Flagellate Phylogeny: A Study in Conflicts

28 DINOFLAGELLATE EVOLUTION

and the DNA-histone antigens in the nuclei of free-living and parasitic Sarcomastigophora. J . Protozool. 14, 225-3 1. 107. Stosch HA, von. 1967. D. Dinophyta, in Ruhland W, ed.,

Handbuch der Pflanzenphysiologie, Springer Verlag, Berlin, 18,

108. - 1972. La signification cytologique de la “cyclose nucleaire” dans le cycle de vie des DinoflagellC. SOC. Bot. Fr. M e m . 1972, 201-12. 109. - 1973. Observations on vegetative reproduction

and sexual life cycles of two freshwater dinoflagellates, Gymno- dinium pseudopalustre Schiller and Woloszynskia apiculata sp. nov. Br. Phycol. J . 8, 105-34. 110. Strain HH, Manning WM, Hardin G. 1944. Xanthophylls

and carotenes of diatoms, brown algae, dinoflagellates, and sea anemones. Biol. Bull. 86, 169-90. 111. - , Svec K, Aitzetmiiller K, Grandolfo MC, Katz

JJ, Kjmen H, Norgird S, Liaaen-Jensen S , Haxo FT, Wegfahrt P, Rapoport H. 1971. The structure of peridinin the characteristic dinoflagellate carotenoid. J. A m . Chern. SOC. 93, 1823-5. 112. Sweeney BM, Haxo FT, Hastings JW. 1959. Action

spectra for two effects of light on luminescence in Gonyaulax polyedra. J. Gen. Physiol. 43, 285-99. 113. Tappan HN, Loeblich AR Jr. 1971. Surface sculpture

of the walls in Lower Paleozoic acritarchs. Micropaleontology 17, 385-410. 114. Taylor DL. 1971. Ultrastructure of the ‘Zooxanthella’

Endodinium chattonii in situ. J . Mar. Biol. Assoc. U. K . 51,

115. Tomas RN, Cox ER. 1973. The symbiosis of Peridinium balticum (Dinophyceae) . I. Ultrastructure and pigment analysis. J. Phycol. 9, 16. 116. -, - , Steidinger KA. 1973. Peridinium bal-

626-36.

227-34.

J. PROTOZOOL. 23 ( 1 ), 28-40 ( 1976).

Flagellate Phylogeny:

ticum (Levander) Lemmermann, an unusual dinoflagellate with a mesocaryotic and a eucaryotic nucleus. J. Phycol. 9, 91-8. 117. Tsenkovskii L. 1881. Otchet” o byelomorskoy ekskursii

1880 z [Lecture on the White Sea exDedition in the year 1880. In R&lan.] Tr. Sankt-Peterburgskagd Obshchestva Eitestvoispy- tateley. 12, 130-71. 118. Tuttle RC. Loeblich AR 111. 1974. The discovery of

genetic recombination in the dinoflagellate Crypthecodinium cohnii. 1. Phycol. 10 (Suppl.), 16. 119. -, - 1974. Genetic recombination in the

dinoflagellate Crypthecodinium cohnii. Science 185, 1061-2. 120. -, - 1975. Sexual reproduction and segrega-

tion analysis in the dinoflagellate Crypthecodinium cohnii. J. Phycol. 11 (Suppl.), 15. 121. -, - , Smith VE. 1973. Carotenoids of

Crypthecodinium cohnii. J. Protozool. 20, 521. 122. Vien C. 1967. Sur l’existence de phCnomZnes sexuels

chez un PCridinien libre, 1’Amphidinium carteri. C . R . Acad. Sci. Paris 26p, 1006-8. 123. - 1968. Sur la germination du zygote et sur un

mode particulier de multiplication v6gCtative chez le Peridinien libre Amphidinium carteri. C. R . Acad. Sci. Paris 267, 701-3. 124. Whittle SJ, Casselton PJ. 1968. Peridinin as the major

xanthophyll of the Dinophyceae. Br. Phycol. Bull. 3, 602-3. 125. Withers N, Haxo FT. 1975. Chlorophyll CI and cg and

extraplastidic carotenoids in the dinoflagellate, Peridinium folia- ceum Stein. Plant Sci. Le t t . 5, 7-15. 126. Woioszyhska J. 1916. Sphaerodinium n. gen. i rozmaianie

plciowe u Sphaerodinium polonicum n. sp. Rozpr. W y d z . Mat . - Przyr., Pol. Akad. Um. , Ser. 3, 15 (B) , 283-91. 127. - 1928. Dinoflagellatae polskiego Baltyku i Brot

nad Pialnica. Arch. Hydrobiol. Rybactwa 3, 153-278.

A Study in Conflicts* P. J. R. TAYLOR

Department of Botany and Institute of Oceanography, University of British Columbia, Vancouver, B.C., Canada V6T I W5

SYNOPSIS. Information relating to the ultrastructure of 4 organellar systems of flagellates-nuclei (including mitosis), flagella, mitochondria and chloroplasts-is examined for bearing on the probable phylogeny of the principal flagellate groups, first considered singly and then in combination. The mitotic mechanism has not proved to be as conservative a character as might be hoped, but still remains characteristic for the average condition in many of the groups. Flagellar features are useful if allowance is made for the reduction or multiplication of the basic pair, and the loss of lateral and terminal hairs seems to have occurred independently several times. The presence of paraxial rods within flagella may be a useful indication of affinity. Rootlet systems are not dealt with in detail here, although the possible similarity between axial microtubular sheets in axostylar flagellates and some members of the green algae containing “manchettes” is noted. The basic patterns of chloroplast internal structure are summarized and their general agreement with other characters is affirmed, noting however that cryptomonads may be closer to the green flagellates (including euglenoids) than is generally accepted. At- tention is drawn to the potential value of internal mitochondria1 morphology as an indicator of large assemblages. Finally, a “tree” based on multiple cell organizational features is presented and discussed.

Index Key Words: Flagellates ; phylogeny, evolution ; organelles; ultrastructure; nuclei; flagella; chloroplasts; mitochondria.

HE phylogeny of the flagellates is of central importance T to an understanding of the early eukaryotic radiation. Ultrastructural studies dealing with flagellates have multiplied considerably in the past decade, clarifying and providing new information on structures and events that could be little more than guessed at with the aid of light microscopy. In particular, ultrastructural details of the mitotic process have become available for at least some members of nearly all the flagellate groups.

*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.

