Species Diversity of Lycoperdaceae (Agaricales) in Israel, with Some Insights into the Phylogenetic Structure of the Family

The diversity of Lycoperdaceae in Israel was studied. Molecular phylogenetic relationships within the family, and genus Lycoperdon in particular, were inferred using original ITS rDNA sequences of 58 samples belonging to 25 species from Israel and six other countries, together with 66 sequences stored in the GenBank database. The current molecular phylogenetic study recovered the family Lycoperdaceae as a monophyletic group, which was supported in both maximum likelihood and Bayesian analyses. The clades corresponding to the genera Apioperdon, Bovista, Calvatia, Disciseda, and Lycoperdon were revealed. The taxonomic structure of the named genera was partially resolved. Within the genus Lycoperdon, some species received significant statistical support; however, their relationships, as well as the problem of the genus monophyly, mostly remained questionable. As a result of a thorough literature survey, extensive sample collection, and studies of the material stored in the Herbarium of the Institute of Evolution, University of Haifa (HAI, Israel), fifteen species representing five genera were found in the territory of Israel. Six species, namely Apioperdon pyriforme, Bovista aestivalis, Calvatia candida, Lycoperdon decipiens, L. niveum, and L. perlatum, are new additions to the diversity of Lycoperdaceae in Israel. Detailed macro- and micromorphological descriptions, ecology, geography, and critical notes, together with light microscopy photos and SEM micrographs, are provided. In-depth discussion on some taxonomically challenging species is presented.


Introduction
The family Lycoperdaceae Chevall.comprises species of Agaricales Underw.with an enclosed (angiocarpic) fruit body development and lack of a true stipe, incorporating so-called "true puffballs"-classic gasteroid genera Lycoperdon Pers., Bovista Pers., Calvatia Fr., Disciseda Czern., together with some recent taxonomic additions.The size of the representatives of the family varies tremendously, from 3-9 (2-15) mm gasterocarps of Bryoperdon acuminatum (Bosc) Vizzini (=Bovista acuminata (Bosc) Kreisel) [1][2][3][4], arguably the smallest puffball, to the gigantic fruit bodies of Calvatia gigantea (Batsch) Lloyd (=Langermannia gigantea (Batsch) Rostk.), reaching more than one meter in diameter and weighing up to 20 kg [5].The shape of the gasterocarps could be globose, subglobose, turbinate, sessile, pyriform, pestle-shape, etc.The spore-forming inner tissue, gleba, is whitish at first, turning to olivaceous, brown, or purplish-colored powdery mass, comprised of mature basidiospores.The gleba is covered with a protective sterile peridium, which usually can be segregated into the exoperidium (the outer layer) and endoperidium (the inner layer).The former is typically ornamented with spines, warts, and granules, and disintegrates after the gasterocarp's maturation, revealing the endoperidium, which either has a more or less regular apical opening, called an ostiole, or just irregularly ruptures and disintegrates, setting free powder containing billions or even trillions of basidiospores [6].Later, the spores are passively dispersed by environmental factors such as wind, raindrops, insects, etc.The family has a cosmopolitan distribution with its members found in temperate, arid, and tropical climates [5].Members of Lycoperdaceae, along with other representatives of the former "Gasteromycetes", have a rich folklore heritage and have been used in folk medicine by different cultures around the world.Burk presented a comprehensive literature review on the usage of puffballs by North American Indians [7].The spectrum of their applications included religious, decorative, hemostatic, dietary, and other purposes.Some members of the family were studied for medicinal properties.Calvacin, calvatan, craniformin, and rubroflavin, all isolated from representatives of the genus Calvatia, demonstrated antitumor activity, based on immunopotentiation [8,9].Altuner et al. observed antimicrobial effects of the ethanolic extract of Bovista nigrescens Pers.against several Gram-positive and Gram-negative microorganisms, especially Bacillus subtilis, Klebsiella pneumonia, and Staphylococcus carnosus [10].
Counting the number of Lycoperdaceae members presents a challenging issue.This is largely due to the lack of a world monograph devoted to the family (this situation also holds true for many other gasteroid taxa).In addition, a considerable number of unresolved taxonomic questions regarding genera structure, synonyms, and even the place of the family within the modern Agaricales further complicates this task.Kirk et al. reported approximately 150 species as members [11].This figure obviously does not represent the real number of species known to date, because many novel species have been recently described [12][13][14][15][16][17][18][19][20][21][22][23][24].The Encyclopaedia Britannica web resource lists 160 species [25], but this number includes the "earthstars"-representatives of the genus Geastrum Pers.Incorporation of puffballs and earthstars into a single family reflects a fairly common taxonomic concept of the past.Krüger et al. demonstrated that the earthstars represent a separate lineage and have evolved independently of Lycoperdales [26].The family Geastraceae Corda is currently placed in the subclass Phallomycetidae K. Hosaka, Castellano & Spatafora, proposed by Hosaka et al. [27] on the basis of molecular phylogenetic studies.In more recent additions to the Dictionary of the Fungi [28], the family Lycoperdaceae is treated as a synonym of Agaricaceae Chevall.
Lycoperdaceae has been traditionally placed in the order Lycoperdales [29,30], which contained earthstars, puffballs, and their allies.Lycoperdales was one of the major orders within "Gasteromycetes" [29,31,32]-an assemblage of taxa, which was characterized by spores maturing inside enclosed fruit bodies and dispersed by environmental factors, due to loss of ballistospori.An evolutionary approach, applied to taxonomical classification of various gasteroid morphologies, as well as numerous molecular phylogenetic studies [27,[33][34][35][36][37], proved that the group represents an artificial assemblage of polyphyletic taxa belonging to different lineages.All the taxa from the "Gasteromycetes" were transferred to the class Agaricomycetes Doweld.As for the family Lycoperdaceae, it is either left intact, preserving its rank, or is incorporated into Agaricaceae sensu lato, which already consumed a number of families of the former "Gasteromycetes", e.g., Battarreaceae, Montagneaceae, Mycenastraceae, Podaxaceae, and Tulostomataceae.The latter view is supported by several studies showing that Lycoperdaceae is nested as a separate clade within the Agaricaceae.Parsimony analysis of nuc-SSU datasets, including sequences of some representatives of Agaricaceae, Lycoperdaceae, Geastraceae, and Phallaceae, demonstrated that the puffballs were nested within the euagarics clade, which received a bootstrap support of 78% [26].Intriguingly, Lepiota cristata (Bolton) P. Kumm. was found to be genetically closer to Mycenastrum corium (Guers.)Desv.and the true puffballs, than to Macrolepiota procera (Scop.)Singer and Macrolepiota konradii (Huijsman ex P.D. Orton) M.M. Moser.Members of Lycoperdaceae grouped in a clade with 72% bootstrap support.Judging from these results, Lycoperdaceae was considered closely related to Lepiotaceae, and the authors suggested the inclusion of the order Lycoperdales in the Agaricales [26].
The analysis conducted by Vellinga, which involved 160 specimens, including some representatives of the genera Bovista, Calvatia, and Lycoperdon, revealed that these species form an independent "Lycoperdaceae" clade within the Agaricaceae [38].However, the resulting trees, derived from individual ITS and nuc-LSU, and combined ITS + LSU datasets, presented different topologies; for instance, in the LSU phylogram, six taxa of Lycoperdaceae formed a distinct clade closely related to the agaricoid genera Agaricus L., Chlorophyllum Massee, Leucoagaricus Locq.ex Singer, and Leucocoprinus Pat., while in the combined ITS + LSU phylogram, a single taxon representing Lycoperdaceae-L.pyriforme (= Apioperdon pyriforme) took a basal position in respect to the core Agaricaceae clade.
Thus, Lycoperdaceae could be treated as a gasteroid lineage within Agaricaceae, losing its family rank [33,39,40].In this case, a broader concept of Agaricaceae is necessary, which, on the other hand, will inevitably lead to the situation in which we will have a family that unites taxa with rather discrepant anatomical characters, while looking artificial from the point of classical taxonomy.The opposite idea is to postpone the final decision regarding the taxonomic status of the family Lycoperdaceae until more advanced phylogenetic reconstructions of Agaricaceae and Agaricales, in general, will be established [41].
Difficulties also occurred when scientists tried to resolve the phylogenetic relations within Lycoperdaceae itself.One can see a somewhat "foggy" concept of genera in the family.Different mycologists had their personal views and opinions regarding the taxonomical composition of Lycoperdaceae, genera richness, and major distinctive characteristics, which would reliably delimit the genera from one another and allow the proper assignment of species.For instance, in the case of Vascellum pratense (Pers.)Kreisel, which is registered for Israel, some researchers believed that the presence of a morphological feature, such as a diaphragm, was sufficient to place the species in the genus Vascellum F. Šmarda [5,42].However, this proved to be unsubstantiated from a molecular perspective: phylogenetic studies [2,21,41,43,44] showed that V. pratense groups with core species of the genus Lycoperdon, including Lycoperdon perlatum Pers.: Pers., L. marginatum Vittad.and L. norvegicum Demoulin, and should be treated as Lycoperdon pratense Pers.
Since the beginning of the 21st century, the family Lycoperdaceae and its members have been the subject of numerous molecular phylogenetic studies [14,15,18,26,40,41,43,[45][46][47].Listed works presented phylogenetic reconstructions, which were based on the following gene datasets: ITS rDNA, nuc-LSU, nuc-SSU, mt-LSU, and mt-SSU rDNA.Larsson and Jeppson provided one of the most comprehensive molecular phylogenetic examinations of the family Lycoperdaceae [41].The authors used a combined ITS and LSU rDNA dataset, and sampled 79 specimens belonging to 47 species, which were mostly of European origin, along with some sequences selected from the GenBank database.Their analysis demonstrated that Lycoperdaceae is a monophyletic group, containing four major clades-Lycoperdon, Calvatia, Bovista, and Disciseda, more or less corresponding to the equally named genera.The structure and boundaries of the genus Lycoperdon, the type genus of the family, presented in the phylograms, are also of great interest.A number of species that were traditionally placed in the related genera were nested within the "Lycoperdon" clade, including Bovista dermoxantha (Vittad.)De Toni, Bovistella radicata Pat., Calvatia cretacea (Berk.)Lloyd, C. turneri (Ellis & Everh.)Demoulin & M. Lange, Handkea excipuliformis (Scop.)Kreisel, H. utriformis (Bull.)Kreisel, and Morganella fuliginea (Berk.& M.A. Curtis) Kreisel & Dring.The "Lycoperdon" clade splits into two subclades.The first subclade contained the type species of the genus, Lycoperdon perlatum, which grouped with representatives of the genera Morganella Zeller and Vascellum, as well as with two other members of Lycoperdon.The second subclade absorbed the main diversity of Lycoperdon species, together with some taxa assigned to Bovista, Bovistella, Calvatia, and Handkea Kreisel, which have already been mentioned above.Larsson and Jeppson [41] chose to accept a broad interpretation of Lycoperdon and proposed to recognize the subgenera Vascellum, Morganella, Bovistella, Utraria, and Apioperdon, in addition to Lycoperdon s. str.The authors noted that allocation of some species remained questionable and suggested subdivision was conditional to some extent.
Vizzini and Ercole [2] conducted a study that aimed to detect the phylogenetic position of Bovista acuminata, a very small puffball, characterized by oblong, conical basidiomes, lack of subgleba, "Lycoperdon" type capillitium with small pores, globose, almost smooth to finely verrucose spores, and bryophilous association.In the analysis [2], both B. acuminata sequences (collections from Italy) clustered in a well-supported clade (BPP-1.00)outside Lycoperdon.The clade was sister to L. pyriforme.Based on morphological, ecological, and molecular data, Vizzini and Ercole erected a new genus Bryoperdon Vizzini, creating a new combination-Bryoperdon acuminatum (Bosc) Vizzini [2].In the same paper, the authors also raised a question regarding the taxonomic placement of L. pyriforme, whose isolated position had already been pointed out in a number of molecular phylogenetic studies [40,41,43,46].Vizzini and Ercole proposed to elevate the rank of Apioperdon, considering it a distinct genus within Lycoperdaceae [2].
Alfredo et al. [14] focused their efforts on the reevaluation of Brazilian species described under Morganella, and examined seventy specimens from Brazilian, NY and PDD herbaria.Analyses of combined ITS and LSU datasets demonstrated that all specimens designated as Morganella (except for M. sulcatostoma C.R. Alves & Cortez) grouped together in a single clade (84% BS and 0.9 BPP) corresponding to the subgenus Morganella within Lycoperdon.The absence of capillitium and reduced, compact cellular subgleba were defined as the most important features of the subgenus.One new species, Lycoperdon oblongatum Accioly, Baseia & M.P. Martín, and six new combinations were proposed.Additionally, the authors questioned the validity of nomenclatural changes introduced by Vizzini and Ercole [2], citing the small number of specimens involved in the mentioned analysis.
The study of the family Lycoperdaceae in Israel began with the work of Reichert and Avizohar-Hershenzon, who published an article on higher fungi of Israel, where they reported on Disciseda cervina (Berk.)Hollós, found near Rehovot (Philistean Plain) [48].The authors gave a very brief description of macro-and micromorphological characteristics of studied specimens, together with black and white photos of their sporocarps.In 1963, Dring and Rayss published a paper focused exclusively on the diversity of gasteroid fungi in Israel, which was unique for that stage of Israeli mycobiota studies [49].As a result, twenty-eight species and intraspecific taxa were new records for the territory of the country, including a novel species for science-Scleroderma multiloculare Dring & Rayss.The article provided a description of the family Lycoperdaceae and four genera, two of which-Lycoperdon and Bovista-were new for Israeli mycobiota.Within these genera, six species were counted-Lycoperdon pratense (as Vascellum pratense), Lycoperdon lividum Pers.(as Lycoperdon spadiceum Pers.), Bovista pusilla (Batsch) Pers.(as Lycoperdon pusillum Batsch ex Schumacher), Bovista plumbea Pers., Bovista nigrescens Pers., and Disciseda bovista (Klotzsch) Henn.Each species was provided with a short description, origin of specimen, pictures of spore and capillitium morphology (some species had photos of gasterocarps), and critical notes.It should be noted that in the same study, Dring and Rayss questioned previous records of D. cervina, and also mentioned the genus Mycenastrum Desv., reporting the presence of Mycenastrum corium in Israel [49].The authors placed the species in Lycoperdaceae, which was typical for that period.Molecular phylogenetic analyses of Krüger et al. [26] and Larsson and Jeppson [41] showed that Lycoperdaceae could remain monophyletic only with the exclusion of M. corium.
Twenty years later, Binyamini, who made a great contribution to the knowledge of fungi in Israel, and gasteroid fungi in particular [50][51][52][53][54], published a book [55] in which he counted several species of Lycoperdaceae that were not known for Israel from the previous studies.These were Lycoperdon molle Pers., Lycoperdon atropurpureum Vittad., and Calvatia gigantea (Batsch) Lloyd (as Lycoperdon giganteum Batsch).Binyamini presented photos of gasterocarps of mentioned species, but he gave only brief descriptions, without any drawings or photos revealing micro-characteristics (spores, capillitium, etc.).The author also did not supply data on the origin of the studied specimens.Unfortunately, the Herbarium of Binyamini, stored in Tel-Aviv University (TELA), has been closed for many years; therefore, the specimens of Lycoperdaceae, collected and identified by Binyamini, were not available for the current study.
Krakhmalnyi presented his PhD thesis on the gasteroid basidiomycetes of Israel, where he gave a thorough treatment of the group, including the family Lycoperdaceae [56].Yet, some information that was provided in the study is currently outdated.

