What do we learn from cultures in the omics age ? High-throughput sequencing and cultivation of leaf-inhabiting endophytes from beech ( Fagus sylvatica L . ) revealed complementary community composition but similar correlations with local habitat conditions

Comparative simultaneous studies of environmental high-throughput sequencing (HTS) and cultivation of plant-associated fungi have rarely been conducted in the past years. For the present contribution, HTS and extinction culturing were applied for the same leaf samples of European beech (Fagus sylvatica) in order to trace both “real” environmental drivers as well as method-dependent signals of the observed mycobiomes. Both approaches resulted in non-overlapping community composition and pronounced differences in taxonomic classification and trophic stages. However, both methods revealed similar correlations of the fungal communities with local environmental conditions. Our results indicate undeniable advantages of HTS over cultivation in terms of revealing a good representation of the major functional guilds, rare taxa and biodiversity signals of leaf-inhabiting fungi. On the other hand our results demonstrate that the immense body of literature about cultivable endophytic fungi can and should be used for the interpretation of community signals and environmental correlations obtained from HTS studies and that cultivation studies should be continued at the highest standards, e.g. when sequencing facilities are not available or if such surveys are expanded into functional aspects with experiments on living isolates.

introduction Fungal endophytes reside in the living tissues of plants without causing visible disease symptoms (e.g. Christian et al. 2015). Particular research interest is given to endophytes of the phyllosphere and other photosynthetic organs due to the enormous availability and environmental importance of leafy habitats (Lindow and Brandl 2003), the complex biochemical processes in these plant tissues and the generally close interconnectivity of leaf mycobiome with their hosts (reviewed in Rodriguez et al. 2009, Peršoh 2015. Within the last decade, significant progress has been made in unraveling plant-associated mycobiomes by using both cultivation (Collado et al. 2007, Unterseher and Schnittler 2009, Gazis and Chaverri 2015 and direct high-throughput sequencing (HTS) techniques (Bálint et al. 2016). To date, most knowledge about endophyte richness, composition or host preferences is still based on traditional culturing approaches (Arnold 2007, Sieber 2007, Albrectsen et al. 2010, Unterseher et al. 2013a). On the other hand, direct environmental assessment of mycobiomes provides unprecedented details of community diversity, composition, taxonomy and interactions (e.g. Peršoh 2013, Jumpponen and Brown 2014, Bálint et al. 2015, Eusemann et al. 2016, Purahong et al. 2016. These improvements go side by side with the ever increasing accuracy of reference sequence databases (Nilsson et al. 2014, 2015, Abarenkov et al. 2016 It is well recognized that interpretation of diversity of endophytes and other fungi depends on the applied methods (Unterseher 2011). On the one hand, cultivation data are often biased by under-sampling and the use of selective media. On the other hand, amplicon library preparation can differ strongly among studies (Salter et al. 2014) and HTS data are generally squeezed through complex and highly customisable bioinformatic pipelines, leading to variable data analysis (Meiser et al. 2014, Bálint et al. 2016, even if the same plant-fungus system is used in independent studies (Cordier et al. 2012, Siddique and. To date, published studies about the comparative assessment of fungal biodiversity are rare. Allmér et al. (2006) demonstrated the advantages and limitations of fruit body observation, mycelial cultivation and T-RFLP identification of wood-inhabiting fungi. Zhang et al. (2014) assessed microbial communities from deep-sea sediments with cultivation and environmental molecular cloning and identified two complementary assemblages. Similar conclusions were made by Langarica-Fuentes et al. (2014) who identified a biased composition of compost fungi by cultivation. Recently, HTS was rated superior over cultivation, given its ability to detect more obligate, slow growing and rare fungi (Al-Sadi et al. 2015, Oono et al. 2015. Whereas the general lack of congruence between mycobiomes generated by cultivation and HTS can be safely postulated nowadays (Pitkaranta et al. 2008), much less is known about environmental correlations of these differing data sets.
In this study, we investigated endophytic phyllosphere fungi with dilution-to-extinction cultivation (and ITS barcoding) and Illumina sequencing of the same DNA region and from the same material. In accordance with existing knowledge, we expected lower OTU (operational taxonomic unit) richness in the cultivation data and a preferential isolation of ubiquitous, primary saprobic taxa. Consequently, we hypothesized that the cultivable mycobiome exhibits different ecological signals compared with the mycobiome obtained by HTS.

Study sites and sampling
The samples were obtained from an experimental site established in 2013 , consisting of two plots of 4 years-old Fagus sylvatica trees from a different origin. The tree seeds originated from the Lustian lowland area (central eastern Germany, HKG 81005) and were grown in a nursery in northern Germany (near Hamburg) for two years. . The plots are located at the same slope of the mountain massif "Untersberg" in Bavaria, Germany at 517 m asl. (above sea level, respectively) (Valley site; Lat: 47.712946 Long: 13.040101) and at 975 m asl. (Mountain site; Lat: 47.683158 Long: 13.002102) (Siddique and Unterseher 2016). It is humus-rich with a well developed topsoil layer (A horizon) at the valley site, while the A horizon is only weakly developed at the mountain site. The ground and understory vegetation of the mountain site was mainly composed of Acer pseudoplatanus, Picea abies, Daphne mezereum, Cardamine (= Dentaria) enneaphyllos, Helleborus niger and Hepatica nobilis (Siddique and Unterseher 2016). At the valley site, ground vegetation was different with dominance of Acer pseudoplatanus, Mentha spp., Petasites hybridus, Equisetum sylvaticum and Rubus sp . Five trees each from each site were selected randomly in October 2014, exactly one year after planting. Ten symptomless green leaves per tree were removed and processed as described in .
Cultivation of endophytes using the dilution-to-extinction method Isolation of endophytes followed the dilution-to-extinction cultivation (Solis et al. 2016). In brief, samples were blended into tiny particles and filtered. Smallest particles (ø < 0.2 mm) were resuspended and strongly diluted before plating onto malt extract agar (MEA, 1.5%) containing 48-well plates (Carl Roth, Karlsruhe, Germany). The plates were inspected regularly for four weeks. Emerging colonies were transferred into Petri dishes containing the same growth medium.

