Testate amoebae is a term used to unite those free-living, unicellular eukaryotes which ameboid cell is covered by an extracellular shell (test), mostly with a single main opening, and which extrude lobose or filose pseudopodia. Testate amoebae are traditionally divided into two main groups based on the morphology of pseudopodia – Arcellinida (with lobose pseudopodia) and Euglyphida (with filose pseudopodia) (Meisterfeld 2002a,b). The progress in the molecular phylogeny and phylogenomics, as a result of accumulation of much new DNA data over the last decade, have led to major revisions in the classification of most groups of organisms, including testate amoebae. Currently, it is known that testate amoebae are a polyphyletic assemblage of at least three major, unrelated taxonomic groups of unicellular eukaryotes: Amoebozoa, Stramenopiles and Cercozoa (Adl et al. 2012, 2019).

Testate amoebae are worldwide distributed and occur from the tropics to polar regions. They are present in most terrestrial and freshwater environments, as well as in brackish and marine habitats, but are especially abundant and diverse in the Sphagnum mosses. The studies on this group have significantly increased over the past two decades due to their increasing use in different applied aspects. Testate amoebae are a good model for taxonomy and evolutionary studies because of their abundance, diversity and presence of a shell, which is a reliable taxonomic feature used in diagnostic of the species. They are considered as valuable bioindicators for ecological and environmental monitoring studies, in particular, as proxies for hydrological changes, and therefore, for paleoclimate reconstruction in peatlands (Charman and Warner 1997, Mitchell et al. 2000, Booth 2002, 2007, Lamentowicz and Mitchell 2005, Qin et al. 2013, Payne et al. 2016). The fact that different species have distinct ecological requirements and after death of the amoebae their shells remain well preserved in peat and sediment makes them excellent microfossils and extremely valuable in micro-paleontological studies for palaeoenvironmental reconstructions (Tolonen 1986, Charman et al. 1998, 2001, Charman 2001, Mitchell et al. 2008, Swindles et al. 2009, 2015). Testate amoebae are very sensitive and quickly respond to environmental changes, such as water table depth, dryness, atmospheric pollution, deforestation and other human activities, and this makes them valuable biomonitors for current environmental health (Booth 2007, Nguyen-Viet et al. 2007, Payne et al. 2012, Nasser et al. 2016). Also, it has been shown that testate amoebae play an important role in the cycling of carbon, nitrogen and silica in terrestrial ecosystems (Schröter et al. 2003, Aoki et al. 2007, Jassey et al. 2015).

The studies on the Sphagnum-dwelling testate amoebae in Bulgaria started in the beginning of the 20th century, when Pateff (1924, 1928) published his works on freshwater Rhizopoda in this country. He recorded a total of 118 rhizopods, of which 52 testate amoebae from the Sphagnum mosses in the Rila, Rhodopes and Vitosha Mountains. Later, Valkanov (1932, 1934) published results of his studies on the fauna of the alpine lakes in the Rila and Pirin Mountains, and recorded many testate amoebae, which are typical inhabitants of the peat mosses. Unfortunately, no distinction was made between the species found in the lakes and in the Sphagnum mosses along the shores of the lakes. In the 1960s and 1970s several reports on the testate amoebae in the Vitosha, Rhodopes and Pirin Mountains were published (Golemansky 1965, 1966, 1968, 1974), including a lot of data on the Sphagnum-dwelling testate amoebae. In the end of the 20th and beginning of the 21th century, the number of publications concerning various aspects of taxonomy, systematics, morphology and ecology of the sphagnicolous testate amoebae in Bulgaria increased considerably (Golemansky and Todorov 1985, 1990, 1993, 2006, Todorov 1993, 2004, 2005, 2010, Todorov and Golemansky 1995, 2000, Golemansky et al. 2006, Todorov et al. 2009, 2010, 2018, Heger et al. 2010, Kosakyan et al. 2012, Bankov et al. 2018).

The aim of this atlas is to present summarised information and to illustrate comprehensively the shell morphology and structure of the majority of recorded Sphagnum-dwelling testate amoebae in Bulgaria. Since most of these species appear to be widely distributed in Europe, as well as in many other regions of the world, the atlas may be of interest to all researchers on testate amoebae and can also be used by specialists in ecology, hydrobiology, palaeoecology and environmental monitoring.

Shells are the main taxonomic feature of testate amoebae and they are extremely diverse in shape, size and composition. Bonnet (1975) classified testate amoebae into sixteen morphological types according to the shape and symmetry of their shells and structure of the aperture (Fig. 1). It was shown that different morphological types are closely related to the nature of habitats and that different types can be used as indicators in ecological studies or to suggest phylogenetic relationships (Chardez 1967, Bonnet 1975, Chardez and Lambert 1981). For example, in aquatic habitats and wet mosses predominate species with a terminal and comparatively large aperture of the arcella (ARC - Arcella), acrostomic (ACS - Difflugia, ACA - Cyphoderia) and trachelostomic (TRAS - Cucurbitella, TRAA - Lesquereusia) morphological types. In habitats where the amoebae are subject to frequent droughts, e. g. aerophilic and epiphytic mosses and soils, the aperture is reduced to a slit-like opening (ACC – Assulina, Heleopera), is covered by an anterior visor or lip (PLS – Centropyxis plagiostoma, PLV – Centropyxis aerophila, CRS – Geoplagiopyxis, CRV – Plagiopyxis callida) or is highly modified, complex structure of the propylostomic (PRO – Lamtopyxis) and diplostomic (DIP – Distomatopyxis) morphological types. Finally, inhabitants of the coastal marine interstitial are adapted to the specific conditions in this environment, they have shells of the cotylostomic type (COT – Psammonobiotidae), with an enlarged collar around the aperture, used for fixation and stabilisation of the shell in the continuous tides and outflow of the water.

