Check-list of microscopic protosteloid amoebae from the Southwest of Europe

Aguilar, M. & Lado, C. 2012. Check-list of microscopic protosteloid amoebae from the Southwest of Europe. Anales Jard. Bot. Madrid 69(2): 217-236. A biodiversity survey for protosteloid amoebae was carried out in Spain, Portugal and France between 2005 and 2010. Samples were collected from three different microhabitats – aerial litter, ground litter, and bark of living plants – in a total of 97 localities. As result 26 to 28 species out of the 33 species of microscopic protosteloid amoebae described to date were recorded. An annotated list that comprises all available data about these organisms from this part of the world, a key to the species, comments on the morphology of the sporocarps and the trophic stages is presented. Photomicrographs of the fruiting bodies of most species, and distribution maps

Their trophic stages vary from uninucleate amoeboid and/or amoeboflagellate cells to multinucleate reticulate plasmodia.Their major defining characteristic is that they form fruiting bodies or sporocarps.Sporocarps develop from a single amoeboid cell and, at maturity, are comprised of a single acellular stalk and one to a few spores (Olive, 1975;Spiegel, 1986Spiegel, , 1990;;Spiegel & al., 2004).They probably have an important role in the regulation of the populations of bacteria present in soils and other microhabitats in terrestrial ecosystems (Feest, 1987;Spiegel & al. 2004), where they take part as predators feeding also upon other decomposers such as yeasts, and filamentous fungi (Olive, 1975;Whitney & Bennett, 1984).These organisms were traditionally classified as occupying a primitive position within the group of slime molds termed Eumycetozoa, that also includes the myxomycetes and the dictyostelids (Olive, 1975;Spiegel, 1986;Spiegel Lee & Rusk, 1995;Baldauf & Doolittle, 1997), but recent molecular data suggest that protosteloid amoebae are polyphyletic and they belong to different groups of Amoebozoa (Shadwick & al., 2009b;Fiore-Donno & al., 2010;Lahr & al., 2011;Adl & al., 2012), not necessarily directly related to other eumycetozoans.
The Iberian Peninsula has previously proved to be an excellent location for other groups of slime moulds, such as Dictyostelids (Romeralo & Lado, 2006) and Myxomycetes (Lado & Pando, 1997), and its special features like an accentuated and varied relief, and its varied vegetation and climate produce a high diversity of ecosystems to be colonized by slime molds.It is also characterized by the longlasting influence of humans, constituting a mosaic of successional stages.The study of protostelids in such a wide variety of habitats can help to increase available information about their diversity patterns in areas with temperate climates.
We present here an annotated list of the microscopic protostelid species (i.e excluding Ceratiomyxa) recorded to date in the southwest of Europe that comprises all available data from this part of the world.Before the beginning of this survey there was no previous information about this group in the study area.This check-list is the result of several sampling efforts carried out between 2005 and 2010 by the authors.Some records have been previously published in Aguilar & al. (2007) and Aguilar & al. (2011); the remainder have not been previously published.

MATERIAL AND METHODS
Samples were collected between 2005 and 2010 in a total of 97 localities (Fig. 1, Appendix 1) in Spain (91), Portugal (4) and France (2).All localities were geo-referenced through the use of a portable GPS unit (model Garmin 12,datum WGS 84).Collections of samples were segregated according to microhabitat type -ground litter (layer of twigs, leaves, and other plant debris extending over the soil surface), aerial litter (assemblage of dead but still attached parts of standing plants) or bark.Samples were placed in separate paper bags, air dried and stored with the codes shown in Appendix 1 in the laboratory of the Real Jardín Botánico.More than 800 primary isolation culture plates were prepared using a modification of the technique described by Olive (1975) and Spiegel & al. (2004), see also Moore & Spiegel (1995), Spiegel & al. (2007) and Aguilar & al. (2011).The material was cut into small (ca.1.5-2 cm) pieces with sterile scissors.Thirty-two pieces from each sample were plated out in 8 lines of four pieces forming a circle on a 9 cm Petri dish with a weakly nutrient medium (wMY: 0.002g malt extract, 0.002g yeast extract, 0.75g K2HPO4, 15g agar/L of distilled water).The material was moistened with a pipette with sterile water just after they were plated out.The plates were incubated at 21 ºC and were surveyed for protostelids during the second week of culture.
Species were identified on the basis of fruiting body morphology under the light microscope using Spiegel (1990), Spiegel & al. (2007) and original descriptions.Nomenclature used herein follows Olive (1975) and Lado (2011).Photomicrographs were taken with a Nikon Eclipse 80i compound microscope using bright field optics and a Nikon Digital Sight DS-5M digital camera head.