DNA extraction and ITS sequencing from axenic cultures
Instead of classifying the fungal cultures according to macroscopic and microscopic characters (Khoyratty et al. 2015) and selecting only a few representative strains per morphotype for downstream processing, genomic DNA was extracted from all isolates using a traditional, chloroform-based protocol (e.g. Solis et al. 2016). The ITS region was amplified with the primer pair V9G -ITS4 (de Hoog and Gerrits van den Ende 1998) using approved amplification kits (MangoTaq; Bioline, Germany) and cycling conditions (Solis et al. 2016). Unpurified PCR products were shipped to Beckman Coulter Genomics (Takeley, UK) for sequencing. Sequences were discarded if their corresponding chromatograms showed pronounced signs of ambiguous base calling after end trimming.

Library preparation for high-throughput Illumina sequencing
Total genomic DNA was extracted with the Charge Switch® gDNA Plant Kit (Invitrogen, Germany) from the same fresh leaf particle mass that was used for cultivation. Library preparation consisted of two consecutive amplification steps in order to add sample-specific tag combination for multiplexing. Please refer to Siddique and Unterseher (2016) and  for the detailed description of this procedure. Amplicons were sequenced in pair-end mode on an Illumina MiSeq platform (Illumina Inc.) at the Genetics Section, Biocentre of the LMU Munich, Germany.

Processing of Illumina reads and Sanger sequences
Demultiplexing and quality filtering of Illumina reads relied on QIIME (Navas- Molina et al. 2013; see Suppl. material 1) and is also detailed in  and Eusemann et al. (2016). Extraction of ITS1 (forward R1 Illumina reads) was done with ITSx (Bengtsson-Palme et al. 2013) followed by reference-based chimera checking (Nilsson et al. 2015), open-reference OTU picking (complete-linkage clustering at 97% similarity; Rideout et al. 2014), selection of representative sequences and taxon assignment (Kõljalg et al. 2013). These last steps were also performed with all high-quality Sanger sequences to guarantee best comparability. Final quality filtering of HTS OTUs consisted of the removal of unique (occurring in only one sample) and rare OTUs (having less than five reads, cf. Brown et al. 2015). In contrast, all OTUs from cultivation data were retained, knowing that they belonged to true fungi. The reasons for using only the ITS1 region for subsequent analyses is comprehensively explained in recent papers (e.g. , Eusemann et al. 2016.

Biodiversity analysis and assessment of functional guilds
The analysis of fungal biodiversity comprised the assessment of OTU richness and further indicators of diversity (Fisher's Alpha, Shannon index and three numbers of Hill's series of diversity, the latter considering different levels of rarity) (Hill 1973). Given the strong positive correlation of OTU richness and read numbers (data not shown, but compare Siddique and Unterseher (2016)) as well as the strongly different sequencing depth between the two approaches, read counts were standardised (divided) by sample totals.
Community composition was assessed with PCoA (principal coordinate analysis) and NMDS (non-metric multidimensional scaling) and tested with PERMANOVA (permutational multivariate analysis of variance). Functional guild analysis was performed according to Nguyen et al. (2016). The entire biodiversity analysis was performed with R (freely available at www.r-project.org, last accessed 11/2016). The corresponding command script and necessary input files are available as "Suppl. material 2". Curated Sanger sequences were taxonomically annotated as far as possible and made available in ENA/GenBank through accession numbers LT604837-LT604881. High-throughput data are available under SRA accession number SRX1211311.

Basic data exploration, diversity and community composition
Data volumes differed strongly between Illumina sequencing and cultivation. Illumina sequencing resulted in 597 OTUs from 170480 curated ITS1 reads and cultivation revealed 70 OTUs from 438 culture-based Sanger sequences with the same settings for OTU clustering. The combined data set comprised 630 OTUs (+ 33 OTUs compared with Illumina data). Thirty-seven OTUs were detected with both methods (see Table  S1 on Suppl. material 3 and Suppl. material 4).
An insignificant trend of lower fungal diversity at the mountain site across all indexes was observed for HTS data (Fig. 1A). Richness analysis of cultivation data was partly contradictory with significant differences of Fisher's Alpha between valley and mountain samples (p <0.01, see Table S2 and S3 "Suppl. material 3") but nearly identical accumulation curves (Fig. 1B). In addition and contrary to HTS data, Hill numbers N2 and N3 were significantly different for cultivation data (Fig. 1B, for statistical details see Table S2 and S3; "Suppl. material 3"). The accumulation curves for HTS data (Fig. 1A) revealed a clearly lower fungal richness for mountain than for valley samples, whereas the cultivation data failed to show such differences (Fig. 1B).

Taxonomic composition of HTS and cultivation data
Three of the five most abundant orders from Illumina data were also most abundant in cultivation data (Capnodiales -both methods, Helotiales -both methods, Saccharomy-cetales -Illumina only, Pleosporales -both methods; all Ascomycota) (Fig. 3). In addition, HTS revealed further and abundant orders from both Asco-and Basidiomycota, which were not recovered during cultivation (Fig. 3), such as the yeast fungi Saccharomycetales (Ascomycota) and Malasseziales (Basidiomycota). The Xylariales (Ascomycota, present with one isolate) was the only order from the cultivation data that was not detected with HTS.

Composition of trophic guilds revealed by HTS and cultivation
The five main guilds (pathotrophs, patho-saprotrophs, patho-symbiotrophs, saprotrophs and symbiotrophs) were all detected by HTS. Cultivation largely failed to detect pathotrophs (including patho-saprotrophs and -symbiotrophs). The relative abundance of saprotrophs was clearly higher in cultivation than in HTS data (Fig. 4B, C).
When analysing the influence of locality for the occurrence of different ecological guilds, it turned out that the abundance of pathotrophs was significantly higher in leaves of mountain trees than of valley trees (Fig. 5A). Saprotrophs were also more abundant in leaves of mountain trees, whereas symbiotrophs were more abundant in valley than in mountain trees (Fig. 5A, B).
Results from HTS and cultivation data were congruent in as much as saprotrophs and symbiotrophs revealed similar abundance patterns for both methods.  Principal coordinate analysis (PCoA) of fungal leaf-inhabiting endophytes of beech display strongly differing assemblages obtained with Illumina sequencing and cultivation. Both methods revealed differing mycobiomes from valley and from mountain leaves, although these differences were less pronounced for cultivation data. Abbreviations: IM = Illumina data from mountain samples, IV = Illumina data from valley samples, CM = cultivation data from mountain samples, CV = cultivation data from valley samples

