Laboulbeniales is one of the most peculiar orders of Ascomycota. These fungi are characterized by an ectoparasitic life-style on arthropods, determinate growth, lack of an asexual stage, high species richness, and intractability to culture. The order Laboulbeniales, sister to Pyxidiophorales, has only recently been assigned a separate class, the Laboulbeniomycetes, based on very few ribosomal DNA sequences. So far, DNA isolations and PCR amplifications have proven difficult. Here, we provide details of isolation techniques and the application of commercially available kits that enable efficient and reliable genetic analyses of these fungi. We provide 43 newly generated Laboulbeniales ribosomal DNA sequences, among which are the first published sequences for species in the genera Gloeandromyces, Herpomyces, Laboulbenia, Monoicomyces, and Polyandromyces. DNA extractions were possible using from 1 to 30 thalli from hosts preserved in ethanol (70-100 %). In two cases, we successfully isolated DNA from thalli on dried insect collections. Laboulbeniales molecular systematics could be substantially enhanced through these improved methods by allowing more complete sampling of both taxa and gene regions.

Modern taxonomy of yeasts is mainly based on phylogenetic analysis of conserved DNA and protein sequences. By far the most frequently used sequences are those of the repeats of the chromosomal rDNA array. It is generally accepted that the rDNA repeats of a genome have identical sequences due to the phenomenon of sequence homogenisation and can thus be used for identification and barcoding of species. Here we show that the rDNA arrays of the type strains of Metschnikowia andauensis and M. fructicola are not homogenised. Both have arrays consisting of diverse repeats that differ from each other in the D1/D2 domains by up to 18 and 25 substitutions. The variable sites are concentrated in two regions that correspond to back-folding stretches of hairpin loops in the predicted secondary structure of the RNA molecules. The substitutions do not alter significantly the overall hairpin-loop structure due to wobble base pairing at sites of C-T transitions and compensatory mutations in the complementary strand of the hairpin stem. The phylogenetic and network analyses of the cloned sequences revealed that the repeats had not evolved in a vertical tree-like way but reticulation might have shaped the rDNA arrays of both strains. The neighbour-net analysis of all cloned sequences of the type strains and the database sequences of different strains further showed that these species share a continuous pool of diverse repeats that appear to evolve by reticulate evolution.

Vipera ursinii graeca is a restricted-range, endemic snake of the Pindos mountain range in the southwestern Balkans. The subspecies was previously reported from eight localities in Greece and one locality in southern Albania. We used species distribution modelling based on climate data from known localities in Greece to estimate the potential distribution of the subspecies. The model predicted suitable areas for eleven mountains in southern Albania, which we visited in ten field expeditions in four years. Based on 78 live individuals and 33 shed skins, we validated the presence of the snake on eight of the eleven mountains. Six populations (Dhëmbel, Llofiz, Griba, Shendelli, Tomorr and Trebeshinë Mountains) are reported here for the first time. Morphological characters undoubtedly supported that all individuals found at these new localities belong to V. u. graeca . Genetic analysis of mitochondrial DNA sequences also confirmed the identity of the snakes as V. u. graeca and a low number of identified haplotypes suggested low genetic variability among populations despite significant spatial isolation. All localities were subalpine-alpine calcareous meadows above 1600 m. These high montane habitats are separated by deep valleys and are threatened by overgrazing, soil erosion, and a potential increase in the elevation of the tree line due to climate change. Our surveys increased the number of known populations by 60% and the known geographical range of the subspecies by approximately 30%. Our study serves as a baseline for further ecological research and for conservation measures for one of the least known European viperid snakes.