One result of this burgeoning information has been an affirmation of the qualitative conservativeness and versatility that characterizes living systems, many structures described under a legion of different terms being found to be elaborations of essentially similar structures. Unfortunately, the initial simpli- fication in terminology resulting from this new insight is being replaced by a host of new terms as subtle distinctions are made at the new level. Furthermore, as usual with breakthroughs of this sort, new insight has not simplified all questions relating to the flagellates, new puzzles arising to replace older ones.

In this contribution an attempt will be made to sum up some current problems in the use of ultrastructural information for phyletic speculation. In particular, I shall attempt to sum- marize dilemmas which arise when information on more than

FLAGELLATE PHYLOGENY: A STUDY IN CONFLICTS 29

TABLE 1. Principal flagellate groups.* nology is reduced to a minimum where possible. “Closed,” in which the nuclear membrane remains intact throughout mitosis,

“PHY TOFLAGELLATES” “ZOOFLAGELL ATES” and “open,” in which it breaks down sooner or later. are

Dinoflagellates (incl. ebriids)

Cryptomonads Euglenoids Chloromonads

C hrysomonads

Xanthophytes Eustigmatophytes Haptophytes

(incl. coccolithophorids) Prasinophytes Chlorouhvtes (Volvocales)

(= Raphidophytes)

(incl. silicoflagellates)

Choanoflagellates (= Craspedo- monads)

Bodonids Trypanosomes ] Kinetoplastids Retortamonads Oxymonads Diplomonads

* The names used in the table are informal and not all of them

t Honigberg (29a). are at the same hierarchical level.

one system is taken into account. For present purposes data on mitosis, flagellation, chloroplast ultrastructure, all of which have been reviewed at least once recently, and mitochondria1 structure (which has been largely neglected as a phyletic tool, with the notable exception of the kinetoplastids) will be em- phasized. While it is obvious that many more features should be taken into consideration, including biochemical ones, this selection should serve to accomplish the main goals of this presentation.

The list in Table 1 has been kept informal to avoid complications in the recognition of the same group at slightly different levels by different authors, serving to illustrate the range of examples and the names used for them. For example, 2 of the polymastigid subgroups (Parabasalia) are listed (for which most ultrastructural information is available), others such as the calonymphids and pyrsonymphids not being referred to here, without, I hope, producing a distorted view of the group. This grouping of the more elaborate zooflagellates follows Grell ( 18).

Any undertaking such as this, which attempts to discern broad features within such a motley assemblage of uneven data, must inevitably be viewed as a highly subjective exercise on the part of the author. In particular, there is a constant difficulty in selecting the most meaningful levels of generalization. No doubt many will differ with those used here. T h e option to deal with “a little about a lot,” rather than vice versa, was chosen to show the amount of focussing still needed to get the over-all picture clear, and to pinpoint apparent conflicts in the apparent affinities of groups as suggested by one system, with that of another.

Nucleus and Mitotic Apparatus

- - convenient major subdivisions. In addition there are many protists in which spindle tubules enter the nucleus from outside through “polar fenestrae,” referred to as “semi-open” here. The presence or absence of kinetochores, their structural complexity, and location (on the envelope or internal) also seem to be useful. The appearance of the chromosomes when condensed (during or before mitosis) is often group distinctive. Features of the spindle, such as the association of the poles with visibly organized centers or the lack thereof, and the relative lengthening or shortening of the “continuous spindle” (axial, interzonal) us chromosome-associated microtubules, also seem significant, a t least in some instances.

As a starting point in comparative studies the determination of the most probably primitive condition is helpful. The only objective criterion for this purpose is a resemblance to prokaryotic genophore separation, making the generally accepted assumption that eukaryotes are from a prokaryotic stock. Typical dinoflagellate nuclear features give the strongest indication of primitiveness using this criterion : although they have chromosomes, nucleoli, and nuclear envelopes they lack normal eukaryotic histones (57) , have a fibrillar appearance of the chromosomes throughout the cell cycle, contact of the chromosomes with the nuclear membrane with the kinetochores located on the membrane (27, 49, 56) , and possible (although not established) mediation of the membrane in chromatid separation. A metaphase plate is not formed. I t has been suggested that the chromosomal DNA may be circular (20) , although this has not been established yet. Within some possibly more advanced members of the group (64) chromosomal appearance can vary through the cell cycle, not appearing to be fibrillar in the condensed state or dispersing at interphase.

In virtually all dinoflagellates the spindle tubules are entirely extranuclear (Fig. 2A) and not associated with centrioles, but even in these features there are exceptions; in one species of the parasitic genus Amoebophrya an intranuclear “pseudo- centriole” is associated with intranuclear tubules (9), and paired centrioles participate in the formation of the extranuclear spindle of Syndinium (56, 61) .

If one assumes that the nuclear features of dinoflagellates can be used also as an indication of spindle primitiveness, it might be concluded that an extranuclear spindle is more primitive than those in other locations. Closed mitosis, in which the nucleoplasm is continuously isolated from the surrounding cytoplasm, has been previously considered primitive (38, 53) , occurring in several other groups thought to be primitive for a variety of other reasons, but in these the spindle is intranuclear (e.g. euglenoids, 38, and oxymonads, 7 ) . The absence of clear rneta- phase plate formation (“pleuromitosis”) is also thought to

In view of the crucial importance of apportioning the primary genome to daughter cells it was not unreasonable to hope, only a few years ago, that the system involved might be highly conservative and, providing recognizable small differences are present among groups, might be useful as a reliable indication of phyletic position. Relatively recent reviews of the mechanisms in prokaryotes, algae (including phytoflagellates), zooflagellates and other “protozoan” groups, and fungi are available (10, 21, 27, 38, 53, 70). A study of this literature and more recent papers reveals that this hope has been only partially fulfilled.

Some of the principal mitotic variations to be found in the flagellates are summarized diagrammatically in Fig. 2. Several superimposed variations are not shown (see below), and termi-

be primitive. The Only group, Other than dinof’age’lates, with closed

mitosis and wholly extranuclear spindles are the parabasalian PolYmastigote flagellates ( t~idmmonads and hypermastigids) . Hollande (28) has noted that in Some hypermastkids the mitosis is similar in nearly every respect to the ‘‘Syndinium-type’’ of dinoflagellate mitosis, although chromatid separation may occur at an earlier stage (assumed actively to involve the nuclear membrane as well as microtubules in Spirotrichonympha, 29, and Trichonyrnpha, 34) . A metaphase plate does not form, as in the dinoflagellates (and also possibly in euglenoids and kinetoplastids ) .

