Towards a new perspective in protozoan evolution

Europ. J. Proti sto!' 30, 365- 371 (1994) November 25, 1994

European Journal of

PROTISTOLOGY

Towards a New Perspective in Protozoan Evolution Norbert HOlsmann and Klaus Hausmann Institute of Zoology, Division of Protozoology, Free University of Berlin, Berlin, Germany

SUMMARY As in other biological disciplines, in prot ozool ogy the field of phylogeneticall y oriente d systematics is currently experiencing a vita l popularity. Th is is not only cau sed by the discovery of new species with attributes or featur es of missing links or by the elab oration of ultr astructural and molecular feature s of little-known protozoa during the last 20 years, but mainl y by the recent establ ishment of new quantitati ve approaches and meth ods of classification and of phylogeny reconstruction. Among them , the concepts of cladi stic tax onomy app ear most not ab le. They allow us to differentiate between homol ogous characters developed in different histor ical horizon s and to evalu ate their impact on the development of new stra tegies.

Phylogenetic System As prerequisitory supposition for the evalua tion of historical event s, the follow ing fundamental assumption s or hypotheses have to be noted: 1) All eukaryotic organisms are descendants of one single common ancestr al species. This species is lost by splitting up into two succeeding daughter species; however, its historical existence can only be acknowledged by a scientific name, * Protozoon eukaryoticum, whereby the star indicates the hypothetical nature of the naming. Therefore, thi s species and all of its successors form a natural group or a holophylum, the Eukaryota. Thi s monophyletic group include s not only all of the recently recognized euk ar yotic species but also those that are up to date unknown as well as the ancestr al ur-species and its successors arising fro m speciation proce sses. 2) Th e structural and organizational diversity among all eukaryotic organisms is the result of evolutionary processes such as genetic variations, topographic separation and species-splitting. On the other hand, the unambiguous similarities of distinct characters between them are caused by sharing a common heritage, by con vergent evoluti on, or by parallelism. Wh en recon structing the phylogenetic relationships betw een th e eukaryotes, how ever, only those attributes or features can be used which have the same evolutionary © 19 94 by G us tav Fischer Verl ag, St u ttga r t

ongin and the same genetic basis. The se so-called hom ologi es comprise ob servable products of the genotype (as morphological, ethol ogical , embryological, biochemical , or other characters) as well as the genome itself or part s of it. 3) Recently conducted tree-formations mostly utilize data derived from mol ecular characters leading to distan ce tree s [8]. Morphological feature s which are useful and necessary in the direc t contact with the surro unding elements such as water, oxygen, parasites or others remain rather underestimated in thi s context. However, they are said to play an important role for the sur vival of the successful species and thu s should also be taken into account for recon struction of tre es.

Intrinsic and Extrinsic Apomorphies For the reconstruction of an evolutionary tree we need characters representing evolutionary novelties , so-called apomorphic characters or apomorphies. One group of these characters is repr esented by innovations regarding the intern al organization (such as amelioration of mitotic division techniques, or evolution of adapted digestion pathways). Such features we would like to designate as intrinsic characters. In a second group, the novelties repre sent characters moderating and interacting with ext ernal factors (such as 0932-4739-94-0030-0365 $3 .50-0

366 . N. Hiilsmann and K. Hausmann

pseudopods used for crawling on a substratum or flagellar mastigonemata useful for modulating the locomotion velocity or swimming direction). The latter group of extrinsic features are more appropriate for use in an ecological context when evolution is seen as a direct process of exposition of the members of a given species to different abiotic parameters on the

one side and biotic non-conspecific competitors on the other side. To us, it seems important to evaluate the impact of those organelles or physiological topics on the general evolution which are present in an undoubtedly complex cell type (such as a representative of the Euglenozoa) and to compare the situation in this group with that in other, less complex taxa. We will try

Fig. 1. Simplified and tenrativc phylogenetic system of eukaryotic organisms. - I. Firsr cukarvoric level wirh endoplasmic rericulum (cr) and nuclucar envelope (ne) and sexuality a aporno rphic characrer s; branching taxon: Mi crospora (M sp). - II. Level of monomastigor-flagel-

