﻿Recommendations on approving the name “ Entomosporium”, with a new species, E.dichotomanthes from China (Leotiomycetes, Drepanopezizaceae)

﻿Abstract The phytopathogenic genus, Entomosporium can cause serious leaf diseases worldwide. Entomosporium has long been regarded as a synonym of Diplocarpon. However, different morphologies between Entomosporium and Diplocarpon make this doubtful. Based on morpho-phylogenetic analyses, the placement of the genus was re-evaluated in this study. The combined the internal transcribed spacer gene region (ITS) and the 28S large subunit ribosomal RNA gene region (LSU) phylogenetic analysis shows that Entomosporium is an independent clade within Drepanopezizaceae and formed a sister clade to the generic type Diplocarpon. Moreover, Hymenula and Pseudopeziza do not cluster in Drepanopezizaceae. We propose to resurrect the name Entomosporium, and exclude Hymenulacerealis and Pseudopezizamedicaginis from Drepanopezizaceae and propose to treat them under Ploettnerulaceae. A new species, E.dichotomanthes is also introduced from China based on morpho-molecular analyses which is associated with Dichotomanthestristaniicarpa.


Introduction
Entomosporium Lév, a synonym of Diplocarpon F.A. Wolf, is a member of the strongly plant-pathogenic family Drepanopezizaceae (Holtslag et al. 2003;Nunes et al. 2016;Johnston et al. 2019;Wöhner and Emeriewen 2019).The Entomosporium species causes entomosporium leaf disease worldwide and frequently occurs as an epidemic (Bogo et al. 2018).As many species are described without molecular data, the relationship with Diplocarpon species remains unclear.Although Diplocarpon species are common and widespread, studies on Diplocarpon have predominantly focused on their phytopathology, with the taxonomy utilizing molecular markers being largely overlooked (Wijayawardene et al. 2017;Ekanayaka et al. 2019).
Although genera, such as Entomopeziza, Entomosporium and Morthiera have morphological similarities to Diplocarpon, it is perplexing that they are considered as synonyms.For example, 15 epithets of Entomosporium were regarded as D. mespili, as the sexual stage of Entomosporium morphologically resembles Diplocarpon (Naoui 2013;Johnston et al. 2014).However, Entomosporium produces cruciform, insect-like, 2-6-celled conidia, which is distinct from the conidia of Diplocarpon (Stowell and Backus 1966).Moreover, Entomosporium species are widely distributed in Argentina, Australia, Brazil, Canada, China, India, Israel, Italy, Japan, New Zealand, North America, Pakistan, and South Africa, on a wide host range of Rosaceae (Stowell and Backus 1966;Cariddi et al. 2009;Batool et al. 2014).Diplocarpon on the other hand, is mostly or specifically parasitic on herbaceous Rosaceae or low shrubs.The proposal to adopt Diplocarpon over Entomosporium is doubtful (Horie and Kobayashi 1980;Wijayawardene et al. 2021).The hypothesis that Diplocarpon mespili did not speciate with its worldwide spread should be re-evaluated (Chethana et al. 2021).An example of evidence is that Entomosporium sp. from Japan has more lateral cells (2-4) (Horie and Kobayashi 1979).Chen et al. (2022) introduced a new species with insect-like conidia but under the name "Diplocarpon".In Index Fungorum (https://www.indexfungorum.org,23 Nov 2023), 12 Diplocarpon species are recorded, namely D. alpestre, D. coronariae, D. earlianum, D. fragariae, D. hymenaeae, D. impressum, D. mali, D. mespili, D. mespilicola, D. polygoni, D. rosae and D. saponariae.We are studying the pathogens of urban and forest tree species in Yunnan Province (Thiyagaraja et al. 2024) and in this study Entomosporium leaf disease was found in Dichotomanthes tristaniicarpa and has not been reported before.Dichotomanthes is endemic to Yunnan and Sichuan provinces in China (Zhou et al. 2000).It belongs to Rosaceae, with only one species D. tristaniicarpa which is a rare evergreen shrub tree, and is used as ornamental and medicinal plants (Tang et al. 2010;Yang et al. 2018).The ITS sequence blastn search of the newly generated sequences showed the close hits to Diplocarpon, and identified it as a new species based on the evidence from both morphology and phylogeny.Since the increasing number of members and updating molecular data of Diplocarpon, this study has provided an opportunity for a better understanding of the taxonomy of the genus.In this study, we interpret the relationship between Entomosporium and Diplocarpon, and further re-evaluate the taxonomy of Drepanopezizaceae.

