Multigene Phylogeny Reveals Haploanthostomella elaeidis gen. et sp. nov. and Familial Replacement of Endocalyx (Xylariales, Sordariomycetes, Ascomycota)

During our investigation of palm fungi in Thailand, two interesting taxa from Elaeis guineensis and Metroxylon sagu (Arecaceae) were collected. Based on phylogenetic analyses of a combined dataset of ITS, LSU, rpb2, and tub2 nucleotide sequences as well as unique morphological characteristics, we introduce the new genus Haploanthostomella within Xylariales, and a new species Endocalyx metroxyli. Additionally, in our study, the genus Endocalyx is transferred to the family Cainiaceae based on its brown conidia and molecular phylogenetic evidence.


Introduction
Palm trees represent a family of perennial lianas and consist of many diverse species worldwide, with the fossil record indicating around 65 million years of evolutionary history [1]. Microfungi on palms have been studied, but only a few have been analyzed using morphology and DNA sequence data. Several fungal species are currently unknown to science, with the total number estimated at somewhere between 2.2 and 3.8 million [2]. Thus, palms are a particularly interesting plant family for studying microfungi species unknown to science.
The subclass Xylariomycetidae has recently been updated to contain three orders (Amphisphaeriales, Delonicicolales, and Xylariales) and 35 families [3]. Recently, the family Induratiaceae was introduced in this subclass by Samarakoon et al. [4] with an updated phylogeny of Xylariales. Cainiaceae is a family of particular interest, as all members in this family tend to be found on monocotyledons, the majority of which are grasses [5]. In previous studies, Cainiaceae was accepted in the Xylariales [3,6]. Later, Hongsanan et al. [7], and Wijayawardene et al. [8] assigned Cainiaceae to the Xylariomycetidae as an incertae sedis family.
Since 2014, fungal research in Thailand has revealed a high diversity of novel species [12][13][14]. In this study, we found fungal species unknown to science from Thailand. The phylogeny results show that Endocalyx grouped within Cainiaceae, and so we transferred Endocalyx from Apiosporaceae (Amphisphaeriales) to Cainiaceae (Xylariales) based on both morphology and multigene phylogeny. We also introduce the new species Endocalyx metroxyli, collected from the economically important oil palm host (Elaeis guineensis). Lastly, we introduce the new genus Haploanthostomella associated with true sago palm (Metroxylon sagu).

Collection, Isolation, and Identification
Saprobic fungi growing on dead leaves, petioles and rachis of Elaeis guineensis and Metroxylon sagu were collected in Krabi and Surat Thani Provinces of Thailand, placed in ziplock bags and brought to the mycology laboratory at the Center of Excellence in Fungal Research, and morphological characteristics were observed. Specimens were examined following the methods provided by Konta et al. [15]. Single spore isolates were obtained following the method of Senanayake et al. [16], using malt extract agar (MEA) and incubating at 25-28 • C overnight. Germinating conidia were transferred to new MEA media and pure cultures were kept at 25-28 • C. Specimens and cultures were deposited in the herbarium of Mae Fah Luang University (MFLU) and Mae Fah Luang University Culture Collection (MFLUCC), Chiang Rai, Thailand, respectively. Faces of Fungi and Index Fungorum numbers were registered as outlined in Jayasiri et al. [17] and Index Fungorum [18].