General
This study is based on the investigation of material stored in the Herbarium of the Institute of Evolution, University of Haifa (HAI, Israel) and the University of Liege Herbarium (LG), and on new fungal samples collected during expeditions to different botanicalgeographical regions of Israel from October 2011 to March 2013.
During sample collection, the following information was recorded: the possible field identification of the specimen, locality, date of collection, collector's name, and additional information (relevant descriptive notes, nearby vegetation, etc.).For color interpretation "Flora of British Fungi: Colour Identification Chart" was used [57].The photos of gasterocarps were taken with an Olympus EPL-1 digital camera.Collected samples were dried at 50 • C, and then transferred to separate polyethylene zip bags for storage.
Distribution of species in Israel is based on phytogeographic districts (Figure 1) proposed by Feinbrun-Dothan and Danin [66].

Microscopy Studies
Some characteristics of studied specimens (structure of exo-and endoperidium, gleba and subgleba, etc.) were examined under a ZEISS Stemi DV4 stereo microscope (Carl Zeiss AG, Oberkochen, Germany), and pictures were taken with a Canon Power Shot G10 digital camera.Micromorphology, including spore and capillitium hyphae structure, was observed under ZEISS Axiostar 1122-100 (Carl Zeiss AG, Oberkochen, Germany) and Olympus BX51 (Olympus LS, Tokyo, Japan) light microscopes.Measurements were made using an UPlanFLN 100x/1.30(Oil) immersion objective, and ≥30 spores of each specimen were measured for the statistical calculation.Melzer's reagent, 3% aqueous KOH, and Lactophenol Cotton Blue were used as mounting media.Spore size is given as follows: (min) average minus standard deviation-average plus standard deviation (max).Microscopic photos were taken with Canon Power Shot G10 and Canon EOS 5D Mark III digital cameras.
Specimens were also studied under a field emission scanning electron microscope (FE-SEM) Mira 3 LMU (TESCAN, Brno, Czech Republic) with integrated Oxford instruments INCA energy (Oxford instruments, Abingdon, UK), and the following scanning electron microscopes (SEMs): Philips SEM 515 (Philips Electronics N.V., Eindhoven, The Netherlands) and JSM-6060 LA (JEOL, Ltd., Tokyo, Japan).A small dried gleba sample with spores of each specimen was mounted on a metal stub using electroconductive glue,

Microscopy Studies
Some characteristics of studied specimens (structure of exo-and endoperidium, gleba and subgleba, etc.) were examined under a ZEISS Stemi DV4 stereo microscope (Carl Zeiss AG, Oberkochen, Germany), and pictures were taken with a Canon Power Shot G10 digital camera.Micromorphology, including spore and capillitium hyphae structure, was observed under ZEISS Axiostar 1122-100 (Carl Zeiss AG, Oberkochen, Germany) and Olympus BX51 (Olympus LS, Tokyo, Japan) light microscopes.Measurements were made using an UPlanFLN 100x/1.30(Oil) immersion objective, and ≥30 spores of each specimen were measured for the statistical calculation.Melzer's reagent, 3% aqueous KOH, and Lactophenol Cotton Blue were used as mounting media.Spore size is given as follows: (min) average minus standard deviation-average plus standard deviation (max).Microscopic photos were taken with Canon Power Shot G10 and Canon EOS 5D Mark III digital cameras.
Specimens were also studied under a field emission scanning electron microscope (FE-SEM) Mira 3 LMU (TESCAN, Brno, Czech Republic) with integrated Oxford instruments INCA energy (Oxford instruments, Abingdon, UK), and the following scanning electron microscopes (SEMs): Philips SEM 515 (Philips Electronics N.V., Eindhoven, The Netherlands) and JSM-6060 LA (JEOL, Ltd., Tokyo, Japan).A small dried gleba sample with spores of each specimen was mounted on a metal stub using electroconductive glue, then coated with a thin layer of gold in fine coat ion sputter JFC-1100 (JEOL, Ltd., Tokyo, Japan), and subsequently analyzed using FE-SEM and SEM.

