New records of Lobaria amplissima ( Lobariaceae , Ascomycota ) in Poland

The current knowledge on the occurrence of Lobaria amplissima, a very rare old-growth forest lichen in Poland, is discussed. Both previous and new localities are presented. The Białowieża Forest, from which L. amplissima is reported, is a refuge site of L. amplissima in the lowland forests of Central Europe. The most important data on the ecology and the general distribution of the species are given. Diagnostic characters related to the morphology, anatomy and chemistry of L. amplissima differentiating it from similar species are described.

Lobaria virens was erroneously reported from Poland.It was noted only at one locality in a well-preserved forest community (Cieśliński, Tobolewski 1988).This species was included into the Polish red-list in CR category (Cieśliński et al. 2006), and into the group of IND-lichens (Czyżewska, Cieśliński 2003).
The current list of Polish Lobaria species still consists of three taxa but L. virens was replaced by L. amplissima (Scop.)Forssell after a revision of the herbarium material deposited in BSG by M. Kukwa (Kukwa et al. 2008).The specimen collected in the Białowieża Forest by S. Cieśliński and Z. Tobolewski and published as Lobaria laetevirens (Lightf.)Zahlbr.= L. virens (With.)J.R. Laundon (Cieśliński, Tobolew-Tobolewski 1988;Cieśliński 2003) in fact belongs to L. amplissima.The new determination was based on the morphology and ascospore features.Further localities of Lobaria amplissima were recently discovered by A. Bohdan during field investigations on rare macrolichens in the Białowieża Forest.
This rare species has distinct, characteristic morphological and chemical features that help to distinguish it from other similar taxa.Despite its special ecological demands it is relatively poorly known in Poland (Nowak, Tobolewski 1975).The aim of the study was to present the species and to indicate the most important diagnostic characters.Habitat requirements of L. amplissima identified to date suggest that few further localities of the species are likely to occur in the least human-affected forest communities in Poland, especially in the north-eastern part of the country and probably in the Bieszczady Mts.The available scarce data on the occurrence of Lobaria amplissima in Poland are given in this study, including new records.
Several detailed morphological features described here (e.g., concerning rhizines) based on new specimens and older specimens stored in KRAM were not published before.

MATERIAL AND METHODS
The material was collected in 2010 and 2011.Specimens were identified with standard morphological and anatomical methods as well as using spot colour reaction tests and TLC to differentiate secondary metabolites (Smith et al. 2009;Orange et al. 2001).The square number of the modified ATPOL grid (Cieśliński, Fałtynowicz 1993) is given for the localities.The nomenclature of lichens follows Fałtynowicz (2003) and Diederich et al. (2009).The material was deposited in the herbarium of the Society "Workshop for All Beings", Podlaskie Branch in Białystok, and in the herbarium of the Institute of Biology, University of Białystok.
Morphology and anatomy.Thallus foliose, leathery, thick (up to 490-645 μm acc. to Tønsberg and Jørgensen 2007), often exceeding 30 cm in diam., forming rosettes or irregularly branched, wavy, discrete at apices, contiguous and overlapping towards centre.The size of the Polish material is usually smaller than that given in the literature (Rose and Purvis 2009).Thalli were mostly irregular, rarely rosette-forming 1-5(10-30) cm in diam.Lobes 5-10(15-20) mm wide, with rounded or truncated apices, minutely indented or sinuate-indented; some margins slightly downturned.Upper surface matt, grey-white or silvery-white when dry (yellow-brown in old herbarium materials), greenish-grey when wet, smooth or slightly to very strongly wrinkled, especially in the centre of thallus; margins of young lobes having fine crystalline frosting when dry. Lower surface pale brown, paler towards margin and dull brown in the centre; tomentose without naked spots; "hairs" whitish or brownish 0.15-0.2mm long; margins of downturned lobes with white rim 0.5-1.0mm wide without tomentum, sinuate margins sometimes with tomentum up to the edge ("hairs" are visible from the upper side of the thallus); in the centre of thallus scattered groups of rhizines, up to 2(4) mm long, 0.10-0.15mm diam., rhizines fasciculate, partly glued at base, ends untangled, the base of thicker rhizines with whitish, short perpendicular outgrows, bottle brush-like.Photobiont in thallus chlorococcoid 7-14 μm diam.and cyanobacteria (Nostoc) in frequently occurring cephalodia.Cephalodia internal and often also superficial, globose on the lower side or shrub-like, densely branched on the upper side, blue-green-brown or brownish black, up to 2 cm in diam.Secondary foliose thalli sometimes attached to branches of cephalodia (Tønsberg, Jørgensen 