A Phylogenetic Assessment of Endocalyx (Cainiaceae, Xylariales) With E. Grossus Comb. et Stat. Nov.

The phylogenetic anities of four representative Endocalyx taxa, including three species and two varieties, 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) conrmed 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 unrelated or distant ascomycetous lineages. Novel morphological and cultural features of Endocalyx taxa based on new isolates are commented. The recently described E. metroxyli is reduced to synonym with E. melanoxanthus. This study represents a comprehensive assessment of the phylogenetic anities of four Endocalyx taxa employing molecular data obtained from 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) conrmed that Endocalyx belongs to the order Xylariales (Sordariomycetes) and its familial position is resolved within the Cainiaceae. All species treated formed a distinct monophyletic lineage and they grouped with representative members of the family including Cainia graminis, the type species of Cainia, type genus of the family. Molecular data along with distinct morphological, cultural and ecological features support well 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 (Fig. 1) and all treated Endocalyx species were consistent with their morphological circumscription mainly based on conidiomata morphology, presence or absence of a carbonaceous hyphal cylinder at the base of conidiomata, colour of the peridial hyphae and conidial mass, conidial wall ornamentation, and palm host preferences (Okada and Tubaki 1984). Texas


Introduction
The anamorphic 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 lamentous conidiogenous cells. They produce dry, unicellular, lenticular, elliptical, almost round in one plane, but sometimes slightly angular, dark brown, blackish brown or almost black conidia, with smooth or minutely to moderately echinulate surface, rarely with hair-like projections, and often with a hyaline slit 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 speci city for these hosts while others also grow on dead vines, lilies or twigs of woody trees (Hughes 1953a(Hughes , 1978Okada and Tubaki 1984 Petch (1908) and later con rmed by Hughes (1953a), was described according to Petch (1908) on dead leaves 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) lectotypi ed 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, i.e., E. indicus J.N. Kapoor  ]. 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. These detailed 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, a further 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) rst 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 anamorphic 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 by 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, Arx (1985) considered that genera with basauxic conidiogenesis and pigmented, often oblate or bilaterally attened conidia growing mainly on litter of monocotyledons, especially grasses and palms, represented a phylogenetic entity. Kendrick and Murase (1994) assembled informal groups of anamorphs with shared features and 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 con rmed or rejected by molecular data. The hypothetical placement of Endocalyx in Apiosporaceae and its close relationship with Arthrinium has been widely accepted ( Konta et al. (2021) recently reassigned Endocalyx to the family Cainiaceae (Xylariales) employing molecular data for the rst time based on a limited taxon sampling including only sequences of E. cinctus available in GenBank (http://www.ncbi.nlm.nih.gov/genbank/) 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 con rm 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 including two ex-holotype strains. Other unpublished specimens and cultures of Endocalyx species obtained by another author (G.O.) mainly in Japan (Sato et al. 1991) were also used in this study for a more comprehensive assessment. Results are presented here and a new combination at species level is introduced based on morphological and molecular evidence.

Materials And Methods
Morphological and cultural studies of specimens and isolates Two fresh specimens of E. melanoxanthus var. melanoxanthus were collected on dead in orescences of the palm tree Sabal minor (Jacq.) Pers. (Arecaceae), the dwarf palmetto, during eldwork carried out by G.D. in southeastern Texas in 2020. Conidiomata were recognized in the eld using a hand lens and pieces of substrate showing colonies were brought to the lab for processing. They were brie y 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 spore 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 BX45 microscope (Olympus, Tokyo, Japan). Minimum, maximum, 5th and 95th percentile values were calculated based on 50 measurements of each structure at 1000× magni cation and outliers are given in parenthesis. Voucher specimens are deposited in ILLS (Illinois Natural History Survey Fungarium, Champaign, Ilinois, USA) and living strains in CBS (Westerdijk Fungal Biodiversity Institute, Utrecht, Netherland). 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://combinezp.software.informer.com/). The abbreviation FSCI is used to further refer to them in gure legends.