TAXONOMIC TREATMENT
A total of 26-28 species of protosteloid amoebae were recorded.Species and comments are listed below.The number of species is given as a range because it is likely the presence of various species indistinguishable from their fruiting body morphology (see comments below).Measures and descriptions of trophic stages are based on existing literature, so we recommend consulting the original papers, Spiegel (1990), and Spiegel & al. (2007) for further information and illustrations.Organisms denoted with an asterisk (*) were previously published as new records for Europe in Aguilar & al. (2007) or Aguilar & Lado (2012), while species with two asterisks (**) are previously unpublished new records for the European continent.Distribution maps (Fig. 2) and photomicrographs of the fruiting bodies of most species (Fig. 3) are also provided.See also Appendix 1 for locality details and Table 1 species classification based on Spiegel (1990), Shadwick & al. (2009), Lahr & al. (2011), and Adl & al. (2012).

Figs. 2a, 3a
Sporocarps.Sporocarps 8-23 µm tall.Very short stalks, 3.2-10.5µm long, with a distinct, cup-like apophysis measuring from one third to more than one half of the total length of the stalk.The apophysis is usually wider than the base of the stalk, but sometimes it is narrower and the stalk seems to be equally thick for its entire length.Spores rough, colorless, nearly spherical, 4.8-12.6µm diam., with spines and warts on their surface.Prespore cells are circular in outline (Spiegel & al., 2007).
Trophic stages.It grows well on bacterial cultures including Serratia liquefaciens strain Florida 20 on wMY agar.The amoeboflagellates typically have one single nucleus and one to several contractile vacuoles, and their sub-pseudopodia are filose.When amoeboflagellates are in water, they can develop usually one but quite often two (more rarely three or four) flagella, cells mostly 5-13 × 14-32.5 µm (Olive, 1964).The amoeboflagellate state eventually develops into an obligate amoeba that is unable to produce flagella (Spiegel & Feldman, 1985).Obligate amoebae are uninucleate and flat, elliptical to fan-shaped, and typically angular in outline.They may form acutely pointed, narrow pseudopodia.When they move, they can be divided into branches (Spiegel & Feldman, 1985).The cysts are spherical to oval or somewhat irregular in shape, 4.3-23 µm in diam.(Olive, 1964).Encystment is infrequent in obligate amoebae.See Spiegel & Feldman (1985) for illustrations.
Though it is usually a common species in the tropics and relatively uncommon in temperate climates (Spiegel & al., 2007;Ndiritu & al., 2009), it was quite abundant in our study area.In Europe, it has been also cited from Russia (Kosheleva & al., 2009).Sporocarps.Sporocarps 20-42 µm tall, with two spores at the tip of a bipartite, recurved stalk.Stalk with a short apiculate base and a longer inflated upper portion that bursts to disperse the spores.The spores are smooth, hemispherical to subglobose, 7.2-12 µm in diam.(Olive & Stoianovitch, 1977a).Prespore cells circular in outline (Spiegel & al., 2007).
It is a relatively uncommon species that appears to be more frequent in the tropics (Spiegel & al., 2007), and we found it only once in our samples.

