Short-spored Subulicystidium (Trechisporales, Basidiomycota): high morphological diversity and only partly clear species boundaries

Abstract Diversity of corticioid fungi (resupinate Basidiomycota), especially outside the northern temperate climatic zone, remains poorly explored. Furthermore, most of the known species are delimited by morphological concepts only and, not rarely, these concepts are too broad and need to be tested by molecular tools. For many decades, the delimitation of species in the genus Subulicystidium (Hydnodontaceae, Trechisporales) was a challenge for mycologists. The presence of numerous transitional forms as to basidiospore size and shape hindered species delimitation and almost no data on molecular diversity have been available. In this study, an extensive set of 144 Subulicystidium specimens from Paleo- and Neotropics was examined. Forty-nine sequences of ITS nuclear ribosomal DNA region and 51 sequences of 28S nuclear ribosomal DNA region from fruit bodies of Subulicystidium were obtained and analysed within the barcoding gap framework and with phylogenetic Bayesian and Maximum likelihood approaches. Eleven new species of Subulicystidium are described based on morphology and molecular analyses: Subulicystidium boidinii, S. fusisporum, S. grandisporum, S. harpagum, S. inornatum, S. oberwinkleri, S. parvisporum, S. rarocrystallinum, S. robustius, S. ryvardenii and S. tedersooi. Morphological and DNA-evidenced borders were revised for the five previously known species: S. naviculatum, S. nikau, S. obtusisporum, S. brachysporum and S. meridense. Species-level variation in basidiospore size and shape was estimated based on systematic measurements of 2840 spores from 67 sequenced specimens. An updated identification key to all known species of Subulicystidium is provided.


Introduction
The genus Subulicystidium was created by Parmasto (1968) to accommodate corticioid fungi with long subulate or sword-like cystidia with a unique morphology. The smooth thick crystalline sheath of cystidia is covered with two chains of the bow-tie-shaped crystals, which are seen in the light microscope as four chains of rectangular crystals along the cystidium body (Jülich 1975, Keller 1985. Other morphological characters of the genus are resupinate arachnoid fruit-bodies, loosely interwoven hyphae with constant clamps and suburniform basidia (Oberwinkler 1977, Duhem andMichel 2001). Repetobasidia were also noted by some authors (Jülich 1968. The genus belongs to the order Trechisporales K.H.Larss., though its relationship with the other genera within the family Hydnodontaceae Jülich remains unclear (Larsson 2007. Fruit-bodies of Subulicystidium are found on decayed wood or other plant debris at the forest floor but exact nutrition mode of the genus is not known (Hibbett et al. 2014).
Species delimitation in Subulicystidium has remained challenging. Basidiospore size and shape were traditionally used as the main discriminating characters, while other microscopic structures of fruit-bodies were considered as generally invariable (Oberwinkler 1977, Boidin and Gilles 1988, Duhem and Michel 2001. However, overlap of spore size between species is reported, as well as high morphological variability of the spores within single collections (Liberta 1980, Hjortstam andRyvarden 1986). This has led to doubts on the identity of some taxa. For example,  regarded S. longisporum as a highly variable "species complex" and that S. brachysporum (P.H.B. Talbot & V.C. Green) Jülich and S. meridense Oberw. should not be accepted until additional data for species limit evaluation became available (Oberwinkler 1977. Despite the general progress in molecular identification of fungi during the last three decades (Kõljalg et al. 2013), almost no data on the genetic diversity within Subulicystidium have been published and the genus remains poorly represented in all kinds of molecular studies. Currently available public sequences are usually identified to genus level only or even just named "Trechisporales". Public sequences from fungal fruit-bodies annotated to the species level are few (Volobuev 2016).
During recent decades, extensive collections of Subulicystidium were made by us in Paleo-and Neotropics. In this paper, 11 new species of Subulicystidium are reported based on morphological and molecular evidence (similarities and phylogenies based on rDNA ITS and 28S sequences). The concepts of five previously known species are clarified and the possibility of species presence on several continents is verified. In the current study, we focus on rich material with relatively short basidiospores, i.e. nonacicular and often less than 10 µm long, thus leaving out S. longisporum-like material for a future study.

Assembling dataset
In this study, we examined 144 herbarium specimens of the genus Subulicystidium, which were collected in several regions of Paleotropics (Réunion Island, Madagascar, Africa, South-East Asia) and Neotropics (Caribbean region, various countries of South America). This material was collected during the last six decades, with the oldest collection (PDD13816) from 1954 and the most recent ones from 2015 (e.g. KAS:L 1860). Collections are preserved in the following herbaria: O (Natural History Museum, Oslo University, Norway), GB (Gothenburg University, Sweden), MG (Museu Paraense Emílio Goeldi, Belém, Brasil), SP (Instituto de Botânica, São Paulo, Brasil), KAS (University of Kassel, Germany), FR (Senckenberg Research Institute and Natural History Museum, Frankfurt am Main, Germany) and LY (University of Lyon, France). We examined also holotype specimens of Subulicystidium meridense Oberw. (TUB, Tübingen University, Germany), S. nikau (G. Cunn.) Jülich (PDD, New Zealand Fungal Herbarium, Landcare Research, Auckland) and the collection of S. allantosporum Boidin and Gilles ad interim (Boidin and Gilles 1988) from LY. Attempts to obtain the type specimen of S. brachysporum from PREM (Plant Protection Research Institute, Queenswood, South Africa) were not successful.
For a better biodiversity data availability and reusability, in Suppl. material 1, the table with detailed information on all 144 specimens examined is provided. If missing on the original specimen labels, data on higher-and lower rank administrative units were mined and added to the corresponding columns in Suppl. material 1. The table also includes geographic coordinates for each specimen in decimal degrees format (DD) with minus signs used to indicate southern and western hemispheres. Originally, geographic coordinates were available for 65 specimens. For the other 69 specimens, an attempt to estimate the coordinates from a map was made using resources Google Maps (via http://www.gpskoordinaten.de/), OpenStreetMap (https://www.openstreet-map.org) and the georeferencing calculator of Wieczorek and Wieczorek (2015). In ten specimens, the locality was not precisely indicated to estimate the coordinates. The manner, in which coordinates were obtained, was specified for each specimen in the Suppl. material 1.
Field data and photos of recent collections from Réunion Island (stored in FR and KAS) are accessible via PlutoF workbench (Abarenkov et al. 2010) under the project "Ordynets_Fungi of Reunion Island" and as a part of GBIF occurrence dataset of the Senckenberg herbarium FR (Senckenberg 2018).

