Some noteworthy records of Helvella from Turkey based on morphology and DNA sequence data

In the present study, current information based on morphological characters and DNA sequence (nrITS) data is given on some uncommon or noteworthy Ascomycetes species collected from different geographic regions of Turkey. First phylogenetic analysis based on nrITS data of Helvella fibrosa and H. macropus the rare and little-known species was performed in Turkey by this study. Also, the nrITS sequence of H. fibrosa was uploaded to current databases for the first time. Detailed morphological description, macro photographs and detailed drawings of micro structures of the two species studied have been presented, and the ecology and distributions of each species has been given.


Introduction
Fungi are the second largest group of eukaryotic organisms in the world, with approximately 100 000 species described (Kirk et al. 2008). The total number of species is estimated to be between 1.5 and 5.1 million (Hawksworth 1991, 2001, O'Brien et al. 2005, Blackwell 2011). According to various studies, the Ascomycota constitute about half of all fungal species (Bass & Richards 2011). In addition to genera with a simple structure such as Peziza Dill. ex Fr. and Aleuria Fuckel (orange peel fungus) with their characteristic cup-shaped apothecia, the Pezizales group of Ascomycota includes other genera producing more complex and larger ascocarps like the wellknown Morchella Dill. ex Pers., Helvella L. and Tuber P. Micheli ex F.H. Wigg. (Larsen 1980, Hansen & Pfister 2006.
Members of the Helvella genus generally are characterized by auriculoid, cupulate to saddleshaped apothecia varying from white to black in color, the presence of ribs on the stipe and the structure of their ascospores (Dissing 1966a, b, Abbott & Currah 1997. So far, approximately sixty Helvella with these characteristics have been described (Wijayawardene et al. 2017). A large number of new entities belonging to Helvella have been described from different parts of the world thanks to recent multigenic DNA studies (Nguyen et al. 2013, Hwang et al. 2015, Zhao et al. 2015, Tibpromma et al. 2017, Skrede et al. 2017. Many Helvella taxa are widely distributed in shadow areas of deciduous and coniferous forests (Wang & Chen 2002, Zhao et al. 2015. Members of this  (Sesli & Denchev 2008, Nguyen et al. 2013, Solak et al. 2015, Hwang et al. 2015, Skrede et al. 2017, Hansen et al. 2019, Uzun 2019, Løken et al. 2020. Helvella fibrosa was previously recorded from the Kahramanmaraş Province in the Mediterranean Region (Kaya 2009) and Helvella macropus from Trabzon Province in the Black Sea Region of Turkey (Akata & Kaya 2012). Our collections of H. fibrosa is given from İstanbul Province and H. macropus is recorded from Artvin Province which are new locality records of these macrofungi (Sesli & Denchev 2008, Solak et al. 2015. The purpose of this study is to provide up-to-date information on rare and noteworthy taxa H. fibrosa and H. macropus identified from different geographical regions of Turkey, based on their morphology and DNA sequence (nrITS) data.

Specimens and morphological studies
The field works were undertaken in different geographical regions of Turkey between the years 2013 and 2015. Photographs of the fruiting bodies were taken in their natural habitats and their ecological notes were recorded.
The macroscopical descriptions and images of the ascomata were obtained by observation of fresh or dried specimens. For microscopical analyses, the dried materials were rehydrated in distilled water and 3% NaOH or 5% KOH, and subsequently stained with Congo Red or Melzer's solution. The following abbreviations are used in the descriptions: L m : for the average length of all the measured ascospores, W m : for the average width of all the measured ascospores, Q: for the quotient of length and width of all the measured ascospores, and Q m : for the average of all calculated Q values for all ascospores measured. At least thirty mature ascospores from each ascoma were measured. The specimens were deposited at the fungarium of Isparta University of Applied Sciences (Turkey).

DNA extraction, PCR amplification and sequencing
Total genomic DNA was extracted from fresh or dried materials using the ZR Fungal/Bacterial DNA MiniPrep kit (Zymo research, Irvine, CA, USA). Protocols for DNA extraction, PCR and sequencing were the same as those outlined in Kaygusuz et al. (2019). PCR and sequencing of the complete internal transcribed spacer (ITS) region from the ribosomal DNA (rDNA) was performed using ITS1F and ITS4 (White et al. 1990, Gardes & Bruns 1993. Primers ITS1F/ITS4 were used to amplification, using the touchdown PCR protocol described in Kaygusuz et al. (2019). All PCR products were sequenced in Sanger DNA sequencing service (Source Bioscience, Berlin, Germany), with the same primers used in the PCR reactions. The raw DNA sequencing files were edited with Chromas Lite 2.1.1 (http://technelysium.com.au/wp/chromas/) and assembled with BioEdit 7.2.5 (Hall 1999). The edited sequences were then used for BLAST searches in GenBank (www.ncbi.nlm.nih.gov). The newly generated sequences were deposited in GenBank with corresponding accession numbers (MF228805 and MF228806).