IV

III

II

larcd eukaryorcs : cvolurion of flagellum (9x2+2) and kine tosome; branching taxon: Archamocbaca (A a), - III. Level of dimas rigor-organized Eukaryora: kincrosomcs arc paired (pks); branc hing taxa: the primarily tetramasrigor flagellares Rcrorta rnonadca (Rm), Oxyrno nadea (Om) and Pa ra basalea (Pb). - IV. Level of co nvergently evolving endomemhranous systems, especially of classical and atyp ical dicryosorncs (d). - V. Level of Meraka ryora: evolurion of cucvtic cells (= meraka ryorcs } by cndosy rnbiosis wirh prokaryotic organisms which later on evolve into rnirochondria and chloroplasts. Typical taxa appear: Euglenozoa (Eu), Alvcolara (AI), Biliphyra

(Bi), Chlorophyta

(Ch),

Metazoa (M z), Choano-

flagellara (Cfl, Ascomyceres (A) and Basidiorn ycetes (B), Chrornobion-

ra (Chr), chizopyrcnida ( c). Boxes wirh © arc reserved for taxa which a rc exti nct or might bc defined in the future. After

141·

Protozoan Evolution . 367

to discuss the following parameters: flagella, dictyosomes, mitochondria, plastids, ribosomes, and sexuality.

Flagella Flagella are doubtlessly extrinsic organelles according to the definition given above. Within the most primitive metakaryotes, the Euglenozoa, they represent plesiomorphic characters (Fig. 1) which this taxon shares with other flagellates and which is derived from an ancestral species that has evolved the singular 9x2+2 architecture of the axoneme. This ancestral species, * Eukrayoticum mastigotum, is therefore splitted from the KINGDOM MICROSPORA as the stem of the KINGDOM MASTIGOTA (Tab. 1). It has a flagellum as apomorphy and is the precursor of all other flagellated organisms including those which have lost this character secondarily in adaption to conditions in water-deprived biotopes and/or when included in massive cell walls (such as terrestrial amoebae, higher fungi, and green plants). However, in the case of Euglenozoa, the appendices to flagella (mastigonemes) or the additional rigidification by an axonemal rod represent apomorphies usable for erecting them as a holophyletic taxon. The number of flagella in a given cell type reaches from zero to several (ten) thousands, such as in ciliates or parabasalians. The number of flagella can therefore serve as more than only a diagnostic character. In case of absent flagella or their basal bodies (kinetosomes), we cannot decide a priori whether this is a primitive or a derived character. In such cases, however, the possibility exists to search for other features which can indicate the exolutionary level. In case of parallel absence of other organelles such as mitochondria, dictyosomes, or 80S-ribosomes, the probability of a general primitive organisation appears to be appropriate. This is the reason why the phylum Microspora (with an otherwise highly developed penetration apparatus as apomorphic feature) represents a lineage branching off from the rest of Eukaryota before the evolution of flagella [3,4, 7], or why amoeboid organisms such as Amoeba proteus or Arcella vulgaris (with all the characters of Euglenozoa except for the flagellar apparatus) have to be seen ari sen from a lineage evolved later than the first species of the mitochondriated Metakaryota (Fig. 1). When flagella or at least kinetosomes are present, the grouping characters become important. So far known, only two general patterns are realised: that of the unpaired or single and that of the paired or twofold situation. Without any doubt, the unpaired flagella document their primitive nature when occurring in combination with other primitive characters as in the subkingdom Archamoebaea [1]; however, uniflagellation (or the existence of only one single kinetosome) has to be designated a posteriori as derived in the case of secondary complete loss of the second flagellum (such as in the swarm cells of Chlorarachnion reptans, in monokinetids of ciliates or possibly in some cercomonads).

In contrast, the doubling of flagellar structures into pairs of flagella arranged in typical compositions serves as indicator for the presence of an apomorphic character. It is therefore justified to erect the SUBKINGDOM DIMASTIGOTA (Tab. 1) as a sister group of the primitive Archamoebaea. As additional apomorphic character, the Dimastigota have developed, for example, the reduction of untypical axonemal microtubular arrangements and special karyomastigont microtubular associations [1]. In the evolution of flagellated cells, the establishment of two flagella instead of a single one opened up the liab ility to differentiate between the two and the potential for a division of function: one flagellum might serve as locomotion organelle or for food acquisition whereas the other serves as holdfast organelle (such as in Bicoeca). In general, the evolution of biflagellation may be seen as one of the most successful radiation points: this character is present from flagellates to multicellular organisms up to man, at least in sperm cells or mitotic cells with duplicated centrioles. Doubling of biflagellates to tetraflagellates may be seen as an independent, convergently evolved process occurring at least in two separated groups: within the Chlorophyta and within the remaining ami tochondriate zooflagellates. The latter could easily form a natural group, primitive by the absence of mitochondria and advanced by the (at least basically) tetraflagellation with sepa ration into one recurrent and three forward directed flagella . This constellation justifies the erection of the SUPERPHYLUM TETRAMASTIGOTA (Tab. 1). Based on this organisation type we find a secondary variation of flagellar number: reduction down to zero (Dientamoeba) and multiplication to several tens of thousand (Parabasalia) . The presence of a kinetosome-derived structure, the axostyle, serves as apomorphic character. The uniqueness of this microtubular organelle justifies the erection of the PHYLUM AXOSTYLATA within the taxon Tetramastigota.