Discussion
The methodology of biodiversity assessment influences the interpretation of community composition and community ecology The most abundant orders were the same for both cultivation and HTS, namely Capnodiales, Helotiales and Pleosporales (Fig. 3). Ascomycota clearly dominated the cultivation data thus confirming results of many earlier cultivation studies (e.g. U' Ren et al. 2012, Scholtysik et al. 2013, Unterseher et al. 2013b). Many of own isolates, such as those with highest sequence similarities to the genus Mycosphaerella and its anamorphs, are often described in literature as frequent asymptomatic inhabitants of living leaves. In this study, taxa generally known as saprotrophs were the dominant trophic guild of the cultivable part (see below) and, in terms of relative abundance, surpassed this guild as detected with HTS by far (Fig. 4B, C).
The compositional difference in the two mycobiomes also corresponded to the presence of parasitic taxa (Taphrinales, Erysiphales) and yeast-like fungi (Saccharomycetales and Tremellales) in the HTS data, whereas the cultivation data were devoid of fungi with obligate parasitic, biotrophic or pathogenic lifestyle. The latter guilds usually cannot be cultivated, and yeasts are often detected only during cultivation studies when growth of . Relative abundance of fungal leaf-inhabiting endophytes of beech among the five main trophic guilds as revealed by analysis with FUNGuild (Nguyen et al. 2016). A compares the two methods for each trophic guild and unassigned data. B displays the trophic guilds and unassigned taxa for Illumina data, C for cultivation data. Abbreviations in [B and C]: U = Unassigned, P = Pathotrophs, PSa = Patho-Saprotrophs, PSy = Patho-Symbiotrophs, Sa = Saprotrophs, Sy = Symbiotrophs Figure 5. Relative abundance distribution of fungal leaf-inhabiting endophytes of beech among the five main trophic guilds as revealed by analysis with FUNGuild (Nguyen et al. 2016). A compares the two localities for each trophic guild on the basis of Illumina data B compares the two localities for each trophic guild on the basis of cultivation data. filamentous fungi is slowed down with low-temperature incubation. In this study, HTS retained a wide range of taxa (compare Al-Sadi et al. 2015) and all guilds available in the FUNGuild reference data base (Nguyen et al. 2016) as expected (Figs 4,5).
A poor comparability of cultivation and HTS data, as it is presented here, was recently reported for a microbiome study (Eevers et al. 2016). It was caused by the fundamentally different sample coverage with the most abundant OTU counting 28621 reads for HTS (Mycosphaerellaceae, Sphaerulina) and 102 isolates for cultivation (Hyaloscyphaceae, Lachnum) (Suppl. material 4).

The two methods revealed consistent signals of both community data to environmental conditions
On the one hand side our results clearly demonstrate the limitations and biases of cultivation approaches for comprehensive biodiversity assessments. On the other hand, the results did not meet our expectations (see above), because significant correlations to environmental parameters (here, it was the difference between valley and mountain samples) were still recognized. The present cultivation data are in concordance with similar studies (Unterseher et al. 2013b) and confirm general knowledge about the community ecology of leaf endophytes (Cordier et al. 2012, Zimmermann and Vitousek 2012, Meng et al. 2013, Glynou et al. 2015, Rojas-Jimenez et al. 2016. Moreover, similar results were observed in the present comparative study on the basis of HTS data (see also Siddique and).

Conclusions
Our results clearly justify the co-existence of cultivation and high-throughput approaches. Despite the fast improvement and diversification of HTS technologies with many undeniable advantages in microbial biodiversity assessments (Bálint et al. 2016), cultivation should be retained at highest standards (e.g. Gazis and Chaverri 2015), given, among others, the availability of living cultures for genome, metabolome or bioprospecting analyses (Gazis et al. 2016, Kolarik et al. 2016.
Our results suggest that the immense body of literature about cultivable endophytic fungi can and should be consulted for the interpretation of community signals obtained from HTS studies.
to the MIxS-Built Environment standard -a report from a May 23-24, 2016 workshop (Gothenburg, Sweden). introduction Podaxis has been collected from numerous arid regions around world; approximately 44 species have been described to date (Conlon et al. 2016). This genus encompasses a wide range of morphological characters such as variation in color, size and shapes in fruit body morphology, as well as a wide range of spore length, width and wall thickness, and has often been confused with Coprinus comatus (Morse 1933;Morse 1941;Herrera 1950). Earlier classifications have placed Podaxis within the family Podaxaceae (Morse 1933); however, modern taxonomic classification places it within the family Agaricaceae (Kirk et al. 2008). Recently, Conlon and collaborators (2016) studied 45 specimens labeled as Podaxis pistillaris, mainly from South Africa, and based on combined internal transcribed spacer (ITS) region and LSU rDNA phylogeny analyses demonstrated that the genus contained at least six clades (A-F) representing different putative Podaxis spp. In Mexico, Podaxis was reported for the first time in 1893 as P. mexicanus from Agiabampo, Sonora (Ellis 1893). Then, in 1908, N. T. Patouillard identified P. farlowii also from Sonora (Morse 1933), and in 1938, D. H. Linder identified P. farlowii from Hipolito, Coahuila (http://mycoportal.org). Since then, all the specimens, including the ones deposited at the fungarium of the Herbario Nacional de Mexico (MEXU), have been described as P. pistillaris (Herrera 1950;Guzmán and Herrera 1969;Guzmán and Herrera 1973;Urista et al. 1985;Esqueda et al. 2010;Esqueda et al. 2012). The reduction of names of all specimens in the MEXU fungarium to P. pistillaris has not been previously investigated in light of molecular data.
Despite the occurrence of Podaxis in arid regions of Mexico, the ethnomycological use of this mushroom in the country is undocumented. This is particularly important since Podaxis spp. have been widely utilized for its culinary value by indigenous people, particularly in the Tehuacán-Cuicatlán Biosphere Reserve (RBTC) in the south of Mexico. In this context, the goals of this study were: 1) to analyze via ITS sequencing newly collected and fungarium specimens of Podaxis from Mexico to better predict their molecular phylogenetic placement and thus establish if one or more phylogenetic species of Podaxis exist in Mexico; and 2) to describe the traditional use, handling and economic importance of Podaxis spp. in the RBTC by observations and interviews with the local people.

