- Email: [email protected]
Molecular phylogeny of eukaryotes
REVIEWS
Molecularphylogenyof eukaryotes Martin Schlegel
he unicellular eukaryotes or Comparisons of ribosomal RNAs and These related proteins are found protists have been studied various protein codihg genes have in organisms that represent each for more than 300 years. contributed to a new view of eukaryote of the three lines. If the gene Besides their extreme mor- phylogeny. Analyses of paralogous protein duplication preceded the diverphological variation they show coding genes suggest that archaebacteria gence, then there should be more great ultrastructural, physiologiand eukaryotes are sistergroups. differences between the duplical and biochemical diversity, far Sequence diversity of small subunit rRNAs cated, paralogous genes within exceeding that of multicellular in protlsts by far exceeds that of any one taxon than within one ortholoorganism+3. Despite our increasmulticellular or prokaryote taxon. gous gene family, even if members ing knowledge, the phylogenetic Remarkably, a group of taxa that lack of the three different lineages are relationships of protists and also mitochondria fhst branches off in the compared. Thus, one family of the origin of the eukaryote ancessmall subunit rRNA tree. The later orthologous genes can be used to tor remained unclear, resulting in an radiations are formed by a series of clades infer the root of the other gene increasing number of often conthat were once thought to be more family, and vice versa. This reflicting classification scheme+s. ancestral. Furthermore, tracing of the sults in mirror trees (Fig. lb). This picture changed dramatievolutionary origin of secondary Such an analysis for the EF-1(Tu) cally when PCR (polymerase chain endobiontic events is now possible and EF-2 (G) sequences puts the reaction) techniques6 became with sequence comparisons. root into the eubacterial line of available, allowing the rapid amdescent, which means that Archae plification and sequencing of bacteria and Eukaryota share a Martin Schlegel is at the Universittit Ttibingen, informative genes from minute more recent common ancestor14. Zoologisches Institut, Abteilung Zellbiologie, Auf der amounts of DNA. By far the most An analysis of duplicated genes Morgenstelle 28, D-72076 Ttibingen, Germany. comprehensive data are available of the ATPase subunit has given from small subunit rRNAs,of which an identical resulW5. These results more than 900 complete sequences (200 eukaryotic) have have been widely acceptedr6. Furthermore, the eubacterial been determined7. Increasingly, protein coding genes are rooting has been used to support the hypothesis that life revealing new insights into eukaryote evolution. The trees evolved at high temperatures, since hyperthermophiles inferred from the different kinds of molecules are largely appear early in the two branches of the universal treenJ8. The discrepancy between the small subunit r-RNAphycongruent, but marked differences also appear. Another issue of debate concerns the correct alignment of the logeny and the protein phylogenies could be explained by different rates of evolution for 16S-like r-RNAand the prosequences (i.e. the juxtaposition of the homologous sites of the molecule) and the conversion of sequence in- tein coding genes in the different lineages. An alternative formation into phylogenetic trees. The different methods scenario was proposed by Soginrl, who postulated an RNAthat are currently used are summarized in Box 1. dependent proto-eukaryote that engulfed an ancestral archaebacterium with a DNAgenome, which gave rise to the eukaryote nucleus. Thus, archaebacteria are more closely The nature of the universal ancestor and evolution of related to eukaryotes in the protein phylogenies but are the eukaryotic cell closer to eubacteria in the r-RNAphylogenies because of Sequence analyses of small subunit rRNAs have retheir longer common evolutionary history. vealed the existence of three primary lines of descent: However, the approach using duplicated genes to Eubacteria, Archaebacteria and Eukaryotaa. Similarities in these three lineages regarding the components of the ma- infer the root of life and its placement in the eubacterial chinery of RNA and protein synthesis, together with the line of descent has been criticized recently, and indeed existence of many metabolic pathways common to eu- things turn out to be much more complicated. Two new bacteria and archaebacteria, indicate that the universal an- eubacterial ATPases were discovered recentlylgJ0, which are more similar to the archaebacterial and eukaryote cestor was probably already an elaborated cellsJO.However, V-type ATPases than to the eubacterial F,/F, ATPases, the root of the universal tree of life seems to be obscure. which were used to represent the eubacteria in the dupliA higher sequence similarity can be observed in the small cated treesr4J5. Therefore, not one but two gene duplisubunit rRNA of Eubacteria and Archaebacteria when compared to Eukaryotarl. By contrast, DNA-dependent RNA cations occurred in the universal ancestor before divergence. This suggests that V-type and F,/F, ATPases are polymerase genes of Eukaryota and Archaebacteria are paralogous and that the trees are composed of orthologous more alike than either is to EubacterialzJ3. and paralogous proteins, which may result in a false rootOne way to root a phylogenetic tree is to use an outing (Fig. 2)2*. group. Logically, however, there are no outgroups for all In contrast, only one family of each elongation factor contemporary life forms. As an alternative, gene families gene has been found in each line of descent (Fig.lb), and that duplicated before the divergence of the three primary lines of descent can be used to infer the root of the uni- this still supports the eubacterial rooting of the universal versal tree (Fig. la and Box 2). For example, elongation fac- tree. An analysis of the duplicated glutamate dehydrogenase genes22 has not resolved the question. Here, the root tor EF-1 (Tu) and EF-2 (G) are duplicated, conserved prowas placed either inside the archaebacterial branch when teins that play an important role in the translation process.
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distance methods were applied, or into the eukaryotic branch when parsimony methods were used. In the tree of CDH family I, the eubacterium Anacystis nidulans groups consistently in the eukaryote lineage with the eumycete Neurospora crassa, whereas the eukaryote diplomonad Ciardia lamblia is placed within the eubacteria. In family II, the bovine Bos groups with chicken Callus rather than with other mammalia. These results throw severe doubt on the value of this molecule for phylogenetic analyses. In a phylogenetic (‘cladistic’) analysis23 of the elongation factor and glutamate dehydrogenase genes, Forterre et al.“’ found three synapomorphies (see Glossary, Box 2) for a sistergroup relationship of Archaebacteria and Eukaryota, and one synapomorphy for a Eubacteria-Archaebacteria clade out of 493 sites! The problems in identifying the root correctly may lie in the increase of multiple substitutions in genes that have been separated for very long periods. This reduces the number of informative sites and hence the value of the gene for phylogenetic analysis of distantly related organisms. And, there is growing evidence that the eukaryote nucleus is truely chimeric and even contains eubacterial genes. Sequence analysis of nuclear-encoded glyceraldehyde-3phosphate dehydrogenase (GAPDH) genes revealed that not only the plastid GAPDH, but also the GAPDHenzyme of glycolysis in plants, animals and fungi evolved from a gene that was laterally transferred from a eubacterial end@ biont which gave rise to the mitochondria or plastids24. Nonetheless, to tackle the root of the universal tree of life, it is necessary to continue the search for conserved duplicated genes in the three primary lines of descent. As pointed out by Forterre et al., it is striking that sequence information is especially lacking from protistszl. Small subunit rRNA diversity and eukaryote phylogeny A comprehensive database exists for the small subunit (‘16S-like’) rRNA. The sequence diversity in protists by far exceeds that of any prokaryote or multicellular taxon (Fig. 3). It has to be emphasized that data from many groups, such as cryptomonads, haptophytes, and most of the ‘amoebae’ are still lacking. The current view of protist phylogeny derived from 16S-like rRNAs is shown in Fig. 4. far/y branchings are represented by amitochondriate
taxa Remarkably, at the base of the tree, taxa that lack mitochondria emerge. The 16%like rRNA tree does not resolve unambiguously the branching order within these groups that lack mitochondria. However, the group as a whole is always placed at the bottom of the tree, regardless of the method of analysis applied (see Box 1)25.Since all members of this group are mostly parasites that live in
(4
