Cladoniaceae of Montenegro

As a result of collections made in 61 locations during the year 2018, the number of Cladonia taxa is enlarged to 42, and ten new records of Cladonia are provided to Montenegro, Cladonia cariosa, C. coccifera, C. cyathomorpha, C. digitata, C. diversa, C. homosekikaica, C. imbricarica, C. merochlorophaea, C. novochlorophaea and C. subulata. The distribution of many taxa previously known for the territory is extended. The chemical variation and the distribution of each species are discussed.


Introduction
The lichenological exploration of Montenegro started with Biasoletto (1841), being Cladonia fimbriata one of the first reports for the country, as  pointed out. Several generalistic lichenological studies were carried out in Montenegro up to date. Out of the 850 taxa of lichenized fungi published, 33 species belong to the genus Cladonia, but no monographic study of the genus has been carried out (Mayrhofer et al. 2017). However, some authors must be highlighted because they contributed with a greater number of species of the genus Cladonia, such as Kušan (1953) who publishes all the information known to date about the territories of the former Yugoslavia, Bilovitz et al. (2008), Strasser et al. (2015).
Although the genus Cladonia has been well studied in many regions of Europe, especially in some bordering countries such as Albania, which has 27 species (Burgaz et al. 2019), Bosnia-Her-zegovina with 25 species, or Croatia with 45 species (Burgaz & Pino-Bodas 2012;Burgaz et al. 2017), others are still poorly explored, especially the countries of southeastern Europe.
The aim of this contribution is to summarize the results of floristic survey from Montenegro in the framework of a project on the family Cladoniaceae in the Mediterranean area of Eurasia.

Description of studied area
Montenegro is a small mountainous Mediterranean country limited by the Adriatic Sea and sourrounded by Croatia, Bosnia-Herzegovina, Serbia and Kosovo (from the old Yugoslavian territories), and Albania. It is located in the SE of Europe, where the Dinaric Alps run parallel to the sea, with many peaks above 2000 m high. The Durmitor massif, as part of the Dinaric Alps, are located in the NW, with the highest altitude at Bobotov peak (2522 m), while the Bjelasica mountains range in the NE, reaching 2139 m at Crna Glava peak. The dominant substrate is constituted by limestones and dolomites, and more than two-thirds of the country is karstic, intersected by river canyons and gorge stretches (Radulovic & Radulovic 1997), although there are some locations with siliceous rocks.
In general, the country has a Mediterranean climate, but with many variations related to the altitudinal elevation and exposures. There are three main climatic zones: Mediterranean, temperate continental and mountains (Horvat et al. 1974), with very variable annual precipitations. This climatic variation places Montenegro among the biological hotspots of European and world biodiversity (NBSAP 2010). The Mediterranean vegetation consisting of maquis and garrigue is located at lower terrains, while the mixed deciduous woodland, beech forests, mixed beech-fir and spruce forests are situated in localities higher than 1000 m altitude. Above the timberline, subalpine grassland covers large areas.
The result of collections made in 61 locations during the summer of 2018 and the analysis of 352 specimens can be found in Annex 1. The specimens were deposited in Madrid (MACB) and Helsinki (H). Numerous specimens of each species were analysed by means of thin-layer chromatography (TLC) to identify the major lichen substances (Orange et al. 2001). The main secondary metabolites identified from Cladonia are listed in Table  1. Distribution maps are presented according to the administrative divisions of Montenegro ( Figure 1). Figure 2 shows the most probable altitude where the species can be found as the average with the standard deviation values and the extreme values are the maximum and minimum where the species was found. The species morphologically variable in southern Europe and those in which we have found chemical variability are discussed in greater depth.

