A phylogenetic assessment of Endocalyx (Cainiaceae, Xylariales) with E. grossus comb. et stat. nov.

The phylogenetic affinities of four representative Endocalyx taxa at the species and variety levels are studied based on materials collected on different palm hosts in Japan and the states of Hawaii and Texas, USA. They include specimens and their isolates belonging to E. cinctus, E. indumentum, E. melanoxanthus var. grossus, and E. melanoxanthus var. melanoxanthus. Phylogenetic analyses of nuclear ribosomal DNA sequence data (ITS-LSU nrDNA) confirmed that Endocalyx belongs to the order Xylariales (Sordariomycetes) where all species and varieties treated form a strongly supported monophyletic lineage within the family Cainiaceae. They were also phylogenetically well resolved and consistent with their morphological and ecological circumscription. Species status is proposed for E. melanoxanthus var. grossus under the name E. grossus comb. et stat. nov. on the basis of its distinct morphological, molecular, cultural, and ecological characteristics. The putative placement of Endocalyx within the family Apiosporaceae (Amphisphaeriales), based on the presence of basauxic conidiophores, is rejected considering that all species treated clustered within the distant Cainiaceae (Xylariales). This characteristic mode of conidiophore elongation is determined to have evolved independently within distantly related ascomycetous lineages. Novel morphological and cultural features of Endocalyx taxa based on new isolates are described and commented. The recently described E. metroxyli is reduced to a synonym with E. melanoxanthus.


Introduction
The genus Endocalyx Berk. & Broome is characterized by sporodochial or synnematous, funnel-shaped, cupulate, cylindrical to pancake-shaped conidiomata arising from an annulus and containing a mass of conidia that is enclosed by yellow or brown sterile peridial hyphae (Petch 1908;Hughes 1953a;Morris 1963;Ellis 1971;Okada and Tubaki 1984;Seifert et al. 2011). Conidiophores are basauxic, hyaline to subhyaline, thread-like and branched, bearing monoblastic or polyblastic, integrated, terminal and intercalary filamentous conidiogenous cells. They produce dry, unicellular, lenticular or elliptical conidia, almost round in one plane but sometimes slightly angular. They range from dark brown, blackish brown to almost black in color, with a smooth or minutely to moderately echinulate surface, rarely with hairlike projections, and often with a hyaline so-called germ slit. Some Endocalyx species such as E. melanoxanthus (Berk. & Broome) Petch var. melanoxanthus and E. cinctus Petch are pantropical in distribution and saprobic on dead plant materials. They are usually found colonizing palm tree debris apparently showing a strong specificity for these hosts, whereas others also grow on dead vines, lilies or twigs of woody trees (Hughes 1953a(Hughes , 1978Okada and Tubaki 1984).
The type species, E. thwaitesii Berk. & Broome [= E. psilostoma Berk. & Broome (Berkeley and Broome 1877)], was first described according to Petch (1908) on dead leaves Section Editor: Hans-Josef Schroers of Oncosperma sp. (Arecaceae) from Sri Lanka (formerly Ceylon). Hughes (1953a), however, expressed doubts whether the scanty collection deposited in IMI was a palm, originally referred to as "dead sticks" in the protologue where no generic type was designated. Petch (1908) lectotypified the genus with E. thwaitesii and amended the original description based on a duplicate of the original specimen. He also described E. cinctus and transferred Melanconium melanoxanthum Berk. & Broome to Endocalyx, collected as well in Sri Lanka on petioles of another palm tree, Caryota urens L. (Berkeley and Broome 1875). Subsequently, four more species and a variety of E. melanoxanthus have been described: E. indicus J.N. Kapoor & Munjal on dead twigs of a woody dicotyledonous plant in India (Kapoor and Munjal 1966); E. indumentum G. Okada (Mena and Mercado 1984); E. amarkantakensis U.S. Patel, A.K. Pandey & R.C. Rajak on dead twigs of Shorea robusta C.F. Gaertn. (Dipterocarpaceae) in India (Patel et al. 2002). Vitoria et al. (2011) accepted these seven species and two varieties although Seifert et al. (2011) recognized only five species and considered E. collantesis a synonym of E. cinctus [see also Index Fungorum (http:// www. index fungo rum. org) and MycoBank (https:// www. mycob ank. org/)]. In a comprehensive study of the genus based on several Japanese specimens collected on palm trees, Okada and Tubaki (1984) described and isolated in pure culture E. melanoxanthus var. melanoxanthus, E. melanoxanthus var. grossus, E. cinctus and E. indumentum. They also conducted detailed morphological investigations on conidiogenesis and conidiomata of these and a representative collection of E. thwaitesii from Ghana (Hughes 1953a) using light and scanning electron microscopies (LM, SEM). These morphological studies on Endocalyx species and other conidioma-producing fungi were later expanded to assess the taxonomic implications of conidiomatal anatomy in synnematous hyphomycetes (Okada and Tubaki 1987;Seifert and Okada 1990). Recently, the eighth species, named E. metroxyli Konta & K.D. Hyde, was described from a dead petiole of the palm tree Metroxylon sagu Rottb. in Thailand using both morphological and molecular data (Konta et al. 2021).