If mitotic features are considered of preeminent phyletic

30 FLAGELLATE PHYLOGENY: A STUDY IN CONFLICTS

The

Chryso

Crypto

Choano

Fla igellates . A I richo

Trypano

Hapto

fq ............. R .,:: :::,:.

..p /

ugleno C hloroph

Prasino 0 \ Di no Fig. 1. Simplified diagrams of flagellate organizational types. Bodo: bodonids; Chloro: chlorophytes (Volvocales) ; Chlorom:

chloromonads ; Choano : choanoflagellates ; Chryso : chrysomonads ; Crypto : cryptomonads; Din0 : dinoflagellates ; Diplo : diplomonads ; Eugleno : euglenoids ; Eustigm : eustigmatophytes ; Hapto : haptoflagellates ; Hypermast: hypermastigids ; Oxymo : oxymonads ; Prasino: prasinophytes; Retorta : retortamonads; Tricho: trichomonads ; Trypano: trypanosomes; Xantho: xanthophytes.

importance, these polymastigote flagellates should be the most closely related group to the dinoflagellates. I n most other features, however, this is not corroborated, the flagella being quite different, paraxial rods, amphiesmal vesicles, trichocysts,

condensed interphasic, fibrillar chromosomes (and mitochondria) being absent from the polymastigotes, and no sign of axostylar development occurring in the dinoflagellates. The long, banded roots (atractophores) , associated with spindle centers in tricho-

* -. *c--

A

FLAGELLATE PHYLOGENY:

B C ,

D E . .

. ’ / :.<:- .-:<.. ,- . . . . .. ’ ...

2. : . , _. :*

. . . . . . . . . . . . . , . . . . . . . . . . . . . . _ . I . , . . . . , . . . \, , . . . ’ , . ,. , . . . < ..’. . . ,: , i. .-‘.‘d

Fig. 2. Five major variations in mitotic mechanisms of flagellates. A. Dinoflagellates, trichomonads, hypermastigids : spindle totally extranuclear, kinetochores in the nuclear envelope if present, with or without visible spindle inducers. B. Euglenoids, trypanosomes and probably bodonids: totally intranuclear spindle. C. Xanthophytes, prasinophytes, chlorophytes, and probably chloromonads: intranuclear spindle with associated extranuclear elements. D. Cryptomonads, chrysomonads, diplomonads : “semi- open” mitosis-entry of extranuclear tubules through polar fenestrae. E. Eustigmatophytes, haptophytes, prasinophytes, chlorophytes. Oxymonads may have type B or C (not clear at present), and the type( s ) in choanoflagellates and retortamonads is unknown. Tubules shown as continuous across the nucleus are probably not, interdigitating at least in some cases.

monads (6a, 9a; Mattern et al., unpublished), and hypermastigids (29) are not found in most dinoflagellates, but have some resemblances to the rhizoplast participation in spindle formation in chrysomonads (59) and prasinophytes (54).

The degree of variability present within the mitotic mechanisms of groups, genera, or even species, may serve as a guide to the conservativeness or plasticity of the system, therefore being indicative of the weight which might be placed on any particular feature(s).

I t is already apparent from the above discussion that mitotic mechanisms are fairly diverse in the dinoflagellates, although the majority of the species examined have a common type of nuclear organization ( “dinokaryotic”) and, probably, mitosis. Among members of the green algae, including the flagellated forms (Volvocales) , a considerable variation has been noted, including the presence or absence of centrioles, kinetochores, degree of chromosomal condensation during mitosis, degree of nuclear envelope breakdown [some, such as those of Chlamy- domonas moewusii being totally closed (68)l and involvement of interzonal elongation in chromatid movement. Pickett-Heaps (54) has reviewed the current state of ultrastructural information on this group, and has pointed to some general trends. In particular, closed mitoses are most commonly associated with volvocalean members or those with Chlamydomonas-like zoospores and “phycoplast” wall formation in which the spindle remnants become reorganized perpendicular to the plane of the spindle during cytokinesis. Polar fenestrae, however, may be present in some, e.g. Chlamydomonas reinhardii. Open mitoses are more commonly associated with prasinophytes and filamentous green algae with bryophyte-like zoospores (asym- metrically inserted flagella, with a microtubular band running

A STUDY I N CONFLICTS 31

the length of the cell) and a “phragmoplast,” the continuous spindle elements persisting during cytokinesis, often even after wall formation.

One of the most interesting insights into the potential for variability within the mitotic apparatus, although not found in flagellates, is worth repeating here as a “cautionary tale,” as Leedale (38) , has put it.

Within the life cycle of the slime mold Physarum flavicomum there is an alternation between a closed mitosis, with an intranuclear spindle in the diploid plasmodia1 nuclei, and an open mitosis with a centriole-associated spindle in the haploid myxameboid stage ( 1 ) . This does not necessarily mean that the presence or absence of centrioles, or the degree of breakdown of the nuclear envelope, are worthless as phyletic indicators. For each group there seems to be a predictable “most common” state and a range of variation, this slime mold having the greatest plasticity observed so far in the mitotic system. I t warns, however, against simplistic judgements based on scanty observa- tion, and some of the apparent contradictions between data for this system and those for others (see Discussion) may be due to an inadequate appreciation of the spectrum of variability.

Some insights are beginning to emerge which may aid in evaluating mitotic data. For example, in some filamentous organisms centriolar homologs may form only before the formation of the motile phase. I n some of the volvocalean green algae the flagellar basal bodies may migrate to the nuclear membrane after the flagella are shed, resembling centrioles (53) .

The interphase appearance of the nucleoplasm, as seen with transmission electron microscopy can be indicative of the group in some instances. The best known examples are the dinoflagellates, as noted above, and the euglenoids, in most representatives of which the chromosomes remain condensed (37). Although in other groups the chromosomes disperse, the density and distribution is not the same in all cases and may also be fairly predictable. For example, in chloromonads and cryptomonads the dense material may appear as scattered bodies, possibly corresponding to the choromosomes whereas in kinetoplastids it often lies around the periphery of the nucleus and nucleolus (8, 60), a more common condition resembling the heterochromatin distribution of metazoans. Polymastigote flagellates often have very little heterochromatin visible during interphase. This aspect requires further exploration to evaluate its usefulness as a phyletic indicator.