2007).Fruticose cephalodia sometimes occurring independently as a blue-green morphotype (Stenroos et al. 2003) earlier known as Dendriscocaulon umhausense (Auersw.)Degel.External cephalodia occasionally absent, as in the material from Poland (Figs 1, 2).Soralia and isidia absent.Apothecia occasional, usually not present on the thallus of specimens with fruticose cephalodia (Tønsberg, Jørgensen 2007), lecanorine, (0.2)0.5-0.7 mm diam., constricted at base, disc red-brown, shiny, concave; margin rather thick, entire or slightly crenulated; very young apothecia sometimes "erumpent" from the layers lying beneath, with cracked rim of upper cortex tissues.Ascospores 36-65 x 3.5-6(7) μm, 1-3 septate, fusiform -straight or slightly sigmoid, colourless at first, becoming pale yellowish-brown.Apothecia in the Polish specimens very rare, young or older and partly damaged.Pycnidia frequent, scattered, ca.150 μm diam., immersed in conical or rather crater-like warts 0.7-1.2mm diam., ostiolum black, concave; ostiolum often opened in the form of an elongated crack, not as a rounded hole; upper part of pycnidium wall two-layered as described by Tønsberg and Jørgensen (2007): inner part of greyish tissues K+ aeruginose and outer part brownish, K-.Conidia simple, bacilliform 4-5 x 1-1.5 μm.Chemistry.According to Ellix and Tønsberg (2006), the species produces depsides: m-scrobiculin (major), p-scrobiculin (submajor), an orselinate derivative -pseudocyphellarin A (minor) and an unknown scrobiculin derivative (trace).Tønsberg and Jørgensen (2007) additionally noted atranorin.Both form of scrobiculin were detected by TLC in all of Polish examined specimens (A.Zalewska 2011).Atranorin was observed only in thalli from two localities (see below).Other additional substances did not occur.Spot test reactions: upper cortex: K+ bright yellow, PD-, C-, KC-; medulla: K+ dull yellow, PD-, C-, KC+ deep pink or reddish pink (reaction clearly visible through the cortex, only from the lower side of the thallus).Ecology.The occurrence of Lobaria amplissima in Poland is confined to well-preserved forest communities.The species was recorded mostly in wet alder forests, rarely in wet mixed forests.It was found exclusively on the bark of fallen Fraxinus excelsior, recently broken and gradually dying.Thalli were observed on the trunk near the tree crown and on the thick branches.
According to literature sources, Lobaria amplissima prefers the bark of old deciduous trees in woodland tree stands.It was observed on the bark of Fraxinus, Ulmus, Acer pseudoplatanus and Quercus (Rose, Purvis 2009).The species is also known from the bark of Alnus (Suza 1933), Castanea, Fagus (Nimis 1993) and Larix (see the data on the exsiccates analysed).L. amplissima was rarely recorded on mossy acidic rocks (e.g., Tønsberg, Jørgensen 2007).Aptroot and Zielman (2004) described interesting records of the species growing on bare lava rocks.
Lobaria amplissima is a member of Lobarion communities (Barkman 1958).Its occurrence in woodlands is confined to the best preserved forest communities with the structure, microclimate and ecological processes of the highest natural quality.It has indicator properties and is used to identify forests of special value for protection purposes (Rose 1992;Arup 1997;Nitaré 2000;Coppins, Coppins 2002;Czyżewska, Cieśliński 2003).
Remarks.Lobaria amplissima is a stenotopic lichen species and its survival in woodlands depends on the continuity of specific ecological factors conditioned by old-growth forests, especially the presence of old (mossy) trunks, high air humidity and, to some extent, the access of light (Nimis 1993;Rose, Purvis 2009).As the number of its localities has been decreasing mostly due to air pollution and forest management, L. amplissima is considered to be vulnerable in many European countries except Great Britain and Norway.It is mostly classified as critically endangered or endangered e.g., in Germany (Wirth et al. 1996), Austria (Türk, Haffelner 1999), Switzerland (Scheiddeger et al. 2002) and the Czech Republic (Liška et al. 2008).
Many localities previously known from European lowlands may be vanishing, as in Latvia (Anonymus 1996) and Denmark (Søchting, Alstrup 2002), where L. amplissima was classified as an extinct species.It seems that the Białowieża Forest in Poland is the last refuge of this species in the lowlands of Central Europe.

DISCUSSION
Lobaria amplissima is a fairly characteristic species although some forms can be confused with other members of the genus Lobaria, especially when the thallus is wet (and greenish in colour) and black-brown fruticose cephalodia are lacking.The species is distinguished by a matt, grey-whitish upper side of the thallus (when dry), sinuate-indented small lobes, the upper cortex reacting with K+ bright yellow and medulla spot colour reaction tests P-, KC+ deep pink or reddish pink.It produces m-scrobiculin and p-scrobiculin and sometimes pseudocyphellarin and/or atranorin as the accessory substances.
