Multi-Gene Phylogeny and Morphology Reveal Haplohelminthosporium gen. nov. and Helminthosporiella gen. nov. Associated with Palms in Thailand and A Checklist for Helminthosporium Reported Worldwide

Palms (Arecaceae) are substrates for a highly diverse range of fungi. Many species are known as saprobes and many are important plant pathogens. Over the course of our studies of micro-fungi from palms in Thailand, two new taxa were discovered. Morphological characteristics and phylogenetic analyses of combined ITS, LSU, SSU, and tef1-α sequence data revealed their taxonomic positions within Massarinaceae. There are currently ten genera identified and accepted in Massarinaceae, with the addition of the two new genera of Haplohelminthosporium and Helminthosporiella, that are introduced in this paper. Each new genus is provided with a full description and notes, and each new taxon is provided with an illustration for the holotype. A list of identified and accepted species of Helminthosporium with morphology, host information, locality, sequence data, and related references of Helminthosporium reported worldwide is provided based on records in Species Fungorum 2021. This work provides a micro-fungi database of Haplohelminthosporium, Helminthosporiella, and Helminthosporium which can be modified and validated as new data come to light.


Introduction
In Thailand, a large number of novel fungi from a variety of hosts have been recently described, adding to the region's highly known fungal diversity [1,2]. This diversity is supported by various factors, including host-plant species relationships, geography, seasons, air humidity, and temperature. Many interesting fungi from Thai monocotyledons such as bamboo (Poaceae) and Pandanaceae have been described in previous studies, and some new taxa and records of microfungi on palms have been published, especially from the southern region of Thailand [3][4][5][6][7][8][9][10][11]. However, more research on fungal diversity on palms in Thailand is needed.
Few previous studies have investigated the Helminthosporium-like taxa from plants, particularly palms, in Thailand. In this study, we were able to isolate Helminthosporiumlike taxa from palms collected in Thailand. Morphology and multi-gene phylogenetic analyses showed two Helminthosporium-like taxa are novel in Massarinaceae. In addition, we provide a checklist of Helminthosporium and the name for Helminthosporiella stilbacea is also validated.

Collection, Isolation, and Identification
The plant materials containing the fungal structures were collected from Krabi and Prachuap Khiri Khan Provinces, Thailand, from living and dead parts of palm trees (Calamus sp. and Cocos nucifera). Samples were taken to the laboratory for morphological study following the methods provided by Konta et al. [9]. Single spore isolates were obtained following the method of Senanayake et al. [38]. Measurements were taken using an Image Framework program. Illustrations were made in Adobe Photoshop CS6. Specimens and cultures were deposited in the herbarium of Mae Fah Luang University (MFLU) and Mae Fah Luang Culture Collection (MFLUCC). Faces of Fungi and Index Fungorum numbers were registered as outlined in Jayasiri et al. [39] and Index Fungorum [40], respectively.

DNA Extraction and Amplification (PCR)
DNA extraction was performed using the Biospin Fungus genomic DNA extraction kit-BSC14S1 (Bioflux, P.R. China) according to Dissanayake et al. [41]. Partial nucleotide genes were subjected to PCR amplification and sequencing of the large subunit (28S, LSU) [42], the internal transcribed spacer (ITS) [43], the small subunit (18S, SSU) [43], and the translation elongation factor 1-alpha (tef1-α) was performed [44,45]. For primers and conditions, see Table 1. PCR amplification and sequencing were carried out following Konta et al. [9]. The resulting fragments were sequenced in both forward and reverse directions, the generated DNA sequences were analysed, and the consensus sequences were computed using SeqMan software. New sequences generated in this study were deposited in GenBank (Table 2).

