A. The families Hartmannellidae and Vahlkampfiidae
The heterogeneity of the family Hartmannellidae (originally subfamily Hartmannellinae Volkonsky, 1931) was eliminated, as far as possible with light microscopic observations, by the redefinition proposed by Page (1974) and subsequent removal of the genus Rhizamoeba (Page 1980). The limits thus established are further confirmed by the present results, especially by the discovery of similar sucker-like elements on all amoebae assinged to the Hartmannellidae. Differences amongst hartmannellids with be discussed later. The strong evidence that the Vahlkampfiidae (with some preservations about Heteramoeba) are a natural, monophyletic grouping was summarised most recently by Page and Blanton (1985). Their most distinctive ultrastructural character, apart from the mitotic process, is the possession of mitochondria with flattened cristae, these mitochondria being usually closely associated with and even enveloped in rough endoplasmic reticulum. This character is shared with the rest of the Heterolobosea. Another important ultratructural character is that in general the Golgi systems of Heterolobosea seem to be represented by clusters of vesicles as illustrated by Fig. 23 of Page and Blanton (1985), rather than organised in bodies of the classical dictyosome structure, i.e., stacks of flattened saccules with associated vesicles. Such dictyosomes were found in all hartmannellids examined in the present study (as in other Amoebida) but in none of the vahlkampfiids. The saccule-like structures in Figs. 76 and 77 appear to be parts of the rough endoplasmic reticulum, though unlike the flattened elements of that system surrounding the mitochondria. Saccules more like the usual Golgi elements have been described by Stevens et al. (1980) in Naegleria lovaniensis and more recently by De Jonckheere et al. (1984) in Willaertia magna. Although rather loosely associated, the structures found in N. lovaniensis and W. magna resemble the usual dictysomes more than do any other inclusions found in vahlkampfiids. The problem is compounded in the case of W. magna by the fact that the mitochondria of this species appear to differ from those of other vahlkampfiids. The problem of the Vahlkampfiid Golgi system will eventurally require a detailed cytochemical investigation. Comparisons between Hartmannellidae and Vahlkampfiidae have been listed several times as further data accumulated (Page 1974, 1978, 1983). The most significant further information presented here is the difference in surface structure. The more fundamental differences between mitochondria and Golgi systems of the Heterolobosea, to which the Vahlkampfiidae belong, and the Lobosea, to which the Hartmannellidae belong, are obvious from the above results and the earlier report (Page and Blanton, 1985).
B. The genera of Hartmannellidae
The original generic distinctions were based on light microscopic observations (Page 1974). Some characters, such as persistence of a hyaline cap, seemed rather trivial but were employed as possible indicators of more fundamental differences. How much agreement is there between the boundaries drawn by light microscopy and those which might be drawn from electron microscopic results? Amongst the three species of Hartmannella investigated, H. vermiformis and H. abertawensis appear more similar to each other than either is to H. cantabrigiensis, on the basis of their nuclei and the appearance of their surface elements. The sizes of the surface elements of H. vermiformis and H. abertawensis differ; other differences have been mentioned in the Observations. H. cantabrigiensis has a quite different nucleus, which with its paranucleolar body strongly resembles that Cashia limacoides. However, any idea that H. cantabrigiensis and C. limacoides might be congeneric is refuted by the peculiar mitochondrial cristae of the latter. H. cantabrigiensis and C. limacoides also differ in their surface elements. H. cantabrigiensis resembles H. vermiformis and H. abertawensis in the shape (elongation) of their mitochondria and the organization of the their rough endoplasmic reticulum, which differ in C. limacoides, though it coud be argued that these characters are of specific rather than generic significance. (It shoud be noted that amongst all the hartmannellids only H. cantabrigiensis and Saccamoeba limax contained crystals). It is therefore concluded that the genera Hartmannella and Cashia as defined earlier (Page 1974) should stand. In addition, in order to be classified in the genus Cashia, any species shoud have mitochondrial cristae like those of the type species Cashia limacoides. If an amoeba with such cristae does not meet the light microscopic criterion of lacking a hyaline cap in continuing locomotion, mitochondrial structure must be regarded as more fundamental. In the absence of electron microscopic data on either cysts or nuclear processes during encystment (Glaser 1912; Page 1974), only the fine structure of the amoeboid stage is available at this time as a source of further information on Glaeseria. The nuclear structure of G. mira is different from either of the patterns (paranucleolar body or conspicuous chromatin masses) found in Hartmannella; its mitochondria never become elongate as in Hartmannella; and its surface elements are more cup-like and larger than those of Hartmannella. Although smaller than the two species of Saccamoeba studied and differing from them light microscopically, G. mira has surface elements resembling in form and approaching in size those of S. limax, and its mitochondria closely resemble those of S. limax. However, the distinctive light microscopic characters of Saccamoeba would in themselves prevent assimilating Glaeseria to the genus, whatever the significance of the peculiar nuclear processes reported for Glaeseria and the absence of endocytic bacteria from G. mira. Pending an electron microscopic study of the nuclear process accompanying encystment in Glaeseria, it continues to stand as a separate genus. The familial position of Saccamoeba and the integrity of the genus itself seem to present greater problems than do the positions of the other genera.
The generic problem can be considered first. Already on the light microscopic level, S. stagnicola and S. limax were found to differ from each other in the presence or absence of crystals and of an encysted stage (Page 1974). However, their similarities were so great that those two characters seemed insufficient for a generic separation, especially since S. limax sometimes lacks crystals in culture and S. stagnicola may suffer a reduction in its encystment capability. The electron microscope has revealed an immediately noticeable difference in the shape of the mitochondria of the two species, suggesting a generic differetiation. On the other hand, the degree of difference in surface structure may be considered of only specific significance. Furthermore, all four strains of Saccamoeba, two strains of S. stagnicola from sites about six miles apart and two strain of S. limax from different hemispheres, contained endocytic bacteria, and, of all the hartmannellids examined, only these strains contained such inclusions. It appears therefore that both species belong to a single genus. The discovery that Saccamoeba possesses surface elements closely resembling those of other hartmannellids supports the classification of this genus in the Hartmannellidae. The further similarity of the mitochondria of Glaeseria mira and S. limax has also been pointed out. Electron microscopic studies of the mitotic processes in hartmannellid genera are needed. They might shed light on such questions as the generic position of H. cantabrigiensis and the relationships of Saccamoeba with non-hartmannellids such as the Amoebidae. However, such studies will be much more difficult than similar investigations of vahlkampfiids, in which mitotic figures are usually found more easily.