[ref. ID; 5753 (Frederick C. Page & Richard L. Blanton, 1985)]

Sarcodina

Superclass Rhizopodia

Class Heterolobosea n. cl.

Diagnoses

Amoebae cylindrical, monopodial, usually moving with more or less eruptive, hyaline bulges; closed intranuclear orthomitosis; temporary flagellate stages in majority of genera; mitochondrial cristae flattened, often disc-like; Golgi system not organised as stacks of flattened cisternae.

Order Schizopyrenida Singh, 1952

Diagnoses

No fruiting bodies formed.

Family Vahlkampfiidae Jollos, 1917; Zulueta, 1917

Diagnoses

Nucleolus forming polar masses in mitosis (promitosis); known species normally uninucleate, except in one genus, sometimes with strong tendency to supernumerary nuclei; flagellate stages in all but two genera.

Genus

Vahlkampfia, Pseudovahlkampfia, Naegleria, Adelphamoeba, Paratetramitus, Tetramitus, Heteramoeba, Tetramastigamoeba.

Family Gruberellidae n. fam.

Diagnoses

Nucleolus disintegrating during mitosis; known species usually multinucleate; no flagellate stage known.

Genus

Gruberella

Order Acrasida Schroter, 1886, emend.

Diagnoses

Fruting bodies formed.

Family Acrasidae Van Tieghem, 1880, emend. L.S. Olive, 1970

Diagnoses

Nucleolus forming polar masses or disintegrating during mitosis; amoebae typically uninucleate, but cells with supernumerary nuclei common in some species; flagellate cells in some strains or species; cells or sorocarp differentiated into morphologically distinct spores and stalk cells.

Genus

Acrasis, Pocheina.

A) Relationship between the orders

The separation of non-fruting and fruting amoebae a high taxonomic levels (class and above) is a relic of the treatment of slime moulds fungi. The slime moulds (as Eumycetozoa and Acrasea) are now poperly considered protozoa (Olive 1975; Levine et al. 1980), but the two classes are still artificial groups classifying together clearly unrelated organisms (Olive 1975). Organisms like the sorogenic ciliate Sorogena stoianovitchae demonstrate that 'true' protozoa can form fruting bodies (Blanton and Olive 1983). Similarly, a variety of amoeboid organisms have evolved the ability to form fruting bodes. Instead of grouping them on the basis of a character possessed also by non-amoeboid organisms (fruting body formation), it is more logical and useful to classify these fruting amoebae on the basis of their trophic stage. Olive (1975) did this when he separated the Acrasea from the Eumycetozoea. We have gone one step further by aligning the Acrasida with the non-fruting Schizopyrenida. The evidence for placing Acrasida and Schizopyrenida in the same class necessarily relies on the trophic cells. Acrasia and Pocheina amoebae are very similar to those of the Schizopyrenida. Their locomotive pattern is similar, they lack dictyosomes, their mitochondria have discoid cristae and the mitochondria are surrounded by rough endoplasmic reticulum. One noticeable difference between acrasids and schizopyrenids is that the amoebae of the former have pink pigmentation. (Acrasis granulata spores were described as brownish-violet, but no indication was given of the pigmentation of the amoebae (Van Tieghem 1880).) Flagellate cells are produced by some strains or species of Pocheina and Acrasia. These superficially resemble most vahlkampfiid flagellates in having a distinct spiral to the cell body and two apical, non-mastigonemate flagella of equal length (Olive and Stoianovitch 1974; Olive et al. 1983; F.W. Spiegel, personal communication). The ultrastructure of these acrasid flagellate cells will be of great interest. The fate of the nucleolus during mitosis differs somewhat among different acrasids as well as between the two families placed in the Schizopyrenida and between most Acrasida and most Schizopyrenida. However, this character does not appear fundamental compared with the intranuclear origin of the spindle and the persistence of the nuclear membrane in mitosis, characters in which all Heterolobose agree.

B) Characters of families

1) Vahlkampfiidae

Characters of the Valkampfiidae and component genera were summarised by Page (1974) and Darbyshire, Page and Goodfellow (1976), and some further comparisons with the Hartmannellidae were made later (Page 1978). Recently a comparison of flagellate stages supported a relationship of those genera (Balamuth et al. 1983). The present study of 11 species (Adelphamoeba galeacystis CCAP 1506/1, Heteramoeba clara CCAP 1536/1, Naegleria gruberi CCAP 1518/1e, Paratetramitus jugosus CCAP 1588/3a, Tetramitus rostratus CCAP 1581/1, Vahlkampfia aberdonica CCAP 1588/4, Vahlkampfia avara CCAP 1588/1a, Vahlkampfia damariscottae CCAP 1588/7, Vahlkampfia enterica CCAP 1588/5, Vahlkmapfia inornata CCAP 1588/2, Vahlkampfia ustiana CCAP 1588/6) belonging to six genera confirms the homogeneity of this family, with some reservation for Heteramoeba. The characteristics of the Vahlkampfiidae as now known (not all included in the formal diagnosis) can be summarised thus, with emphasis on the amoeboid stage:

