Poaceae).The habitat where it was discovered is terrestrial and typically covered with snow throughout the year.which are hyaline, smooth, and doliiform.These cells exhibit prominent periclinal thickening or tightly aggregated percurrent proliferations.The conidia are solitary, brown, smooth, fusoid-ellipsoidal to subcylindrical, (1-)3 septate, and slightly constricted at the septa, with a subobtuse apex and a truncate base.They measure (8-)12-14(-16) × (2.5-)3(-3.5)μm.Notes: The asexual form of Phaeosphaeria poagena was isolated from Poa sp.(Poaceae) and was introduced by Crous et al. [36].Phaeosphaeria poagena is described as a new species because its small conidial dimensions do not match any of the asexual forms previously documented on Poa [32,33,37].In our study, two strains from a sexually reproductive fungus formed a monophyletic clade with the type strain of Phaeosphaeria poagena (CBS 136771) with 99% MLB and 1.00 BYPP support values.A comparison of the ITS nucleotides between our new strains and the type strain of Phaeosphaeria poagena revealed only four base pair differences.Unfortunately, no protein-coding sequence data from the type strain are available.Instead of introducing a new species, we propose considering the host similarities (Poaceae) and ITS sequences to link these asexual and sexual forms as a single species.Therefore, we revised the species description to update the characteristics of its sexual form.This finding represents a new record of this fungus from bamboo and provides protein sequence data (tef 1 and rpb2) for the species.Please refer to the notes under Phaeosphaeria chengduensis for further details.
It is saprobic on dead Pandanaceae leaves.The sexual state was not observed.The asexual state: conidiomata are 140-170 µm high, 150-200 µm in diam., pycnidial, scattered and immersed, and appearing as slightly raised small black dots on the surface of the host.They are uniloculate and can be globose to subglobose or irregular in shape, with a color ranging from brown to dark brown.The pycnidial wall is 10-15 µm wide and consists of two layers of brown cells.The outer margin is composed of cells arranged in a textura angularis pattern, while the inner layer is made up of hyaline to pale brown cells arranged in a textura angularis pattern.The conidiophores are reduced to conidiogenous cells, which are holoblastic, phialidic, and ampulliform in shape and measure 3-6 × 3.5-5.5 µm (M = 4.6 × 4.4 µm, n = 20).They can be either hyaline or pale brown.The conidia measure 7-10 × 3.5-5 µm (M = 8.4 × 4 µm, n = 30) and are ovoid to ellipsoid in shape, with a conically rounded apex and base.Initially, the conidia are hyaline and aseptate with guttules, but they become pale brown to brown and 1-2 septate as they mature.Occasionally, they may contain guttules.The conidia are not constricted at the septa and have a smooth surface.
Culture characteristics: Colonies on PDA reach a diameter of 2 cm after four weeks at 25 • C. Initially, the colonies are white but become creamy white when mature.They are dense in texture, and slight radiation can be observed.The reverse side of the colonies appears creamy orange at the edges, gradually becoming orange-brown to brown at the center.Notes: In our phylogenetic analysis, two strains (KUNCC 23-13568 and KUNCC 23-13569) from an asexual morphic fungus were found to be grouped with Phaeosphaeria species.Specifically, they formed the basal lineage of a monophyletic sister clade that included Phaeosphaeria ampeli (MFLUCC 19-0150 and MFLUCC 18-1641), P. chengduensis (KUNCC 23-13570 and KUNCC 23-13571), P. chinensis (KUMCC 19-0161 and MFLUCC 19-0217), P. poagena (KUNCC 23-13572 and KUNCC 23-13573), and P. sinensis (NCYUCC 19-0369).Morphologically, the conidia of the new fungus differ from other Phaeosphaeria species in terms of shape and the number of septa.Typically, Phaeosphaeria species produce fusiform conidia with multiple vertical septa [32,33].However, this new fungus has ovoid to ellipsoid conidia with only 1-2 vertical septa.Nevertheless, the ampulliform, holoblastic, and conidiogenous cells of the new species bear resemblance to the asexual morphs in Phaeosphaeria.