Echinosteliopsis oligospora
Trophic stages.It can be cultivated on hay infusion agar along with a food organism, like Flavobacterium sp., Escherichia coli, Aerobacter aerogenes, Serratia liquefaciens strain Florida, Dyadobacter sp.strain Malaya, and a mixture of Phoma conidiigena and Flavobacterium.The spore liberates a single amoeba which is quite broad in movement 28.6-62.1 × 34.5-89.7 µm, and has a distinct hyaloplasmic anterior margin.Posteriorly, fine filose projections are produced.The amoebae are usually uninucleate, but can have up to 4 nuclei with numerous peripheral small nucleoli.No flagellated cells have been observed.The sheath, the spore walls, and the cysts walls give a positive test for cellulose in chloriodide of zinc.The cysts are uninucleate to multinucleate and irregular in outline.See Reinhardt & Olive (1966), and also Lindley & al. (2006) for transmission electron micrographs.
It is common worldwide and sometimes locally abundant (Spiegel & al., 2007).It was also very abundant in some of our cultures.In Europe, this species has been reported previously from Germany (Tesmer & al., 2005) and Russia (Kosheleva & al., 2009).Sporocarps.Sporocarps very small, 19-26 µm long, twospored, with a seath that can be inflated with water in high hu-midity conditions or stuck to the spores in drier stages.Stalk short, 7-13.5 µm long, straight to gently curved, and with a pronounced taper from the base to the tip.The upper part of the spore is flattened.Spores 7-10 µm diam.One spore is directly attached to the stalk and the other is at the top.Prespore cells circular in outline (Spiegel & al., 2007).
Though it is common worldwide showing patches of high local abundance (Spiegel & al., 2007), it was not very common in our study.In Europe, it has been previously reported from Germany (Tesmer & al., 2005).This species was first described as a protostelid by Olive & Stoianovitch (1966a) but it was later included in the myxomycetes (Spiegel & Feldman, 1989;Whitney & al., 1982).However, as protosteloid amoebae are recognised solely on the basis of sporocarp morphology and not on any suggestion of relatedness (Shadwick & al., 2009b), E. bisporum can be considered as a protosteloid member of the myxomycetes.Sporocarps.Sporocarps 95-135 µm tall.Stalk 60-87.6 µm, broad, slightly tappered and with a distinct knob-like apophysis at the tip.Spores uninucleate, irregular, from elliptical to spherical in shape, 33.6-51.5 µm diam.(Olive & al., 1984).Spores appear to be strongly warted, maybe due to the presence of bacteria attached to the external surface of the spore wall.Prespore cells are circular in outline (Spiegel & al., 2007).

Specimens examined
Trophic stages.It grows on lactose-yeast extract and on oak bark pH 6 agar media with Flavobacterium sp.added.The amoeba typically contains a nucleus and a contractile vacuole, and it is uninucleate and surrounded by a sheath that contains small particles, and frequently bacteria.The cysts, 24-38.4µm, are typically globose, usually surrounded by a scabrous sheath (see Olive & al., 1984).
It is a rare species and has been recorded only a few times (Spiegel & al., 2007), and we found it only in one of our cultures.Sporocarps.Sporocarps brownish to yellowish.Stalk 47-120 µm long, beaded, having a chain-like appearance.Spore with a variable shape, from somewhat campanulate to elongated or irregular, 14.5-40.5 × 24-46.5 µm, sometimes with warts that appear to be bacteria stuck to the spore surface.Prespore cells are slightly ellipsoid to round in outline (Spiegel & al., 2007).

Specimens examined
Trophic stages.It grows and sporulates on various bacteria (e.g., Flavobacterium sp.) or on combinations of two food organisms such as Aureobasidium pullulans and a bacterium, or on two bacteria, according to preference of the particular isolate.Its trophic cells are nonpigmented, most frequently uninucleate but also plurinucleate, usually with a single large contractile vacuole.The amoebae are comparatively large, but they exhibit much variation in cell and nuclear size, and they can develop numerous filose pseudopodia in water.Plurinucleate protoplasts are not uncommon in some cultures, with their number of nuclei ranging from 2 to 16 or more.The cysts are very thin-walled, globose to subglobose or slightly irregular in outline, 22-40 µm diam.See Olive & al (1984).
It was originally described as Protostelium zonatum L.S. Olive & Stoian.(Olive & Stoianovitch, 1969).It is quite common worldwide and it is found frequently growing on substrates collected from a relatively dry habitat that is exposed to direct sunlight (Spiegel & al., 2007).It was rare but locally abundant in our cultures.Spores are compressed against each other forming together an ellipsoidal structure slightly flattened in the upper side, 12-18.5 µm in diam., and can be observed through the sheath.Prespore cells are circular in outline (Olive & Stoianovitch, 1977b).