Taxon Sampling for the Phylogenetic Study
The phylogenetic relations between species and genera within the puffball family Lycoperdaceae were investigated using molecular methods, in conjunction with the examination of macro-and micromorphological characteristics, to re-evaluate the systematic position of some controversial species, resolve the taxonomic structure of the family, and reinforce the identification of Israeli material.Fifty-eight samples from Israel and other countries (Belgium, France, Germany, Luxembourg, Ukraine, USA), comprising twenty-five species, which belong to Apioperdon, Bovista, Calvatia, and Lycoperdon genera, deposited in the Herbarium of the Institute of Evolution, University of Haifa (HAI) and the University of Liege Herbarium (LG), together with sixty-six previously published sequences from Gen-Bank database (NCBI, https://www.ncbi.nlm.nih.gov/genbank/),including specimens assigned to Bovista, Bovistella, Calvatia, Disciseda, Handkea, Holocotylon, Lepiota, Lycoperdon, Morganella, Mycenastrum, Tulostoma, and Vascellum were the subject of the current phylogenetic analysis.Information about the specimens and GenBank accession numbers are provided in Table 1.In cases when specimen vouchers in GenBank and published studies do not match, vouchers provided in the original publications are presented.

DNA Extraction
Approximately 30 to 100 mg of glebal tissue from dried herbarium material or fresh samples were placed in 1.5 mL Eppendorf tubes.Two sterile metal beads were added to each tube.The tubes were subsequently submerged in liquid nitrogen for 5-10 s, and immediately after that homogenization of material was carried out using a Qiagen Tissue Lyser II (Retsch, Haan, Germany) for 3 min at 30 Hz.After homogenization, CTAB and SDS (10%) extraction buffers were added directly onto the crushed material, then Eppendorf tubes were incubated in a water bath for 12 h at 65 • C. In the extraction DNA enrichment procedure, phenol:chloroform:isoamyl alcohol 25:24:1 (Sigma-Aldrich Co., St. Louis, Missouri, USA) was used first, and chloroform-isoamyl alcohol 24:1 (SEVAG) solution thereafter.Isolated DNA was cleaned by washing with ≈100% isopropanol (Sigma-Aldrich Co.) and 70% ethanol (Sigma-Aldrich Co.), and then dried.The dried DNA was diluted in 50 µL 0.1 M TE buffer and 1 µL of RNase A (1 mg/mL) (Sigma-Aldrich Co.) was added.The concentration of obtained DNA was measured with a NanoDrop ® ND-1000 spectrophotometer (NanoDrop Technologies, Montchanin, DE, USA) and adjusted to an average value of 20 ng/µL for downstream applications.

PCR Amplification, Purification, Sequencing
Nuclear ribosomal internal transcribed spacer (ITS) was amplified.The amplification of the whole ITS region (ITS1-5.8S-ITS2) of rDNA was carried out using primers ITS1F and ITS4B (Sigma-Aldrich Co.) [70].The polymerase chain reaction (PCR) was mixed in a total volume of 15 µL containing: 7.5 µL of Reddy Mix (PCR Master Mix, Thermo Fisher Scientific, Waltham, Massachusetts, USA), 0.4 µL of each primer, 3.7 µL of molecular water, and 3 µL of genomic DNA (with the usual quantity of 60 ng/per reaction).The PCR amplification was carried out on a GeneAmp ® PCR System 9700 (Applied Biosystems, Inc., Waltham, Massachusetts, USA).The basic PCR cycling conditions were as follows: an initial denaturation step of 95 • C for 5 min, followed by 35 cycles of denaturation at 95 • C for 30 s, primer annealing at 55 • C for 30 s, primer extension at 72 • C for 1 min, and a final elongation step at 72 • C for 7 min.If samples were not responding to this program, some parameters (number of cycles, annealing temperature, quantity of DNA per reaction) were individually adjusted for better output results.
All PCR products were visualized in 1% (w/v) agarose gels, stained with GelRed (Nucleic acid stain, Biotium, Fremont, CA, USA) and viewed under ultra-violet light on a Molecular Imager Gel Doc XR with BioRAD Quantity One 4.5.2Software.Sizes of PCR amplicons were estimated against a GeneRuler 100 bp DNA Ladder (Thermo Fisher Scientific).Prior to DNA sequencing, PCR products were cleaned using an illustra ExoProStar 1-Step (GE HealthCare, Chicago, Illinois, USA) following the manufacturer's instructions.
Cleaned PCR products were sequenced using a Big Dye Terminator V1.1 Cycle Sequencing Kit (ABI, Austin, TX, USA) in two different reactions with ITS1F and ITS4B primers.They were subsequently analyzed on an Applied Biosystems (AB) 3130xl Genetic Analyzer (Hitachi, Ltd., Tokyo, Japan).

Phylogenetic Analysis
The whole ITS rDNA region dataset comprised 124 sequences, 58 of which were obtained during the current research and 66 were acquired from the GenBank database.Sequences were aligned individually for each locus using the MUSCLE software package in Mesquite 2.73 [71], and the alignment was inspected and adjusted manually with ambiguous regions excluded.Total length of the alignment was 704 bp.After exclusion of ambiguous regions, the length of the alignment decreased to 639 bp.Phylogenetic relationships within the family Lycoperdaceae were determined by maximum likelihood (ML) and Bayesian Interference (BI) methods.Phylogenetic support was assessed via the bootstrap method using PAUP* v.4.0a109 [72], and posterior probability for ML and BI analyses, respectively.The software Garli-1.0 [73] for ML and MrBayes 3.1.2[74] for BI methods on the CIPRES Science Gateway V. 3.1 (www.phylo.org)were used.Analyses were conducted applying the GTR + C + I substitution model and rate heterogeneity with unlinked parameters.One thousand bootstrap replicates were computed for ML.For Bayesian phylogenetic estimations, parallel runs of four chains were computed to 20 million generations, with sampling every 1000 generations.Trees were sampled when an equivalent posterior probability plateau was achieved between runs.Statistical support was recognized as significant with >70% bootstrap values (BS) for ML, and >0.95 posterior probabilities (BPP) for BI.Tulostoma kotlabae and Lepiota cristata were selected as the outgroup.Mycenastrum corium was also included in the analysis.

Results and Discussion
As a result of a thorough literature survey, extensive collections of fresh samples, and examination of material stored in the HAI herbarium, fifteen species belonging to five genera were found in the territory of Israel (Table 2).Six species, including Apioperdon pyriforme (Schaeff.)Vizzini, Bovista aestivalis (Bonord.)Demoulin, Calvatia candida (Rostk.)Hollós, Lycoperdon decipiens Durieu & Mont., L. niveum Kreisel, and L. perlatum Pers.: Pers.are new additions to the diversity of the family Lycoperdaceae and the whole gasteroid mycobiota in Israel.