Sampling, isolation and morphological observations
Leaves with lesions of Dichotomanthes tristaniicarpa were collected from Yunnan Province.For single-spore isolation, the fruit bodies were transferred to sterilized water in a centrifuge tube using a syringe needle, then crushed into pieces using pipette tips.Subsequently, 200 μL of the spore suspension was transferred to potato dextrose agar (PDA) using a micropipette (Zhang et al. 2013).For tissue isolation, the leaves were washed with distilled water for 1 minute and then air-dried.The margins of the disease lesions were cut into fragments (0.5 × 0.5 cm) under aseptic conditions.These fragments were surface-sterilized with 75% ethanol for 30 seconds, followed by dipping in 1% sodium hypochlorite for 40 seconds.They were then rinsed three times in sterile demineralized distilled water before being transferred onto a PDA plate, with four fragments per plate (Senanayake et al. 2020).The Petri dishes were incubated in the dark at 25 °C.Specimens were deposited at the Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences (KUN-HKAS).Morphological observations were performed using Nikon SMZ745T dissecting microscope (DM) and Nikon Eclipse 80i compound microscope, equipped with IMG Camera SC2000C.Index Fungorum and Facesoffungi numbers were obtained as in Index Fungorum (https://www.indexfungorum.org/)and Jayasiri et al. (2015) and the details of the fungus were deposited in the Greater Mekong Subregion database (Chaiwan et al. 2021).

DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted by using Lysis Buffer for Microorganism to Direct PCR (Takara), following the user manual.PCR amplifications were performed in T100 Thermal Cycler (T100™, Bio-Rad, USA) with ingredients of 21 µL Golden-Star T6 Super PCR Mix (Tsingke), 1 µL (10 µM) of each primer and 2 µL DNA template.Amplification conditions include 3 min initial denaturation at 95 °C, followed by 35 cycles of 95 °C denaturation for 15 s, 53 °C ~ 56 °C annealing for 15 min, 72 °C extension for 20 s, followed by a final extension at 72 °C for 5 min.The primer set ITS5/ITS4 (White et al. 1990) was used to amplify the internal transcribed spacer gene region (ITS); and LROR/LR5 for the 28S large subunit ribosomal RNA gene region (LSU) (Vilgalys and Hester 1990;White et al. 1990) and 983F/2218R for translation elongation factor 1-alpha gene region (tef-α) (Rehner and Buckley 2005).PCR products were purified and sequenced by Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China.

Phylogenetic analyses
Reverse and forward sequences were assembled using Chromas Pro (2.1.8)and initial identification was subjected to the NCBI (https://www.ncbi.nlm.nih.gov/) using BLAST search.Sequences of similar taxa were retrieved from the NCBI, and additional reference sequence selections based on Johnston et al. (2019) were downloaded from the DataStore (https://datastore.landcareresearch.co.nz/).The alignment was constructed with the online tool MAFFT v.7 (http://mafft.cbrc.jp/alignment/server)(Katoh and Standley 2013), and refined using BioEdit v. 7.7.1 (Hall 1999).The final combined data matrix was converted by the online tool ALTER (https://www.sing-group.org/ALTER/)(Glez-Peña et al. 2010).A quick Phylogenetic analysis was conducted using OFPT (Zeng et al. 2023) following its default protocol.The final Phylogenetic analyses were conducted on the CIPRES Science Gateway platform (https://www.phylo.org),using tools of RAxML-HPC v.8 on XSEDE (8.2.12) for maximum likelihood (ML) and MrBayes on XSEDE (3.2.7a) for Bayesian inference (BI).In the Bayesian inference, the best optimal substitution model was determined by using Mod-elFinder (Kalyaanamoorthy et al. 2017) under the Bayesian information criterion (BIC).The final phylogenetic tree was visualized with FigTree v. 1.4.4 and edited using Adobe Photoshop CS6 version 10.0.Sequences of the new strain generated in this study are deposited in GenBank (Table 1).