Analysing microscopic traits
Sections from dried herbarium specimens were examined in 3% aqueous solution of potassium hydroxide (KOH) mixed with 1% aqueous solution of Phloxine, using 100× immersion oil lens of a Leica DM500 light microscope. Images were captured with a built-in ICC 50 HD Camera using Leica Application Suite EZ V.3.2.1 software (Leica Microsystems Ltd., Switzerland). Measurements were done with the software "Makroaufmaßprogramm" from Jens Rüdigs (https://ruedig.de/tmp/messprogramm. htm) and analysed with the software "Smaff" version 3.2 (Wilk 2012). At least 30 basidiospores per specimen were measured where possible for the sequenced specimens or otherwise important collections. When referring to the basidiospore measurement results, abbreviation L was used for the spore length, W for the spore width, Q for the length to width ratio and N x/y for the x number of spores measured from y specimens.
The raw spore measurements were undertaken as follows. First, for each collection, automated search for size outliers was performed with the "Smaff" software (Wilk 2012). To account for the outliers in both spore length and width simultaneously, the parameters of (i) length multiplied by width and (ii) volume were calculated for each spore by the programme. These values, (i) followed by (ii), were checked to represent the outliers in the sample on a 95% probability level, using simultaneously the tests of Verma and Quiroz-Ruiz (2006), David et al. (1954) and Grubbs (1950). Upon their detection, outliers were excluded from the sample as recommended by Wilk (2012). This procedure usually resulted in a better fit of the spore measurements to the normal distribution. The spore measurements after excluding outliers are provided in the Suppl. material 2.
These filtered spore measurements were used to calculate the spore size range of the species. The main range was presented as the interval into which 90% of non-outlier measurements fall, while 5% of the smallest and 5% of the largest non-outlier measurements were included in parentheses. For the species with more than one specimen available, the filtered spore measurements were pooled together and the main range (90% of the data) with 5% of the smallest and 5% of the largest values were defined for this pooled sample. Calculations were done in R version 3.3.3 (R Core Team 2017) and script is available from Ordynets and Denecke (2018). Additionally, for species with at least three sequenced specimens, hypothetical intervals were calculated within which 90% of all existing individuals' specimen mean values lie. This way of representing basidiospore size variability in species was highly recommended by Parmasto and Parmasto (1987) and Raitviir (1972). These 90% tolerance intervals were calculated for the 90% probability level, with the method of Howe (1969) implemented in the "normtol.int" function of the "tolerance" R package version 1.3.0 (Young 2010). R script for these calculations is available in Ordynets (2018b).
At least 10 basidia and cystidia were measured per specimen and their size variation was presented simply as the range between minimum and maximum values for the pooled measurements of all collections belonging to one species. When basally swollen cystidia was a regular feature, both the largest diameter at the place of swelling and the diameter next to the swelling were noted. The protruding bow-tie crystals were included in the measurements of cystidium diameter. The shape of the cystidium apex followed terminology for sterile hymenial elements of Yurchenko and Wu (2016) and included the following options: tapering, acute and acuminate. Cystidial ornamentation was described as seen under the light microscope.

DNA extraction, amplification and sequencing
Sequences of two nuclear ribosomal DNA regions were considered in our study: internal transcribed spacer (ITS) and ribosomal large subunit-coding DNA (28S). Sequences were obtained from dried herbarium specimens. Total DNA was isolated according to the protocol of Izumitsu et al. (2012). For that, 1-2 mg of fungal fruit body tissue were suspended in 100 µl TE buffer in a 1.5 ml tube. The tubes were microwaved (700 Watt) for 1 min two times, with a 30 seconds pause while keeping the tubes at room temperature. Tubes were cooled at -20°C for 20 min and centrifuged at 10000 rpm for 5 min. The supernatants were 10 or 100 times diluted and in this form used in PCR.
Amplifications were performed in 96-well TGradient Thermocycler (Biometra, Göttingen, Germany). PCR with primer pairs ITS1F/ITS4, ITS1/ALRO and NL1/ NL4 was set as initial denaturation at 94°C for 3 min followed by 29 cycles of dena-turation 94°C for 30 s, annealing 55°C for 45 s and extension 72°C for 60 s; final elongation was done at 72°C for 7 min. PCR with primer pair LR0R/LR5 differed only in having the annealing temperature as 48°C.
PCR products were checked on 1% agarose gel stained with GelRed fluorescence dye (BIOTIUM, Hayward, CA, USA) in the Transilluminator Biometra Ti5 equipped with BioDocAnalyze software (Biometra GmbH, Göttingen, Germany). PCR products were cleaned with QIAquick PCR Purification Kit according to manufacturer's instructions (QIAGEN GmbH, Hilden, Germany). Sanger sequencing of purified products was performed in the facilities of the Senckenberg Research Institute and Natural History Museum (Frankfurt am Main, Germany) and by the company GATC Biotech AG (Constance, Germany). The primers used for sequencing were identical to those used for amplification.
The oldest specimen we succeeded to sequence, with regard to both ITS and 28S regions, was from the year 1978 (LR 15483 in O:F 918488). Attempts of DNA amplification from the type specimens of S. meridense, S. nikau and S. allantosporum Boidin ad interim (Boidin and Gilles 1988) were not successful, as well as an attempt to sequence the type specimen of the new species Subulicystidium ryvardenii Ordynets, Langer & K.H. Larss. sp. nov. We did not succeed in amplifying two protein-coding genes from any of the specimens: a partial segment (511 bp) of the translation elongation factor 1α (TEF1α) with EF-595f and EF-1160r primer pair (Kauserud and Schumacher 2001), as well as the largest subunit of RNA polymerase II gene (RPB1) with RPB1-Af and RPB1-Cr primer pair (Matheny et al. 2002).

