Species composition and diversity of morphotypes in epilithic biotopes of Zhytomyr region, Ukraine

16 species of naked amoebae are recorded in the eplithic biotopes of Zhytomyr region, Ukraine: Saccamoeba sp. (1), Saccamoeba sp. (2), Saccamoeba sp. (3), Cashia limacoides Page, 1974, Thecamoeba striata (Penard, 1890) Schaeffer, 1926, Thecamoeba sp., Stenamoeba stenopodia (Page, 1969) Smirnov et al., 2007, Mayorella cantabrigiensis Page, 1983, Mayorella sp., Korotnevella sp., Vexillifera sp., Vannella sp., Ripella platypodia Smirnov, Nassonova, Chao et Cavalier-Smith, 2007, Acanthamoeba sp. (1), Cochliopodium sp., Vahlkampfia sp. (1). The frequency of occurrence in samples is highest for Thecamoeba sp., Mayorella sp., Vahlkampfia sp. (1), Korotnevella sp., Vannella sp., and is lowest for Saccamoeba sp. (1), C. limacoides, M. cantabrigiensis, Cochliopodium sp., S. stenopodia, R. platypodia, T. striata, Saccamoeba sp. (2). The species and morphotype diversity of naked amoebae of the epilithic mosses and lichens is influenced by the humidity and the height above soil of the substrate. The species and morphotype diversity of naked amoebae decreases with the increase in height above soil and the decrease of humidity of substrate. We recorded naked amoebae of 9 morphotypes: monotactic, dactylopodial, fan-shaped, lens-like, lingulate, mayorellian, striate, acanthopodial, and eruptive. Amoebae of the lens-like and fan-shaped morphotypes disappear with the increase of height of substrate above soil. Skład gatunkowy i zróżnicowanie morfotypów w biotopach epilitycznych regionu żytomirskiego na Ukrainie Słowa kluczowe ameby nagie, morfotypy, biotopy epiliptyczne, region Zhytomyr Streszczenie W eplitycznych biotopach rejonu Żytomierza na Ukrainie odnotowano 16 gatunków nagich ameb: Saccamoeba sp. (1), Saccamoeba sp. (2), Saccamoeba sp. (3), Cashia limacoides Page, 1974, Thecamoeba striata (Penard, 1890) Schaeffer, 1926, Thecamoeba sp., Stenamoeba stenopodia (Page, 1969) Smirnov et al., 2007, Mayorella cantabrigiensis Page, 1983, Mayorella sp., Korotnevella sp., Vexillifera sp., Vannella sp., Ripella platypodia Smirnov, Nassonova, Chao et Cavalier-Smith, 2007, Acanthamoeba sp. (1), Cochliopodium sp., Vahlkampfia sp. (1). Frekwencja w próbach była najwyższa dla Thecamoeba sp., Mayorella sp., Vahlkampfia sp. (1), Korotnevella sp., Vannella sp., a najniższa dla Saccamoeba sp. (1), C. limacoides, M. cantabrigiensis, Cochliopodium sp., S. stenopodia, R. platypodia, T. striata, Saccamoeba sp. (2). #0#


Introduction
The naked amoebae are widely distributed in soils, freshwater and saltwater biotopes. For most protists, humidity is the limiting factor in terrestrial biotopes (Bonnet, 1973). The majority of known species of protists are small, which allows them to live in thin films on substrates (Bonnet, 1973;Schonborn, 1989;Korganova, 2003).
There have been no previous reports of the amoebic fauna of epilithic mosses and lichens. For Ukraine, there are only reports of the species compositions of amoebae in the water bodies and the soils (Patcyuk, Dovgal, 2012;Patsyuk, 2014. Hence, any new data on species of these protists from various biotopes of Ukraine is valuable for science.