Fungal material
Eighteen fungarium and five fresh Podaxis specimens from different arid regions of Mexico were used for the phylogenetic study. The fresh fruiting bodies were obtained from four sites in three communities of the state of Oaxaca (Table 1); all collections were made during rainy season. Sampling, description, digitalization, and drying of mushrooms were performed as recommended by Cifuentes et al. (1986). We analyzed the specimens in the laboratory, and measured macro and microscopic characteristics  (Herrera 1950;Guzmán and Herrera 1969). The collected specimens were deposited in the fungarium of the Herbario Nacional de Mexico (MEXU) of the Instituto de Biología at the Universidad Nacional Autónoma de México (UNAM). In addition, basidiospores we obtained from the center of the dried cap of each of fresh and fungarium fruiting bodies fixed with KOH 5% and photographs were taken (Figure 2 and Suppl. material 1).

DNA extraction, PCR amplification and Sanger sequencing
DNA was extracted from a powder of dried cap (pileus, fresh samples) and the center of the stipe (fungarium samples) from specimens indicated in Table 1. Approximately 5 mg of powdered fungal material was transferred to a bashing bead tube with DNA lysis buffer provided by Zymo research fungal/ bacterial DNA extraction kit. Next, DNA was extracted using the procedures indicated in the Zymo fungal/ bacterial DNA MiniPrep kit. The entire ITS region was PCR-amplified on an Applied Biosystems Veriti thermal cycler using PuReTaq Ready-To-Go PCR Beads with ITS5 and ITS4 primers (Gardes et al. 1991;White et al. 1990). The PCR reaction was carried out in 25 µL containing 3-5 µL template DNA, 2.5 µL BSA, 2.5 µL 50% DMSO, and 1 µL of each 10 µM forward (ITS5) and reverse (ITS4) primers. Molecular biology grade water from Fisher scientific was added to reach 25 µL. The following thermocycling parameters were used for the amplification: initial denaturation at 95°C for 5 min followed by 39 cycles at 95°C for 30 s, 55°C for 15 s, and 72°C for 1 min, and a final extension step of 10 min at 72°C (Schoch et al. 2012). The PCR products were then examined on an ethidium bromide-stained 1% agarose gel (Fisher Scientific) along with a 1 kb DNA ladder (Promega) to estimate the size of the amplified band. PCR products were purified using a Wizard SV Gel and PCR Clean-up System. Sanger sequencing of the purified PCR products was performed at Eurofins Genomics (http://www.operon.com/default.aspx) using BigDye Terminator v3.1 cycle sequencing. The sequencing was accomplished bidirectionally using both strands with a combination of ITS5 and ITS4 primers. Sequences were generated on an Applied Biosystems 3730XL high-throughput capillary sequencer. For both sequencing reactions, approximately 15 µL of PCR template were used along with 2 µM sequencing primers.

Phylogenetic analysis
Sequences were assembled with Sequencher 5.3 (Gene Codes), optimized and then manually corrected when necessary; the latter step was to assure that the computer algorithm was properly assigning base calls. Each sequence fragment was subjected to an individual Basic Local Alignment Search Tool (BLAST) (Altschul et al. 1990) search in NCBI GenBank to verify its identity. Detailed BLAST search using ITS data were conducted utilizing only published sequences as outlined previously (Raja et al. 2017).
The newly obtained ITS sequences (Table 1) were aligned with ITS sequences of authenticated published sequences or from vouchered fungarium samples (Brock et al. 2009;Osmundson et al. 2013), such as those from a recent phylogenetic study on Podaxis spp. (Conlon et al. 2016) using the multiple sequence alignment program MUSCLE (Edgar 2004), with default parameters in operation. Leucoprinus was used as an outgroup taxon based on previous studies (Hopple and Vilgalys 1999;Conlon et al. 2016). MUSCLE was implemented using the program Seaview v. 4.3. (Galtier et al. 1996;Gouy et al. 2010). Maximum Likelihood (ML) analysis was perfomed using RAxML v. 7.0.4 (Stamatakis et al. 2008). The analysis was run on the CIPRES Portal v. 3.3 (Miller et al. 2010) with the default rapid hill-climbing algorithm and GTR model employing 1000 fast bootstrap searches. Clades with bootstrap values ≥ 70% were considered significant and strongly supported .

Spore morphology
All spores were measured using a Carl Zeiss Primo Star microscope (Carl Zeiss, Germany) with a Canon PowerShot G6 camera with a Zeiss universal digital camera adapter d30 M37/52´0.75. For each specimen, 25 spores were measured for spore length and width, and presence or absence of a germ pore ( Table 2). The Mann-Whitney (U-test) was used to determine whether the mean values of the spore lengths and widths were significantly different between the MEXU specimens assigned to the phylogenetic clades.

Ethnomycological study
This study was conducted in the RBTC, which is located in the states of Puebla and Oaxaca in central Mexico (between 17°32'24" and -18°52'55"N, and 97°48'43" and -97°48'43"W; Figure 1), and its mainly characterized by arid and semiarid vegetation (Valiente-Banuet et al. 2009). This region comprises eight ethnic groups: two in Puebla, the Popolocas and Nahuas; and six in Oaxaca, the Mixtecs, Cuicatecs, Mazatecs, Chinantecs, Chocholtecs, and Ixcatecs (SEMARNAT and CONANP 2013). In the regions where this study was conducted, some people spoke an indigenous language but Spanish was the prevalent mean of communication among them (Table 1). Local people from the RBTC were randomly selected for the ethnomycological interview.
Inhabitants of the region, most 18 years and older, shared their knowledge through the following questionnaire: i) personal information (name, age, sex, ethnicity, place of birth, residence, occupation, and number of family members); ii) knowledge of mushrooms from the locality (traditional name, description of the fruiting body, myths, and uses); iii) how they collect the mushrooms (frequency of collection, if they eat it or buy it); iv) importance of mushrooms in their life; v) how many different mushrooms they what kind of problems they have when they collect mushrooms in the field; and viii) what information they need to identify the mushrooms.