Phylogenetic Analyses
The sequences generated in this study were subjected to a BLAST search in GenBank to identify closely related sequences. Sequence data retrieved from GenBank and recent publications were used as references [24]. Sequence data for the ITS, LSU, SSU, and tef1-α regions were analysed both individually and in combination. A total of 93 taxa were used for the combined phylogenetic analyses (ITS, LSU, SSU, and tef1-α) in order to find a natural classification placement. In addition, 103 taxa of ITS and 113 taxa of LSU were used for phylogenetic analyses. For both the individual and combined phylogenetic analyses, Cyclothyriella rubronotata (Cyclothyriellaceae) was selected as the outgroup taxon. Absent sequence data (i.e., ITS, LSU, SSU, tef1-α sequence data) in the alignments were treated with gaps as missing data. Sequence alignments were carried out with MAFFT v.6.864b [46] and were manually improved where necessary. The single gene datasets were combined using Mega7 [47]. Data were converted from fasta to nexus and PHYLIP format with Alignment Transformation Environment online, https://sing.ei.uvigo.es/ALTER/ (accessed on 15 July 2020) [48]. The tree topologies obtained from single gene sequence data were compared prior to the combined gene analysis in order to check for incongruence in the overall topology of the phylogenetic tree. Maximum likelihood (ML) analysis was accomplished using RAxML-HPC2 (v.8.2.12) on XSEDE in the CIPRES Science Gateway platform (http://www.phylo.org) (accessed on 12 May 2020) [49] with GTRGAMMA model and set as 1000 bootstrap replicates. Bayesian analysis was performed at CIPRES using Bayesian analysis on XSEDE (v.3.2.7) as part of the "MrBayes on XSEDE" tool [49][50][51]. GTR+I+G model was selected by using MrModelTest 2.2 [52] under the Akaike information criterion (AIC) as the best-fit models of the combined dataset for maximum likelihood and Bayesian analysis [52]. Bayesian posterior probabilities (BYPP) were determined by Markov Chain Monte Carlo sampling (MCMC) in MrBayes on XSEDE v.3.2.7. Six simultaneous Markov chains were run for 5,000,000 generations and trees were sampled every 1000th generation. An MCMC heated chain was set with a "temperature" value of 0.20. All sampled topologies beneath the asymptote (25%) were discarded as part of a burn-in procedure; the remaining trees (7502) were used for calculating posterior probabilities in the majority rule consensus tree. Bootstrap support values for ML and BYPP are given near to each node (Figures 1 and 2). The phylogenetic trees were configured in   (B) Phylogenetic tree of the dataset for LSU sequence data. Bootstrap support values for maximum likelihood (ML) equal to or higher than 50%, and Bayesian Posterior Probabilities (BYPP) equal to or greater than 0.90 are given above each branch. Novel taxa are in blue. Ex-type strains are in bold. The tree is rooted to Cyclothyriella rubronotata strains TR, TR9 (Cyclothyriellaceae).

Figure 2.
Maximum likelihood majority rule consensus tree for the analyses of Massarinaceae and sister family Perioconiaceae isolates based on a dataset of combined ITS, LSU, SSU, and tef1-α sequence data. Bootstrap support values for maximum likelihood (ML) equal to or higher than 50%, and Bayesian posterior probabilities (BYPP) equal to or greater than 0.90 are given above each branch. Novel taxa are in blue. Ex-type strains are in bold. The tree is rooted to Cyclothyriella rubronotata strains TR, TR9 (Cyclothyriellaceae). Maximum likelihood majority rule consensus tree for the analyses of Massarinaceae and sister family Perioconiaceae isolates based on a dataset of combined ITS, LSU, SSU, and tef1-α sequence data. Bootstrap support values for maximum likelihood (ML) equal to or higher than 50%, and Bayesian posterior probabilities (BYPP) equal to or greater than 0.90 are given above each branch. Novel taxa are in blue. Ex-type strains are in bold. The tree is rooted to Cyclothyriella rubronotata strains TR, TR9 (Cyclothyriellaceae).
The phylogenetic analyses (Figures 1 and 2) showed several topologies of the tree had generally rather low support (ML ≤50% and BYPP ≤0.90). This reflects the relatively high amount of homoplasy in the data. Most Helminthosporium-like taxa did not have SSU and tef1-α sequence data for the phylogenetic analyses. In the future, divergent time estimations will be needed for Helminthosporium-like taxa to resolve taxonomic confusion and placement. Etymology-Haplo in Greek means single, which refers to the single conidium in each conidiophore. It is a close relative of Helminthosporium.