Table 1. Phylogenetic relationships within the Eukaryota. Taxa in bold characters are defined for the first time in this article . EMPIRE EUKARYOTA Kingdom Microspora Phylum Microspora Kingdom Mastigota Subkingdom Archamoebaea Phylum Caryoblasta Subkingdom Dimastigota Superphylum Tetramastigota Phylum Retortamonada Phylum Axostylata Superphylum Metakaryota

368 . N. Hiilsmann and K. Hausmann

Dictyosomes Dictyosomes are considered as apomorphic structures evolved in the stem line of Dictyozoa CavalierSmith [2,3]. Regarding the initial question whether they represent intrinsic or extrinsic organelles, an answer is difficult to give. Basically, dictyosomes represent stacks of flattened vesicles derived from, or at least in connex to, the endoplasmic reticulum (ER). In so far, where present, their occurrence does not represent a true novelty, but merely a new morphological complex of basic elements established already in the stem line of the Eukaryota. Comparing the actual informations known from different taxa, the following situations appear: so-called dictyosomes are present in Metakaryota sensu Cavalier-Smith and, within the amitochondriate eukaryotes, in the Parabasalia; they seem to be, in a lower degree of perfection, also constitutive elements of some Microspora. While for Microspora the informations are rare and therefore not interpretable, dictyosomes of both other assemblages represent two rather different groups of differentiation: (1) the dictyosomes of the Parabasalia appear as large organelles of two dozen or more stacks stabilized by elements of the cytoskeleton and with an intrinsic metabolic pathway [10], whereas (2) the dictyosomes of the Metakaryota represent smaller organelles with normally only 3 -7 cisternae involved in several extrinsic functions. Thus the latter organelle produces scales, mastigonemes, extrusomes and so on, at least primarily with functions in cell defence or cell attack [2], later on also with more intrinsic functions within the function of the GERL complex. Under these circumstances, a parallel or convergent evolution of ER-stacks in Parabasalia and Metakaryota seems to be more probable than a monophyletic origin. Therefore, the taxon Dictyozoa sensu Cavalier Smith seems not to be justified.

Mitochondria Mitochondria are considered as relics of formerly independent prokaryotic endosymbionts. They likely represent extrinsic symbionts in the beginning of coevolution, later on merely intrinsic organelles. The extrinsic relation may be seen in the evolutionary pressure to handle and to absorb the poisonous oxygen produced increasingly by photoautotrophic cyanobacteria. Their function as established organelles, however, lies merely in the utilisation of oxygen for optimized respiration purposes. After establishment, these organelles show a relatively high diversification regarding the structural architecture of the internal cristae [7]. This has no recognizable influence on the fitness of their hosts and sometimes mitochondria of morphologically different types are even present in one and the same species during distinct stages of its life cycle [10]. Therefore, the usefulness of mitochondria for evaluating phylogenetic relationships of their host species seems at least questionable. Even in the small group of non-related anaerobic rumen ciliates, where they evolve or degenerate to hydrogenosomes [6], their unique morphological features arose convergently under the pressure of the same ecological conditions. So they form an ecological community but not a systematic taxon. The fact that free-living Archamoebaea and Tetramastigota, living in almost oxygen-free microhabitats exposed at least temporarily to the atmosphere, do contain extrinsic bacteria as endosymbionts demonstrates that the attempt to host oxygen-consuming bacteria is obviously of convergent origin. But only in the metakaryotes did such an association become manifested permanently. Therefore, a corresponding taxon, Metakaryota sensu Cavalier-Smith, is justified.