Flagella

The number, arrangement, and type of flagellar beat have long been used to indicate relationships. Although lateral “hairs” have been visible with the aid of critical light microscopy, electron-microscopic studies have provided a wealth of information on the widespread occurrence and structure of such hairs, their intracellular formations [sometimes in cisternae of the endoplasmic reticulum (22) , or perinuclear space (40)], the presence of cryptic short flagella or 2nd basal bodies (= kinetosomes) in many supposedly uniflagellate species, and the lack of a “9 + 2” system in the supposed “third flagellum” of haptophyte flagellates. Manton (42) reviewed the impact of earlier electron-microscopic studies on phyletic speculation, and Dodge ( I 1 , 12) has provided a more recent interpretation.

Small scales and club-like appendages, thought to be group specific and phyletically significant, have been found on the surface of some flagella; and banded, fibrillar components running parallel to the axoneme, usually termed “paraxial rods,” have been discovered in several groups, possibly also serving as an indication of affinity. A diagrammatic summary of the


FLAGELLA C d e

a

k I n

P

7

i

0 ?

Fig. 3. Flagella. a : dinoflagellate; b: euglenoid (primitive type) ; c: Cephalothamnium (bodonid) ; d: bodonid; e: trypansome; f : Pedinella (chrysomonad) ; g : chrysomonad; h: chrysomonad/xanthophyte; i : eustigmatophyte; j : chloromonad; k: Pauloua (haptophyte) ; 1: haptophyte; m: prasinophyte; n: cryptomonad; 0 : chlorophyte; p: Micromonas (prasinophyte?) ; q: retortamonad, oxymonad; r : diplomonad; s: trichomonad; t: hypermastigid.


distribution of some flagellar features among the groups is provided in Fig. 3.

The tips of the flagella may be narrowed for a short distance ( a condition referred to as mucronate), the narrow portion containing only the central pair of tubules. The most extreme example of the latter can be found in Micromonas pusilla, in which the narrow portion is more than half the length of the single flagellum (42) .

finer than bacterial flagella in euglenoids and the presumed prasinophyte Pedinomonas (43) ; thicker, curved rods in other prasinophytes and the cryptomonad Chilomonas (42) ; or thick complex structures with differentiated bases, distal portions, and lateral or terminal branches (typically found in the chrysophytes, xanthophytes, brown algae and chloromonads) . Different hair- types may be intermingled on the same flagellum. Bouck ( 4 ) and Leedale (39 ) have summarized information on flagellar scale and hair forniation.

Sparse hairs have been found on many flagella formerly thought to lack them. For example, hairs may be present on the posterior flagellum of some chrysophytes ( 3 ) or dinoflagellates (36) , and even on the isokont flagella of one species of Chlamydomonas (55) , suggesting that the hairy condition was probably more widespread earlier, with an independant reduction of hairs in several groups. Hairs have not been seen in the haptophytes or many of the zooflagellates and green algae. Although most haptophytes have isokont, smooth flagella, their internal organization and pigmentation are more similar to those of heterokont groups, such as the chrysophytes, than to those of the green algae. The flagellar condition, plus the production of organic body scales, is most probably the result of parallelism in these groups. Some haptophytes have unequal flagella, the anterior flagellum possessing small projections in Pavlova ( 17 ) , and thus seem intermediate with chrysophytes.

Paraxial (or paraflagellar) rods are found in both flagella of the euglenoids and bodonids, as well as in the single flagellum of trypanosomes, supporting their suggested affinity (38) . They may also occur in only 1 flagellum, often being associated with a lateral, ribbon-like flattening, such as the dinoflagellate trans- verse flagellum or the anterior flagellum of several chrysophytes [Pedinella, Apedinella (62) ) The undulating membrane associated with the recurrent flagellum of some polyniastigote flagellates, notably trichomonads, is usually a ridge of the cell surface rather than a lateral flagellar extension.

Flagellar swellings are present near the base of one of the flagella of the euglenoids, some trypanosoines [although lacking the paracrystalline infrastructure of euglenoids and certainly of little photosensory value in the bloodstream forms of Trypano- soma (SS)], and on the postero-lateral flagellum of chrysophytes and xanthophyte zoospores (24) . The 2 former groups are probably related, and so are the 2 latter, but no relationships between the 2 pairs are evident. A lateral expansion is present at the base of the hairy anterior (often sole) flagellum of the zoospores of rustigmatophytes ( 25 ) , thus resembling euglenoids more than xanthophytes (as well as the spatially associated eyespot not being located within a chloroplast) and supporting the recent separation of the group from the xanthophytes.

The haptonema, the peculiar flagellar derivative with reduced microtubular content and endoplasmic reticulum intrusion occurring in addition to the isokont flagella of some haptophytes (reviewed in Ref. 11 ), is unfortunately restricted to that group and does not aid in the present endeavour.

The flagellar hairs vary in thickness and complexity:

Flagellar type, number and arrangement is summarized diagrammatically in Fig. 3. In many groups uniflagellate representatives are known, but in most of these vestiges of 1 or more other flagella can be observed, such as the nonemergent 2nd flagellum of many euglenoids, or basal bodies additional to that giving rise to the flagellum (42) . The question has been raised, however, as to whether some of the very small uniflagellate prasinophytes (Micromonas, Mantoniella) might represent a primordial condition (42) . Secondary basal bodies have not been observed in these genera, but this is not conclusive evidence. The general assumption seems to be a fundamentally biflagellate condition of the flagellates, with secondary reduction, or the production of supernumerary flagella.

Quadriflagellate members are common in the prasinophytes (in which all the basal bodies are symmetrically oriented) and in the retortamonads and oxymonads (in which the basal body of the recurrent flagellum is directed at an angle to the other 3 ) . The diplomonads appear to have doubled this arrangement (as well as some of their internal organelles, including nuclei), and the trichomonads can have 3, 4 or 5 exterior flagella additional to the recurrent flagellum. Other polymastigotes have multiplied their flagella still further, to produce large numbers. All these zooflagellates appear to have smooth flagella, whereas the prasinophytes usually have hairs and scales on their flagella (both lacking in Micromonas, scales lacking in Pedinomonas).

On the basis of flagella only, the zooflagellates would seem to form 1 phyletic series, except for the choanoflagellates, in which the single anterior flagellum may be smooth ( 3 5 ) or possibly hairy, and which are thought to be related to chrysophytes, one pigmented genus, Stylochromonas, being variously attributed to either group. O n general cytologic grounds, however, the kinetoplastids, at least, arr probably separate from the others (see Discussion).

