A new level of confidence: biogeography, morphometry and ecology of enigmatic Cyclopyxis puteus Thomas, 1960 (Amoebozoa: Arcellinida: Netzeliidae)

: Testate amoebae Cyclopyxis puteus Thomas (1960) has a characteristic morphology, that makes the possibility of overlook or misidentification unlikely. Although this species has been found worldwide, its morphology, biometrics and ecological preferences are poorly understood. In this study, isolated material from Bulgaria was investigated by using light and scanning electron microscopy. Based on morphological and biometrical data, we provide an improved diagnosis of this enigmatic species. A summary of the geographical distribution and ecological preferences is provided.


Introduction
Genus Cyclopyxis Deflandre, 1929 comprises lobose testate amoebae with shells of agglutinated mineral particles, hemispherical in lateral and circular in ventral view. According to latest taxonomic reviews, it belongs to class Elardia, Order Arcellinida, Suborder Glutinoconcha, Infraorder Sphaerothecina and Family Netzeliidae (Kang et al., 2019;Kosakyan et al., 2016;). Nowadays, genus Cyclopyxis contains approximately 80 species and subspecies (Nicholls, 2005) that can be found in various habitats like soils, leaf litter or mosses. Some of them are widely distributed with high population density in the environment (Cyclopyxis eurystoma Deflandre, 1929 andCyclopyxis kahli Deflandre, 1929), while some rare species with restricted or patchy distribution have been described.
One of those rare and enigmatic species is Cyclopyxis puteus Thomas, 1960. According to the original description "ce cyclopyxis a l'aspect général et la couleur de C. kahli Defl., mais avec une taille bien plus considérable. Une autre particularité réside par la constitution spéciale du pseudostome, qui permettra de toujours le reconnaître facilement. L'ouverture de ce pseudostome est placée à l'extrémité terminale d'un tube conique fortement invaginé à l'intérieur de la thèque. Ce pseudostome, vaguement circulaire, a un contour légèrement crénelé par la bordure granuleuse. En vue ventrale, le tube du pseudostome, étant large à sa base, dessine un double contour très apparent, car ce tube, observé à l'apic, va en se rétrécissant à l'intérieur de la thèque au niveau du pseudostome. Le revêtement ne présente rien de particulier; il est pierreux à grosses particules sur le dôme. Dimensions: diamètre 145-163 μm; hauteur 110-120 μm; pseudostome 25-35 μm; base du tube 45-50 μm; longueur du tube 28-30 μm." (this species has the general appearance and colour of C. kahli, but with much larger size. Another feature is the special construction of a pseudostome, which allows to recognise it easily. The opening is situated at the terminal end of a strongly invaginated inside the shell conical tube. This roughly circular pseudostome has slightly serrated contours due to the grained edge. In ventral view, the tube of the pseudostome, being wide at the base, forms a very distinct double contour, because it narrows inward into the shell at the level of the pseudostome. The coating does not present anything particular; pieces of quartz, larger on the dorsal side. Measurements: diameter 145-163 μm; height 110-120 μm; pseudostome 25-35 μm; base of the tube 45-50 μm; length of the tube 28-30 μm).
The characteristic morphology and large size of this species makes the possibility of overlook or misidentification unlikely. However, the registered data emphasises the difficulty of defining the distribution patterns. So far, C. puteus is reported worldwide but with infrequent findings and low number of specimens. The scarcity of records, on the other hand, leads to a poorly studied morphology, biometry and ecological preferences.
During the investigation of testate amoebae communities from soil mosses and beech's litter from Bulgaria, we found population of C. puteus with a high abundance. We carried out detailed light and scanning electron microscopy analyses with additional morphometric examination of the shells. Our primary aims were: 1) to examine the Bulgarian populations of C. puteus morphologically and biometrically; 2) to clarify its geographic distribution and ecological preferences.