According to the shell composition, there are four main types of shells: proteinaceous, agglutinate, siliceous and calcareous (Ogden and Hedley 1980, Meisterfeld 2002a).

Proteinaceous shells can be divided into three types: 1) shells in which the wall is more or less a flexible membrane (Microcoryciidae); 2) shells with a smooth and rigid homogeneous coat composed of mucoprotein (Hyalosphenia); 3) shells, constructed of numerous regularly arranged, hollow building units or alveoli (Arcellidae) (Fig. 2).

Agglutinate shells may be divided into two types. The shells of the first type have either a structured or sheet-like organic cement matrix in which foreign material from the environment (xenosomes), such as quartz grains or diatom frustules, is incorporated (most of Difflugiidae). It is noteworthy that the structure of the organic cement has valuable importance for the taxonomy and distinction of the species in this group. The second type of agglutinate shells is characteristic for most representatives of the families Hyalospheniidae and Heleoperidae. Their shells are composed of siliceous shell-plates with different shape and size, embedded in a sheet-like organic cement matrix. It is important to note that the species with such shells do not make their own shell-plates but obtain them by predation mainly on small euglyphids (Fig. 3).

Siliceous shells are composed of endogenous siliceous shell-plates (idiosomes) which are produced by the amoebae themselves. These structural elements are formed and stored in the parent’s cytoplasm prior to cell division, and are used in the division process to construct the daughter shell, identical to the parent. The shape, size and arrangement of the siliceous shell-plates (idiosomes) are species specific and have valuable taxonomic significance. The idiosomes are too different in shape and vary from oval or circular in the euglyphids (Euglyphida), through rods in Lesquereusia, and nail-like in Netzelia to quadrangular in Quadrulella (Fig. 4).

Calcareous shells are the least presented and are characteristic to only two genera Paraquadrula and Cryptodifflugia. The first one has quadrangular calcite shell-plates embedded in a sheet-like organic cement matrix. The second has a smooth shell surface and the wall is composed of two layers, a thin organic outer layer and a thick inner layer of amorphous calcium phosphate (Hedley et al. 1977).

Some species of testate amoebae are described only on the basis of empty shells and their biology is totally unknown. Most of the species with agglutinate shells, composed of quartz grains (xenosomes), have poorly studied cell morphology because their shells are opaque and robust, with thick walls, which prevent the study of the cytoplasm by light and transmission electron microscopy. Thus, much of our knowledge on the cell structure of testate amoebae is mainly due to the studies of species with proteinaceous, siliceous or calcareous shells (Charret 1964, Hedley and Ogden 1973, 1974, Hedley et al. 1974, 1977, Netzel 1975, 1983, Harrison et al. 1976, Bonnet et al. 1981, Golemansky et al. 1987, Ogden and Coûteaux 1987, Anderson 1990, Ogden 1991).

In general, the cell of testate amoebae is surrounded by a thin and elastic plasmalemma with numerous microtubules and microfilaments, lying beneath it and having function of a cytoskeletal structure. The cytoplasm is usually divided into two zones. The anterior zone is a granular cytoplasm and includes many food and digestive vacuoles, numerous mitochondria and peripherally located vesicles containing organic cement. The posterior zone is a dense cytoplasm, which contains nucleus, surrounded by a compact mass of granular endoplasmic reticulum, numerous ribosomes, one or more Golgi complexes, as well as several contractile vacuoles located laterally of the nucleus, close to the plasmalemma (Ogden and Hedley 1980, Meisterfeld 2002a). Most species have one nucleus and rarely the nuclei are two (many Arcella species), about ten to twenty (Arcella polypora), up to several tens or hundreds (Arcella megastoma, Difflugia urceolata, Phryganella nidulus, etc.). The nuclei of testate amoebae are two main types, according to the classification of Raikov (1982): vesicular nuclei with one large, central nucleolus and ovular nuclei with several to many small nucleoli (Fig. 5). In the smaller euglyphids (e.g. Corythion, Euglypha, Sphenoderia, Trinema) the cytoplasm usually fills the whole shell, while in larger species (Hyalospheniidae, Difflugiidae) it fills about a half or two-third of the shell and the cell is attached to the posterior part of the shell wall with numerous thin cytoplasmic strands (epipodes) (Fig. 6).

The movement of testate amoebae is accomplished by a steady flow of the cytoplasm, which involves the extension and retraction of pseudopodia through the shell aperture (pseudostome) and creep on the substrate. The pseudopodia have different shape and mode of activity and for a long time they have been used as a main feature in the classification of testate amoebae. There are three main types of pseudopodia: endolobopodia, ectolobopodia (reticulo-lobopodia) and filopodia. The endolobopodia are thick, digitate and granular pseudopodia with rounded ends, characteristic for most species of the order Arcellinida (suborders Sphaerothecina and Difflugina). The ectolobopodia (reticulo-lobopodia) are completely hyaline, conical, pointed, sometimes branched and may anastomose. They are presented in the suborder Phryganellina (Cryptodifflugia, Meisterfeldia, Phryganella, Wailesella). The filopodia are hyaline, filamentous, thin, straight, finely pointed, sometimes branched and are presented in all the Euglyphida (Fig. 7).