1. Flagellae stage in all genera except Vahlkampfia, which is distinguished by absence of that stage, and apparently Pseudovahlkampfia (Sawyer 1980).
2. Promitotic nuclear division, a type of closed intranuclear orthomitosis with division rather than disintegration of the nucleolus.
3. Mitochondrial cristae flattened, usually discoid.
4. Golgi system not organised as stacks of flattened cisternae.
5. Rough endoplasic reticulum usually closely associated with mitochondria (This is not simply part of the encystment process as apparently suggested by Maitra et al. 1974. V. damariscottae and G. flavescens are non-encysting marine amoebae).
6. Amoeboid locomotion usually eruptive.
7. Mean length/breadth ratio of amoebae usually less than 3.0.
8. Maximum relative locomotive rate (um per min: length) often more than 4.0.
9. Supernumerary nuclei (i.e. two or more) more common than in most uninucleate species of amoebae.

Heteramoeba is included in this family despite the divergence of its mitochondrial cristae from those of other vahlkampfiids. The lamellar nature of those cristae ranges the genus with the Heterolobosea rather than the Lobosea. It is aberrant in other ways such as its reported sexuality (Droop 1962), but its similarities to other vahlkampfiids (Carey and Page, in press) suggest a close affinity. Perhaps it is transitional between this group and others. The inclusion of Pseudovahlkampfia Sawyer, 1980, and Tetramastigamoeba Singh and Hanumaiah, 1977, is base entirely on light microscopy. Singh and Hanumaiah (1977) reported division in the flagellate stage of Tetramastigamoeba, which thus resembles Tetramitus and Paratetramitus in that respect, though they found no cytostome.

2) Gruberellidae

The multinucleate marine amoeba Gruberella flavescens strains 301 and 302, with no known flagellate stage, was previously (Page 1983, 1984) left incertae sedis, largely because the disintegration of its nucleolus during mitosis distinguished it from all Vahlkampfiidae, just as a similar phenomenon distinguished Acrasis rosea from the vahlkampfiids (Page 1978). However, the intranuclear origin of the mitotic spindle and the persistence of the nuclear membrane have been demonstrated in Gruberella (Page 1984) as in Naegleria (Schuster 1975), Tetramitus (Balamuth et al. 1983), and Heteramoeba (Carey and Page, in press). The fate of the nucleolus is here considered less fundamental than the origin of the spindle and is retained only as a familial character. A new family Gruberellidae is added to the order Schizopyrenida, sharing other characters of the Vahlkampfiidae. Any uninucleate schizopyrenid in which the nucleolus disintegates during mitosis would also be classified in the Gruberellidae. Multinuclearity is not itself a familial character, since multinucleate genera are justifiably included with uninucleate ones in the families Amoebidae (Page 1976), Thecamoebidae (Page 1981), and Vahlkampfiidae (Sawyer 1980).

3) Acrasidae

The characteristics of the Acrasidae can be summarised as follows, based on observations of the amoeboid stage. Pocheina flagellata amoebae were not available to us but can be assumed to be similar to those of P. rosea (R.L.B. strain CB 84-1, isolated from bark of a Sequoiadendron giganteum on the grounds of the Institute of Astronomy, University of Cambridge). Acrasis granulata, the type species of the genus, has only been isolated and described once (Van Tieghem 1880). The original description gave no information about the amoebae.

1. Flagellate stages are not common in the Acrasidae. They have been described from Pocheina flagellata (Olive and Stoianovitch 1974; Olive et al. 1983) and from an Acrasis isolate (F.W. Spiegel, University of Arkansas, personal communication, but the isolate could not be grown in culture).
2. Nuclear division has been studied only with the light microscope. In nuclear division of Acrasis rosea and Pocheina flagellata, the nuclear envelope remains intact, but the nucloelus disintegrates (Page 1978; Olive et al. 1983). Pocheina rosea has 'promitosis', i.e., both the nuclear membrane and the nucleolus persist (Olive et al. 1983).
3. Mitochondrial cristae flattened, usually discoid.
4. Golgi system not organised as stacks of flattened cisternae.
5. Rough endoplasmic reticulum usually closely associated with the mitochondria.
6. Amoeboid locomotion usually eruptive.
7. Mean length/breadth ratio of amoebae usually less than 3.0 (Page 1978).
8. Maximum relative locomotive rate (um per min: length) usually more than 4.0 (Page 1978).
9. Supernumerary nuclei are not common in Acrasis rosea (Page 1978); they are common in Pocheina rosea, with as many as 34 nuclei in some cells (Olive et al. 1983); and they are common but not as numerous (up to six observed) in P. flagellata (Olive et al. 1983).
10. Pocheina rosea, P. flagellata, and Acrasis rosea amoebae have pigmented (pink to orange) cytoplasm.