DNA sequence-based analyses
All sequences obtained in this study went through the standard quality assessment steps outlined by Nilsson et al. (2012). Raw sequence data were processed with Geneious version 5.6.7 (http://www.geneious.com, Kearse et al. 2012). For various sequence format conversions and alignment viewing, Mesquite version 3.40 (Maddison and Maddison 2018), AliView version 1.19 (Larsson 2014) and Seaview version 4 (Gouy et al. 2010) were used.
In this study, 49 sequences of ITS rDNA region and 51 sequences of 28S rDNA region of Subulicystidium were generated and submitted to GenBank (Benson et al. 2013). They are available as accessions MH041511-MH041559 for ITS and MH041560-MH041610 for 28S region. Additional ten ITS and six 28S sequences of Subulicystidium, earlier available in GenBank and UNITE database (Kõljalg et al. 2013), were downloaded and used in our analyses. Finally, we included sequences of Brevicellicium exile (H.S. Jacks.) K.H. Larss. & Hjortstam and B. olivascens (Bres.) K.H. Larss. & Hjortstam to serve as an outgroup in our sequence-based analyses. All sequences used in the study are listed with brief metadata in Table 1.
Sequences from each locus, ITS and 28S, were pre-aligned in Geneious version 5.6.7 (Kearse et al. 2012) with MUSCLE algorithm (eight iterations) (Edgar 2004). Table 1. Ribosomal DNA sequences used in this study with information on voucher specimens. Most sequences are newly generated for this study and ITS and 28S region were sequenced separately. For specimens GB:KHL 14229 and 16100 and TU 124388, single accession number in each case refers to a sequence containing both ITS and 28S regions. Sequences retrieved from other studies are marked with an asterisk. Abbreviation "na" means sequence is not available. In the species S. brachysporum, "B" means morphological species concept following Boidin and Gilles (1988), while "T" means the species as described by Talbot (1958).  (Abarenkov et al. 2010). The same tool was used to partition ITS into ITS1, 5.8 and ITS2 regions prior to phylogenetic analyses of ITS alignment, to estimate the evolutionary model parameters for each partition separately. The 28S alignment was trimmed manually to produce the sequences of the same lengths and with fewer (if any) gaps at both ends and was not partitioned. Key properties of the final alignments were explored and described using RAxML terminology (Stamatakis 2016).

Species
Morphologically outlined species were compared in terms of genetic distances estimated separately for the trimmed ITS and 28S alignments. For this, raw (also called uncorrected) pairwise dissimilarities of sequences in each alignment were calculated, defined as the percentage of sites that differ between each two full-length sequences including gap positions (Schoch et al. 2012, Kõljalg et al. 2013). This procedure was done with the "dist.dna" function of "ape" R package (Paradis et al. 2004) with option pairwise.deletion=FALSE (i.e. without deleting the sites with missing data in a pairwise way). Results were visualised with the ggplot2 R graphics (Wickham 2009) and R script can be viewed in Ordynets (2018a). Pairwise sequence dissimilarities were further analysed on the intraspecific versus interspecific level. The two levels of sequence variability were segregated with the "sppDist" function of the "spider" R package (Brown et al. 2012) and plotted simultaneously as histograms in a search for the barcoding gap (Meyer et al. 2005). As this classical approach provides only a general overview of sequence variability, i.e. for the pooled dataset, we applied also the recommended alternative which considers the species identity. For each sequence, the maximum intraspecific distance was contrasted with the minimum interspecific distance as recommended by Collins and Cruickshank (2012). Both types of distance for each sequence were estimated, respectively, with the functions "maxInDist" and "nonConDist" of the "spider" R package (Brown et al. 2012) and visualised as a scatterplot. R script for these procedures is available in Ordynets (2018c).
All phylogenetic analyses were performed using the GTR+G evolutionary model. We performed separate analyses of the ITS alignment (partitioned into ITS1, 5.8 and ITS2 regions), unpartitioned 28S alignment and concatenated ITS+28S alignment partitioned into four regions (ITS1, 5.8, ITS2 and 28S). For Bayesian inference of phylogeny, MrBayes 3.2.3 (Ronquist et al. 2012) was used. Two independent MCMC processes, each in 4 chains, were run. Five million trees were generated, sample frequency was set to 1000 and burnin fraction to 0.2. The acceptability of selected settings and mixing sampled trees, were confirmed by the standard deviation of split frequencies, by the potential scale reduction factor, by the sum of average effective sample size in two runs and by tracing likelihood scores of generated trees with Tracer 1.6 (Ronquist et al. 2011, Rambaut et al. 2014. For 8002 sampled trees per analysis (burn-in fraction excluded), a majority rule consensus tree was computed with branch supports representing the relative frequencies of bipartitions (posterior probabilities). Maximum likelihood analyses were performed in RAxML 8.2.10 (Stamatakis 2014). The search for the best-scoring maximum likelihood tree and bootstrap analysis (1000 replicates) were performed in a single run. Both RAxML and MrBayes were run on CIPRES Science Gateway V 3.3 (Miller et al. 2010; http://www.phylo.org). Resulting phylogenetic trees were first viewed in FigTree v. 1.4.2 (Rambaut 2014). Further visualisation and annotation of the phylogenetic trees were done in R version 3.3.3 (R Core Team 2017) and R script is available in Ordynets (2018d). The multiple sequence alignments, details of phylogenetic analyses and trees generated in the study were deposited in TreeBASE: http://purl.org/phylo/treebase/phylows/study/TB2:S22473. Etymology. boidinii, in honour of Jacques Boidin, a great explorer of fungi of Réunion Island, who collected this species and suggested an independent status for it.
Hyphal system monomitic. All septa with clamps. Subiculum thin, with loosely interwoven richly branched hyphae 1.5-2.5 µm wide, thin-walled, hyaline and smooth. Subhymenium thin, with hyphae similar to those in subiculum but occasionally bearing slight amorphous hyaline encrustation. Cystidia subulate, rather narrow, 45-65 × 2.5-3.5 µm including encrustation, projecting up to 30 µm, without basal swelling, terminal or pleural, with thin hyaline cell wall and outer hyaline crystalline sheath covering the whole cystidia except the tapering, thin-walled, acuminate apex. Crystal protrusions on cystidium are small and clearly rectangular and arranged in longitudinal rows.
Remarks on species. Until now, it is the only known Subulicystidium species with such large cylindrical basidiospores. Additionally, large cystidia with regular large protrusions, together with large basidia, make the species remarkable. Etymology. harpagum, from the Latin "harpaga", English "harpoon", a spear with barbs and serrated edges used in fishing. Epithet refers to the cystidium encrustation pattern.
Hyphal system monomitic. All septa with clamps. Subiculum thin, with interwoven richly branched hyphae 2-3 µm wide, thin-walled to very slightly thick-walled, hyaline, often with rough surface because of slight encrustation. In the older fruitbody parts, encrustation represents an up to 1 µm thick sheath over the hypha. Subhymenium thin, with hyphae identical to those in subiculum. Cystidia subulate, 35-62 × 2.5-3.5 µm including encrustation, projecting up to 30 µm, without basal swelling, terminal or pleural, with thin to slightly thickened hyaline cell wall and outer hyaline crystal sheath covering the whole cystidium except the thin-walled, acuminate and particularly narrow, apex. Crystal protrusions on cystidium are formed like short rods that project backwards under acute angle, thus making cystidia resembling a harpoon.
Despite being mixed, the specimen was still selected as type because of the hymenium and subhymenium are better preserved and the ITS and 28S sequences retrieved are of higher quality. Etymology. inornatum (Lat.), without ornament, referring to the almost smooth cystidia.
Hyphal system monomitic. All septa with clamps. Subiculum thin, with loosely interwoven richly branched hyphae 3-4 µm wide, hyaline, thin-walled to slightly thick-walled, covered by a thin hyaline crystal sheath giving them a slightly rough appearance. Subhymenial hyphae similar to those in subiculum, but more compactly arranged and slightly agglutinated. Cystidia subulate, 45-60 × 4-5.5 µm including encrustation, projecting up to 45 µm, occasionally with slight basal swelling (up to 6 µm), terminal, thick-walled and with an outer hyaline crystal sheath covering the whole cystidium except the thin-walled acuminate apex. Surface of the crystal sheath slightly rough, crystal protrusions lacking.
Additional specimens examined. COSTA RICA. Puntarenas: Carrara Biologica Reserva,ca. 50 m,9.7472, Remarks on species. This is the only species in which cystidia and hyphae have a similar surface, which is smooth or slightly rough due to a thin layer of crystalline matter. Etymology. oberwinkleri, named after Franz Oberwinkler, a German mycologist who provided a perceptive view into the species concepts in Subulicystidium and was an early collector of the species in South America.
Hyphal system monomitic. All septa with clamps. Subiculum with interwoven and richly branched hyphae 3-4 µm wide, occasionally swollen up to 6 µm, slightly to moderately thick-walled, hyaline. Subhymenium thin and loose. Subhymenial hyphae richly branched, intricate, regular or occasionally slightly inflated, 3-4 µm wide, thin-walled. Cystidia tubular, 80-150 × 5.5-10 µm including encrustation, projecting up to 70 µm, without basal swelling, with septa having or devoid of clamps, with thin or only slightly thickened hyaline cell wall and outer hyaline crystalline sheath (up to 3.5 µm thick) covering at least the lower half and, at a maximum, almost the whole cystidium except the short, 2-3 µm wide, hyphoid, cylindrical or tapering apex. The crystal protrusions on cystidium are large, plate-like, slightly rhomboid or irregular in outline, somewhat imbricately arranged. Similar encrustation pattern is found also on the subicular and especially subhymenial hyphae and sometimes on the bases of basidia.
Hyphal system monomitic. All septa with clamps. Subiculum thin, with loosely interwoven richly branched hyphae 1.8-3 µm wide, thin-walled, hyaline and smooth. Subhymenium thin, with hyphae similar to those in subiculum but occasionally bearing slight amorphous hyaline encrustation. Cystidia subulate, 45-65 × 2.5-3 µm including encrustation, projecting up to 30 µm, without basal swelling, terminal or pleural, with thin hyaline cell wall and outer hyaline crystalline sheath covering the whole cystidium except the thin-walled, narrow, acuminate apex. Crystal protrusions on cystidium are low but clearly rectangular and arranged in longitudinal rows.
Remarks on species. The few spaced far from each other crystals on cystidium and thick cell wall of cystidium are peculiar. Furthermore, in the single collection studied, cystidia were relatively infrequent and subhymenium was more compact than in other species. Species can be distinguished from Subulicystidium brachysporum also by larger cylindric basidiospores. Etymology. robustius (Lat.), having large cystidia with large crystal protrusions.
Hyphal system monomitic. All septa with clamps. Subiculum thin, with loosely interwoven richly branched hyphae 3-4 µm wide, thin-walled to slightly thick-walled, hyaline and smooth. Subhymenium weakly developed, with hyphae 3-4 µm wide, loosely arranged, slightly thick-walled and often covered with a hyaline crystal sheath. Cystidia subulate, 65-115 × 4.5-6 µm including encrustation, projecting up to 50 µm, with or without a slight basal swelling (up to 6.5 µm diam.), terminal, with thick hyaline cell wall and an outer hyaline crystal sheath covering the whole cystidium except the tapering, thin-walled apex. Crystal protrusions on cystidium are large and mostly rounded and sparsely arranged in longitudinal rows.
Remarks on species. With its hirsute hymenium which has numerous large cystidia, the species is similar to S. robustius, but differs by broader basidiospores and more rounded single crystals on cystidia. Etymology. tedersooi, named after Leho Tedersoo, an Estonian mycologist, the vigorous explorer of the global soil fungal diversity and collector of the type specimen.
Hyphal system monomitic. All septa with clamps. Subicular and subhymenial layer weakly differentiated, consisting of richly branched hyphae 2-3 µm wide, thin-walled, with rough surface due to a subinvisible hyaline crystal sheath. Cystidia subulate, 85-125 × 4.5-5 µm, usually without basal swelling, terminal, with thick hyaline cell wall and an outer hyaline crystal sheath covering the whole cystidium except the acuminate apex. Crystal protrusions on cystidium are rectangular, moderately large, regularly arranged in longitudinal rows.