Materials and methods
The samples were taken on 8.11.2022 from soil mosses, litter (Ao) and soil humus (Ah -0-5 cm) of beech forests (Fagus sylvatica L.) near Plachkovtsi Village (N 42.79821 E 25.49891, 591 a.s.l.) (Fig. 1). The brown mountain-forest soil is the main soil type within the area. The collected material was washed and examined in petri dish. Isolation was made under stereomicroscope at 100x magnification.
Morphological characters and morphometric variables of 350 shells were studied with optical microscope "Amplival" (Zeiss-Jena) using 40x objective and 10x oculars lens. The light micrographs were taken using an Axio Imager M2-Carl Zeiss compound microscope with a digital camera (ProgRes C7) and specialised software (CapturePro Software 2.8). Helicon Focus 8 software was used for the stacked images. The following morphometric measurements were taken: diameter, depth (= height in the original description), external opening (= base of the tube), internal opening (= pseudostome) and invagination (= length of the tube). For each morphometric variable, we calculated following basic summary statistics: arithmetic mean, median (M), standard deviation (SD), standard error of mean (SE), coefficient of variation in % (CV), extreme values (Min and Max). Statistical analysis was conducted using the computer program STATISTICA Software, Version 10.0 (StatSoft 2010).
For the comparative analysis of the morphometric data, we used microscopic slides fixed with Canada balsam from two spatially distant locations in Bulgaria: Borisova Gradina area (litter and humus of oak forest (Quercus spp.)) and the area close to Mechata Dupka Cave on the path to Treskavets Hut (litter and humus of beech forest (Fagus sylvatica)) (unpublished data) (Fig. 1). Additional measurements were taken of single specimens from Eastern Rhodopes (Bulgaria; unpublished data) and North Korea (unpublished data).
Scanning electron microscopy analysis was conducted, as part of the specimens were extracted by using a glass micropipette, washed several times in distilled water, mounted on coverslip and air-dried. The shells were coated evenly with gold in a vacuum coating unit. The micrographs were obtained by using a JEOL JSM-5510, operating at 10 kV.

Biometry
The morphometric characterisations of C. puteus according to out studies are given in Table 1. Dimensions of the shell are: diameter 145-190 μm; depth 93-145 μm; external opening 42-65 μm; internal opening 28-44 μm and invagination 39-57 μm. All measured shell parameters are moderately variable (CV between 5.21 and 10.0%). The most stable characters in this population are shell's diameter and depth (5.21 and 7.41, respectively), while internal opening and invagination have maximal values (9.53 and 10, respectively). External opening has an intermediate value of 8.20.
Analysis of the size frequency distribution shows that C. puteus is a size-monomorphic species with a relatively well-expressed main-size class of the basic shell characters (Fig. 2). For example, about threequarters of all measured individuals (73%) have a diameter of shell within the limits of 160-180 μm, whereas only 17% have a diameter less than 160 μm and 10% -more than 180 μm ( Fig. 2A). Regarding the shell's depth and diameter of external opening, most of the measured individuals also varied within close limits: depth of shell 105-135 μm (91%) and diameter of external opening 46-64 μm (96%) (Fig. 2  B, C). The presence of a well-expressed main-size class of the basic shell characters and the lack of the subsidiary peaks (bell-shaped curves) indicate a normal distribution. The average values of these characters are respectively 169.4 ± 0.47 (diameter), 119.8 ± 0.47 (depth) and 55.4 ± 0.24 (external opening). They fully correlate with the main-size classes of characters and testify to the monomorphism of the species.
Our results were compared with the morphometric dimensions of the other two populations from Bulgaria (Table 1). Mean values indicate overall larger size dimensions of the Plachkovtsi population. When comparing variability of all characters, analysed populations have moderate coefficient of variation: diameter (between 4.63 and 7.29); depth (6.96-9.08); external opening (7.87-11.75); internal opening (9.41-10.03) and invagination (8.46-10 respectively for Borisova Gradina and Plachkovtsi; no invagination was measured for Mechata Dupka). The population with the lowest coefficient of variation was that of Borisova Gradina (with an exception of external opening), while Plachkovtsi's population has intermediate position.
Comparative analysis of the size frequency distribution confirms size-monomorphism of C. puteus, characterised by a main size class and a small size range. In the Fig. 3 is shown frequency analysis of shell diameter, which confirms larger dimensions of Plachkovtsi population, while "apparent" polymorphism of Mechata Dupka population is due to small number of measurements.
The above mentioned conclusions of comparative analysis are supported by the scatter plot of shell diameter versus shells depth (Fig. 4). All three populations have a linear relationship. Plachkovtsi and Mechata Dupka are characterised with moderate strength, while Borisova Gradina has almost horizontal slope that indicate a weak strength.
The summarised data of previous biometric records is given in Table 2. The results of the three examined populations correspond well with those given by other authors (Thomas 1960, Golemansky 1962, Bobrov & Mazei 2004, Vincke et al 2006, Todorov & Bankov 2019) and fall within biometrical framework of previous studies. Must be noted that in Bobrov & Mazei 2004 the values of diameter and depth are probably erroneously shifted.

