Search for Clues to the Evolutionary Meaning of Ciliate Phylogeny

J. PROTOZOOL. 23 ( 1 ) , 48-56 ( 1976).

Search for Clues to the Evolutionary Meaning of Ciliate Phylogeny *t

S. H. HUTNER and JOHN 0. CORLISS Haskins Laboratories at Pace University, Pace Plaza, New York, NY 10038, and Department of

Zoology, University of Maryland, College Park, MD 20742

SYNOPSIS. Progress in ciliatology and in allied fields may demystify ciliate phylogenetics. Concentration on hymenostomes (mainly Tetrahymena and Paramecium) may have obscured directional features of ciliate physiology in phylogenetic problems. Therefore, means are suggested for “domesticating” the presumptively primitive, predominantly marine, sand-dwelling gymnostomes having nondividing diploid macronuclei. The prize quarry is the marine psammophile Stephanopogon whose homokaryotic condition may mark it as a living fossil. Eventual axenic cultivation of these “primitive” ciliates may be aided by use as food of easily grown photosynthetic prokaryotes, some isolated from the marine sulfuretum or adjacent aerobic muds and sands where “karyorelictid” ciliates flourish.

(a ) the macronucleus evolved as a coordinator of chemical and physical signals, for efficient detection of food and toxins; (b) oral structures evolved meanwhile as sensors as well as mechanical food-gatherers. This conjunction enabled complexity of adaptive behavior and evolutionary success. Ciliate origins cannot be considered apart from origin (s) of phagotrophy and its underlying versatile heterotrophy. Because of the well developed heterotrophy in some photosynthetic prokaryotes (including several proposed as food organisms), they are viewed as alternatives to blue-green algae as fore- bears of eukaryotes. Nor can ciliate origins be considered apart from origin(s) of eukaryotes. A check of these assumptions that Stephanopogon and gymnostomes with nondividing macronuclei are primitive-may be forthcoming from sequencing amino acids in certain key enzymes, given an adequate sampling of ciliates, flagellates (especially dinoflagellates and cryptomonads), lower fungi, and photosynthetic prokaryotes other than blue-green algae.

Index Key Words: Evolution ; gymnostomes, hymenostomes; hypotrichs; photosynthetic prokaryotes ; macronucleus.

We assume that:

YSTEMATISTS, in a rare display of amity [see (23)], depict S ciliates as remote from other groups because of their con- stellation of unique characteristics; their origin, accordingly, is placed far back in geologic time. Thus Leedale (81) pictures ciliates, metazoa, flagellates, and the host of amebae emerging from the limbo of a no-extant-protists swamp between pro- and eukaryotes. Taylor ( 131 ) (this symposium) treats their origins in like gingerly fashion. A decade ago, Holz (61) contended that to push Tetrahymena and ciliates generally into pigeonholes labeled plant and animal is folly; that “. . . Tetrahymena is Tetrahymena . . . ”-a creature to be accepted on its own terms as a mosaic of plant and animal characters. Besides singu- larities listed earlier (61), others are now recognized (66); several but not all will be mentioned later. Begging the question of ciliate phylogeny is less acceptable today. Molecular paleontol- ogy, notably that derived by sequencing amino acids in several universal enzymes, reveals that all creatures consist of proteins which are mosaics: they embody both conservative and recent sequences. Presumably the corresponding DNAs and RNAs will have a like melange of conservative and divergent nucleotide sequences. Meanwhile the popularity of Tetrahymena and Paramecium as amenable animals increases; biochemists presumably suppose, tacitly, that if the molecular biologists dare assert that what holds for Escherichia coli holds also for elephants, then ciliates, the super-animals among protozoa, are even better models. Imminent large-scale use of Tetrahymena in food processing, agricultural and medical research, aside from consideration of taxonomy, does not permit this assumption to stand

* Presented as part of a Symposium, “Early Evolution of Protists” (sponsored by the Society of Protozoologists and co- sponsored by the Phycological Society of America and the Society for Invertebrate Pathology), held at the 28th Annual Meeting of the Society of Protozoologists, Oregon State University, Corvallis, August 1975.

t Investigations of the authors and their collaborators reported in this paper were supported by the following Research Grants: a t Haskins Laboratories-2 R 0 1 AM 15137 and FR 05596, from the National Institutes of Health, U.S. Public Health Service; BMS 75-10956, from the National Science Foundation; and a grant from the S. and W. T. Golden Foundation; at the University of Maryland-GB-41172, from the National Science Foundation.

unexamined. Fortunately, testing of Tetrahymena’s relevance as a tool for solving human problems will intensify scrutiny of its physiology and, eventually, of other ciliates, and may disclose characters of taxonomic value indicative of ancient metabolic divergences. This is happening with Euglena, Ochromonas mal- hamensis, and Ochromonas danica, paralleling the attentions lavished on the cultivable protozoal pathogens of man and domestic animals. We submit that recent advances in ciliatology, microbiology, and biochemistry reveal so many promising lines of investigation that one may reasonably sketch the possible filling in of blanks in the phyletic map.

The underlying homogeneity of the ciliates is emphasized in texts (72, 124) : possession of cilia joined by a complex infraciliature; 2 types of nuclei; binary fission typically cutting across the longitudinal rows of cilia; phenomena (e.g. conjugation) involving exchanges of gametic nuclei; and a GC mole ”/o between 25 and 35% (the lowest reported for any taxon)- each unique.

