Molecular methods are increasingly used to identify species that lack conspicuous macro‐ or micromorphological characters. Taxonomic and ecological research teams barcode large numbers of collected voucher specimens annually. In this study we assessed the efficiency of long‐read high throughput sequencing (HTS) as opposed to the traditionally used Sanger method for taxonomic identification of multiple vouchered fungal specimens. We also evaluated whether this method can provide reference information about intraindividual gene polymorphism. We developed a workflow based on a test set of 423 basidiomycete specimens (representing 195 species), the PacBio HTS method, and ribosomal rRNA operon internal transcribed spacer (ITS) and 28S rRNA gene (LSU) markers. The PacBio HTS had a higher success rate than Sanger sequencing at a comparable cost. Species identification based on PacBio reads was usually straightforward, because the dominant operational taxonomic unit (OTU) typically represented the targeted organism. The PacBio HTS also enabled us to detect widespread polymorphism within the ITS marker. We conclude that multiplex DNA barcoding of the fungal ITS and LSU markers using PacBio HTS is a useful tool for taxonomic identification of large amounts of collected voucher specimens at a competitive price. Furthermore, PacBio HTS accurately recovers various alleles and paralogues, which can provide crucial information for species delimitation and population‐level studies.

Lignicolous fungi plays are a vital part of forest ecosystems in Europe. They are involved in Carbon cycle, through decay processes of woody debris. Very fine woody debris (VFWD) forms an important component of this dead wood, being found in any forest in the World. Among European and Romanian forests, Fagus spp. dominating forests are the most important broadleaved ecosystems, of great biotic and abiotic complexity. The present distribution of lignicolous fungi is mainly linked to trees distribution. In the context of climate change, European beech forests will also shift in distribution, structure and composition, triggering changes in lignicolous fungal communities and diversity as well. Considering this background, VFWD lignicolous diversity might be a future beech forests climate change indicator. This will bring the necessity of assessing the main climatic factors that are influencing the lignicolous fungal diversity distribution across European beech forests in Romanian's NorthEast Region. In the present study, our findings confirms the fact that macroclimate have a great influence on lignicolous mycodiversity in beech forests. It seems that minimum temperature and Gams Continentality Index explains approximately 48% of the mycodiversity variation. While dropping minimum temperatures and increasing Gams CI values, the lignicolous fungal richness will rise. While minimum temperature of January might be linked to a complex ecological and phonological framework, Gams CI is a known ecological indicator for optimum habitat of beech forests, which in turn influence lignicolous diversity distribution. Those climatic variables might characterize the relation between plants-fungi-climate in the near future, as increasing atmospheric temperatures will manifest at different scales. Thereafter, VFWD mycodiversity might function as a valuable macroclimatic changing indicator.

Coarse woody debris is often highlighted as the most important microhabitat for numerous saproxylic species, including macrofungi. Providing valuable nutrients, stable microclimatic conditions and development space, logs and large branches are considered of great ecological value for macrofungal diversity conservation. Old forests are especially rich in downed coarse dead wood both at quantity and quality level. Unfortunately, these forests are also affected by human interventions, through wood extraction and forest fragmentation. The main objective of this study was to find the factors that best explain the macrofungal diversity on downed coarse woody debris (DCWD). For this, we sampled 21 plots in forests dominated by beech or oak from Northeastern Romania, where we collected data about fungi, forest structure, and dead wood. We completed the variables set with forest fragmentation and topographic indices. In order to find the best models and predictors, we used generalized linear models (GLM). We found 163 taxa, polypores and agarics being the most frequent. The two most important predictors had a positive effect, increasing macrofungal diversity: 2'nd and 3'rd decay stages DCWD volume and elevation while the third one had a hump-shape effect on diversity. In old forests, downed dead wood quality and quantity is a vital component for numerous species of fungi to survive and develop. Elevation is a known proxy of macroclimatic conditions, furthermore creating new richresources niches because increasing humidity and taxonomic diversification by conifers occurrence. Patch shape can have divergent effects on fungi, as increasing perimeter is associated from one point on, with human deforestation and accessibility. Overall, we believe that Northeastern Romania's old forests hosts a great lignicolous macrofungal richness, which will be protected through silvicultural practices such as keeping valuable dead wood on site.

Molecular methods are increasingly used to identify species that lack conspicuous macro- or micromorphological characters. Taxonomic and ecological research teams barcode large numbers of collected voucher specimens annually. In this study we assessed the efficiency of long-read high throughput sequencing (HTS) as opposed to the traditionally used Sanger method for taxonomic identification of multiple vouchered fungal specimens, and providing reference information about intra-individual allele polymorphism. We developed a workflow based on a test-set of 423 fungal specimens (representing 205 species), PacBio HTS method, and ribosomal rRNA operon internal transcribed spacer (ITS) and 28S rRNA gene (LSU) markers. PacBio HTS had a higher success rate than Sanger sequencing at a comparable cost. Species identification based on PacBio reads was usually straightforward, because the dominant operational taxonomic unit (OTU) typically represented the targeted organism. Unlike the Sanger method, PacBio HTS enabled detecting widespread allele polymorphism within the ITS marker in the studied specimens. We conclude that multiplex DNA barcoding of the fungal ITS and LSU markers using a PacBio HTS is a useful tool for taxonomic identification of large amounts of collected voucher specimens at competitive price. Furthermore, PacBio HTS accurately recovers various alleles, which can provide crucial information for species delimitation and population-level studies.

Background and aims – Lignicolous fungal assemblages perform numerous functions in forest ecosystems, one of the most important being their capacity to decay wood. As a consequence of their belonging to different ecological niches, the forest ecosystem influences the fungal assemblages in terms of species richness and composition. Methods – In this study we analyzed the main lignicolous macrofungal assemblages in some deciduous and mixed deciduous-coniferous forests in the North-East Region of Romania. We searched to find fungal indicator species for a certain forest type and which are the main drivers and their effects on the composition of the lignicolous macrofungal assemblages. Fungal assemblages were identified using a hierarchical agglomerative clustering procedure, while diagnostic species for each cluster were identified based on the indicator value index. Relationships between fungal composition of plots and environmental variables were performed using detrended and canonical correspondence analyses.Key results – A total of 377 fungal taxa in approximately 4600 records (in 59 plots) were identified. Six distinct clusters of lignicolous fungal assemblages were defined and separated three groups: 1) species-rich lignicolous fungal assemblages in beech forests (1 cluster), 2) well defined fungal assemblages in the mixed broadleaved-coniferous forests (2 clusters), and 3) fungal assemblages typical to oak forests (3 clusters). Ordination methods highlighted the forest type as the most important factor influencing the fungal composition of plots. Forestry Aridity Index, tree diversity and large trees basal area were also important factors for fungal assemblages but with a lower contribution. Conclusion – In the studied region, fungal assemblages changed from oak to beech and to mixed, broadleaved-coniferous forests mainly as a consequence of different tree composition. Climate also shaped fungal composition but to a lesser extent.