Morphological and molecular characterization of Helvella L. species growing in Türkiye

Helvella is a genus of fungi within the family Helvellaceae of phylum Ascomycota, characterized by irregularly lobed, saddle-, cup- or goblet-shaped apothecia. Although Türkiye possesses a rich mycobiota, comprehensive studies on the genus Helvella remain limited. It is believed that many species of the genus Helvella remain undiscovered in Türkiye's diverse ecological regions; therefore, a current and comprehensive taxonomic revision of the genus is still needed. In this doctoral dissertation, specimens collected from the field from 2022 to 2024 and obtained from various fungaria were evaluated using both morphological and molecular methods. Species identifications were were made and their phylogenetic relationships were clarified. Morphological descriptions of the collected specimens were carried out by considering their macroscopic and microscopic features. Macroscopic characters such as apothecia, stipe, receptacle, and hymenium structures, and microscopic characters including asci, ascospores, paraphyses, and excipulum were examined. For microscopic analyses, various stains and reagents such as distilled water, melzer's reagent, cotton blue, congo red, and potassium hydroxide (KOH) were used. Additionally, comprehensive morphological descriptions were provided for all Helvella species identified in the study, updating the descriptions of some species that had previously been poorly or inadequately documented. Furthermore, a new identification key based on morphological and ecological characteristics was developed to differentiate Helvella species distributed in Türkiye. As part of the molecular systematic studies, DNA sequences were obtained from five different barcode regions: the internal transcribed spacer (ITS), the large subunit (LSU) of ribosomal DNA, translation elongation factor 1-alpha (TEF1-α), the second largest subunit of RNA polymerase II (RPB2), and heat shock protein 90 (HSP90). After generating separate sequence alignments for each region, combined phylogenies were constructed using concatenated datasets for four-locus (ITS/LSU/TEF1-α/HSP90) and five-locus (ITS/LSU/TEF1-α/RPB2/HSP90) multilocus analyses. Phylogenetic trees were inferred based on Bayesian Inference (BI), revealing species boundaries and clade structures supported by morphological evidence. A total of 50 specimens were analyzed during the study, resulting in the identification of 26 distinct species, which were classified into the Acetabulum, Crispa, Elastica, and Lacunosa clades of the genus Helvella. Among these, H. arctoalpina in the Acetabulum clade; H. capucina, H. pubescens, and H. rivularis in the Elastica clade, and H. alpina, H. dryophila, and H. sublactea in the Lacunosa clade were recorded as new species for the Turkish mycobiota. This thesis presents the most comprehensive assessment to date of the taxonomic diversity and phylogenetic relationships of the genus Helvella in Türkiye. The integration of morphological and molecular data significantly resolved identification issues and indicated that some traditional species delimitations need to be reconsidered in light of molecular evidence. Moreover, the findings provide significant contributions not only to the mycobiota of Türkiye but also to the global systematic positioning of the genus Helvella. In conclusion, this study not only fills critical knowledge gaps but also establishes a robust foundation for future ecological, evolutionary, and conservation-based mycological research. By raising awareness of Türkiye's fungal diversity and updating mycological inventories, this thesis serves as a fundamental reference source. Keywords: DNA barcode, Helvella, HSP90, ITS, LSU, New record, RPB2, TEF1-α