Testate amoebae normally reproduce by binary fission, with a replication of the parent. Firstly, an identical daughter-cell is constructed and after that the fission takes place usually as a closed ortomitosis (Ogden 1979, Ogden and Coûteaux 1987, Raikov and Mignot 1991) (Fig. 8). The doubling time of the amoebae is between two and twelve days depending on the species and environmental conditions, usually shorter in a laboratory than in natural conditions (Heal 1964, Hedley and Ogden 1974, Hedley et al. 1974, 1977, Ogden 1989). It was believed for a long time that testate amoebae are reproduced only asexually. In the late 19^th and early 20^th century the reports in which are provided evidence for the presence of sexuality in testate amoebae are scarce (Jickeli 1884, Blochmann 1887, Penard 1902, Awerintzew 1906, Reukauf 1912, Dunkerly 1923, Dangeard 1900). Later, Valkanov (1962, 1966) studied in detail the reproduction in testate amoebae and distinguished four types of copulation: 1) type Difflugia in which the cytoplasm of one individual passes entirely into the shell of the other individual, where the two cells merge and form a zigocyst (sexual cyst) – widely distributed in the genera Centropyxis, Difflugia, Lesquereusia, Nebela, Pontigulasia, Sphenoderia, Trinema, etc.); 2) type Pyxidicula in which two individuals merge and form a zigocyst in the space between the two shells – in Pyxidicula and Phryganella; 3) type Euglypha, where the cytoplasm of the copulated individuals goes out of the shells, merge and compose a new larger shell, in which the zigocyst remains – in Assulina, Euglypha, Valkanovia, etc.; 4) type Clypeolina which is the most specific and occurs only in Clypeolina, which shell is composed of two valves. The individuals in copulation touch laterally, the inner valves of each of them disintegrate, the cells merge and form the zygocyst, which is covered by a shell, composed of the outer valves of the copulated individuals. Lüftenegger and Foissner (1991) established the presence of plasmogamy and karyogamy with subsequent cyst formation in Paraquadrula. Mignot and Raikov (1992) provided new evidence for the meiosis in testate amoebae, studying in details the meiosis and autogamy in the cyst of Arcella. Iudina and Sukhanova (2000) studied the cytoplasmic fusion and karyogamy in Corythion delamarei and documented division of the synkaryon into four nuclei and formation of four daughter cells as a result of meiosis. Currently, there is clear evidence that testate amoebae, as well as many other microbial eukaryotes, can reproduce also sexually (Lahr et al. 2011, Tekle et al. 2017, Hofstatter et al. 2018).

Besides the reproductive cysts testate amoebae can produce resistant and short-time cysts as a protection against unfavorable conditions in the environment, such as drought, extremal temperatures, lack of food or anaerobiosis. Usually, the resistant cysts are formed within the shell and are contained within a thick organic membrane. During encystment the amoebae reduce the volume of cytoplasm and number of organelles, and in such encysted state they can survive for a long time (several months). In the period of desiccation, many soil- and moss-dwelling species form short-time cysts (precists), which differ from the resistant cysts by their relatively thin membrane enclosing the cell and by their ability to pass quickly into active life when appropriate conditions in the environment occur. Often, testate amoebae forming cysts additionally seal the shell aperture with a plug of siliceous or extraneous particles (Fig. 9).

Testate amoebae are mostly phagotrophic organisms feeding on bacteria, algae and fungi, thus playing an important role in nutrient recycling in the aquatic and terrestrial ecosystems. Some larger species (mostly Hyalospheniidae) are predators preying on other Protozoa, such as the euglyphid testate amoebae and naked amoebae, or small metazoan organisms, such as the nematodes and rotifers (Yeates and Foissner 1995, Ogden 1989, Gilbert et al. 2000, Han et al. 2008). At the same time, testate amoebae can be preyed on by nematodes, earthworms, collembolans and mites, thus re-entering the classical food chain (Bamforth and Lousier 1995, Wilkinson and Mitchell 2010). Significantly less are the mixotrophic testate amoebae, which are capable of building a symbiotic relationship with the phototrophic algae (zoochlorellae) and combine both phagotrophy and phototrophy (e.g. Amphitrema, Archerella, Difflugia and Hyalosphenia) (Fig. 10). The host heterotrophic species usually supply their symbiotic algal cells with nitrogen and CO₂, and in turn, the algae supply their hosts with photosynthetic products, such as maltose and oxygen (Esteban et al. 2010, Summerer et al. 2007). In some Difflugia species this symbiotic relationship can be facultative and the host can shift to heterotrophic nutrition if exposed to prolonged darkness or if the environment lacks symbionts. Most frequently, the symbiotic relationship is obligatory and the host can no longer survive without its symbionts. Schönborn (1965) showed experimentally that Archerella flavum and Hyalosphenia papilio can not survive for long periods in the dark. His study also demonstrated that A. flavum depends entirely on the symbiotic algae for its nutrition requirements, while H. papilio provides up to 40% of the required nutrients from photosynthetic products of the algae and the rest through phagotrophy.

The presented data on species diversity and distribution of the Sphagnum-dwelling testate amoebae were based on the review of all available literature from Bulgaria. In addition, we included unpublished data from our studies in the period 2016 – 2018.

