Taxonomic notes on the Polish Tulostoma species

Tulostoma genus Pers.: Pers. is represented in Poland by five species (Wojewoda 2003). Only two of those: Tulostoma brumale Pers.: Pers. and T. fimbriatum Fr. can be considered as a relatively common while others such as: Tulostoma kotlabae Pouzar, T. melanocyclum Bres. and T. squamosum (Gmelin in L.): Pers. are very rare. Observations of macrofungi of xerothermic habitats at the Nida Basin (Central Poland), demonstrated the existance of many varieties of particular taxa until now not listed in the Polish mycological literature, and also new sites of all so far described Tulostoma species. Recording of lower taxa such as: Tulostoma brumale Pers. var. longipes (Czern.) J. E. Wright, T. brumale Pers. var. pallidum (Lloyd) J. E. Wright, and T. fimbriatum Fr. var. campestre (Morgan) Moreno are identified at the study area.


INTRODUCTION
Species belonging to the genus Tulostoma has been investigated by many researchers and a variety of taxonomic approaches to these fungi have been adopted in monograph studies.The genus Tulostoma initially consisted of two sections: Euty lostoma and Schizostoma (Fries 1921;Fischer 1900Fischer , 1933;;Petri 1909), distinguished by peristome morphology.A more precise classification was proposed by Pouzar (1958), who assigned the species to four sections: Brumalia, Poculata, Fimbriata and Volvulata, based on the morphology and the way the exoperidium breaks off, the morphology of the peristome and the stem.The system proposed by Pouzar was used by Wright (1987) in the current classification based on primary and secondary micromorphological characters.Primary characters include: the shape, size and colour of the endoperidium; the colour, persistence of the exoperidium and the way it breaks off, peristome, spore size and episporium ornamentation.Secondary characters used to identify the species are: the height, colour and morphology of the stem surface, the thickness of capillitial threads, and the morphology of transverse septa produced by them.
During the field investigations, a great number of the genus Tulostoma fruitbodies were collected of which precise identification (especially to the lower taxa) was very difficult.The aim of this study was to compile keys to allow identification to the lower taxa such as for example varieties, within examined species.Keys to macro-and microscopic characters are given, which aim to underline intraspecific variability of selected species growing in Poland.Lower taxa can be fully determined and identified correctly based on the descriptions of macro-and micormorphological features.

MATERIAL AND METHODS
Mycological investigations were conducted in xerothermic habitats in the Nida Basin and the Chęciny District since October 2010.Studies will continue over the next three years.Communities of xerothermic vegetation distributed in protected areas such as Nature 2000 ecological sites, nature reserves and landscape parks have been examined.Preliminary results of studies are presented below.
Fruitbodies of T. fimbriatum were collected at localities in Kielce and in xerothermic psammophilous grasslands in Zajączków village in the Chęciny District.
Carpophores of the genus Tulostoma were collected each time during observations.The number of fruitbodies of a species and organoleptic features, i.e. the shape, size and colour of the endoperidium, the colour and breaking off of the exoperidium, and the stem colour and features, were recorded upon collection.
Laboratory studies were conducted using a light microscope and a scanning electron microscope.Threads of the capillitium, septa and spores whose morphology, size and shape are important taxonomic features were examined by LM.Slides were made from each fruitbody.Ten randomly selected spores were measured in each slide.Microscopic structures were observed using an immersion lens.
Interspecific relationships in the taxonomy of the species were described while determining the species based on the above studies.Notes on the intraspecific differentiation of taxa were based on the material collected by the authors.
The dry material is deposited in the Fungal Herbarium (KTC) of the Faculty of Mathematics and Nature, Jan Kochanowski University, Kielce (Poland).