Ribosomes In protists two kinds of ribosomal types occur, the 80S- and 70S-ribosomes. Whereas the former - so far known - are exclusively found in metakaryotes, the latter are typical constituents of prokaryotes and,

Fig. 2. This suggestion for the evolutionary lineages within the photoautotrophic eukaryotes, which results from molecular and morphological data, resembles a three-dimensional network of relationships. The individual genomes of the host cell and the endosymbiont are symbolized by lines; the pathway of autotrophic lineages is drawn in green and that of the heterotrophic lineages in blue (prokaryotes) or red (eukaryotes). - HM = Level of heterotrophic metakaryotes with two genomes (2) from the starting point of endobiosis between one heterotrophic eukaryote (HE) and one mitochondrial precursor (heterotrophic prokaryote, HP ). - AM = Level of the autotrophic metakaryotes in which, after endosymbiotic incorporation of an autotrophic prokaryote (AP), three genomes are present (3). - HM + AM = Level of endosymbiosis between one heterotrophic metakaryote of the level HM and one of the autotrophic partners of the level AM representing the evolution of autotrophic organisms such as dinoflagellates (D), Chromista (Chr), euglenids without (Eu) and with syrnbionts (Eus), and other taxa; primarily, the numer of genomes is 4 (+ 1 reduced mitochondrial genome). - HM + HM + AM = level of the most complex associations: unicellular organisms of the level HM + AM live as endosymbionts in heterotrophic metokaryotes such as ciliates without (Ci) or with endosymbionts (Cis), foraminifers without (Fo) and with endosymbionts (Fos), "radiolarians" without ("R") or with endosymbionts ("Rs"), or metazoans (Co = corals). In such cases, the number of genomes might reach up to seven (5+1+1), but mostly only five genomes are present. Within the three lower levels, the organisation principle of unicelluarity is evolved independently into multicellularity which is symbolized by left- or right-handed arrows: multicellular organisms originate within the Biliphyta (Bi), Chlorobionta (Ch), Phaeophyta (Ph), higher fungi (A / B = Ascomycetes / Basidiomycetes), and higher Metazoa (hMz). I-V: compare Fig. 1.

~

Protozoan Evolution· 369

Fos

Bi

Ch

AP

fiRs"

370 . N. Hiilsmann and K. Hausmann

within the lower eukaryotes, also of the Microspora and some Tetramastigota (Giardia) . Even at this early stage of knowledge, the decision is justified ,t hat both kinds of these intrinsic organelles are constituents of the eukaryotic cell and that they obviously had ~o ~m­ portant influence on the furthe~ success ~f speciation processes. The existence of 70S-nbosomes m some l?wer Eukaryota may indicate that over a rather l0!lg. time of evolution both kinds were present as synergistic organelles in the same species, before the evolutio.n~ry pressure towards simplification favoured a decision for only one of the two possibilities. The appearance of 70S-ribosomes in eukaryotes is interpreted as a crypto typic appearance or atavism.

Plastids All existing plastidomes are exclusively found in eukaryotic cell lines which belong to the Metakaryota. They therefore depend on the pre-existence of functioning mitochondria. This constellation supports the t~e­ ory of mitochondrial origin from oxygen-consuming and later on oxygen-depending endosymbionts. Fro,m the evolution of life, this does not mean the pre-existence of animal-like versus plant-like organisms, but only the pre-existence of. eukaryotic ~eterotr~phic organisms versus eukaryotic autotrophic orgam~ms, [9). The original intracellular impact could be an mt~ms!c one: to allow the oxygen-depending cells to survive m oxygen-free but light-flooded microhabitats. I~ ~ore stationary organisms a dependence on the nutritional aspect, the use of metabolites, is more f~v0l!rable. The establishment of symbiosis and organellisation between two prokaryotic cyanobacterial lineages and tw,o biflagellated metakaryotes led to two , autotrop~Ic lineages (Biliphyta and Chlorophyta, FIg. 2). which must be considered as holophyletic groups either at the moment of origination or when excluding per definitionem all those unicellular individuals (not species!) which later on the~selves ?ecome ~ndosy~­ bionts of other heterotrophic organisms. ThIS special situation of speciation by chimera formation h~s not yet been considered by the experts of p.hy~ogenet!c, ~ys­ tematics. On the other hand, the symbiotic acquisition of phototrophic organisms belonging to the gr:e~ algae or the red algae by certain metakaryotes within ,the groups of Euglenozoa, Dinoflagellata and Chromista (and others, compare [2,3,5,9]) does not mean ~hat the phyletic nature of the host-line must be put into question. In a subsequent step of more recent ~nd theref~re rather loose chimera formations, organisms containing four genomes such as autotrophic, dinoflagell~t~s (zooxanthelles) and rhizopodial ~rga!lIsms (f?rammIfers and radiolarians), but also cnidarians are mvolve? (Fig. 2). The mutual dependence seems to be more u,mlateral (favouring the hosts more t.han the guests WhI,C~ can survive independently). Only in the case of the CIliate Mesodinium rubrum is the guest divided into several compartments.