Are Tetrahymena and other hymenostomes representative ciliates? A terrain is surveyed by building up a network of triangulation

points (“benchmarks”) firmly anchored to other triangles. The reference baseline or sight line for the ciliate terrain is obviously Tetrahymena c-) Paramecium. How wobbly is this reference line? Besides consideration of other ciliate triangulation points, one must make triangulations outside the ciliates. Rather than concocting a family tree, with dispute over twigs and branches us main trunk-that is to say, cladistics-we thus content our- selves with 2-dimensional mapping while exposing, by inference, grounds for our preferences for root, trunk, branches, and topiary manicuring of twigs or species.

Of some 8,000 described ciliate species, research concentrates, as noted, on Tetrahymena pyriformis, Paramecium aurelia, and Paramecium caudatum, thanks to their abundant yields in mass cultures, and improvements in culture technics, culminating in defined media. We defer mention of the heterotrich Blepharisma and the hypotrichs Euplotes and Stylonychia, biochemically largely unexplored but of keen interest due to thc extraordinary reorganization of the new macronucleus formed after conjugation.

As expected, others besides Holz have questioned the typicality

48

CILIATE PHYLOGENY 49

of T . pyriformis as a ciliate; in the present context, how much wobble is there at the T . pyriformis end of the baseline? Much, judged from the differences among DNAs from different syngens, based on DNA hybridization; the differences are comparable to those between rodent genera or of Drosophila species (3) . An alternative reference point for surveying tetrahymenids is in focus: comparative studies of strains ( 6 in all) resembling Colpidium campylum, Colpidium colpoda, and Colpidium kleini (94). The C . campylum strains grew axenically in skim-milk- tryptone-yeast extract; the other 2 species could not be grown axenically. Of 10 enzyme systems studied electrophoretically in “C. campylum,” every strain shared at least 70% of the enzyme systems with one other strain, and they shared an esterase with one in Tetrahymena.

As for other Tetrahymena “species,” their variations in nutritional patterns suggest that the aforementioned differences in DNA extends to them, phenotypically judged, even from sparse knowledge of their metabolism and behavior: differences in tendency to conjugate, temperature optima, minimal essential nutritional requirements, pathogenicity, and cannibalistic ten- dencies. Evaluation of these differences would be systematized by use of a high-precision, efficient, defined medium. Because of employment of T . pyriformis WH-14 (syngen 1 ) to assay the biologic value of proteins (41, 73), we are developing a yardstick medium-one following Ham’s guidelines for optimization of defined media for metazoan cell cultures (52) . As an example, such a medium might serve to gauge divergence from the amicronucleate strain W used for protein assay (73); it would be a start in applying nutritional criteria to the phenotypic grouping of both micro- and amicronucleate strains derived from comparisons of 8 isoenzyme systems ( 11 ) .

One could thus develop a reliable baseline for the 1st sub- triangulation: that which includes tetrahymenas besides T . pyrif ormis, especially those having proclivities towards parasitism (21) and whose nutrition comprises some oddities (60) . This projected standardization might provide leads to improving media for Paramecium; work remains to overcome difficulties reported in applying the defined medium for P. aurelia (156) to grow single cells of P. aurelia (54). I t is not clear why this defined medium falls short in satisfying P. bursaria ( 138). Media for P . aurelia did lead to defined media for the hymenostome Uronema nigricans among other marine forms (127). The resemblance to P. aurelia media was well documented for one devised for Uronema marinum (53) .

Returns soon diminish if one seeks to refine media for each newly available ciliate (60) . Still, concentration of effort on ciliates of special genetic, biochemical, or other value should become more productive as a result of advances in maintenance methods and formulation of basal media as frozen dry mixes.

Do Tetrahymena-Paramecium Media Embody Features of Potential Taxonomic Value?

Lipoic acid requirement.-This requirement of all ciliates examined to date, occurs also in the gram-positive bacterium Listeria.

Steroids.-The absolute requirement of P. aurelia for “plant” sterols, e.g. the 24-ethylsterols stigmasterol and sitosterol, is shared by the guinea pig (136). Evidently neither requirement is specific. A role for “plant” sterols in insect metabolism sur- faces as stimulation of the growth of an insect by cholesterol plus stigmasterol (1 16) . T o interpret the apparent absence of sterols in Tetrahymena (63) as meaning that Tetrahymena cannot synthesize any sterol is to misapprehend biochemical findings, as has been pointed out (60) . The very existence of plant-sterol