Taxonomy
Saprobic on living leaves and petioles of Calamus sp. On living leaves, small spots, circular to irregular, yellow in the beginning, later becoming red brown surrounded by yellow. Colonies on natural substrate forming black patches on the upper leaf, petiole surfaces. Sexual morph: Undetermined. Asexual morph: Hyphomycetous. Colonies on natural substrate forming black patches on the upper leaf, petiole surfaces. Mycelium mostly immersed, partly on the surface forming small stroma-like aggregations of red brown pseudoparenchymatous cells. Conidiophores arising singly or fasciculate from stroma cells, erect, simple, unbranched, straight, curved and swollen at apex, septate, thick-walled, cylindrical, smooth, bulbous at base, hyaline in the middle, brown to yellow-brown at 1-2-cells above the base, pale brown to yellow-brown at apical cell. Conidiogenous cells monotretic, terminal, determinate, cylindrical, wide and yellow-brown with a well-defined, small, noncicatrized pore at the apex. Conidia one for each conidiophore, obpyriform to lageniform, straight or curved, smooth, olive-brown, distoseptate, with a dark scar at the base.
Type species-Haplohelminthosporium calami Konta & K.D. Hyde Notes: Haplohelminthosporium is established as a monotypic genus with Hap. calami as the type species. ITS phylogenetic analyses separated this genus from other genera, while in the LSU and multigene analyses it clustered with Helminthosporium and Helminthosporiella, but both without good statistical support (Figures 1 and 2). Haplohelminthosporium is presented herein as an asexual morph (hyphomycete) similar to Helminthosporium and Helminthosporiella in that it is hyphomycete with an erect conidiophore, monotretic conidiogenous cell and distoseptate conidia [19,22,63]. The type species of Helminthosporium has pale to dark brown, septate conidiophores, with terminal and intercalary polytretic conidiogenous cells, noncicatrized pores at the apex and upper 3-4 cells, solitary or short catenate conidia that are subhyaline to brown, distoseptate, and is dark brown to black scar at the base [19]. Helminthosporiella has brown to red-brown conidiophores with terminal, polytretic conidiogenous cells, with catenate and easily disarticulating chains of conidia that are medium brown, striated at surface and distoseptate [63]. However, Haplohelminthosporium is distinguished by its unbranched conidiophores arising solitarily or fasciculate from the stroma-like bulbous basal cells that are hyaline in the middle, brown to red-brown at 1-2-cells above the base, pale brown to red-brown and curved at the apical cell with well-defined non-cicatrized small pores and with a single olive-brown conidium arising from each conidiophore ( Figure 3). In the BLAST search of GenBank, the closest match of the LSU, ITS, and SSU sequence data were identical to Helminthosporium spp. Based on distinguishing morphological characteristics together with single/multigene phylogenetic analyses we introduce the newly described strain as a new genus Haplohelminthosporium in Massarinaceae. µm (x = 160 × 6 µm, n = 50), wide at the base and apex, macronematous, mononematous, arising singly or fasciculate from the stroma cells, erect, simple, unbranched, straight, curved and swollen at the apex, thick-walled, cylindrical, smooth, bulbous at base, hyaline in the middle, brown to red-brown at 1-2-cells above the base, pale brown to red brown at the last cell of the apex, (3-)4-5(-6) septa. Conidiogenous cells monotretic, terminal, determinate, cylindrical, with well-defined small noncicatrized pores at the apex, wide and yellow-brown at the apex. Conidia (55-)70-100(-120) × (13-)17-20(-23) µm (x = 80 × 20 µm, n = 60), one on each conidiophore, obpyriform to lageniform, straight or curved, smooth, olive-brown, (3-)4-6(-7)-distoseptate, with a dark scar at the base.  Culture characteristics: Culture on PDA, colony yellow-gray-brown at the center, turning dull creamy white toward to margin, smooth, dense, zonate at the margin ( Figure 3X).
Notes: BLAST search of the ITS sequence of the newly described strain (Haplohelminthosporium calami) shows 88.89% similarity with Helminthosporium juglandinum (L118), the LSU sequence shows 98.75% similarity with H. aquaticum (MFLUCC 15-0357), and the SSU sequence shows 99.52% similarity with H. quercinum (L90). Based on ITS phylogenetic analysis, Haplohelminthosporium calami formed a single branch at the basal clades of Helminthosporiella and Helminthosporium ( Figure 1A Table 3. Other H. endiandrae strains (AKMR1, CBS 138902; ex-type from the holotype, and SM61) grouped together in Helminthosporium, as the other strains have an ITS region, but the H. endiandrae (CBS 138902, MH878637) strain that grouped with our new collection lacks the ITS region. Therefore, we compared the morphology of these two species and found that Hap. calami differs from H. endiandrae with respect to its smaller conidiophores (    [63]. In this paper we accept Helminthosporiella as a distinct genus, presently with a single species Helminthosporiella stilbacea. Since a Latin diagnosis is no longer required, we provide an English diagnosis and priority was given to the previous genus and species names. Furthermore, this study provides the holotype to validate the genus and species, and reports the first host record of Hel. stilbacea associated with coconut tree (Arecaceae) in Thailand. In particular, based on the present morphology and DNA sequence data, Helminthosporiella is identified as a monotypic genus, with Hel. stilbacea as the type species. The members of Helminthosporiella were found associated with leaf spots on oil palm (Arecaceae) [64]. Saprobic on dead petiole of Cocos nucifera.Sexual morph: Undetermined. Asexualmorph: Colony on natural substrate black, hairy. Mycelium mostly immersed, at the surface forming small stroma-like aggregations of dark brown pseudoparenchymatous cells (6-)11-15(-25) µm diam (x = 14 µm). Conidiophores (60-)165-270(-310) × (5-)7-9(-12) µm (x = 200 × 8 µm, n = 30), macronematous, wide at the apex and base, arising singly from the stroma cells, erect, simple, unbranched, straight or flexuous, thick-walled, cylindrical, smooth-walled, dark brown, becoming pale brown at the apex, (4-)12-15-septate. Conidiogenous cells terminal and intercalary, polytretic, with well-defined thick, pale brown pores. Conidia (30-)45-60(-70) × 6-9 µm (x = 50 × 7 µm, n = 30), obpyriform to lageniform, straight or curved, smooth-walled, subhyaline to light brown, 5-8-distoseptate, with a thick scar at the base.