The material for the present study was extracted from wet Sphagnum mosses, collected at the mountains Western Stara Planina, Rhodopes, Vitosha, Rila and Pirin. A total of 126 samples of testate amoebae from 23 localities were collected and examined. Testate amoebae were extracted from fresh Sphagnum mosses at the sampling site and concentrated by sieving (350 μm). The resulting fraction (50 ml) was observed with optical microscope “Amplival” (Zeiss-Jena) using 40x objective and 10x oculars lens. The light micrographs (LM) were taken using an Axio Imager M2-Carl Zeiss compound microscope with a digital camera (ProgRes C7) and specialised software (CapturePro Software 2.8). Most of the pictures for illustration of the pseudopodia and nuclei of the live individuals were taken with differential interference contrast (DIC).

The morphometric characterisation of the species was made using our own measurements of individuals isolated from the wet Sphagnum mosses, collected during this study. The following basic characters were measured: arithmetic mean; median (M); standard deviation (SD); standard error of mean (SE); coefficient of variation in % (CV); and extreme values (Min and Max). The statistical analysis was performed using the STATISTICA Software, Version 10.0 (StatSoft 2010).

For the scanning electron microscopy (SEM) the specimens were isolated by searching through small isolates of material in a petri dish. The specimens were extracted using a glass micropipette, washed several times in distilled water, and then individual shells were positioned with a single-hair brush on a previously mounted double-sided adhesive tape on a standard aluminium stub and air-dried. The shells were coated evenly with gold in a vacuum coating unit. The photomicrographs were obtained using scanning electron microscopes JEOL JSM-5510 and LYRA/TESCAN 5007, operating at 10 kV.

The classification of testate amoebae at higher ranks follows Adl et al. (2019). Taxonomic revision of some genera and families, as well as numerous taxonomic and nomenclature changes, based on recent molecular studies and subsequent phylogenetic reconstructions, have also been taken into account (Lara et al. 2007, Mazei and Warren 2012, 2014, 2015, Kosakyan et al. 2012, 2016a, 2016b, Chatelain et al. 2013, Gomaa et al. 2013, 2017, Lahr et al. 2013, 2019, Bobrov 2016, Blandenier et al. 2017, Dumack et al. 2017, Duckert et al. 2018).

Eukaryotes

Domain Amorphea Adl et al., 2012

Supergroup Amoebozoa Lühe, 1913, sensu Cavalier-Smith, 1998

Phylum Tubulinea Smirnov et al., 2005

Class Corycida Kang et al., 2017

Genus Amphizonella Greeff, 1866

Genus Diplochlamys Greeff, 1888

Class Elardia Kang et al., 2017

Order Arcellinida Kent, 1880

Suborder Glutinoconcha Lahr et al. 2019

Infraorder Sphaerothecina Kosakyan et al., 2016

Family Arcellidae Ehrenberg, 1843

Genus Antarcella Deflandre, 1928

Genus Arcella Ehrenberg, 1830

Family Netzeliidae Kosakyan et al., 2016

Genus Cyclopyxis Deflandre, 1929

Genus Netzelia Ogden, 1979

Incertae sedis Sphaerothecina, Genera: Cornuapyxis Coûteaux et Chardez, 1981; Cucurbitella Penard, 1902; Distomatopyxis Bonnet, 1964; Ellipsopyxella Bonnet, 1975; Ellipsopyxis Bonnet, 1965; Geopyxella Bonnet et Thomas, 1955; Lamtopyxis Bonnet, 1974; Protocucurbitella Gauthier-Lièvre et Thomas, 1960; Suiadifflugia Green, 1975; Trigonopyxis Penard, 1912.

Infraorder Longithecina Lahr et al., 2019

Family Difflugiidae Wallich, 1864

Genus Difflugia Leclerc, 1815

Genus Pseudonebela Gauthier-Lièvre, 1953

Family Lesquereusiidae Jung, 1942

Genus Lesquereusia Schlumberger, 1845

Genus Microquadrula Golemansky, 1968

Genus Paraquadrula Deflandre, 1932

Genus Pomoriella Golemansky, 1970

Infraorder Excentrostoma Lahr et al., 2019

Family Centropyxidae Jung, 1942

Genus Centropyxis Stein, 1857

Genus Proplagiopyxis Schönborn, 1964

Family Plagiopyxidae Bonnet et Thomas, 1960

Genus Bullinularia (Penard, 1907) Deflandre, 1953

Genus Geoplagiopyxis Chardez,1961

Genus Hoogenraadia Gauthier-Lièvre et Thomas, 1958

Genus Paracentropyxis Bonnet, 1960

Genus Plagiopyxis Penard, 1910

Genus Planhoogenraadia Bonnet, 1977

Genus Protoplagiopyxis Bonnet, 1962

Incertae sedis Excentrostoma, Genera: Conicocassis Nasser and Patterson, 2015; Oopyxis Jung, 1942.