Box 1, Methods for inferring phylogenetic trees from sequence datas4y43 Distance matrix methods - (1) Genetic distance values are calculated Pairwise on the basis of the nucleotide or amino acid differences between the sequences corn pared (including correction factors for multiple substitutions). (2) Distance values are then converted into phenetic dendrograms. (3) The goodness-of-fit of the observed distances in the tree to the calculated distances is measured, and the tree with the minimum discrepancy is preferred. This method is largely rateindependent, but is sensitive to sampling set. Maxlmum parsimony methods - (1) The trees that need the minimum number ot steps (mutations) to convert the sequences into each other are found. (2) Hypo thetical ancestral sequences are calculated. Maximum parsimony is compatible with the concept of ‘phylogenetic systematics ‘23; any extra count of site change on a tree gives the number of ancillary hypotheses that must be erected to explain evolution in the group, and the method favours the tree with the mlnimum number of hypotheses (‘Occam’s Razor’). These methods are rate-sensitive. Fast-evolving nonrelated sequences tend to be grouped together. A rate-insensitive variant IS Lake’s evolutionary parsimony44 or ‘invariants? Sites where predominantly transitions occur are excluded from the analysis. However, since it uses only part of the sequence information, less-significant branches may result. In some parsimony methods (Hennig86; see Box 3) ancestral states can be defined. Maximum likelihood methods -These have a statistical approach. (1) A concrete model of the evolutionary process that converts one sequence into another IS specified. (2) The likelihood that the given evolutionary model will yield the observed sequences is calculated. The inferred phylogenles are those with the highest likelihood. This method uses the largest amount of sequence information. but is computationally very intensive. Bootstrapping - (1) Repeated samplings (up to 1000 and more) of the alignec data set are made, whereby parts of the sequence are excluded; other parts are included several times in order to yield a slightly different data set. (2) All modified data sets can then be analysed with one of the tree-building methods and the number of times a branch appears In the different trees be visualized in a consensus tree. Although computationally very time-consuming. this method is frequently used to estimate the significance of branchings in a tree. Analysis of signature sites - This is a phylogenetic approach. Signature sites mean the presence of the same character (nucleotide. amino acid, insertion. deletion) in all outgroup sequences (or at least predominantly). The presence of the same character in the ingroup indicates a plesiomorphy, and shared deviations indicate synapomorphies; these are used to argue for a relationship. Usually, however, only a few sites in a sequence are signature sites. -
low-oxygen habitats, such as in the gut of cockroaches and termites, it was long assumed that these organisms had lost their mitochondria in adaptation to their environment. In contrast, the rRNA tree strongly supports the view that these organisms were primarily without mitochondria. Thus, it seems reasonable that amitochondriate organisms were common and widely distributed in the early evolution of eukaryotes. After the emergence of the mitochondriate cells, they may have remained competitive only in lowoxygen habitatszfi. Mitochondriate taxa branching off in the middle part of the tree The uptake of a bacterial endobiont that gave rise to the mitochondrion happened before the next taxa branched off in the eukaryote tree (Fig. 4). This part of the tree is
(b)
E
Archaebacteria
Eubacteria
Archaebacteria
Archaebacteria
Eukatyota
Eubacteria
Eubacteria
Eukaryota
Eukaryota Fig. 1. (a) Gene duplication occurred before the divergence groups Archaebacteria and Eukaryota together.
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Box 2. Glossary Homology: characters (e.g. genes, proteins, hairs, setae, feathers, bones) which descend from a common ancestor are homologous. Paralogous genes: homologous genes which originated from a gene duplication. Orthologousgenes: one of the duplicated genes being compared between different species (see also Fig. 1). For phylogeny inference orthologous gene or protein sequences have to be compared. Differences between orthologous sequences are those which occurred after speciation, whereas paralogous sequences show differences which accumulated after the duplication event and are thus not informative for reconstruction of a branching pattern. Plesiomorphy: ancestral state of a character, e.g. the primary winglessness of insects. Apomorphy: derived character state, e.g. wings of ptetygote insects. Autaoomorphy: derived character which accounts for the monophyly of a taxon. Synapomorphy: shared derived character which accounts for the sistergroup relationship of two taxa. The terms plesiomorph and apomorph are relative, depending on the taxonomic level looked at. For example, hairs are an autapomorphyforthe mammalia, a synapomorphy for the two sister taxa Monotremata and Theria, and a symplesiomorphy for the Marsupialia and Placentalia within the Theria.
Ancestral
ATPase
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& First duplication into alpha and beta subunit
L4 Second duplication V-type and F-type
into
Tree reconstruction paralogous genes
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False outgroup
False
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and
(the other subunit) Archaebacteria
V-type
Eubacteria
V-type
Eukaryota
V-type
Archaebacteria
?F-type
Eubacteria
?F-type
Eukaryota
F-type
Fig. 2. The ATPase gene-tree may be composed of orthologous and genes and thus does not reflect the true phylogenetic relationships of Archaebacteria and Eukaryota. The thick lines indicate the compared The correct outgroups would probably be the F-type ATPases for ATPases, and vice versa.
paralogous Eubacteria. molecules. the V-type
statistically well-supported: in 94% of bootstrap replicates (see Box 1) this branch appears. The following branches are not so strongly supported, except the monophyly of the Euglenozoa, which first branches off after the acquisition of mitochondria. With approximately 2000 species, the Euglenozoa are the most important group in this middle part of the tree. They comprise the heterotrophic Kinetoplastida (free-living Bodonida and parasitic Trypanosomatida) and the free-living Euglenida. A part of the Euglenida is aute trophic. However, their chloroplasts are not primary prokaryotic endobionts. They are remnants of a eukaryotic endobiont that is related to the chlorobiontsl. Since green 332
algae appeared much later in eukaryote evolution (see below), as suggested by electron microscopical analyses and the occurrence of chlorophylls A and Bl, it is evident that the autotrophic euglenids are a late emergence and that the Euglenozoa evolved as a primarily heterotrophic group. The acellular slime molds and several taxa of the ‘old’ amoebae follow next: the Entamoebae, the Schizopyrenida (amoeboflagellates), together with one group of the cellular slime molds, the Acrasida. A relationship between the latter two was already assumed because of similarities in the life cycle and in trophic amoebae. Interestingly, the Entamoebae have no mitochondria. Their position in this part of the tree suggests, however, that these organelles were secondarily lost in adaptation to their parasitic way of life (but also see below). Rapid proliferation of major groups later in eukaryote evolution The next branch leading to a radiation (Fig. 4) is also statistically well-supported by bootstrap analysis. A series of clades separated about one billion years ago. This was calibrated with red algae, which appear to branch off first in the radiation; their oldest known fossils (bangiophytes) were found in 1.25 billion year old rock.+. This rapid radiation is difficult to resolve. However, a sistergroup relationship between Metazoa and Fungi is suggested28. There is also some evidence that the Chlorobionta (green algae and metaphytes) are the sistergroup to Metazoa and FungP. The remaining taxa provide an impressive example of the power of molecular systematics: the ‘stramenopiles’ contain a variety of quite different groups, such as the former heterokont algae (brown algae, diatoms and chryse monads), water molds, opalinids and others. The sequence data support a grouping of these taxa that originally was based on the presence of thin tripartite hairs on the membrane surface, especially on one of the two flagellazg.With respect to the ‘alveolates’ the sequence analyses revealed the phylogenetic relationships of ultrastructurally welldefined but isolated taxa. The alveolates encompass the Dinoflagellata, a group with mostly free-living and a few parasitic species. Their next relatives are the Apicomplexa, which consist solely of endoparasitic species and which are flagellated only during sexual stages in their life cycle. Together, Apicomplexa and Dinoflagellata are the sistergroup of the primarily phagotrophic Ciliophora30. These three taxa are so different in ultrastructure and ecology that their relationship was not accepted before the sequence information, although there were ultrastructural hints, namely their possession of the submembraneous alveolar structures, which led to the name ‘alveolates’. In 1976, Taylor pointed out a possible relationship between Dinoflagellata and Ciliophora31. Not all of the branches are significant. Thus, it is necessary to search for additional information that might help to resolve these parts of the tree better, and moreover, to check the total branching order of the IGS-like rRNA tree. For this purpose, protein coding genes are increasingly used. Comparison of ribosomal data with protein trees A tree inferred from elongation factor EF-1 gene sequences using a maximum likelihood method is largely congruent with the rRNA tree32. Especially, there is strong support for a metazoan-fungal clade. But it differs markedly in the position of Entamoeba histolytica and Dictyostelium discoideum: E. histolytica branches off before the emergence TREE vol.