Specimens examined
Trophic stages.It grows and sporulates on soft oak bark agar (at pH 6-6.6) or lactose-yeast extract agar (at pH 6), with a mixture of Flavobacterium sp. and Dyadobacter sp.strain Malaya.The amoebae are uninucleate and they usually have a single contractile vacuole.They have lobose pseudopodia with filose subpseudopodia.No flagellates have been ob-served.The cysts are spherical to subspherical, 10.8-20.4µm diam.See Olive & Stoianovitch (1977b).
It is an uncommon species worldwide (Spiegel & al., 2007), and it was very rarely found during present study.Two described species share this morphotype, but differ in details of ther life cycles.Studies on this complex must be carried out to clarify whether they are truly distinct (Spiegel & al., 2007).
Trophic stages.N. gracile can be cultivated on wMY agar with mixtures of Kitani yeast with Dyadobacter sp.strain Malaya, or of Cryptococcus laurentii with Malaya bacterium.C. tahitiensis grows on malt-yeast extract agar or hay infusion agar (pH 6-7.3) with Dyadobacter sp.strain Malaya and the Kitani yeast at room temperature or in an incubator at 23 ºC.They produce a thin, multinucleate, non-reticulate or reticulate plasmodium (Spiegel & Feldman, 1985).The plasmodium divides into irregular multinucleate masses before fruiting.C. tahitiensis in water produces anteriorly uniflagellate or occasionally bi-flagellate cells, with or without supernumerary flagella.N. gracile does not form flagellates.The cysts are round to irregular in outline.See Spiegel & Feldman (1986, 1991) for illustrations.
This species complex is very frequently found on samples.It is also common in temperate regions but it is almost absent at high latitudes, and above 2500 m (Spiegel & al., 2007).It was very common in our cultures.It has been previously recorded in Europe: in Germany (Tesmer & al., 2005) and in Russia (Kosheleva & al., 2009).Sporocarps.Stalks 30-220 µm long, thick and robust, with a distinct knob-like apophysis.Spores ovoid to ellipsoid, 10-18.5 × 13-29 µm in diam., deciduous, that have a distinct ringshaped hilum with a raised edge that fits with the apophysis of the stalk.Prespore cells round from above and hat-shaped from the side.

Specimens examined
Trophic stages.It grows on wMY agar on Flavobacterium sp or with mixtures of Kitani yeast with Dyadobacter sp.strain Malaya, or of Cryptococcus larentii with Dyadobacter sp.strain Malaya.When spores germinate, they produce a thin, multinucleate, branching to reticulate plasmodium, that divides into irregular multinucleate masses before fruiting.The cysts are round to irregular in shape.See Olive & Stoianovitch, (1966c).
This species was originally described as Schizoplasmodium ovatum L.S. Olive & Stoian..It is quite common in temperate areas, and less frequent but also abundant in tropical localities (Spiegel & al., 2007).It was locally common in the Iberian Peninsula.In Europe, this species has been previously recorded in Germany (Tesmer & al., 2005) and Russia (Kosheleva & al., 2009).(1 long and 1 short).The amoeboid cells have 1 or a few nuclei, but up to 21 nuclei have been observed within a single cell.The amoebae are flattened or slightly raised, and circular to elliptical in outline (Spiegel & Feldman, 1985).They do not return to the flagellate stage when placed in water.It has also been observed a vermiform stage, but it is less common than in other members of the genus.The cysts contain 1-5 nuclei, and are globose to oval or occasionally irregular.See Spiegel & Feldman (1984) and Spiegel & al. (1986) for illustrations.