Sequence dissimilarities and barcoding gap
The aligned ITS dataset included 59 Subulicystidium sequences and two outgroup sequences of Brevicellicium. The dataset consisted of 671 characters (gaps included) and contained 477 distinct alignment patterns, namely 238 in ITS1, 28 in 5.8S and 211 in ITS2 region. The proportion of gaps and completely undetermined characters in this alignment was 20.02%.
Aligned ITS sequences fell into several dissimilarity categories. All the Subulicystidium sequences were at least 10% different from two Brevicellicium sequences (outgroup), as well as from single sequence of Subulicystidium oberwinkleri (Fig. 1a). The sequences of S. robustius were at least 3% and maximum 10% different from the rest of the genus. The sequences of S. harpagum and S. parvisporum were most distant from S. robustius (7-10%) and 3-7% distant from the rest of the genus. Sequences mostly belonging to morphospecies S. meridense and S. brachysporum formed four groups within which they were all 0-3% (in many cases only up to 1%) dissimilar. One of these groups included also sequences of S. fusisporum and S. tedersooi, another group-S. longisporum and S. grandisporum and the third-single sequence of S. obtusisporum. Therefore, both easier and harder distinguishable species, in terms of ITS region identity, were found in the dataset.
The pattern seen through a visual inspection of the ITS sequence dissimilarity matrix was confirmed by the barcoding gap analysis. Throughout the dataset, intraspecific and interspecific distances strongly overlap and no universal for the genus Subulicystidium barcoding gap could be detected (Fig. 2b). Mean and maximal intraspecific distances were 2.87 and 7.73%, while mean and minimal interspecific distances were 5.06 and 0%, respectively. At the level of individual species, a barcode gap existed for S. fusisporum, S. parvisporum, S. robustius and S. tedersooi (Fig. 2a).
The aligned 28S dataset included 57 Subulicystidium sequences and two outgroup sequences of Brevicellicium. The dataset consisted of 617 characters (gaps included) and contained 246 alignment patterns, while the proportion of gaps and completely undetermined characters was 7.54%.
Pairwise 28S sequence dissimilarities were structured differently compared to the ITS dataset (Fig. 1b). The most distinct species in terms of 28S identity was S. oberwinkleri. The dissimilarity of its two sequences from the rest of Subulicystidium and two Brevicellicium sequences was 10-20%. The next most distinct group was formed by the sequences of S. harpagum and S. parvisporum which were 7-10% dissimilar from the rest of the genus except one group containing S. meridense and S. brachysporum sequences (2-3%). The majority of dissimilarities lay in the range 1-5% and were clearly grouped (Fig. 1b).
In a whole 28S dataset, intraspecific and interspecific distances strongly overlapped and thus showed no universal for the genus Subulicystidium barcode gap (Fig 2d). Mean and maximal intraspecific distances were 2.52 and 12.5%, while mean and minimal interspecific distances were 5.58 and 0%, respectively. At the level of individual species, the barcode gap was evident for S. oberwinkleri, S. fusisporum, and S. robustius (Fig 2c).