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REVIEWS of the Euglenozoa, while Eukatyota D. discoideum groups with the Stramenopiles Chlorobionta. How can these differences be explained? Fungi c\ v /3 Apicomplexans Some of the problems enCiliates, Dinoflagellates countered with sequence data Red algae may be revealed by these contradicting results, such as the proper alignment of Slime molds I, the sequences, which is especially a problem for distantly related taxa and which Eubacteria is ultimately always a subjecI \ tive decision of the researcher doing the analysis. Different people can arrive at different alignments and consequently different trees even with the very same sequences. Second, different genes evolve at different rates, and the same gene may evolve at different rates in different lineages. Diplomonads Thus, the same gene may have reached a point of saturation, containing no more phylogenetic information in one taxon (because of too many back mutations), while it still Archaebacteria is informative in another. Third, a bias in C/C content Fig. 3. Molecular phylogeny of Eubacteria, Archaebacteria and Eukaryota inferred from 16Slike rRNAs. Lengths of branches correspond to genetic distances. The genetic distance among protists by far exceeds that of any multicellular taxon and that can influence the position of of both Eubacteria and Archaebacteria. Redrawn, with permission, from Ref. 11. a sequence in the tree, as was the problem with the early branching of the C. lumblia of a serial endosymbiosis is strongly supported by rRNA sequence in the 16S-like rRNA tree*s. Fourth, rapidly and protein sequence data. This situation, however, is evolving sequences tend to group together and branch more deeply in a sequence tree33. This is especially the complicated by repeated endobioses, or secondary intertaxonic recombinations39, which frequently occurred in case for trees obtained with maximum parsimony method+. In the case of Entumoeba histolytica, it is tempting to protist evolution. In this case, a heterotrophic organism engulfed a phototrophic eukaryote, which, by reduction of speculate that the protein tree is correct. This would offer the eukaryotic endobiont, led to a complex plastid with the more parsimonious explanation that it never had mitothree or four membranes - for example, in various taxa of chondria rather than that they were secondarily lost. Euglenida, Dinoflagellata and Cryptomonadina. The best However, other protein data neither support the position proof for such a secondary intertaxonic recombination is of E. histolytica nor that of D. discoideum. Actin protein provided by protists endowed with eukaryotic endosymcomparisons group both Dictyosteliumand Entamoeba with the Metazoazs. The most severe argument against the as- bionts that are partially but not fully reduced to plastids”9. sumption that the rRNA tree is wrong and the elongation This is apparently the case in Cryptomonadina and the plasmodial protist Chlorarachnion reptarS. Their plastids factor tree is correct, however, is that the two analyses are complex, and there is a narrow plasmatic space behave used completely different and thus uncomparable data sets. In the protein tree, sequences other than Meta- tween the two pairs of envelope membranes. This space zoa and Fungi are missing, especially those of the other contains a small nucleus-like organelle, the nucleomorph, which is assumed to be the vestigial nucleus of the eukary(primary) amitochondriate groups, which branch off first in the rRNA tree. As long as these data sets are not comotic endobiont. Its plastid structure led Gibbs40to suggest parable, I will rely more on the rRNA data, since protein that this eukaryotic endobiont might be a red alga. Two recoding genes always harbor the danger of comparing parsearch groups independently isolated and sequenced both alogous genes (see previous section), whereas the ribothe host nuclear gene and the endobiont 16.5like r-RNAgene somal gene copies are homogenized by concerted evolution, of the cryptomonad species Cryptomonas $41and fyrenowhich results in low heterogeneity between the repeated monas salina39, respectively. The results obtained are ribosomal genes”G.37.Of course, the database of protein congruent with respect to the host cell: the cryptomonad coding genes has to be enlarged considerably, both in num- nuclear rRNAs group with the chlorobionts. However, the bers of species and genes investigated. Cell division cycle results differ with respect to the position of the nucleo(CDC) proteins and cyclins may be good candidates. morph DNA. In the tree of the Halifax group41, the nucleomorph groups with the red algae, whereas in the tree Tracing secondary events in eukaryote evolution of the Freiburg group39 it does not, but branches off after It is well-known that mitochondria and plastids have the red algae. In addition, there is no evidence from other their phylogenetic origins in prokaryotes38. This concept published plastid sequences for a phylogenetic link
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REVIEWS between the cryptomonad endobiont chloroplast and the red algal chloroplastQ. Thus, we have to emphasize that at least for the moment the exciting new sequence data on the cryptomonad nucleomorph are not as conclusive as had been hoped. Conclusions Molecular data such as rRNA and protein sequence data are valuable sources which have substantially contributed to a new and comprehensive view of eukaryote phylogenetic relationships. One can be confident that we will achieve a virtually complete picture of eukaryote phylogeny in the not too distant future. However, to reach this goal it is necessary to investigate more genes as well as various protist taxa that are still not represented. In addition, the data sets for all molecules have to be made as comparable as possible, so that there is convergence on the organisms used for analysis. Molecular characters are in my view no better a priori than other characters. All characters and approaches have their advantages and disadvantages, and we will get the best possible answer to the question of eukaryote phylogeny when we put our results together, in the sense of a reciprocal illumination. This will enable us to tackle questions of both molecular and morphological evolution, for which the phylogenetic framework is the indispensable prerequisite.
Trichomonads
Acknowledgements I wish to thank Dieter Ammermann, Alexandra Stechmann, Detlef Bernhard and Denis Lynn for critical reading of the manuscript. I also thank Mitchell Sogin and Detlef Leipe for the permission to use modified drawings of their dendrograms in Figs 3 and 4.
and
References
Fig. 4. Molecular phylogeny section of the tree contains section comprises species boxed numbers indicate the from Ref. 26.
of Eukaryota inferred from 16Slike rRNAs. The lower taxa that have primarily no mitochondria. The upper descending from a mitochondriate eukaryote. The per cent bootstrap values. Redrawn, with permission,
Box 3. Selection of software packages available for phylogeny reconstruction PHYLIP: Phylogeny Inference Package (Joseph Felsenstein, Dept of Genetics SK-50, University of Washington, Seattle, WA 98195, USA). PHYLIP is a collection of about 30 programs including maximum likelihood, parsimony, Invariants, distance and bootstrap methods, plus some utility programs. PHYLIP is generally distributed as Pascal source code which can be easily implemented on most computer systems. PAUP: Phylogenetic Analysis Using Parsimony (David L. Swofford. Illinois Natural History Survey, 607 E. Peabody Drive, Champaign, IL 61802, USA) performs parsimony analyses under a variety of models including Lake’s evolutionary parsimony and bootstrapping. PAUP is distributed in precompiled form for IBM-PC and Macintosh microcomputers, and as C-source code for workstations, minicomputers and mainframes. However, although announced for IBM-compatible computers for several years, the improved later versions (from 3.0 on) are, at present, available for Macintosh computers only. Hennig66: (James S. Farris, 41 Admiral Street, Port Jefferson Station, New York, NY 11776, USA) Hennig86 is a small, fast and effective program for parsimony analyses, though it is not very user-friendly. The program is distributed as an executable program file for IBM-compatible computers only.