Materials and methods
Material was collected during two field trips in the forests of Zhytomyr region (near Zhytomyr city, Denyshi village, Novohrad-Volynsky district, and Olevsk district) in 2019. 162 samples were analyzed. The species composition of naked amoebae was studied in soils and in the mosses and lichens growing on the soils and the rocks near water bodies. Sphagnum spp. dominated in the moss cover, Hypogymnia spp. in that of lichens. The collected samples were stored in zip-lock bags and transferred to the laboratory. 5 g of substrate sample was placed in a flask of 150 ml with 100 ml of distilled water for 2 hours, after which the mixture was shaken. 10 ml of the mixture was evenly distributed in a Petri dish with non-nutrient agar. The amoebae were cultured after Page's method (Page, 1991).
The amoebae cultures were maintained at 20°С. Alive protist cells were observed in water droplets on slides, using Axio Imager MI light microscope with differential interference contrast.
Current methods do not allow to assess the abundance of amoebae, thus we've analyzed the frequency of occurrence of amoebae in the samples from the epilithic biotopes of Zhytomyr region (calculated as ratio of the samples containing amoebae to all samples). The substrate humidity was established by weight method.
The diversity of the amoeba species and morphotypes were analyzed using the Chekanovsky-Sørensen index, constructing the dendrogram with bootstrap support of nodes in PAST 1.18 (Hammer, 2001).

Results and discussion
In total, 16 species of the naked amoebae were found in the epilithic biotopes of Zhytomyr region (Tables 1, 2). The systematics of those protists is given below following the current systematics of eukaryotes (Adl et al., 2019 By the frequency of occurrence in the epilithic biotopes of Zhytomyr region, the most common species were Thecamoeba sp.  We also analyzed the distance of epilithic biotopes to soil (height) as a factor influencing the species diversity of the naked amoebae. With increasing height, the species richness parameters decreased for those protists. The highest number of species was recorded in substrates at the soil level (Table 2) (Table 1).  The diversity of amoebae did not change significantly with the distance between soil and the epilithic mosses and lichens increasing to 0.5 m (Tables 1, 2). At that height, we recorded Vahlkampfia  (Table 1).   3) were not numerous and preferred the epilithic mosses and lichens at the soil level. At the height of 0.5 m these species were not found.
The species diversity of amoebae from the epilithic biotopes of Zhytomyr region was compared to that in soil at the ground level and that of epigeic mosses in order to determine the biotope-specific features. The soil-living species included Vahlkampfia sp. (1), C. limacoides, T. striata, S. stenopodia, M. cantabrigiensis, Vannella sp., Acanthamoeba sp. (1), for all of them the frequency of occurrence did not exceed 38%. In the epigeic mosses, Vahlkampfia sp. (1) (15%), Saccamoeba sp. (1) (3.6%), Acanthamoeba sp. (1) (12%) were found. That points to the similarity of the species diversity of amoebae of the epilithic mosses and lichens and that of soils. Besides that, most of the species of our study have been found before in the water bodies of Ukraine (Patcyuk, Dovgal, 2012;Patsyuk, 2014. The similarity of species composition of the naked amoebae from various epilithic biotopes was analyzed using the Chekanovsky-Sørensen index. At the dendrogram, two clusters can seen ( Figure 2). One of the clusters represented the species diversity of amoebae of the epilithic lichens at different height, and that of the epilithic mosses at 0.5 m above soil. The bootstrap support of that cluster is 47%. The other cluster (bootstrap support 100%) represented the amoeba species sampled at 1.0 m above soil. Its specifics were determined by Vahlkampfia sp. (1), Thecamoeba sp., Mayorella sp., and Korotnevella sp. The index of faunistic similarity was higher than 50% (0.50-0.96) for the species diversity of amoebae collected in the various epilithic biotopes of Zhytomyr region. The species richness of amoebae was defined by the levels of microbiotope humidity. It was relatively high in the moist mosses and lichens (68-78%). Low values of species richness were found in the driest conditions of the epilithic mosses and lichens (41-44%). The humidity levels of the epilithic biotopes decreased with the increasing height above soil.