Molecular Phylogeny of Lycoperdaceae
Of all the tested algorithms, the maximum likelihood tree of the family Lycoperdaceae had the highest number of supported clades, and the Bayesian tree showed, in general, a similar topology (Figure 2).In the current study, Lycoperdaceae was recovered as a monophyletic group with significant statistical support in both ML and Bayesian analyses (88% BS and 1.00 BPP).The outgroup was comprised of T. kotlabae together with L. cristata.The position of M. corium relative to the members of the family is still an open question.The monotypic genus Mycenastrum for a long time had been placed in Lycoperdaceae, although Zeller [75] considered that distinctive macro-and micromorphological characteristics, including large subglobose to irregularly shaped gasterocarps, a thick endoperidium, the lack of an ostiole, pitted basidiospores, and thick-walled, elastic, non-poroid, non-septate capillitial threads with spinose branches were sufficient to establish a separate family, i.e., Mycenastraceae.In the study of Bates et al., M. corium was treated as a basal member of Lycoperdaceae, although the authors expressed their awareness that in terms of its unique morphological features, the species is clearly separated from other representatives of the family [43].The current study recovered M. corium as a sister taxon to the rest of the species composing the Lycoperdaceae ingroup, which agrees with results presented by Krüger et al. [26] and Larsson and Jeppson [41].The question of M. corium assignment either to Lycoperdaceae or Mycenastraceae becomes even more entangled and somewhat incorrect in the situation when both families tend to be incorporated into the Agaricaceae s.l., which will inevitably lead to the reduction in their taxonomic rank.
J. Fungi 2023, 9, x FOR PEER REVIEW 13 of 48 awareness that in terms of its unique morphological features, the species is clearly separated from other representatives of the family [43].The current study recovered M. corium as a sister taxon to the rest of the species composing the Lycoperdaceae ingroup, which agrees with results presented by Krüger et al. [26] and Larsson and Jeppson [41].
The question of M. corium assignment either to Lycoperdaceae or Mycenastraceae becomes even more entangled and somewhat incorrect in the situation when both families tend to be incorporated into the Agaricaceae s.l., which will inevitably lead to the reduction in their taxonomic rank.Within Lycoperdaceae, six main clades were revealed.Clade A contained two species of the genus Disciseda-D.bovista (GenBank no.DQ112627, Sweden) and D. candida (Gen-Bank no.EU833654, USA), and represented an unsupported sister clade to the remaining species of the family.The genus Disciseda has a worldwide distribution and is characterized by the exoperidium persisting as a disc at the top of the gasterocarp, a basal position of the mouth, and a peculiar inversion of mature gasterocarps [3].
Clade B comprised six sequences of Apioperdon pyriforme representing the material from Israel (OR594093, OR594096), Ukraine (OR594094, OR594095, OR594097), and one sequence obtained from the Swedish specimen by Larsson and Jeppson [41].The clade had significant statistical support (100% BS and 1.00 BPP) and was located more basally within the family, supporting the data of Bates et al. [43] and Kim et al. [21], which, however, do not agree with the topologies presented in a number of studies [2,14,41,44].The sequences obtained from Ukrainian and Israeli specimens clustered together, while the sequence GenBank no.DQ112558 (Sweden) occupied the sister branch.BLAST analysis showed that the sequence of A. pyriforme OR594096 is 99% similar to the sequence of Morganella pyriformis GenBank no.DQ112557 [41], having one base insertion, one base deletion, and four substitutions difference, while the sequence of L. pyriforme GenBank no.AY854075 demonstrated the closest match, with the difference in just two substitutions.
Clade B was a sister group to the remaining species within Lycoperdaceae.The basal position of A. pyriforme, observed both in the present analysis and in the analysis of Bates et al. [43], was neither supported by ML nor by Bayesian methods.Although A. pyriforme was incorporated into the lignicolous genus Morganella by Krüger and Kreisel on the basis of molecular data and ecological features [45], in the current study it did not cluster with sampled representatives of the genus-M.fuliginea (AF485065) and M. subincarnata (AJ237626).The same picture was observed in the investigations of Krüger et al. [26], Larsson and Jeppson [41], Bates et al. [43], and Alfredo et al. [14].Apioperdon pyriforme is similar to members of Lycoperdon in having a true capillitium (which is, however, not pitted), and to the genus Morganella by its lignicolous habitat.At the same time, it differs from both by possessing a distinctly white cellular subgleba and by the structure of exoperidium sphaerocysts.Based on these distinctive characteristics, Krüger and Kreisel proposed a new subgenus Apioperdon Kreisel et D. Krüger within the genus Morganella, to accommodate M. pyriformis [45].In the analysis of Larsson and Jeppson, A. pyriforme occupied a basal position relative to the genus Lycoperdon, and the authors chose to distinguish Apioperdon (Kreisel & D. Krüger) Jeppson & E. Larss.as a monotypic subgenus within Lycoperdon [41].Later, Vizzini and Ercole made a decision to elevate the position of Apioperdon to the genus level, based on cumulative morphological, ecological, and molecular evidence, and proposed a new combination-Apioperdon pyriforme (Schaeff.)Vizzini [2].Our analysis solidifies the status of Apioperdon as a separate genus within Lycoperdaceae.
Clade C represented the genus Bovista in the current analysis and was supported only by the Bayesian method (0.95 BPP).The clade splits into two subclades, C1 and C2, corresponding to the subgenera Globaria and Bovista, respectively.The Globaria subclade united species, which are characterized by intermediate to "Lycoperdon" types of capillitium, whereas the Bovista subclade incorporated species with dichotomously branched capillitium and pedicelate spores.Neither of the subclades had significant ML support, but both "Globaria" and "Bovista" received Bayesian support of 0.97 BPP and 0.96 BPP, respectively.The phylograms presented by Larsson and Jeppson showed a similar topology with the same species composition of each subclade [41].However, in their analysis, the subclades Globaria and Bovista were well supported by both maximum parsimony and Bayesian methods, while the whole Bovista clade had only Bayesian support.The Globaria subclade in the current study was represented by B. aestivalis, B. furfuracea, B. polymorpha, and B. promontorii.Sequences of two Israeli specimens of B. aestivalis (OR594144 and OR594145) clustered together with the Swedish specimen (GenBank no.DQ112620), along with two sequences assigned to B. promontorii (GenBank no.DQ112621) and B. polymorpha (GenBank no.AJ237613), in a well-supported clade.BLAST analysis showed that the sequence of B. aestivalis OR594145 is 99% similar to the sequence of B. aestivalis (GenBank no.EU833650) from the study of Bates et al. [43], with a difference in four substitutions, and is also 99% similar to the mentioned sequence of B. aestivalis GenBank no.DQ112620 [41], with a difference in two deletions (one base pair each) and one base substitution.
The sequence of B. pusilla (GenBank no.AJ237631) from the study of Krüger et al. [26], which also nested within the Globaria subclade, clustered with two sequences of B. furfuracea (specimens from Belgium and Sweden) and, more likely, represents the same species.Larsson and Jeppson [41] noted that Krüger et al. [26]  Clade D accommodated members of the genus Calvatia, including C. gigantea (= Langermannia gigantea).Main characters of the genus are irregular rupturing of the peridium and medium-to large-sized gasterocarps.The clade received significant support according to both ML (70% BS) and BI methods (1.00 BPP).The topology of the clade within Lycoperdaceae suggested a close relationship with the genus Lycoperdon.The same picture was observed in the phylogenetic reconstructions of Bates et al. [43].However, this position of the Calvatia clade did not receive significant statistical support in either analysis.Phylogenetic trees presented in the studies of Larsson and Jeppson [41] and Alfredo et al. [14] showed a more basal position of the genus Calvatia.The Calvatia clade splits into subclades D1 and D2, both receiving significant ML and Bayesian support-88% BS/1.00 BPP and 98% BS/1.00 BPP, respectively (Figure 2).The subclade D1 contained sequences belonging to C. candida, C. chilensis, C. craniiformis, C. cyathiformis, and C. fragilis.BLAST analysis showed that the sequence of C. candida obtained from Israeli material (OR594136) is 99% similar to the sequence of C. candida presented in the study of Larsson and Jeppson [41] GenBank no.DQ112624, with the difference in only one base marked as "N" in the latter sequence.These two sequences of C. candida acquired from Israeli and Hungarian specimens clustered in a well-supported clade.The final subclade within D1 that received significant support from BS and BI analyses was comprised of two C. cyathiformis sequences-one obtained during the current study (OR594134, Corse, France) and the other GenBank sequence no.AJ486873, along with the third sequence (GenBank no.AJ617493), which was deposited in the GenBank database under the name "C. fragilis".There is no clear understanding whether C. cyathiformis and C. fragilis are two separate species.Some mycologists considered the latter as a form of C. cyathiformis [76], and molecular data support their close relationship.However, there is a number of macro-and micromorphological characteristics on the basis of which these species can be delimited.For instance, Bates et al. observed a significant difference in spore ultrastructure between the specimens of C. cyathiformis and C. fragilis under SEM [43].Thus, the question of their demarcation remains open.
The subclade D2 contained two sequences belonging to C. gigantea and C. bicolor.These two species also clustered together in the phylogram presented by Bates et al. [43], although their position in relation to other species of the genus Calvatia was not statistically supported.In the study of Kim et al., C. gigantea, C. bicolor and "C.pachydermica" (C.pachyderma, voucher AN014692) formed a well-supported clade (95% BS / 1.0 BPP) [21].In the current analysis, the subclade D2 received support by both ML and BI methods (98% BS and 1.00 BPP).Larsson and Jeppson suggested retaining the wide concept of the genus Calvatia with Langermannia taking a position of a subgenus [41].
Clade E presented a rather puzzling finding.The clade contained three sequences designated as "B.dermoxantha" (GenBank no.HQ235047 and no.HQ235050) and "L.pusillum" (GenBank no.AB067724), and received only Bayesian support (1.00 BPP).These three samples collected in the USA (NCP34 and SCP2) and Japan (Lp1, mycelia) during the studies of Miller et al. [67] and Terashima et al. [69] were related to the similar habitatsputting greens in golf courses.Intriguingly, the sequences clustered neither with B. pusilla in the Bovista clade C, nor with the sequences of B. dermoxantha (GenBank no.DQ112579) acquired by Larsson and Jeppson [41] and B. dermoxantha (OR594143) from the current research, which were nested within the major Lycoperdon clade (Figure 2).BLAST analysis showed that the sequences GenBank no.HQ235047, HQ235050, and AB067724 are fairly distant from other identified Lycoperdaceae sequences deposited in the GenBank database (less than 97% similarity), suggesting that they present a distinct species.Furthermore, the fact that the clade E took a position of a sister group to the Lycoperdon clade F reinforces this statement and even makes an argument for a separate subgenus/genus within Lycoperdaceae.However, this topology of the clade E was not statistically supported.Unfortunately, due to an obvious lack of morphological data regarding these three samples, we can only speculate on their taxonomical position.
Clade F in the current analysis represented the genus Lycoperdon s.l.The clade did not receive statistical support from either the ML or the Bayesian analyses.Thus, the problem of the genus monophyly remains under question.The clade splits into two subclades, F1 and F2, which also had no statistical support.The subclade F1 accommodated the type species of the genus, L. perlatum, with closely related L. marginatum and L. norvegicum.BLAST analysis showed that the sequence of L. perlatum (OR594101) obtained from the Israeli specimen is 99% similar to the sequence of L. perlatum GenBank no.DQ112630 [41], having one deletion, two substitutions, and one unidentified base pair difference.Sequences presenting Israeli and Swedish material of L. perlatum clustered in a clade with 100% BS and 1.00 BPP support.Additionally, within the subclade, a former representative of the genus Vascellum-V.pratense, two species both classified as belonging to the genus Morganella-M.fuliginea and M. subincarnata, one representative of the genus Handkea-H.subcretacea, and L. caudatum were nested.The close relationship of L. perlatum, L. marginatum, and L. norvegicum was supported by both ML (80% BS) and BI (0.99 BPP) methods.The subclade F2 united the major number of species within the Lycoperdon clade F. Apart from the species, which have been traditionally placed in the genus Lycoperdon, the subclade incorporated sequences assigned to the genera: Calvatia-C.cretacea, C. turneri; Handkea-H.excipuliformis, H. fumosa, H. utriformis; Bovistella-B.radicata; and the species Holocotylon brandegeeanum.The latter proved to be closely related to the members of the genus Lycoperdon by Bates et al. [43].The sequences of B. dermoxantha (Swedish and German material), L. rupicola-the species recently described by Jeppson et al. [15], and H. brandegeeanum formed a well-supported clade in both ML and Bayesian analyses (72% BS/1.00 BPP).The subclade F2 corresponds with the subgenus Utraria (Quél.)Jeppson & E. Larss.proposed by Larsson and Jeppson [41], with the exception of H. utriformis and B. radicata.
In the light of the current research and previous molecular studies [14,15,21,41,43,44], Kreisel's concept of the genus Handkea [77] appears to have a polyphyletic origin and presents an artificial taxon [78].The main distinguishing characteristic of the genus, on the basis of which it was segregated from Calvatia, is slit-like pores in the capillitium.All the sequences under Handkea involved in the analysis, including H. utriformis, the type species of the genus, were nested within Lycoperdon clade F (Figure 2).The sequences of B. radicata (AJ237624 and OR594137), C. utriformis (OR594133), and H. utriformis (EU833659, DQ112607) clustered together in a well-supported clade (96% BS/1.00 BPP).We agree with Demoulin and Rebriev [78] and believe that the sequence GenBank no.AJ237624 [26] represents C. utriformis (= H. utriformis).A close phylogenetic relationship between H. utriformis and B. radicata was previously reported by Larsson and Jeppson [41].The authors recovered them as members of Lycoperdon and proposed a separate subgenus Bovistella (Morgan) Jeppson & E. Larss. to accommodate L. utriforme and L. radicatum.The subgenus is characterized by medium-sized gasterocarps and the presence of a distinct pseudo-diaphragm.Analysis of Alfredo et al. supported this allocation [14].Demoulin and Rebriev considered B. radicata and C. utriformis to be so closely related, both in terms of morphology and ribosomal locus, while at the same time being distinct enough from Lycoperdon s. str.that they should belong to the genus Bovistella, and introduced a new combination-Bovistella utriformis (Bull.: Pers.)Demoulin et Rebriev [78].However, we believe that the exclusion of these two species from the genus Lycoperdon was rather premature, and this question requires more thorough molecular examination based on a wider taxonomical and geographical sampling, including Bovistella japonica Lloyd, B. poeltii Kreisel, B. sinensis Lloyd, etc.
Lycoperdon atropurpureum is probably the most common representative of the genus Lycoperdon in Israel.It is widespread in the woods throughout the northern region of the country, including the Carmel Mountain, the Western and the Eastern parts of Upper Galilee, the Golan Heights, and even the Hula Valley.Surprisingly, there were no specimens from the Lower Galilee region.Kreisel accepted a wide concept of L. atropurpureum and treated it and L. decipiens as synonyms [60].Jeppson and Demoulin, however, demonstrated that there is a sufficient number of distinguishing macro-and micromorphological characteristics to consider L. atropurpureum and L. decipiens as separate species [79].The authors included in their paper a comparative table, pointing out the differences between the two species.Pegler et al. also recognized L. atropurpureum, L. decipiens, and L. molle as distinct species, although very similar and often confusing [5].Molecular phylogenetic reconstructions provided an additional support for their demarcation [21,41,44].Depending on the method of tree construction, L. atropurpureum nested closer either to L. decipiens or L. molle.During the current study, fifteen sequences of L. atropurpureum were obtained (seven were included in the analysis): fourteen presented Israeli material and one originated from the French specimen (OR594124).BLAST analysis of all these sequences showed the closest match of 99% to the sequence of "Lycoperdon cf.decipiens" (GenBank no.DQ112586), which was obtained from the specimen of L. atropurpureum (M.Jeppson 3269) collected in Sweden [41].The current analysis also recovers L. atropurpureum, L. decipiens, and L. molle as separate species, solidifying results of the earlier studies.
Lycoperdon niveum was described by Kreisel from the specimens collected in the Himalayas [80].Later, the species was reported from Iceland, Norway, and Sweden (Gotland Island).Larsson and Jeppson noted that L. niveum belongs to the L. molle morphological species complex and its distribution is limited to arctic-alpine environments [41].Alfredo provided molecularly verified findings of L. niveum from the Republic of Macedonia and Spain [81].BLAST analysis showed that the sequence of Israeli specimen OR594103 is 99% similar (difference in two substitutions and one base marked as "W") to the sequence of "L.cf.niveum" (GenBank no.DQ112571) acquired by Larsson and Jeppson [41] from L. niveum specimen (M.Jeppson 4068, Iceland).Both sequences of L. cf.niveum (OR594102 and OR594103) representing the material from Israel clustered with the sequence from Iceland in a clade with 74% BS and 0.99 BPP support (Figure 2).After analyzing both molecular and macro-and micromorphological data, the current Israeli material was identified as L. niveum.Thus, it must be noted that the range of L. niveum is broader than expected and its ecological and chorological data requires re-evaluation.
The specimen of Lycoperdon from Koncha-Zaspa, Ukraine (OR594091), collected and identified by Demoulin as "L.cf.molle Pers.", turned out to be almost 100% similar (difference in only one base substitution) to GenBank sequence no.DQ112602 ("L.lambinonii" voucher MJ6371) representing recently described L. subumbrinum [15].The species was originally reported from Sweden and Slovakia.Alfredo examined and sequenced L. subumbrinum specimens from France, the Republic of Macedonia, Spain, and the United Kingdom [81].Lycoperdon subumbrinum is probably a widely distributed species, which had been misidentified in the past (as L. molle, L. lambinonii, etc.).The current study presents the first finding of the species from the territory of Ukraine.The species L. subumbrinum, L. muscorum, and L. ericaeum formed a clade with 98% BS and 1.00 BPP support.The same clade was recovered in the phylogenetic reconstructions of Larsson and Jeppson [41] and Jeppson et al. [15].These species also clustered together in the analysis of Kim et al., along with newly described Lycoperdon albiperidium C.S. Kim [21].
In the course of the current study, six sequences from Israeli specimens (OR594118, OR594119, OR594120, OR594121, OR594122, and OR594123), presenting small puffballs, characterized by a compact subgleba, globose spores with short pedicels, and "Lycoperdon" type capillitium with abundant spherical to ellipsoid pores, were obtained.BLAST analysis revealed that the sequence OR594123 was the closest match to L. cf.dermoxanthum (GenBank no.FJ438478) from the molecular phylogenetic study of Bates et al. [43], demonstrating 99% identity, with a difference in one base insertion and three substitutions.The similarity with the sequence of L. dermoxanthum (= B. dermoxantha, GenBank no.DQ112579) acquired by Larsson and Jeppson [41] was only 98%-the sequence OR594123 had three insertions (one base each) and fourteen substitutions difference.The sequence from the specimen OR594119, which turned out to be the closest to L. cf.dermoxanthum (GenBank no.FJ438478), having one base deletion and one substitution difference, showed only 97% similarity with L. dermoxanthum (GenBank no.DQ112579).In the phylogenetic reconstructions presented by Bates et al., L. cf.dermoxanthum nested in a clade with "L.niveum" (L.lividum GenBank no.DQ112599) and L. echinatum [43].The authors chose to treat the specimens from Arizona as L. cf.dermoxanthum until more information concerning this species is acquired.High similarities between the sequences obtained from the material collected in the USA and Israel resulted in a confusion, and Israeli samples were preliminarily identified as L. cf.lividum.Current molecular analysis recovered a clade uniting sequences of L. cf.lividum (Israel) and the sequence of L. cf.dermoxanthum (Arizona, USA) with the sequences of L. lividum (specimens from Belgium-GenBank no.OR594117 and Nepal-DQ112599).The clade was supported by both ML (80% BS) and BI (1.00 BPP) methods.The study of Bates et al. provided very similar descriptions and line drawings of L. lividum and L. cf.dermoxanthum [43].Quality SEM micrographs of the material from Arizona also demonstrated the high similarity of these species in terms of basidiospore ultrastructure.Unfortunately, the authors did not include any sequences of L. lividum in their phylogenetic tree.More careful examination of Israeli material supported its earlier preliminary identification.Additionally, we believe that the specimens designated as L. cf.demoxanthum [43] most likely represent L. lividum.
In summary, phylogenetic relations within the family Lycoperdaceae remain only partly resolved.However, the data accumulated during the current and previous molecular studies [2,14,15,18,21,26,40,41,[43][44][45][46][47]78] allow us to draw some preliminary conclusions.Lycoperdaceae appears to be a monophyletic group comprised of puffball-like species, including the type genus Lycoperdon and its allies.Within Lycoperdaceae, clades corresponding to the genera Apioperdon, Bryoperdon, Bovista, Calvatia, Disciseda, and Lycoperdon can be distinguished.Yet, their relative position is still debatable.Apioperdon seems to present a distinct monotypic genus within the family, although its exact placement is also questionable.The main uncertainty lies within the genus Lycoperdon, which most likely represents a polyphyletic entity.The current analysis supported the inclusion of the following species commonly assigned to the related genera into Lycoperdon s.l.: Handkea fumosa, Holocotylon brandegeeanum, Lycoperdon cretaceum, L. dermoxanthum, L. excipuliforme, L. fuligineum, L. pratense, L. radicatum, L. subincarnatum, L. subcretaceum, L. turneri, and L. utriforme.This shows that Lycoperdon is a taxon with higher morphological variability than was formerly accepted, and it can be left intact in a form of a single unit only if a broader genus concept is proposed.Another option would be to establish the monophyletic Lycoperdon by reducing its diversity to the generic type and limited number of related species [41].The polyphyletic nature of the genus requires more in-depth investigations, because we are still lacking a solid statistically supported picture regarding the species composition and relationships within Lycoperdon.Similar problems arise when dealing with the family Lycoperdaceae.If it is incorporated into the family Agaricaceae, the rank of Lycoperdaceae could potentially be lowered to the tribe level, as was proposed by Larsson and Jeppson [41].Mycenastrum corium appears to be a sister taxon to Lycoperdaceae and could be treated as a monotypic tribe within the family Agaricaceae.
Both individual ITS and combined ITS + partial nuc-LSU datasets proved to be insufficient to generate phylograms that fully resolve phylogenetic relations within Lycoperdaceae, while providing significant statistical support.It is noteworthy to mention that ITS and nuc-LSU sequences of different Lycoperdaceae representatives demonstrated very high similarities.The difference between related species in the ITS gene is usually less than 1%.This is even more relevant for the nuc-LSU region, where such dissimilarity could come down to several base pairs.It is now clear that future investigations of gasteroid lineages within the Agaricales, and the "puffball" lineage in particular, will depend on the multigene phylogenies, which will give greater resolution and provide higher levels of statistical support.RNA polymerase II gene subunits RPB1 and RPB2, translation elongation factor 1 alpha (TEF-1α), intergenic spacer (IGS), and mitochondrial rDNA genes are the most likely candidates for additional molecular phylogenetic studies.Furthermore, RAPD (Random Amplified Polymorphic DNA) and microsatellite analyses can be used for the purpose of resolving phylogenetic relations within Lycoperdaceae.The studies of Jeppson et al. [82,83] dealing with phylogenetic relationships within the gasteroid family Geastraceae and the genus Tulostoma Pers., both based on combined full ITS, partial LSU, and partial TEF-1α datasets, are examples of modern tendencies in fungal molecular systematics.The contribution of morphological and molecular features in creating a self-consistent taxonomic picture of both individual genera, and Lycoperdaceae in general, remains debatable and requires further studies.Description: Gasterocarps growing on wood in clusters, pyriform, 2.5-3 cm in height and 1.5-2 cm in width, with white rhizoids.Exoperidium 12 (Fulvous) to 17 (Snuff brown) with minute warts, somewhat granulose, which is more distinct down towards the base.Endoperidium papery, light brown to reddish brown.Pseudocolumella present.Gleba first white 4D-5E, then olive to grey-brown.Subgleba whitish 2B.Capillitium from 2-3 µm (wall 0.4-0.5 µm), yellow, to 4-5.5 (up to 6 µm), reddish brown in Melzer's reagent (wall 0.6-0.7,rarely up to 1 µm).Pores not observed.Spores globose to subglobose (3) 3-3.6 (4) µm in diam.(n = 30), under light microscopy appear smooth, light reddish brown in Melzer's reagent, some with a small pedicel attached (usually less than 1 µm long, sometimes up to 2-3 µm).Sphaerocysts 35-45 × 25-30 µm, subglobose to ovoid-pyriform.