1 Margulis, L., Corliss. J.O., Melkonian, M. and Chapman, D., eds (1989) Handbook offrotoctista, Jones and Bartlett 2 Ragan, M.A. and Chapman, D.J. (1978) A Biochemical Phylogeny of frotists, Academic Press 3 Rothschild, L.J. and Heywood, P. (1987) in Progress in frotisto/ogy,
(Vol. 2) (Corliss, J.O. and Patterson, D.J., eds), pp. l-68, Biopress Lee, J.J., Hutner, S.H. and Bovee, E.C. (1985) An Illustrated Guide to the Protozoa, Society of Protozoologists 5 Cavalier-Smith, T. (1993) Microbiot. Rev. 57, 953-994 6 Innis, M.A.,Gelfand, D.H., Sninsky, J.J. and White, T.J. (1990) PCR Protocols: A Guide to Methods and Applications, Academic Press 7 De Rijk, P., Neefs, J-M., Van de Peer, Y. and De Wachter, R. (1992) 4
Nucleic Acids Res. 20, 2075-2089 8 9 10 11 12 13
Pace, N.R., Olsen, G.J. and Woese, CR. (1986) Ce/l45,25-26 Cavalier-Smith, T. (1987) Ann. NY. Acad. Sci. 504, 17-54 Zillig, W. (1991) Curr Opinion Genet. Dee. 1,544-551 Sogin, M.L. (1991) Gun: Opinion Genet. Deu. 1,457-463 Piihler, G. (1989) froc. NattAcad. Sci. USA 86,4569-4573 Iwabe, N., Kuma, K., Kishino H., Hasegawa, M. and Miyata, T. (1991) J Mol. Euol. 32, 70-78 14 Iwabe, N., Kuma, K., Hasegawa, M., Osawa, S. and Miyata, T. (1991) Proc. Nat1 Acad. Sci. USA 86,9355-9359 15 Gogarten. J.P. et al. (1990) froc. Nat1 Acad. Sci. USA 87,4576-4579 16 Woese, CR., Kandler, 0. and Weelis, M. (1990) Proc. Nat/ Acad. Sci. USA 8734576-4579 17 18 19 20
Pace, N.R. (1991) Cell 65,531-533 Gottschal, J.C. and Prins, R.A. (1991) Trends Ecol. Evol. 6, 157-162 Tsutsumi, S. et a/. (1991) Biochim. Biophys. Acta 1098, 13-20 Kakinuma, Y., lgatashi, K., Konishi, K. and Yamoto, I. (1991) Fed. Eur.
Biochem. Sot. Lett. 292, l-2 21 Forterre, P. et al. (1993) BioSystems 28, 15-32 22 Benachenhou-Lahfa, N.. Forterre, P. and Labedan, B. (1993) J. MO! Evol. 36,335-346 23 Hennig, W. (1966)fhylogenetic Systematics, University of Illinois Press 24 Martin, W., Brinkmann, H., Savonna, C. and Cerff, R. (1993) Proc. Nat/ Acad. Sci. USA 90,8692-8696 TREE
uol.
9,
no.
Y
September
1994
REVIEWS 25 Leipe, D.D., Gunderson. J.H., Nerad, T.A. and Sogin, M.L. (1993) Mol. Biochem.Parasifol. 59, 41-48 26 Leipe, D.D. and Hausmann, K. (1993)Siologie in UnsererZeif 23,178-183 27 Butterfield, N.J., Knoll, A.H. and Swett, K. (1990) Science 250, 104-107 28 Wainright, P.O., Hinkle, G., Sogin, M.L.and Stickel, SK. (1993) Science 260,340-342 29 Patterson, D.J. (1989) in The Chromophyfe Algae: Problems and Perspectioes (Systematic Association Special Vol. 38) (Green, J.C., Leadbetter, B.C.S.and Diver, W.L.,eds), pp. 357-379, Clarendon Press 30 Cajadhar, A.A. et al. (1991) Mol. Biochem. Parasirol. 45,147-154 31 Taylor, F.J.R. (1976) J. Protozool. 23, 28-40 32 Hasegawa, M., Hashimoto, T., Adachi, J., Iwabe, N. and Miyata, T. (1993) J. Mol. Euol. 36,380-388 33 Wolters, J. (1991) BioSystems 25, 75-83
34 Felsenstein, J. (1988)Annu. Rev. Genet. 22,521-565 35 Loomis, W.F. and Smith, D. (1990) Proc. Nat!Acad. Sci. USA 87, 9093-9097
36 37 38 39 40 41
Dover, G. (1982) Nature 299.111-117 Schlegel, M. (1991) Eur, J. Protisfol. 27. 207-219 Gray, M.W.(1989) Trends Genet. 5,294-299 Sitte, P. (1993) Eur. J. Protislol. 29, 131-143 Gibbs, S.P. (1981) Ann. N. Y Acad. Sci. 361, 193-208 Douglas, SE., Murphy, CA., Spencer. D.F. and Gray, M.W. (1991) Nature 350,148-151
42 Maerz, M., Wolters, .I., Hofmann, C.J.B., Sitte. P. and Maier, U-G. (1992) Curr Genet. 21,73-81 43 Hillis, D.M.and Moritz, C., eds (1990) Molecular Systemafics, Sinauer 44 Lake, J. (1987) Mol. Bio/. Euol. 4, 167-191
The use and abuseof pollinatorsby fungi B.A. Roy ing dark olive and white (e.g. Rust fungus spermatia (non-motile sperm) are borne in solutions containing sugarsr7, and sugars are also found in the spore mass (gleba) of stinkhorn+ (Fig. lc). Many rust fungi emit an odor - some species smell pleasant and flower-likes,lT722 whereas other species smell ‘like carriona. The stinkhorns stink: to our noses they are nasty and repellent rather like rotting meat with an B.A. Roy is at the Center for Population Biology, overlay of cloying sweetness. Section of Evolution and Ecology, University of Many rust fungi and flowers California, Davis, CA 95616, USA. attract flying insects. Thus, it is not too surprising that they have evolved similar ways for doing so. However, some rust fungi are clearly parasitizing the relationship between flowers and their pollinators. An example of a rust fungus The mechanics of pollinator attraction that takes advantage of its host’s pollinators is Uromyces Some fungi do not attract pollinators on their own, but instead take advantage of the insect-attracting ability of cladiF (also see Patt, J.M., unpublished dissertation, Rutgers University, USA, 1992). Early in the spring, before flower+. For example, the violet-spored smut fungi (some species of Ustilago and Microbotryumg; see Box 1) sporuthe aroid Peltandra flowers, U. cladii erupts on its leaves. late in the anthers of dicot flowers (Fig. lb). While forag- At this time, the rust exudes spermatia in a sugary fluid ing for pollen and nectar, pollinators visit both infected which smells like the host’s flowers. The host-specific and uninfected flowers and transmit smut spores to new pollinators of feltandra - flies in the genus Elachiptera host+5.10. Despite the apparent passiveness of this sysvisit and feed on the spermatia and, in the process, aid in outcrossing the fungus. Later, as the flowers of its host tem, anther smuts partially or completely sterilize their hostss,l’.r2 and may alter their hosts in ways that enhance begin to open, the fungus produces aeciospores, which spore production and transmissior$3. For example, smutare infectious. The flies then act as vectors, transporting infected plants often have more flowers than uninfected the aeciospores to Peltandra flowers, where they germioneG5, infected plants flower earlier than uninfected one&, nate and infect a new host. Pattrs suggests that the reand infected flowers can remain open longer than un- lationships between Uromyces, Peltandra and Elachiptera infected ones”. may be mutualistic. The fungus provides a food source Instead of using existing flowers, other fungi have for the host-specific pollinators of Peltandra before they flower. This may facilitate population growth of the flies, evolved their own pollinator attraction systems14-19,though these systems often share some of the characteristics of leading ultimately to more Peltandra pollination later. true flowers such as bright coloration and/or the proThe flies are critical for fertilization and for dispersal of duction of some kind of food reward and scent. Sexually the fungus, and infection causes little reduction in host survival. receptive rusts, for example, are typically red, orange or yellow (Fig. la)1T.20,and the stinkhorns (Fig. lc) can be A more complicated example of fungal exploitation of pollinators is found in the mustard rusts, Puccinia monoica bright orange-red (e.g. Muthus. Aseriie) or starkly contrastungi can use flower-visiting insects such as bees, flies and butterflies in at least three distinct ways. First, some rust fungi use pollinators the same way as flowers, that is, to ferry gametes between different fungal individuals for sexual reproduction (Fig. la). Second, some pathogenic fungi use their hosts’ pollinators to transfer infectious spores to new hosts; these are the ‘sexually transmitted’ plant diseases such as the anther smuts (Fig. lb). And third, a few non-pathogenic fungi, such as the stinkhorns (Phallales, Fig. lc), use pollinators to disperse their spores.
F
Some fungi use flower-visiting insects to facilitate sexual reproduction or to disperse spores. These fungi have evolved elaborate techniques, such as floral mimicry and the Invasion of extant flower parts, for attracting ‘pollinators’. Recent research shows that fungal exploltatlon of pollinators has the potential to affect floral evolution, polllnation ecology, plant life history traits, as well as disease-transmission dynamics and fungal evolution.
Dictyophora).