Infraorder Hyalospheniformes Lahr et al., 2019

Family Hyalospheniidae Schultze, 1977, emend. Kosakyan and Lara, 2012

Genus Alabasta Duckert et al., 2018

Genus Alocodera Jung, 1942

Genus Apodera Loeblich and Tappan, 1961

Genus Certesella Loeblich and Tappan,1961

Genus Cornutheca Kosakyan et al., 2016

Genus Gibbocarina Kosakyan et al., 2016

Genus Hyalosphenia Stein, 1859

Genus Longinebela Kosakyan et al., 2016

Genus Mrabella Kosakyan et al., 2016

Genus Nebela Leidy, 1874

Genus Padaungiella Lara and Todorov, 2012

Genus Planocarina Kosakyan et al., 2016

Genus Porosia Jung, 1942

Genus Quadrulella Cockerell, 1909

Infraorder Volnustoma Lahr et al., 2019

Family Heleoperidae Jung, 1942

Genus Heleopera Leidy, 1879

Suborder Organoconcha Lahr et al., 2019

Family Microchlamyidae Ogden, 1985

Genus Microchlamys Cockerell, 1911

Genus Spumochlamys Kudryavtsev and Hausmann, 2007

Genus Pyxidicula Ehrenberg, 1838

Suborder Phryganellina Bovee, 1985

Family Phryganellidae Jung, 1942

Genus Phryganella Penard, 1902

Family Cryptodifflugiidae Jung, 1942

Genus Cryptodifflugia Penard, 1890

Genus Meisterfeldia Bobrov, 2016

Genus Wailesella Deflandre,1928

Incertae sedis Arcellinida, Genera: Argynnia Vucetich, 1974; Awerintzewia Schouteden, 1906; Geamphorella Bonnet, 1959; Jungia Loeblich and Tappan, 1961; Lagenodifflugia Medioli and Scott, 1983; Lamtoquadrula Bonnet, 1974; Leptochlamys West, 1901; Maghrebia Gauthier-Lièvre et Thomas, 1958; Pentagonia Gauthier-Lièvre et Thomas, 1958 ; Physochila Jung, 1942; Pontigulasia Rhumbler, 1896; Pseudawerintzewia Bonnet, 1959; Schoenbornia Decloitre, 1964; Schwabia Jung, 1942; Sexangularia Awerintzew, 1906, Zivkovicia Ogden, 1987.

Incertae sedis Amoebozoa, Genera: Microcorycia Cockerell, 1911; Parmulina Penard, 1902; Penardochlamys Deflandre, 1953; Zonomyxa Nüsslin, 1882

Domain Diaphoretickes Adl et al., 2012

Supergroup Sar Burki et al., 2008, emend. Adl et al., 2012

Phylum Stramenopiles Patterson, 1989, emend. Adl et al., 2005

Class Labyrinthulomycetes Dick, 2001

Order Amphitremida Poch, 1913, emend. Gomaa et al., 2013

Family Amphitrematidae Poch, 1913

Genus Amphitrema Archer, 1869

Genus Archerella Loeblich and Tappan, 1961

Genus Paramphitrema Valkanov, 1970

Supergroup Rhizaria Cavalier-Smith, 2002

Phylum Cercozoa Cavalier-Smith, 1998, emend. Adl et al., 2005; emend. Cavalier-Smith, 2018

Class Thecofilosea Cavalier-Smith, 2003, emend. Cavalier-Smith, 2011

Order Cryomonadida Cavalier-Smith, 1993

Family Rhogostomidae Dumack et al., 2017

Genus Capsellina Penard, 1909

Genus Rhogostoma Belar, 1921

Genus Sacciforma Dumack et al., 2017

Order Tectofilosida Cavalier-Smith, 2003

Family Chlamydophryidae de Saedeleer, 1934

Genus Chlamydophrys Cienkowsky, 1876

Genus Clypeolina Penard, 1902

Genus Diaphorodon Archer, 1869

Genus Lecythium Hertwig and Lesser, 1874

Genus Leptochlamydophris Belar, 1921

Family Pseudodifflugiidae de Saedeleer, 1934

Genus Pseudodifflugia Schlumberger, 1845

Class Silicofilosea Adl et al., 2005, emend. Adl et al., 2012

Order Thaumatomonadida Shirkina, 1987

Family Thaumatomonadidae Hollande, 1952

Genus Penardeugenia Deflandre, 1958

Order Euglyphida Copeland, 1956, emend. Cavalier-Smith, 1997

Family Assulinidae Lara et al., 2007

Genus Assulina Ehrenberg, 1872

Genus Placocista Leidy, 1879

Genus Valkanovia Tappan, 1966

Family Euglyphidae Wallich, 1864, emend. Lara et al., 2007

Genus Euglypha Dujardin, 1841

Genus Scutiglypha Foissner and Schiller, 2001

Family Sphenoderiidae Chatelain et al., 2013

Genus Deharvengia Bonnet, 1979

Genus Sphenoderia Schlumberger, 1845

Genus Trachelocorythion Bonnet, 1979

Family Trinematidae Hoogenraad and De Groot,1940, emend Adl et al., 2012

Genus Corythion Taranek, 1881

Genus Pileolus Coûteaux et Chardez, 1981

Genus Playfairina Thomas, 1961

Genus Puytoracia Bonnet, 1970

Genus Trinema Dujardin, 1841

Family Cyphoderiidae de Saedeleer, 1934

Genus Campascus Leidy, 1877

Genus Chardezia Golemansky, 1970

Genus Corythionella Golemansky, 1970

Genus Cyphoderia Schlumberger, 1845

Genus Messemvriella Golemansky, 1973

Genus Pseudocorythion Valkanov, 1970

Genus Schaudinnula Awerintzev, 1907

Family Paulinellidae de Saedeller 1934, emend. Adl et al. 2012

Genus Micropyxidiella Tarnawski and Lara, 2015

Genus Ovulinata Anderson, Rogerson & Hannah, 1997

Genus Paulinella Lauterborn, 1895

Incertae sedis Euglyphida, Genera: Ampullataria van Oye, 1956; Euglyphidion Bonnet, 1960; Heteroglypha Thomas et Gauthier-Lièvre, 1959; Matsakision Bonnet, 1967; Pareuglypha Penard, 1902; Tracheleuglypa Deflandre, 1928

