The revision of specimens of the Cladonia pyxidata-chlorophaea group ( lichenized Ascomycota ) from northeastern Poland deposited in the herbarium collections of University in Bialystok

In northeastern Poland, the chemical variation of the Cladonia chlorophaea-pyxidata group was much neglected, as TLC has not been used in delimitation of species differing in the chemistry. As a great part of herbal material of University in Bialystok from NE Poland was misidentified, I found my studies to be necessary. Based on the collection of 123 specimens deposited in Herbarium of University in Bialystok, nine species of the C. pyxidata-chlorophaea group are reported from NE Poland. The morphology, secondary chemistry, and ecology of examined lichens are presented and the list of localities is provided. The results revealed that C. fimbriata is the most common species in the northeastern Poland, comprising around 33% of the studied specimens. Cladonia conista, C. cryptochlorophaea, and C. merochlorophaea are known only from very few locations. This study shed light on the role of the lichens substances to diagnosis of the species of C. pyxidatachlorophaea group.


Introduction
Lichens of the Cladonia pyxidata-chlorophaea group are characterized by primary thallus squamulose and scyphose podetia.The podetia are covered with farinose to granular soredia, corticated granules, and/or more or less areolate cortex.Apothecia are brown and rare [1,2].
The species of this group have a diverse secondary chemistry.Fourteen chemotypes and 35 lichen secondary metabolites have been identified within the group from the world [3].However, the species status of chemically different entities has been frequently questioned.
Some authors recognize chemical variability only at the chemotypes of species, varieties and subspecies level [4][5][6], others support chemically defined taxa at the species level [2,[7][8][9].Many authors accept the later viewpoint about chemically species [2,[9][10][11][12].The C. pyxidata-chlorophaea group requires molecular investigation with a larger number of samples and more variable gene regions [10,11].The recent molecular studies have indicated that at least all the chemically different taxa of the C. chlorophaea group do not form a separate subclade and their morphological similarity is rather a result of convergent evolution [10].
The aim of the present study is the revision of specimens of the C. pyxidata-chlorophaea group (lichenized Ascomycota) from the area of NE Poland deposited in Herbarium of University in Bialystok.For each specimen examined, the occurrence of the lichens of the C. pyxidata-chlorophaea group from tested area along with their morphology, chemistry, ecology, and distribution of each species is described.The chemical variation of the C. pyxidata-chlorophaea group from NE Poland was not sufficiently recognized because thin layer chromatography (TLC) has not been used during species identification.The area of study includes protected areas (e.g., Wigry National Park, Biebrza National Park, Narew National Park, Podlaski Przełom Bugu Landscape Park, Puszcza Knyszyńska Landscape Park) and areas not protected in the vicinity of the villages or small towns (e.g., Suchowola, Kaniuki, Ciechanowiec, Boćki) and surrounding the Siemianówka Reservoir in the Upper Narew Valley and forests (e.g., Romincka Forest).

Results
Nine species of the C. pyxidata-chlorophaea group were found in the examined material.Cladonia fimbriata appeared to be the most common species of the investigated lichen group in NE Poland.Three taxa, C. conista, C. cryptochlorophaea, C. merochlorophaea are known only from very few locations.
Cladonia chlorophaea (Flörke ex Sommerf.)Spreng., Syst.Veg.4: 273(1827) A characteristic species with regular podetia covered soredia.Podetia are up to ca. 3.0 cm tall.Scyphi are up to 7 mm wide, with irregular or dentate margins, usually gradually expanded.Scyphi and surface of podetia covered by granular soredia and corticated granules.Apothecia are brown and stalked, on cup margin.Pycnidia occur on scyphal margins.For a detailed description see [5,[14][15][16].The species is characterized by the production of fumarprotocetraric acid complex only.The specimens of C. chlorophaea are often morphologically similar to C. grayi and C. merochlorophaea.
The species is common and inhabits a many substrata, namely soil (12 specimens), bark (six specimens) and wood (four specimens).Corticolous specimens were collected from Betula spp.(two specimens), Pinus sylvestris (one specimen), Salix spp.(one specimen), Fraxinus excelsior (one specimen), and Robinia pseudoacacia (one specimen).Robinia pseudoacacia is a new phorophyte of C. chlorophaea in NE Poland [2].A similar habitat requirement was also discovered from Poland by Kowalewska et al. [2] and Belarus by Tsurykau and Golubkov [9].Location of investigation sites of C. chlorophaea in NE Poland from the herbarium collections of University of Bialystok has been presented in Fig. 1.Cladonia conista is distinguished by the podetia tall, up to ca. 2.5 cm high, with regular, ± goblet-shaped cups.The upper part of the stalk and the cups with soredia.The lower part of the stalk is corticated.Soredia are farinose rather than granular.The detailed description of the species is presented elsewhere [2,7].Secondary metabolites of C. conista include bourgeanic and fumarprotocetraric acids.Cladonia conista is morphologically very similar to C. humilis.In contrast to this species, C. conista produces bourgeanic acid (K−).Cladonia humilis produces fumarprotocetraric acid and atranorin (K+ yellow).Pino-Bodas et al. [12] have stated that the species are morphologically indistinguishable, but the taxa differ in their geographical distribution and C. conista and C. humilis are recognized as distinct species.
The species was found on soil in pine woodland (one specimen).The observed habitat requirements agreed with those reported for the species by Kowalewska et al. [2].All the Polish specimens were found on soil.Distribution of stands of C. conista in NE Poland from the herbarium collections of University of Bialystok has been presented in Fig. 2.