The phylogenetic position of Endocalyx has been the subject of speculation since the introduction of the genus. Berkeley and Broome (1877) first considered it was closely allied to Alwisia Berk. & Broome, a genus of myxomycetes (Mycetozoa, Amoebozoa). Later, Petch (1908) rejected this hypothesis and compared Endocalyx with Graphiola phoenicis (Moug. ex Fr.) Poit. to conclude that in all essential details they were dissimilar although he did not exclude some reminiscences with other Graphiola species. Corte (1963), however, included the genus within the family Graphiolaceae, at that time belonging in the now defunct "Fungi Imperfecti", and provided a key for the three species known at the time. This genus and particularly G. phoenicis are now well resolved within the Exobasidiales (Ustilaginomycotina, Basidiomycota) based on morphological, ultrastructural, life cycle and molecular data (Begerow et al. 2006). Hyde et al. (1998), on the other hand, suggested that Endocalyx as well as other genera may belong to the family Apiosporaceae (Amphisphaeriales) due to the presence of basauxic conidiophores. The criterion for this tentative placement probably followed Hughes (1953b) who included Arthrinium Kunze, Endocalyx, Dictyoarthrinium S. Hughes, Spegazzinia Sacc., Graphiola and Papularia Fr. in his section VIII. This group was characterized by basauxic conidiophores that elongate at a basal growing point and arise from a swollen "conidiophore mother-cell", with the oldest conidia towards the apex and the youngest towards the base of the conidiophores. Minter (1985) reviewed conidial development in Arthrinium and morphologically related genera such as Endocalyx and Nigrospora Zimm. to conclude that they were indeed closely related. He predicted that any teleomorphs found in fungi belonging to the "Arthrinium group" will occur in and around the genus Apiospora Sacc. Similarly, von Arx (1985) considered that genera with basauxic conidiogenesis and pigmented, often oblate or bilaterally flattened conidia growing mainly on litter of monocotyledons such as grasses and palms, represented a phylogenetic entity. Kendrick and Murase (1994), on the other hand, assembled informal groups of anamorphs with shared features. They speculated whether the relatively rare basauxic development in conjunction with other unusual features such as thick, darkly pigmented septa of the conidiogenous axis arising from a phialide-like mother cell and conidia with germ slits represented a monophyletic group. Nevertheless, they considered that their putative groupings may ultimately have to be confirmed or rejected by molecular data. The hypothetical placement of Endocalyx in Apiosporaceae and its close relationship with Arthrinium has been widely accepted (Taylor and Hyde 2003;Senanayake et al. 2015;Wijayawardene et al. , 2018Wijayawardene et al. , 2021Hyde et al. 2020). However, Konta et al. (2021) recently reassigned Endocalyx to the family Cainiaceae (Xylariales) employing molecular data for the first time. Their study was based on a limited taxon sampling including only two sequences of E. cinctus available in GenBank (http:// www. ncbi. nlm. nih. gov/ genba nk/) and those of a novel species named E. metroxyli described from Thailand.
During recent surveys of saprobic microfungi carried out by one of the authors (G.D.) in subtropical Texas, USA, specimens of E. melanoxanthus var. melanoxanthus were collected on palm tree debris and isolated in pure culture. In order to confirm the phylogenetic placement and taxonomic status of Endocalyx on the basis of a more extensive taxon sampling, DNA sequence data was generated from these and the morphologically and culturally well-characterized voucher specimens and strains described by Okada and Tubaki (1984) in their seminal paper about the genus. Further unpublished specimens and cultures obtained by another author (G.O.), mainly in Japan (Sato et al. 1991), were also included for a more comprehensive assessment. Results are presented here and a new combination at the species level is introduced based on morphological and molecular evidence.

Morphological and cultural studies of specimens and isolates
Two fresh specimens of E. melanoxanthus var. melanoxanthus were collected on dead inflorescences of the palm tree Sabal minor (Jacq.) Pers. (Arecaceae), the dwarf palmetto, during fieldwork carried out by G.D. in southeastern Texas in 2020. Conidiomata were recognized in the field using a hand lens and pieces of substrate showing colonies were brought to the lab for processing. They were briefly washed off under tap water and incubated in a moist chamber at room temperature (23-25 °C) for three weeks followed by periodical examinations under the stereoscope to observe the development of conidiomata and to take images. Single conidium isolations from conidiomata were made following Choi et al. (1999). Germinated conidia were transferred aseptically to 2% Malt Extract Agar with 0.01% chloramphenicol (MEA: Hardy Diagnostics, Santa Maria, California, USA) and incubated at 25 °C. Colony features were recorded after two weeks under similar conditions. Fungal structures were mounted in lacto-cotton blue for examination under an Olympus BX45 microscope (Olympus, Tokyo, Japan). Minimum, maximum, 5th and 95th percentile values were calculated based on 50 measurements of each structure at 1000 × magnification and outliers are given in parenthesis. Images of conidiomata in Texas specimens were obtained with an Olympus E-520 digital camera attached to an Olympus SZ61 stereomicroscope. They were stacked using the CombineZP focus stacking software v.1.0 (https:// combi nezp. softw are. infor mer. com/). The abbreviation FSCI is used to further refer to focus stacking composite images in figure legends. Voucher specimens are deposited in ILLS and living strains in CBS. Herbaria and culture collection acronyms throughout the text are cited following Index Herbariorum (http:// sweet gum. nybg. org/ scien ce/ ih/) and the Culture Collections Information Worldwide of the WFCC-MIRCEN World Data Center for Microorganisms (CCINFO; http:// www. wfcc. info/ ccinfo/), respectively (See also the  footnotes of Tables 1, 2).