General morphology of Podaxis
All specimens studied (Table 1) share the typical morphological characteristics of the genus Podaxis (Figure 2 and Suppl. material 1): white or grayish-white fruit body when young and brown in old or dry specimens, with a long bulbous stem, traversing the gleba as a columella supporting the pileus at the apex. Pileus enveloping a large portion of the stipe, including most of the upper part, with a peridium of two layers and a well-developed capillitium. Exoperidium scaly, most of the scales deciduous at maturity. Endoperidium persistent, membranous, when dehiscing, becoming free from the stipe at the base and by longitudinal fissures. Capillitium threads simple, eventually branched and septate, hyaline or pigmented, and flattened. Spores smooth, pigmented, apical pore present, wall of two layers. Basidia fasciculate with 1-4 spores on short sterigmata (Figure 2 and Suppl. material 1).

Variation in basidiospore size and morphology
The length and width, ranges and standard deviation (SD) of basidiospores are outlined in Table 2. MEXU specimens were grouped into two clades (see molecular phylogenetic analysis; Figure 4). Clade D: size of basidiospores in this clade ranged from 9-13 × 8-12 µm (mean = 11-12 × 9-10 µm); and clade E, size of basidiospores in this clade ranged from 9-17 × 9-16 µm (mean = 10-15 × 10-14 µm). Overall the basidiospores in clade D were smaller than basidiospores in clade E (Table 2). Based on the Mann-Whitney (U-test), we found that spore length (p < 0.001; Figure 3A) and width (p < 0.001; Figure 3B), were significantly different between clades D and E, which supports their molecular phylogenetic placements based on the ITS phylogeny ( Figure  4). The color of spores in clade D was generally lighter when compared to those in clade E, which were dark reddish-brown (Figure 2 and Suppl. material 1).

Phylogenetic analysis of molecular data
Eighteen new ITS sequences from different specimens of Podaxis from Mexico were obtained; these included four from freshly collected specimens, and 14 from samples in the MEXU fungarium (Table 1). High quality DNA and PCR products were obtained for all specimens, including MEXU 1191, a collection made in 1948. We were unable to obtain DNA from MEXU 11887, 21635, and 5015 while MEXU 1148 and 27844 produced a PCR band, but resulted in mixed signals perhaps due to low or poor quality of DNA. The ITS alignment consisted of 55 taxa of Podaxis and one outgroup taxon (Leucocoprinus birnbaumii). The original ITS alignment consisted of 848 nucleotides, after ambiguous regions were limited and removed via GBlocks, the final ITS alignment contained 681 nucleotides. RAxML analysis of the ITS dataset produced a single most likely tree (Figure 4). We recovered the same six clades (A, B, C, D, E, and F) that were revealed in Conlon  (8424, 8422, 8426, 8425, 8423, 22610 and 27843) are placed in (clade E, sensu Conlon et al. 2016), with 95% RAxML boostrap support and grouped together with a sequence of P. pistillaris (GenBank: U85336), which has been reported in previous molecular phylogenetic studies of Agaricales fungi (Johnson 1999;Vellinga 2004), while 11 other MEXU specimens (10805, 5772, 12338, 12808, 5015, 27845, 7217, 27558, 1191, 27557 and 7023) were nested within clade D (sensu Conlon et al. 2016); however this clade did not receive significant RAxML bootstrap support. All ITS sequences generated from this study were deposited in the GenBank and accession numbers are provided in Table 1 (KY034673-KY034690).

Ethnomycology
Ethnomycological importance lies in the fact that people from this region eat the fruiting body of Podaxis, commonly known as "hongo" (mushroom), "hongo blanco comestible" (white edible mushroom), or "soldadito" (little soldier), almost daily during rainy season ( Figure 5A-C). They cook the mushroom and mix it with green peppers, onions and "epazote" (Dysphania ambrosioides), and then make "empanadas" (stuffed corn tortilla with the mix) ( Figure 5D-E). It is considered a tasty mushroom, and according to the habitants of the region, as "one of the tastiest and most nourishing products the land gives us". The local people consider this fungus similar to a "piece of chicken" because of it taste. They also eat it raw, mixed with zucchini, or incorporated in chicken soup and the typical dishes "tesmole", "caldillo" and "mole".
Through the years, the local people have acquired the necessary knowledge to easily locate, harvest and select this mushroom from the land. Although this mushroom is mainly used for personal consumption, some people collect it and sell it in the community. They have also acquired the knowledge about the phenology and ecology of Podaxis spp., and they relate the "acidity of rain" with the germination of its spores. In addition, most of the people agree on the following: "when there are constant rains, the fungi starts to grow", "small mushrooms show up after it rains, the sun comes out and the sky is clear", "in order for it to grow, the mushroom needs sunlight for one or two days". Concerning the habitat and soil, they indicate that: "mushrooms grow mainly on the river bank or on sandy soil" but also "mushrooms are produced throughout the mountain slopes, even on agricultural production areas". They also say: "if you find one, you will find two" or "they are born in pairs". Finally, when a mushroom fruiting body has "aged", the local people spread the spores in places where they want the fungi to grow next rain season, and they say: "if they don't grow this season, they'll grow during the next one".