requirement in Paramecium implies that Tetrahymena can synthesize a t least C-24 sterols. Besides, Tetrahymena corlissi, Tetrahymena paravorax, and Tetrahymena setifera have sterol requirements satisfied by plant sterols or cholesterol. A crucial experiment might be to determine whether these requirements are satisfied by the triterpenoid tetrahymanol which has been isolated from the oleander. Steroid biochemistry illustrates how findings in comparative biochemistry erode old phyletic assumptions, only for new complexities to emerge. The idea had been prevalent that prokaryotes lacked sterols, and that sterols first appeared in membranes newly developed in eukaryotes, perhaps the nuclear membrane. But bacteria proved to have sterols (46) , as did blue-green algae (47) which have both cholesterol and 24-ethylsterols-now a familiar duality. The concentration range is vast between sterols as structural components of membranes in contrast to their activities as hormones. Moreover, the multiplicity of steroid hormones in eukaryotes would mark the absence of any sterol synthesis in Tetrahymena as extraordinary indeed. One may here adduce ergosterol functioning as precursor of the ecdysones of arthropods and higher plants ( 1 15); cholesterol and “phytosterols” as precursors of the female hormone antheridiol from Achlya, perhaps via fucosterol (45); and, in man, outcropping of another aspect of sterol activity a t the vitamin-hormone level of vitamin D ( a precursor of the familiar ergosterol + ultraviolet light). How easily this steroid activity could have been missed was shown by the discovery that conventional vitamin D must be hydroxylated on (2-25 in the liver, then on C-1 in the kidney, producing la-25-dehydroxyvitamin D,, considered the hormonally active vitamin D, (101). Connections between cholesterol and plant sterols are revealed by the effectiveness of cholesterol and plant steryl glycosides in counteracting the growth inhibitions of Tetrahymena by hypocholesteremic steroids (62) , recently by 3p-( p-dimethylamino- ethoxyl) and androst-5-en-17-one (59) . The pathway in Tetra- hymena of dealkylation of 24-ethylsterols to cholestatrienol is novel and different from that recognized in arthropods (and, by reversal of the biosynthetic pathway in plants), but in view of vast ignorance of sterol metabolism, it seems too soon to con- clude that the C-24 dealkylation pathway in Tetrahymena is “. . . outside of a direct line to the animal kindgdom” (100).

Glyoxylate cycle.-The active glyoxylate cycle in Tetrahymena (88), with concomitant high activity of acetate as substrate for respiration (114) and growth (92) is primitive since the cycle occurs in many bacteria, acetate flagellates [represented by Euglena gracilis, the cryptomonad Chilomonas paramecium, the volvocines Polytoma and Polytomella, and the phagotrophic colorless dinoflagellate Oxyrrhis (34)]. This cycle, however, does not separate Tetrahymena entirely from the metazoa, for Ascaris has the cycle (8).

Biotin and Thiamine.-These requirements are shared with all animals examined-protozoan and metazoan. Elucidation of the pathway of biotin biosynthesis (36) reveals several stable inter- mediates, hence it may be feasible to ascertain whether synthesis of biotin is blocked a t the same point in all animals. An identical block might argue that animals, ciliates included, are monophyletic. In principle, similar analysis could be applied to any exogenously essential metabolite and, in totality, be phylogenetically helpful.

Lysine.-Lysine synthesis may yet be of clear-cut phyletic meaning. Vogel, in his much-reviewed work (137), presented evidence that Euglena, most phycomycetes, and higher fungi, employed the a-aminoadipic synthetic pathway; bacteria, green algae, some phycomycetes, and higher plants employ the diaminopimelic pathway. Can the biosynthetic block for ciliates be

50 CILIATE PHYLOGENY

located even though they cannot synthesize lysine? Apparently their ancestral synthesis was by the diaminopimelic pathway because rumen ciliates can form lysine from diaminopimelic acid (103, 104). Before thinking that this again points to a remote origin of ciliates, bypassing many eukaryotic groups having extant members, notice should be taken that both lysine pathways occur in a higher plant (97) . Man perhaps can use the a- aniinoadipic pathway, if only for a hardly detectable trickle of synthesis of lysine by a theoretically possible reversal of the route of lysine catabolism (27). Degradation of lysine in Neurospora similarly leads to pipecolate and a-aminoadipate ( 12). Diamino- pimelic acid can substitute for exogenous lysine in Trypanosomati- dae (49). Evidently, data for many more organisms are needed before phyletic conclusions can be drawn from lysine pathways; missing are data for axenic ciliates, phytoflagellates besides Euglena, and the newly isolated photosynthetic prokaryotes described later. A practical matter arises: If Tetrahymena should satisfy its lysine requirement from diaminopimelic acid, it would give falsely high “lysine” values, assuming that verte- brates do not respond to diaminopimelic acid. Use of bacteria in foodstuffs for domestic animals and man is nearing industrial reality (123), hence shortcomings of Tetrahymena for assay of bacterial protein will compel resolution of this question.

Folic acid and biopterin.-Tetrahymena’s folate requirement resembles man’s in molecular specificity but differs from it in lack of obvious interrelation with vitamin B,, or methionine (60). Unlike that in bacteria, Tetruhymena’s folate requirement is spared but not replaced by thymidine (143). Tetra- hymena sharply differs from Trypanosomatidae, whose members have an unphysiologically high folate requirement, lowered to physiologic levels by a minute concentration of biopterin. In- activation of folk acid but stability of biopterin on acid hydrolysis permits the growth response of Crithidia fasciculata to serve as a sensitive, specific assay of biopterin (6). Biopterin-sparing by folic acid occurs in P . aurelia (126). Perhaps this apparent back-synthesis of biopterin from folate in 2 remotely related protozoan groups (it has not been found elsewhere) denotes an outcropping of an ancient eukaryotic pathway. (For discussion of this pathway and the occurrence of extraordinarily high concentrations of biopterin in blue-green algae, see Ref. 40.) Should a prokaryote be found that harbored the pteridine ciliapterin (threobiopterin) , so far seen only in Tetrahymena (76) , or displayed the folic-biopterin-sparing phenomenon, it could point to eukaryote origins. Antifolics, e.g. methotrexate, figure prominently in recent successes in cancer chemotherapy (19) , hence elucidation of the role of pteridines in ciliate metabolism has a high priority. As noted later, findings in this area may well help enlarge knowledge of microtubule function in ciliates.