A set of twenty-two Endocalyx strains, currently deposited at JCM and two others hosted at NBRC, were also studied ( Table 1). Sixteen of them were originally collected and isolated by G.O. (Okada and Tubaki 1984) on decaying petioles and peduncules of palm hosts at different locations throughout Japan between the years 1981 and 1983. They were morphologically characterized in detail on natural substrates and eight different culture media, and originally deposited at TKB as TKBC and IFO. They are currently preserved at NBRC with identical IFO accession numbers. Most duplicate strains except NBRC 31306 and NBRC 31299 are available at JCM and some at CBS. The corresponding voucher specimens, originally deposited at TKB as TKBF, were incorporated in 2012 into TNS with TNS-F accession numbers. They are available for search at the NMNS Collection Database of Specimens and Materials (http:// db. kahaku. go. jp/ webmu seum_ en/). Some duplicates were also deposited at IMI and CBS-H.
Eight specimens and strains not originally studied by Okada and Tubaki (1984) but collected and isolated later by G.O. following identical procedures were included. Potato Dextrose Agar (PDA; Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) was mainly used for isolating and culturing these strains. To attempt sporulation, some Japanese isolates were incubated on autoclaved wet petioles of suitable palm hosts placed on thick water agar plates in an unsealed glass Petri dish (or similar with high sides) at room temperature under prolonged incubation for several months or over a year. To obtain focus stacking composite images of conidiomata in Japanese specimens or isolates, a Z16 APO macroscope (Leica Microsystems, Wetzlar, Germany) with installed CombineZP software was used. An ORTHOPLAN microscope with a phase contrast device (Leitz, Wetzlar, Germany) and a BIOPHOT microscope with a differential interference contrast device (Nikon, Tokyo, Japan) were also employed (PC & DIC; abbr. used in figure legends). Photos were recorded using Leica MC190 HD and Nikon DS-5 M/ DS-Fi1 digital cameras, and some photos were prepared as composite images (CI; abbr. used in figure legends). Voucher specimens collected by G.O. are deposited mainly in TNS and partly in ILLS. Strains are available for search at the JCM On-Line Catalogue of Strains (https:// jcm. brc. riken. jp/ en/ catal ogue_e). A total of twenty-six Endocalyx strains were included in this study (Table 1). Fungal names follow MycoBank and host plant names follow the International Plant Names Index (https:// www. ipni. org).

DNA extraction, PCR amplification, and sequencing
Genomic DNA was extracted from fungal mycelia grown on MEA using a modified NaOH extraction method  (Osmundson et al. 2013), which consisted of adding 200 µL 0.5 M NaOH to ~ 75 mg of tissue, grinding with a micropestle, centrifugation at 14,000 RPM for 2 min, and adding 5 μL of the resulting supernatant to 495 μL 100 mM Tris-HCl buffered with NaOH to pH 8.5-8.9 (Tris-HCl-DNA extraction solution). The complete nrDNA internal transcribed spacer (ITS) region and the first 1,100 bp of the 5' end of large subunit nrDNA (LSU nrDNA) were amplified as two overlapping regions. PCR amplification using a GoTaq® Green Master mix (Promega, Madison, Wisconsin, USA) consisted of the following: 12.5 μL GoTaq® Green Master mix, 2.5 µL BSA, 2.5 µL 50% DMSO, 2 μL of each 10 μM primer ITS1F/LR3 or LR0R/LR6, and 3 μL DNA. PCR amplification was completed on a Bio-Rad PTC 200 thermal cycler under the following parameters: initial denaturation at 94 °C for 2 min, followed by 40 cycles of 94 °C for 30 s, 47 °C for 30 s, 72 °C for 1 min with a final extension step of 72 °C for 10 min. Gel electrophoresis (1% TBE agarose gel stained with ethidium bromide) was used to verify the presence of a PCR product. PCR products were purified using a Wizard® SV Gel and PCR Clean-Up System (Promega), and template DNA was used in 10 μL sequencing reactions with BigDye® Terminator v3.1 (Applied Biosystems, Foster City, California, USA) using a combination of the following primers: ITS1F, ITS4, LR0R, LR3, LR3R and LR6 (Vilgalys and Hester 1990;White et al. 1990;Gardes and Bruns 1993;Rehner and Samuels 1995). Sequences were generated on an Applied Biosystems 3730XL highthroughput capillary sequencer at the W.M. Keck Center at the University of Illinois Urbana-Champaign. In the case of the JCM strains, they were first grown on PDA, and genomic DNA extraction, PCR amplification and sequencing were performed following the previously reported method (Hashimoto et al. 2021

Taxon sampling and datasets
The novel Endocalyx sequences obtained from the Japanese, Hawaiian and Texan strains were subjected to megablast searches in GenBank database to first explore their identity and phylogenetic position. The closest hits from blast searches were representatives of Xylariales, particularly members of the families Cainiaceae and Xylariaceae, which were selected and downloaded to assemble individual datasets.  dataset. Other sequences from recent phylogenetic studies of Xylariales (Jaklitsch et al. 2014(Jaklitsch et al. , 2016Senanayake et al. 2015;Hyde et al. 2020) were also included (Table 2). To test the hypothesis whether Endocalyx species are putative members of Apiosporaceae, sequences of representative taxa of Arthrinium (= Apiospora) and Nigrospora Zimm. obtained from Crous and Groenewald (2013) and Wang et al. (2017) were also incorporated in the datasets. The strains Achaetomium macrosporum CBS 532.94, Chaetomium elatum CBS 374.66 and Sordaria fimicola CBS 723.96 (Sordariales) were selected as outgroups.