Incertae sedis Cercozoa:

Family Psammonobiotidae Golemansky, 1974, emend. Meisterfeld, 2002

Genus Alepiella Golemansky, 1970

Genus Centropyxiella Valkanov, 1970

Genus Edaphonobiotus Schönborn, Foissner and Meisterfeld, 1983

Genus Micramphora Valkanov, 1970

Genus Micropsammella Golemansky, 1970

Genus Nadinella Penard, 1902

Genus Ogdeniella Golemansky, 1982

Genus Psammonobiotus Golemansky, 1967

Genus Propsammonobiotus Golemansky, 1991

Family Volutellidae Sudzuki, 1979

Genus Pseudovolutella Sudzuki, 1979

Genus Volutella Chardez, 1977

Incertae sedis Cercozoa, Genera: Feuerbornia Jung, 1942; Frenzelina Penard, 1902; Lesquerella Chardez et Thomas, 1980; Rhumbleriella Golemansky, 1970

Prior to our investigation, the number of known Sphagnum-dwelling testate amoebae in Bulgaria was 171 (Bankov et al. 2018). Present study increases the number with 4 species and this group currently comprises 175 species classified into 45 genera, 20 families, six orders, five classes and three phyla. The species are presented in a systematic order, according to the classification in the previous chapter.

Varieties and forms recorded from Bulgaria are not included separately in the atlas, despite the fact that, according to the International Code of Zoological Nomenclature, article 45.6.3, when the name was published before 1961 using the abbreviation ‘var.’ or ‘f.’, it is deemed to be subspecific rather than infrasubspecific. However, due to the fact that many of these taxa have not sufficiently detailed descriptions and in many cases they are described on a basis of traits that do not have much taxonomic significance (small differences in size, the presence or absence of spines/cornes and their number, the number of lobes of the aperture, etc.) these taxa remain with unclear taxonomic status. So, until the clarifying of their status with the help of combined morphological and molecular approaches and full confirmation of their validity, we prefere to adopt a conservative position and consider these taxa as the product of the phenotipic plasticity of a nominal species. Nevertheless, in the next chapter “Species descriptions and illustrations” to each nominal species, in ‘Notes’, we have included all the records for these infrasubspecific taxa, in the event that some of them may be raised in rank in the future.

Amoebozoa

Family Arcellidae

Genus Arcella

• Arcella arenaria Greeff, 1866
• Arcella bathystoma Deflandre, 1928
• Arcella catinus Penard, 1890
• Arcella dentata Ehrenberg, 1838
• Arcella discoides Ehrenberg, 1871
• Arcella gibbosa Penard, 1890
• Arcella hemisphaerica Perty, 1852
• Arcella intermedia (Deflandre, 1928) Tsyganov and Mazei, 2006
• Arcella rotundata Playfair, 1918
• Arcella vulgaris Ehrenberg, 1830

Family Netzeliidae

Genus Cyclopyxis

• Cyclopyxis arcelloides (Penard, 1902) Deflandre, 1929
• Cyclopyxis eurystoma Deflandre, 1929
• Cyclopyxis kahli Deflandre, 1929
• Cyclopyxis pirini Golemansky, 1974
• Cyclopyxis puteus Thomas, 1960

Genus Netzelia

• Netzelia oviformis (Cash, 1909) Ogden, 1979
• Netzelia tuberculata (Wallich, 1864) Netzel, 1983

Incertae sedis Sphaerothecina

Genus Trigonopyxis

• Trigonopyxis arcula (Leidy, 1879) Penard, 1912

Family Difflugiidae

Genus Difflugia

• Difflugia acuminata Ehrenberg, 1838
• Difflugia ampullula Playfair, 1918
• Difflugia angulostoma Gauthier-Lièvre and Thomas, 1958
• Difflugia bacillariarum Perty, 1849
• Difflugia brevicolla Cash, 1909
• Difflugia bryophila (Penard, 1902) Jung, 1942
• Difflugia distenda Ogden, 1983
• Difflugia elegans Penard, 1890
• Difflugia glans Penard, 1902
• Difflugia globulosa Dujardin, 1837
• Difflugia hiraethogii Ogden, 1983
• Difflugia lanceolata Penard,1890
• Difflugia lobostoma Leidy, 1874
• Difflugia lucida Penard, 1890
• Difflugia mammillaris Penard, 1893
• Difflugia mica Frenzel, 1892
• Difflugia microclaviformis (Kourov, 1925) Ogden, 1983
• Difflugia minuta Rampi, 1950
• Difflugia molesta Penard, 1902
• Difflugia oblonga Ehrenberg, 1838
• Difflugia penardi Hopkinson,1909
• Difflugia petricola Cash, 1909
• Difflugia pristis Penard, 1902
• Difflugia pulex Penard, 1902
• Difflugia pyriformis Perty, 1849
• Difflugia rotunda (Chardez, 1956) Ogden, 1983
• Difflugia rubescens Penard, 1891
• Difflugia stoutii Ogden, 1983
• Difflugia subaequalis Penard, 1910
• Difflugia urceolata Carter, 1864
• Difflugia ventricosa Deflandre, 1926
• Difflugia viscidula Penard, 1902

Family Lesquereusiidae

Genus Lesquereusia

• Lesquereusia epistomium Penard, 1902
• Lesquereusia gibbosa Thomas et Gauthier-Lièvre, 1859
• Lesquereusia modesta Rhumbler, 1896
• Lesquereusia spiralis (Ehrenberg, 1840) Bütschli, 1880