Taxonomy
Under SEM spores appear globose to subglobose, with a prominent ornamentation of low apprised warts irregular in shape and with rounded and flattened tips.Some warts are merged together or rarely connected by low, thin anastomoses.The spore surface between warts looks rough and rugged, and has numerous small warts and ornamentation, comprised of somewhat short strands.Apiculus usually 0.5-0.7 µm long is observed.
Under SEM spores appear globose to subglobose, with a prominent ornamentation of low apprised warts irregular in shape and with rounded and flattened tips.Some warts are merged together or rarely connected by low, thin anastomoses.The spore surface between warts looks rough and rugged, and has numerous small warts and ornamentation, comprised of somewhat short strands.Apiculus usually 0.5-0.7 µm long is observed.Habitat: Lignicolous species found in broad-leaved, coniferous and mixed conifer forests, parks and gardens on decaying wood (logs, stumps, etc.), mostly hardwood, but also softwood.Sub-cosmopolitan species present in all continents, except Africa and Antarctica.
General distribution: Europe: Andorra, Austria, Denmark, Germany, Finland, Italy, Ireland, Luxembourg, Norway, Poland, Romania, Russian Federation (Central European part), Slovenia, Spain, Sweden, Switzerland, United Kingdom.Middle East: Israel.Asia: Japan.North America: Canada, Mexico, USA.Central America: Costa Rica.South America: Argentina.Arctic Territories: Iceland.Australasia: Australia, New Zealand.[43], also demonstrated that L. pyriforme is not phylogenetically related to representatives of Morganella, and the authors chose not to follow Krüger and Kreisel [45] in placing the species in the latter genus, although they totally agreed with the fact that the lignicolous habit of L. pyriforme could be a strong case for putting it into Morganella.The current phylogenetic analysis supports the earlier results of Vizzini and Ercole [2].
The SEM micrographs supplied by Krüger and Kreisel show spores with low isolated warts with rounded apices, and low conical processes [45].SEM photos of Bates et al. demonstrated similar morphology, with spores having ornamentation of very low warts [43].Spores of current samples have notably denser ornamentation of more pronounced warts, in addition to the whole spore surface being rough and rugged.
Under SEM, spores almost perfectly globose, covered with low, irregularly shaped warts, usually with rounded apices and ridges.Warts almost evenly dispersed throughout the spore surface, sometimes merged together or rarely connected by low thin anastomoses.Apiculus 0.5-1.7 µm in length and 0.5 µm in diam., with a distinct terminal pore, is observed.Area surrounding apiculus usually less crowded with warts or completely free of any ornamentation.Additionally, in the current research, spores and capillitium of some specimens were studied in epifluorescence mode.In all the samples, it was observed that spores shine intensively under ultraviolet light; as for the hyphae, they appear almost dark.
Habitat: The species prefers warm and dry habitats, can be found in open areas on calcareous or, less frequently, sandy soils and sand fields, sand dunes, sand steppes, and dry meadows vegetation in the communities of Festucetalia vaginatae (records from Hungary), or grows amid leafy debris under Abies spp., Juniperus spp., Pinus spp., etc.
Material examined: Israel.UG.Mt.Meron National Park.On the ground, under Pinus sp.19  General distribution: Europe: Estonia, France (South and Central), Germany, Hungary, Netherlands, Scandinavia (includes three kingdoms of Denmark, Norway, Sweden, Finland), Spain, United Kingdom.Arctic Territories: Iceland.Middle East: Israel, Turkey.Asia: China, Japan, Mongolia.(Jeppson (2001) noted that records from North America, Africa, Australia, and New Zealand should be treated carefully and may refer to closely related taxa).
Notes: Bovista aestivalis is a very polymorphous species with a wide range of distribution and habitats, which consequently resulted in many issues related to its identification, synonyms and demarcation from morphologically similar species.A complicated situation with synonyms is still misleading in terms of creating a global map of its distribution.Demoulin synonymized B. pusilliformis (Kreisel) Kreisel and B. polymorpha, creating a new combination-B.aestivalis (Bonord.)Demoulin [84].He found that observed morphological differences between the named taxa mostly depend on the environmental factors, and on the portion of gleba from which the sample was taken for the examination of the capillitium structure.Some findings of B. aestivalis were recorded as Lycoperdon furfuraceum Hollós [85].Jeppson, in his survey of B. aestivalis occurrence in northern Europe, presented a very wide discussion on variations in macro-and micro-characteristics of the studied material [86].He demonstrated that gasterocarps of B. aestivalis could be small and subglobose, possessing a very small, reduced sterile base (subgleba), or, to the contrary, some samples could have well developed subgleba, forming a pseudostipe, which comprises almost half a length of the gasterocarp.The sample HAI-G-73 has a subglobose shape of the gasterocarp with a small-to-reduced subgleba, and the sample HAI-G-103 is pyriform with a developed pseudostipe.Additionally, the color of the gasterocarps widely varies.
Moyersoen and Demoulin studied a large number of B. aestivalis specimens from Corsica under SEM, and presented photomicrographs reveal some polymorphism of spores' ultrastructure [63].In their specimens, spores tend to be slightly ovoid in shape (in the current material, spores are globose), a larger number of spores have numerous meshes of low, thin ornamentation between warts.Some of the specimens from Corsica have spores with denser ornamentation of warts merging together and forming irregularly shaped conglomerates; also, some spores tend to have a pattern of small warts surrounding larger cylindrical in shape warts with rounded or flattened tips.Current SEM photos are in agreement with SEM micrographs and descriptions of Rimóczi et al. [65].General distribution: Europe: Andorra, Austria, Belgium, Denmark, France, Germany, Greece, Hungary, Ireland, Italy, Luxembourg, Norway, Poland, Slovenia, Romania, Russian Federation (European part), Spain, Sweden, Switzerland, United Kingdom.Mediterranean Islands: Balearic Islands (Spain).North America: Canada, USA.Carib- General distribution: Europe: Andorra, Austria, Belgium, Denmark, France, Germany, Greece, Hungary, Ireland, Italy, Luxembourg, Norway, Poland, Slovenia, Romania, Russian Federation (European part), Spain, Sweden, Switzerland, United Kingdom.Mediterranean Islands: Balearic Islands (Spain).North America: Canada, USA.Caribbean region: Do-Modern methods of molecular biology helped to resolve this situation.In 2008, Larsson and Jeppson published their study on the phylogenetic relations within the family Lycoperdaceae [41].The sequence of B. dermoxantha (GenBank no.DQ112579), one sequence of B. pusilla (GenBank no.AJ237631) originated from the study of Krüger et al. [26], and the sequence of B. furfuracea (GenBank no.DQ112622) were included in the analysis.The study clearly demonstrated that sequences of B. dermoxantha, B. furfuracea and B. aestivalis present distinct species.The last two sequences nested in a clade corresponding to the subgenus Globaria within the genus Bovista, while B. dermoxantha appeared in the Lycoperdon clade separately to all Bovista species.High similarity between the sequences of B. pusilla and B. furfuracea were discussed and the authors suggested that Krüger et al. [26], in their identification, followed a concept of B. pusilla used by Kreisel.The sequence of B. furfuracea (GenBank no.DQ112622) showed a huge separation from B. dermoxantha supporting morphological demarcation of the species by Moyersoen and Demoulin [63].Later, Larsson et al. demonstrated that B. pusilla and B. limosa represent two distinct, yet closely related species, which nested within the subgenus Bovista [47].