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Molecularphylogenyof eukaryotes Martin Schlegel
he unicellular eukaryotes or Comparisons of ribosomal RNAs and These related proteins are found protists have been studied various protein codihg genes have in organisms that represent each for more than 300 years. contributed to a new view of eukaryote of the three lines. If the gene Besides their extreme mor- phylogeny. Analyses of paralogous protein duplication preceded the diverphological variation they show coding genes suggest that archaebacteria gence, then there should be more great ultrastructural, physiologiand eukaryotes are sistergroups. differences between the duplical and biochemical diversity, far Sequence diversity of small subunit rRNAs cated, paralogous genes within exceeding that of multicellular in protlsts by far exceeds that of any one taxon than within one ortholoorganism+3. Despite our increasmulticellular or prokaryote taxon. gous gene family, even if members ing knowledge, the phylogenetic Remarkably, a group of taxa that lack of the three different lineages are relationships of protists and also mitochondria fhst branches off in the compared. Thus, one family of the origin of the eukaryote ancessmall subunit rRNA tree. The later orthologous genes can be used to tor remained unclear, resulting in an radiations are formed by a series of clades infer the root of the other gene increasing number of often conthat were once thought to be more family, and vice versa. This reflicting classification scheme+s. ancestral. Furthermore, tracing of the sults in mirror trees (Fig. lb). This picture changed dramatievolutionary origin of secondary Such an analysis for the EF-1(Tu) cally when PCR (polymerase chain endobiontic events is now possible and EF-2 (G) sequences puts the reaction) techniques6 became with sequence comparisons. root into the eubacterial line of available, allowing the rapid amdescent, which means that Archae plification and sequencing of bacteria and Eukaryota share a Martin Schlegel is at the Universittit Ttibingen, informative genes from minute more recent common ancestor14. Zoologisches Institut, Abteilung Zellbiologie, Auf der amounts of DNA. By far the most An analysis of duplicated genes Morgenstelle 28, D-72076 Ttibingen, Germany. comprehensive data are available of the ATPase subunit has given from small subunit rRNAs,of which an identical resulW5. These results more than 900 complete sequences (200 eukaryotic) have have been widely acceptedr6. Furthermore, the eubacterial been determined7. Increasingly, protein coding genes are rooting has been used to support the hypothesis that life revealing new insights into eukaryote evolution. The trees evolved at high temperatures, since hyperthermophiles inferred from the different kinds of molecules are largely appear early in the two branches of the universal treenJ8. The discrepancy between the small subunit r-RNAphycongruent, but marked differences also appear. Another issue of debate concerns the correct alignment of the logeny and the protein phylogenies could be explained by different rates of evolution for 16S-like r-RNAand the prosequences (i.e. the juxtaposition of the homologous sites of the molecule) and the conversion of sequence in- tein coding genes in the different lineages. An alternative formation into phylogenetic trees. The different methods scenario was proposed by Soginrl, who postulated an RNAthat are currently used are summarized in Box 1. dependent proto-eukaryote that engulfed an ancestral archaebacterium with a DNAgenome, which gave rise to the eukaryote nucleus. Thus, archaebacteria are more closely The nature of the universal ancestor and evolution of related to eukaryotes in the protein phylogenies but are the eukaryotic cell closer to eubacteria in the r-RNAphylogenies because of Sequence analyses of small subunit rRNAs have retheir longer common evolutionary history. vealed the existence of three primary lines of descent: However, the approach using duplicated genes to Eubacteria, Archaebacteria and Eukaryotaa. Similarities in these three lineages regarding the components of the ma- infer the root of life and its placement in the eubacterial chinery of RNA and protein synthesis, together with the line of descent has been criticized recently, and indeed existence of many metabolic pathways common to eu- things turn out to be much more complicated. Two new bacteria and archaebacteria, indicate that the universal an- eubacterial ATPases were discovered recentlylgJ0, which are more similar to the archaebacterial and eukaryote cestor was probably already an elaborated cellsJO.However, V-type ATPases than to the eubacterial F,/F, ATPases, the root of the universal tree of life seems to be obscure. which were used to represent the eubacteria in the dupliA higher sequence similarity can be observed in the small cated treesr4J5. Therefore, not one but two gene duplisubunit rRNA of Eubacteria and Archaebacteria when compared to Eukaryotarl. By contrast, DNA-dependent RNA cations occurred in the universal ancestor before divergence. This suggests that V-type and F,/F, ATPases are polymerase genes of Eukaryota and Archaebacteria are paralogous and that the trees are composed of orthologous more alike than either is to EubacterialzJ3. and paralogous proteins, which may result in a false rootOne way to root a phylogenetic tree is to use an outing (Fig. 2)2*. group. Logically, however, there are no outgroups for all In contrast, only one family of each elongation factor contemporary life forms. As an alternative, gene families gene has been found in each line of descent (Fig.lb), and that duplicated before the divergence of the three primary lines of descent can be used to infer the root of the uni- this still supports the eubacterial rooting of the universal versal tree (Fig. la and Box 2). For example, elongation fac- tree. An analysis of the duplicated glutamate dehydrogenase genes22 has not resolved the question. Here, the root tor EF-1 (Tu) and EF-2 (G) are duplicated, conserved prowas placed either inside the archaebacterial branch when teins that play an important role in the translation process.
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distance methods were applied, or into the eukaryotic branch when parsimony methods were used. In the tree of CDH family I, the eubacterium Anacystis nidulans groups consistently in the eukaryote lineage with the eumycete Neurospora crassa, whereas the eukaryote diplomonad Ciardia lamblia is placed within the eubacteria. In family II, the bovine Bos groups with chicken Callus rather than with other mammalia. These results throw severe doubt on the value of this molecule for phylogenetic analyses. In a phylogenetic (‘cladistic’) analysis23 of the elongation factor and glutamate dehydrogenase genes, Forterre et al.“’ found three synapomorphies (see Glossary, Box 2) for a sistergroup relationship of Archaebacteria and Eukaryota, and one synapomorphy for a Eubacteria-Archaebacteria clade out of 493 sites! The problems in identifying the root correctly may lie in the increase of multiple substitutions in genes that have been separated for very long periods. This reduces the number of informative sites and hence the value of the gene for phylogenetic analysis of distantly related organisms. And, there is growing evidence that the eukaryote nucleus is truely chimeric and even contains eubacterial genes. Sequence analysis of nuclear-encoded glyceraldehyde-3phosphate dehydrogenase (GAPDH) genes revealed that not only the plastid GAPDH, but also the GAPDHenzyme of glycolysis in plants, animals and fungi evolved from a gene that was laterally transferred from a eubacterial end@ biont which gave rise to the mitochondria or plastids24. Nonetheless, to tackle the root of the universal tree of life, it is necessary to continue the search for conserved duplicated genes in the three primary lines of descent. As pointed out by Forterre et al., it is striking that sequence information is especially lacking from protistszl. Small subunit rRNA diversity and eukaryote phylogeny A comprehensive database exists for the small subunit (‘16S-like’) rRNA. The sequence diversity in protists by far exceeds that of any prokaryote or multicellular taxon (Fig. 3). It has to be emphasized that data from many groups, such as cryptomonads, haptophytes, and most of the ‘amoebae’ are still lacking. The current view of protist phylogeny derived from 16S-like rRNAs is shown in Fig. 4. far/y branchings are represented by amitochondriate
taxa Remarkably, at the base of the tree, taxa that lack mitochondria emerge. The 16%like rRNA tree does not resolve unambiguously the branching order within these groups that lack mitochondria. However, the group as a whole is always placed at the bottom of the tree, regardless of the method of analysis applied (see Box 1)25.Since all members of this group are mostly parasites that live in
(4