Septoriella Oudem., Ned.Kruidk.Arch.ser.Notes: Septoriella was initially described by Oudemans [38], but it received limited attention, and only a few species were recognized.However, a recent study by Crous et al. [39] redefined the genus and established an epitype (CBS H-22281) and an ex-epitype strain (CBS 140065) for the type species, Septoriella phragmitis.The genus exhibits a wide distribution and encompasses a diverse array of species.Septoriella species are commonly associated with various host plants, including grasses, cereals, and other herbaceous plants [40].Over the years, the taxonomy and classification of Septoriella have undergone significant revisions, with the discovery of new species and re-evaluation of existing ones using molecular and morphological data [40][41][42][43].In the study of Marin-Felix [40], the ex-type strains of the sexual genera Allophaeosphaeria, Poaceicola, and Vagicola were found within the clade representing the genus Septoriella.Consequently, these genera were synonymized with Septoriella in their research.Subsequent to the research conducted by Crous et al. [39], later studies have contributed to the addition of 29 recognized species to the genus Septoriella [2].However, it is crucial to note that this number is subject to change, as new species are continually being discovered and identified.Taxonomic studies have utilized various approaches, including molecular phylogenetics, morphology, and host specificity, to delineate and classify different species within Septoriella.In this study, we propose the synonymization of Amarenographium ammophilicola, Amarenomyces dactylidis, Dactylidina shoemakeri, Hydeopsis verrucispora, Loratospora luzulae, Phaeopoacea asparagicola, P. festucae, and P. muriformis under Septoriella.
Septoriella Descriptions and illustrations.See Dayarathne et al. [44].Notes: Dayarathne et al. [44] introduced Amarenographium ammophilicola based on its morphological characteristics and phylogenetic analysis.In our phylogenetic analysis, the type strain of A. ammophilicola clustered with other Septoriella species within the Phaeosphaeriaceae (Figure 1).Therefore, we propose the transfer of Amarenographium ammophilicola to the genus Septoriella, and subsequently it should be recognized as Septoriella ammophilicola, comb.nov.
Descriptions and illustrations: See Hyde et al. [19].Notes: Thambugala et al. [42] established the genus Phaeopoacea to accommodate P. festucae and P. phragmiticola (≡Phaeosphaeria phragmiticola).The type species, P. festucae, was previously known only from its asexual morph, which forms pycnidial conidiomata that are globose to subglobose or linear in rows on the host, producing brown to dark brown, oblong conidia [42].Meanwhile, Phaeosphaeria phragmiticola was previously considered a synonym of Septoriella leuchtmannii by Crous et al. [39].However, based on multi-gene phylogenetic analyses conducted by Li et al. [41] and Thambugala et al. [42], Phaeosphaeria phragmiticola was placed within Phaeopoacea.Hyde et al. [19,45] later included two additional species, P. asparagicola and P. muriformis, in this genus.The phylogenetic analysis by various authors revealed that Phaeopoacea does not form a clearly defined clade and consistently clusters with the genera Allophaeosphaeria, Amarenographium, Amarenomyces, Dactylidina, Poaceicola, Septoriella, and Vagicola [35].In our phylogenetic analysis, the three type strains of Phaeopoacea, P. asparagicola (MFLUCC 16-0379), P. festucae (MFLUCC 17-0056), and P. muriformis (MFLUCC 17-0372) were found to be distantly related to each other within Septoriella.To provide better clarity and avoid unresolved taxonomic circumscriptions, we propose the synonymization of these three species under Septoriella.As a result, we   (j,l) 10 µm, scale bar of (d) applies to (e), scale bar of (g) applies to (g-i), scale bar of (j) applies to (k), and scale bar of (l) applies to (l-o).