Redescription
The original description of C. puteus (Thomas, 1960) is based on light microscopy with notes on the general appearance and structure of the pseudostome (Fig. 5). The number of measured individuals is not mentioned. The most comprehensive study to Mechata Dupka -------improve this taxonomic characterisation was conducted by scanning electron microscopy and gives first data of species ultrastructure (Todorov & Bankov, 2019). Nevertheless, the number of examined specimens weren't enough to scope the phenotypic plasticity range. In addition, the analysed data was from microscopic slides, where no information about degree of invagination was collected. The results of this study support and refine previous description. To clarify the description and avoid ambiguous terminology, we refer base of the tube (Thomas, 1960) and diameter of aperture (Todorov & Bankov, 2019) as external opening. Shell circular in ventral and dorsal views, hemispherical in lateral, yellowish or brownish, composed mainly of quartz particles (larger on the dorsal side) (Fig. 6), embedded in a layer of organic cement with numerous small pores on the ventral and lateral surface (Fig. 7). The outline has a smooth surface. Internal opening circular, central and situated at the terminal end of a strongly invaginated inside the shell conical tube. External opening with larger diameter. Different diameters of external and internal openings, form a very distinct double contour with slightly serrated borders due to the grained edge.
It must be noted that most of the records are based on single individuals or populations with very low density. This global distribution pattern must be interpreted with caution, given recent molecular studies that reveals species complexes with hidden (pseudo)cryptic diversity (Kosakyan et al., 2012, Singer et al., 2015 and different geographical distribution .
Additional data associated with geographical distribution is available at the Supplementary material 01 -Table A1 �.

Ecology
The type locality of C. puteus is a sandy soil of pine forest. Indeed, subsequent studies confirmed this species as inhabitant of forest litter, soils and mosses (Puytorac et al., 1970;Bonnet, 1977aBonnet, , b, 1979Bonnet, , 1987Bonnet, , 1992Chardez & Lambert, 1981, Lousier, 1982Golemansky & Todorov, 1991, 2000Balik, 1992Balik, , 1994Balik, , 1997Bobrov, 2001;Todorov, 2001, unpublished data;Bobrov & Mazei, 2004;Vincke et al., 2004;Bobrov et al., 2010;Payne et al., 2020). Records of solitary specimens came from calcimorphic soil on karst (Bonnet, 1992), humic soil without plant cover (Balik, 1992), humus collected from bamboo roots (Bonnet, 1987), flooded savannah (Bonnet, 1997), rhizosphere of epiphytic plants and area with cut off forest, which was subse-quently burned (Coûteaux & Chardez, 1981). Additional information about its ecological preferences is provided by Bonnet (1974), who registered it in both acidic and carbonated soils in Québec province, Canada. Accidental findings of C. puteus in Sphagnum mosses (Chardez et al., 1987;Golemansky & Todorov, 1991;Bankov, 2022) and freshwater habitats like a small swamp in rice field (Golemansky & Todorov, 1991) or forest stream (Golemansky & Todorov, 2000) may be due meteorological conditions like wind and/or heavy rain that brought single specimens from adjacent areas. Unfortunately, no differentiation has been made between live and dead specimens in previous studies, therefore the ecological characteristics of the habitats with single specimens or low population densities may not give us additional information about the ecology of the species. In the studied populations from Bulgaria, the number of living individuals varies between 30-40 %. Samples were taken from beech (Plachkovtsi and Mechata Dupka) and oak (Borisova Gradina) forests, confirming conclusion that Cyclopyxis puteus is a soil and litter-dwelling species with occasional findings in peat bogs and freshwater habitats. The next steps involve additional studies from different geographic areas to establish the phenotypic plasticity and ecological preferences of the species. Genetic characterisation is needed to provide evidences of its cosmopolitan distribution and/or the potential presence of (pseudo)cryptic diversity within Cyclopyxis puteus.