Has Phagotrophy a Unique Origin?

Debate as to whether eukaryotes arose symbiotically as suggested by Margulis (91) can temporarily be set aside in favor of a question more immediately pertinent to that of ciliate origins: Did phagotrophy and pinocytosis have a single origin?

The evolution, in flagellates, of pharyngeal stiffening structures at the canal shaped by the invaginated area of flagellar emergence presents little difficulty, granted the exceptional plasticity of the microtubular cytoskeleton for which evidence is presented in many papers on euglenoids and kinetoplastids. Development of phagotrophy as life style would appear to go back at least as far as photosynthetic euglenoids, for E. gracilis strain Z takes up exogenous peroxidase in the vesicles adjacent to the contractile vacuole, which suggests that uptake of macromolecules is a

way to recover useful material from the effluent of the contractile vacuole (78). Still another mechanism is implied by how scales are released by the prasinophycean flagellate Pyramimonas; formed in the Golgi body, the scales perhaps are released to the exterior by passageways to the flagellar pit and by a duct system extending posteriorly and anteriorly from a storage depot ( 102). The phagotrophy of many chrysomonads, conspicuous in the voracious yet fully photosynthetic Ochromonas danica, implies that phagotrophy played a part early in phytoflagellate evolution. Support for the reabsorption theory is provided by the resorption of food-vacuole membrane by vesicles at the posterior cytoproct of P . caudatum, with involvement of microtubules in the cytoproct region ( 2 ) .

Cavalier-Smith ( 18) emphasizes the pre-eminence of endocytosis in the origin of eukaryotes-loss of the cell wall in a bacterium, producing an L-form, promoted exocytosis and eventually re-fusion of the phagosome membrane with the plasma membrane, an ability foreshadowed by the propensity of vesicles to fuse. Replacement of the rigid cell wall by an actin-myosin- like microfilament system, which controlled cell division as well as endocytosis, would have led also to cell fusion; the subsequent gene redundancy would permit rapid evolution once chromosomes became linear, leading in turn to more efficient recombina- tion and further acceleration of evolution.

Possession of biliproteins and 2-layered thylakoids could signify that the most primitive of extant flagellates are the cryptomonads. Cyathomonas, a cryptomonad which has a cytopharynx and well developed endozoic habit (94), would be, assuming retention of archaic characters, among the most primitive among extant animals, indispensable for tracing the evolution of phagotrophy. Bacterized cultures are available. But: cryptophyte biliproteins differ from those of blue-greens and rhodophytes (129); biliproteins from the latter 2 have extensive homologies (139). The need to postulate that the primitive eukaryote contained biliproteins derived from blue-green algae is weakened by discovery of an alga having prokaryotic organization, no phycobilin pig- ments, and possessing chlorophylls a and b (84) ; unfortunately it is not yet in culture. Another surprise: a cyanophyte has been described lacking thylakoids ( 117). When considered together with some of the food organisms mentioned later, the assemblage supports Stanier’s ( 129) surmise that non-blue-green 0,-producing prokaryotes may still be around. In the present context, that amounts to saying that the prokaryotic abutment for the bridge spanning the gulf between them and eukaryotes need not rest on conventional blue-greens with their heterotrophy, which is dismayingly poor (74).

T h e Gymnostomes: Predatory and Primitive? Some Strategies for Cultivation-Hints from Hymenostome Predators for Rearing Raptorial Gymnostomes and Hypotrichs

“Upon what meat doth this our Caesar feed that he is grown so great?” [Shakespeare: Julius Caesar, Act I, Scene 21

Corliss, to highlight postulated primitivity persisting among gymnostomes, exalted some in rank (23, 24) for reasons of ( a ) apicality or subapicality of the mouth and absence of oral membranelles; (b) occurrence in many of nondividing diploid macronuclei; and ( c ) occurrence in Stephanopogon of mono- morphic nuclei. These characters are thought to justify erection, under subclass Gymnostomata, of the new suborders Primociliatida to include Stephanopogon, and Karyorelictida for forms with a dual nuclear apparatus, macronuclei diploid but nondividing, in contrast to the macronuclear autonomy seen, for example, in amicronucleate tetrahymenas.


We believe, looking ahead, that rearranging higher taxa in this manner is justifiable heuristically: it challenges us to cultivate gymnostomes (predominantly raptorial rather than those hymenostomes ( essentially bacteriophagic, herbivorous or omnivorous) that, as noted, now attract major attention along with the grazing hypotrichs Stylonychia and Euplotes. How else can taxonomic ideas be tested if possibly key organisms are neglected? An opening gambit might be to resume work with parasitic or carnivorous tetrahymenas and their Glaucoma and Colpidium kin. One might, beginning with the aforementioned nutritional yardstick, once adequate for bacteriophagic strains of T . pyriformis, define the biochemical basis of predatory predilections (21 ), perhaps thereby detecting biochemical evidences of ten- dencies and incitements to cannibalism and the macrostome condition (14) . It might be helpful, also, to clarify oddities such as why T. setifera meets its sterol requirement, satisfied by cholesterol, only if the cholesterol is dissolved in ethanol (64) , and whether the requirements of amino acids and vitamins as well as lipids for Glaucoma scintillans, C. campylum (75) , Glaucoma chattoni A (65) , exceed those for orthodox T. pyriformis strains. Also helpful would be intensified interest in what it is in Tetrahymena as prey that promotes cannibalism and giantism in T . uorax (14, 7 7 ) . Cox (25) has reported that a Tetrahymena isolated by Dr. Martin Shapiro, from moribund mosquito larvae, grew in defined media. One of us (S.H.H., unpublished) has confirmed this; the vigor of this isolate makes it a candidate reagent for protein assays; a search for such predation-promoting enzymes as chitinase might reveal cor- relations with potential or actual pathogenicity.