Culture characteristics: The ascospores showed germination on PDA within 24 h.After a two-week incubation period at 25 • C, the colonies on PDA medium grew to a diameter of 4 cm.These colonies had an undulate margin and initially appeared whitish grey, transitioning to a grey shade with an irregularly raised texture at the center.Towards the periphery, the color became greenish grey.When observed from the reverse side, the colonies displayed a blackish brown color at the center and a creamy grey coloration towards the periphery.Notes: Our phylogenetic analyses have revealed that the new strain KUNCC 23-14219 is closely related to the other isolates of Septoriella paradactylidis (=Amarenomyces dactylidis), including the type strain (MFLU 17-0498).Further investigations comparing our isolate to the type species have revealed a similar size range of the ascomata, asci, and ascospores, as well as the ascospore septation [45].Therefore, we are now reporting the presence of S. neodactylidis in Lolium perenne, which represents the first record of this fungus in that host and provides protein sequence data (tef 1 and rpb2) for this species.Since the name 'Septoriella dactylidis' is already in use within the Septoriella genus, we have utilized a nomenclature novelty (Septoriella paradactylidis) for this synonymization.Descriptions and illustrations: See Wanasinghe et al. [48].Notes: In Wanasinghe et al. [48], they described Dactylidina shoemakeri based on its morphological traits and a conducted phylogenetic analysis.However, it should be noted that this species has not been validly published (Nom.inval., Art.35.1 (Shenzhen)).In our phylogenetic analysis, we found that the strain MFLUCC 14-0963, which was previously invalidly published as Dactylidina shoemakeri, grouped with Septoriella species and showed a close affiliation to S. neodactylidis (MFLUCC 14-0966).Therefore, we propose reassigning Dactylidina shoemakeri to the genus Septoriella, and going forward, it should be referred to as Septoriella shoemakeri.

Septoriella shoemakeri
Septoriella verrucispora (J.F.Descriptions and illustrations: See Zhang et al. [49].Notes: The monotypic genus Hydeopsis, typified by H. verrucispora, was introduced by Zhang et al. [49].It exhibited a close phylogenetic relationship with Dactylidina and Phaeopoacea within the family Phaeosphaeriaceae.The authors differentiated Hydeopsis from these genera based on differences in pigmentation and the number of septa in ascospores.Our combined sequence phylogenetic analysis reveals that Hydeopsis verrucispora is phylogenetically positioned within the genus Septoriella.Additionally, pigmentation and the number of septa appear to be highly informative at the species level, although these characteristics alone cannot reliably distinguish between genera.Species of Septoriella also share more similar characteristics.Therefore, Hydeopsis is determined to be congeneric with Septoriella, and as a result, we propose assigning it to the genus Septoriella as a new combination.

Discussion
The delimitation of species in Phaeosphaeriaceae is typically based on a combination of morphological and molecular characteristics.Some of the major morphological features used to delimitate species in this family include fruiting body type, peridium, spores, asci, conidiogenous cells, and colony morphology [4].The shape, size, and color of ascospores and conidia can vary between different species of Phaeosphaeriaceae.However, the usefulness of these morphological features for species delimitation can vary depending on the species and the taxonomic group being studied.There are several challenges in using morphological features for species delimitation in Ascomycota, including in Phaeosphaeriaceae.Morphological variation within species of Phaeosphaeriaceae can pose challenges in distinguishing closely related species.For instance, in the case of Septoriella species, there are variations in ascospore features [40].These include the arrangement of 1-3 seriate in an ascus, colors ranging from hyaline to yellowish-brown, pale brown, brown, golden brown, or reddish-brown, and shapes that can be narrowly or broadly fusiform, oblong, or narrowly oblong.The surface can be smooth-walled or echinulate, with transverse or longitudinal septa and sometimes enlarged medium cells.The ends of the ascospores can be conical, obtuse, or rounded, and they may or may not have sheaths.Furthermore, even the conidia morphology of the asexual morphs within these species can vary [39], with shapes ranging from cylindrical to subcylindrical, fusiform, or subfusiform.The apex can be obtuse or subobtuse, the base truncate, and the conidia can be straight or curved.They are euseptate, pale brown to brown, and can have thin-walled, smooth surfaces or minutely verruculose surfaces.Additionally, they may bear mucoid appendages at both ends.
Cryptic or polyphyletic species within Phaeosphaeriaceae are morphologically similar but genetically distinct, making it challenging to differentiate them based solely on morphology.This can be observed in sexual morphs resembling Phaeosphaeria or Ophiobolus, as well as asexual morphs with a camarosporium-like resemblance (i.e., Melnikia, Camarosporioides, and Dlhawksworthia).Convergence, where different species develop similar traits due to similar ecological pressures, can contribute to this morphological similarity.Moreover, variations in methods used to observe and measure morphological features can lead to inconsistencies in species delineation.Careful examination of old literature is crucial to avoid mistakenly introducing existing species as new ones.Additionally, limited information on the morphological features of certain Phaeosphaeriaceae species in old-type materials makes accurate delimitation based on morphology alone difficult.To overcome these challenges, a combination of morphological and molecular approaches is essential for species delineation in Phaeosphaeriaceae.Molecular data can provide valuable insights into genetic variation and evolutionary relationships, aiding in the resolution of taxonomic uncertainties and differentiation between closely related species with similar morphology.