A set of twenty-two Endocalyx strains currently deposited at the Japan Collection of Microorganisms (JCM), RIKEN BioResource Research Center, Tsukuba, Japan, and two others hosted at the Biological Resource Center (NBRC), National Institute of Technology and Evaluation (NITE), Kisarazu, Japan, were also studied ( Table 1). Sixteen of them were originally collected and isolated by Okada and Tubaki (1984) on decaying petioles and peduncules of palm hosts at different locations of 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 the Institute of Biological Sciences, University of Tsukuba, Tsukuba (TKB) as TKBC and the Institute for Fermentation (IFO), Osaka. They are currently preserved at NBRC with identical IFO accession numbers. Most duplicate strains except NBRC 31306 and 31299 are available at JCM and some at CBS. The corresponding voucher specimens were originally deposited at TKB as TKBF, but they were incorporated in 2012 into the Herbarium of the National Museum of Nature and Science, Tsukuba (TNS) with a TNS-F accession number. They are available for search at the NMNS Collection Database of Specimens and Materials (http://db.kahaku.go.jp/webmuseum_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 that with high waist) 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 v.1.0 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 gure legends). Photos were recorded by Leica MC190 HD and Nikon DS-5M/DS-Fi1 digital cameras, and some photos were prepared as composite images (CI; abbr. used in gure 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/catalogue_e). A total of twenty-six Endocalyx strains were included in this study and their speci c information is available in Table 1. Fungal names follow MycoBank and the International Plant Names Index (https://www.ipni.org) for host plant names. Herbaria and culture collection acronyms are cited according to Index Herbariorum (http://sweetgum.nybg.org/science/ih/) and Culture Collections Information Worldwide (CCINFO; http://www.wfcc.info/ccinfo/) of the WFCC-MIRCEN World Data Center for Microorganisms (WDCM), respectively.

DNA extraction, PCR ampli cation and sequencing
Genomic DNA was extracted from fungal mycelia grown on MEA using a modi ed NaOH extraction method (Osmundson et al. 2013), which consisted of adding 200 µL 0.5M NaOH to ~75 mg of tissue, grinding with a micropestle, centrifugation at 14,000 RPM for 2 minutes, 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 rst 1,100 bp of the 5' end of large subunit nrDNA (LSU nrDNA) were ampli ed as two overlapping regions. PCR ampli cation 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 ampli cation was completed on a Bio-Rad PTC 200 thermal cycler under the following parameters: Initial denaturation at 94 °C for 2 minutes, followed by 40 cycles of 94 °C for 30 seconds, 47 °C for 30 seconds, 72 °C for 1 minute with a nal extension step of 72 °C for 10 minutes. Gel electrophoresis (1% TBE agarose gel stained with ethidium bromide) was used to verify the presence of a PCR product. PCR products were puri ed 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 high throughput capillary sequencer at the W.M. Keck Center at the University of Illinois Urbana-Champaign. In the case of the JCM strains, they were rst grown on PDA and genomic DNA extraction, PCR ampli cation and sequencing were performed following the previously reported method (Hashimoto et al. 2021). Consensus ITS-LSU sequences were assembled with Sequencher 5.4 (Gene Codes Corp., Ann Arbor, Michigan, USA) and deposited in GenBank.

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 rst 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 the combined ITS-LSU dataset. Exception was the only available LSU sequence of Seynesia erumpens (Berk. & M.A. Curtis) Petr. (AF279410) which produced incongruent results during subsequent analyses and was removed from the nal dataset. Additionally, ITS-LSU sequences of E. cinctus JCM 7946 available in GenBank and those belonging to the recently described E. metroxyli (Konta et al. 2021) were also added to the datasets. An additional ITS sequence from a specimen identi ed as Endocalyx sp., collected in the Ogasawara  (Hillis and Bull 1993). The best t-substitution model for the combined ITS-LSU dataset as determined in MEGA using the corrected Akaike Information Criterion value was the GTR+G+I. Bayesian inference analysis 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 rst 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 diagnosed in Tracer v1.6.0 (Rambaut et al. 2014).