It also seems worthwhile, if only as an exercise, to renew the attempt to cultivate Didinium nasutum, the ferocious predator of Paramecium, on axenic paramecia and killed bacteria or other prokaryotic food-a problem whosr difficulty can hardly be gauged today, yet whose previous near-intractibility may be mitigated by recent advances in Paramecium nutrition.

The same tactics may work for hypotrichs. Efforts to rear them on ciliate prey revealed a snag to be expected from gout in man and feeding single-cell protein to domestic animals: the nucleic acid content of the large macronucleus. The capacity of the predator to cope with nucleic acid may be overtaxed- causing the equivalent of gout-as well as setting up imbalances in purine entry, so there are even circumstances where anti- purines helped (85, 86) . This promising but necessarily pains- taking work has not been pursued; an incentive to do so is provided by studies of the postconjugation reorganization band in the macronucleus, site of a drastic reduction in DNA, probably by elimination of spacer DNA (110), seen also in oligo- trichs (120). Genetic analysis would aid in deciding whether ciliate macronuclear DNA-polyploid or polytenic-is homologous with polyteny in the giant polytenic chromosomes in Diptera and in amphibian oocytes, or the apparently polytenic chromosomes in dinoflagellates (50, 118, 119, 128). While preparation of antigenic, morphologic, behavioral, and mating- type markers do not require axenic media, such media obviously would enable application of mutagenesis to provide biochemical markers as well as aid analysis of the conjugation process. Rigorous genetic proof is lacking for a 1 gene-1 band relation between bands and genes in the giant banded chromosomes in Diptera (83) . Availability of defined media for Stylonychia and Euplotes thus becomes highly desirable, for genetic analysis of their macronuclear reorganizations and clarification of their polytenic or at least polygenonlic nature.

Another strategy, perhaps experimentally simpler though devious, might he to rrar gymnostome5 carrying algal symbionts

(most commonly zoochlorellae) . Such ciliates are not uncom- mon; one locality in Wisconsin yielded, among gymnostomes, a Frontonia and a Prorodon; the heterotrich Climacostomum; and the hypotrichs Euplotes and Holotricha ( 130). Green Prorodon may be common; it has also very recently been recorded again from Germany ( 106). The symbiote-bearers might be more amenable than their nongreen counterparts, although studies of P. bursaria indicate that one cannot take success for granted. Climacostomum virens (green) is fed the phytoflagellate Chloro- gonium elongatum (grown in a complex medium); the ciliate appears colorless when grown in the dark, thus avoiding the excessive growth of endosymbiotes (107), an intriguing system to set alongside P. bursaria. The occasional persistence of ingested chloroplasts in ciliates may make it difficult to differentiate it from true endosymbiosis ( 1 1 ) .

Approaches to Rearing “Karyorelictid” Ciliates; Psammophiles; S o m e Potentially Valuable Prokaryotic Food Organisms f rom the Marine Sulfuretum and Adjacent Sands and M u d s

Stephanopogon and many if not most “karyorelictid” gymno- somes are restricted to interstitial spaces in sands and muds. These habitats in turn comprise many subenvironments, some high in H,S, the “sulfuretum,” which intergrades with aerobic environments. Severe physicochemical and tactile constraints keep them in these zones, helped in all likelihood by specialized requirements for food organisms. As previously mentioned, newly available photosynthetic prokaryotes may provide exceptionally useful food organisms as well as prokaryotic organisms for extending triangulations. The engaging ciliates in these environments have been described by Dragesco (23) , Fenchel (38) , and Fenchel & Riedl (39) ; the latter authors note that many of the bacterivores apparently prefer various photosynthetic bacteria; many ciliates are carnivorous, i.e. feeding on ciliates. How fascinating to rear, for example the cosmopolitan Tracheloraphis dogieli, with numerous nondividing diploid macronuclei with large chromocenters and a body up to 2 mm long, apparently having the beginning of inclusion of the separate micro- and macronuclei within a common membrane ( l l l ) ! We take Raikov’s use here of the expression “lower ciliates” as concurrence in viewing these ciliates as primitive-a short-hand terminology like that for “lower” (monogenetic) and “higher” (digenetic) Trypanosomatidae. Choice of possible food organisms might well take into account ecologic, biochemical and as well triangulation values. One might begin, from the aerobic side, with the red halophiles, e.g. Halobacterium halobium and H . cutirubrum, which can live anaerobically by a photophosphorylation without chlorophyll carried out by a light-dependent, vitamin A-containing purple membrane like rhodopsin of the retina (38, 80) . Aerobically, aside from the extreme halophilism, they behave like ordinary pseudomonads but with requirements for amino acids and nucleotides. They disintegrate in less than 14% NaCI; defined media are available. Purple bacteria, which live just above the H,S zone, may prove to provide superior food for ciliates because the hardy Rhodopseudomonas spheroides, and the denitrifier Paracoccus denitrificans, more studied respirometrically, have features of respiratory control uniquely resembling those in mitochondria ( 70). In keeping with this respiratory resemblance between these 2 bacteria, a denitrifying strain, almost certainly a R. spheroides, has been isolated (121). Taken together with the conclusion, based on comparisons of large oligomers in RNA, that the Athiorhodaceae, represented by R . spheroides, and the related enteric-Vibrio group “. . . are on one side of a very primitive phylogenetic divide, while Bacillaceae and Cyanophyta are on another” ( 144), it reinforces our suspicion


that undue phylogenetic significance has been attributed to the conventional blue-greens. In any event, the prediction that many more ecologic restrictions will be discovered with respect to habitats of protozoan species (22), reinforces emphasis on extreme environments as likely sources of relict organisms, ciliates among them.

