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A three-gene data set was generated to explore species diversity and delimitations within the stalked puffballs (Tulostoma, Agaricales) in Europe. Data on species from other parts of the world were included for comparison of species concepts and distribution ranges. Sequence data from 26 type specimens are included. The phylogenetic analyses support Tulostoma as monophyletic. Eleven major clades, 37 minor clades, and 20 single branches were recovered and found to correspond to 30 described species and 27 species without scientific names.Five species are here described as new to science: Tulostoma calcareum, T. calongei, T. eckbladii, T. grandisporum, and T. pannonicum. In total we report 26 described, and 19 undescribed, species from Europe. An epitype for T. fimbriatum with ITS sequence data is selected to fix the name.The recovered tree topology was not in congruence with the current infrageneric classification of Tulostoma, suggesting that many of the morphological characters used for segregation of taxa are plesiomorphic or homoplasious. Spore ornamentation and hyphal structure of the peridium are found to be reliable characters for delimitation of species. RESEARCH ARTICLEMikael Jeppson et al. / MycoKeys 21: 33-88 (2017) 34The majority of the species occur in the dry, arid areas of southern and east central Europe but a few are shown to be restricted to humid temperate regions in the North. The study confirms that species with smooth or sub-smooth spores are restricted to dry and arid habitats whereas species with more strongly ornamented spores occur in humid habitats.Areas with steppe vegetation in Hungary and Spain are here identified as hot spots for Tulostoma species diversity.

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Non-denaturing FISH (ND-FISH) was used to compare the distribution of four simple sequence repeats (SSRs)-(AG) n , (AAG) n , (ACT) n and (ATC) n -in somatic root tip metaphase spreads of 12 barley (H. vulgare ssp. vulgare) cultivars, seven lines of their wild progenitor H. vulgare ssp. spontaneum, and four lines of their close relative H. bulbosum, to determine whether the range of molecular diversity shown by these highly polymorphic sequences is reflected at the chromosome level. In both, the cultivated and wild barleys, clusters of AG and ATC repeats were invariant. In contrast, clusters of AAG and ACT showed polymorphism. Karyotypes were prepared after the identification of their seven pairs of homologous chromosomes. Variation between these homologues was only observed in one wild accession that showed the segregation of a reciprocal translocation involving chromosomes 5H and 7H. The two subspecies of H. vulgare analysed were no different in terms of their SSRs. Only AAG repeats were found clustered strongly on the chromosomes of all lines of H. bulbosum examined. Wide variation was seen between homologous chromosomes within and across these lines. These results are the first to provide insight into the cytogenetic diversity of SSRs in barley and its closest relatives. Differences in the abundance and distribution of each SSR analysed, between H. vulgare and H. bulbosum, suggest that these species do not share the same H genome, and support the idea that these species are not very closely related. Southern blotting experiments revealed the complex organization of these SSRs, supporting the findings made with ND-FISH.

The genetic relationships between several wild species and subspecies of the genus Hordeum were assessed using fluorescence in situ hybridization (FISH). Plant material included natural populations of wild barley growing in Spain of the annual species, H. marinum ssp. marinum (2n = 14) and gussoneanum (2n = 14), and H. murinum ssp. murinum (2n = 28), and leporinum (2n = 28) and the perennial species H. bulbosum (2n = 14) and H. secalinum (2n = 28), plus the South American perennial species H. chilense (2n = 14). FISH was used to locate the chromosomal sites of two rDNA multigene families 5S and 18S-26S (pTa71 and pTa794) and three repetitive DNA sequences (pSc119.2, pAs1 and pHch950) isolated from different species and genera. The seven chromosomes of the diploid species were readily distinguished by their external morphology and hybridization patterns to pTa71, pTa794, pSc119.2 and pAs1. These DNA probes were also useful for the identification of homologous chromosomes and in differentiating these from unidentified chromosomes in the tetraploid taxa. The use of the probe pHch950 permitted intergenomic differentiation in tetraploids and supports the diphyletic origin of H. murinum and H. secalinum. The in situ experiments yielded the following conclusions: (1) differences between the sub-species marinum and gussoneanum; (2) close relationships between the subspecies murinum and leporinum; and (3) major differences in physical mapping between H. bulbosum and the remaining taxa. The genomic and phylogenetic relationships between taxa, as inferred from the results, are discussed.