Phylogenetic analyses
Bayesian analysis (BA) of the ITS alignment was finished with the standard deviation of split frequencies of 0.008 (equals average) and was characterised by the average potential scale reduction factor 1.00 (maximal 1.002) and pooled effective sample size from two MCMC runs 4151.3494. Maximum likelihood analysis (ML) resulted in a tree with a final optimisation log likelihood of -7019.372. BA produced a tree with a partly similar topology to ML tree but contained large polytomy at one of the basal nodes. Hereinafter we present and discuss the topology of the BA tree plotted with both posterior probabilities (pp) from BA and bootstrap supports (bs) from ML.
The phylogenetic tree, generated for the ITS dataset, contains monophyletic and polyphyletic taxa as well as several species represented by a single sequence (Fig. 12). Subulicystidium oberwinkleri is the most basal member of the ingroup. Other singletons descending from the basal nodes are S. nikau, the most deviating sequence of the morphospecies S. brachysporum (TU 110416) (see Discussion for the explanation) and S. rarocrystallinum. The clade dominated by Reunionese collections (pp=1, bs=87%) contains the new species S. parvisporum (pp=1, bs=99%) and S. harpagum (pp=1, bs=98%). The latter includes also L. Ryvarden's collection from Colombia (LR 15736 in O:F: 918487). Two sequences of S. perlongisporum are also placed in the basal part of the tree but do not form a separate clade and, moreover, one sequence (from LE 302156) forms a clade with S. boidinii (KAS:L 1584a). S. robustius is recovered as a distinct clade (pp=1, bs=100%) of four sequences from Neotropics subtended by a long branch. S. fusisporum, represented by three sequences from the Caribbean region (pp=1, bs=98%), is a sister species to S. tedersooi represented by two sequences from Vietnam (branch support pp=1, bs=98%).
The remaining three clades each contain a mixture of sequences belonging to the morphospecies S. brachysporum and S. meridense with their likes. One clade contains also single sequences of S. obtusisporum from Germany (FR: W213-3-I) and S. harpagum from Jamaica (GB:KHL 10733). This clade is joined by three sequences of S. brachysporum: first by two sequences from Réunion (KAS:L 1498 and 1795) and at the next ancestor node with one sequence from Costa Rica (GB:KHL 11216). Another large clade is roughly equally rich in sequences of S. brachysporum and S. meridense (pp=1, bs=100%) and joined by a single sequence of S. inornatum (pp=0.88, bs=41%). One large clade (pp=1, bs=99%) included more collections of S. brachysporum, mostly sensu Boidin and Gilles (1988) and less of S. meridense, but also a single sequence of S. grandisporum (LR 29162 in O:F 506781). One more sequence of S. brachysporum (GB:KHL 10411) formed a weakly supported clade with the three sequences of S. longisporum from Europe (pp=0.84, bs<50%).
Bayesian analysis (BA) of the 28S alignment was finished with the standard deviation of split frequencies of 0.004 (equals average) and was characterised by the average potential scale reduction factor 1.00 (maximal 1.005) and pooled effective sample size from two MCMC runs 4673.55. Maximum likelihood analysis (ML) resulted in a tree with a final optimisation log likelihood of -2209.83. BA produced the tree with the topology highly similar to that of the ML tree. Hereinafter we present and discuss the topology of the BA tree plotted with both posterior probabilities (pp) from BA and bootstrap supports (bs) from ML, mostly focusing on differences from the results obtained for the ITS dataset.
The most basal ingroup members on the 28S tree were S. oberwinkleri (clade with two sequences, pp=1, bs=100%), S. rarocrystallinum and S. harpagum from Jamaica (GB:KHL 10733) (Fig. 13). The species S. harpagum and S. parvisporum remained in the single clade (pp=1, bs=92%) but were recovered as polyphyletic due to the placement of sequence from the KAS:L 1226 amongst the sequences of S. harpagum. They were joined by the clade containing sequences from S. obtusisporum (GB: KHL 10622) and S. brachysporum (TU 110416). The remaining sequences, mostly belonging to S. brachysporum and S. meridense, occupy upper nodes of the tree without clear grouping by morphospecies. 28S dataset was importantly enriched by the sequences from specimens, for which the ITS region could not be sequenced: Bayesian and Maximum likelihood phylogenetic analyses of the concatenated alignment (ITS+28S) resulted in a tree topology which was contributed by both ITS and 28S regions (Fig. 14). In line with the ITS-based tree, S. boidinii and S. harpagum were recovered as polyphyletic but S. parvisporum as monophyletic (cf. Fig. 12). Furthermore, the German sequence of S. obtusisporum was found in the brachysporummeridense clade and not between the sequences of S. longisporum (cf. Fig. 13). On the other hand, similarly to 28S-based tree, S. fusisporum and S. tedersooi intruded the brachysporum-meridense clades. Additionally, the 28S portion of concatenated alignment contributed to resolving a polytomy at one of the basal node containing sequences of S. nikau and S. rarocrystallinum.