Sequence alignment and phylogenetic analyses
For this study, two new sequences of nrITS were generated. Further twenty-four related sequences used in phylogenetic analysis were downloaded from the NCBI (National Center for Biotechnology Information, Rockville Pike, Bethesda MD, USA) database. All sequences were aligned by MAFFT (version 7.110) programs (Katoh & Standley 2013). In addition, final alignments were manually corrected via BioEdit and MEGA X v.10.0.5 (Kumar et al. 2018 (Ronquist et al. 2012). Markov chains were run for 10 6 generations, saving a tree every 1000 th generation, with two runs per analysis. The initial 25% trees recovered were excluded as a burn-in, and a 50% majority consensus tree of the remaining trees was then used to calculate the posterior probabilities (PP) of the group. The phylogram inferred from both analysis were displayed with FigTree v.1.4.3 (Rambaut 2016).

Molecular phylogeny
Our phylogenetic analysis has involved on twenty-six nucleotide sequences. The final dataset contained 1141 nucleotide sites. The resulting phylogram with the lowest BIC (Bayesian Information Criterion) value (-16015.88) and highest log likelihood (-7976.45) are presented. We selected the topology resulting from the first iteration to present here ( Fig. 1, -InL = 7745.88). Ascoma 5-13 mm high. Apothecia 3-6 mm high, 3-4 mm wide, cup-shaped at first, becoming saddle-shaped to irregularly lobed, typically initially laterally compressed, sometimes with a deflexed margin, pinched in at the apex; hymenium pale grey or dark gray when young, fading to greyish-brown to pale gray, smooth; exterior surface light-grey to pale-grey or pearl grey, very hairy, densely pubescent. Stipe 4-12 mm long, 1-2 mm broad, central, cylindrical, solid, hairy, densely pubescent, whitish to cream, becoming creamy when dried, basal mycelium white. Context very thin and fragile, light to pale gray. Smell and Taste indistinct. Spore print white.
Comments -Characteristic features such as an apothecia varying from blackish to grey in color, a cup-shaped apothecia, a thin stipe and lower parts with a pubescent form are useful in identifying members of the genus Helvella. Of these, Helvella fibrosa has been recorded from different regions of the world and is seen to have a wide distribution. However, this species has long been confused with H. macropus, and wrong identifications have been made. Korf (2008) summarized the outlines of the historical name changes of H. fibrosa, and how its current name has been accepted. H. fibrosa is morphologically similar to H. macropus, but they can be distinguished by the darker hymenium and less villose apothecia surface of H. fibrosa (Skrede et al. 2017). Also, the ascospores of H. fibrosa are ellipsoidal and not acuminate.
Ecology -Solitary or gregarious from late September to early October, generally present at over 300 m of elevation, on the ground, among mosses, in temperate deciduous mixed forests, especially on acidic noncalcareous clay-loam soils. Reported also to grow under tea bushes (Camellia sinensis).
Comments -The species of the Helvella genus can generally be distinguished from one another by the dimensions, form, configuration and colour of the stipe and apothecia. Helvella macropus is characterized by a villose apothecia and stipe, and by verrucose ascospores.
Morphologically H. macropus and H. brevis (Peck) Harmaja have similar features, however the ascospores of H. macropus are broader than those of H. brevis (Peck 1902, Weber 1972. Also, the stipe length of H. macropus is greater than that of H. brevis (8-16 mm long) (Harmaja 1974, Beug et al. 2014). Finally, the outside surface of the apothecia of H. brevis is much less villose than that of H. macropus.
Our phylogenetic analysis shows that H. macropus is phylogenetically closely related to but distinct from H. fibrosa, H. chinensis and H. cf. corium based on the ITS data. In addition, the result that H. macropus clustered with collections of H. macropus from South Korea and H. cf. macropus from China as a whole gets high statistical support, and 100% bootstrap and 1.0 bayesian PP support. Similar phylogenetic results were observed by Landeros et al. (2015) and Skrede et al. (2017) in their phylogenetic analysis. H. macropus is presented from Turkey for the first time based on ITS data in this study. In future studies, it will be necessary to include analyses of sequences from Europe and other areas in order to determine the position of H. macropus in relation to other species.
In conclusion, our survey presents the new DNA sequences of the interesting and noteworthy species Helvella fibrosa and H. macropus. In addition, the nrITS sequence of H. fibrosa was uploaded to current databases by us for the first time as a result of this study. The two species studied were presented with detailed morphological descriptions, macro photographs and drawings of micro-structures. Our study confirms the existence in Turkey of certain uncommon and important fungi, and contribute to future studies on the phylogeny, morphology, taxonomy and distribution of the species recorded.