The taxonomy of the Phaeosphaeria genus is continuously evolving, with ongoing research and the utilization of molecular techniques aiding in the identification and classification of new species.Molecular phylogenetic analyses, in conjunction with morphological studies, have contributed to the clarification of species boundaries and relationships within the genus.The diversity of Phaeosphaeria is evident through the wide range of hosts, substrates, and ecological roles displayed by its various species.Recent phylogenetic analyses have revealed that Phaeosphaeria is polyphyletic, leading to the reclassification of many Phaeosphaeria sensu lato species into different genera within Phaeosphaeriaceae [37,40,43,45,46,50,51].
Based on our multi-gene phylogenetic analysis, we observed that Paraloratospora is closely related to Loratospora aestuarii (JK 5535B), Sulcispora pleurospora (CBS 460.84), S. supratumida (MFLUCC 14-0995), and Wingfieldomyces cyperi (CBS 141450), forming a sister relationship.However, it is important to note that these species were represented by single strains.Therefore, further evaluation of their inter-generic relationships should be conducted with additional species/collections and through DNA-based sequence data analyses.Currently, only three species are accepted in Paraloratospora, viz., P. camporesii, P. gahniae, and P. marina [52].In our analysis, two type strains of Phaeosphaeria breonadiae (CPC 25944) and P. fructigena (FMR 17808) were grouped among these three Paraloratospora species.Additionally, two of our new strains (KUNCC 23-14218 and HKAS 129218) also clustered with Paraloratospora species.Consequently, we synonymized Phaeosphaeria breonadiae and P. fructigena under Paraloratospora, and introduced our new strains as belonging to a new species.Furthermore, strains provisionally named as Phaeosphaeria avenaria f. sp.tritici (CBS 289.52),P. caricicola (CBS 603.86),P. eustoma (CBS 307.71 and CBS 724.92), P. glyceriae-plicatae (CBS 101261), P. juncophila (CBS 575.86),P. norfolcia (CBS 593.86), and P. parvula (CBS 260.49 and CBS 605.86) were also grouped within the genus Paraloratospora.However, since these strains are not related to their type materials, we did not transfer these species to Paraloratospora in our analysis.Further studies are needed to resolve the phylogenetic classification of the aforementioned species.
In our analysis, we primarily utilized single-gene phylogenetic analyses but obtained a more robust topology through the combined gene analysis (SSU + LSU + ITS + tef 1 + rpb2).For molecular comparisons, ITS sequences were available for 97.7% of the species in the family, while LSU sequences were available for 98.8%.However, the availability of sequence data for protein-coding genes, such as tef 1 and rpb2, was limited in previous studies, with tef 1 available for 54.6% of the species and rpb2 available for 32.7%.By incorporating more informative gene data and conducting extensive sampling worldwide, it is anticipated that most of the monotypic species will be transferred to a few genera within the family.One example of this is the synonymization of Hydeopsis under Septoriella in this study.Based on morphological, phylogenetic, and ecological evidence, we have introduced four new species, seven new combinations, and two new names in the genera Paraloratospora, Phaeosphaeria, and Septoriella.Despite the advancements in our understanding of these genera, several challenges still persist.Differentiating closely related species remains a significant obstacle, necessitating further investigation and the integration of multiple data sources.Moreover, the limited knowledge regarding the life cycles, host range, and geographic distribution of many of these species hinders our comprehension of their ecological roles.To overcome these challenges, future research should prioritize the utilization of advanced molecular techniques such as genomics, transcriptomics, and metagenomics.These approaches can provide insights into the genetic diversity, evolutionary history, and ecological functions of Paraloratospora, Phaeosphaeria, and Septoriella.By employing these cutting-edge methods, we can unravel the intricate complexities within these genera and gain a more comprehensive understanding of their significance in the ecosystem.

