Cladistic analysis of ribosomal RNAs — the phylogeny of eukaryotes with respect to the endosymbiotic theory

BioSyetems, 21 (1988) 209-214

209

Elsevier Scientific Publishers Ireland Ltd.

Cladistic analysis of ribosomal RNAs -- the phylogeny of eukaryotes with respect to the endosymbiotic theory J~rn Wolters and Volker A. Erdmann Inititut flit Biochemie, Fachbereich Chemic, Fie@ Universitdt Berliz~ Thielallee 63, D-IO00 Berlin 33/Dahlend (F.R.G.] (Revision received October 17th, 1987) A strict cladlstie analysis of FxS and 16S rRNA secondary and primary structure confirms particular hypotheses concerning the phylogeny of eukaryotos: plastids of Euglena, green algae and land plants, and the cyaneUe of Cyemophora share a specific character and are closely related to eyanobecterla of the Synechococcus-type. Angiosperm mitoehondria share specific signatures with the alpha subdivision of rhodobaetoria. Cyanophora is a member of the Eugienozoa, the Oomycetes are derived from a group of heterokont algae.

Keywords: Cladlsties; 5S rRNA; 16S rRNA; Plastids; Mitochondrla; Endosymbiotic theory.

1. Introduction When cladistics are considered in the classical sense morphological characters appear in only two stages: primitive, called plesi~ morphic; and derived, apomorphic. The same data set can be subjected to different methods of analysis (parsimony, compatibility, etc.) and will yield different phylogenetic trees, which is primarily due to the different treatment of homoplasy (back and parallel mutations) and extremely rapid evolution found especially in symbionts and parasites. With respect to nucleic acid and protein sequences the high degree of homoplasy complicates the analysis. Simple computer methods like average linkage clustering can handle hundreds of species but yield similarity diagrams (phenc~ grams) which are rather unsatisfactory, conversely, sophisticated methods like additive trees are limited to about 30 species. In this paper we employ a strict cladistic approach using 470 5S and 66 16S rRNA sequences and show that meaningful phylogenetic trees can be obtained. 2. Materials and methods 1,

The BERLIN RNA DATABANK as of July 1987 comprises 265 eukaryotic, 168

eubacterial, 15 plastid, 4 mitochondrial and 18 archaebacterial sequences, which are biannually published in Nucleic Acids Research (Erdmann and Wolters, 1986). A total alignment of 470 5S rRNAs has been obtained by using the program ALIGNSTAT (technoma GmbH, Heidelberg) designed for the IBM XT/AT and compatibles which performs multisequence alignments of up to 150 sequences with 120 residues length by the method of Kriiger and Osterburg (1983). It was manually adjusted to account for common secondary structure elements, some remaining uncertainties could be eliminated by using an iterative approach, i.e. hierarchical alignment according to a preliminary phylogeny. For 16S rRNA the alignment of Huysmans and DeWachter (1986) is used omitting areas of hypervariability V1--V8 in which no reliable alignment is possible. An application of the cladistic approach to structural RNA requires characters of very low variability, one of the alternatives should be easily definable as plesiomorphic: (1) Three types of characters are defined: (a) insertions/deletions of single nucleotides; (b) odd base-pairs versus Watson-Crick basepairs; (c) single nucleotides. GC, AU and GU are considered Watson-Crick base-pairs. Odd

0303-2647/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

210

base-pairs are defined to be able to replace W a t s o n - C r i c k base-pairs without disturbing helical conformation. This has been shown for AC and UU odd base-pairs (Digweed et al., 1986) and might also hold for other combinations. (2) A character is considered plesiomorphic if it is present in the majority of higher taxa in either (a) archaebacteria and eubacteria or (b) archaebacteria and eukaryotes, thus assuming the archaebacterial phenotype to be the most primitive. The third combination (c) eubacteria and eukaryotes in 5S rRNA does not actually arise without including archaebacteria. Although archaebacterial 5S rRNAs have a lower rate of evolution in comparison to eubacteria and eukaryotes, exemplified in its conservative Helix C, structural diversity is clearly reflected in its Helix D (Erdmann and Wolters, 1986). (3) A character of extreme low variability (called a signature) with top priority in constructing the phylogeny is defined by having only changed up to three times when considering all 470 sequences. If, and only if, a signature has been found, the criterion can be applied to a lower taxonomic level. (4) In cases where signatures are incompatible they are hierarchically weighted according to their overall variability in preliminary clustering analyses: insertions/deletions > odd base-pairs > single nucleotides. (5) In order to address signatures to higher taxa these must be represented by at least two species. The signature must occur in 1000 of the species within the predefined higher taxa, exceptions are obvious sequence errors or if a species is a symbiont or parasite. (6) Every effort has been made to cite only monophyletic groups from the systematic literature, in eukaryotes these can be clearly defined by morphological characters, and are usually of phylum or class rank (Strasburger et al., 1983; Wurmbach and Siewing, 1985). For eubacteria phenetic classifications are used, which are derived from 16S rRNA oligonucleotide catalogues and usually define phlya or divisions and subdivisions (Woese et al., 1985; Stackebrandt, 1985). The same holds

true for archaebacteria, whose classifications are derived from r R N A / D N A hybridization studies and yield taxa with the rank of an order (Schnabel et al.,1983).