Phylogenetic analyses
Sequences were aligned with MAFFT v.7.475 (Katoh and Standley 2013) on the online server which automatically selected the FFT-NS-i iterative refinement strategy (Katoh et al. 2002) for both the ITS and LSU datasets. Maximum Likelihood (ML) and Bayesian inference (BI) approaches were first conducted for each individual dataset using RAxML v.8.2.12 (Stamatakis 2014) and MrBayes v.3.2.7a (Ronquist and Huelsenbeck 2003;Ronquist et al. 2012), respectively, on the CIPRES Science Gateway server (Miller et al. 2010). ML analyses were run under the GTR + CAT model using the rapid bootstrapping algorithm and 1,000 bootstrap iterations to estimate branch support with bootstrap support (BS) values ≥ 70% considered significant (Hillis and Bull 1993). BI analyses consisted of two independent runs of 10 million generations with four (one cold and three heated) Markov Chain Monte Carlo chains each starting from different random trees with default prior values and trees sampled every 100th generation. The first 25% of trees were discarded as burn-in prior to convergence and the remaining trees were used to compute a 50% majority rule consensus tree and estimate posterior probabilities (BPP) for each node. Analyses were set to stop when the standard deviation of split frequencies decreased below 0.01 and convergence of runs was further confirmed in Tracer v.1.6.0 (Rambaut et al. 2014). Clades were considered statistically significant when BPP ≥ 0.95 (Alfaro et al. 2003). No significant topological differences were observed between individual ITS and LSU trees (see Results). Therefore, datasets were concatenated in MEGA v.6.06 (Tamura et al. 2013) for further ML and BI analyses using the above settings. The best-fit substitution model for individual and concatenated datasets, as determined in MEGA using the corrected Akaike Information Criterion, was the GTR + G + I. Trees were edited in MEGA and further refined using Inkscape (https:// inksc ape. org). All alignments and resulting trees are deposited in TreeBASE (https:// treeb ase. org/, study number S19717).

Morphology on the hosts and in culture
On the hosts and in culture, the morphology of conidiomata and conidia of the Endocalyx species newly collected in Japan and USA agreed well with those in previous descriptions (Petch 1908;Hughes 1953a;Okada and Tubaki 1984). A synopsis of key diagnostic features is provided in Table 3. In addition to a new taxonomy for E. melanoxanthus var. grossus (proposed below as "E. grossus") and the description of E. melanoxanthus var. melanoxanthus (reported below as E. melanoxanthus) based on the Texas specimens, novel observations and comments on other Endocalyx species are also included below. As a general remark, all species treated produced subspherical pycnidioid conidiomata on autoclaved palm/wooden chips and agar media such as PDA and others (Okada and Tubaki 1984;this paper;Figs. 3C, D, G, 4). These conidiomata lacked ostioles on growth media (Table 3), and conidia were released by random cracking of the conidiomatal outer walls (Fig. 4C, F).

Molecular analyses
Intraspecific pairwise comparisons of the ITS and LSU sequences belonging to the Japanese, Hawaiian and Texan specimens of E. melanoxanthus var. melanoxanthus showed they are nearly identical except for one C-T transition at position 421 of the ITS alignment in strains JCM 5156, JCM 5159, JCM 5161, JCM 7948 and NBRC 31299, and one G-A transition at position 396 of the LSU alignment in strain JCM 5159. Similarly, strains MFLUCC 15-0723A, B and C belonging to E. metroxyli collected in Thailand differed only by one T-C transition at position 49 of the ITS alignment, whereas their LSU sequences were identical to those of the studied E. melanoxanthus var. melanoxanthus strains. In contrast, the strain Endocalyx sp. MAFF 244025 showed a total of 15 bp and one gap differences compared with the remaining ITS sequences. In the case of E. melanoxanthus var. grossus, all sequences were identical except for the LSU of JCM 5167 which contained a C-T transition at position 430 of the alignment. The ITS and LSU sequences of E. cinctus strains each showed three nucleotide differences between them, whereas they were identical between E. indumentum strains.