Box 1, Methods for inferring phylogenetic trees from sequence datas4y43 Distance matrix methods - (1) Genetic distance values are calculated Pairwise on the basis of the nucleotide or amino acid differences between the sequences corn pared (including correction factors for multiple substitutions). (2) Distance values are then converted into phenetic dendrograms. (3) The goodness-of-fit of the observed distances in the tree to the calculated distances is measured, and the tree with the minimum discrepancy is preferred. This method is largely rateindependent, but is sensitive to sampling set. Maxlmum parsimony methods - (1) The trees that need the minimum number ot steps (mutations) to convert the sequences into each other are found. (2) Hypo thetical ancestral sequences are calculated. Maximum parsimony is compatible with the concept of ‘phylogenetic systematics ‘23; any extra count of site change on a tree gives the number of ancillary hypotheses that must be erected to explain evolution in the group, and the method favours the tree with the mlnimum number of hypotheses (‘Occam’s Razor’). These methods are rate-sensitive. Fast-evolving nonrelated sequences tend to be grouped together. A rate-insensitive variant IS Lake’s evolutionary parsimony44 or ‘invariants? Sites where predominantly transitions occur are excluded from the analysis. However, since it uses only part of the sequence information, less-significant branches may result. In some parsimony methods (Hennig86; see Box 3) ancestral states can be defined. Maximum likelihood methods -These have a statistical approach. (1) A concrete model of the evolutionary process that converts one sequence into another IS specified. (2) The likelihood that the given evolutionary model will yield the observed sequences is calculated. The inferred phylogenles are those with the highest likelihood. This method uses the largest amount of sequence information. but is computationally very intensive. Bootstrapping - (1) Repeated samplings (up to 1000 and more) of the alignec data set are made, whereby parts of the sequence are excluded; other parts are included several times in order to yield a slightly different data set. (2) All modified data sets can then be analysed with one of the tree-building methods and the number of times a branch appears In the different trees be visualized in a consensus tree. Although computationally very time-consuming. this method is frequently used to estimate the significance of branchings in a tree. Analysis of signature sites - This is a phylogenetic approach. Signature sites mean the presence of the same character (nucleotide. amino acid, insertion. deletion) in all outgroup sequences (or at least predominantly). The presence of the same character in the ingroup indicates a plesiomorphy, and shared deviations indicate synapomorphies; these are used to argue for a relationship. Usually, however, only a few sites in a sequence are signature sites. -
low-oxygen habitats, such as in the gut of cockroaches and termites, it was long assumed that these organisms had lost their mitochondria in adaptation to their environment. In contrast, the rRNA tree strongly supports the view that these organisms were primarily without mitochondria. Thus, it seems reasonable that amitochondriate organisms were common and widely distributed in the early evolution of eukaryotes. After the emergence of the mitochondriate cells, they may have remained competitive only in lowoxygen habitatszfi. Mitochondriate taxa branching off in the middle part of the tree The uptake of a bacterial endobiont that gave rise to the mitochondrion happened before the next taxa branched off in the eukaryote tree (Fig. 4). This part of the tree is
(b)
E
Archaebacteria
Eubacteria
Archaebacteria
Archaebacteria
Eukatyota
Eubacteria
Eubacteria
Eukaryota
Eukaryota Fig. 1. (a) Gene duplication occurred before the divergence groups Archaebacteria and Eukaryota together.
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(paralogous
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Box 2. Glossary Homology: characters (e.g. genes, proteins, hairs, setae, feathers, bones) which descend from a common ancestor are homologous. Paralogous genes: homologous genes which originated from a gene duplication. Orthologousgenes: one of the duplicated genes being compared between different species (see also Fig. 1). For phylogeny inference orthologous gene or protein sequences have to be compared. Differences between orthologous sequences are those which occurred after speciation, whereas paralogous sequences show differences which accumulated after the duplication event and are thus not informative for reconstruction of a branching pattern. Plesiomorphy: ancestral state of a character, e.g. the primary winglessness of insects. Apomorphy: derived character state, e.g. wings of ptetygote insects. Autaoomorphy: derived character which accounts for the monophyly of a taxon. Synapomorphy: shared derived character which accounts for the sistergroup relationship of two taxa. The terms plesiomorph and apomorph are relative, depending on the taxonomic level looked at. For example, hairs are an autapomorphyforthe mammalia, a synapomorphy for the two sister taxa Monotremata and Theria, and a symplesiomorphy for the Marsupialia and Placentalia within the Theria.
Ancestral
ATPase
gene
& First duplication into alpha and beta subunit
L4 Second duplication V-type and F-type
into
Tree reconstruction paralogous genes
with a mixture
False outgroup
False
root
A&
of orthologous
and
(the other subunit) Archaebacteria
V-type
Eubacteria
V-type
Eukaryota
V-type
Archaebacteria
?F-type
Eubacteria
?F-type
Eukaryota
F-type
Fig. 2. The ATPase gene-tree may be composed of orthologous and genes and thus does not reflect the true phylogenetic relationships of Archaebacteria and Eukaryota. The thick lines indicate the compared The correct outgroups would probably be the F-type ATPases for ATPases, and vice versa.
paralogous Eubacteria. molecules. the V-type
statistically well-supported: in 94% of bootstrap replicates (see Box 1) this branch appears. The following branches are not so strongly supported, except the monophyly of the Euglenozoa, which first branches off after the acquisition of mitochondria. With approximately 2000 species, the Euglenozoa are the most important group in this middle part of the tree. They comprise the heterotrophic Kinetoplastida (free-living Bodonida and parasitic Trypanosomatida) and the free-living Euglenida. A part of the Euglenida is aute trophic. However, their chloroplasts are not primary prokaryotic endobionts. They are remnants of a eukaryotic endobiont that is related to the chlorobiontsl. Since green 332
algae appeared much later in eukaryote evolution (see below), as suggested by electron microscopical analyses and the occurrence of chlorophylls A and Bl, it is evident that the autotrophic euglenids are a late emergence and that the Euglenozoa evolved as a primarily heterotrophic group. The acellular slime molds and several taxa of the ‘old’ amoebae follow next: the Entamoebae, the Schizopyrenida (amoeboflagellates), together with one group of the cellular slime molds, the Acrasida. A relationship between the latter two was already assumed because of similarities in the life cycle and in trophic amoebae. Interestingly, the Entamoebae have no mitochondria. Their position in this part of the tree suggests, however, that these organelles were secondarily lost in adaptation to their parasitic way of life (but also see below). Rapid proliferation of major groups later in eukaryote evolution The next branch leading to a radiation (Fig. 4) is also statistically well-supported by bootstrap analysis. A series of clades separated about one billion years ago. This was calibrated with red algae, which appear to branch off first in the radiation; their oldest known fossils (bangiophytes) were found in 1.25 billion year old rock.+. This rapid radiation is difficult to resolve. However, a sistergroup relationship between Metazoa and Fungi is suggested28. There is also some evidence that the Chlorobionta (green algae and metaphytes) are the sistergroup to Metazoa and FungP. The remaining taxa provide an impressive example of the power of molecular systematics: the ‘stramenopiles’ contain a variety of quite different groups, such as the former heterokont algae (brown algae, diatoms and chryse monads), water molds, opalinids and others. The sequence data support a grouping of these taxa that originally was based on the presence of thin tripartite hairs on the membrane surface, especially on one of the two flagellazg.With respect to the ‘alveolates’ the sequence analyses revealed the phylogenetic relationships of ultrastructurally welldefined but isolated taxa. The alveolates encompass the Dinoflagellata, a group with mostly free-living and a few parasitic species. Their next relatives are the Apicomplexa, which consist solely of endoparasitic species and which are flagellated only during sexual stages in their life cycle. Together, Apicomplexa and Dinoflagellata are the sistergroup of the primarily phagotrophic Ciliophora30. These three taxa are so different in ultrastructure and ecology that their relationship was not accepted before the sequence information, although there were ultrastructural hints, namely their possession of the submembraneous alveolar structures, which led to the name ‘alveolates’. In 1976, Taylor pointed out a possible relationship between Dinoflagellata and Ciliophora31. Not all of the branches are significant. Thus, it is necessary to search for additional information that might help to resolve these parts of the tree better, and moreover, to check the total branching order of the IGS-like rRNA tree. For this purpose, protein coding genes are increasingly used. Comparison of ribosomal data with protein trees A tree inferred from elongation factor EF-1 gene sequences using a maximum likelihood method is largely congruent with the rRNA tree32. Especially, there is strong support for a metazoan-fungal clade. But it differs markedly in the position of Entamoeba histolytica and Dictyostelium discoideum: E. histolytica branches off before the emergence TREE vol.