DNA Extraction and Amplification (PCR)
Genomic DNA was extracted from fruiting bodies of Haploanthostomella elaeidis and fungal mycelium of Endocalyx metroxyli. DNA extraction and amplification were followed Dissanayake et al. [19]. Konta et al.'s method [16] was followed for PCR amplification of ITS, LSU, SSU, tef1-α and rpb2, while O'Donnell and Cigelnik's method [20] was followed for PCR amplification of the tub2 region. Amplification was done using the primers ITS5 and ITS4 for the internal transcribed spacer regions and intervening 5.8S rDNA (ITS), the primers LR5 and LR0R for the large subunit (LSU) rRNA gene, the primer pair fRPB2-5f and fRPB2-7cR for the RNA polymerase II second largest subunit (rpb2) gene, and the primers T1 and T22 for the partial gene β-tubulin (tub2). PCR amplifications were performed using 1× PCR buffer with 8.5 µL ddH 2 O, 12.5 µL 2× Easy Taq PCR SuperMix (mixture of Easy Taq TM DNA Polymerase, dNTPs and optimized buffer (Beijing Trans Gen Biotech Co., Beijing, China)), 2 µL of DNA template, and 1 µL each of forward and reverse primers (10 pM) in a final volume of 25 µL. The cycle conditions in the initiation step were started at 95 • C for 3 min, followed by 35 cycles at 95 • C for 30 s, 55 • C for 50 s, 72 • C for 30 s (for ITS, LSU); 95 • C for 5 min, followed by 35 cycles at 95 • C for 1 min, 54 • C for 2 min, 72 • C for 1:5 min (for rpb2); 95 • C for 5 min, followed by 35 cycles at 94 • C for 1 min, 52 • C for 1 min, 72 • C for 1:5 min (for tub2); a final elongation step at 72 • C for 10 min and a final hold at 4 • C were done as the last steps. Purification and sequencing were performed by Sangon Biotech Co., Shanghai, China. Consensus sequences were computed using SeqMan software, and new sequences generated in this study were deposited in GenBank (Table 1).

Phylogenetic Analyses
The consensus sequences were put through a BLAST search in the NCBI GenBank nucleotide database to search for the fungal sequences of closest relatives that have been deposited in the NCBI database. Dissanayake et al.'s study [19] was followed for the phylogenetic analyses. Voglmayr and Beenken's study [79] was used as a reference of the dataset. Both individual and combined ITS, LSU, rpb2, and tub2 nucleotide sequences were analyzed. A total of 151 taxa were used for the phylogenetic analyses in order to find the taxonomic placement of each species. Three genera viz. Delonicicola, Furfurella (Delonicicolaceae), and Leptosillia (Leptosilliaceae) in Delonicicolales were used as the outgroup taxa.
The MAFFT online program was used to obtain initial alignments for each locus [94]. Alignments were manually edited and single gene sequence data sets were combined using MEGA7 [95]. The Alignment Transformation Environment online program was used to convert the file format [96]. MrModeltest [97] was used to find the best model for maximum likelihood (ML) and Bayesian analyses (BYPP). The six simultaneous Markov chains were run for 20,000,000 generations and trees were sampled every 1000th generation. Bayesian posterior probabilities from MCMC were evaluated with a final average standard deviation of the split frequency of <0.01. Bootstrap values for ML equal to or greater than 50% and BYPP equal to or greater than 0.90 are given at the nodes (Figure 1). Fig Tree v1