Phylogenetic affiliations of MEXU specimens based on molecular and morphological comparison
Podaxis pistillaris sensu lato has been collected and reported from numerous semi-arid regions around the world, fruiting mainly in the rainy seasons. In Mexico, it has widely been collected from north to south (Herrera 1950;Dennis 1960;Guzmán and Herrera 1973;Urista et al. 1985;Aparicio-Navarro et al. 1991). Despite its wide geographical distribution, the identification of P. pistillaris remains confusing mainly because the type specimen of P. pistillaris collected and described from India has not been sequenced (Linnaeus 1771), making a true molecular phylogenetic assessment difficult. It is likely that cryptic speciation is rampant in this widely distributed species.
All the studied specimens from the MEXU fungarium were named as P. pistillaris based on its morphological characteristics (Figure 2 and Suppl. material 1); however, molecular phylogenetic analysis of the ITS region of these specimens, along with ITS sequences from a recent study of Podaxis spp. from South Africa (Conlon et al. 2016), placed the MEXU specimens into two clades: D and E (Figure 4). Therefore, our analysis indicate there are at least two phylogenetic species of Podaxis in Mexico, and not all species of Podaxis collected from Mexico should be identified as P. pistillaris.
Interestingly, all specimens in clade E (Figure 4) have been reported from North America, including Mexico. In our ITS phylogeny seven MEXU specimens (8424, 8422, 8426, 8425, 8423, 22610 and 27843) were grouped with an ITS sequence of P. pistillaris (GenBank: U85336; Johnson 1999; Vellinga 2004) with significant bootstrap support (95%). However, at this time it is not possible to name this clade. This is because there are other species from the new world, including southwestern US, Mexico and Argentina such as P. argentinus Speg., P. longii McKnight, P. microporus McKnight (McKnight, 1985), P. farlowii Massee (Morse 1933), and P. mexicanus (Ellis 1893), which need to be examined in light of molecular phylogenetic analysis.
Clade E (Figure 4) is entirely comprised of specimens from the new world and all of these occur as free-living in desert-like semi-arid regions (Table 1). There have been reports of symbiotic association of Podaxis spp. with termites in Australia (Priest and Lenz 1999;Young et al. 2002), Nigeria (Alasoadura 1966), South Africa (Bottomley 1948;Conlon et al. 2016), and Bolivia (Rocabado et al. 2007). In this context, it is worth to mentioning that in the RBTC (Oaxaca, Mexico) such a symbiotic association with termites has not yet been reported. Further molecular studies of Podaxis specimens collected from the new world are required to test the hypothesis of loss or gain of termite symbiosis in this clade.
We examined the spore sizes and morphology of MEXU specimens from clade E and compared them to the measurements obtained from the type material of P. pistillaris in the LINN fungarium (Priest and Lenz 1999). The spore size of 10-14 × 9-12 µm from the type material fits the average measurements (10-15 × 10-14 µm) obtained from the MEXU specimens in clade E (Table 2 and Figure 3). The spore color of most specimens in clade E is also reddish-brown with thick-walls (Figure 2 and Suppl. material 1). These attributes are in agreement with the type specimen examined by Priest and Lenz (1999). However, the type specimen from the LINN herbarium needs to be sequenced to corroborate the morphological data.
Eleven of the eighteen specimens (10805, 5772, 12338, 12808, 5015, 27845, 7217, 27558, 1191, 27557, and 7023) were nested within clade D (sensu Conlon et al. 2016), but without significant RAxML bootstrap support (Figure 4). Other members in clade D include seven sequenced specimens from GenBank both labeled as Podaxis sp. and/or Podaxis pistillaris and mostly included specimens collected from desert-like arid regions in western India (Singh et al. 2006). When we removed all other Gen-Bank data from our analysis and only included sequences from our study and those of Conlon et al. (2016), we found that clade D had significantly high bootstrap support (82%; data not shown). All specimens from clade D were reported to be free-living with the exception of PREM 34405 from South Africa (Conlon et al. 2016). The average spore measurements of MEXU specimens in clade D were 11-12 × 9-10 µm (Table 2 and Figure 3), which was well within the range of those reported in clade D by Conlon et al. (2016). Specimens in clade D were reported from both the New World (MEXU) and the Old World (South Africa and India), suggesting that species in this clade are widely distributed geographically.
Based on the fruiting body morphology, it was difficult to separate MEXU specimens in clade D and E (Figure 2 and Suppl. material 1). This observation agrees with the results from Conlon et al. (2016) as they reported that fruiting body morphology of Podaxis spp. does not significantly differ between the termite associated and free-living clades. The spores in clade D (free-living and termite associated) and E (free-living only) were both thick-walled (Figure 2 and Suppl. material 1); this result agrees with the observations made by Conlon et al. (2016), who reason that free-living, desert dwelling species have thick-walled spores as it may help prevent desiccation in desert-like dry environments. Due to the lack of molecular data from type specimens except for P. rugospora (Conlon et al. 2016), currently it is not possible to name specimens in either clade D or E. Based on our preliminary molecular phylogenetic analysis of MEXU specimens, it seems highly unlikely that all MEXU specimens represent P. pistillaris.

Ethnomycology
In Mexico, the use of Podaxis species for food consumption has not been reported. Our study includes data from interviews that state the consumption and farming of this mushroom within the RBTC. In this area, the species is greatly valued by the local people, who sell the fungus for 1-1.5 USD per kilogram or consume young fruiting bodies of Podaxis in typical dishes from the region, particularly as "empanadas" ( Figure  5), a favorite among the people of the region. They also have developed the ability to locate and harvest the mushrooms, as well as farming (proto-cultivation) is considered very important during rainy/wet season. To consistently obtain more fruiting bodies, the locals scatter the spores in the soil where they want the fungus to grow and emerge in the following wet season. This method of spore spreading helps them to locate and collect the mature fungus more quickly.
On the other hand, Podaxis has also been catalogued as a non-edible mushroom (Guzmán 1977) and has been referred as being toxic in Nigeria and South Africa (Alasoadura 1966); contrastingly, it has been reported as edible in Afghanistan, Pakistan, India, and Australia (Batra 1983). People from the Sind Province of Pakistan are familiar with the commerce of "Khumb" or "Khumbi" (fungus P. pistillaris). Khumbi is also a term used by rural communities in Haryana, India, who also refer to this fungus as "Saanp ki chhatri" (umbrella of a snake or snake's cap) (Mridu and Atri 2015). In this region, the mushroom is much appreciated as it is considered a delicacy with medicinal properties for the "Hakims", the dispensers of folk medicine (Khan et al. 1979).
In Yemen and South Africa, Podaxis is used for its medicinal properties and antibacterial activity against Staphylococcus aureus, Micrococcus flavus, Bacillus subtilis, Serratia marcescens, Escherichia coli, Pseudomonas aeruginosa, and Proteus mirabilis (Al-Fatimi et al. 2006;Panwar and Purohit 2002). In Australia, it has been used as hair dye (Batra 1983), while in West Africa, P. pistillaris is used to produce baby-powder (desiccative) as an anti-abortive (Gérault and Thoen 1992). Such medicinal properties arise from the chemical constituents of the fruiting body, which include nitrogen, crude protein, true protein, carbohydrates, lipids, and ash content (Gupta and Kapoor 1990;Gupta and Singh 1991;Khaliel et al. 1989 and.