The H2S-rich photic zone is characterized microbiologically by its anaerobic rhodobacteria and the more anaerobic chlorobacteria, the latter more or less dependent on H2S as the H- donor in photosynthesis. It had been generally thought that evolution proceeded much as in the zonation of a Winogradsky column, upward towards aerobiosis, i.e. chlorobacteria + rhodobacteria (red + purple) + cyanobacteria. Ciliatologists thus might have to grow several obligately anaerobic bacteria in light under critical control of H2S or sulfides lest the ciliates be killed. These difficulties may be substantially lessened by the blue- green alga Oscillatoria limnetica which can carry out both a conventional oxygenic photosynthesis or an anoxygenic photosynthesis with H2S, thus removing H2S (20). Even more startling, the filamentous, gliding bacterium Chloroflexus, with optimum temperature of 55 C (89) carries out a chlorobacterial- type photosynthesis anaerobically, yet grows like the usual gliding bacteria aerobically in the dark, i.e. heterotrophically, bypassing the rhodobacterial steps toward the cyanobacteria and algae. Supply of H2S might be regulated by adjustment of conditions of growth of a Desulfovibrio. Evidently, organisms are at hand for attempting construction of a model sulfuretum. A difficult technical problem may be finding a way to grow Chloroflexus within the temperature range of the ciliates. I t seems fitting that “relict” ciliates should be reared with “relict” photosynthetic forms, all surviving in extreme environments, which suggests that other phylogenetic prizes are extant; occurrence of the red alga Cyanidium caldarium in hot, salty, acid waters is a case in point. The problem seems on its pm-scale much like rearing flamingoes-a relict bird-also confined, as may be many lower ciliates, to warm soda lakes.

Amino Acid Sequencing; Antigenic Analysis; Ribosomal Com- ponents

Mass cultures once at hand, the decisive instrument for research on evolution can be the amino-acid sequencer. The leading enzymes for sequencing are cytochrome c (29) , superoxide dismutases (mitochondrial and cytoplasmic) (42), the ferredoxins (5 1 ) , and, among eukaryotes, histones. Since noise-random- ness-in sequencing a small number of proteins can be as high as 15% (132), the phylogeneticist should sequence as many homologous proteins as possible from obviously related forms rather than from only 1 or a few representatives of a group. For another gauge of affinities of a group one may resort to antigenic analysis, for evidence cited by Crawford (26) for several proteins from eukaryotes reveals ‘ I . . , a correlation between strength of immunological cross-reaction and amino acid sequence resemblance, therefore evolutionary distance, but application to the tryptophan synthetase brought out a difficulty: the method . . . may be too sensitive for determining the natural relationships between the major bacterial groups.” Again it argues for every available possible link between ciliate groups being “domesticated” for the purpose, at least in antigen-free media as a stage in axenic cultivation.

The axonemic proteins tubulin and dynein may provide the least ambiguous connection between the ciliates and the prokaryotes since the biochemistry and consequent morphogenesis of cilia and kinetosomes form ciliate organization. Antigenic resemblances here may serve the purpose of revealing which

extant flagellate group is nearest the ciliates, which would focus attention on the currently perhaps more rewarding problem of flagellate origins. No relationships are yet recognized between the proteins and overall organization of bacterial and eukaryote flagella (1,125). The remarkable sensitivity of inhibition of microtubule assembly, especially by colchicine, Vinca alkaloids, and perhaps even more conveniently, the herbicide trifluralin (55), puts powerful pharmacologic probes ( 140) into the hands of the phylogeneticist-comparable to the way penicillin sensitivity tests for the prokaryotic cell wall or, as with mycoplasmas, confirms absence of a cell wall. Such probes of ciliate microtubule function have been applied to Stentor coeruleus; the pineal hormone melatonin, as in vertebrate skin, made the ciliate lighten, apparently by interfering with normal cortical position of pigment granules (141).

Another promising phylogenetic probe is co-electrophoresis of the 55 different ribosomal proteins in prokaryotes and the -70 proteins in eukaryotes. Aside from the expected gulf (along with a few probably random coincidences), the distance between E. coli and Bacillus stearothermophilus was of the same order, i.e. approximately same number of overlaps in proteins (5-6), as that between E. coli and the guinea pig, while there were - 25 overlaps between guinea pig and higher plants (30). The high resolution of this method makes its extension to ciliates eagerly awaited. Cytochromes c of Euglena and Crithidia are closer to each other than to those of metazoa, e.g. the fewest mutations need be postulated to connect them. Other similarities between these mitochondrial cytochromes include in all likelihood, the heme’s having only one thioether group instead of 2 and the presence of e-N-trimethyllysine ( 108). Ciliate cytochrome c has not been sequenced.

The Ciliate Psyche: An Emerging Taxonomic Character?