So far no comment has been made on the internal structures associated with flagella, i.e. microtubular bundles or sheets, and microfibrillar roots (often banded). The arrangement of these may well be of value in determining phylogenies when their basic patterns and probable modified equivalents can be determined for the groups. A start along these lines has been made for the zooflagellates and some phytoflagellates (32) , but it is too early to use this information satisfactorily.

Manton (42) has noted a basic similarity between the root structures of members of the green algae and uni-, bi- or quadriflagellate prasinophytes. Pickett-Heaps (54) has indicated that the hiflagellate Chlamydomonas condition, with its 2 additional, nonfunctional basal bodies, and cruciate rootlet system, resembles the replicated flagellar apparatus of the prasinophyte genus Pedinomonas, suggesting that the latter may be close to the ancestral stock for the green algae.

While many roots probably serve as anchoring devices for the flagella (or possibly conductive mechanisms), some roots are associated with spindle formation in the chrysophytes, trichomonads, and hyperniastigids (29) . In some phytoflagellates a banded root may make contact with ihe nuclear envelope, or terminate close to it, e.g. in chloromonads (46) .

The sheet of microtubules (single or multiple rows) forming the axostyle runs from the vicinity of the flagellar bases to the posterior end of the cells of several zooflagellate groups. It may serve to indicate affinities among those groups possessing an axostyle (oxymonads, trichomonads, and hypermastigids) . This axial band of microtubules is somewhat reminiscent of those seen in some green algae thought to be in the phyletic line leading to the higher plants ( 5 4 ) , and in the spermatozoids of the latter. This complex has been termed a manchette. It is difficult to


A B C

D E F

Fig. 4. Chloroplast structure. A. Chlorophytes and prasinophytes (hatched areas indicate internal starch). B. Euglenoids. C. Dino- flagellates. D. Cryptomonads (NE : nuclear envelope; CER: chloroplast endoplasmic reticulum). E. Haptophytes. F. Chrysomonads, xanthophytes, chloromonads. Eustigmatophytes have chloroplasts resembling type E, but lack chloroplast endoplasmic reticulum. The nonflagellated brown algae and diatoms possess type F, and red algae resemble type A but have only single thylakoids, producing starch external to the chloroplast.

judge if this resemblance is only superficial or not (see Dis- cussion).

Additional systems of tubules and fibrils may be associated with the flagella (such as the striated parabasal filaments or costae of trichomonads and hypermastigids), but there are also many which are not, a discussion of which is beyond the limits of this paper.

Chloroplasts These organelles occur only in phytoflagellates (by definition)

and derived photosynthetic stocks. The photosynthetic pigments located within them provided the basis for early attempts at sub- dividing the macroscopic lower plants into green, red, and brown algae. Subsequent detailed analyses of these pigments (chlorophylls, carotenes, xanthophylls and phycobilins) have largely supported this subdivision, with complications arising in some less well known groups.

Ultrastructural studies, in addition to affirming the basic similarity of all chlorophyll a-containing organelles (and thus leading many to drop the discrimination between “chloroplasts” for green bodies, and “chromatophores” for those of other hues), have also shown a relatively good agreement between pigment arrays, thylakoid arrangements, presence or absence of a girdle lamella (peripheral band of thylakoids) , arrangement of DNA regions, number of envelope membranes, association with a special form of endoplasmic reticulum surrounding the chloroplast (and hence referred to as “chloroplast endoplasmic reticulum” or “CER’) , location of the phycobilin pigments if present, production of solid storage bodies internal or external to the

chloroplast envelope, and pyrenoid type. The principal organizational types found in flagellates are presented diagrammatically in Fig. 4. Detailed reviews of these matters have been recently published (2, 11, 16).

Only in the green flagellates (prasinophytes, chlorophytes) is the chloroplast envelope single and not intimately associated with encircling endoplasmic reticulum (Fig. 4A) , a feature they share with the red algae (nonflagellated) . These flagellates deposit starch within their chloroplasts, this perhaps being one cause for the thylakoids becoming bunched together. When the membranes are in contact they are said to be “stacked” or “appressed,” and may represent an early stage in the formation of grana.

In the dinoflagellates and euglenoids the envelope appears to be double-layered and is thought to be the result of the CER becoming appressed to the chloroplast envelope. In both, the thylakoids are most commonly observed in groups of 3. This resemblance between the 2 groups is a t odds with the types of chlorophyll present in each (a + c in dinoflagellates; a + 6 in euglenoids) , and their xanthophyll spectra also differ. Bisalputra ( 2 ) considers that stacking occurs in euglenoid chloroplasts although, to judge from published micrographs, this process must be variable within the group (see also 25). Both groups produce solid reserve bodies external to the outer envelope, but in dinoflagelletes this is starch-like, in euglenoids it is a Pl-3 glucan (paramylon) .

In cryptomonads, and in the prasinophyte Pedinomonas, the thylakoids most commonly occur in pairs. The resemblance, however, ends there, for the former also possess phycobilins

FLAGELLATE PHYLOGENY: A STUDY I N CONFLICTS 35

A C

B

\kDNA Fig. 5. Mitochondria. A. Flattened cristae (cryptomonads,

prasinophytes, chlorophytes) . B. Flattened cristae with constricted bases (euglenoids) . C. Tubular cristae (microvilli : dinoflagellates, chloromonads, xanthophytes, chrysomonads, choanoflagellates, as well as nonflagellated brown algae, diatoms, many fungi). D. Kinetoplast-mitochondrion (bodonids, trypanosomes) .

(apparently located within the thylakoid cysternae, a unique condition), produre starch outside their chloroplasts, and have well developed CER confluent with the nuclear envelope. The starch-like product in this group is usually located between the chloroplast envelope and the CER.

In the remaining phytoflagellates the thylakoids are usually arranged in unappressed groups of 3, and CER encircles the chloroplasts, often connecting with the nuclear envelope. In most xanthophytes, chrysophytes, and the nonflagellated diatoms and brown algae a peripheral girdle lamella, consisting of 3 thylakoids, is present, and the DNA typically occurs in a ring at an angle and internal to the girdle lamella. A girdle lamella is lacking in the eustigmatophytes, haptophytes, and a few xanthophytes.