Conclusions
Podaxis is considered a very important mushroom in arid regions of the world due to its culinary and medicinal values. Further taxonomic and molecular phylogenetic studies of this genus are urgently required to better understand species boundaries and provide accurate names on specimens of Podaxis, particularly the ones used as food in Mexico and worldwide. Better understanding of Podaxis spp. might be possible when mycologists work closely with local communities in different parts of both the Old and New World. Our study provides preliminary morphological and molecular data from Podaxis specimens collected in Mexico along with its ethnomycolgy use. We anticipate our study will encourage future phylogenetic diversity analyses on this widely distributed yet taxonomically poorly studied genus of Agaricomycetes.  (Li and Cui 2013). The genus mainly grows on fallen or dead angiosperm branches which have not decayed much and causes a white rot (Li and Cui 2013). It used to be considered that only a few species were in Megasporoporia s.l. (Ryvarden et al. 1982), but the species diversity in the genus is very rich in subtropical and tropical Asia, with 12 species having been described from the region (Li and Cui 2013). In addition, four species, Megasporoporia cavernulosa (Berk.) Ryvarden, M. hexagonoides (Speg.) J.E. Wright & Rajchenb., M. mexicana Ryvarden and M. setulosa (Henn.) Rajchenb., have been found in subtropical and tropical America, but none has been recorded from Europe (Ryvarden et al. 1982, Ryvarden andMelo 2014).
During the study of polypores from southern China, six specimens collected on fallen angiosperm branches were examined, phylogenetic relationships were analyzed based on ITS and nLSU rDNA sequences data, and three new species of Megasporia were discovered. The aim of this work demonstrates the diversity of Megasporia in China. Illustrated descriptions of these species and a key to known species in the genus are provided in the present paper.

Morphological studies
Specimens examined were deposited in the herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC). Macro-morphological descriptions were based on field notes and herbarium specimens. Color terms follow Petersen (1996). Micromorphological data were obtained from dried specimens, as observed under a light microscope. Sections were studied at a magnification of up to ×1000 using a Nikon E 80i microscope with phase contrast illumination. Drawings were made with the aid of a drawing tube. Microscopic characters, measurements and drawings were made from slide preparations stained with Cotton Blue (CB) and Melzer's reagent (IKI). Spores were measured from sections cut from the tubes. To represent variation in the size of spores, 5% of measurements were excluded from each end of the range, and are given in parentheses. The following abbreviations are used: KOH = 5% potassium hydroxide, IKI-= both non-amyloid and non-dextrinoid, CB-= acyanophilous, L = mean spore length (arithmetic average of all spores), W = mean spore width (arithmetic average of all spores), Q = variation in the L/W ratios between the specimens studied, n (a/b) = number of spores (a) measured from a given number (b) of specimens.

Molecular study and phylogenetic analysis
A CTAB rapid plant genome extraction kit (Aidlab Biotechnologies Co., Ltd, Beijing) was used to obtain total genomic DNA from dried specimens, according to the manufacturer's instructions with some modifications (Chen et al. 2016). The DNA was amplified with the primers: ITS5 and ITS4 for ITS (White et al. 1990), and LR0R and LR7 for nLSU (http://www.biology.duke.edu/fungi/mycolab/primers.htm). The PCR procedure for ITS was as follows: initial denaturation at 95°C for 3 min, followed by 35 cycles at 94°C for 40 s, 54°C for 45 s and 72°C for 1 min, and a final extension of 72°C for 10 min. The PCR procedure for nLSU was as follows: initial denaturation at 94°C for 1 min, followed by 35 cycles at 94°C for 30 s, 50°C for 1 min and 72°C for 1.5 min, and a final extension of 72°C for 10 min. The PCR products were purified and sequenced in Beijing Genomics Institute, China, with the same primers.
Maximum parsimony phylogenetic analysis followed Li and Cui (2013). It was applied to the combined dataset of ITS and nLSU sequences using PAUP* version 4.0b10 . Sequences of Cinereomyces lindbladii (Berk.) Jülich and Sebipora aquosa Miettinen were used as outgroups to root trees following Li and Cui (2013). All characters were equally weighted and gaps were treated as missing data. Trees were inferred using heuristic search option with TBR branch swapping and 1,000 random sequence additions. Max-trees were set to 5,000, branches of zero length were collapsed and all parsimonious trees were saved. Clade robustness was assessed using bootstrap analysis with 1,000 replicates . Descriptive tree statistics tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC), and homoplasy index (HI) were calculated for each maximum parsimonious tree generated.
MrModeltest2.3 (Nylander 2004) was used to determine the best-fit evolution model for the combined dataset of ITS and nLSU sequences for estimating Bayesian inference (BI). Bayesian inference was calculated with MrBayes3.1.2 (Ronquist and Huelsenbeck 2003). Four Markov chains were run for 2 runs from random starting trees for 1 million generations, and trees were sampled every 100 generations. The first one-fourth generations were discarded as burn-in. Majority rule consensus tree of all remaining trees was calculated. Branches that received bootstrap support for maximum parsimony (MP) and Bayesian posterior probabilities (BPP) greater than or equal to 75% (MP) and 0.95 (BPP), respectively, were considered as significantly supported.