Results
A total of 50 ingroup taxa from Drepanopezizaceae, Hyaloscyphaceae, Ploettnerulaceae and Vibrisseaceae were used in the phylogenetic tree analysis, of which 20 species were from the type (Fig. 1).In total, 31 isolates contained all extant species that have available molecular data within Drepanopezizaceae.The combined LSU and ITS yield a 1409 bp alignment, with the best substitution models for each summarised as TIM2e+I+G4 and TIM2+F+R3, respectively.
Phylogenetic analysis demonstrated that Diplocarpon divided into two phylogenetically close relative clades, Diplocarpon and Entomosporium.Diplocarpon clade is composed of D. coronariae (from China, Japan, Korea and the USA), D. earlianum (unknown country) and D. rose (from China, Germany and an unknown country).Those three species have common characteristics of two-celled conidia.The new species Entomosporium dichotomanthes (from China), along with E. mespili (from England, Korea and an unknown country) and E. mespilicola (from China) consisted of clade Entomosporium, which showed insect-like conidia.Moreover, Hymenula cerealis and Pseudopeziza medicaginis were within Ploettnerulaceae.
Sequence comparison reveals the intergeneric and interspecific variation (Fig. 2).ITS sequence shows a high nucleotide variation within Diplocarpon, with an average of 58.1, compared to Diplocarpon, the Entomosporium, Blumeriella, Drepanopeziza and Thedgonia have an average of 62.6, 68, 71.3 and 76, respectively.The sequence comparison results align with the phylogenetic analysis, indicating that the closely related species exhibit less nucleotide variation.The LSU sequences have a lower variation.The Diplocarpon has an average of 28, and the Drepanopeziza, Blumeriella, Entomosporium and Thedgonia have an average of 35.5, 37, 40, and 44, respectively.The interspecific variation of Drepanopeziza and Entomosporium is 26.8 and 41.3.

Taxonomy Drepanopezizaceae Baral
MycoBank No: 828889 Facesoffungi Number: FoF05864 Fig. 3 Type.Drepanopeziza (Kleb.)Jaap 1914.Description.Sexual morph: Ascomata small-sized, up to 2 mm in diameter, apothecial, cupulate, margin often protruding, with or without lobes, sessile and mostly immersed.Excipulum is composed of cells of textura angularis.Paraphyses hyaline, thin-walled, aseptate or septate, apically swollen.Asci 4-8-spored, clavate or cylindrical, apex obtuse to conical, with or without apical Notes.Drepanopezizaceae was described with sexual and asexual morphs.Both life morphs were found as parasitic on leaves of various dicotyledons, and rarely on herbaceous (Johnston et al. 2019).The sexual morph is recognized by the cupulate, apothecial ascomata, and the paraphyses with swollen apical (Harada et al. 1974;Williamson and Bernard 1988;Spiers and Hopcroft 1998).The asexual morph is acervular but varies in conidial shape among genera (Crous et al. 2009;Khodadadi et al. 2022).The family name was first time used by Batista and Maia (1960), but was invalid because of unavailable diagnosis or description (Johnston et al. 2019).It was difficult to trace back the history of members accommodated in the family until Johnston et al. (2019), validated the family name based on the phylogenetic analysis (    (Stowell and Backus 1966;Horie and Kobayashi 1980).Historically, Entomosporium is composed of multiple morphologically indistinguishable species.Sivanesan and Gibson combined all species to E. mespili, but they did not mention their taxonomic basis (Horie and Kobayashi 1980).Atkinson recorded the process by which ascospores from a cupulate fungi formed the conidia of E. maculatum, and named the species as Fabraea maculata, while he later proposed that F. maculata may be identical to E. mespili (Atkinson 1897(Atkinson , 1909)).Taxonomic status changed for the morphologically similar taxa, viz.Diplocarpon, Entomopeziza, Fabraea and Marssonina (Stowell and Backus 1967;Johnston et al. 2014). After versions, Jørstad (1945) combined Diplocarpon and Entomosporium, and recognized Atkinson's collection as the type.This opinion was also discussed by Stowell and Backus (1967), but they failed the verification through experiments.The mystery of Entomosporium associated with sexual morph is still not confirmed by molecular data, since no new collection was found in sexual stage in recent decades.
Notes.Entomosporium dichotomanthes is characterized by having three to four cells of conidia.Its morphology resembles D. mespili and D. mespilicola, but has different host plants association and distribution.E. dichotomanthes is easily detectable on the host substrate in the mountains around the lake of Longchuanqiao Park.However, we couldn't find this fungus on nearby plants of the host, or on other plants in the mountains.We also failed to isolate the culture by using both single spore isolation and tissue isolation methods which indicates E. dichotomanthes strictly rely on D. tristaniicarpa.