Figure 1 .
Figure 1.RAxML tree based on a combined dataset of partial SSU, LSU, ITS, tef1, and rpb2 DNA sequence analyses in Phaeosphaeriaceae.The bootstrap support values for ML (MLB) greater than or equal to 70% and Bayesian posterior probabilities (BYPP) greater than or equal to 0.95 are indicated as MLB/BYPP above the corresponding nodes.The newly analyzed isolates are highlighted in blue.The scale bar in the figure represents the expected number of nucleotide substitutions per site.

3. 2
. Taxonomy Pleosporales Luttr.ex M.E.Barr, Prodromus to class Loculoascomycetes: 67 (1987).Phaeosphaeriaceae M.E.Barr, Mycologia 71: 948 (1979).Paraloratospora Bundhun, Tennakoon, Phookamsak & K.D. Hyde, Fungal Diversity 100: 101 (2020).Notes: This study presents an updated and comprehensive phylogenetic classification of the genus Paraloratospora, incorporating SSU, LSU, ITS, tef1, and rpb2 DNA sequence analyses.By combining morphological and phylogenetic considerations, we have identified a new species, Paraloratospora sichuanensis, as well as proposed two new combinations, Paraloratospora breonadiae (=Phaeosphaeria breonadiae) and P. fructigena (=Phaeosphaeria fructigena), within the genus.Detailed information regarding these taxonomic changes can be found in the note sections, where an additional discussion and supporting evidence are provided.The discovery of Paraloratospora sichuanensis adds to the known diversity of Paraloratospora species and expands our knowledge of the ecological and morphological characteristics within the genus.The two newly proposed combinations, Paraloratospora breonadiae and P. fructigena, reflect the revised taxonomic understanding based on the integration of molecular and morphological data.These taxonomic changes contribute to the overall refinement and accuracy of the classification system for Paraloratospora.Paraloratospora breonadiae (Crous & Jol.Roux) Maharachch.& Wanas.comb.nov.MycoBank: MB 849359.

Figure 1 .
Figure 1.RAxML tree based on a combined dataset of partial SSU, LSU, ITS, tef 1, and rpb2 DNA sequence analyses in Phaeosphaeriaceae.The bootstrap support values for ML (MLB) greater than or equal to 70% and Bayesian posterior probabilities (BYPP) greater than or equal to 0.95 are indicated as MLB/BYPP above the corresponding nodes.The newly analyzed isolates are highlighted in blue.The scale bar in the figure represents the expected number of nucleotide substitutions per site.Paraloratosporabreonadiae (Crous & Jol.Roux) Maharachch.& Wanas.comb.nov.MycoBank: MB 849359.≡Phaeosphaeria breonadiae Crous & Jol.Roux, Persoonia 36: 399 (2016).Holotype: South Africa, Limpopo Province, Wolkberg, on leaves of Breonadia microcephala, January 2015, J. Roux (CBS H-22631, holotype), ex-type, CPC 25944, CBS 141334.Descriptions and illustrations: See Crous et al.[30].Notes: The phylogenetic analysis conducted in this study yielded interesting results regarding the relationship between Phaeosphaeria breonadiae and P. fructigena with the species of Paraloratospora.The analysis revealed that type strains of these two species clustered together with Paraloratospora, forming a distinct group separate from the Phaeosphaeria sensu stricto (Figure1).Based on these findings, it was determined that a reclassification of these two species within the genus Paraloratospora was warranted.Consequently, we propose the new combinations Paraloratospora breonadiae comb.nov.and P. fructigena comb.nov.for Phaeosphaeria breonadiae and P. fructigena, respectively.This reclassification aligns with the phylogenetic evidence and provides a more accurate taxonomic placement for these species within the Paraloratospora genus.The newly proposed combinations reflect the closer relationship and shared characteristics observed between these species and other members of Paraloratospora.Paraloratospora sichuanensis Maharachch.& Wanas.sp.nov.(Figure2).

Author
Contributions: Conceptualization, D.N.W. and S.S.N.M.; resources, S.S.N.M.; writingoriginal draft preparation, D.N.W.; writing-review and editing, S.S.N.M.; funding acquisition, S.S.N.M.All authors have read and agreed to the published version of the manuscript.