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REVIEWS of the Euglenozoa, while Eukatyota D. discoideum groups with the Stramenopiles Chlorobionta. How can these differences be explained? Fungi c\ v /3 Apicomplexans Some of the problems enCiliates, Dinoflagellates countered with sequence data Red algae may be revealed by these contradicting results, such as the proper alignment of Slime molds I, the sequences, which is especially a problem for distantly related taxa and which Eubacteria is ultimately always a subjecI \ tive decision of the researcher doing the analysis. Different people can arrive at different alignments and consequently different trees even with the very same sequences. Second, different genes evolve at different rates, and the same gene may evolve at different rates in different lineages. Diplomonads Thus, the same gene may have reached a point of saturation, containing no more phylogenetic information in one taxon (because of too many back mutations), while it still Archaebacteria is informative in another. Third, a bias in C/C content Fig. 3. Molecular phylogeny of Eubacteria, Archaebacteria and Eukaryota inferred from 16Slike rRNAs. Lengths of branches correspond to genetic distances. The genetic distance among protists by far exceeds that of any multicellular taxon and that can influence the position of of both Eubacteria and Archaebacteria. Redrawn, with permission, from Ref. 11. a sequence in the tree, as was the problem with the early branching of the C. lumblia of a serial endosymbiosis is strongly supported by rRNA sequence in the 16S-like rRNA tree*s. Fourth, rapidly and protein sequence data. This situation, however, is evolving sequences tend to group together and branch more deeply in a sequence tree33. This is especially the complicated by repeated endobioses, or secondary intertaxonic recombinations39, which frequently occurred in case for trees obtained with maximum parsimony method+. In the case of Entumoeba histolytica, it is tempting to protist evolution. In this case, a heterotrophic organism engulfed a phototrophic eukaryote, which, by reduction of speculate that the protein tree is correct. This would offer the eukaryotic endobiont, led to a complex plastid with the more parsimonious explanation that it never had mitothree or four membranes - for example, in various taxa of chondria rather than that they were secondarily lost. Euglenida, Dinoflagellata and Cryptomonadina. The best However, other protein data neither support the position proof for such a secondary intertaxonic recombination is of E. histolytica nor that of D. discoideum. Actin protein provided by protists endowed with eukaryotic endosymcomparisons group both Dictyosteliumand Entamoeba with the Metazoazs. The most severe argument against the as- bionts that are partially but not fully reduced to plastids”9. sumption that the rRNA tree is wrong and the elongation This is apparently the case in Cryptomonadina and the plasmodial protist Chlorarachnion reptarS. Their plastids factor tree is correct, however, is that the two analyses are complex, and there is a narrow plasmatic space behave used completely different and thus uncomparable data sets. In the protein tree, sequences other than Meta- tween the two pairs of envelope membranes. This space zoa and Fungi are missing, especially those of the other contains a small nucleus-like organelle, the nucleomorph, which is assumed to be the vestigial nucleus of the eukary(primary) amitochondriate groups, which branch off first in the rRNA tree. As long as these data sets are not comotic endobiont. Its plastid structure led Gibbs40to suggest parable, I will rely more on the rRNA data, since protein that this eukaryotic endobiont might be a red alga. Two recoding genes always harbor the danger of comparing parsearch groups independently isolated and sequenced both alogous genes (see previous section), whereas the ribothe host nuclear gene and the endobiont 16.5like r-RNAgene somal gene copies are homogenized by concerted evolution, of the cryptomonad species Cryptomonas $41and fyrenowhich results in low heterogeneity between the repeated monas salina39, respectively. The results obtained are ribosomal genes”G.37.Of course, the database of protein congruent with respect to the host cell: the cryptomonad coding genes has to be enlarged considerably, both in num- nuclear rRNAs group with the chlorobionts. However, the bers of species and genes investigated. Cell division cycle results differ with respect to the position of the nucleo(CDC) proteins and cyclins may be good candidates. morph DNA. In the tree of the Halifax group41, the nucleomorph groups with the red algae, whereas in the tree Tracing secondary events in eukaryote evolution of the Freiburg group39 it does not, but branches off after It is well-known that mitochondria and plastids have the red algae. In addition, there is no evidence from other their phylogenetic origins in prokaryotes38. This concept published plastid sequences for a phylogenetic link
A
REVIEWS between the cryptomonad endobiont chloroplast and the red algal chloroplastQ. Thus, we have to emphasize that at least for the moment the exciting new sequence data on the cryptomonad nucleomorph are not as conclusive as had been hoped. Conclusions Molecular data such as rRNA and protein sequence data are valuable sources which have substantially contributed to a new and comprehensive view of eukaryote phylogenetic relationships. One can be confident that we will achieve a virtually complete picture of eukaryote phylogeny in the not too distant future. However, to reach this goal it is necessary to investigate more genes as well as various protist taxa that are still not represented. In addition, the data sets for all molecules have to be made as comparable as possible, so that there is convergence on the organisms used for analysis. Molecular characters are in my view no better a priori than other characters. All characters and approaches have their advantages and disadvantages, and we will get the best possible answer to the question of eukaryote phylogeny when we put our results together, in the sense of a reciprocal illumination. This will enable us to tackle questions of both molecular and morphological evolution, for which the phylogenetic framework is the indispensable prerequisite.
Trichomonads
Acknowledgements I wish to thank Dieter Ammermann, Alexandra Stechmann, Detlef Bernhard and Denis Lynn for critical reading of the manuscript. I also thank Mitchell Sogin and Detlef Leipe for the permission to use modified drawings of their dendrograms in Figs 3 and 4.
and
References
Fig. 4. Molecular phylogeny section of the tree contains section comprises species boxed numbers indicate the from Ref. 26.
of Eukaryota inferred from 16Slike rRNAs. The lower taxa that have primarily no mitochondria. The upper descending from a mitochondriate eukaryote. The per cent bootstrap values. Redrawn, with permission,
Box 3. Selection of software packages available for phylogeny reconstruction PHYLIP: Phylogeny Inference Package (Joseph Felsenstein, Dept of Genetics SK-50, University of Washington, Seattle, WA 98195, USA). PHYLIP is a collection of about 30 programs including maximum likelihood, parsimony, Invariants, distance and bootstrap methods, plus some utility programs. PHYLIP is generally distributed as Pascal source code which can be easily implemented on most computer systems. PAUP: Phylogenetic Analysis Using Parsimony (David L. Swofford. Illinois Natural History Survey, 607 E. Peabody Drive, Champaign, IL 61802, USA) performs parsimony analyses under a variety of models including Lake’s evolutionary parsimony and bootstrapping. PAUP is distributed in precompiled form for IBM-PC and Macintosh microcomputers, and as C-source code for workstations, minicomputers and mainframes. However, although announced for IBM-compatible computers for several years, the improved later versions (from 3.0 on) are, at present, available for Macintosh computers only. Hennig66: (James S. Farris, 41 Admiral Street, Port Jefferson Station, New York, NY 11776, USA) Hennig86 is a small, fast and effective program for parsimony analyses, though it is not very user-friendly. The program is distributed as an executable program file for IBM-compatible computers only.