Morphology and Phylogeny
The combined dataset comprised 151 taxa from selected taxa in Amphisphaeriales, Delonicicolales, and Xylariales (Table 1). The RAxML analyses of the combined dataset yielded the best-scoring tree ( Figure 1)  Saprobic on dead leaves and rachis in terrestrial habitats. Sexual morph: Ascomata immersed in the host epidermis, beneath a clypeus, visible as slightly raised blackened areas, dark brown to black, coriaceous, solitary or aggregated into clusters, scattered, with an ostiolar canal. Peridial wall thick, comprised of several layers of cells, outwardly comprising dark brown cells of textura prismatica and inwardly comprising hyaline cells of textura angularis. Paraphyses septate, tapering hyphae-like, hyaline. Asci eight-spored, unitunicate, clavate to cylindric, short pedicellate, with J-, apical ring. Ascospores unibiseriate into the asci, unicellular, obovoid, fusoid, hyaline or brown to dark brown, verrucose with a mucilaginous cap at apex. Germ slit straight, less than spore-length. Asexual morph: Not observed.
Type species: Haploanthostomella elaeidis Konta & K.D. Hyde. Notes: Anthostomella species were proven to be polyphyletic, and it is of no surprise that a new genus with anthostomella-like characteristics was discovered in this study [99]. Phylogenetic analyses based on a single dataset of ITS (supporting information section) and combined sequence data indicated that Haploanthostomella belongs to Xylariales genera incertae sedis, separating well from other genera but with low bootstrap values ( Figure 1). According to the phylogenetic tree (Figure 1), seven genera (Ceratocladium, Circinotrichum, Gyrothrix, Idriella, Neoanthostomella, Virgaria and Xenoanthostomella) are closely related to our new genus, but morphological characteristics of these genera are different. The genera Neoanthostomella, Virgaria, and Xenoanthostomella were compared morphologically since they are similar to our new taxon. Haploanthostomella differs from Virgaria, Neoanthostomella, and Xenoanthostomella in having a J-apical ring, fusoid-obovoid ascospores, and verrucose with a mucilaginous cap at the apex, while Virgaria has asci with a J+ apical ring and smooth-walled elliposidal ascospores lacking of a mucilaginous sheath; Neoanthostomella smooth-walled elliposidal ascospores surrounded by a thick mucilaginous sheath; Xenoanthostomella has unilocular ascoma, and ascospores lacking germ slits and mucilaginous sheaths [13,72,89]. Therefore, Haploanthostomella is described here as a new genus based on phylogeny coupled with morphology. In addition, we provide a key to genera with Anthostomella-like characteristics.   Etymology: Referring to the genus of palm trees Elaeis Jacq. Holotype: MFLU 20-0522. Saprobic on dead leaves and rachis of Elaeis guineensis.
Notes: A BLAST search of H. elaeidis ITS sequence shows 83.87% similarity with Gyrothrix oleae (CPC 37069); LSU sequence shows 95.95% similarity with Gyrothrix eucalypti (CPC 36066); and rpb2 sequence shows 80.95% similarity with Lopadostoma meridionale (LG). Only the sexual morph of H. elaeidis was found in nature, and we could not obtain a pure culture from fresh samples. Therefore, the morphological characteristics of H. elaeidis were not compared with Ceratocladium, Circinotrichum, Gyrothrix, and Idriella, as they only had asexual morphs found in nature. Hence, the morphological features of H. elaeidis were only compared with Neoanthostomella, Virgaria, and Xenoanthostomella, as they have sexual morphs.
Key to genera related to Anthostomella-like genera 1. Hyaline ascospores Alloanthostomella 1. Brown ascospores 2 2. Asci with a J-apical ring 3 2. Asci with or without J+ apical ring 5 3. Ascospores with or without germ slit 4 3. Ascospores with germ slit Xenoanthostomella 4. Ascospores with a germ slit and the length less than spore length with a mucilaginous cap at the apex Haploanthostomella 4. Ascospores with or without germ slit, with mucilaginous sheath Neoanthostomella