Specimens examined
Our material shows sporocarps bearing two spores in most cases, but also four-spored sporocarps were observed.They grow frequently on bark and wood.It appears to be a species that is often associated with arid habitats, and it occurs at higher elevations (>3000m) than most protostelids (Spiegel & al., 2007).It was fairly common in our cultures.In Europe, it was previously found in Russia (Kosheleva & al., 2009), France, and England (Olive, 1975).Trophic stages.It grows and sporulates on oak bark agar (pH 8), with Dyadobacter sp.strain Malaya bacterium and a moniliaceous fungus (Goetricum sp.).The fungus is generally necessary for sporulation, but it may also sporulate in the vicinity of Penicillium sp.Single spores give rise to 8 flagellate cells on germination, while spores in pairs produce 4 flagellate cells.Flagellate cells typically have a single anterior flagellum, but occasionally two of equal length are present, and only rarely it is possible to find a short flagellum paired with the longer one.Pseudoflagella (ephemeral filose extensions of the flagellate cell) are commonly seen.Plurinucleate protoplasts that do not develop flagella or become reticulate may be found in cultures several days after spore germination.

Specimens examined
M. Aguilar & C. Lado Protoplasmodia divide by plasmotomy, which tends to limit their size and nuclear number.Under certain conditions, the plurinuceate protoplasts become converted into worm-like structures.This vermiform phase has an almost segmented appearance, and undulates changing the shape of the swellings constantly.At one or both ends of the protoplast there are knob-like areas with short filose pseudopodia.Cysts round to oval or somewhat irregular, 16.3-33.8 × 22.5-53.8µm.See Olive & Stoianovitch (1972).This is a very uncommon species and it is usually found on bark of living trees, sometimes forming dense patches (Spiegel & al., 2007), and it was found only once during this study.4.3-5.5 × 5.3-7.5 µm, in groups of four, forming structures, 7.5-11 in diam.(Olive & Stoianovitch, 1972).Prespore cells unknown.

Specimens examined
Trophic stages.It grows on malt-yeast extract agar with an unidentified bacterium, isolated from hickory bark, as its food source.The species has a restricted pH tolerance in culture and fails to grow if the pH deviates significantly from 5.1.Each spore germinates giving rise to 2 flagellate cells.The trophic stage consists of uninucleate or plurinucleate ameboid cells, that can produce filose pseudopodia when the cells are placed in water.They can also form a vermiform stage, readily reversible to the flattened ameboid phase.The flagellate cells commonly have either 1 long anterior flagellum, paired with a short reflexed one, or a pair of long flagella.The short flagellum tends to lie against the side of the cell and usually is inconspicuous.Pseudoflagella commonly appear at the apical end of the cell and migrate to the posterior end where they disappear.The nucleus, containing a small central nucleolus, is situated in the more or less hyaloplasmic anterior 1/3 of the cell.The cysts are globose, oval, oblong, or occasionally irregular, 10-27 × 13.8-35 µm.See Olive & Stoianovitch (1972).
It is an uncommon species found in most cases growing on bark of living trees or on rotting wood (Spiegel & al., 2007), and it was found only once during this study.
Trophic stages.It grows on lactose-yeast or wMY agar, with Flavobacterium sp..The fanshaped amoebae have filose subpseudopodia and a single distinct nucleus.They present a contractile vacuole, and a scalloped, hyaline anterior border when migrating across the agar surface.The microcysts are spherical and thin-walled, 10-2.8 µm diam.See Olive (1962).
This fairly common species is more abundant in the tropics than in temperate areas.It probably represents a species complex and is unlikely to be a member of the eumycetozoans (Spiegel & al., 2007).It was quite uncommon in our cultures, and it has been previously recorded in Europe in Germany (Tesmer & al., 2005) and Russia (Kosheleva & al., 2009).This species has been excluded from the genus Protostelium (Spiegel & al., 1994), but has yet to be assigned a new genus (see Table 1).Sporocarps.Most individuals have sporocarps that move easily in air currents, others have piliform sections in their stalks that float and curl continuously, even in the absence of evident air currents, while others present stiffer stalks.Stalks less than 70 µm long, tapered at maturity, flexuous, and flexible, often presenting a small apophysis at their tips.Spores spherical to slightly obpyriform, 8.8-13.8µm in diam., smooth (Olive & Stoianovitch, 1969).Prespore cells are elliptical when viewed from above.