Family Centropyxidae

Genus Centropyxis

• Centropyxis aculeata (Ehrenberg, 1830) Stein, 1857
• Centropyxis aerophila Deflandre, 1929
• Centropyxis cassis (Wallich, 1864) Deflandre, 1929
• Centropyxis constricta (Ehrenberg, 1838) Penard, 1902
• Centropyxis cryptostoma Bonnet, 1959
• Centropyxis discoides (Penard, 1890) Deflandre, 1929
• Centropyxis ecornis (Ehrenberg, 1841) Leidy, 1879
• Centropyxis elongata (Penard, 1890) Thomas, 1959
• Centropyxis gibba Deflandre, 1929
• Centropyxis hirsuta Deflandre, 1929
• Centropyxis laevigata Penard, 1890
• Centropyxis minuta Deflandre, 1929
• Centropyxis orbicularis Deflandre, 1929
• Centropyxis plagiostoma Bonnet et Thomas, 1955
• Centropyxis platystoma (Penard, 1890) Deflandre, 1929
• Centropyxis spinosa (Cash, 1905) Deflandre, 1929
• Centropyxis sylvatica (Deflandre, 1929) Bonnet et Thomas, 1955

Family Plagiopyxidae

Genus Bullinularia

• Bullinularia indica (Penard, 1907) Deflandre, 1953

Genus Plagiopyxis

• Plagiopyxis callida Penard, 1910
• Plagiopyxis declivis Thomas, 1955
• Plagiopyxis glyphostoma Bonnet, 1959
• Plagiopyxis labiata Penard, 1910
• Plagiopyxis minuta Bonnet, 1959
• Plagiopyxis oblonga (Bonnet et Thomas, 1955)

Family Hyalospheniidae

Genus Alabasta

• Alabasta militaris (Penard, 1890) Duckert, Blandenier, Kosakyan and Singer, 2018

Genus Gibbocarina

• Gibbocarina galeata (Penard, 1890) Kosakyan, Lahr, Mulot, Meisterfeld, Mitchell and Lara, 2016

Genus Hyalosphenia

• Hyalosphenia elegans (Leidy, 1874) Leidy, 1879
• Hyalosphenia papilio (Leidy, 1874) Leidy, 1875

Genus Longinebela

• Longinebela ampulla Todorov, Bankov and Ganeva, 2018
• Longinebela golemanskyi (Todorov, 2010) Kosakyan, Lahr, Mulot, Meisterfeld, Mitchell and Lara, 2016
• Longinebela penardiana (Deflandre, 1936) Kosakyan, Lahr, Mulot, Meisterfeld, Mitchell and Lara, 2016
• Longinebela speciosa (Deflandre, 1936) Kosakyan, Lahr, Mulot, Meisterfeld, Mitchell and Lara, 2016
• Longinebela tubulosa (Penard, 1902) Kosakyan, Lahr, Mulot, Meisterfeld, Mitchell and Lara, 2016

Genus Nebela

• Nebela aliciae Mitchell et Lara, 2013
• Nebela collaris (Ehrenberg 1848) Leidy, 1879
• Nebela flabellulum Leidy, 1874
• Nebela guttata Kosakyan and Lara, 2013
• Nebela pechorensis Kosakyan et Mitchell, 2013
• Nebela rotunda Penard, 1890
• Nebela tincta (Leidy, 1879) Awerintzew, 1906

Genus Padaungiella

• Padaungiella americana (Taranek, 1882)
• Padaungiella lageniformis (Penard, 1890) Lara et Todorov, 2012
• Padaungiella nebeloides (Gauthier-Lièvre et Thomas, 1958) Lara et Todorov, 2012
• Padaungiella tubulata (Brown, 1911) Lara et Todorov, 2012
• Padaungiella wailesi (Deflandre, 1936) Lara et Todorov, 2012

Genus Planocarina

• Planocarina carinata (Archer, 1867) Kosakyan, Lahr, Mulot, Meisterfeld, Mitchell and Lara, 2016

Genus Quadrulella

• Quadrulella longicollis (Taranek, 1882)
• Quadrulella symmetrica (Wallich, 1864) Cockerell, 1909
• Quadrulella tubulata Gauthier-Lièvre, 1953
• Quadrulella variabilis Kosakyan, Lahr, Mulot, Meisterfeld, Mitchell and Lara, 2016

Family Heleoperidae

Genus Heleopera

• Heleopera petricola Leidy, 1879
• Heleopera rosea Penard, 1890
• Heleopera sphagni (Leidy, 1874)
• Heleopera sylvatica Penard, 1890

Family Microchlamyidae

Genus Microchlamys

• Microchlamys patella (Claparede and Lachmann, 1859) Cockerell, 1911

Genus Pyxidicula

• Pyxidicula patens (Claparede and Lachmann, 1858)

Family Phryganellidae

Genus Phryganella

• Phryganella acropodia (Hertwig and Lesser, 1874) Hopkinson, 1909
• Phryganella hemisphaerica (Penard, 1890) Penard, 1902
• Phryganella nidulus Penard, 1902
• Phryganella paradoxa Penard, 1902

Family Cryptodifflugiidae

Genus Cryptodifflugia

• Cryptodifflugia compressa Penard, 1902
• Cryptodifflugia oviformis Penard, 1890