Similar findings have been observed in the studies of relationship between the species composition of other invertebrates (testate amoebae, heterotrophic flagellates, mites, etc.) and the distance from soil (Miller et al., 2007;Lindo et al., 2008).
All nine morphotypes of amoebae were found in the epilithic mosses of the study region. In the epilithic lichens, the lingulate amoebae were absent. At 0 to 1.5 m above soil, we found the monotactic, dactylopodial, fan-shaped, mayorellian, striate, acanthopodianl, eruptive morphotypes, making up 78% of all the recorded morphotypes. Amoebae of the lens-like and the lingulate morphotypes, 22% of all recorded morphotypes, were not collected at 1.5 m above soil.
Similarly, all nine morphotypes of amoebae were present in the epilithic mosses at soil level (0 m). Seven morphotypes of amoebae were observed in the epilithic lichens at that level, with the exception of the lens-like and lingulate morphotypes. In the epilithic mosses at soil level, the most common amoeba were of acanthopodial (98%), eruptive (95%), striate (67%), mayorellian (67%), dactylopodial (67%) morphotypes; the least common were amoebae of monotactic (5.5%) and lingulate (17%) morphotypes; for fan-shaped and lens-like amoebae, the frequency of occurrence was 33%. In the epilithic lichens at soil level, the most common were the amoebae of striate (67%) and eruptive (70%) morphotypes. Frequency of occurrence of amoebae of other morphotypes did not exceed 37% (Figure 3).
At 0.5 m above soil, the frequency of occurrence and the diversity of amoebae of certain morphotypes did not change essentially (Figure 3). In the epilithic mosses, seven of the morphotypes were represented (78% of all morphotypes). In the epilithic lichens, the amoebae of eight morphotypes (89%) were present.
The diversity of amoeba morphotypes was analyzed using Chekanovsky-Sørensen index. The resulting two groups are shown on Figure 4. The first group included the morphotypes of amoebae found in epilithic mosses and lichens (0-0.5 m), with 76% bootstrap support. The second group included the morphotypes of amoebae from the epilithic mosses at 1 m above soil. That complex was defined by the amoebae of dactylopodial, mayorellian, striate and eruptive morphotypes. The results of clustering analysis of the amoeba species diversity were in line with these findings. Vahlkampfia sp. (1), Thecamoeba sp., Mayorella sp., Korotnevella sp. formed a separate complex of species, and their morphotypes were grouped similarly.  Thus, 16 species of naked amoebae of the molecular clusters of Tubulinea, Discosea and Discoba were identified in present study of the species composition of naked amoebae in the epilithic mosses and lichens of Zhytomyr region, Ukraine. Most species grouped together in clusters are eurybionts found in the water bodies, soils, and epilithic biotopes. The changes in species composition of amoebae in the epilithic mosses and lichens occur at 0.5-1 m above soil. Factors that determine the species composition of amoebae in such biotopes are distance to soil and humidity levels ( Figure 5). According to the cluster analysis, the species diversity of amoebae of the epilithic biotopes of Zhytomyr region consists of two complexes. One of them includes the amoebae of the epilithic lichens at varying height above soil and epilithic mosses at 0.5 m above soil (47% bootstrap support), the other is formed by of amoebae of the epilithic mosses at 1.0 m above soil (100% bootstrap support). In the epilithic mosses of the study region we found amoebae of monotactic, dactylopodial, fan-shaped, lens-like, lingulate, mayorellian, striate, acanthopodial and eruptive morphotypes. The amoebae of lingulate morphotype are not found in the epilithic lichens. At 1.5 m above soil, the amoebae of the lingulate and lens-like morphotypes are not present. The cluster analysis of the morphotype diversity results in two groups of the amoeba morphotypes similar to the clusters of amoeba species diversity. The species and morphotype compositions of the naked amoebae are similar in structure according to the results of cluster analysis, that is, the representatives of different morphotypes prefer similar ecological conditions.