Results and discussion
Cladonia is the only genus of Cladoniaceae represented in Montenegro. The species studied occur between sea level and 2400 m of altitude, taking into account both our data and other bibliographic references published by different authors ( Figure 2). The Montenegro forests located at the main mountainous areas of the center and north of the country contain the greatest diversity of species between 1000 and 2000 m altitude. The fir and spruce forest, present in altitudes above 1500 m, harbours the largest number of species. Altitudes above 2000 m, where Pinus mugo prevails accompanied by Juniperus communis, are also important growth areas for the species studied. Beech and other deciduous forests host too a high number of species, although in a smaller quantity, decreasing with altitude. The areas with Mediterranean vegetation type maquis are very scarce, therefore the number of species found in these formations is low. Although basic substrates dominate in Montenegro, there are scattered locations of acidic rocks which allow the entrance of many acidophilic species.
The commonest species are: Cladonia cariosa, C. fimbriata, C. foliacea, C. furcata, C. pyxidata, C. rangiformis and C. symphycarpa. Most of the species (64%) have NW-NE distribution along the Dinaric Alps, which mostly enjoys a temperate continental climate (Vuksanović et al. 2016), being 43% of them located in the eastern subregions of Montenegro and only 7% growing in the northern subregions; while C. chlorophaea, C. coniocraea, C. conista, C. cyathomorpha, C. hosekikaica and C. rei also grow in the mountainuous locations of SW areas (Figure 3). Cladonia cervicornis is the only Mediterranean species s.str. in Montenegro following the geographical regionalization of   Seven species are chemically variable (Table  1), C. cariosa with 4 chemotypes, C. furcata with 3, C. macilenta with 2, C. macroceras with 2, C. rangiformis with 2, C. rei with 2 and C. symphycarpa with 5. A complex combination of chemotypes was found in the so-called C. cariosa group. Most of the specimens of C. cariosa have podetia but not C. symphycarpa, which difficults the adecuate morphological identification. Similar chemical variability was published of Poland (Osyczka & Skubala 2011) and Albania (Burgaz et al. 2019). Pino-Bodas et al. (2012a) confirmed that morphological or chemical characters were insufficient to delimit the species boundaries in the group and considered the existence of four phylogenetics lineages. At present, a molecular study on this group is underway including specimens from other European and American countries, but we do not yet have definitive results (Pino-Bodas et al. in prep). (Wallr.) Flot., in Wendt, Thermen Warmbrunn 94 (1893) A scattered terricolous species reported of Plav and Rožaje municipalities (Strasser et al. 2015) and Plužine municipality (Kušan 1953;) growing at 2065-2403 m altitude.  (1827) It is a common species found in Andrijevica, Berane, Cetinje, Herceg Novi, Nikšić, Plav, Plužine, Rožaje and Žabljak municipalities growing at 858-1947 m altitude. It was collected on bare soils of limestone and calcareous rock substrate on shrubs and pasture of several woodland formations, fir, spruce, coniferous, beech and mixed forests. Most of the specimens have the characteristic fissured podetia. The specimens analysed are chemically variable (Table 1), most of them belong to the chemotype 1, containing atranorin, norstictic and connorstictic acids. The chemotype 2, with atranorin and rangiformic acid, and some with additionally zeorin was also common; less common are the chemotypes with only atranorin or atranorin and fumarprotocetraric acid, in both cases also with zeorin. 1995-2065 m altitude on coniferous lignum and siliceous soils.

Cladonia crispata (Ach.) Flot., in Wendt, Thermen Warmbrunn: 93 (1839)
A very rare species only reported by Savić  (1796) This uncommon species was collected in Rožaje municipality growing on Picea abies stump in a Picea-Abies woodland. One speci-men analysed contains thamnolic acid and the other specimen fumarprotocetraric acid additionally.

21.
Cladonia gracilis (L.) Willd., Fl. Berol. Prodr.: 363 (1787) A rare species only reported of Mojkovac (Bilovitz et al. 2009), Plav (Strasser et al. 2015 and Rožaje municipalities (Bilovitz et al. 2008) on siliceous and calcareous soils. Nuno, J. Jap. Bot. 50: 294 (1975) This common species of the C. chlorophaea group, containing fumarprotocetraric and homosekikaic acids, was collected in Cetinje, Gusinje, Kolašin, Plav, Plužine, Rožaje and Žabljak municipalities. It grows on limestone and acid rock substrates, on pastures with deciduous and coniferous woodlands. Kristinsson, Lichenologist 6: 143 (1974) This is a rare species containing sphaerophorin and in one specimen additionally fumarprotocetraric acid. It was collected in Nikšić and Žabljak municipalities growing at 1140-1761 m altitude, on limestone substrate and shrublands with Ostrya carpinifolia, Pinus mugo, Juniperus communis and Pinus nigra. This species is common in high mountains of the Neotropic and was also recorded from northern Europe (Ahti & Stenroos 2013), it was recently reported from Slovakia at 616 m altitude (Palice et al. 2018) in the western Carpathians. This species is difficult to identify without chemical analysis and has probably been overlooked.  Nyl., Flora 58: 447 (1875) Only one specimen of this species was found in Plav municipality at 1532 m altitude, on Picea abies forest and acid rock substrate. It contains atranorin and fumarprotocetraric acid. Previously Strasser et al. (2015) reported this species from Plav at 2300 m altitude on siliceous soil. Asahina, J. Jap. Bot. 16: 713 (1940) This is a very rare species collected at 1532 m altitude in Plav municipality, on Picea abies forest and acid rock substrate. It contains fumarprotocetraric and merochlorophaeic acids. Sandst., Cladon. Exs. nº 55 (1918) It was only reported of Plav municipality at 2065 m altitude (Strasser et al. 2015).