Two species of true morels (the genus Morchella, Ascomycota) recorded in the Ojców National Park (south Poland)

In this paper, we present results of survey on the occurrence of true morels, conducted in the Ojców National Park (ONP). The first data about true morels from the area of the ONP date back to the nineteenth century. However, despite multiple surveys in the next decades, their presence has not been confirmed. Therefore, true morels were thought to be extinct in the ONP. In 2014 and 2015, two morphotypes of true morel fruiting bodies were discovered in two sites, identified as black morels and yellow morels. In 2016, we collected three fruiting bodies for detailed morphological characterization and molecular identification. According to our results, these morels were identified as Morchella deliciosa and Morchella esculenta. Our finding is the first confirmation of the occurrence of both species (as they are presently recognized) in Poland.


Introduction
The Ojców National Park (ONP) is the smallest national park in Poland, it has only 2146 ha, however it is characterized by unique biodiversity and the occurrence of almost 10.4 thousand species of fungi, plants, and animals that have been reported here [1]. Unfortunately, for many species of fungi these records are outdated and not confirmed recently. Therefore, these species have been deemed extinct in the ONP [2]. True morels Morchella spp. are among interesting and rare fungi, which occurrence have not been confirmed in the ONP, for more than 100 years.
True morels are sac fungi belonging to Morchellaceae family. Their fruiting bodies appear generally in spring, albeit sometimes even from early spring to mid-winter [3][4][5]. They are saprotrophs or form biotrophic relationships with trees [6]. True morels belong to the most valuable and widely recognized, edible fungi [7]. Five species of true morels have been reported from Poland: Morchella esculenta, M. elata, M. conica, M. gigas, and M. crassipes, all of them are under partial protection [8,9].
True morels are highly polymorphic, what impedes their taxonomic distinction [10][11][12]. They can differ in head shape and color, ridges and pits arrangement, and stalk to head ratio [11,13]. General agreement exists that two groups can be morphologically distinguished: the first comprises taxa with darken, grey or black regular ribs ("black morels", Elata clade), while the second one encompasses taxa with yellow or cream to tan, irregular ribs ("yellow morels", Esculenta clade) [14,15].
There are only two reports from the nineteenth century of true morels from the area of the ONP, i.e., Morchella esculenta and M. deliciosa [16]. Because there were no further reports of the occurrence of true morels from this area, they were considered extinct in the ONP [2,17,18]. On the list of protected and endangered fungi of Poland [19] there is Digital signature This PDF has been certified using digital signature with a trusted timestamp to assure its origin and integrity. A verification trust dialog appears on the PDF document when it is opened in a compatible PDF reader. Certificate properties provide further details such as certification time and a signing reason in case any alterations made to the final content. If the certificate is missing or invalid it is recommended to verify the article on the journal website. and a yellow morel Morchella esculenta (L.: Fr) Pers. occurring at two sites. All three target fragments for both species were identical with their respective GenBank accessions (Tab. 1).
Tab. 1 Results of molecular identification of specimens with ITS, LSU, and rpb2 sequences.
Paraphyses ramified at the base, septate, slightly broadened at the top. Despite slight morphological difference between fruiting bodies on Site 1 and 2, they proven to be genetically uniform for all three target sequences, ITS, LSU, and rpb2.   (Fig. 2e). On the superficies of some colonies stromatic structures were formed (as shown in Fig. 1d