Molecular analyses
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 ITS 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) resulted from the ML analysis is shown in Fig.  1 with E. melanoxanthus var. melanoxanthus, E. melanoxanthus var. grossus and E. metroxyli as E. melanoxanthus, "E. grossus" and E. melanoxanthus, respectively. The ML tree topology was similar in topology to the 50% majority rule consensus tree of the 6,688 sampled trees from the Bayesian analysis. Effective sample size values of all relevant parameters were >200 as veri ed in Tracer, indicating adequate sampling of the posterior distribution (Drummond et al. 2006;Drummond and Rambaut 2009). The twenty-six Endocalyx strains formed a strongly supported monophyletic group (97% BS, 1.0 BPP) within the family Cainiaceae. Each species and variety were well resolved, and they split in two subclades: one including E. melanoxanthus var. melanoxanthus, E. indumentum and E. cinctus and the other containing only E. melanoxanthus var. grossus. The Japanese, Hawaiian and Texan strains of E. melanoxanthus var. melanoxanthus formed a strongly supported monophyletic group (100% BS, 1.0 BPP). The three strains belonging to E. metroxyli from Thailand (MFLUCC 15-0723A, B and C, obtained from the same single specimen) and that one of Endocalyx sp. MAFF 244025 from Japan also clustered within this clade indicating they are conspeci c with E. melanoxanthus var. melanoxanthus. They grouped sister to the two available strains of E. indumentum including an ex-holotype (JCM 5171) without signi cant BS support but showing signi cant BPP = 0.98. Strains of E. indumentum, E. cinctus and E. melanoxanthus var. grossus including an ex-holotype (JCM 5164) each formed highly supported clades (99% or 100% BS, 1.0 BPP). The four taxa of Endocalyx shown in Fig. 1 were clearly recognized at species level and not at variety level. Therefore, E. melanoxanthus var. melanoxanthus and E. melanoxanthus var. grossus should be treated as E. melanoxanthus and "E. grossus" and a new taxonomic treatment is proposed below for the latter. The Endocalyx lineage was sister to a moderately supported monophyletic group including Cainia graminis (Niessl) Arx & E. Müll., the type species of Cainia Arx & E. Müll., and other members of Cainiaceae (Xylariales) belonging to three different genera. The family was recovered as a highly supported monophyletic clade (99% BS, 1.0 BPP). Arthrinium and Nigrospora species grouped together in a highly supported clade (98% BS, 1. 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 (Fig. 3G) and conidia (Fig. 4A) on autoclaved petioles of L. chinensis var. subglobosa after three month-incubation period that were almost the same as those on palm hosts (Fig. 3F). The conidial surface was slightly rough especially in young conidia (Fig. 4A). Endocalyx cinctus is the best species for inducing conidiomata and conidia on autoclaved petiole of palms (Okada and Tubaki 1984; this paper). After further prolonged incubation on autoclaved petiole of L. chinensis var. subglobosa, irregularly shaped conidiomata including subspherical ones were produced (TNS-F-91425). Notes: Okada and Tubaki (1984) morphologically distinguished E. melanoxanthus var. grossus from E. melanoxanthus var. melanoxanthus based on the brown to yellowish brown colour 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 light microscopy but scanning electron microscopy (SEM) clearly revealed a thin outer layer that cracks and forms coarse-relief islets evenly distributed over the conidial surface. Moreover, the colour of conidial mass in E. melanoxanthus var. melanoxanthus is somewhat glistening blackish than the variety grossus. Differences in conidiomata were less evident, except the colour 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, while 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 best-known cold-hardiest species of Arecaceae. Conidia of 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 much obvious in the variety melanoxanthus, and growth was observed at 5 °C after three 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 an ex-holotype strain, grouped together with high support in our concatenated analyses of ITS and LSU sequence data distant from those of the variety melanoxanthus. Considering all the morphological, ecological, cultural and molecular evidence based on multiple specimens and cultures (Seifert and Rossman 2010), the variety grossus is elevated here to species rank distinct from E. melanoxanthus and the remaining Endocalyx species.
In culture, strain JCM 32998 produced conidial columns (Fig. 4G arrows) on autoclaved petiole of T. fortunei. They consisted of almost rough conidia (Fig. 4I) and emerged from an annulus-like structure (Fig. 4G white arrowheads) associated with hemispherical to subspherical conidiomata ( Fig. 4G black arrowheads). In the case of strain JCM 5164 (ex-holotype) 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. 4H). 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. 5A). Dark brown conidial mass appeared from broken conidiomata (Fig. 5B, C arrowheads), and conidial columns (Fig. 5D, E arrows) were produced from the lower part of broken conidiomata (Fig. 5D, E arrowheads), which were very similar to the annulus-like structure formed on autoclaved petiole of T. fortunei (Fig. 4G white arrowheads). 