On the basis of chloroplast structure alone 2 main series seem evident in the flagellates, In one (associated with chlorophylls a and b ) single thylakoids become aggregated and stacked (with varying numbers of thylakoid per stack), internal starch is produced, and a single chloroplast envelope is present. In the 2nd, usually developed among groups with chlorophylls a and c , thylakoids are loosely associated in groups of 3, CER is usually present, and girdle lamellae are common.

The cryptomonads and euglenoids form the principal anom- alies. The former occasionally have triple thylakoid groupings, and the combination of chlorophylls a and c, plus CER, suggests a closer affinity to the 2nd main type described above, but the presence and location of their phycobilins, and starch location, are unusual. The euglenoids also appear quite similar to this series (16) especially as they produce an external, nonstarch storage product, but, if stacking is present, this may be consistent with a phyletic position close to other chlorophyll a + b-containing organisms.

Although the spacing of the thylakoids is known to vary under environmental conditions, the type of change is relatively predictable and can be taken into account when variability is observed. Nevertheless, in some groups, although the commonest grouping can be anticipated, there can be considerable variation. For example, in dinoflagellates groupings from 2 or more than 10 can be seen, and the arrangement, although usually longitudinal, may be almost radial, and pyrenoids may be internal or stalked and ocrasionally perforated (13) . The presence of girdle lamellae in 2 species may be due to contamination by a chrysophyte-like rndosyinbiote (67) .

Taylor (63) has raised the question as to whether some anom- alies may be due to earlier symbioses or other exchanges in organelles, particularly with regard to euglenoids. So far the invocation of this mechanism does not seem necessary.

Mitochondria The internal structure of mitochondria is known to vary, not

only in the degree of development of the cristae (internal projections of the inner mitochondrial membrane) but also, in insect and mammalian tissues, from tubular (microvilli) to flattened cristae (66).

I t is not surprising, in view of these observations, that little use has been made of mitochondrial structure in the discussion of probable relationships. For example, Dodge (11 , 12) has made no mention of particular mitochondrial structures associated with algal groups (including the phytoflagellates) . A notable exception to this neglect has been the recognition of the order Kinetoplastida (30) among the zooflagellates, uniting the bodonids with trypanosomes due to the presence of the kinetoplast, a mitochondrial feature (see below), as well as other compatibilities.

Taylor et al. (65) used mitochondrial structure as one means of distinguishing functioning organellar complexes of cryptomonad origin within a ciliate.

In fact, it is striking to this author how consistently predictable most protist groups are in the presence of either tubular microvilli (Fig. 5C, the commonest condition among the protists) or flattened, plate-like cristae (Fig. 5A, B ) in their mitochondria. In some groups a finer distinction can be made. For example, euglenoids seem consistently to have flattened cristae which are constricted near their bases (37). In broad view they often appear paddle- or heart-shaped, but the constriction can also be seen in narrow view.

In general, flattened cristae appear in the phytoflagellates containing chlorophylls a + b (the prasinophytes and chlorophytes) and in one group with chlorophylls a + c, the cryptomonads, already noted as occupying an anomalous position with regard to their chloroplast ultrastructure. The euglenoid condition could be considered a possible derivative of this type. Other flagellates possessing mitochondria, and most other protists, have microvilli.

The mitochondria of the kinetoplastids contain a swollen region, the kinetoplast, usually located near the flagellar basal bodies and containing an extraordinarily large amount of mitochondrial DNA (customarily designated kDNA) . This situa- tion has attracted considerable attention and some recent summaries of these studies are available (26, 47, 58). In some electron micrographs of kinetoplastids the cristae of the mitochondria appear to be flattened, a t times appearing to be pinched at the base somewhat like those of euglenoids. This latter feature might support the suggested affinity between euglenoids and kinetoplastids (38) . Some tubular cross-sections, however, are visible in sections of the mitochondrion of the bloodstream forms of Trypanosoma, e.g. congolense (69) , and they are also apparently tubular in the unusual bodonid Trypanophis grobbeni (10) . They may be flattened in Bod0 curvifilus (8) , but the published pictures do not provide enough resolution to be certain.

Among the wholly nonphotosynthetic flagellates there are several groups, mostly digestive tract endobionts, which appear to lack mitochondria entirely [retortamonad; ( 5 ) , diplomonads ( 14), oxymonads (45) , trichomonads (41), and hypermastigids (19)]. It has not been unreasonable to attribute this to in-

36 FLAGELLATE PHYLOGENY: A STUDY I N CONFLICTS

FLAGELLATE ORGANIZATION

Fig. 6. Analytical diagrams representing flagellate organizational types, disregarding flagellar insertional differences. A. Dinoflagel- lates. B. Cryptomonads. C. Euglenoids. D. Prasinophytes. E. Chlorophytes. F. Chrysomonads. G. Chloromonads H. Haptophytes. I. Bodonids (trypanosomes with reduction of 1 flagellum). J. Oxyrnonads. K. Diplomonads (retortamonads have half this). L. Trichomonads. M. Hypermastigids. The position of wall elements is indicated by dashed lines (or a continuous wall in E) , and paraxial rods present in flagella are indicated by striations. Also shown are spindle positions (variable in some, see text), mitochondria with tubular (circular symbols) or flattened cristae (square symbols), thylakoid number in chloroplasts, kinetoplasts (k) , and axostyles (a ) .

dividual responses to low oxygen environments, especially in view of the response of yeast to anaerobic conditions.

Eyden & Vickerman (14) have reported that even some free- living diplomonads ( T r e p o n o n a s agilis and Hexamita spp.) found in aquaria together with mitochondria-containing bodonids, lack mitochondria. This raises the intriguing possibility that the amitochondrial flagellates (with other similarities to one another, noted in other sections here) may have arisen from a common stock in which mitochondria were irreversibly lost.

Against this it must be noted that Hollande & Carruette- Valentin (29) have referred to the presence of bodies assumed to be degenerated mitochondria in some hypermastigids. Some of these have only a single bounding membrane and resemble microbodies, but others have a double membrane and might be vestigial mitochondria, although it is difficult to judge from their pictures.

Despite variability in a few groups and species, mitochondria1

structure appears to be a useful supplementary guide to phyletic position and deserves more detailed study.