Specimens
Substrate of the specimen includes soil.The species prefers pine forests.Almost three-quarters of the records of C. cryptochlorophaea in Poland [2] are represented by epigeic specimens.The similar ecology notes are given by Holien and Tønsberg [7], Ahti [15].Distribution of investigation site of this species in NE Poland from the herbarium collections of University of Bialystok has been presented in Fig. 3.
In Poland, it is rare species [2], but its known localities are scattered, mostly in northeastern and southern part of Poland [2,19].The species is known from all continents, except Antarctica.Many specimens have been recorded mostly in boreal zone [9].This species is distinguished by the podetia up to 3.0 cm tall, simple, with scyphi.Scyphi are up to 6 mm wide, regular.Podetia with goblet-shaped.The surface of podetia and scyphi coated with soredia.Soredia are farinose.Apothecia are rare, brown and simple.For the description of the species see [2,5,[14][15][16].The species produces fumarprotocetraric acid complex only.Cladonia fimbriata may resemble C. chlorophaea.It has more regular and extended scyphi, granular soredia and smaller stalks.Cladonia fimbriata is most similar to C. conista, but C. conista differs by its usually distinctly corticate podetial stalk [2,20].

Specimens
The species is ubiquitous and inhabits a wide range of substrata.In NE Poland, C. fimbriata prefers well-lit open pine, oak and birch forests and urban areas.The species was found on soil (30 specimens), wood (eight specimens), Betula spp.(one specimen), Pinus sylvestris (one specimen), rock (one specimens).Location of investigation sites of C. fimbriata in NE Poland has been presented in Fig. 4. Cladonia fimbriata is common species in Poland [2].Many localities of this species were reported in NE Poland by Kowalewska et al. [2].Outside Poland, the species has been recorded from many regions.It is known from all continents except for tropic regions [8,18].The squamulose of primary thallus are shrub-like.The podetia are up to 4.0 cm tall.In the upper part, the podetia are covered by granular soredia, with goblet or trumpetshaped scyphi up to ca. 1 cm wide, often with proliferations.Apothecia are brown to dark brown and stalked.For a detailed description see [2,5,7].One chemotypes is recognized in NE Poland: grayanic acid always and additionally substances of the fumarprotocetraric acid complex (chemotype II).In Poland, Kowalewska et al.
Cladonia grayi is mostly found in pine forests.The species inhabits soil (20 specimens) and wood (six specimens).Distribution of stands of C. grayi in NE Poland has been presented in Fig. 5.
In Poland, C. grayi is common species [2].Many localities of this species were reported in NE Poland by Kowalewska et al. [2].World distribution data of C. grayi are most widely, it has been reported from many continents: Europe, Asia, North America, Central America, South America, Australia, and New Zealand [2,8].Squamules of primary thallus are greenish-grey, large and thick.Podetia are up to 1.5 cm tall, with regular cups.Scyphi are gradually flaring, inside covered with bullate corticate plates inside.Apothecia are brown, simple or occur in glomerulose accumulations.For detailed descriptions see Tsurykau and Golubkov [9], Aptroot et al. [21], and Kowalewska and Kukwa [22].The species is characterized by the production only fumarprotocetraric acid complex.Cladonia monomorpha is similar to C. pyxidata and C. pocillum.All those taxa are esorediate and contain fumarprotocetraric acid as the mayor secondary metabolite, while they differ morphologically.For a detailed description see Kowalewska et al. [2] and Aproot et al. [21].

