Biodiversity of phototrophs in illuminated entrance zones of seven caves in Montenegro

The biodiversity of the entrance zones of the Montenegro caves is barely studied, therefore the purpose of this study was to assess the biodiversity of several caves in Montenegro. The samples of phototrophs were taken from various substrates of the entrance zone of 7 caves in July 2017. A total of 87 species of phototrophs were identified, including 64 species of algae and Cyanobacteria, and 21 species of Bryophyta. Comparison of biodiversity was carried out using Jacquard and Shorygin indices. The prevalence of cyanobacteria in the algal flora and the dominance of green algae were revealed. The composition of the phototrophic communities was influenced mainly by the morphology of the entrance zones, not by the spatial proximity of the studied caves. the phototrophic epibiotic communities of the cave entrance zones. taken substrates calcite, were bryophytes and ferns were and herbarized, algae and cyanobacteria (including samples from the gametogophytes of bryophytes) were collected into sterile vials. Phototrophs from the epibiotic communities were examined using a Leica light microscope (Germany). Specimens for examination were prepared by separating small fragments from the communities (biofilms) and placing them in water droplets. and abundance of species in the samples were determined, and the data were extrapolated to the entire epibiose area.


Introduction
The subterranean karst forms represent habitats that considered more climatically stable than the surface. Generally, cave climate is characterized by the low positive temperatures, high relative humidity and lack of lighting (Vanderwolf et al. 2013). Seasonal and daily fluctuations of these parameters can be observed at the entrance zones of the caves, yet they remain more stable than on the surface (Prous et al. 2015). In the deep zones of the caves there is no natural light and ecosystems are able to function due to the introduction of organic matter from the outside or the activity of chemolithoautotrophic communities (Engel et al. 2004;Pentecost & Zhaohui 2001). The entrance areas of caves, include habitats with low levels of photon fluxes, are characterized by a light gradient that allows phototrophs to develop.
Considered close to the communities of the entrance zone of the caves, the lampenflora communities represent photosynthetic species growing in the excursion caves near the lamps. There were not enough studies on the flora in Montenegro prior to 2018, when Mazina & Kozlova (2018) have researched lampenflora and phototrophic communities of entrance zone of Lipska Cave in the southern part of Montenegro.
The territory of Montenegro is of great interest for the study of hypogean karst formations and has both scientific and cultural significance (see Barović et al. 2018). Most of Montenegro is covered by mountain ranges, composed mainly of limestone, with well-developed surface and subterranean karst topographic forms. The largest number of biological studies is focused on the cave fauna of troglophiles and troglobionts (see Pešić et al. 2018 for an overview).
In this paper, we studied phototrophic communities of seven caves located in the southern part of Montenegro characterized by the different morphology of entrance zones. The objective of this study was to describe the biodiversity of phototrophic communities at the entrance zones of these caves and to examine the species composition of the selected caves using standard similarity indices.
Fieldwork for the study was carried out during July 2017. We studied the phototrophic epibiotic communities of the cave entrance zones. Samples were taken from each epibiose area: substrates (limestone, calcite, clay sediments) were sampled, bryophytes and ferns were collected and herbarized, algae and cyanobacteria (including samples from the gametogophytes of bryophytes) were collected into sterile vials. Phototrophs from the epibiotic communities were examined using a Leica DMLS light microscope (Germany). Specimens for examination were prepared by separating small fragments from the communities (biofilms) and placing them in water droplets. The occurrence and abundance of species in the samples were determined, and the data were extrapolated to the entire epibiose area.
Samples of soils and cave communities were incubated on selective culture media. Bristol and Gromov №6 media were used for algae and cyanobacteria. The exposure temperatures were 12 and 24°C and the illumination level was 30-40 μm × m -2 × s -1 . The methods of growth slides and incubation in a liquid medium were used. For a more complete identification of the phototroph species composition from the illuminated zones, representative samples from communities were incubated in Gromov liquid medium №6. Systematics of cyanobacteria and algae used in the paper correspond to the database available at http://www.algaebase.org (Guiry & Guiry 2019). Samples were identified using several field guides: bryophytes with Ignatov & Ignatova (2004), ferns with Mayevsky (2014), and lichens with Andreev (2008). The systematics of bryophytes is given according to Ignatov & Ignatova (2004), ferns according to Cherepanov's report (1995), the lichen according to the database available at mycobank.org. The Jacquard floristic similarity coefficient and the Shorygin community structure similarity index (Rozenderg et al. 2000) were used to analyze the similarity of community composition and structure. Cluster analysis was carried out and tree diagrams were constructed using the Euclidean distance in the STATISTICA 7.0 program. Relative abundance was used as a basis for the determination of the dominant species; dominants were also determined in each systematic group: bryophytes, algae, and cyanobacteria.
Dominant species of cyanobacteria were the same in the following caves: Golubinja and Vrbačka jama (Gl. compacta), Vrbačka jama and Jama ER-1 (P. foveolarum). The diatom S. bacillaris was dominant in the caves Jama ER-1 and Vrbačka jama. The chlorophyte Chl. vulgaris was dominant in the caves Njegoš Pećina, Veluštica Pećina, Golubinja Pećina and Obodska Pećina. Bryophyte dominants were different in each cave.
We assessed the similarity of the species composition and community structure of the studied caves using standard similarity indices. We evaluated biodiversity as a whole and diversity of algal flora (algae and cyanobacteria) separately. Analyses indicated that the similarity of the cave phototrophs is low (Fig. 2). On the other hand, the cave algal flora demonstrated greater similarity. Flora of the caves, which are similar in morphology of the entrance zone (e.g., Golubinja Pećina and Obodska Pećina having a large grotto-like entrances, and Vrbačka jama and Jama ER-1 having entrances in a form of vertical wells) showed the greatest similarity. We predicted that the caves located close to each other would have high Jaccard and Shorygin indices, but that assumption was not confirmed.  -10 -----