The success of freshwater and soil ciliates vis-i-vis the karyo- relict forms invites speculation as to the role of each feature of ciliate ecology and organization in the development of behavior or, what we here call the “ciliate psyche.” A few years ago this would hardly be an acceptable way of putting it, but now it is known that even bacteria have myriad chemosensing receptor proteins, with correspondingly complex behavior (1, 9 ) . Moreover, some of the prerequisites for adaptive behavior, i.e. compounds mediating neural activity, occur in ciliates, e.g. catecholamines (68), serotonin (69), and cyclic AMP (90). Like metazoa, changes in ciliary activity are mediated by Mg2+- and Caz+-generated action potentials (98). What in the environ- ment spurred the radiative evolution of the higher ciliates? A hint is the remarkable discrimination of some ciliates in se- lecting bacterial food, rejecting poisonous ones (32). This competition between prey and predator might be intense, especially in soils and freshwater muds; a theme elaborated by Singh (122) for amebae preying on soil bacteria, with bacteria and higher fungi developing poisonous principles, among them antibiotics, countered by the amebae developing greater selectivity, including ability to cope with these toxins. Ciliates could provide parallels. If so, one would expect a proliferation of chemoreceptors in ciliates inhabiting the biotically rich environments of moist soils and waters, reaching its apogee in ciliates inhabiting polluted waters.

The array of ciliates serving as reliable indicators of biologically polluted waters (10) can be viewed as a collective success story from the standpoint of numbers and cosmopolitanism, both for bacteriophagic and omnivorous forms and their predators, conspicuous among the latter being hymenostomes, heterotrichs, and, above all, peritrichs and hypotrichs. The over-urbanized


protozoologist need go no further than a sewage works, preferably an activated-sludge plant, to find ciliates to domesticate, including predatory gymnostomes besides the aformentioned Didinium, above all the voracious gymnostome Dileptus.

Perhaps, as surmised earlier, the sensors were localized in the oral region, leading to their concentration first in the cytostome- cytopharyngeal area of the gymnostomes, later, more efficiently, in the buccal membranelles of the higher ciliates. Hyman (67) long ago attributed sensory functions to such organelles. As circumstantial evidence, cilia occur commonly in metazoan sense organs (4, 7 ) . Association and fusion of cilia in membranelles and cirri may denote concentration of sensors. More immediate evidence of the essentiality of microtubules for feeding, perhaps indicative of more than contributing to mechanical stiffening of buccal structures, is that the microtubule-inhibitor cytochalasin reduces food vacuole formation in Tetrahymena, an inhibition overcome by glucose + nucleosides (57) . Pitelka (109) has discussed Lwoff’s idea of the multiple potentialities of the basal bodies. Besides the postulated sensory function, another function is associated with the oral region, implied by the participation of microtubules in cellular transport. Supporting evidence is being amassed that microtubules mediate transport of soluble materials as well, thus helping to explain why vinblastin is synergistic with the antifolic methotrexate in several types of cancer-vinblastin causes a one-way entry of methotrexate into cells with consequent accumulation of therapeutic levels of methotrexate (44). This transport function extends to streaming of nerve axon protoplasm (142). It may not be rash to assume that, as ciliate evolution proceeded, elaboration of microtubule-rich buccal structures per- mitted more efficient sensing, endocytosis, and intracellular transport of large molecules such as the folates; this may not be very different from the (as yet largely unknown) mechanisms which mediate the stockpiling and transport of the lipid components of pellicular membranes (133). The idea that coordinated ciliature evolved as a means of increasing the efficiency of filter feeding has occurred to Rasmussen et al. (113). The beginning analysis of proteins of the oral apparatus (43) should be applied to this line of development.

Genetic and electrophysiologic analysis of the behavior of Paramecium, well under way (79) , should provide a measure of the versatility of adaptive behavior in other ciliates. Another index might be the concentration of biopterin in the ciliate body, for hiopterin mediates the hydroxylation of tyrosine leading to DOPA, dopamine, epinephrine, and of tryptophan to serotonin and melatonin. Such an analysis of isolated membrane fractions in gymnostomes, hymenostonies, and other “higher” ciliates, might provide a parallel index of the concentration and localization of these or equivalent neurohumoral compounds. Mapping of hiopterin in nerve tracts has not yet begun for mammals; a beginning, by an isotope method, has been made for a culture of a line of neuroblastoma cells (13).

Notes on the Molecular Makeup of Ciliate DNA

Emphasis on nuclear organization, especially on increased macronuclear polyploidy as a major taxonomic character, raises the question as to whether evolution of ciliate DNA at the molecular level proceeded in a direction noticeably away from that of other eukaryotes. If so, it would underscore the isolated position of the ciliates and support those zealous investigators who agitate for elevation of Ciliata to phylar rank. A sharp distinction between pro- and eukaryotes is possession of histones, among the most conservative of all eukaryotic proteins (37, 48) . Tetrahymena and Stylonychia have all the normal histones of higher eukaryotes (37)-a conclusion documented by details of