DISCUSSION The preceding highly abbreviated summaries of organellar

ultrastructural variability are not intended to be the inade- quately detailed, encapsulated versions of larger, more exhaustive and authoritative reviews that they surely are. They should serve, however, to show that in each of the systems considered, although there are predictable group/organelle-structure associ- ations, and relationships which can be suggested, based on the presence of similar features in different groups (sometimes with apparent intermediates), there are instances in each in which a close affinity suggested by the features of one system, is contra- dicted by the aggregate of the other features of the cells. Fur- thermore, the demonstration of striking variability within certain species shakes one’s confidence in the reliability of the character

FLAGELLATE PHYLOGENY: A STUDY I N CONFLICTS 37

HYPERMASTIC IDS Phaeophytes

\ TRICHOMONADS \f

DI PLOMONADS

RETORTAMONADS

.*... .....

Diatoms

-.*. HAPTOPHVTES

CHOANOFLAGELLATES

?

Higher Plants

EUSTIGMATOPHYTES

CHLOROPHYTES I

. . Fig. 7. An arrangement of the flagellates based on the features discussed in this paper. Some nonflagellated groups are included

(lower case names). Organisms 1 and 2 (circled) are thought to have resembled Pedinomonas and Nephroselmis, respectively. This scheme, in which no one feature (such as nuclear type or chlorophyll composition) is considered of preeminent importance, is not radically different from recent treatments of the algal flagellates (e.g. Ref. 12) , except for the link considered to be most probable for cryptomonads, and the integrated polyphylety of the zooflagellates.

in question as an indicator of phyletic position, even though the ability of certain groups to undergo considerable change does not mean that other members share this ability.

Character similarities between groups which are not apparently closely related, can be the result of either the retention of primitive features shared by a distant common ancestor, or by independent convergent evolution. At this stage in the knowledge of the flagellates it is often difficult to distinguish between these.

Where similarity might have been caused by loss, such as the absence of chloroplasts, mitochondria, flagella, or smaller features such as flagellar hairs, the probability of independence of events is high. Thus, the hairless isokont flagella of the haptophytes are not considered to be a highly significant factor indicating close affinity with other isokont flagellates, especially when the other cell features are considered. Some anisokont haptophytes are known, and the lateral appendages of the anterior flagellum of Paulova ( 1 7 ) may correspond to the hairs of chrysophytes, but expressed in another form.

Nonphotosynthetic members are present in nearly all the

the permanent loss of mitochondria by a common ancestor, as a few free-living diplomonads lack them ( 1 4 ) . A summary of the most common co-occurring features of each group, omitting cell shape and details of flagellar arrangement, as a 1st step in considering the systems together, is provided in Fig. 6.

The phytoflagellates provide the greatest number of features for use in suggesting probable phyletic affinities among the protists. This does not necessarily make the task easier, but does provide a more detailed general organization on which to base judgments.

The larger groupings of flagellates and probable lineages which result from this author’s interpretation of the following considerations are shown in Fig. 7.

Hibberd & Leedale (24) have suggested that the predominantly flagellated chrysophytes form a natural grouping with the partly flagellated xanthophytes (sensu stricto, excluding the eustigmatophytes) , the nonflagellated diatoms, and brown algae. The haptophytes and eustigmatophytes, long grouped with the chrysophytes and xanthophytes respectively. are thought to be somewhat apart from the above assemblage, although still related

phytofiagellates (bearing in mind that some groups might be unrecognizable without their chloroplasts!), and it is therefore This view has held up well in the light of more recent studies not necessary to assume that the wholly nonphotosynthetic (25) . Ultrastructurally there is now almost no clear distinction flagellates are inonophyletic. between the chrysophyte and motile xanthophyte cells, and the

The lack of mitochondria among many of the nonkinetoplastic brown algae and diatoms are also very similar, sharing chloroplast zooflagellates, as noted above, can he readily attributed to their ultrastructure, nuclear envelope-associated CER (incomplete in usually low-oxygen environments, but might also be due to some brown algae), mitochondria1 villi, heterokont flagella (ex-

to them more closely than other pigmented groups.

38 FLMBILATE PHYLOGENY: A STUDY IN CONFLICTS

cept for uniflagellate diatom spermatozoids) , and biochemical features.

Another, but more unexpected, grouping seems to be emerging from recent studies. Cryptomonads and euglenoids have long been of problematic position. The former have been grouped with other chlorophyll c-containing classes, long considered close to the dinoflagellates until ultrastructural studies, particularly of the nuclei and flagella, revealed major differences between the two. Similarly, although the presence of chlorophyll b in euglenoids has been taken to indicate a closeness to green algae, most of their ultrastructural features and the formation of a PI-3 glucan reserve, seem to contradict this suggestion (37, 63) .

As increasing information has become available on the prasinophyte flagellates (long grouped with the green algae but now separated at the level of class or higher); some possibly intermediate features between the above groups and the more conventional green algae have been found. For example, prasinophytes have flagellar hairs as well as the more extraordinary flagellar scales. The hairs may be stiff and slightly curved, resembling some found on the flagella of the nonphotosynthetic cryptomonad Chilomonas (42) , or very fine in Pedinomonas, resembling those of euglenoids (43) . The flagellar arrangement of Nephroselmis, with 2 lateral flagella instead of the 4 apical flagella more commonly found in prasinophytes, is very similar to that of some simple cryptomonads such as Protochrysis (= Hemiselmis). The cell wall, or theca, of prasinophytes, is non- cellulosic, with some amino acids present (41), thus resembling the proteinaceous pellicle of euglenoids (37) , and probably proteinaceous platelets of cryptomonads (15). In the 2 latter groups, however, the strips and plates are intracellular, whereas the prasinophyte theca, if formed, is extracellular. All have mitochondria with flattened cristae.

An unusual possible link between prasinophytes and cryptomonads is suggested by the recent discovery of coiled, ribbon- like ejectosomes, in 2 species of Pyramimonas (48) , similar to those previously known only for cryptomonads.

Among flagellate groups examined so far, mitosis involves varying degrees of breakdown of the nuclear envelope in prasinophytes (52) and cryptomonads (49) , whereas it is per- sistent in the euglenoids, and nearly so in their probable nonphotosynthetic relatives, the kinetoplastids (60) . The chloroplasts and surrounding membrane ultrastructural features differ, but then they are unique in cryptomonads. Thus, the only principal difficulty in suggesting a possible derivation of the cryptomonads from a common ancestor to Nephroselmis and Pedinomonas, is the presence of chlorophyll c. There is no com- pelling reason why this pigment should be a more reliable indicator of probable affinity than the general organization of the cells. Strictly speaking, it is a chlorophyllide and occurs in two forms, c1 and c2. Most groups containing c have both, but c1 is absent from cryptomonads (31) , possibly testifying in favor of their separate position.