Bovista nigrescens
Due to the objective lack of data, it is very difficult to say now what species was really observed by Dring and Rayss [49]: it could have been B. furfuracea, L. dermoxanthum, B. aestivalis or, maybe, something similar.Thus, the current study chose to treat the finding as B. pusilla until more data will be obtained.
Under SEM, spores are covered with ornamentation of conical to irregularly shaped warts.Some warts appear sharper, while others have more rounded apices and ridges; also, some warts are merged together to form irregularly shaped complexes.Apiculus short ≈0.7 µm, with a terminal opening.
Habitat: Xerothermophilous species, preferring dry continental regions, can be found in forest edges, grasslands and even sand steppe communities.
Notes: Spores of the current specimen correspond well with illustrations and descriptions presented by Rimóczi et al. [65] and Bates [46], although are, on average, 1 µm larger.Description: Gasterocarps commonly pestle-shaped with well-developed pseudostipe, sometimes subglobose-turbinate, tapering towards the base with wrinkles and folds.Young fruit bodies almost subglobose-sessile, with underdeveloped pseudostipe; during maturation, pseudostipe becomes larger and comprises up to 2/3 of gasterocarps' length, with white branching mycelium (1) 2-2.5 cm long at the base, binding soil particles.Pestle-shaped specimens have the following dimensions: "cap" from 4-4.5 to 6.5-7 cm diam., 2.5-3 cm in height, pseudostipe (2.5) 3-3.5 (4) cm in diam., (3) 3.5-4 (4.5) in length, with full length of gasterocarp 5.5-7.5 cm.Subglobose-turbinate and depressed Description: Gasterocarps commonly pestle-shaped with well-developed pseudostipe, sometimes subglobose-turbinate, tapering towards the base with wrinkles and folds.Young fruit bodies almost subglobose-sessile, with underdeveloped pseudostipe; during maturation, pseudostipe becomes larger and comprises up to 2/3 of gasterocarps' length, with white branching mycelium (1) 2-2.5 cm long at the base, binding soil particles.Pestleshaped specimens have the following dimensions: "cap" from 4-4.5 to 6.5-7 cm diam., 2.5-3 cm in height, pseudostipe (2.5) 3-3.5 (4) cm in diam., (3) 3.5-4 (4.5) in length, with full ground.22  Notes: The species is one of the two most commonly distributed members of the family Lycoperdaceae in Israel, along with L. lividum.Lycoperdon atropurpureum is morphologically similar to L. molle and L. decipiens (for a wider discussion see the "Notes" under L. decipiens).Most of the studied Israeli samples of L. atropurpureum do not possess the purplish color of gleba, which is one of the diagnostic characteristics of the species, and have pestle-shaped gasterocarps.Spores under SEM differ from current specimens of L. decipiens by the shape and density of warts-L.atropurpureum has a very dense ornamentation of cylindrical, somewhat "phalliform", and irregularly shaped processes.The spore surface between the warts is much rougher and more rugged, probably, due to remains of the perisporium.Some spores from the current SEM examination show similarities with SEM micrographs of L. decipiens by Rimóczi et al. [65], while others have ornamentation of cylindrical warts with rounded tips, resembling spores' micrographs of L. molle from the studies of Bates [46] and Bates et al. [43].SEM photos of Moreno et al. display ornamentation of high (more than 1 µm) conical processes with sharp or rounded tips [89].All of the abovementioned lead to the assumption that spore ornamentation of L. atropurpureum can demonstrate great variability in structure, density, and arrangement of warts.Description: Gasterocarps subglobose to subglobose-turbinate, subglobose-sessile, compressed vertically, usually wider than high, ranging from 2 cm in width and 2-2.5 cm in height, to 5-6 cm in width and 3-3.5 in height, tapering towards the base with folds and wrinkles, without pseudostipe and with poorly developed subgleba, with 0.5-1 (up to 1.5) cm basal turf of mycelium binding substratum.Exoperidium almost smooth at the top, with minute, fragile spines 6F to 52 (Buff) more abundant near the base of the gasterocarp.Some mature specimens have cracks and wrinkles of exoperidium in the upper part.Color of the exoperidium from 52 (Buff) at the base, gradually becoming 32 (Clay buff) and 34 (Smoke grey) towards the top.In young specimen, gleba from 56 (Yellowish green) to 57 (Greenish yellow), becoming in mature specimens 61 (Grey olivaceous), 62 (Olivaceous), 16 (Cigar brown) to 27 (Hazel).Subgleba, first white, then becoming olivaceous to greyish brown.Spore deposit brown to greyish brown.Spores (4) 4.4-5.6 (6) µm in diam.(n=60) excluding ornamentation, globose to subglobose, strongly warted, yellow to light brown, mixed with sterigmal remnants.Some spores with pedicel attached up to 3 µm in length.Capillitium of "Lycoperdon" type, brown, fragile, from (2) 2.5-4 µm (wall 0.5-0.6) to 5-6 (up to 7) µm (wall 0.7-1 µm).Pores rarely seen ≈1 µm circular to ovoid in shape, usually in thin hyphae.Exoperidium of simple sphaerocysts.