Discussion
The taxonomic status and the phylogenetic relationship of Diplocarpon and Entomosporium in Drepanopezizaceae were assessed in this study.We included all extant species with molecular data in Drepanopezizaceae, as well as most genera of its sister family Ploettnerulaceae for the first time.Upon molecular phylogenetic analysis, Diplocarpon divided into two distinct clades representing Entomosporium and Diplocarpon.Sequence comparison reveals the average nucleotide variation of Blumeriella, Drepanopeziza, Entomosporiummm, and Thedgonia is higher than Diplocarpon which means intergeneric variation is greater than interspecific variation.Moreover, Diplocarpon and Entomosporium have a high nucleotide variation compared to the more speciose genus Drepanopeziza.Consequently, Entomosporium recovered separately from Diplocarpon and should not assign all species to E. mespili.On the plant host (Table 3), D. rose is commonly reported on Roses, D. earlianum on strawberries and D. coronaria on apple trees.However, Entomosporium has a wide host range of woody plants like shrubs and trees, such as apple, hawthorn, saskatoon and pear (Holtslag et al. 2004;Nunes et al. 2016;Thurn et al. 2019).Likewise, the morphology features of conidia sustained the difference, Entomosporium displays insect-like conidia while Diplocarpon produces 2-celled conidia.Johnston et al. (2014) stated that Entomosporium and Diplocarpon are conspecific due to the linked asexual and sexual morphs, but this was not confirmed by molecular data.Conclusively, our study based on morphology coupled with molecular data supported the division of these genera.Entomosporium leaf disease is mainly associated with Entomosporium species (Holtslag et al. 2003;Seo et al. 2010;Nunes et al. 2016).
Hence, the classification system of Diplocarpon was revised.We propose to recover the validity of the genus name "Entomosporium", to accommodate species that have insect-like conidia species in Drepanopezizaceae.Furthermore, we introduced a new species E. dichotomanthes from China.Its taxonomical placement is basal in the "Entomosporium" clade supported by high bootstrap.
The disease symptom appeared as black spots or irregular black stripes on the upper side of the mature leaf of Dichotomanthes tristaniicarpa, which was easily recognizable.We also generated the first sequence of the tef1-α gene for Entomosporium, from E. dichotomanthes.However, we used only LSU and ITS sequences data in our study, since scant tef1-α sequences data are available for reference taxa that cannot be used in this phylogenetic analysis.The blast against NCBI shows the tef1-α sequences have highest similarity with D. coronariae (MT674914) and Hyaloscypha fuckelii (MT254572), gained the value of 884/948(93%) and 860/948(91%), respectively.Correspondingly, Hymenula cerealis and Pseudopeziza medicaginis were not clustered in Drepanopezizaceae in our phylogenetic tree.However, Hymenula was recovered within Drepanopezizaceae in Zhu et al. (2023).Further, the type material of H. cerealis (= Cephalosporium gramineum, CBS 132.34) was used in their study and obtained a good statistical support (MLBP/BIPP = 96%/100%), in the phylogenetic analyses conducted based on the combined five-gene data set.However, only H. cerealis as well as a small group of taxa from both Drepanopezizaceae and Ploettnerulaceae were applied in their phylogenetic analysis.The disease caused by Hymenula is cephalosporium stripe on herbaceous plants that is different from Drepanopezizaceae (Wiese and Ravenscroft 1978;Zhu et al. 2023).Similar situations with Hymenula, Pseudopeziza cause black spot disease mostly found on Alfalfa and Red clover (Fabaceae), not on Rosaceae plants (Jones 1919;Meyer and Luttrell 1986;Yuan et al. 2007).The taxonomy of Pseudopeziza is confusing (Meyer and Luttrell 1986).There are 135 species epithets that have been linked to Pseudopeziza in Index Fungorum (https://www.indexfungorum.org), of which many names have been transferred  (2009) to other families, such as Diaporthaceae, Ploettnerulaceae and Rhytismataceae.Only three sequences labeled as Pseudopeziza were accessible in the Gen-Bank, and P. medicaginis (CBS 283.55) was used in this study.Morphologically, Hymenula was only found in the asexual stage.Meanwhile, the asexual morph of Drepanopezizaceae does not share highly persuasive common morphological characteristics for delimiting its generic members.The morphology of P. medicaginis fits Drepanopezizaceae (Jones 1919), but differs in having indistinctive swollen apical paraphyses (Meyer and Luttrell 1986).Thus, we propose to exclude H. cerealis and P. medicaginis from Drepanopezizaceae and to treat them under Ploettnerulaceae.