The combined ITS-LSU alignment consisted of 102 sequences and 1,683 positions including the outgroups, 897 from the ITS alignment and 786 from the LSU. The single best RAxML tree (ln = −17725.715920) generated  Okada and Tubaki (1984); this study a cf. Seifert and Okada (1990) b Growth media used: Czapek-Dox agar, malt extract agar, potato dextrose agar, cornmeal agar, and sterilized wooden chips. Details mentioned in Okada and Tubaki (1984)  Wide variety of palms including T. fortunei Petch (1908); Okada and Tubaki (1984); this study from the ML analysis is shown in Fig. 1 Wijayawardene et al. (2018). In general, phylogenetic reconstructions between individual loci (ITS/LSU) recovered similar topologies despite some minor differences in position for families in Xylariales such as Lopadostomataceae and Xylariaceae. Nevertheless, the Endocalyx clade and the family Cainiaceae grouped with strong support and identical consistency in all analyses (Figs. S1, S2).

Taxonomy
Endocalyx cinctus Petch, Ann. Bot. (London) 22: 394 (1908). (Figs. 2F, G, 3A). For a detailed description on natural substrates and in culture together with key diagnostic features see Okada and Tubaki (1984) and Table 3. Notes: Morphologically, the specimen studied (TNS-F-91424) agrees well with the description provided by Okada and Tubaki (1984). In culture, strain JCM 7946 produced conidiomata and conidia (Figs. 2G, 3A) on autoclaved petioles of L. chinensis var. subglobosa after a 3-month incubation period that were almost the same as those on palm hosts (Fig. 2F). The conidial surface was slightly rough especially in young conidia (Fig. 3A). Conidiomata of E. cinctus are unique in morphology among Endocalyx species and consist of synnemata with a bilayered cortex on natural substrate (Fig. 2F) or with a spherically swollen carbonaceous base in culture Tubaki 1984, 1987;Seifert and Okada 1990). Endocalyx cinctus is also the best species for inducing conidiomata and conidia on autoclaved petioles of palms (Okada and Tubaki 1984;this paper). After further prolonged incubation on an autoclaved petiole of L. chinensis var. subglobosa, irregularly shaped conidiomata including subspherical ones were produced (TNS-F-91425). These morphologically unique features of E. cinctus might become the subject of a future research project analyzing its whole genome available in GenBank (BCKC00000000). MycoBank MB817907. Basionym: Endocalyx melanoxanthus var. grossus G. Okada & Tubaki, Mycologia 76: 303 (1984).
For a detailed description on natural substrate and in culture together with key diagnostic features see Okada and Tubaki (1984) and Table 3.
Typification: Japan, Ibaraki, Tsukuba, Higashioka, 36°05′46.6"N 140°07′34.2"E, on dead petiole of Trachycarpus fortunei, 6 Aug. 1982, leg. G. Okada, TNS-F-18281 (TKBF 5027, holotype); ex-type strain NBRC 31308 = JCM 5164 = CBS 105.86 = G. Okada OFC 1116). Notes: Okada and Tubaki (1984) morphologically distinguished E. melanoxanthus var. grossus from E. melanoxanthus var. melanoxanthus based on the brown to yellowish brown color of the peridial hyphae enclosing the conidial mass, which is vivid yellow to greenish yellow in the latter, and the verrucose conidial wall ornamentation. This is relatively smooth to tuberculate under LM, but SEM clearly revealed a thin outer layer that cracks and forms coarse-relief islets evenly distributed over the conidial surface. Moreover, the color of the conidial mass in E. melanoxanthus var. melanoxanthus is somewhat more glistening blackish than in the variety grossus. Differences in conidiomata were less evident, except the color of the peridial hyphae, but the variety grossus forms mostly cupulate, rarely cylindrical and usually smaller conidiomata, 0.2-0.3 mm and up to 3 mm high, whereas those of the variety melanoxanthus are 0.5-1 mm and can reach up to 7 mm high. Ecologically, this taxon is apparently restricted and known so far only from T. fortunei, the windmill palm, one of the bestknown, cold-hardiest species of Arecaceae. Conidia of the variety grossus developed readily in culture compared to the variety melanoxanthus (Okada and Tubaki 1984). Clavate chlamydospore-like conidia were also formed in culture, but more obvious in the variety melanoxanthus, and growth was observed at 5 °C after 3 months, whereas other Endocalyx taxa did not grow under these conditions. In addition to these differences, the eleven strains belonging to the variety grossus, including the ex-type strain, grouped together with high support in our individual and concatenated analyses of ITS and LSU sequence data distant from those of the variety melanoxanthus. The variety grossus is therefore elevated here to the species rank distinct from E. melanoxanthus and the remaining Endocalyx taxa following the guidelines and recommendations outlined for description of fungal species (Seifert and Rossman 2010;Aime et al. 2021).