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 autoclaved petiole of L. chinensis var. subglobosa after three month-incubation period. Hemispherical to subspherical conidiomata were produced on the surface of the substrate after further prolonged incubation, and dark brownish conidial masses in columns or not emerged from inside of burst conidiomata (Fig. 4C, D arrow & arrowheads; TNS-F-91433). Conidia with hair-like projections (Fig. 4E, F) were the same as those on palm hosts (Okada and Tubaki 1984, Fig. 23). The strain JCM 5171 (ex-holotype) did not produce conidiomata in the same conditions this time. On the other hand, strain JCM 8042 developed subspherical conidiomata on PDA under prolonged incubation and without any autoclaved substrate (Fig. 5F). In the broken conidiomata (Fig. 5F arrowheads), small immature conidia without lamentous ornamentation were observed as reported by Okada and Tubaki (1984, Fig. 24). Conidiomata scattered or aggregated in small to large groups and emerging from an annulus-like, black, circular pustules, at rst 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 fructi cations 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 of conidia. Conidiophores micronematous, liform, exuous, 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 attened 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 nely 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, at 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 super cial 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 exuous to curved, pale brown to brown or dark brown, thick-walled, smooth, 0(-1)-septate, at times slightly Notes: Our specimens agree well with previous descriptions of the fungus in having distinct annular, vivid or greenish yellow fructi cations 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 fructi cations. Okada and Tubaki (1984) also obtained morphologically similar, well-developed conidiomata in 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 and tears laterally in several places or apically, opening up and releasing spores on the substrate (Fig. 2D-F). Wall ornamentation of conidia was con rmed to be very nely roughened and more visible around the paler wall of the germ slits in agreement with Okada and Tubaki (1984), who reported a ne dust-like layer covering conidia which cracks and creates islets as seen under SEM. In culture, the Texas strains did not sporulate after three months incubation on MEA, but they produced abundant chlamydospores in the same time period (Fig.  2J-L). This also agrees with Okada and Tubaki (1984) who reported solitary or catenate, terminal or intercalary, dark brown, similar in size and shape chlamydospores, with a paler germ slit and super cially resembling conidia. They also obtained immersed or super cial, pycnidioid conidiomata on sterilized wooden chips that produced conidia similar to those on natural substrate, but this technique to induce sporulation was not attempted this time for the Japanese, Hawaiian and Texan strains.

Discussion
This study represents a comprehensive assessment of the phylogenetic a nities of four Endocalyx taxa employing molecular data obtained from 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) con rmed that Endocalyx belongs to the order Xylariales (Sordariomycetes) and its familial position is resolved within the Cainiaceae. All species treated formed a distinct monophyletic lineage and they grouped with representative members of the family including Cainia graminis, the type species of Cainia, type genus of the family. Molecular data along with distinct morphological, cultural and ecological features support well 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 (Fig. 1) and all treated Endocalyx species were consistent with their morphological circumscription mainly based on conidiomata morphology, presence or absence of a carbonaceous hyphal cylinder at the base of conidiomata, colour of the peridial hyphae and conidial mass, conidial wall ornamentation, and palm host preferences (Okada and Tubaki 1984).
On the hosts and in culture, the morphological and cultural features of conidiomata and conidia of Endocalyx species newly collected in Japan and USA agree quite well with Okada and Tubaki (1984) and other previous papers (Petch 1908;Hughes 1953a). In addition to a new taxonomy for E. melanoxanthus var. grossus and the description of E. melanoxanthus based on the Texas specimens, novel observations and comments on other Endocalyx taxa are also added. In culture, Endocalyx grossus, E. indumentum, E. cinctus, and E. melanoxanthus produce subspherical pycnidioid conidiomata on autoclaved palm/wooden chips and agar media such as PDA and others (Okada and Tubaki 1984; this paper; Fig. 4C, D, G, Fig. 5). These conidiomata lack ostioles resembling "asexual cleistothecium-like conidiomata", and conidia are released by cracking of the outer walls (Fig. 5C, F). In the case of E. cinctus, conidiomata are unique in morphology among Endocalyx species and consist of synnemata with a bilayered cortex on natural substrate (Fig. 3F) or with a spherically swollen carbonaceous base in culture Tubaki 1984, 1987; Seifert and Okada 1990). These morphologically unique features of E. cinctus might become the subject of a future analysis research project using its whole genome available in GenBank (BCKC00000000).
Although Endocalyx is revealed as a phylogenetically, morphologically and ecologically well-de ned genus based on these several specimens and isolates, E. twaithesii, the generic type, is not sequenced yet. Therefore, the generic position revealed by Konta et al. (2021) and this study, although including ex-holotype strains of species such as E. grossus and E. indumentum, still requires con rmation through sequences from E. twaithesii. An online search on the IMI database (http://www.herbimi.info/herbimi/home.htm) shows that two specimens of E. twaithesii, 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 recollection of fresh authentic materials of E. twaithesii at the type locality (Sri Lanka) is needed to further evaluate the phylogenetic status 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 centre enclosed by yellow peridial hyphae, and conidia nearly identical in size. The only distinctive features to separate them 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 con rmed by the specimen collection date that took place during the tropics drier season (December) and no mention of subsequent incubation procedure was made. 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). Example is the visible differences in conidiomata development seen in specimen E. melanoxanthus ILLS 121433 on the host before and after incubation in moist chamber [ Fig. 2C (before), D-F (after)]. Moreover, thread-like conidiophores are usually di cult to nd and especially in a poorly sporulated specimen but they can be detected with due diligence (Fig. 2I). 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.