Discussion
The exact number of Morchella species is not clear. Index Fungorum lists 332 records within Morchella genus, and the MycoBank 296 records [26,27]. In the morphologybased classifications of the genus, the number of species varies from 3 to 52 (discussed in [10] and [12]). Some doubts concern two taxons: Morchella conica and M. elata, both of them regarded in Polish literature and legal regulations as separate species [8,9]. This status is, however, disputed as some authors regard Morchella conica as a distinct species [28][29][30][31], while the other as a synonym of Morchella elata (e.g., [32]). The uncertainty described above is an example of problems arising from attempts to accommodate morphologically defined species names to molecularly defined monophyletic groups. The inaccuracy in using of morphologically defined binomial species names to describe genetic diversity detected with molecular methods resulted in adoption of provisional system of species designations (see [7,24,[33][34][35][36]), where M. deliciosa is referred to as Mel-26 and M. esculenta as Mes-8. According to the newest data provided by Richard et al. [12], the name Morchella conica is illegitimate at the species rank, while the Morchella elata status needs further study to be definitively resolved. Morchella deliciosa is regarded a valid species, formerly sometimes under different names: M. conica var. deliciosa, M. conica var. flexuosa, M. conica var. nigra, M. conica var. violeipes, M. conica, or M. intermedia [12].
According to our results, two species of true morels, M. deliciosa, and M. esculenta, were identified. This finding is the only report of the occurrence of true morels in the area of the Prądnik Valley, including ONP, for more than 100 years, as they were recorded previously only by Berdau [16]. For studied specimens macroscopic and microscopic features: sizes of fruiting bodies, asci, and ascospores, does not deviate from descriptions reported in literature for M. conica var. deliciosa and M. esculenta [28,33,37]. There are also no differences between these two species in microscopic features. Similarly, all sequences for all three target fragments for the studied specimens do not deviate from the previously reported. Additionally, the sequences detected in our specimens occurred also (100% match) in specimens collected in: Czech Republic, France, Montenegro, Turkey, Spain, and Ukraine -M. esculenta [7,10,12,24,38] and in: China, France, and Turkey -M. deliciosa [7,12,24,[33][34][35][36]. This fact indicates that both morels species found in the ONP belong to widely distributed genotypes.
As for habitat requirements, the ONP morels generally follow the usual habitat pattern: yellow morels occur predominantly in deciduous forest, while black morels in conifer forest, however, both can also grow in different habitats [13,35,39,40]. Our observation supports this pattern, as two sites of M. esculenta were located in C. betulus dominated or C. betulus, F. excelsor, A. pseudoplatanus mixed stands, and M. deliciosa site was located in A. alba / P. abies dominated stand. It is known that some morels are considered saprotrophs, while other can form biotrophic relations with plants, some researchers even believe that morels can form mycorrhiza-like interactions with trees [6,41,42]. While we did not consider that aspect in our study, it could be, however, interesting subject for future studies.
Güeler and Arkan [43] pointed out that Morchella mycelium is the fastest growing of all mushrooms and has the same nutritive content and aroma as its ascocarps. The fast growth of the cultures was confirmed in our experiments for cultures isolated from the ONP specimens. Some researchers informed also about the formation of sclerotia in pure cultures of Morchella spp. [43,44]. Buscot [44] observed two types of sclerotia: the first type involves numerous sclerotia formed along periphery of the colony, 2 days after the colony reached the edge of a Petri dish, while the second type comprises the sparse sclerotia formed after 12 days and randomly distributed over the medium. However, the colony structure and development of Morchella spp., including sclerotia formation, depends strongly on the incubation temperature and medium composition [43,44]. Güeler and Arkan [43] used three medium types: potato dextrose agar, malt extract agar, and complete yeast medium prepared with casein, casamino acids, peptone, and sodium nitrate, while Buscot [44] used malt extract agar and yeast extract agar. We used only malt extract agar to culture our isolates. Perhaps for this reason, in colonies of both analyzed Morchella species sclerotia, as described above, were not observed. On the surface of some colonies, however, stromatic structures were formed, but dissimilar to described by Buscot [44].
The interesting observation is that in some M. deliciosa cultures, conidia were formed. The first author who observed the formation of anamorphic state was Molliard [45,46], who classified it in Costantinella genus. Later, Paden [47] obtained the Costantinella state for M. elata cultures. Another observation of conidial state found in Morchella cultures was reported by Buscot [44], while Barron [48] indicated the close relationship of Costantinella and Nodulosporium asexual states. The next step was the observation of Costantinella colonies in nature: on soil, moss, and woody debris in fall in the inland Pacific Northwest USA; from these colonies, pure cultures of Costantinella were isolated [49]. According to these authors, the widespread occurrence of asexual states of morels, false morels, and allied taxa suggests that their life cycles are more complex than previously thought. Conidiophores and conidia observed on M. deliciosa cultures from the ONP corresponded to features of Costantinella [50].
The survival of true morels in the ONP seems to be unthreatened. Some of the sites are located next to tourist trails, however, mushroom picking in the ONP is forbidden [51]. Despite small area of the ONP and more than 200 years research history, it is still not totally explored place, especially concerning fungi. There is no doubt that the community of fungi in the ONP requires further studies.