Genus Wailesella

• Wailesella eboracensis (Wailes and Penard, 1911) Deflandre, 1928

Incertae sedis Arcellinida

Genus Argynnia

• Argynnia bipes (Carter, 1870) Murray, 1870
• Argynnia dentistoma (Penard, 1890)
• Argynnia vitraea (Penard, 1899)

Genus Awerintzewia

• Awerintzewia cyclostoma (Penard, 1902) Schouteden, 1906

Genus Lagenodifflugia

• Lagenodifflugia bryophila (Penard, 1902) Ogden, 1987
• Lagenodifflugia montana (Ogden and Zivkovic, 1983) Ogden, 1987
• Lagenodifflugia vas (Leidy, 1874) Medioli and Scott, 1983

Genus Pontigulasia

• Pontigulasia elisa (Penard, 1893) Schouteden, 1906
• Pontigulasia rhumbleri Hopkinson, 1919

Genus Zivkovicia

• Zivkovicia compressa (Carter, 1864)
• Zivkovicia spectabilis (Penard, 1902) Ogden, 1987

Incertae sedis Amoebozoa

Genus Microcorycia

• Microcorycia flava (Greeff, 1866), emend. Penard, 1902

Stramenopiles

Family Amphitrematidae

Genus Archerella

• Archerella flavum (Archer, 1877) Loeblich and Tappan, 1961

Cercozoa

Family Chlamydophryidae

Genus Lecythium

• Lecythium mutabilis (Bailey, 1853)

Family Pseudodifflugiidae

Genus Pseudodifflugia

• Pseudodifflugia fascicularis Penard, 1902
• Pseudodifflugia gracilis Schlumberger, 1845

Family Assulinidae

Genus Assulina

• Assulina muscorum Greeff, 1888
• Assulina seminulum (Ehrenberg, 1848) Leidy, 1879

Genus Placocista

• Placocista glabra Penard, 1906
• Placocista spinosa (Carter, 1865) Leidy, 1879

Family Euglyphidae

Genus Euglypha

• Euglypha acanthophora (Ehrenberg, 1841) Perty, 1849
• Euglypha aspera Penard, 1899
• Euglypha bryophila Brown, 1911
• Euglypha ciliata (Ehrenberg, 1848), Leidy, 1878
• Euglypha compressa Carter, 1864
• Euglypha cristata Leidy, 1874
• Euglypha denticulata Brown, 1912
• Euglypha filifera Penard, 1890
• Euglypha laevis (Ehrenberg, 1845) Perty, 1849
• Euglypha rotunda Wailes and Penard, 1911
• Euglypha strigosa (Ehrenberg, 1871) Leidy, 1878
• Euglypha tuberculata Dujardin, 1841

Genus Scutiglypha

• Scutiglypha crenulata (Wailes, 1912) Foissner and Schiller, 2001

Family Sphenoderiidae

Genus Sphenoderia

• Sphenoderia fissirostris Penard, 1890
• Sphenoderia labiata Thomas et Gauthier-Lièvre, 1959
• Sphenoderia lenta Schlumberger, 1845
• Sphenoderia minuta Deflandre, 1931
• Sphenoderia ovoidea Jung, 1942
• Sphenoderia splendida (Playfair, 1917) Deflandre, 1931

Genus Trachelocorythion

• Trachelocorythion pulchellum (Penard, 1890) Bonnet, 1979

Family Trinematidae

Genus Corythion

• Corythion constricta (Certes, 1889) Jung, 1942
• Corythion delamarei Bonnet et Thomas, 1960
• Corythion dubium Taránek, 1881

Genus Playfairina

• Playfairina valkanovi Golemansky, 1966

Genus Trinema

• Trinema complanatum Penard, 1890
• Trinema enchelys (Ehrenberg, 1838) Leidy, 1878
• Trinema galeata (Penard, 1890) Jung, 1942
• Trinema grandis (Chardez, 1960) Golemansky, 1963
• Trinema lineare Penard, 1890

Family Cyphoderiidae

Genus Campascus

• Campascus minutus Penard, 1899
• Campascus triqueter Penard, 1891

Genus Cyphoderia

• Cyphoderia amphoralis (Wailes and Penard, 1911)
• Cyphoderia ampulla (Ehrenberg, 1840) Leidy, 1878
• Cyphoderia major (Penard, 1891)

Family Paulinellidae

Genus Paulinella

• Paulinella chromatophora Lauterborn, 1895

Incertae sedis Euglyphida

Genus Pareuglypha

• Pareuglypha reticulata Penard, 1902

Genus Tracheleuglypha

• Tracheleuglypha acolla Bonnet et Thomas, 1955
• Tracheleuglypha dentata (Moniez, 1888) Deflandre, 1928

A total of 120 species are described in the atlas, including most of the characteristic sphagnicolous testate amoebae, as well as some rare or accidentally found in Sphagnum mosses species. The following information is given for each species: description; ecology; geographical distribution; distribution in Sphagnum mosses in Bulgaria and relevant literature sources; morphometric characterisation; taxonomic notes (if necessary); synonymous names (if available); original publication and publication where last revision is made (if any).

Each species is illustrated by nine micrographs, primarily on scanning electron microscope (SEM), to receive information about the shell ultrastructure. Micrographs taken on light microscope (LM) are additionally given for most of them to illustrate the cytoplasm and pseudopodia of live individuals.

In measurements of the species in which the aboral protuberances or horns (spines) are present, the length includes these appendages when they are part of the shell matrix (e.g. Centropyxis, Difflugia, Pareuglypha), but excludes them when they are specialised shell components (e.g. Euglypha).