Specimens examined
Trophic stages.The amoebae are reported to feed upon bacteria (Flavobacterium sp. and Aerobacter aerogenes) as well as fungi, Rhodotorula mucilaginosa and Cryptococcus laurentii (Olive & Stoianovitch, 1969).However, according to F.W. Spiegel (personal communication) many strains will not fruit if any bacteria are present in culture.They are uninucleate, with a prominent nucleolus in interphase.Amoebae contain one to three prominent contractile vacuoles and numerous pink to orange lipid droplets.Migrating amoebae produce broad, lamellate pseudopodia, with some blunt to acutely pointed subseudopodia extending from them.More elongated, pointed subpseudopodia are found under wetter conditions.The shape of the amoebae varies from irregularly circular to elongate to occasionally flabellate.Amoebae may move by the gently eruptive production of pseudopodia that subsequently appear to pull the cell along, or they may glide along the substratum.Gliding is faster than pseudopodial crawling.They have distinct three-dimensional relief when viewed on the surface of a culture plate (Spiegel & al., 1994).

Protostelium nocturnum
Trophic stages.It grows on wMY agar or hay infusion agar with Xanthomonas fragariae (Fla-20 isolate of Olive) or Rhodotorula mucilaginosa, and on CM+ agar with Rhodotorula.It grows well but fruits poorly on CM+ with X. fragariae.The amoebae are small, uninucleate, with a single central nucleolus, and one or more contractile vacuoles and many food vacuoles.Orange pigmented lipid droplets are also present.They are relatively smooth in outline on dry agar, but acutely pointed pseudopodia and lamellopodia become increasingly prominent as the medium becomes more liquid.The microcysts are spherical or ellipsoidal.See Spiegel (1984).
This species fruit most heavily after sunset until early morning.It is relatively common worldwide (Spiegel & al., 2007) and also common in the Iberian Peninsula.This species has been previously cited in Europe for Germany (Tesmer & al., 2005).Sporocarps.Sporocarps 15-25 µm tall, ballistosporous.Stalk bipartite, with two segments separated by an articulation, apophysis spherical to ovoid present.Spore nearly spherical, (7.2)9.5-10.5 µm in diam.When intact, the spore and the apophysis flag at the articulation point.The spore is actively shot with the disappearance of the apophysis and only the rigid basal portion of the stalk remains, resembling "beard stubble".Prespore cells are elliptical (Spiegel & al., 2006).
It is a rare and recently described species, and it was found only two times during our study.
It is usually a common species, more abundant in the tropics than in temperate regions (Spiegel & al., 2007), but it was not very common in this study.In Europe, this species has been previously reported from Germany (Tesmer & al., 2005), and Russia (Kosheleva & al., 2009).This species is probably excluded from the genus Protostelium and has to be assigned a new genus (see Table 1).Sporocarps.Sporocarps 18-30 µm tall.Stalks 6-14.4 µm, straight, suddenly thinner towards their apex forming a sharp point.Spores nearly spherical, 12-22 µm in diam., single, proportionally big, globose, uninucleate, and non-deciduous, with a minutely punctate surface (Olive & Whitney, 1982).Prespore cells oval to round in outline.