Discussion
In this study, 51 taxa of phototrophs were recorded from seven caves located in the karstic region of southern Montenegro is reported. In terms of taxonomic richness, the communities composition of studied caves was dominated by Cyanobacteria and algae (Bacillariophyta, Chlorophyta and Ochrophyta) and which is consistent with many studies. For example Mazina & Kozlova (2018) found that flora assemblage of Lipska cave include 29 phototrophic species of which 17 species belong to algae and Cyanobacteria, and 12 to Bryophyta. Diatom Humidophila contenta was the only species found in all the caves and had a high score of abundance (more than 7) in the caves-wells. Green algae Chlorella vulgaris and Stichococcus bacillaris were found in most caves. It is interesting to note that, as a rule, these species dominated in the studied communities (scores of relative abundance were 7-12). However, in the case when both species were simultaneously found in the cave, their relative abundance decreased to 3-5 points.
The cyanobacterium Aphanothece saxicola was found in all the caves except the wells; this species is usually found in the form of biofilm on limestone and calcite. The species of the genus Microcystis are found in all caves, except the Golubinja Pećina. It should be noted that this cave is rich in organic matters and protonema. A similar abundance of protonema is found in the Vrbačka jama. The genus Gleocapsa is represented in all caves.
Aerophilous diatom H. contenta (formerly Diadesmis contenta) was registered at the entrance areas of all the studied caves. Most members of the Humidophila genus are considered cosmopolitan due to their wide distribution (Poulíčková & Hašler 2007). H. contenta also had the highest occurrence in the caves of Poland (Czerwik-Marcinkowska et al. 2015); and was found in the caves of Central Moravia (Czech Republic) (Poulíčková & Hašler 2007), Canada (Lauriol et al. 2006) and in the Urals (Abdullin 2007).
The largest number of bryophyte species were registered at the entrance area of the Golubinja Pećina. This is presumably due to the specific morphological structure of the entrance and the presence of a large amount of organic matter (pigeon droppings). The species Amblystegium serpens and Campylidium calcareum have been documented by Mulec & Kubešová (2010) as part of the lampenflora in Slovenian caves. A. serpens has been also found in the lampenflora of the caves in Italy (Castello 2014) and the Czech Republic (Kubešová 2001). Ditrichum flexicaue and Fissidens taxifolius have been also recorded Kubešová (2001) as a part of the lampenflora. Conardia compacta and Plagiopus oederianus have been registered in the lampenflora of the show caves in Abkhazia and the Krasnodar Territory (Mazina 2016). A. serpens, Tortella tortuosa and a lichen of the genus Lepraria has been registered Pentecost & Zhaohui (2001) in the flora of cave in England (North Yorkshire).
The fern Asplenium trichomanes, which we registered in the Veluštica Pećina, is widely distributed and can be found in well-lit entrance areas, as well as in the lampenflora (Castello 2014).
The results of our study showed that the spatial proximity of the investigated caves is not an indicator of assemblage similarity since we did not find any evidence that the species of neighboring caves had coincided more than in the distant ones. Probably, the assemblage similarity caused by the morphology of the entrance zones of the studied caves.