Among the species which can grow in similar habitats, Lobaria virens bears the greatest resemblance to L. amplissima.The former differs from Lobaria amplissima by having a thinner thallus (up to 300 μm acc. to Tønsberg and Jørgensen 2007) with a green to pale greyish-brown upper side when dry, often shiny, smooth or rarely wrinkled (the thallus is green when moist, as in L. amplissima).Apothecia of L. virens are always present and are bigger (to 11 mm in diam.) with a brown (Tønsberg, Jørgensen 2007) or rose-to orange-red disc (Wirth 1995).Its ascospores are shorter and wider, 32-47 x 6-8(10) μm.The morphology of pycnidia and the size of conidia are similar in both species but the shape of conidial cells is different.Conidia of L. virens are widening at the ends in contrast to bacilliform conidia of L. amplissima.L. virens has only internal cephalodia with cyanobacteria.The photobiont in the thallus is a green algae.This species does not produce secondary substances.L. virens is a very rare lichen, endangered in Central Europe.It does not occur in Poland but it may be possible to find its sites in well-preserved forests.
Lobarina scrobiculata and Lobaria pulmonaria are distinguished from Lobaria amplissima by the thallus with depressions and ridges, broader lobes up to 4.5-5 cm, and by vegetative, symbiotic propagules (isidioid consoredia and typical isidia and soredia, respectively) lacking in L amplissima.
Lobarina scrobiculata also varies in having only one type of photobiont: cyanobacteria (Nostoc).Due to the presence of cyanobacteria the wet thallus of this species is dark grey-blue (yellowish-grey to blue-grey with a brownish margin when dry).The thallus of Lobaria pulmonaria contains other green algae: Dictyochloropisis reticulata with big cells (up 20 μm in diam.) and cyanobacteria inside the internal cephalodia.Its thallus is vivid green when wet and yellowish grey or brown when dry.
The two species have also different chemical properties.Similarly to Lobaria amplissima, also Lobarina scrobiculata contains m-scrobiculin as a major compound, p-scrobiculin as a submajor substance and additionally usnic acid (minor) and the stictic acid complex, including norstictic acid (I chemotype) or usnic acid without the stictic complex (II chemotype).Its medulla reacts C-and KC+ deep orange-pink as in L. amplissima, but unlikely to this species, P+ orange and K+ yellow (acc.to Rose and Purvis (2009).Tønsberg and Jørgensen (2007) give other data on colours for Lobarina scrobiculata -I chemotype: C-, KC-, P+ orange, K+ yellow to orange, II chemotype: C-, KC-, P-, K-.Lobaria pulmonaria produces stictic acid as a major substance and constictic acid (submajor or minor), as well as other acids of the stictic complex in minor quantities and salazinic acid (minor).The spot colour reaction test for the medulla according to Rose and Purvis (2009) is: C-, KC+ yellow-orange, P+ orange, K+ yellow-orange, and according to Tønsberg and Jørgensen (2007): C-, KC-, P+ orange, K+ yellow to orange.
Flavoparmelia caperata (L.) Hale can also be similar to Lobaria amplissima.Both species have foliose, rosette-forming wrinkled thalli with a greenish tinge when moist, but when dry, F. caperata has a brighter yellow-green thallus, and moreover it produces soralia, lacks cephalodia and the tomentum on the lower side which is much darker (blackish).Its medulla reacts C-, KC+ red, P+ orange-red, and contains protocetraric, caperatic and usnic acids (Wirth 1995).This species grows mainly in deciduous, especially oak-hornbeam forests.It prefers open stands, forest margins and free-standing trees of oak, ash, hornbeam and linden (Wirth 1995).Lobaria amplissima and Flavoparmelia caperata can grow together at more open sites in regions with high precipitation and high air humidity (e.g., oceanic sites in West Europe and in mountains).Indeed, the two lichens were observed at the same localities inside natural wet alder forests in the Białowieża Forest.Lobaria amplissima was recorded there exclusively on the bark of Fraxinus excelsior, while Flavoparmelia caperata also occurred on the trunks of Alnus glutinosa (Cieśliński 2003).
Lobaria quercizans Michx., known mostly from North America, is externally very similar to L. amplissima.Hale (1957) stressed their resemblance and different distribution.L. quercizans has not been recorded in Europe but L. amplissima has only recently been discovered in Alaska and Carolina (Tønsberg and Goward 2001).It is difficult to assess whether L. quercizans is likely to be found in Europe.It favours old-growth forests with a mixed vertical structure.This species has been observed mainly on the trunks of old Betula alleghaniensis, Thuja occidentalis and Acer saccharum, rarely on Fraxinus spp.and on rocks, both with and without moss (Wetmore 2002).Its thallus is brown to grey in colour, smooth, without reticulate ridges.It lacks cephalodia, soredia and isidia, and contains green algae.Brown apothecia are common (Lobaria quercizans.2011.Minnesota Department of Natural Resources).The medulla is pink when tested with C due to the presence of gyrophoric acid (Wetmore 1981).
Based on the current knowledge on the identification of L. amplissima as well as characteristic habitats preferred by this lichen, rather few further localities of the species are likely to be discovered in Poland, and this only in the least human-affected forest communities.Detailed observations of old tree stands within large forest complexes in NE Poland and probably in the Bieszczady Mts should be conducted.It may be possible to find Lobaria amplissima on trunks (especially at the base of the crown) of living, standing or fallen old ashes, oaks or beeches and sycamores in the mountain forests.