Phylogenetic analysis
The combined ITS and nLSU dataset included 45 sequences of ITS and 44 sequences of nLSU regions from 45 fungal samples representing 37 species. The dataset had an aligned length of 1919 characters in the dataset, of which 1330 characters are constant, 178 are variable and parsimony-uninformative, and 411 are parsimony-informative. Maximum parsimony analysis yielded 6 equally parsimonious trees (TL = 2082, CI = 0. 451, RI = 0.625, RC = 0.282, HI = 0.549), and one of the maximum parsimonious trees is shown in Figure 1. The best model for the combined ITS and nLSU sequences dataset estimated and applied in the BI was GTR+I+G. BI resulted in a similar topology with an average standard deviation of split frequencies = 0.007691 to MP analysis, and thus only the MP tree is provided. Both bootstrap values (≥50%) and BPPs (>0.90) are shown at the nodes (Figure 1). Basidiocarps. Annual, resupinate, corky, without odor or taste when fresh, becoming hard corky and cracked upon drying, up to 17 cm long, 4 cm wide, and 0.4 mm thick at centre. Sterile margin thinning out, white when fresh, cream when dry, very narrow to almost lacking. Pore surface white to cream when fresh, cream when dry; pores angular, 3-4 per mm; dissepiments thick, entire. Subiculum pale buff, corky, up to 0.1 mm thick. Tubes cream, paler than subiculum, corky, up to 0.3 mm long.
Basidiocarps. Annual, resupinate, corky, without odor or taste when fresh, becoming hard corky to leathery upon drying, up to 5 cm long, 3 cm wide, and 1.5 mm thick at centre. Sterile margin thinning out, cream when dry, up to 1 mm wide. Pore surface clay-pink to fawn when dry; pores round, 2-3 per mm; dissepiments thin, entire to lacerate. Subiculum cream, corky, up to 0.5 mm thick. Tubes clay-pink, slightly darker than subiculum, corky, up to 1 mm long.
Additional specimen ( Diagnosis. Differs from other Megasporia species by brownish tints on pore surface and lacking tetrahedric or polyhedric crystals. Basidiocarps. Annual, resupinate, corky, without odor or taste when fresh, becoming hard corky upon drying, up to 3 cm long, 2 cm wide, and 2 mm thick at centre. Sterile margin thinning out, white when dry, up to 1 mm wide. Pore surface white to cream but with brownish tints when dry; pores round, 2-3 per mm; dissepiments thin, lacerate. Subiculum white, corky, up to 1 mm thick. Tubes cream, corky, up to 1 mm long.
Spores Among the accepted Megasporia species, M. hexagonoides and M. major have big basidiospores (16.6-21.8 × 5.2-6.8 µm, 15.2-20 × 5.5-7.1 µm, Li and Cui 2013). The three new species described in the current paper have similar basidiospores as M. hexagonoides and M. major, but they have distinct smaller pores than those in M. hexagonoides and M. major (2-4 per mm vs. 0.5-1.5 per mm). Furthermore, hyphal pegs are present in M. hexagonoides and M. major, while they are absent in the three new species. In addition, M. rimosa differs from other species in Megasporia by its extremely thin basidiocarp (less than 0.5 mm thick) and cracked when dry, while fruiting bodies in other species are more than 1 mm thick, and not cracked when dry (Dai and Wu 2004, Du and Cui 2009, Li and Cui 2013. Megasporia tropica is distinguished from other species in the genus by lacking dendrohyphidia, cystidioles and hyphal pegs. Megasporia yunnanensis has brownish tints on its pore surface and lacks tetrahedric or polyhedric crystals, while other species in Megasporia have abundant tetrahedric or polyhedric crystals but lack brownish tints on their pore surfaces. It seems that species of Megasporia prefer small branches rather than big logs; all specimens of the genus having been collected mostly on fallen branches and dead branches on living trees, and such branches being not strongly decayed. The basidiocarps of the genus are usually not very big and usually form small patches, although some patches may be merged finally. All the species of Megasporia have been found on angiosperm wood (never on gymnosperms), and they have a distribution in subtropical and tropical forests, especially in open environments, e.g. fallen branches along roads or paths. In addition, the species diversity of the genus is very rich in subtropical and tropical Asia, many more undescribed taxa are found from our samples based on phylogenetic analyses, but all these samples are sterile as a common feature of the genus, and the best season for producing basidiospores on these taxa are unknown.
Although Megasporoporiella, Megasporoporia and Megasporia are very similar, we found some difference among these genera both in morphology and ecology. The main difference is that Megasporoporia has di-trimitic hyphal structure and strongly dextrinoid skeletal hyphae, while dimitic hyphal structure and weakly to moderately dextrinoid skeletal hyphae are in Megasporoporiella and Megasporia. In addition, Megasporoporiella has a distribution in temperate region, while Megasporia in subtropical to tropics. large, morphologically heterogenous genus that includes more than one hundred and twenty species , Parmasto 2004, Parmasto and Gilbertson 2005, He and Li 2011a, b, He and Dai 2012 , Parmasto 2005, Parmasto and Gilbertson 2005. In 2015 and 2016, several field trips were carried out in southern China, northern Thailand and central Taiwan, and many corticioid fungal specimens including those of Hymenochaete on bamboos were collected. Morphological and molecular studies of the specimens revealed six species of Hymenochaete on bamboos, two of which, H. bambusicola and H. orientalis, are described here as new.

Materials and methods
Morphological studies. Voucher specimens are deposited in the herbaria of Beijing Forestry University, Beijing, China (BJFC), the Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, Thailand (MFLU), and the National Museum of Natural Science, Taichung, Taiwan (TNM). Samples for microscopic examination were mounted in cotton blue and 2% potassium hydroxide (KOH). The following abbreviations are used: L = mean spore length, W = mean spore width, Q = L/W ratio, n (a/b) = number of spores (a) measured from given number of specimens (b). Color codes and names follow Kornerup and Wanscher (1978).
DNA extraction and sequencing. A CTAB plant genome rapid extraction kit-DN14 (Aidlab Biotechnologies Co., Ltd) was employed for DNA extraction and PCR amplification from dried specimens. The ITS region was amplified with the primer pair ITS5 and ITS4 (White et al. 1990) using the following procedure: initial denaturation at 95 °C for 4 min, followed by 34 cycles at 94 °C for 40 s, 58 °C for 45 s and 72 °C for 1 min, and final extension at 72 °C for 10 min. The nrLSU gene region was amplified with the primer pair LR0R and LR7 Hester 1990, Lapeyre et al. 1993) using the following procedure: initial denaturation at 94 °C for 1 min, followed by 34 cycles at 94 °C for 30 s, 50 °C for 1 min and 72 °C for 1.5 min, and final extension at 72 °C for 10 min. DNA sequencing was performed at Beijing Genomics Institute, and the sequences were deposited in GenBank (Table 1).
Maximum likelihood (ML) and maximum parsimony (MP) analyses were conducted for the dataset. MP analysis were performed using PAUP* 4.0b10 . Gaps in the alignments were treated as missing data. Trees were generated using 100 replicates of random stepwise addition of sequence and tree-bisection reconnection (TBR) branch-swapping algorithm, with all characters given equal weight. Branch supports for all parsimony analyses were estimated by performing 1000 bootstrap replicates  with a heuristic search of 10 random-addition replicates for each bootstrap replicate. Max-trees were set to 5000, branches of zero length were collapsed and all parsimonious trees were saved. The tree length (TL), consistency indices (CI), retention indices (RI), rescaled consistency indices (RC) and homoplasy index (HI) were calculated for each generated tree. RAxML v.7.2.6 (Stamatakis 2006) was used for ML analysis. Default setting were used for all parameters in the ML analysis, and statistical support values were obtained using nonparametric bootstrapping with 1000 replicates .