Helminthosporiella stilbacea
Culture characteristics: Culture on MEA, colony yellow-green at the center, turning dull green, pale yellow next, becoming dull green again, pale yellow, and white toward the margin. Colony smooth, dense at the middle, zonate, fluffy at the margin ( Figure 4P   Notes: Crous et al. [63] introduced a new genus Helminthosporiella with a new combination of Hel. stilbacea based on fresh collections from oil palm (Elaeis oleifera) in Colombia and the second collection of Hel. stilbacea was also collected from oil palm (Elaeis guineensis) in Brazil by Rosado et al. [64]. The full descriptions, illustrations, and sequence data are provided with interesting information as this species causes elliptical necrotic spots with a yellowish halo on living leaves of commercial oil palm plantations [63,64]. However, the type species was invalid because of the basionym lacked a Latin diagnosis [63]. From these, our fresh collection was collected from dead petiole of coconut (Cocos nucifera) and in phylogenetic analysis (Figures 1 and 2), three strains of Hel. stilbacea, including our strain, are grouped together with high bootstrap support. In this study, we therefore provide a holotype from our specimen, and introduce a new species Helminthosporiella stilbacea, complete with an English diagnosis, and validated by using the same name while linking to the valuable information provided from the previous publication of this species.
A BLAST search of the ITS sequence of our isolate showed 90.19% similarity with H. velutinum (L131), the LSU sequence showed 97.05% similarity with H. aquaticum (MFLUCC 15-0357), the SSU sequence showed 99.15% similarity with H. quercinum (L90), and the tef1-α sequence showed 92.61% similarity with H. tiliae (L88). These blast results do not match the results of the phylogenetic analyses.
The comparison between three strains of Hel.stilbacea (see Table 4) from three collections showed that our collection MFLU 20-0521 has several differences when compared with the other two strains CPHmZC-01 and COAD 2126. Our collection was obtained from a dead petiole, while the two other strains were isolated from living leaves [63,64]. Therefore, our new collection has been provided as a holotype for Hel. Stilbacea. It is also the first geographical record from Thailand, and is a new record of the species from a coconut host (Cocos nucifera).

Conclusions
In this study, we introduce the new genus Haplohelminthosporium,with Hap. calami as the type species. In multigene phylogenetic analyses, Hap.calami clustered together with Helminthosporium endiandrae (CBS 138902) without strong good bootstrap support (other H. endiandrae (AKRM1, CBS 138902 (ex-type), SM61) groups together in Helminthosporium). Moreover, we were unable to synonymize H. endiandrae (CBS 138902) under Haplohelminthosporium because H. endiandrae has only LSU sequence data available [60]. In the future, H. endiandrae needs more collections and sequence data to confirm taxonomic placement.
Another newly described isolate clusters together with Helminthosporiella stilbacea. Helminthosporiella was introduced by Crous et al. [63] but was invalidated as the type species was not provided with a Latin diagnosis. In this study, we validate Helminthosporiella with Hel. stilbacea as the type species. Moreover, the newly described strain from this study is the first saprobic report of Hel. stilbacea, as this was reported in previous studies as a pathogenic fungus on leaves [63,64]. Moreover, topological nodes in phylogenic analyses showed conflicting results (Figures 1 and 2). Probably, using only single gene ITS or LSU analyses will preclude the establishment of taxonomic placements, while combined gene analyses (including protein coding genes) provide sufficient molecular data to determine the placements.
Helminthosporium is generally described as a common saprobe found on leaf or twig litter, and it appears to have a diverse distribution. Occasionally, members of this genus are also described as pathogens, occurring on a wide range of hosts. Comparison of morphology is important for fungal identification [79]. In this study, we provide a checklist for Helminthosporium species reported worldwide including details of each species based on records from Species Fungorum [80] (Table 5). We noted that ten Helminthosporium species have been found on palm substrates (Arecaceae). Although Helminthosporium conidia superficially resemble many genera, such as Drechslera, Bipolaris, and Exserohilum, phylogenetic analyses have provided different results [19,33,[81][82][83]. Furthermore, we recommend revision of the genus Helminthosporium with fresh collections and DNA sequence data (specifically the ITS region and protein coding genes).              Conidiophores (175-)215-325(-455) µm long, 11-23 µm wide at the base, 8.5-14 µm wide near the slightly inflated apex, fasciculate, erect, simple, straight or flexuous, thick-walled, sub-cylindrical, smooth, brown to dark brown, darker to black at the apex, the latter with a well-defined apical pore; Conidia