In culture, strain JCM 32998 produced conidial columns (Fig. 3G arrows) on an autoclaved petiole of T. fortunei. They consisted of almost rough conidia (Fig. 3I) and emerged from an annulus-like structure (Fig. 3G white arrowheads) associated with hemispherical to subspherical conidiomata (Fig. 3G black arrowheads). In the case of strain JCM 5164 (ex-type) and JCM 32997, many smaller conidiomata were produced on the autoclaved substrate under prolonged incubation. Moreover, it was observed that under the same conditions JCM 32997 developed a few conidial columns with brown peridial hyphae from an annulus-like structure (Fig. 3H). This conidiomatal structure is basically the same as that on the palm host. On the other hand, strain JCM 5166 abundantly developed similar subspherical conidiomata of various sizes on PDA under prolonged incubation and without any autoclaved substrate (Fig. 4A). Dark brown conidial masses appeared from broken conidiomata (Fig. 4B, C arrowheads), and conidial columns (Fig. 4D, E arrows) were produced from the lower part of broken conidiomata (Fig. 4D, E arrowheads), which were very similar to the annulus-like structure formed on the autoclaved petiole of T. fortunei (Fig. 3G white arrowheads). For a detailed description on natural substrates and in culture together with key diagnostic features see Okada and Tubaki (1984) and Table 3. Notes: Morphological details of the specimen studied (TNS-F-91432) agree well with the description of Okada and Tubaki (1984). In culture, strain JCM 8042 formed primordium-like structures of conidiomata on an autoclaved petiole of L. chinensis var. subglobosa after a 3-month-incubation period. Hemispherical to subspherical conidiomata were produced on the surface of the substrate after further prolonged incubation, and dark brownish conidial masses sometimes in columns emerged from inside of burst conidiomata (Fig. 3C, D arrow & arrowheads; TNS-F-91433). Conidia with hair-like projections (Fig. 3E, F) were the same as those on palm hosts (Okada and Tubaki 1984, Fig. 23). The strain JCM 5171 (ex-type) did not produce conidiomata under the same conditions. On the other hand, strain JCM 8042 developed subspherical conidiomata on PDA under prolonged incubation and without any autoclaved substrate (Fig. 4F). In the broken conidiomata ( Fig. 4F arrowheads), small immature conidia without filamentous ornamentation were observed as reported by Okada and Tubaki (1984, Fig. 24 Conidiomata scattered or aggregated in small to large groups and emerging from annulus-like, black, circular pustules, at first short-cylindrical or short-cupulate becoming long cylindrical, subcylindrical, long conical or cup-shaped after incubation for several days, reaching up to 3 mm high in well-developed fructifications and consisting of a black, glistening mass of conidia enclosed by a yellow to greenish yellow or orange yellow, annular mass of sterile peridial hyphae which remain at the base once the conidioma expands and grows upwards surrounding the column Fig. 4 Subspherical conidiomata of Endocalyx grossus JCM 5166 (A-E) and E. indumentum JCM 8042 (F) on PDA after prolonged incubation at room temperature. A. Blackish mature conidiomata of different sizes on a PDA disk. B, C. Dark brown mass of conidia (arrowheads) filled in subspherical conidiomata and conidia smeared on the medium (arrow). D, E. Conidial columns (arrows) produced from lower part of broken conidiomata (arrowheads). F. Broken conidiomata (arrowheads) in which small immature conidia without filamentous ornamentation were produced inside. Scale bars: 1 mm of conidia. Conidiophores micronematous, filiform, flexuous, hyaline, septate, smooth, anastomosing, 1-2.5(-3.5) µm wide. Conidiogenous cells holoblastic, monoblastic, integrated, terminal or intercalary, cylindrical, minutely denticulate. Conidia solitary, dry, globose, subglobose or broadly ellipsoidal, slightly polygonal and flattened in front view, (10-)12-16 × 9-12(-13) µm, ellipsoidal, lenticular or rarely oblong in lateral view, (7-)8-9(-10) µm thick, with a paler equatorial germ slit, aseptate, brown, dark brown or blackish brown, thick-walled, smooth to finely roughened, often with a central or nearly central attachment scar.
Colonies on MEA moderately fast growing reaching 25-35 mm diam. after 10 days at 25 °C, cottony, white, up to 5 mm high at the center, flat and dull-white toward the edge, margin entire, sometimes with visible yellow bands or yellowish spots; reverse dull white, yellowish at the center. Sporulation not observed after 3 months. Chlamydospores present, abundant in gray or blackish patches immersed under the superficial mycelium, terminal or intercalary, solitary or catenate and in short chains of up to 6, globose, subglobose, ellipsoidal, long ellipsoidal, subcylindrical, long cylindrical or elongated, rarely narrowly clavate or pyriform when terminal, straight or flexuous to curved, pale brown to brown or dark brown, thick-walled, smooth, 0(-1)-septate, at times slightly constricted around the center, sometimes with a paler equatorial germ slit, 8-24(-29) × 5-9(-11) µm.  Notes: The description above refers to the freshly collected Texas specimens and serve to document the presence of E. melanoxanthus for the first time in the state. These materials agree well with previous descriptions of the fungus in having distinct annular, vivid, greenish yellow fructifications surrounding a black mass of subglobose, more or less angular, dark brown to blackish brown, aseptate conidia with a paler germ slit. Conidiomata readily developed after incubation for a few days and the conidial mass together with the yellow peridial hyphae expand upward forming long cylinders up to 3 mm high in the longer fructifications. Okada and Tubaki (1984) also obtained morphologically similar, well-developed conidiomata in a moist chamber that reached up to 7 mm high. The moisture conditions induced abundant sporulation that cannot be held by the outer layer of peridial hyphae, which tears laterally in several places or apically, opening up and releasing spores on the substrate ( Fig. 5D-F). Wall ornamentation of conidia was confirmed to be very finely roughened and more visible around the paler wall of the germ slits in agreement with Okada and Tubaki (1984), who reported a fine dust-like layer covering conidia that cracks and creates islets as seen under SEM. In culture, the Texas strains did not sporulate after 3 months incubation on MEA, but they produced abundant chlamydospores in the same time period (Fig. 5J-L). This also agrees with Okada and Tubaki (1984) who reported solitary or catenate, terminal or intercalary, dark brown, chlamydospores similar in size and shape, with a paler germ slit and superficially resembling conidia. They also obtained immersed or superficial, pycnidioid conidiomata on sterilized wooden chips that produced conidia similar to those on the natural substrates, but this technique to induce sporulation was not attempted in this study for the Hawaiian and Texan strains. Additional details of Japanese specimens growing on the natural substrates and in culture are also found in Okada and Tubaki (1984), and key diagnostic features given in Table 3.