5.
Asci with a J+ apical ring, ascospores with germ slit, with or without mucilaginous sheath 6 5. Asci with J+ or J-apical ring, ascospores with or without germ slit (straight or spiral), and also with or without appendages or mucilaginous sheath Anthostomella 6. Ascospores with germ slit less than spore length, with or without mucilaginous sheath 7 Saprobic on various plants. Colonies on host plant, pustules nearly flat or raised, circular, discolored, dark brown to black, at last bursting, the conidiomata developing. Sexual morph: Undetermined. Asexual morph: Conidiomata scattered, erect, cupulate to cylindrical; peridial hyphae enclosing the inner conidial mass, nonsporiferous, brown to yellowish brown; some species consisting of two parts of conidioma: (1) a basal cylinder covering a central column, rough-walled, carbonaceous, composed of black hyphae which are sometimes branched and are adherent to one another; (2) a slender central column, synnematous, expanding radially apically, high, enclosed by the peridial hyphae which are nonsporiferous, orange-yellow to lemon-yellow. Peridial wall thick, comprising dark brown, thick-walled cells of textura angularis. Conidiophores thread-like, septate, with or without short pegs bearing the conidia, meristematic at the base, colorless basally and gradually turning brown apically, 1-2 µm wide; peridium thick, comprising dark brown, thick-walled cells of textura angularis. Conidiogenous cells holoblastic, integrated, determinate. Conidia solitary, unicellular, flattened, round, oval or slightly polygonal in face view, at first pale, dark brown to fuscous black at maturity, with or without guttules, often with a longitudinal hyaline straight germ slit extending the full-length (adapted from [99][100][101]).
Ghana [133] Sri Lanka [133] Oncosperma sp. (Arecaceae; clustering, rarely solitary palm) Ghana [133] Sri Lanka (Holotype) [133] * Have molecular data.  Recently, Longiappendispora was introduced under Cainiaceae, with seven genera in total included in the family by Mapook et al. [11]. In our study, detailed molecular analyses were done for Endocalyx and its placement in Cainiaceae (Xyalriales) was confirmed. Previously, Endocalyx was classified in Apiosporaceae (Xylariales, Sordariomycetes) based on morphological evidence. As the first detailed molecular data of Endocalyx cinctus have been made available from a Japan laboratory [32], their current placement is supported (Figure 1). However, there are no recent publications referring to the molecular data of this genus yet. Thus, in this study, we present the placement of Endocalyx based on multigene phylogenetic analyses with recent sequence data from the Japan collection as well as the Thailand collection. In addition, we accept eight genera in Cainiaceae (Alishanica, Amphibambusa, Arecophila, Atrotorquata, Cainia, Endocalyx, Longiappendispora, and Seynesia), and seven species by including our new species in the genus Endocalyx (Table 2). In addition, we provide a key for the members of Cainiaceae. Conidiophores reduced to conidiogenous cell, hyaline to pale-brown, unbranched, smooth. Conidia 13-16 × 7-10 µm (x = 13 × 10 µm, n = 30), unicellular, ellipsoid-globose, brown to dark brown, with short pegs bearing conidia, with germ slit, smooth-walled.
Culture characteristics: Colonies on MEA, at first white, raised, effuse, velvety to hairy, circular, smooth at the margin, white from above, pale-brown from below. Endocalyx metroxyli is morphologically similar to E. melanoxanthus. However, Endocalyx metroxyli does not have erect conidiomata developing from the pustules, as was mentioned by Petch [100], Okada and Tubaki [101], and Vitoria et al. [102,131]. In this study, we found only a black raised pustule structure with ostiole surrounded by a yellow hyphae ring, and hyaline conidiophore, unicellular, dark brown conidia with a longitudinal germ slit. Endocalyx melanoxanthus was collected and described from palm hosts (Arecaceae), and a few collections were collected from other host plants ( Endocalyx metroxyli is morphologically similar to E. melanoxanthus var. melanoxanthus, in having black raised pustules surrounded by yellow hyphae and smooth-walled conidia with no significant size differences [100][101][102]. However, our new taxon lacks cupulate or cylindrical conidiomata [101,102]. On the other hand, E. metroxyli differs from E. melanoxanthus var. grossus by lacking the production of ornamented conidia [100,101].

Discussion
Based on phylogeny and morphological characteristics, the new monotypic genus Haploanthostomella (type species: Haploanthostomella elaeidis) and the new species Endocalyx metroxyli have been established. The former new species was isolated from a dead rachis of Elaeis guineensis, and the latter from a dead petiole of Metroxylon sagu (Arecaceae) in Thailand. Phylogenetic analyses of combined datasets together with morphological characteristics revealed that Haploanthostomella belongs to Xylariales incertae sedis, while Endocalyx belongs to the Cainiaceae (Xylariales).
Endocalyx metroxyli is similar to other species by having dark brown conidia with a fulllength germ slit, it but differs from other species by not having conidiomata produced from the pustulate and no thread-like structure of conidiophores. Morphological characteristics of species in the genus are mostly flat or raised pustules, capsule or slender conidiomata with or without branches at the apex, and brown to dark brown conidia with smooth walls (E. amarkantakensis, E. collantesis, E. indumentum, E. melanoxanthus, E. melanoxanthus var. melanoxanthus), while some species are verrucose-walled (E. cinctus, E. indumentum, E. melanoxanthus var. grossus, E. thwaitesii). We referred to previous publications for morphological comparisons to the taxa in this study, as we did not observe all holotype specimens [100][101][102].
According to the literature, there are also strains derived from another two species and two varieties. Excluding E. cinctus, no sequence data are available for generic types of Endocalyx and other species, and their morphology and host substrates are closely related to our novel taxon. Endocalyx species have been reported in several countries, especially in tropical and subtropical regions. Furthermore, palm trees (Arecaceae) have most commonly been reported as the host, while several species have been presented from other hosts ( Table 2).