DISCUSSION
Tulostoma exhibit high morphological similarity between species, which causes several taxonomic problems.They are often similar in shape, size and colour of the endoperidium, the morphology and colour of the peristome, pigmentation, persistence and breaking off of the exoperidium, and the size, colour and morphology of the stem.The preservation of morphological features in specimens of individual species also affects correct identification of important diagnostic differences between fruitbodies as some traits become similar when fruitbodies mature and age.Features that are not persistent include exoperidium pigmentation, scaling on the stem, the shape and attachment of scales to the stem, the shape and the size of the mouth, and the pigmentation of the peridium and the stem.
Problems discussed in this chapter were especially challenging in the case of T. brumale var.brumale and T. kotlabae.The taxa are very similar and can be easily mistaken.They are also similar to other species.A list of similarities and differences is given in Table 2.
Many authors, including Wright (1987) and Pouzar (1958), have pointed out diagnostic problems between Tulostoma brumale var.brumale and T. melanocyclum.Authors encountered such problems while identifying fruitbodies of these species.The colour of the peristome is an especially important feature used to determine fruitbodies of T. brumale var.brumale.Like in T. melanocyclum, fruitbodies of T. bru male var.brumale have a darkly pigmented collar around the peristome.However, the peristome is light-coloured in T. brumale var.brumale while it is dark brown in T. melanocyclum similarly to the collar around the mouth (Fig. 3 A, B).The two species   also differ by the colour of the stem.The stem is intensively dark brown or dark chestnut in T. melanocyclum unlike T. brumale var.brumale which is distinguished by a light brown stem.They differ radically by spore size, morphology, shape and ornamentation.Spores in T. melanocyclum are larger than in T. brumale var.brumale and their ornamentation is visible not as verrucae but as small spikes appearing as coalesced columns (Fig. 4 A, B).Another interesting species is T. simulans.The name refers to the fungus simulating other species.According to Wright (1987) the species can be confused with T. brumale var.brumale.However, only slight similarity between T. brumale var.bru male and T. simulans was observed in our studies.Both species may be mistaken due to similar microscopic features: verrucose spores and thickened transverse septa.The external structure allows the two species to be distinguished as appropriate.
More distinct morphological similarity was observed between fruitbodies of T. simulans and those of T. squamosum.The endoperidium in both T. squamosum and T. simulans is light ochraceous or light brown and both have a characteristic stem.The stem is covered with detached squamules in the two species; T. simulans differs by a light brown stem while it is brown reddish in T. squamosum (Fig. 5).
Another problem arose during the ongoing process of identification of Tulostoma sp.Fruitbodies initially determined as Tulostoma brumale var.brumale in the field are too large and too massive which is rarely observed in this species.The endoperidium ochraceous-coloured at base and a pale stem with distinct pale squamules were not consistent with the diagnosis of the species.SEM revealed high similarity to spores of Tulostoma giovanellae.The ornamentation appeared as tiny verrucae, fusing and forming fine chains especially conspicuous towards the apex (Fig. 6A, B).Spores ornamentation appears as coalesced crests forming a distinct net over their entire surface in T. cyclophorum under SEM.Ornamentation appears as dense, uncoalesced verrucae in T. fimbriatum under SEM.* based on: Pouzar (1958); Wright (1987); Moreno (1990); Jeppson (2005); Jordal and Johansen (2009) This feature is not observed in spores in T. brumale var.brumale, but, as literature data show (Wright 1987;Altés et al. 1999), it is characteristic of T. giovanellae spores.These authors also report that capillitial threads are an important diagnostic trait that makes misdetermination of the species impossible.The walls of capillitial threads are conspicuously thick and they are distinctly irregularly undulate.This feature was observed in our studies; however, only individual threads were undulate while the majority were straight and thin-walled, like most species discussed here (Fig. 7).It is uncertain whether this variability of capillitial threads is probable for the species and further studies are required.
Although the comparative material of Tulostoma brumale and T. fimbriatum available for our study was quite extensive, intraspecific taxonomic identification of these taxa was very difficult due to high differentiation of episporium ornamentation, spore shape and size, and the morphology of capillitial threads.Co-occurring spores collected in a single sample of the spore mass of T. brumale differed by size, shape and ornamentation (Fig. 8).Great differentiation was also observed in capillitial threads.Individual hyaline threads were distinguished not only by thickness but also by transverse septa (Fig. 9).The species also varied considerably by external features of the fruitbodies.Some fruitbodies differed by the shape, size and colour of the endoperidium and the stem, peristome morphology, and the persistence and pigmentation of the exoperidium.
Two varieties of Tulostoma brumale were distinguished based on the variability and differentiation of the fruitbodies.In his world monograph of the genus Tulos toma, Wright (1987) lists a typical variety Tulostoma brumale var.brumale and two other varieties: Tulostoma brumale var.pallidum and Tulostoma brumale var.longipes exhibiting a long stem.
Three varieties of the species were distinguished in our material.Fruitbodies lacking dark pigmentation around the peristome mouth, characteristic of the typical variety T. brumale var.brumale, and having a lightly pigmented endoperidium and stem were recognized as T. brumale var.pallidum.Spores of this variety appear under SEM as rather thick verrucae and the external surface of capillitial threads is not covered with crystals which are present in T. brumale var.brumale.Fruitbodies having a long stem growing over 45 mm with spore ornamentation visible as low and fine verrucae were identified as the variety T. brumale var.longipes.Morphological features observed for lower taxa of the species agreed with the descriptions of the varieties of T. brumale var.brumale reported by Wright (1987).
Intraspecific differentiation of Tulostoma fimbriatum was approached in a similar way.The typical variety, T. fimbriatum var.fimbriatum, as well as a new variety, previously not reported in the Polish mycological literature and described by Moreno (1980), T. fimbriatum var.campestre, were observed in our studies.They mostly differed by the size and stoutness of the fruitbodies, the morphology, shape and colour of the stem, and, less importantly, the size and colour of the peridium.Fruitbodies of T. fimbriatum var.fimbriatum were conspicuously smaller and more delicate, which is rarely observed in T. fimbriatum var.campestre.The stem in T. fimbriatum var.fim briatum is slender and can reach a maximum length of 50 mm, with the most usual colour dark brown, while the stem length can reach even 100 mm in T. fimbriatum var.campestre.It also forms a bulbous ending at base.T. fimbriatum var.campestre also differs from the typical variety by being slightly light brown.

FINAL REMARKS
Two species, Tulostoma brumale and T. fimbriatum, are especially interesting.Although they have been reported from relatively many sites in Poland and the comparative material is rich, their taxonomic interpretation is not clear.Further investigations into intraspecific differentiation of these fungi would help overcome problems arising during the determination of these taxa.Only the competent identification of their features at the level of macro-and micromorphological decreases the doubts of proper taxa identification, and makes it possible to correctly identify the species at the levels of intra-and interspecific.This in turn, gives the possibility to present the list of taxa in the complete taxonomic aspect, and results in giving signal to the presence of new species and lower taxa, which are extremely rare and valuable fungi of Polish mycobiota.

Table 1 A
list of taxa and their new localities

Table 2
Selected species of the genus Tulostoma and other species recorded in Europe Peristome elongated and tubular in T. niveum, without a dark brown zone which is often present in T. brumale var.brumale.