Figure 1 .
Figure 1.Maximum likelihood phylogenetic tree inferred from combined LSU and ITS sequence data of Drepanopezizaceae and its closely related families.The tree is artificially rooted with Leuconeurospora capsici (CBS:176.44)and Leuconeurospora pulcherrima (AFTOL-ID 1397).Maximum likelihood bootstrap values ≥65% and Bayesian Posterior Probabilities (BYPP) ≥ 0.90 are given at the nodes.Novel taxon is in bold.Type sequences are labeled asterisk (*).

Figure 2 .
Figure2.Intergeneric and interspecific variation analysis A mean of ITS sequence variation within genera B means of LSU sequence variation within genera C ITS sequence variation of the query sequence and the subject, "S" is the subject, "x" is the mean value of nucleotide variation within species.

Table 1 .
GenBank accession numbers used in the phylogenetic analyses.
Type strains are marked with "T", and strains from the present study are in black bold.

Table 2 .
Main versions of classification of Drepanopezizaceae and its accepted genera.
thick-walled, unequal, the upper cell slightly lager.Asexual morph: Conidiomata solitary to gregarious or confluent, mostly epiphyllous, acervulus.Conidiogenesis hyaline, cylindrical, holoblastic.Conidia 2-6-celled, hyaline, thin-walled, cruciform or insect-like, basal cell developed from the conidiogenous cell, cylindrical, globose to obovate, and other cells attached basal cell in both upper sides and apex, apical cell larger, globose to subglobose, lateral cells globose to ellipsoidal, smaller than the apical and basal cells, the apical and basal cells with a tubular appendage.Notes.Entomosporium was erected by Leveille in 1856, based on E. maculatum from leaves of Pyrus communis (Rosaceae), and was characterized by 4-celled, cross-like conidia

Table 3 .
Diplocarpon species documented from different countries and plant host.