Spore-based species comparisons
We measured in total 2840 basidiospores from 67 specimens of Subulicystidium. We defined three groups of species according to the principal basidiospore shape: species with fusiform, cylindric and allantoid basidiospores. We found that some of the species could be delimited based on the basidiospore morphology solely, while, for other species, this was not possible and additional morphological characters had to be considered.
The species with fusiform basidiospores are barely distinguishable according to the basidiospore length. It varied generally from 8 to 11 µm, while the mean value did not exceed 10 µm (Fig. 11a). The only exception was S. fusisporum which had spores 10.7-12.3 µm long (main range, i.e. 5-95% quantiles of measurements data) and 11.5 µm long in average. Three species, viz. S. robustius, S. inornatum and S. fusisporum were indistinguishable in the spore width which varied for three of them between 2.5 and 3.5 µm. In contrast, S. ryvardenii had broader basidiospores with the main range 3.5-4.2 µm and mean value 3.8 µm. S. naviculatum was distinguished by the broadest fusiform basidiospores (main range 4.3-5.0 µm, mean value 4.6 µm), while S. tedersooi by the narrowest fusiform basidiospores (main range 2.1-2.6 µm, mean value 2.4 µm). The spore length to width ratio was as useful as spore width to discriminate the species and was remarkably the lowest in S. naviculatum (2.0-2.5, mean 2.2) and the highest in S. tedersooi (3.5-5.0, mean 4.2).
Under allantoid basidiospores, we considered those with adaxial side clearly concave and having length to width ratio around 2, thus looking rather as reniform or phaseoliform. Amongst the species with such spores, S. oberwinkleri had distinctly the longest and the broadest spores: mean length and width were 9.2 and 4.7 µm, respectively (Fig. 11b). S. nikau could be distinguished from S. boidinii by broader spores. The spore width in S. nikau was 3.9-4.6 µm (mean 4.2 µm) and length-width ratio is 1.6-2.0 (mean=1.8), while S. boidinii has spores 2.8-3.3 µm broad (mean 3.1 µm) and length-width ratio 1.9-2.4 (mean=2.1). Comparing our own data with the Cunningham's specimen of S. nikau (holotype) and Boidin's specimen of S. boidinii showed that we have the same understanding of the respective species as the mentioned authors. S. harpagum and S. parvisporum had rather overlapping spore width and length to width ratio but differed in the spore length: 5.6-8.3 µm (mean 6.7 µm) in the former versus 5.0-6.2 µm (mean 5.6 µm) in the latter.
Species with cylindric basidiospores (Fig. 11c) were characterised by the average length to width ratio of at least 2.8 and were well distinguished by the mean spore length: 12.7 µm in S. grandisporum, 10.7 µm in S. obtusisporum and 9.2 µm in S. cylindrosporum. The collections of the meridense-brachysporum morphogroup were also characterised by cylindrical basidiospores but of the smaller length and were less clearly distinguishable. Examples of single collections representing each morphogroup (Fig. 11d) showed that S. brachysporum sensu Boidin and Gilles (1988) had on average slightly longer basidiospores than S. brachysporum sensu Talbot (1958), viz. 7.9 µm versus 7.3 µm. Curved spores of the classical S. meridense were on average shorter than straight spores of S. aff. meridense, viz. 6.9 µm versus 8.2 µm. Spores in the type specimen of S. meridense were of the intermediate average length compared to the two former examples (7.4 µm). The spore width and length to width ratio were very much overlapping in the material of meridense-brachysporum morphogroup.
With reference to the newly obtained data, in the next section we present the key to the genus Subulicystidium. We used the successful key of Gorjon et al. (2011) as the basis and a source of the information on the long-spored taxa.
Morphological key to the genus Subulicystidium

General remarks
In this study, we describe 11 new species of Subulicystidium based on morphological evidence and rDNA ITS and 28S sequence analyses. Ten of these species are characterised by a unique combination of basidiospore and cystidium morphology and rDNA sequence identity. One species (S. ryvardenii) could not be sequenced but the morphological evidence itself was sufficient for describing it as a new species. With our contribution, the number of the known species in the genus Subulicystidium now totals 20. The provided morphological key to all known species should facilitate identification of specimens previously treated as highly variable S. longisporum or left without species name. Such literature is urgently needed to assist in tropical fungal inventories. We revised also the morphological and genetic borders of the five previously known species. One of them, S. naviculatum, could not be sequenced, while for S. nikau, only one specimen with amplifiable DNA was available. For the two morphospecies, S. brachysporum and S. meridense, numerous sequences from different localities were obtained. Our data show that, despite differences in the protologues, the species are hard to separate morphologically and molecularly. They share, to a large extent, basidiospore size and shape as well as highly similar ITS and 28S sequences, leading to strongly intermixed clades in phylogenetic trees. Therefore, S. brachysporum and S. meridense, in current understanding, are highly polyphyletic.