the histones from pure macronuclei of Stylonychia mytilis: they markedly resembled calf thymus histone fractions (47, 77). If otherwise, polyteny of the macronucleus might well prove nonhomologous with polyteny in metazoa. Correspondingly, the unique sparsity in dinoflagellates of the acid-soluble-protein fraction of chromosomes (which contains the histones) (1 18, 119) may denote primitivity. While wider surveys of histones in phytoflagellates may affirm the primitivity of dinoflagellates, as evidenced by other nuclear characters, possession of a metazoan- like array of histones by ciliates argues that the search for origins of ciliates might profitably center on those phytoflagellates and lower fungi having metazoan-like histones. These findings en- courage the belief that use of ciliates as models to arrive at the molecular basis of aging, as genetically programmed in Para- mecium and, less clearly, in Tetrahymena (99) , may be relevant; hence a character linking ciliates to metazoa. Noteworthy is the occurrence of N6-methylcytosine in Tetrahymena macronuclear DNA, while eukaryote DNA commonly has 5-methylcytosine (48). A comparison of the 3 RNA polymerases isolated from the macronucleus of an amicronucleate strain of Tetra- hymena led to the conclusion that the polymerases were of eukaryotic rather than bacterial type (56) . Finally, do the lower ciliates have the same low GC % as the higher ciliates?

The gap between ciliates and metazoa may be narrowed with the discovery of uniflagellate spermatozoa in the littoral turbel- larian Nemertoderma, which lines it up with the spermatozoa, all uniflagellate, of Bilateria ( 134). Here again is a psammophile, but this time a metazoon, to domesticate.

Miscellaneous Potential Markers

Blepharisma garnones.-Gamone 1 of Blepharisma intermedium is a glycoprotein blepharmone, excreted by mating type 1, which stimulates mating type 2 to excrete gamone 2 [calcium-3-(2’- formylamino-5’hydroxybenzoyl) lactate], enabling conjugation (95, 96) . I t is too soon to tell whether a similar chain of triggerings of matings by gamones, similar or different, occurs in other heterotrichs, let alone other ciliate groups.

Mitochondria.-The finding that Trypanosomatidae have a single, branched mitochondrion did not long remain isolated. Sometimes Euglena gracilis has a single reticulate mitochondrion (82, 105), as is true also of the volvocine Polytomella agilis ( 15), Chlorella ( 5 ) , and yeast (58). Evidently possession of conven- tionally discrete mitochondria cannot be taken for granted as an attribute of the ur-ciliate.

Circadian Rhythms.-This property is found only in eukaryotes and has consequently inspired speculation that it depends on eukaryotic DNA being multirepliconic, e.g. having redundant nucleotide sequences; Tetrahymena displays it well (35). Com- parisons of amicronucleate and micronucleate strains and of lower ciliates may provide insights into the rhythm. Conceivably, ease of induction of circadian rhythms could become a measure of the extent of polyploidization with its attendant increased DNA redundancy; and its photoinducibility points up the de- sirability of media in which such putative photoreceptors as porphyrins, riboflavin, and pteridines are under close control.

Concluding Reflections

“Who are the ciliates?”-What is their phylogenetic significance? These questions may become less vaporous as data ac- cumulate on: ( a ) phyletic origin of the axoneme (traced by its proteins) and discovery of stages between the simple flagellar apparatus of the flagellates and the ciliate infraciliature; ( b ) genetic analysis of progressive polyploidization and increasing autonomy of macronuclei; ( c ) demonstration of presence or


absence of coordination between elaboration of specialized oral structures, on the one hand, and of sensory receptors on the other. Phylogenetic dendrograms based on low-noise sequencing of cytochrome c , ferredoxins, and histones might prove super- imposable on schemes based on antigenic analysis of the aforementioned enzymes, electrophoresis of ribosomal proteins, and RNA and DNA hybridizations. If these superimpositions in turn rest on a wide sampling of ciliates, flagellates, other unicellular eukaryotes, and chemo- and photosynthetic prokaryotes, the phylar relationships of ciliates should become clearer.

In totality, this is a vast enterprise, but it may save us from constructing phyletic guideposts which point in wrong directions. Today’s taxonomic faith is founded on a gross structure only sketchily aligned with micro-structure, i.e. biochemistry. Our taxonomic and phylogenetic schemes are, we trust, governed by the principle of parsimony, but the facts are still too parsimonious. Old technical obstacles are dissolving. The advent of stable broad-spectrum antibacterial antibiotics, e.g. chloramphenicol and gentamicin, used in conjunction with the newer broader- spectruin penicillins should ease axenization even in the notori- ously tough situation of removal of marine bacterial and yeasts ( 7 1 ) . Another significant advance is identification of nucleosides replacing particulates as stimulants for food-vacuole formation in T . fyriformis ( 112). Advances in inorganic nutrition (several too recent to be mentioned here) should ease the replacement of natural crude media by more reproducible and efficient defined media, since there will be less dependence on supply of trace elements as contaminants in “C.P.-grade” biochemicals of natural origin. Many ciliates, e.g. Paramecium and Uronema, prefer higher concentrations of purines and pyrimidines, supplied as nucleotides, than are required by Tetrahymena. I t may now lie possible to analyze the reasons for this discrepancy, which may apply to many other exacting ciliates, perhaps especially to carnivores. Does this requirement result from a higher threshold for induction of food vacuoles, a higher rate of catabolism, greater contamination with trace elements, or all or none of these?

Our dread of forcing “facts” to fit fancy, in which dread we attempt to follow Canella (16, 17), is as applicable in all kinds of approaches as it is a t all levels of classification. Our attempt here has been to point out advantages of a broad and far-ranging attack in viewing ciliate phylogeny overall, making the “con- stellation of characters” hypothesis more of a reality than a starry-eyed hope.

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