The Acrasida as here delimited are a sound, probably monophyletic group. There have been several discussions of the relatedness of Acrasis and Pocheina (Olive 1970, 1975; Blanton 1982; Olive et al. 1983), and the recent discovery of a flagellate Acrasis (F.W. Spiegel, personal communication) emphasises further the close relationship between these genera.

C) Similar but excluded organisms

Some organisms sharing certain similarities with the Schizopyrenida must be excluded from the Heterolobosea. Nolandella hibernica (Page 1980, as Hartmannella hibernica; 1983) has eruptive locomotive behaviour and mitochondria enveloped in rough endoplasmic reticulum. However, its mitochondrial cristae are tubular, it has a prominent dictyosome, and it mitotic pattern (examined with the light microscope) differs from that of schizopyrenids. Sappinia diploidea (Hartmann and Nagler 1908) has mitotic pattern (not studied with the electron microscope) superficially resembling promitosis (Hartmann and Nagler 1908; Goodfellow, Belcher and Page 1974), but the mitochondrial cristae are tubular and there are other important resemblances to the genus Thecamoebae, though dictyosomes were not found in the first electron microscopical study of Sappinia (Goodfellow, Belcher and Page 1974). The order Acrasida has been strictly defined and limited to the family Acrasidae in order to be included in the Heterolobosea. The family Copromyxidae is excluded because of its members' tubular cristae; other characters, such as a different type of fruiting structure, also support the exclusion. The Guttulinopsidae are excluded with less certainty. They have mitochondria with plate-like cristae, and the amoebae lack dictyosomes (Erdos and Raper 1978), but the mitochondria contain helical inclusions and are not surrounded by rough endoplasmic reticulum (Dykstra 1977; Erdos and Raper 1978). There are also significant differences in fruiting body development and type between the Guttulinopsidae and the Acrasidae. Another perplexing organism is Fonticula alba, which produces sorocarps that appear similar to those of the Guttulinopsidae. However, the amoebae are not of the limax, eruptive type, though the mitochondria have discoid cristae (Worley et al. 1979; Dykstra 1977). The amoebae of the families Guttulinopsidae and Copromyxidae are currently being examined in an attempt to understand the relatioships of the genera to one another and to other, nonfruiting amoebae. Until such relationships are clarified, the families are incetae sedis. A tentative treatment would be to consider them as separate classes (Guttulinopsea and Copromyxea) in superclass Rhizopoda.

D) Wider relationships

At this stage relationships with other protozoa can be little more than sepculative. With the wide distribution of flagellate and amoeboid cells, the possession of one flagellate and amoeboid characters alternately or at the same time is insufficient indication of relationship. For example Pseudospora has tubular cristae and dictyosomes (Swale 1969). Brugerolle (1982) found no dictyosome in Mastigina hylae, but mitochondrial structure is of no help because that endozoic species lacks mitochondria. Amongst rhizopods with no known flagellate stage, Nuclearia has mitochondrial cristae which appear similar to those of the Heterolobosea (Mignot and Savoie 1979; Patterson 1983), but these filose amoebae differ both light- and electron-microscopically from the Heterolobosea, one difference being the possession of dictyosomes, another a very different locomotive pattern. The dichotomy of the Mastigophora on the basis of cristal structure proposed by Seravin in Krylov et al. (1980) suggests a search for flagellate relatives, especially since the Heterolobosea occupy a position in the borderland between Sarcodia and Mastigophora. An obvious possibility is the Euglenida, but such a relationship is still on the outer edge of spiculation. The Heterolobosea are one more indication that the vexatious border between Mastigophora and Sarcodina is little more than convention. Without prejudicing eventual changes at a higher level, the Heterolobosea are proposed at this time as a class of the Rhizopoda, whether the latter are considered a superclass (Levine et al. 1980) or even a separate phylum. The Schizopyrenida are transferred from the class Lobosea and the Acrasida from the class Acrasea, which thus ceases to exist.