Diversity and distribution of testate amoeba (Testaceae) in the moss Pleurozium schreberi

Study area

Lithuania is in the  eastern part of Europe on the coast of the Baltic Sea in the temperate climatic zone (56°00'N and 24°00'E). Moss was sampled in the central part of Lithuania, dose to the  largest cities Vilnius and Kaunas. Presumably polluted sites were chosen along motorways with intensive traffic, while clean sites for sampling were selected in parks and forests located far away from pollution (Table 1).

Laboratory procedures

Two moss samples from the each of the nine sites were collected in September-October 2018 from the ground (approximately 3 g of moss at each site). All sampling data are presented in Table 1.

Before analysis, moss samples were stored at the temperature of 4°C. Later 2 g of moss were used to measure pH using the Mettler Toledo SevenMulti pH meter S40-K (Sigma-Aldrich, Germany).

The mosses (1  g) were chopped finely by scissors and covered with distilled water. After 1-2 min. of intensive shaking in a jar with a  lid (for separation of testate amoebae from the moss), the moss was squeezed and filtrated through the  planktonic sieve. The  filtrate was concentrated with a  centrifuge at 1500  rpm for 2 min. The supernatant was removed and the debris was analysed with a compound microscope (BM2000) equipped with a  digital camera (Moticam 3.0 MP). No fewer than 150 specimens of live individuals and empty shells were counted in each sample and identified at 100×, 400× magnifications. The  obtained values of the number of amoebae were evaluated in 1 g of the substrate. Identification of specimens was based on the  keys of Ogden, Hedley (1980), Patterson, Headley (1992), Charman et al. (2000), and Clarke (2003).

Numerical analyses

The diversity and distribution of testate amoebae were evaluated using relative abundance of species (p_i), species richness (S) and Shannon-Wiener's (H) index (Magurran, 2004). The relative abundance of species (p_i) was calculated by the formula:

p_i = n_i/N × 100%

where n_i is the number of individuals of a testate amoebae species found in the studied sample, N is the total number of all individuals. Species that had p_i > 5% were considered as dominant.

The Shannon index (H) measures the diversity of a species in a community and is calculated thus:

H = -Σp_i * ln(p_i)

where p is the proportion (n/N) of individuals of one species found in community (n) divided by the total number of individuals found (N).

The Simpson index (D) is a dominance index as it gives more weight to common or dominant species:

D = 1 - ∑n_i (n_i - 1)/N (N - 1)

where n is the number of individuals of each species and N is the total number of individuals of all species.

Overall results: species composition

A total of 19 taxa were found in the 18 samples analysed (Table 2). Species Hyalosphenia papilio and Amphitrema flavum were represented by few individuals found in the moss from beside the road Vievis-Trakai, which were only included in the general list of species but not analysed further. Some amoebas from the genus Euglypha and Nebela tincta species complex were identified only to the  genus level. The taxa of Nebela collaris-tincta group and Tracheleuglypha dentata were considered occasional, occurring in one to two sampling sites. The dominant species in the moss Pleurozium schreberi were Corythion dubium (21.15%), Trinema enchelys (20.34%), Euglypha strigosa (16.46%), Centropyxis aerophila-complex (11.40%), and Euglypha laevis (10.77%). The genus Euglypha was represented by five species.

However, it should be kept in mind that, due to the cryptic taxa and adaptive polymorphism, taxonomic uncertainty is valid for some taxa of TA listed below (Schönborn, 1992; Mulot et al., 2017; Bobrov, Mazei, 2004; Roland et al., 2017). Thus, it is possible that Corythion dubium identified in this study may represent not just one but several taxa. The abundance of Trinema enchelys presents a similar case: its abundance identified in this work can be shared with very similar T. lineare (Roland et al., 2017).

A complete list of taxa is presented in Table 2.

All testate amoebas found are mostly registered eurybionts living in soils, moss, and ponds (Chardez, 1967; Mazei, Belyakova, 2011; Mieczan, Adamczuk, 2015; Souto et al., 2021). Testate amoebae of the families Trinematidae and Euglyphidae prevailed in the moss Pleurozium schreberi. They represented typical soil testate amoebae communities (Bobrov et  al., 1999; Mitchell et al., 2008). TA identified as Centropyxis aerophila complex (Foissner, Korganova, 2000) was the most frequent taxa in the moss Pleurozium schreberi in all investigated sites. The taxa of the  genus Nebela, which usually are inhabitants of Sphagnum (Gilbert, Mitchell, 2006), were found in moss Pleurozium schreberi as well, but only in habitat, covered by mixed forest (Trakai National Park) and containing humid and fertile soil.

Distribution and diversity of testate amoebae in polluted and clean sites

Species richness of testate amoebae in moss P.  schreberi varied from 6 to 15. The  highest richness (15 species) was established in the samples of Trakai National Park, meanwhile the samples collected beside the Vilnius-Utena motorway were represented only by six taxa.

Sixteen species (and three groups of testate amoebae identified to the genus level) of testate amoebae were found in clean sites. The following seven species dominated (constituted >5%) here: Corythion dubium (21.99%), Euglypha strigosa (15.87%), Centropyxis aerophila-complex (10.91%), Euglypha laevis (9.88%), Assulina muscorum (9.80%), Cyclopyxis eurystoma (6.78%), and Trinema enchelys (6.62%).

Fourteen species (and two groups of testate amoebae identified to the  genus level) of testate amoebae were found in the moss in polluted sites. The composition of the taxa of testate amoebae was similar to that of clean sites, but the prevalence of the species was different (Fig. 1).

For example, Corythion dubium constituted 21.98% in the  moss of clean sites, meanwhile their abundance decreased almost by half in contaminated moss and constituted only 10.10%. Trinema enchelys composed 6.62% in clean sites, and dominated (35.90%) in contaminated sites. In agreement with other authors (Nguyen-Viet et al., 2008; Mieczan, 2009; Qin et  al., 2016; Heinemeyer, Swindles, 2018; Freitasa  et  al., 2022), Corythion dubium and Trinema enchelys occur in sites contaminated with heavy metals.

The Shannon diversity index varied from 1.39 to 2.32, with the  highest value in Trakai National Park and the  lowest (H = 1.39) in the  moss collected beside the  motorway Vilnius-Utena with intense traffic (Table 2, Fig. 2).

The obtained results are in agreement with other authors who report that TA diversity was lower in the most polluted places with higher concentrations of Cu, Pb and Zn (Wanner et al., 2020; Payne, 2010; Nguyen-Viet et al., 2007).

Environmental acidity impacts TA diversity and distribution (Charman, Warner, 1992; Booth, 2002; Mitchell  et  al., 2000 Lamentowicz, Mitchell, 2005), but in this study, pH of the moss was quite similar in all sites, and thus it did not have a decisive impact on diversity. Rather, it is possible that the  variation of diversity in the communities of testate amoebae were determined by the fertility of the soil and the humidity level, which is closely related to the site being open or hidden in shadow. Obviously, the canopy of trees in Trakai National Park creating shadow for the  moss helps to maintain humidity, while fallow leaves fertilise the soil. These conditions create a suitable habitat for the moss and its inhabitants, testates amoebae. Simultaneously, the shadowless habitats on the roadsides, with fluctuating humidity and contaminated by heavy metals, could have been an important factor determining lower richness and diversity of TA species.