Lycoperdon decipiens
Spores under SEM subglobose, strongly verrucose.Relatively dense ornamentation consisting of irregularly shaped verrucae, usually closer to the conical shape, was observed.Most of the warts merged together in groups, forming complexes with separated bases and connected tips, tending to be conical in shape.Some warts connected together by low thin anastomoses.Spore surface between warts appears almost smooth.Apiculus usually less than 1.5-2 µm in length with a terminal pore can be observed.Habitat: Thermophilous species, can be found in dry, mostly calcareous, grasslands of the forest steppe and light deciduous forests.
General distribution: Europe: Andorra, Austria, Denmark, Germany, Great Britain (England and Ireland), Hungary, Slovenia, Spain, Sweden.Middle East: Israel.North America: Canada and USA.
Material examined: Israel.CM.Mt.Carmel National Park, "Little Switzerland".On the ground.21  Notes: Lycoperdon decipiens can be easily confused with L. atropurpureum and L. molle.These three species form the so-called Lycoperdon atropurpureum-molle-decipiens species complex, because they show high similarity in macro-and micromorphological characteristics, and also turned out to be related in terms of molecular phylogenetics.Prior to the present study, only L. atropurpureum and L. molle were known for Israel; both were mentioned for the first time in the work of Binyamini [55].
All three species have thin exoperidium spines and show great variability in color, shape, and size of gasterocarps.Current specimens of L. decipiens have subglobose-turbinate gasterocarps, compressed vertically, while Israeli specimens of L. atropurpureum usually possess very well-developed pseudostipe and are pestle-shaped, resembling specimens of Lycoperdon excipuliformis (= Handkea excipuliformis).
Current SEM photos differ from the specimens examined by Rimóczi et al. [65], which had denser ornamentation of cylindrical, conical, and somewhat "phalliform" processes with remains of the perisporium, covering regions of the spore surface.Spores in the present study have less dense ornamentation of conical processes, which tend to merge together in groups, forming conical structures with separated bases and connected tips.
Under SEM, spores globose, covered with conical or irregularly shaped processes with rounded apices.Some warts merged together, or have connected tips, rarely connected together by low meshes.Spore surface between the warts covered with low, irregularly shaped (somewhat granulate) verrucae.Apiculus 0.7-1 (up to 2) µm in length with a terminal pore is observed.Habitat: Terricolous and found on weakly acid to calcareous humus or soil in dry grassland sites, steppes, pastures, meadows, dry lawns, also on sandy soils, dunes, limestone, and gypsum; in subarctic to subtropical climates.
General distribution: Europe: Austria, Belgium, Bulgaria, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ireland, Italy, Lithuania, Netherlands, Norway, Poland, Portugal, Romania, Russian Federation (European Part), Slovakia, Spain, Sweden, Switzerland, United Kingdom, former Yugoslavia.Mediterranean Islands: Balearic Islands (Spain), Corse (France).Macaronesia: Canary Islands (Spain).Arctic Territories: Greenland, Iceland, Svalbard (Norway).Middle East: Armenia, Israel.Asia: Japan.North America: USA.Australasia: Australia, New Zealand.Description: Gasterocarps 3 cm in width and 3 cm in height, subglobose-turbinate, tapering towards the base, with a compact subgleba and 1-1.5 cm long strands of basal mycelium, mixed with soil particles.Exoperidium from 52 (Buff) at the base to light brown towards the top.The upper part of the gasterocarp is covered with short dark brown spines with connected tips, and cracks, forming some kind of a reticulum, while near the base small fragile light-colored spines were observed.Gleba, first 3C-5E, then 27 (Hazel), 61 (Grey olivaceous), and 62 (Olivaceous).Subgleba compact, poroid, 4D.Spores globose, (4) 4.1-5.4(6) µm in diam.(n = 41) (wall ≈0.5 µm thick) excluding ornamentation, distinguishably verrucose, hyaline to light green, with an oil droplet, some- Description: Gasterocarps 3 cm in width and 3 cm in height, subglobose-turbinate, tapering towards the base, with a compact subgleba and 1-1.5 cm long strands of basal mycelium, mixed with soil particles.Exoperidium from 52 (Buff) at the base to light brown towards the top.The upper part of the gasterocarp is covered with short dark brown spines with connected tips, and cracks, forming some kind of a reticulum, while near the base small fragile light-colored spines were observed.Gleba, first 3C-5E, then 27 (Hazel), 61 (Grey olivaceous), and 62 (Olivaceous).Subgleba compact, poroid, 4D.Spores globose, ( 4   Description: Gasterocarps solitary or in small groups, turbinate to pestle-shaped, 2.5-4.5 cm in height and 2-4 cm in width, with a strongly developed sterile subgleba.Pseudostipe from 1/2 reaching to 2/3 of gasterocarp's height, either cylindrical and even throughout its length, or tapering towards the base.Rhizoids 0.5-1 cm (up to 2 cm) long, binding substrate.Exoperidium 5E to 52 (Buff) at the base, gradually becoming darker towards the top-27 (Hazel) to 17 (Snuff brown), with the darkest spot on the place of ostiole.Exoperidium in the apical portion of the gasterocarp bears 0.5-1 mm long conical spines 17 (Snuff brown) to 24 (Date brown), which do not coalesce, each surrounded by a circular row of smaller warts.After gasterocarp's maturation, large spines wear off, leaving scars surrounded by warts, that produce a characteristic well-defined reticulate pattern on the endoperidium.Down towards the pseudostipe, a pattern of fine even warts 5E to 52 (Buff) was observed; their abundance decreased from top to bottom.Some specimens with almost smooth pseudostipe, free of any ornamentation.The apex of immature gasterocarps bears a prominence or papilla, characterized by darker coloring, after maturation becoming an apical ostiole, with orbicular to irregularly shaped open- Description: Gasterocarps solitary or in small groups, turbinate to pestle-shaped, 2.5-4.5 cm in height and 2-4 cm in width, with a strongly developed sterile subgleba.Pseudostipe from 1/2 reaching to 2/3 of gasterocarp's height, either cylindrical and even throughout its length, or tapering towards the base.Rhizoids 0.5-1 cm (up to 2 cm) long, binding substrate.Exoperidium 5E to 52 (Buff) at the base, gradually becoming darker towards the top-27 (Hazel) to 17 (Snuff brown), with the darkest spot on the place of ostiole.Exoperidium in the apical portion of the gasterocarp bears 0.5-1 mm long conical spines 17 (Snuff brown) to 24 (Date brown), which do not coalesce, each surrounded by a circular row of smaller warts.After gasterocarp's maturation, large spines wear off, leaving scars surrounded by warts, that produce a characteristic well-defined reticulate pattern on the endoperidium.Down towards the pseudostipe, a pattern of fine even warts 5E to 52 (Buff) was observed; their abundance decreased from top to bottom.Some specimens with almost smooth pseudostipe, free of any ornamentation.The apex of immature gasterocarps bears a prominence or papilla, characterized by darker coloring, after maturation becoming an apical ostiole, with orbicular to irregularly shaped opening.Endoperidium, first yellow 6F, then becoming papery and shiny olive-grey, olive-brown.Gleba, first white or light yellow 6F, 8G, 50 (Straw), becoming olive-brown to grey-brown.Subgleba strongly developed, alveolate, first white to 6F, 8G, then olive-brown to brown.Pseudocolumella present.Spore deposit yellow-brown, olive-brown to grey-brown.
Under SEM, spores globose to slightly subglobose, showing distinct ornamentation of conical processes, more or less equal in size (about 0.3 µm), with either sharper, or with rounded and flattened apices.The ornamentation irregularly dispersed.Most of the warts connected together by low, thin meshes, forming a somewhat reticulated pattern.Some warts merged together, or occasionally connected by tips.The spore surface between warts and meshes not perfectly smooth, and appears rough and rugged.The apiculus 0.5-0.7 µm long, with a terminal pore 0.3-0.5 µm wide, is observed.
Habitat: Occurs in deciduous and coniferous woods, cosmopolitan species present in all continents, except Antarctica.
Material examined: Israel.CM.Mt.Carmel National Park, Nahal Nesher.On the ground.28  Notes: Cosmopolitan species found in all continents, except Antarctica.Taking into consideration its wide distribution, it is not surprising that L. perlatum is now registered for Israel.Some amateur mycologists and mushroom hunters reported its presence in the country, but this could not be proven until the material previously stored in the HAI Herbarium and collected during additional field trips was studied.Currently, L. perlatum is known from two localities-Mt.Carmel and Mt.Meron.
The type species of the genus Lycoperdon is fairly easy to distinguish by its pestle-shape and reticulate pattern of exoperidium with large spines, encircled by small warts.In the current samples, gasterocarps with two pseudostipe morphologies were observed.Samples HAI-G-69 and HAI-G-80 have almost cylindrical, wide pseudostipe (≈80% of gasterocarp's width), with very little tapering towards the base.Moreover, this type of pseudostipe is almost free of any warts.Gasterocarps of the second type (HAI-G-171, HAI-G-173) have a notably thinner and slender pseudostipe, which shows a very strong tapering towards the base (it can constitute less than 35-40% of gasterocarp's width), and bears an ornamentation of fine even warts.
A molecular phylogenetic study carried out by Larsson and Jeppson demonstrated that L. perlatum clusters with L. marginatum and L. norvegicum, along with some species of Morganella and Vascellum in a clade with 59 BS/1.00 BPP support [41].The current analysis recovered the same topology of the species.
Bates [46] and Bates et al. [43] presented high-quality SEM micrographs of investigated representatives of the family Lycoperdaceae.Examination and a comparison of L. perlatum spores' micrographs, showed that spores of current specimens are very similar in terms of size and shape, and also have a distinct ornamentation of conical processes, yet differ in some aspects.The number of meshes between warts in Bates et al. [43] specimens is much lower than in current samples, and additionally the spore surface between warts and meshes appears smoother compared to Israeli material.
Lycoperdon pratense Pers., Neues Mag.Bot.1: 87 (1794).Figures 14 and A2E  Bates [46] and Bates et al. [43] presented high-quality SEM micrographs of investigated representatives of the family Lycoperdaceae.Examination and a comparison of L. perlatum spores' micrographs, showed that spores of current specimens are very similar in terms of size and shape, and also have a distinct ornamentation of conical processes, yet differ in some aspects.The number of meshes between warts in Bates et al. [43] specimens is much lower than in current samples, and additionally the spore surface between warts and meshes appears smoother compared to Israeli material.