Specimens examined
Check-list of protostelids Trophic stages.It can be cultivated on oak bark agar made with three times the usual amount of oak bark, on supplemented cornmeal agar with half the usual amounts of dextrose and yeast extract, or on wMY agar, grown with bacteria isolated from the original substrate, Escherichia coli, Flavobacterium sp., or Serratia liquefaciens strain Florida 20 and Dyadobacter sp.strain Malaya bacteria as food organisms.The amoebae are thin, uninucleate (sometimes binucleate) and they produce many filose subspeudopodia.They can fuse to produce large, sometimes reticulate plasmodia, but with no nuclear fusion observed.The cysts are round to irregular or reticular, 10-175 × 10-475 µm.See Olive & Stoianovitch (1975).
It is very rare but has been encountered worldwide (Spiegel & al., 2007), and it was identified in only one of our cultures.In Europe, it has been recovered from cultures from Russia (Kosheleva & al., 2009).Spiegel et al. (1995) suggested that this may possibly be a morphotype of Tychosporium acutostipes.
Trophic stages.They can be cultivated on hay infusion agar or wMY agar with a mixture of Flavobacterium sp. and Escherichia coli, or Serratia liquefaciens strain Florida 20.Spore germination liberates a single, uninucleate, elongate amoeba, with long and filose pseudopodia.The amoebae can form plasmodia by nuclear division with no plasmotomy, or by fusion of small amoebae.Plasmodia are often branched and anastomosing and may be several millimeters wide.They fragment into uninucleate prespore cells or into cysts.See Olive (1967).
It is one of the smallest but most frequent species, and tends to fruit in big, dense patches.This organism is one of the most common protostelids worldwide, very frequently found in temperate and tropical regions (Spiegel & al., 2007), and it was also very abundant in our samples.In Europe it has been cited from Germany (Tesmer & al., 2005), Ukraine (Glustchenko & al., 2002) and Russia (Kosheleva & al., 2009).Sporocarps.Sporocarps very variable in length, 45-110 µm.Gracile stalks, 38-90 µm long.Spores spherical, 7-20 µm in diam., with a reticulum of ridges on their surface (Olive & Stoianovitch, 1975).Prespore cells round, formed from single amoebae or segments of plasmodia.

Specimens examined
Trophic stages.It can be cultivated on oak-bark agar (Olive, 1975) or wMY agar, in association with bacteria isolated from its original substrate or bacterium Serratia liquefaciens strain Florida 20.The spores produce a single uninucleate amoeba, which is branched with numerous, often branched filose subpseudopodia.They can fuse to produce large plasmodia but no nuclear fusion is observed.The cysts are globose to oblong or irregular, 7-32 × 7-58 µm.See Olive & Stoianovitch (1975).This is a relatively rare species, but it is widespread.It occurs in situations wherever S. vulgare is likely to be found (Spiegel & al., 2007).It was found only once during our study.

Figs. 2j, 3t
Sporocarps.Stalk relatively thick and very variable in length, 9-70 µm long, tapering to a blunt apex but not tapering to a fine point.Spores, 8-16(37) µm in diam., nearly spherical and coarse, with low ridges formed by a reticulum of spore wall thickenings that appear as slight bumps (Olive & Stoianovitch, 1975).Prespore cells circular in outline, and usually many of them are formed simultaneously due to fragmentation of the plasmodium.
Trophic stages.It can be grown on hay infusion agar, oak bark agar, lactose-yeast agar or wMY agar on association with pregrown bacterium Florida 20 or Flavobacterium sp..Each spore gives rise to a single, thin, branched amoeba with filose subpseudopodia and several contractilve vacuoles.They can fuse to produce large plasmodia with no nuclear fusion observed.The cysts are round to irregular, uninucleate to plurinucleate, 5-66 × 7-300 µm.See Olive & Stoianovitch (1975).
This species has been cited in Europe for England (Olive 1975b), Germany (Tesmer & al., 2005), and Russia (Kosheleva & al., 2009).It is a common species worldwide, and in cool, moist habitats, it is often one of the few species encountered (Spiegel & al., 2007).It was quite common in our cultures from the Iberian Peninsula.Sporocarps.Sporocarps ballistosporous.Stalk relatively short, 4.3-8 µm, thick, with a distinct goblet-shaped apophysis.Spore almost spherical, relatively big, 11-20.5 µm in diam., smooth, typically multinucleated, attached to the stalk by a ring-shaped hilum that fits the apophysis (Olive & Stoianovitch, 1966b).Just before the spore discharge, a swelling of the sheath, interpreted as a gas bubble or a liquid droplet, appears.Prespore cells are round from above and hat-shaped from the side.

