Checklist of Sphagnum-dwelling testate amoebae in Bulgaria

Abstract Background Until now, a complete checklist of Sphagnum-dwelling testate amoebae in Bulgaria has never been published. Records for species diversity and distribution in the country were scattered in many faunistic and ecological publications. The aim of the present study is to summarise all data for the species distribution at the level of country by reviewing the existing literature and by additional data obtained in our research over the past two years. New information The checklist comprises 171 species, classified into 43 genera, 20 families, three orders, three classes and three phyla. We present data for 16 new Sphagnum-dwelling testate amoebae in Bulgaria and new distribution data for 134 species. Of them, 99 species are recorded from Stara Planina Mt., for which there was no available data to date. Additionally are recorded 69 new species for Pirin Mt., 21 for Vitosha Mt. and 18 for Rila Mt. Thirty six species are synonymised according to the latest taxonomic changes. Two misidentified taxa (Euglypha brachiata Penard, 1902 and Difflugia compressa var. africana Gauthier-Lièvre et Thomas, 1958) are transferred into valid species E. acanthophora and Zivkovicia compressa, respectively. Three of the recorded species have not been included in the checklist, because they are currently not refering to testate amoebae (Cochliopodium bilimbosum (Auerbach 1856) and Cochliopodium echinatum Korotneef, 1879 are gymnamoebae (naked amoebae) and Microgromia elegantula (Penard 1904) = Paralieberkuehnia elegantula (Penard 1904) is freshwater foraminifera).


Introduction
Testate amoebae are a polyphyletic assemblage of at least three major, unrelated taxonomic groups of eukaryotes (Adl SM et al. 2012). They are present in most terrestrial and freshwater environments and more marginally in brackish and marine habitats. They are especially abundant and diverse in Sphagnum mosses. Research on testate amoebae have increased significantly over the past two decades due to their increasing use in different applied aspects: as a bioindicators for palaeoecological studies, in environmental monitoring, pollution hazards, ecotoxicology, studies on their role in the cycling of elements in terrestrial ecosystems, biogeographical and evolutionary studiesetc. (Aoki, Yoshiyuki et al. 2007, Booth 2002, Charman 2001, Charman et al. 1998, Lamentowicz and Mitchell 2005, Nguyen-Viet et al. 2007, Swindles et al. 2008, Swindles et al. 2015, Qin et al. 2013).
Studies on Sphagnum-dwelling testate amoebae in Bulgaria started at the beginning of the 20th century, when Pateff (1924), Pateff (1928) published his works on freshwater Rhizopoda in the country. He recorded a total of 118 rhizopods, of which 52 testate amoebae were from Sphagnum mosses in the Rila, Rhodopes and Vitosha Mountains. Later, Valkanov (1932), Valkanov (1934) published results of his studies on the fauna of alpine lakes in the Rila and Pirin Mountains and recorded many testate amoebae, which are typical inhabitants of peat mosses. Unfortunately, they were recorded along with all other species and no distinction was made amongst the species found in the lakes themselves and in the Sphagnum mosses from the lake shore. In the 60s and 70s of the 20th century, several publications on testate amoebae from the Vitosha, Rhodopes and Pirin Mountains were published (Golemansky 1965, Golemansky 1966, Golemansky 1968, Golemansky 1974, including a lot of data on Sphagnum-dwelling testate amoebae. At 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 sphagnicolous testate amoebae in Bulgaria increased considerably (Golemansky and Todorov 1990, Golemansky and Todorov 1993, Golemansky and Todorov 2006, Golemansky and Todorov 1985, Golemansky et al. 2006, Heger et al. 2010, Kosakyan et al. 2012, Todorov 1993, Todorov 2004, Todorov 2005, Todorov and Golemansky 1995, Todorov and Golemansky 2000, Todorov et al. 2009). All data in the abovementioned publications have never been summarised in one place and that is the reason why the main goal of the present paper is to unite this scattered information and, together with the new data obtained from our research over the past two years, to present a checklist of all known to date Sphagnum-dwelling testate amoebae in Bulgaria. Data on the distribution, habitats, regions, localities, altitude, authors and sources of information are provided in Suppl. material 1. Some typical and a few rare sphagnicolous testate amoebae, recorded from the country, are illustrated in Figs 1, 2, 3, 4.