I t is easier to make a case for a link between the primitive prasinophytes (54) and euglenoids, if the presence of a different- linked glucan as a storage product (paramylon), produced ex- ternally to the chloroplast, is not considered of major signifi- cance. The only record of paramylon outside the euglenoids is for the haptophyte Pavloua mesolychnon (33) .

The chloromonads are an enigmatic small group of phytoflagellates. Because of their lack of a secondary chlorophyll additional to a they were thought to be closely related to xanthophyceans. The latter are now considered to be very close to the chrysophytes, their ultrastructure being consistent

with that of the latter group [since the recognition and removal of the eustigmatophytes as a separate class or division (25)] and they possess small amounts of chlorophyll c. Kinetochores have been reported for Vacuolaria ( 2 3 ) , occurring in some xanthophytes (5 1 ) .

Chloromonads also have some ultrastructural similarities to the chrysophytes, particularly in their heterokont flagella, mitochondria with tubular cristae, chloroplast thylakoids associated in groups of 3 and possession of a girdle band. They also show, however, some notable differences, such as the development of an intranuclear spindle, presence of kinetochores, lack of direct connection between endoplasmic reticulum surrounding the chloroplasts and the nuclear envelope, and the possession of trichocysts. Reports on the apparent persistence of the nuclear envelope during mitosis are equivocal (23, 46) . I n the present discussion they are considered as possibly more primitive members of a line leading to the chrysophyte complex, but occupying a somewhat isolated position.

Dinoflagellates are perhaps among the most phyletically para- doxical of the flagellates. Although their nuclear features may be the most primitive of any eukaryotic group, their general cell organization is not noticeably so (64) , being essentially similar to the chrysophyte lineage (chloroplast, mitochondrial and flagellar organization). The production of wall plates in internal vesicles is found only in this group but the manufacture of cellulose and starch suggests a link to the green flagellates. They are perhaps best placed as an early offshoot, close to the di- vergence between the chlorophyll a + b-containing groups and those with a only, or a + c. They have been reported to lack chlorophyll c l , like the cryptomonads ( 3 1 ) , although this may not hold for all. Dodge (12) has suggested an isolated position for both the dinoflagellates and cryptomonads, a view that ac- cords with that put forward here.

Among the zooflagellates, one of the more apparent relationships is that between the bodonids and trypanosomes, linked together by the presence of kinetoplasts within their rnito- chondria, paraxial rods in the flagella, and intranuclear spindles. The euglenoids also possess paraxial rods in their flagella, lacking kinetoplasts but possibly having similar mitochondrial cristae and intranuclear spindles. Furthermore, euglenoids and kinetoplastids have no extranuclear microtubules associated with the intranuclear spindle (24) .

The diplomonads have certain similarities to the retortamonads, effectively doubling not only the flagella but also the nuclei and some rootlets. They appear to be literally “double organisms” (18). Polar fenestrae are present in the nuclear membrane of diplomonads ( 6 ) .

In searching for the possible affinities of the axostyle-containing zooflagellates one is thwarted by the relatively few features that are not found exclusively within them, especially as mitochondria may be almost universally absent. Most axostylar flagellates have closed mitotic mechanisms. The spindle is extranuclear in the trichomonads and hypermastigids (29, 34) and this, combined with the presence of kinetochore equivalents in the nuclear membrane, is similar to the dinoflagellates (49) . However, no other features of these polymastigote flagellates resemble dinoflagellates particularly. Oxymonads and pyrsonymphids have intranuclear, instead of extranuclear, spindles (7).

One intriguing similarity between the above groups and members of the green algae and higher plants has been drawn attention to earlier in this text. The elaborate microtubular arrangements in the axostylar flagellates, with essentially a “pre- nuclear” component, the pre-axostyle (e.g. Monocercomonoides, Ref. 7 ) and the axostyle sheet running from the nucleus to


the posterior end, are well known. In the zoospores of 2 filamentous green algal genera, Coleochaete and Klebsormidium, as well as those of the Charales, bryophytes, and higher plants forming zoospores, there is also usually a band formed of microtubules, which courses from the vicinity of the flagellar bases to the antapex just beneath the surface (see summary and illustrations in Ref. 5 4 ) . An additional component, a laminated “multi-layered structure,” is present near the anterior end. Mitosis is open in forms with zoospores containing these microtubular bands. Although not suggested previously, this possible affinity seems worth exploring, especially as there are so few features to use in polymastigote phylogenetic speculation.

A less radical derivation of the nonkinetoplastid zooflagellates is from non-photosynthetic chrysophyte-like ancestors. I t has already been suggested that choanoflagellates may have origi- nated from this diverse group, but these seem to occupy an isolated position relative to the other zooflagellates. An argument in favor of a chrysophyte-like ancestry for at least some others is the basic flagellar arrangement, with 1 or more anteriorly directed flagella not yet shown to possess lateral hairs and a posterior “recurrent” flagellum. In chrysophytes there is a participation of banded roots in spindle formation (e.g. Ochro- monas, Ref. 5 9 ) , this also being the case in trichomonads (34) , and hypermastigids (29) . Banded structures, however, are also associated with the flagellar bases of green flagellates, particularly in prasinophytes (c.g. Pyramimonas, Refs. 52, 54 ) and in at least one species, Platymonas subcordiformis, the roots seem to be involved in spindle formation. They appear to become “used up’’ in the process.

The origin of the trichomonads and hypermastigids from chrysophyte-like ancestors would also place them closer to the dinoflagellates. The axostylar flagellates with an intranuclear spindle may not be as close to the others as their axostyles sug- gest.

No mention has heen made here of the opalinids or amebo- flagellates. This has been due to the lack of the type of information used here for the former, as well as their paraflagellate status, and the latter are probably not a single phyletic unit, but ameboid forms of various colorless protists.

The general conclusion one reaches from an exercise of this sort is that, although each system seems to provide a guide to probable phyletic position in most cases, there are notable exceptions which would not be obviously apparent if only one system were being considered. This emphasizes the necessity of ronsidering as many aspects of cellular organization as are feasible, in conjunction with one another, when attempting to determine phyletic positions in the flagellates (or for that matter, any other organisnis). I t is hoped that a future combination of the type of insight provided by comparisons at this level, with more quantitative measures of relatedness a t the molecular level, will provide a clear phylogenetic picture of the flagellates. It is doubtful that either approach alone can achieve it.

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Flagellate Phylogeny: A Study in Conflicts

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