1 Margulis, L., Corliss. J.O., Melkonian, M. and Chapman, D., eds (1989) Handbook offrotoctista, Jones and Bartlett 2 Ragan, M.A. and Chapman, D.J. (1978) A Biochemical Phylogeny of frotists, Academic Press 3 Rothschild, L.J. and Heywood, P. (1987) in Progress in frotisto/ogy,
(Vol. 2) (Corliss, J.O. and Patterson, D.J., eds), pp. l-68, Biopress Lee, J.J., Hutner, S.H. and Bovee, E.C. (1985) An Illustrated Guide to the Protozoa, Society of Protozoologists 5 Cavalier-Smith, T. (1993) Microbiot. Rev. 57, 953-994 6 Innis, M.A.,Gelfand, D.H., Sninsky, J.J. and White, T.J. (1990) PCR Protocols: A Guide to Methods and Applications, Academic Press 7 De Rijk, P., Neefs, J-M., Van de Peer, Y. and De Wachter, R. (1992) 4
Nucleic Acids Res. 20, 2075-2089 8 9 10 11 12 13
Pace, N.R., Olsen, G.J. and Woese, CR. (1986) Ce/l45,25-26 Cavalier-Smith, T. (1987) Ann. NY. Acad. Sci. 504, 17-54 Zillig, W. (1991) Curr Opinion Genet. Dee. 1,544-551 Sogin, M.L. (1991) Gun: Opinion Genet. Deu. 1,457-463 Piihler, G. (1989) froc. NattAcad. Sci. USA 86,4569-4573 Iwabe, N., Kuma, K., Kishino H., Hasegawa, M. and Miyata, T. (1991) J Mol. Euol. 32, 70-78 14 Iwabe, N., Kuma, K., Hasegawa, M., Osawa, S. and Miyata, T. (1991) Proc. Nat1 Acad. Sci. USA 86,9355-9359 15 Gogarten. J.P. et al. (1990) froc. Nat1 Acad. Sci. USA 87,4576-4579 16 Woese, CR., Kandler, 0. and Weelis, M. (1990) Proc. Nat/ Acad. Sci. USA 8734576-4579 17 18 19 20
Pace, N.R. (1991) Cell 65,531-533 Gottschal, J.C. and Prins, R.A. (1991) Trends Ecol. Evol. 6, 157-162 Tsutsumi, S. et a/. (1991) Biochim. Biophys. Acta 1098, 13-20 Kakinuma, Y., lgatashi, K., Konishi, K. and Yamoto, I. (1991) Fed. Eur.
Biochem. Sot. Lett. 292, l-2 21 Forterre, P. et al. (1993) BioSystems 28, 15-32 22 Benachenhou-Lahfa, N.. Forterre, P. and Labedan, B. (1993) J. MO! Evol. 36,335-346 23 Hennig, W. (1966)fhylogenetic Systematics, University of Illinois Press 24 Martin, W., Brinkmann, H., Savonna, C. and Cerff, R. (1993) Proc. Nat/ Acad. Sci. USA 90,8692-8696 TREE
uol.
9,
no.
Y
September
1994
REVIEWS 25 Leipe, D.D., Gunderson. J.H., Nerad, T.A. and Sogin, M.L. (1993) Mol. Biochem.Parasifol. 59, 41-48 26 Leipe, D.D. and Hausmann, K. (1993)Siologie in UnsererZeif 23,178-183 27 Butterfield, N.J., Knoll, A.H. and Swett, K. (1990) Science 250, 104-107 28 Wainright, P.O., Hinkle, G., Sogin, M.L.and Stickel, SK. (1993) Science 260,340-342 29 Patterson, D.J. (1989) in The Chromophyfe Algae: Problems and Perspectioes (Systematic Association Special Vol. 38) (Green, J.C., Leadbetter, B.C.S.and Diver, W.L.,eds), pp. 357-379, Clarendon Press 30 Cajadhar, A.A. et al. (1991) Mol. Biochem. Parasirol. 45,147-154 31 Taylor, F.J.R. (1976) J. Protozool. 23, 28-40 32 Hasegawa, M., Hashimoto, T., Adachi, J., Iwabe, N. and Miyata, T. (1993) J. Mol. Euol. 36,380-388 33 Wolters, J. (1991) BioSystems 25, 75-83
34 Felsenstein, J. (1988)Annu. Rev. Genet. 22,521-565 35 Loomis, W.F. and Smith, D. (1990) Proc. Nat!Acad. Sci. USA 87, 9093-9097
36 37 38 39 40 41
Dover, G. (1982) Nature 299.111-117 Schlegel, M. (1991) Eur, J. Protisfol. 27. 207-219 Gray, M.W.(1989) Trends Genet. 5,294-299 Sitte, P. (1993) Eur. J. Protislol. 29, 131-143 Gibbs, S.P. (1981) Ann. N. Y Acad. Sci. 361, 193-208 Douglas, SE., Murphy, CA., Spencer. D.F. and Gray, M.W. (1991) Nature 350,148-151
42 Maerz, M., Wolters, .I., Hofmann, C.J.B., Sitte. P. and Maier, U-G. (1992) Curr Genet. 21,73-81 43 Hillis, D.M.and Moritz, C., eds (1990) Molecular Systemafics, Sinauer 44 Lake, J. (1987) Mol. Bio/. Euol. 4, 167-191
The use and abuseof pollinatorsby fungi B.A. Roy ing dark olive and white (e.g. Rust fungus spermatia (non-motile sperm) are borne in solutions containing sugarsr7, and sugars are also found in the spore mass (gleba) of stinkhorn+ (Fig. lc). Many rust fungi emit an odor - some species smell pleasant and flower-likes,lT722 whereas other species smell ‘like carriona. The stinkhorns stink: to our noses they are nasty and repellent rather like rotting meat with an B.A. Roy is at the Center for Population Biology, overlay of cloying sweetness. Section of Evolution and Ecology, University of Many rust fungi and flowers California, Davis, CA 95616, USA. attract flying insects. Thus, it is not too surprising that they have evolved similar ways for doing so. However, some rust fungi are clearly parasitizing the relationship between flowers and their pollinators. An example of a rust fungus The mechanics of pollinator attraction that takes advantage of its host’s pollinators is Uromyces Some fungi do not attract pollinators on their own, but instead take advantage of the insect-attracting ability of cladiF (also see Patt, J.M., unpublished dissertation, Rutgers University, USA, 1992). Early in the spring, before flower+. For example, the violet-spored smut fungi (some species of Ustilago and Microbotryumg; see Box 1) sporuthe aroid Peltandra flowers, U. cladii erupts on its leaves. late in the anthers of dicot flowers (Fig. lb). While forag- At this time, the rust exudes spermatia in a sugary fluid ing for pollen and nectar, pollinators visit both infected which smells like the host’s flowers. The host-specific and uninfected flowers and transmit smut spores to new pollinators of feltandra - flies in the genus Elachiptera host+5.10. Despite the apparent passiveness of this sysvisit and feed on the spermatia and, in the process, aid in outcrossing the fungus. Later, as the flowers of its host tem, anther smuts partially or completely sterilize their hostss,l’.r2 and may alter their hosts in ways that enhance begin to open, the fungus produces aeciospores, which spore production and transmissior$3. For example, smutare infectious. The flies then act as vectors, transporting infected plants often have more flowers than uninfected the aeciospores to Peltandra flowers, where they germioneG5, infected plants flower earlier than uninfected one&, nate and infect a new host. Pattrs suggests that the reand infected flowers can remain open longer than un- lationships between Uromyces, Peltandra and Elachiptera infected ones”. may be mutualistic. The fungus provides a food source Instead of using existing flowers, other fungi have for the host-specific pollinators of Peltandra before they flower. This may facilitate population growth of the flies, evolved their own pollinator attraction systems14-19,though these systems often share some of the characteristics of leading ultimately to more Peltandra pollination later. true flowers such as bright coloration and/or the proThe flies are critical for fertilization and for dispersal of duction of some kind of food reward and scent. Sexually the fungus, and infection causes little reduction in host survival. receptive rusts, for example, are typically red, orange or yellow (Fig. la)1T.20,and the stinkhorns (Fig. lc) can be A more complicated example of fungal exploitation of pollinators is found in the mustard rusts, Puccinia monoica bright orange-red (e.g. Muthus. Aseriie) or starkly contrastungi can use flower-visiting insects such as bees, flies and butterflies in at least three distinct ways. First, some rust fungi use pollinators the same way as flowers, that is, to ferry gametes between different fungal individuals for sexual reproduction (Fig. la). Second, some pathogenic fungi use their hosts’ pollinators to transfer infectious spores to new hosts; these are the ‘sexually transmitted’ plant diseases such as the anther smuts (Fig. lb). And third, a few non-pathogenic fungi, such as the stinkhorns (Phallales, Fig. lc), use pollinators to disperse their spores.
F
Some fungi use flower-visiting insects to facilitate sexual reproduction or to disperse spores. These fungi have evolved elaborate techniques, such as floral mimicry and the Invasion of extant flower parts, for attracting ‘pollinators’. Recent research shows that fungal exploltatlon of pollinators has the potential to affect floral evolution, polllnation ecology, plant life history traits, as well as disease-transmission dynamics and fungal evolution.
Dictyophora).
335