Specimens
In NE Poland, the species was found on soil (one specimen).In Poland, the frequency on different substrata is as follows: soil, rocks, and tree bark [2].This agrees with the habitat spectrum published by Aproot et al. [21].Location of investigation site of C. monomorpha from the herbarium collections of University of Bialystok has been presented in Fig. 7.
Most of the records come from the northern and southern part of the country [2].Several localities of C. monomorpha were reported in NE Poland by Kowalewska et al. [2].The species is known from Europe, Asia, and North America [2,21].Squamules are brown-grey.Podetia are brownish or blackish, up to 1.4 cm tall.Scyphi are up to 0.8 cm wide, simple to proliferating from margins.Surface of podetia are corticate, verruculose, squamulose, inner part of the scyphi eroded, covered with cortical plates.Apothecia are stalked, brown.For detailed descriptions see Brodo and Ahti [5], Tsurykau and Golubkov [9], and Ahti [14].The species is characterized by the production homosekikaic, sekikaic, and fumarprotocetraric acids.Cladonia novochlorophaea is morphologically similar to C. merochlorophaea, but it is never clearly sorediate.It has a similar chemistry to C. homosekikaica.
In NE Poland, C. novochlorophaea has been found on soil (six specimens), wood (three specimens) and bark of Picea abies (one specimen) in open habitats and forests.Distribution of stands of this species in NE Poland has been presented in Fig. 8.
It is known from northern and central part of the Poland.It is rare species.Three localities of C. novochlorophaea were reported in NE Poland by Kowalewska et al. [2].The species has been reported from Europe, North and South America, New Zealand, and the Antarctic region [8,23].In NE Poland, the species was recorded on soil, mainly in well-lit pine forests (12 specimens).One specimen was collected from wood.Location of stands of C. pyxidata in NE Poland from the herbarium collections of University of Bialystok has been presented in Fig. 9.

Specimens
This species is rare in north part of Poland, but it is common in southern Poland [2].The species is known worldwide [8,18].It is common in the arctic and temperate zones [15].

Discussion and conclusion
The taxa from C. pyxidata-chlorophaea group are probably the earliest designated group of chemical taxa [9].The importance of secondary lichen metabolites to the taxonomy and species discrimination within the C. pyxidata-chlorophaea group has been confirmed based upon simultaneous analyses of DNA sequences and morphological and chemical data by Stenroos et al. [10].
In NE Poland, the chemical variation of the C. pyxidata-chlorophaea group was much neglected, as TLC has not been used in delimitation of species differing in the chemistry.As a great part of herbarium collections of University of Bialystok from NE Poland was misidentified, I found my research highly needed.Except for the samples of C. fimbriata, many studied specimens were misidentified.
So far, the lichens from the group C. pyxidata-chlorophaea and allied species has been reported from different regions of Poland mostly based on the identification using methods of classical taxonomy [19].The information about the species recognizable only by secondary substances is rather sparse.In northeastern part of Poland, only a few studies have used these laboratory techniques for the identification of C. pyxidata-chlorophaea group so far [2].Altogether, nine species of the C. pyxidatachlorophaea group have been recognized in the examined materials.Three taxa (C.asahinae, C. humulis, and C. pocillum) have not been reported up to now from NE Poland [2].Cladonia asahinae was found in the mountains.Cladonia humilis was known from a few localities along the Baltic coast, and C. pocillum from southern Poland [2].
Cladonia fimbriata is the most common species in Poland [2] and NE Poland.For comparison, C. grayi is the commonest species in Belarus (ca.40% of the studied specimens) and C. pyxidata is uncommon in Belarus, known only from 10 localities [9].In Poland, C. homosekikaica Nuno has not reported from C. pyxidata-chlorophaea group.This species was reported in the neighboring countries of Poland, in Belarus [9] and Lithuania [24].
The lichens from the group C. pyxidata-chlorophaea were found in NE Poland on soil, in open and sun-exposed sites, and in pine forest.Three species (C.chlorophaea, C. fimbriata, C. novochlorophaea) have been reported on bark of trees.Cladonia chlorophaea, C. fimbriata, C. grayi, and C. novochlorophaea inhabit wood.Only one specimens of C. fimbriata has been reported on rock.Similar habitat requirements for many species were reported by Kowalewska et al. [2].
The paper presents a number of new localities of species of C. pyxidata-chlorophaea group for NE Poland [2].Additionally, a bark of Robinia pseudoacacia as a new substrate to C. chlorophaea has been found.
The use of TLC method for the identification of lichens collected in this region of Poland showed several specimens of rare species: C. conista, C. cryptochlorophaea, C. monomorpha, and C. novochlorophaea.
The studies have shown that species C. pyxidata-chlorophaea group differ in their chemical characteristics.In four species of this group, the only chemical component is fumarprotocetraric acid complex (C.chlorophaea, C. fimbriata, C. monomorpha, C. pyxidata).In other species occur fumarprotocetraric acid and other acids.Bourgeanic acid is detected in one species (C.conista).Gyrophoric acid has been found only in C. grayi.Cryptochlorophaeic and paludosic acids have been detected in C. cryptochlorophaea.In C. merochlorophaea, substances detected by TLC include merochlorophaeic and 4-O-methylcryptochlorophaeic acids.Homosekikaic and sekikaic acids have been detected in C. merochlorophaea.Similar chemical content for many species were reported by Kowalewska et al. [2].Presented study confirmed utility of chemical methods in the diagnosis of the species of C. pyxidata-chlorophaea group.