Specimens examined
Trophic stages.It grows well on hay infusion agar with Kitani, or mixture of Kitani and Dyadobacter sp.strain Malaya bacterium.It forms plasmodia that can be reticulate, eventually fragmenting into few to many multinucleate prespore cells.The cysts are very variable in size and shape, 12-30 × 13.5-50 µm.See Olive & Stoianovitch (1966b).
Check-list of protostelids It is a fairly common species in temperate areas and also common in the tropics (Spiegel & al., 2007), and was not uncommon in present study.This species has been reported previously only from Germany (Tesmer & al., 2005) in Europe.Sporocarps.Sporocarps 32.5-45 µm tall, ballistosporous.Stalk bipartite with a broadly tapered basal section and a uniformly thin apical section, usually sharply reflexed at the junction of the two sections.Spores spherical, single, 11.3-17.4µm diam., forcibly discharged with the disappearance of the stalk (Olive & Stoianovitch, 1981).Prespore cells "fried-egg" shaped.

Specimens examined
Trophic stages.It can be cultivated on oak bark agar (at pH 7) or wMY with Xanthomonas sp. and Dyadobacter sp.strain Malaya as food organisms.It produces amoeboid and nonflagellate cells, usually flabellate during migration.They are typically uninucleate, and occasionally binucleate.Their nucleolus is irregular and often multilobed.The cysts are round, 9-33.6 µm diam., oval, or irregular in shape, 7.2-22.8× 9.6-26.4µm, uninucleate.The nucleolus of the cyst nucleus generally has a more regular shape than those of amoeboid cells.See Spiegel & al. (1994).
It was originally described as Protostelium expulsum L.S. Olive & Stoian.It is not uncommon and somewhat more abundant in the tropics than in temperate habitats (Spiegel & al., 2007), but it has been found only once during our study.Sporocarps.Spores nearly spherical, 13.9-22.5µm in diam.Stalks proportionally long, 30-127 µm, straight, gently tapered with a hastate apophysis (Olive & Stoianovitch, 1969).Spore deciduous, that can adhere to the side of the stalk after falling, and becomes "American football"-shaped when dryed.Prespore cells are "fried-egg" shaped.

Specimens examined
Trophic stages.It feeds on Flavobacterium sp. or Escherichia coli.Amoebae typically uninucleate (but also plurinucleate).The nucleolus is divided into many small, phase dense subunits, and the nucleus may be irregular in outline.The amoebae are very thin, almost invisible on the agar surface when viewed with bright field optics, and slightly bigger than those of S. expulsum.The amoebae have numerous, small contractile vacuoles and many food vacuoles when they are feeding.They are typically flabellate when migrating, with a broad lamellopodial front.There are numerous acutely pointed subseudopodia which can become quite elongated under very moist conditions.Motility appears to be solely by pseudopodial crawling and gliding has not been observed.See Spiegel & al. (1994).
It is one of the most common species in temperate areas and worldwide (Spiegel & al., 2007), and it was also quite common in our cultures.In Europe, this species have been cited from Germany (Tesmer & al., 2005) and Russia (Kosheleva & al., 2009).Sporocarps.Stalks (6.9)35-64 µm long, with a somewhat undulate surface, stiff, gradually thinner towards their apex, sharp-poited, undulated.Spores turbinate to nearly spherical, 8.0-12.5 µm in diam., uninucleate, relatively indeciduous (Spiegel & al., 1995), sometimes "American football"-shaped when dryed.In air currents spores can incline to one side, but remaining attached to the stalk.Prespore cells are ellipsoidal.

Specimens examined
Trophic stages.It grows and fruits well on wMY agar with either Fla-20 or Flavobacterium sp. as its food source.The amoebae are typically uninucleate and unpigmented, though they may become plurinucleate in older cultures.See Spiegel & al. (1995).
Though it is usually a relatively uncommon species, it is found worldwide (Spiegel & al., 2007) and was abundant in our cultures.In Europe, this species has been cited for Germany (Tesmer & al., 2005) and Russia (Kosheleva & al., 2009).APPENDIX 1

Specimens examined
Sampled localities and their characteristics.