In general, isolates of E. melanoxanthus showed a surprisingly low genetic divergence despite originating from three disjunct tropical or subtropical locations such as 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 specimen examination. The Ogasawara Islands is an isolated archipelago in the northwestern Paci c Ocean 1000 km south of Tokyo with a high level of endemism of its ora 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 the Mycology Collections data Portal [MyCoPortal (http://mycoportal.org/portal/index.php) accessed 26 February 2021] shows a total of 106 records of the fungus in seventeen countries. Its long-distance dispersal is probably favored by its peculiar conidiomata morphology as well as its host association (including tree plantation), but previous knowledge about its intraspeci c variability is currently lacking. However, in 1993 G.O. collected some E. melanoxanthus-like fungi on bamboos, as well as E. melanoxanthus on palms (cf., Vitoria et al. 2011), in Brazil, in which the colours of peridial hyphae of the former were slightly different from the latter (Okada, unpublished).
Other species of Endocalyx apparently have more restricted 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 E. grossus evolved later from other tropical or subtropical Endocalyx species (Fig. 1). Similarly, E. indumentum, a species distinct in having conidia densely covered by a hair-like, lamentous ornamentation and dark brown, larger conidiomata compared with other taxa (Okada and Tubaki 1984), is known so far only 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 Indonesia in 2011 on Carpentaria acuminata Becc. (Okada, unpublished; E. melanoxanthus was also found coexisting on the same sample of C. acuminata), and therefore it is likely that more collections will show a wider distribution and genetic divergence. Similarly, 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., ILLS 121502) and E. indumentum (e.g., ILLS 121501 and an Indonesian collection) on the same palm debris. Endocalyx grossus, on the other hand, only colonizes T. fortunei as far as we know although E. melanoxanthus rarely occurs on this host (Okada and Tubaki 1984, p. 301). Future morphological, ecological and phylogenetic studies using more specimens, isolates and additional molecular markers will help increase our limited knowledge of their intraspeci c 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' used by Ellis (1971) to be applied to conidiogenesis in Endocalyx and based on light microscopy and SEM ultrastructural studies they only found basauxic elongation of conidiophores and holoblastic conidiogenesis, but no conidiophore mother-cells. 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 fructi cations which elongate and anastomose to produce thread-like geniculate conidiophores. Other anamorphic genera considered putative members of Apiosporaceae were Spegazzinia Sacc. and Dictyoarthrinium S. Hughes. Recent phylogenetic studies, however, assigned them to the family Didymosphaeriaceae in Pleosporales (Dothideomycetes) (Tanaka et al. 2015;Samarakoon et al. 2020a, b) and therefore they are phylogenetically distant from Endocalyx and Arthrinium in Xylariales and Amphisphaeriales, respectively. Anamorphs having basauxic conidiophores are clearly not a uniform group and the absence of conidiophore mother-cells in Endocalyx is phylogenetically signi cant among them similar to the differences noted by Kirschner et al. (2017) between modes of conidiogenesis in Spegazzinia and Arthrinium. Anamorphic fungi with this mode of conidiophore elongation do not represent a natural group and this feature is shown to have evolved independently within unrelated or even distant ascomycetous lineages.
The family Cainiaceae (Krug 1977) was recently de ned in the last outline of the class Sordariomycetes (Hyde et al. 2020) and accepted within Xylariales along with fteen other families. Konta et al. (2021) keyed out eight genera in Cainiaceae including the anamorphic Endocalyx and refrained from comparing the genus with other members of the family as they have been described solely based on their teleomorphs. However, a morphological connection between some of them and Endocalyx is supported by the presence of germ slits in both their ascospores and conidia. Other features such as the very dark brown ascospores at maturity, globose but also ellipsoidal in shape, unicellular to 1-septate with ornamented walls having reticulations or longitudinal striations (Kang et al. 1999;Senanayake et al. 2015) may also be considered reminiscent of conidia of some Endocalyx species. Seynesia erumpens, for example, has two-celled ascospores with a full-length germ slit in each cell (Hyde 1995). Together with S. nobilis (Welw. & Curr.) Sacc., the type species of the genus currently lacking molecular data, they are also pantropical in distribution and found as saprobic colonizing petioles and stems of various palm trees and bamboos. Other Arecophila species without available sequence data are also known to colonize rachides or dead trunk and wood of palm species (Hyde 1996). In contrast, Cainia species are mainly saprobic or pathogenic on grasses (Poaceae) (       (arrowheads) lled 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 lamentous ornamentation were produced inside. Scale bars: 1mm.