The material for the present study (an aggregate sample of about 500 g) was collected in October 2013 from the organic horizon (litter, twigs and woody material) of rainforests at the Maromizaha Protected Area (Madagascar). This Protected Area is located in Central Madagascar, east of the capital Antananarivo and south of the village Anevoka, on the eastern slopes of the Central Highlands (18°57'S, 48°27'E, 950 m a.s.l.). At the laboratory, the material was dried for one day in a thermostat at 60˚C. Then was soaked and mixed in chlorinated tap water for about ten minutes, after which was filtered through a sieve with 500 μm mesh to remove large organic and mineral particles. The resulting filtrate was allowed to precipitate for two hours, the sediment was removed and the shells that floated on the surface were collected for examination. The study was carried out 12 hours after the flotation, when the gas bubbles inside the shells completely disappeared and the structure of the shells was well visible. All filtrate was examined in a petri dish at 100X magnification with Stereomicroscope “STEMI” Citoval-2 (Carl Zeiss Jena). A total of 81 individuals of L. callistoma were determined and isolated. The basic morphometric characters of 75 of them were measured by an optical microscope “Amplival” (Zeiss-Jena, Germany) at 400X magnification. The isolated specimens were transferred in a drop of glycerol in order to maintain them in a given position during the measurements.

Remaining six specimens were studied by scanning electron microscope (SEM). They were extracted using a glass micropipette, washed several times in distilled water, and then individual shells were positioned with a single-hair brush onto a small drop of Araldite on a previously cleaned 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.

The biometric description was made according to Schönborn et al. (1983). Frequency distribution analysis was carried out in order to describe variation of characters. Statistical analysis was performed using the computer program STATISTICA, version 7.0 (StatSoft 1999).

Until present, data on the ultramorphology and biometry of L. callistoma are almost entirely lacking. In the period from its description to the present time, this species was found only in four countries - Ivory Coast, Papua New Guinea, Philippines and Indonesia (Bonnet 1974, Bonnet 1976, Bonnet 1980a, Bonnet 1980b, Bonnet 1992). Data on the species sizes are given only in its original description. On the basis of measurements of 18 specimens the author gives the next sizes: average shell diameter (Mean ± SD = 175 µm ± 8.1), shell depth (2/3 of the diameter) and apertural invagination (1/3-2/3 of the depth). The morphological data and illustrations of the species are also given only in its original description. In other three publications, where L. callistoma was recorded from Papua New Guinea, Philippines and Indonesia data on the species dimensions and illustrations are not given. However in these papers the author published information on the ecology and biogeography of the species and indicated that it is characteristic inhabitant of the humus of acid forest soils. In the biogeographical aspect, the species has been referred to the association of species of the “Gondwanan-tropical” group of soil testate amoebae. In some other publications (Bonnet 1981a, Bonnet 1981b, Bonnet 1984) discusses mainly the biogeographical and palaeogeographic significance of soil testate amoebae. He also included in these discussions information and illustrations about L. callistoma based on his previous studies. In these publications the author traces the line Cyclopyxis-Lamtopyxis-Distomatopyxis in an evolutionary aspect, emphasizing the trend of flattening on the teeth and reducing of pseudostome. He separated species of the genus Lamtopyxis on the basis of the number of teeth and their flatness, as well as of the shape of the internal apertural opening. In almost all papers the author indicated that L. callistoma is usually with four flattened teeth and oval internal apertural opening. From the foregoing it is seen that data on the morphology and biometry of species are quite scarce, and in fact the illustrations of the species are based mainly on the drawings, except LM photos of ventral surface of L. callistoma given by Bonnet 1979a, Bonnet 1981a.

Biometrical characteristic of the species, based on relatively rich material, was made. In addition to the diameter of the shell, six other shell characters were described biometrically for the first time. Our data for the shell diameter (Mean ± SD) fully corresponds to those given by Bonnet (1974) in the original description of the species (175.8 ± 7.3 (n=75) and 175 ± 8.1 (n=18), respectively). Furthermore, it should be noted that three of the characters measured show high variability (length of teeth, large and small axis of the internal opening). The variability of the last two characters is the reason for the great variability in the shape of the internal opening – from a broad oval to nearly circular.

Surprisingly, our study showed that the population of L. callistoma from Madagascar is comprised of both, specimens with four teeth and specimens with three teeth, in ratio of about 60% to 40%. This largely contradicts to the conclusion of Bonnet, who in most of his works emphasized that this species has four teeth. However, even in the original species description the author pointed at that, though rarely, specimens with five teeth have also occurred. Moreover, in his publication for the soil testate amoebae of Papua New Guinea, Bonnet (1980a) stated that in the populations of L. callistoma and L. cassagnaui specimens with two teeth can be observed very rarely. From the above facts it is seen that the number of teeth cannot be the most reliable taxonomic character for the species separation in the genus Lamtopyxis. Moreover, the individuals with three teeth of the external opening are the most typical and are found in all five species of this genus. Maybe for L. callistoma specimens with 4 teeth are more frequent, but specimens with three, five and rarely two teeth can also be observed. So, the separation of the species must be made on the base of a complex assessment of many characters: diameter and depth of shell; number, structure and shape of the teeth; sizes and shape of internal aperture. In this regard, from the five known species of the genus Lamtopyxis the most distinctive is L. sarocchii, with its largest dimensions (Dm = 278-290 µm) and the structure of the apertural opening which is closer to the genus Cyclopyxis than to genus Lamtopyxis (Coûteaux and Chardez 1981). L. travei has similar sizes as L. callistoma (Dm = 168-171 µm), but differs from the last by its characteristic elongated shape of the internal opening and by less-developed teeth of the external opening (Bonnet 1977b). The other two species – L. cassagnaui and L. trifoliata are characterized by three highly developed, flattened and sometimes nearly touching teeth on the external opening and differ from L. callistoma by significantly smaller dimensions – an average Dm of about 100 µm (Bonnet 1977b, Bonnet 1979a). L. trifoliata has a similar elliptical shape of the internal opening as L. callistoma, while L. cassagnaui differs significantly from all other species of the genus by its triangular shape of the internal opening.

Another interesting feature observed in our SEM studies of L. callistoma is that the thick layer of organic cement, of which the apertural shell surface is composed, extends over the teeth which are made mainly by organic cement. This finding differs from that in the original description of the species, where it is stated that the opening in the base of apertural invagination is surrounded by four large, thickened and rounded “siliceous” teeth.

In respect to the biogeography of L. callistoma it is evident that this is a very rare species of the Gondwanan-tropical group of testate amoebae, which has been found only in soils of tropical forests from countries situated south of the Tropic of Cancer. Despite numerous studies on soil testate amoebae carried out in the Northern Hemisphere, it has never been reported from Europe, North America and Asia. Taking into account the restricted geographical distribution, the large sizes and characteristic apertural morphology of L. callistoma we assume that this species, like some bryophilic ‘Nebelas’ with circumaustral distribution (e.g. Apodera vas, Alocodera cockayni, Certesella certesi, Certesella martiali, etc.), can be used as an example that in free-living microbial eukaryotes ‘not everything is everywhere’.