Materials and methods
The data presented in the checklist are based primarily on published information concerning Sphagnum-dwelling testate amoebae in Bulgaria. In addition, data from our research conducted over the past two years are also included. We have tried to explore the diversity and distribution of testate amoebae in the main areas of the country where peat mosses are more widespread. Genus Sphagnum in Bulgaria numbers 27 species (Natcheva and Ganeva 2005) distributed in mires in mountain areas of the country above the tree belt or in mires in coniferous forest, these being an important component of habitat 91D0 (Bog Woodland) of Directive 92/43/EEC. Some of the species (e.g. Sphagnum capillifolium (Ehrh.) Hedw., S. subsecundum Nees, S. girgensohnii Russ., S. centrale C.E.O.Jensen, S. platyphyllum (Lindb. ex. Braithw.) Sull. ex Warnst., S. squarrosum Crome, S. teres (Schimp.) Ängstr.) are more widespread in mires of Western Stara Planina, Rhodopes, Vitosha, Rila and Pirin Mountains, where, with other bryophytes and sedges, they comprise the specific mire flora.
The material for the present study was extracted from wet Sphagnum mosses, gathered at Vitosha and Western Stara Planina Mountains in 2016 and Rila and Pirin Mountains in 2017. A total of 109 samples from 18 localities were sampled and examined. All data, concerning sampling localities including date, region, altitude, coordinates, Sphagnum moss species, as well as many physical and chemical parameters of the sampling sites, are provided in Suppl. material 2.
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 an optical microscope "Amplival" (Zeiss-Jena) using 40x objective and 10x ocular lens. For scanning electron microscopy (SEM), specimens were isolated by searching through small isolates of material in a petri dish. 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 a JEOL JSM-5510, operating at 10 kV. Characteristic Sphagnum-dwelling testate amoebae from the order Arcellinida. The checklist is annotated with information regarding the synonymous names (if available), species distribution in Sphagnum mosses in Bulgaria, the relevant literature sources and notes (mainly taxonomic). The higher classification used here follows Adl SM et al. 2012 andMeisterfeld (2002a), Meisterfeld (2002b). Taxonomic revision of some genera and a b c d e f  families, as well as numerous taxonomic and nomenclature changes, based on recent molecular studies and subsequent phylogenetic reconstructions, have also been taken into account (Blandenier et al. 2017, Chatelain et al. 2013, Gomaa et al. 2013, Gomaa et al. 2017, Heger et al. 2010, Kosakyan et al. 2016a, Kosakyan et al. 2012, Kosakyan et al. 2016b, Lahr et al. 2013, Mazei and Warren 2012, Mazei and Warren 2014, Mazei and Warren 2015. The list does not include separate varieties and forms recorded from Bulgaria, 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, which in many cases are based on signs that do not have much taxonomic significance (small differences in size, the presence or absence of spines/horns 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 nominal species. Nevertheless, in 'Notes' to each nominal species, 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.

Notes:
The species has been recorded both as nominal species and as synonym D. tuberculata (Rhodopes Mt.).

Notes:
The species has been recorded both as nominal species and as synonym D. venusta (Rhodopes Mt.).

Notes:
The species has been recorded both as nominal species and as synonym D. gassowskii (Rhodopes Mt., Rila Mt., Vitosha Mt.).

Notes:
The species has been recorded both as nominal species and as synonym D. amphoralis (Rila Mt., Vitosha Mt.).

Notes:
The species has been recorded both as nominal species and as synonym D. globularis (Rhodopes Mt., Rila Mt., Vitosha Mt.).

Notes:
The species has been recorded both as nominal species and as the infrasubspecific taxon Difflugia oblonga var. microclaviformis (Vitosha Mt.).

Notes:
The species has been recorded both as nominal species and as synonym D. manicata (Rhodopes Mt., Rila Mt., Vitosha Mt.).

Notes:
The species has been recorded both as nominal species and as synonym P. compressa (Rhodopes Mt., Rila Mt., Vitosha Mt.).

Notes:
The species has been recorded both as nominal species and as synonym P. bigibbosa (Pirin Mt., Vitosha Mt.). Furthermore, in several publications on the testate amoebae from Vitosha Mt. (Golemansky 1965, Golemansky and Todorov 1990, Todorov 1993, Todorov and Golemansky 1995, the infrasubspecific taxa Difflugia compressa var. africana Gauthier-Lièvre et Thomas, 1958 has erroneously been recorded, because the description of the found individuals fully corresponds to Z. compressa.

Notes:
The species has been recorded both as nominal species and as synonym P. spectabilis (Rhodopes Mt.).

Notes
Notes: So far, all records for the species have been as N. galeata.

Notes:
The species has been recorded as N. golemanskyi.
Notes: So far, all records for the species have been as N. tubulosa.

Notes:
The species has been recorded both as nominal species and as infrasubspecific taxon E. ciliata f. glabra Wailes, 1915 (Rhodopes Mt., Rila Mt.).

Notes:
The species has been recorded both as nominal species and as infrasubspecific taxon E. compressa f. glabra Wailes, 1915 (Rhodopes Mt.).

Notes:
The species has been recorded as infrasubspecific taxon E. crenulata var. minor Wailes, 1912.

Notes:
The species has been recorded both as nominal species and as synonym E. alveolata (Rhodopes Mt., Vitosha Mt.).