Discussion
This study represents a comprehensive assessment of the phylogenetic affinities of four Endocalyx taxa employing molecular data obtained from the specimens and strains collected in Japan, Hawaii and continental USA. Our phylogenetic analyses using a more extensive taxon sampling than that of Konta et al. (2021) confirmed that Endocalyx belongs to the order Xylariales (Sordariomycetes) and its familial position is resolved within the Cainiaceae. All species formed a distinct monophyletic lineage and they grouped with representative members of the family including Cainia graminis, the type species of Cainia and type genus of Cainiaceae. Molecular data along with distinct morphological, cultural and ecological features support the recognition of E. grossus, originally described as a variety of E. melanoxanthus, as a separate species. The remaining three taxa, E. cinctus, E. indumentum and E. melanoxanthus, were also phylogenetically well-resolved (Figs. 1, S1, S2) and all treated Endocalyx species were consistent with their morphological circumscriptions mainly based on conidiomatal morphology, presence or absence of a carbonaceous hyphal cylinder at the base of conidiomata, color of the peridial hyphae and conidial mass, conidial wall ornamentation, and palm host preferences (Okada and Tubaki 1984).
Although Endocalyx is revealed as a phylogenetically, morphologically and ecologically well-defined genus based on the sampled specimens and isolates, E. twaithesii, the generic type species, lacks sequence data. Therefore, the generic position revealed by Konta et al. (2021) and this study, although including the ex-type strains of species such as E. grossus and E. indumentum, still requires confirmation by the inclusion of sequences from E. twaithesii. An online search on the IMI database (http:// www. herbi mi. info/ herbi mi/ home. htm) shows that two specimens of E. twaithesii, one a type specimen from the original collection (Thwaites 1408 in Sri Lanka; IMI 48588a) and another authentic specimen collected by S. Hughes in Ghana (formerly Gold Coast) [IMI 43614c, on twigs of Cissus oreophila Gilg & M. Brandt (Vitaceae)], are currently deposited in IMI. Future collection of fresh authentic material of E. twaithesii at the type locality (Sri Lanka) is needed to further evaluate the phylogenetic position of the genus.
The recently described E. metroxyli collected in Thailand on a dead petiole of M. sagu (Konta et al. 2021) clustered within the E. melanoxanthus clade (Fig. 1), and therefore it was reduced to its synonym (see the taxonomic part of E. melanoxanthus). Morphologically, the authors recognized that E. metroxyli was very close to E. melanoxanthus in having similar black annulus-like pustules, the fertile center enclosed by yellow peridial hyphae, and conidia nearly identical in size. The only distinctive features used to separate both species were the lack of cupulate or cylindrical conidiomata and the absence of thread-like conidiophores in E. metroxyli. In our experience, however, after examining several specimens of E. melanoxanthus on natural substrate before and after incubation in moist chamber, conidiomata of the Thailand specimen MFLU 15-1454 (Konta et al. 2021, Figs. 3C-E) seem to be poorly developed and sporulated probably due to its surrounding environmental conditions. This is confirmed by the specimen collection date that took place during the tropics drier season (December) and no mention of subsequent incubation was made by the authors. The expansion and development of fully cupulate, cylindrical or funnel-shaped conidiomata in E. melanoxanthus, E. grossus and E. indumentum seem to be enhanced under conditions of high moisture (Okada and Tubaki 1984;this paper). An example in this paper is the clear differences in conidioma development seen in the specimen E. melanoxanthus ILLS00121433 on the host before and after incubation in a moist chamber [ Fig. 5C (before), D-F (after)]. Moreover, thread-like conidiophores are usually difficult to find especially in a poorly sporulated specimen, but they can be detected with due diligence (Fig. 5I). Prior to Konta et al. (2021) and the present work, only two unpublished nrDNA sequences and the master record of a whole genome shotgun sequencing project belonging to E. cinctus JCM 7946 (BCKC00000000) were available in GenBank. A large number of anamorphic genera such as Endocalyx currently lack molecular data, and therefore thorough review of past literature together with careful examination of authentic specimens and cultures is still essential to avoid redescribing old and well documented taxa as new (Koukol and Delgado 2021).