Species distributions
Our study is based on examination of a large set of specimens from numerous localities in Paleo-and Neotropics. Upon this, we could show that the diversity of the short-spored Subulicystidium species is much higher than previously known. The newly described S. robustius is in fact a frequently occurring species in the Caribbean region and in South America. Furthermore, we could report a multicontinental distribution for several species, verified by DNA sequence data. S. brachysporum, S. boidinii, S. harpagum and S. oberwinkleri are typified by material from Paleotropics (South Africa in the first species and Réunion in three others), but were found by us also in South America. The morphospecies S. meridense, described from Venezuela (Oberwinkler 1977) and later found in Costa Rica (Kisimova-Horovitz et al. 1997), was also found on Réunion Island by Boidin and Gilles (1988). In addition to sequenced collections from a few more countries in South America, we confirmed the species presence on Réunion by sequencing collections of Boidin and Gilles (1988). In this study, we also report S. meridense for the first time from South-East Asia (Taiwan). The new species S. fusisporum was first considered by us as a Caribbean endemic. Re-identification as S. fusisporum of the specimen collected by G. Gilles in Côte D'ivoire (LY 7375, originally labelled as S. longisporum, DNA could not be amplified) may suggest that the species is present in West Africa as well.
It was surprising for us to find the species occurring on more than one continent or on islands separated by thousands of kilometres. For fungi with spores carried by wind, dispersal limitation was shown to act strongly even at small spatial scales (Peay et al. 2010, Norros et al. 2012. Given the architecture and location of Subulicystidium fruit-bodies (next to the ground, not rarely underside of the logs), one would expect prevailing spore dispersal distance smaller than 1 m (Galante et al. 2011). However, in macrofungi, there remains a probability of spore travel on a distance of kilometres (Nordén and Larsson 2000, Peay et al. 2012, Norros et al. 2014) and also overseas (Geml et al. 2012). Spore morphology traits have been recently discussed in a connection with dispersal and arrival success of a species (Norros et al. 2014, Calhim et al. 2018. In this regard, the genus Subulicystidium, with a high diversity of spore size and shape between species, is an interesting object to correlate spore traits and biogeography in future studies. Notes. Unfortunately, we were not able to study the type specimen of Peniophora longispora var. brachyspora (Talbot's No. 40683 in PREM, Mycology Division, ARC-Plant Protection Research Institute, Queensland, South Africa). However, reviewing taxonomic literature on Subulicystidium brought us to delineating two morphogroups in the species S. brachysporum, according to the views of the earlier authors. Talbot (1958), while describing Peniophora longispora var. brachyspora, characterised its basidiospores as "elliptic-fusoid, 6.4-8 × 2.2-3.2 µm … sometimes with a faint band about the middle". Boidin and Gilles (1988) described the basidiospores of S. brachysporum from Réunion as elliptic in frontal face and bananiform (cylindric with slightly attenuated apex, slightly curved) in lateral face, 7.5-10 × 2-2.5(-3) µm. Therefore, we differentiated groups of (i) S. brachysporum sensu Talbot, i.e. sensu typi, with straight oblong-elliptic basidiospores having long attenuated base, with the mean length below 7.5 µm and mean length to width ratio hardly reaching 3 (see Fig. 10m); and (ii) S. brachysporum sensu Boidin and Gilles with cylindric and slightly curved basidiospores with the mean length over 7.5 µm and length to width ratio between 3 and 4 (Fig. 10l). In our dataset, S. brachysporum sensu Boidin and Gilles was represented by far more specimens than S. brachysporum sensu Talbot.
When describing S. meridense, Oberwinkler (1977) stressed the importance of allantoid, i.e. clearly curved, basidiospores. We adhered to this concept assigning our collections to S. meridense (see Figs 10 n, o). We named those with similar spore size but with straight cylindric spores "Subulicystidium aff. meridense" (Fig. 10p). However, molecular level sequence similarity analyses and phylogenetic reconstructions did not support the presence of two distinct groups.
In addition to the South American and Reunionese specimens, we examined also specimens of S. meridense from India and Taiwan. Boidin and Gilles (1988) reported LY 12456 and LY 12772 from Réunion as S. meridense with the note that the spores are larger and more elongated than in the Venezuelan type material. Our morphological examination (and DNA sequence data for LY 12772) showed that the Reunionese collection represent S. brachysporum sensu Boidin and Gilles. However, we concur with Boidin and Gilles that LY 12816 (sequenced) from Réunion and LY 9144 from Gabon should be named S. meridense. The description of the specimen TMI 25520 from Vanuatu (Maekawa 2002) corresponds to our concept of Subulicystidium aff. meridense. Notes. We examined a single collection from Costa Rica (KHL 11566 in GB) which had broad fusiform basidiospores (8.6-)8.8-11.2(-11.6) × (4.0-)4.3-5.0(-5.3) µm, i.e. slightly shorter than in the holotype specimen FO 12778 (TUB) from Venezuela: 10-12 × 4.5-5 µm (Oberwinkler 1977). Furthermore, the cystidia in our specimen were covered with rows of rectangular crystals while the ornamentation pattern of cystidia in the holotype resembled that of S. harpagum, i.e. short rod-like protrusions that project backwards under an acute angle, giving cystidia the resemblance of a harpoon (see fig. 32 in Oberwinkler 1977). Unfortunately, the holotype in TUB could not be located. Kisimova-Horovitz et al. (1997) reported a collection from Costa Rica (207a-I, =FO 42968 in TUB) as S. naviculatum. However, we re-identified the collection as S. robustius.