Sexuality Sexuality must be seen as a mainly intrinsic feature developed from mitosis and using cytoskeletal elements such as micro tubules and microfilaments necessary for chromosome transport or for cell division . In a genetic and phylogenetic context, sexuality accomplishe.s se~­ eral important tasks: 1. the regulatIOn .bet~een dIpl~Id and haploid life stages (whereby the diploid stage w!th two alleles functions simultaneously as conservative storage and as an experimental platform for mutations) 2. the conservation of a diploid status, 3. the new combination of genes, 4. the elimination of genetic malinformations, 5. the most successful kind of distribution of new informations within a conspecific population (with important impac.t ?~ speciation ,processes). This also implies the possibility to ~st.a?hsh a sexual polymorphism necessary for the division ~f funct ion and/or the avoidance of intraspecific compention between male and female organisms. In many protozoa sexuality is not easy to detect; however, using electron microscopy, synaptonemal complexes are the morphological proof for sexuality as demonstrated in all major groups from Microspora to Metakaryot.a during the last two decades. We may assume that diploidy and sexuality are apomorphic characters established during the evolution of eukaryotes and therefore not suited for the construction of phylogenetic trees. Only the differentiation into male and female gamonts, observable in Metakaryotes such as Chlorophyta and Apicomplexa, can serve as indicators of the le,vel of evolution. Similarly the loss of sexual reproduction in favour of vegetative clonal repr~duction s~rateg.ies cannot be used to elucidate the quality of relationships between major groups.

Acknowledgements We wish to thank Prof. Dr. W. Sudhaus, Institute of Zool ogy, Division of Evolutionary Biology, F~ee University of Berlin for critical reading of the manuscnpt and for valuable discussions and suggestions. Peter Adam, scientific illustrator of the Institute of Zoology, is thanked for his immeasurable patience during the preparation of the graphics.

References 1 Brugerolle G. (1991): Flagellar and cytoskeletal systems in amitochondriate flagellates: Archamoeba, Metamonada and Parabasala. Protoplasma, 164, 70-90. 2 Cavalier-Smith T. (1991): Cell diversification in heterotrophic flagellates . In: Patterson D. J., and ~arsen J. (eds.): The biology of free-living heterotrophic flagellates, pp. 113-131. Clarendon Press, Oxford. 3 Cavalier-Smith T. (1993): Kingdom Protozoa and its 18 phyla. Microbiol. Rev., 57, 953-994. , 4 Leipe D. and Hausmann K. (1993): Neue Erkenntnisse zur Stammesgeschichte der Eukaryoten. BIUZ, 23, 178-183.

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5 Margulis L. (1993): Symbiosis in cell evolution. Freeman, New York. 6 Muller M. (1993): The hydrogenosome. J. Gen. Microbiol., 139,2879-2889. 7 Patterson D. J. (1994): Protozoa: Evolution and systematics. In: Hausmann K. and Hulsmann N. (eds.): Progress in protozoology, proceedings of the IX International Congress of Protozoology Berlin 1993, pp. 1-14. Gustav Fischer, Stuttgart. 8 Schlegel M. (1991): Protist evolution and phylogeny as discerned from small subunit ribosomal RNA sequence comparison. Europ. J. Protisto!' , 27, 207-219.

9 Sitte P. (1993): Symbiogenetic evolution of complex cells and complex plastids. Europ. J. Protisto!., 29, 131-143. 10 Vickerman K., Brugerolle G. and Mignot J.-P. (1991): Mastigophora. In: Harrison F. W. and Corliss J. O. (eds.): Protozoa. In: Harrison F. W. (ed.): Microscopic anatomy of invertebrates, vo!. 1, pp. 13-159. WileyLiss, New York.

Key words: Evolution - Microspora - Mastigota - Dimastigota - Tetramastigota - Axostylata Norbert Hiilsmann and Klaus Hausmann, Institute of Zoology, Division of Protozoology, Free University of Berlin, KoniginLuise-Str, 1-3, D - 14195 Berlin, Germany