In general, isolates of E. melanoxanthus showed a surprisingly low genetic divergence despite originating from three disjunct tropical or subtropical locations in Japan, Hawaii and Texas. Sequences belonging to "E. metroxyli" from Thailand, synonymized here under E. melanoxanthus, were also nearly identical to the Japanese, Hawaiian and Texan collections. The only exception was the strain Endocalyx sp. MAFF 244025 which showed a considerable genetic variation in its ITS and may represent a distinct population of the fungus growing on the same palm host in the Ogasawara Islands, although further conclusions remain pending in the absence of additional molecular data or specimens examination. The Ogasawara Islands is an isolated archipelago in the northwestern Pacific Ocean 1000 km south of Tokyo with a high level of endemism of its flora and fauna (Kobayashi and Ono 1987;Ito 1998). Endocalyx melanoxanthus is a common colonizer of palm debris with a wide distribution that has been recorded so far from several tropical or subtropical countries including Taiwan (Okada, unpublished) on many different palm hosts (Ellis 1971;Taylor and Hyde 2003;Vitoria et al. 2011;Konta et al. 2021). An online search in MyCoPortal (MyCoPortal 2021) shows a total of 106 records of the fungus in seventeen countries. In USA, E. melanoxanthus has been previously recorded only in the states of Florida and Hawaii and now for the first time from Texas. Its long-distance dispersal is probably favored by its peculiar conidioma morphology as well as its host association (including tree planting), but previous knowledge about its intraspecific variability was lacking. However, G.O. collected in Brazil in 1993 some E. melanoxanthus-like fungi on bamboos, as well as E. melanoxanthus on palms (cf., Vitoria et al. 2011), in which the colors of peridial hyphae of the former were slightly different from the latter (Okada, unpublished).
Other species of Endocalyx apparently have more restricted hosts and distributions. Endocalyx grossus, elevated here to the species rank, is known only from T. fortunei, a cold-hardy palm native to Japan and other Asian countries. We speculate that probably E. grossus evolved independently from other studied tropical or subtropical Endocalyx species (Fig. 1). Similarly, E. indumentum, a species distinct in having conidia densely covered by a hair-like, filamentous ornamentation and dark brown, relatively larger conidiomata (Okada and Tubaki 1984), is so far only known from the endemic palm tree L. chinensis var. boninensis in the Ogasawara Islands. However, G.O. collected this species in Brazil in 1993 on bamboos and in Indonesia in 2011 on Carpentaria acuminata Becc. (Okada, unpublished; E. melanoxanthus was also found coexisting on the same sample of C. acuminata), suggesting that more collections will show a wider host range, distribution and genetic divergence. On the other hand, E. cinctus has been collected also from distant locations such as Argentina (Capdet and Romero 2012), Ghana (Hughes 1953a), Japan (Okada and Tubaki 1984), Sri Lanka (Petch 1908, type locality) and Brazil (Okada, unpublished). It is interesting to note that E. melanoxanthus was found growing together with E. cinctus (e.g., ILLS00121502) and E. indumentum (e.g., ILLS00121501 and an Indonesian collection) on the same palm debris. In contrast, Endocalyx grossus only colonizes T. fortunei as far as we know, although E. melanoxanthus rarely occurs on this host (Okada and Tubaki 1984). Future morphological, ecological and phylogenetic studies using more specimens, isolates and additional molecular markers will increase our limited knowledge of Endocalyx intraspecific variability as well as their species boundaries.
The putative placement of Endocalyx within the family Apiosporaceae (Hyde et al. 1998(Hyde et al. , 2020Taylor and Hyde 2003;Senanayake et al. 2015;Wijayawardene et al. 2021) was not supported in our analyses. The four Endocalyx species treated in this paper clustered within the distant family Cainiaceae in Xylariales in agreement with Konta et al. (2021). Okada and Tubaki (1984) previously pointed out that the term "basauxic" refers to the nature of conidiophores, and conidiogenesis in Endocalyx was not the same as in Arthrinium or Spegazzinia. They even rejected the term "sympodial proliferation", applied by Ellis (1971) to conidiogenesis in Endocalyx, and based on LM and SEM studies they only found basauxic elongation of conidiophores and holoblastic conidiogenesis, but no conidiophore mothercells. These structures were not found during examination of the Texas specimens. Neither Hughes (1953a) nor Ellis (1971) described conidiophore mother-cells in Endocalyx. They stated that conidiophores are continuations of the core of hyphae at the base of the fructifications which elongate and anastomose to produce thread-like geniculate conidiophores. Other anamorphic genera having conidiophore Funding Many samples were obtained by G.O. during the Second General Survey on Natural Environment of the Ogasawara (Bonin) Islands (1990Islands ( -1991 supported by the Tokyo Metropolitan University (Japan), a field trip to Brazil in 1993 as part of a cooperative program to develop a culture collection in Campinas funded by the Japan International Cooperation Agency (JICA, Japan), and a field trip to Indonesia in 2011 as part of a cooperative program to support a LIPI culture collection in Cibinong funded by JICA and Japan Science and Technology Agency (JST, Japan).
Data availability Sequence data are available in the NCBI GenBank (https:// www. ncbi. nlm. nih. gov) under the accession numbers given in Table 1; sequence alignments are available in TreeBASE (https:// www. treeb ase. org/); new combination name was registered in MycoBank (https:// www. mycob ank. org/); specimens and strains were deposited in herbaria TNS or ILLS and culture collections JCM, CBS or NBRC, respectively, with acronyms explained in the text.

Declarations
Consent to participate Not applicable.