Data Availability Statement:
The DNA sequence data that had been obtained during the current study were deposited at the GenBank database.The accession numbers are provided in Table 1 and Figure 2.

Acknowledgments:
The authors thank Vincent Demoulin (University of Liège, Belgium) for sending samples of the representatives of the family Lycoperdaceae, stored in the Herbarium of Liège University, and for his important advice and suggestions concerning the manuscript.We would like to thank Ellen Larsson (University of Gothenburg, Sweden) and Scott Thomas Bates (University of Minnesota, USA) for their consultations and literature.We thank Alona Biketova for her contribution to sample collection and some assistance during molecular studies.The authors express their gratitude to Alexander Krakhmalnyi for the help with SEM examinations.We are grateful to Robin Permut (Institute of Evolution, University of Haifa) for her editorial assistance, and to Paul A. Volz for a critical review of the early draft of the manuscript.We express our gratitude to Olga Godorova, Zohar Shafranov, Alexandr Gibkhin, Yona Cherniavsky and other amateur mycologists and enthusiasts for helping with organization of field trips, and providing us with fungal samples collected in different regions of Israel.

Conflicts of Interest:
The authors declare no conflict of interest.

Figure 2 .
Figure 2. Maximum likelihood tree of the family Lycoperdaceae based on the whole ITS sequence dataset.The topology of the Bayesian tree, in general, was congruent with the ML tree, but had more supported branches.Thick black lines indicate branches with significant statistical support (>70% bootstrap value for ML and >0.95 posterior probability for Bayesian analysis).Thick grey

Figure 2 .
Figure 2. Maximum likelihood tree of the family Lycoperdaceae based on the whole ITS sequence dataset.The topology of the Bayesian tree, in general, was congruent with the ML tree, but had more supported branches.Thick black lines indicate branches with significant statistical support (>70% bootstrap value for ML and >0.95 posterior probability for Bayesian analysis).Thick grey lines indicate branches significantly supported only by the Bayesian method.Respective BS and BPP values are given near the branches.All sequences are provided with full taxonomic names, GenBank accession number, and country of origin.The sequences obtained during the current study are marked with a dot.
Material examined: Israel.UG.Mt.Meron National Park.On wood.7 January 2004.Leg.Y. Ur, det.M. Krakhmalnyi (HAI-G-11).GH.Forest Odem.On wood.8 December 2012.Leg.Z. Shafranov, A. Biketova, det.M. Krakhmalnyi (HAI-G-159).Notes: For a long time, the taxonomic position of the species remained debatable.In 2003, Krüger and Kreisel published an article in which they transferred L. pyriforme to the lignicolous genus Morganella, creating a new combination-Morganella pyriformis (Schaeff.: Pers.)Kreisel & D. Krüger [45].The authors also proposed a new subgenus Apioperdon Kreisel & D. Krüger, to accommodate M. pyriformis.The subgenus took a somewhat intermediate position between Lycoperdon and Morganella subgen.Morganella in terms of its morphological features and habitat.Subsequent studies pertaining to molecular phylogenetics of the family Lycoperdaceae were not congruent with the results of Krüger and Kreisel [45].In the reconstructions of Larsson and Jeppson, L. pyriforme occupies a basal position to the Lycoperdon clade.Additionally, the species did not cluster with sequences of M. fuliginea (Berk.& M.A. Curtis) Kreisel & Dring and M. subincarnata (Peck) Kreisel & Dring, which represented the genus Morganella in the analysis.Bates et al.

Funding:
The Graduate Studies Authority of the University of Haifa, which supported M.A. Krakhmalnyi Ph.D program at the Institute of Evolution and Department of Evolutionary and Environmental Biology, Faculty of Natural Sciences, University of Haifa (Haifa, Israel).The standard government funding of the National Academy of Sciences (NAS) of Ukraine (Kiev, Ukraine).Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.

Table 1 .
Data on specimens involved in the phylogenetic study.

Table 2 .
Species composition of the family Lycoperdaceae in Israel.