Subulicystidium nikau (G. Cunn.) Jülich Figs 5c,d; 10u
Notes. The species was described by Cunningham (1955) as Peniophora sororia based on material from the midribs of the dead leaves of nikau palm (Rhopalostylis sapida) which is endemic to New Zealand. Later Cunningham (1963) noticed that the name was occupied (Bourdot and Galzin 1912;p. 386) and provided the legitimate name Peniophora nikau.
As the basidiospores are similar, S. nikau has been confused with S. oberwinkleri. The former has cystidia with regular ornamentation (rows of rectangular crystals) typical for the genus Subulicystidium and the generitype S. longisporum. In contrast, S. oberwinkleri has larger cystidia with large, irregularly shaped crystalline plates. Cunningham (1955) illustrated cystidia of S. nikau correctly, while the characterisation by Stalpers and Buchanan (1991) as "covered with plate-like crystals" is misleading. Oberwinkler (1977) and Maekawa (1998) realised the discrepancies in cystidial ornamentation but did not provide a solution. We re-identified the record of S. nikau from Reunion, LY12488 (Boidin and Gilles 1988) as S. oberwinkleri. We also collected and sequenced a Reunionese specimen with regularly ornamented cystidia (KAS: L1296). Though sampled on dead wood and far from locus classicus, we keep it under the name S. nikau for the time being. Réunion is thus the second known locality of the species after New Zealand. The record from Venezuela reported by  has not been studied and is hard to interpret because no illustration of cystidia was provided.
Specimens Notes. Duhem and Michel (2001) identified a collection from Venezuela (Oberwinkler 1977, Fig. 29, FO15970 in TUB) as S. obtusisporum, while we regard the same specimen as S. brachysporum sensu Boidin and Gilles (1988). Maekawa (2002) reported S. obtusisporum from Vanuatu and Ghobad-Nejhad et al. (2009) from Russian Caucasus. Here we report the species from East Asia and Caribbean region.
The first sequenced material of S. obtusisporum is our collection from Frankfurt am Main, central Germany (FR:W213-3-I). Fruit-body morphology, as well as microhabitat (exposed dead wood) agree with the data for the type specimen and related collections from southern France (Duhem and Michel 2001). Another sequenced specimen GB:KHL 10622 from Jamaica was very distant from the German specimen in terms of ITS and 28S sequence identity and position on the phylogenetic tree, which means S. obtusisporum is polyphyletic. Sequencing additional specimens, not least from Asia, is needed to clarify the taxonomy of this morphospecies. Which morphological characters are useful for the species delimitation in Subulicystidium?
In general, spore size and shape are of crucial importance for the taxonomy of fungi (Parmasto et al. 1987). In our study, however, we found that basidiospore morphology itself may be insufficient for species-rank identifications in Subulicystidium. In this regard, the usefulness of other morphological characters is worth discussing. Jülich (1975) studied cystidia of S. brachysporum and S. longisporum under a scanning electron microscope and concluded the identity of their ornamentation pattern. Jülich (1975) hypothesised, after observing cystidia of S. nikau under a light microscope, that this ornamentation pattern was universal at the genus level. Regarding the shape of cystidia, Jülich (1975) noticed the less prominent basal swelling in S. brachysporum compared to S. longisporum. Oberwinkler (1977) characterised cystidia of Subulicystidium as uniform, but his remarks and especially illustrations displayed several deviations from the common pattern regarding both size and ornamentation. We further developed the idea of the importance of cystidial morphology and showed the presence of interspecific size differences as well as species-specific types of cystidial ornamentation (S. oberwinkleri, S. harpagum, S. robustius, S. rarocrystallinum). The important finding of Jülich (1975) is that the shape of single crystals can vary within a collection. The sharpness of the crystals is reduced with age, resulting in rounded instead of rectangular crystals as observed in a light microscope.
Beside cystidia, also hyphae and hymenial elements can have encrustation. Oberwinkler (1977) illustrated crystalline collars on the bases of basidia in the holotype of S. meridense and in the specimen of S. brachysporum sensu Boidin and Gilles (Figs 30 and 29. respectively in Oberwinkler 1977) but not in other species. Kisimova-Horovitz et al. (1997) noticed a nearly ubiquitous presence of hymenial encrustation in Subulicystidium. We share this opinion after examining our collections. Jülich (1968) and  observed repetobasidia and considered them to be a criterion of the genus Subulicystidium. On the contrary, Eriksson et al. (1984) did not observe any repetobasidia in the North European collections of S. longisporum. In our large set of tropical specimens we did not find any repetobasidia. Thus we suppose that repetobasidia observed by Jülich and Liberta are simply basidia with a well-developed crystalline collar. Oberwinkler (1977) illustrated slightly thick-walled subicular hyphae in S. meridense but thin-walled in the rest of the species. We confirm this pattern for S. meridense but also observed similar deviating subicular hyphae in several other species, viz. S. brachysporum, S. robustius, S. ryvardenii and S. oberwinkleri. However, in all these cases, we found that the hyphal surface is rough and the wall is highly light-refractive, which means they are covered by crystalline material. In S. harpagum, this crystalline sheath around hyphae can reach a thickness of one and in S. oberwinkleri several micrometres. Under a light microscope, it is not possible to decide to what extent thickness depends on the cell wall or is due to the deposition of crystalline material. Bourdot and Galzin (1912) reported a series of collections deviating from typical S. longisporum in fruit-body thickness and colouration and cystidial uniformity. The several varieties proposed by the French authors were not accepted by Jülich (1975) who considered them to represent normal variation and different developmental stages of S. longisporum. In line with Jülich (1975), after examining our specimens, we conclude that fruit-body thickness and density is variable within the species and thus of little value for the species-level identifications. Nevertheless, an experienced eye can differentiate the hirsute hymenial surface of the species with more robust cystidia (S. robustius, S. ryvardenii) from the velutinous hymenial surface of the rest of the genus. In a single species, S. robustius, we consistently observed slightly yellowish fruiting bodies, which was due to a yellowish hue of the hyphal and cystidial walls.

Morphology complements molecular data for species delimitation
Partitioning sequence dissimilarity of both ITS and 28S into interspecific and intraspecific components revealed a clear barcode gap for some of the species but problems to delimit others. Therefore, both cases when morphology and available molecular information are congruent and cases when they are in conflict were found. This points to the importance of careful morphological examination and the need to combine morphology, rDNA barcode data and other DNA markers when defining species in Subulicystidium. Figure 1. Raw pairwise dissimilarities (proportion of the differing sites, %) between Subulicystidium sequences of (a) ITS and (b) 28S region. Three-letter code before each specimen's number corresponds to a species epithet as explained in the legend.  , d) regions. a, c Maximal intraspecific divergence compared with minimal interspecific distances between the aligned rDNA sequences in ITS (a) and 28S (c) datasets. Specimens falling above 1:1 line indicate the presence of the barcoding gap (molecular distinctness of the species) b, d Frequency distributions of intra-and interspecific distances without referring to particular species in ITS (b) and 28S (d) datasets. In the legend, the capital "B" following epithet in S. brachysporum means morphological species concept following Boidin and Gilles (1988), while "T" means the species as described by Talbot (1958). Three-letter code before each specimen's number corresponds to a species epithet as explained in the legend         . Basidiospore size range in the short-spored species of Subulicystidium. Only measurements from sequenced or important historical collections were included in calculations (in total 67 specimens, 2840 basidiospores). Boxes (with median inside) delimit the range between 5% and 95% data quantiles, while the whiskers show minimum and maximum values without considering outliers (see Materials and Methods for details on excluding outliers). If more than one sequenced specimen was available for species, raw measurements without outliers were pooled to calculate basidiospore size range of the species. In S. brachysporum, the capital "B" following epithet means morphological species concept following Boidin and Gilles (1988), while "T" means the species as described by Talbot (1958).

Figure 12.
Phylogenetic relationship of Subulicystidium based on ITS nrDNA sequences. 50% majority-rule consensus tree from Bayesian analysis is shown, with posterior probabilities above the branches and bootstrap support values from the maximum likelihood estimation below the branches. Tips of the tree are annotated according to morphological identification and marked with colours in non-monophyletic taxa (see legend). In the legend, the capital "B" following epithet in S. brachysporum means morphological species concept following Boidin and Gilles (1988), while "T" means the species as described by Talbot (1958). Figure 13. Phylogenetic relationship of Subulicystidium based on 28S nrDNA sequences. 50% majority-rule consensus tree from Bayesian analysis is shown, with posterior probabilities above the branches and bootstrap support values from the maximum likelihood estimation below the branches. Tips of the tree are annotated according to morphological identification and marked with colours in non-monophyletic taxa (see legend). In the legend, the capital "B" following epithet in S. brachysporum means morphological species concept following Boidin and Gilles (1988), while "T" means the species as described by Talbot (1958).

Figure 14.
Phylogenetic relationship of Subulicystidium based on concatenated ITS+28S nrDNA alignment. 50% majority-rule consensus tree from Bayesian analysis is shown, with posterior probabilities above the branches and bootstrap support values from the maximum likelihood estimation below the branches. Tips of the tree include GenBank/UNITE accession numbers of ITS followed by 28S region. Tips are annotated according to morphological identification and marked with colours in non-monophyletic taxa (see legend). In the legend, the capital "B" following epithet in S. brachysporum means morphological species concept following Boidin and Gilles (1988), while "T" means the species as described by Talbot (1958).