3. Results and discussion 3.1. Origin of plastids Cyanobacteria and plastids together clearly define a monophyletic group by the insertion of position 30.1 in the 5S rRNA, using the generalized numeration of Erdmann and Wolters (1986) (Fig. 1). The deletion of position 36 unites all plastids and cyanobacteria of the

120.2 120.1 120

Ur-

5S rRNA

A B

,,

104,1

b

b'

~04.2

.

.

.

.

.

(D)

(~,°

. IN

c

I "

,,,

c

~)041 e~.lI

~V ez e

Fig. 1. Secondary structure model of the Ur-SS rRNA, the ancestor of all present-day 5S rRNA molecules. Squares indicate conserved base-patting, dreles unpaired nucle~ tides. Where assignment was possible, plesiomorphie bases are indicated by letters at particular positions. Helices are designated A - E , loops a--e. Position numbering is according to the general system of Erdmann and Wolters (1986).

211 Prochloron

....I

speC.

Anacystis nidulans

INS.

Synechococcus lividus III Synechococcus llvidus II pt Cyanophora

3e DEL I

I~G~IU~

[ .... [~" G Y ~ C- G-[ /

pt Euglen. (Euglenida~ pt Chlamydom. . . . ~Volvophyta) pt Chlorella ~Chlorophyta~

pt DryopterJs (~Pteridophyta) I~e G_U ~ p t

Spermatophyta C4) 1

Pig. 2. Cladogram of cyanobacteria and plastids as deducod from 58 rRNA secondary and primary structures. pt, plastid; INS, insertion; DEL, deletion; colon denotes base-pairing. Position numbering as in Fig. 1. For the higher t~ffi~ the number of species sequenced are shown in parentheses.

genus Synechococcus, thus inferring a secondary loss of position 30.1 in Euglena plastids {Fig. 2). In order to resolve the ineompatibilty of positions 36 and 30.1, a reversal (in position 30.1) is favoured over two parallel events (in position 36) (see also Wolters and Erdmann, 1986). Position 79 must have changed twice within this phylum: (1) a CG to GY transversion defines a subgroup consisting of all plastids and cyanobacteria except Prochloron; (2) a back mutation is common to all plastids. The latter makes all plastids monophyletic ineluding those from Euglena and the cyanelle of Cyanophora. Owing to a treeing algorithm based on average linkage, the Euglena plastid is always placed way out on the phenogram (Dams et al., 1987) and a mixed analysis of Delihas et al. (1987) also results in an early origin of the Euglena plastid. Both latter misplacements are typical for organisms with a high rate of evolution. It is clear that additional plastid sequences of rhodophytes, chrysophytes, dinophytes and cryptophytes are urgently needed as well as more sequences from cyanobacteria to prove or disprove the monophyly of plastids. The presence of chlorophyll b as an accessory pigment and the lack of phycobilins in the prokaryotic genus Prochloron has misled

some biologists to claim a Prockloron-like ancestor for plastids. Proctdoron might, of course, be a model system for an endosymbiotic uptake of a photosynthetic bacterium (in this case by an ascidian), but this 5S rRNA analysis confirms the 16S oligonucleotide catalogue comparisons in detecting no specific relationships to plastids (Seewaldt and Stackebrandt, 1982). To determine the closest relative of plastids, the vast numbers of Synchococcus.like species should be investigated and the possibility considered that the primitive eubacteria (and also primitive cyanobacteria) might have been thermophilic (Achenbach-Richter et al., 1987). 3.2. Origin of mitochondria Angiosperm mitochondria are claimed to be derived from rhodobacteria {purple bacteria TABLE 1 58 rRNA signature nueleotides for elucidation of the phylogeny of eubacteria. Phylum

27

56

59

Plesiomorphi¢ Ocotopus 2/3 Th/omd#m Act/nobeeteria Firmibeeterla mycoplasmas

A A A A A Ab

U U U U U" Uc

G G G G G G

Desulfovibrio Herpetosiphon

A C

U U

G G

Cyanobarterla plastids Thermi Rhodobacteria gamma beta alpha-4 alpha-1 mitochondria alpha-2 alpha-3

C C C

G G G

A A A

C C C A A G A

G G G G G G/U U

A A A A A A G

Ezceptio~.~G in Bac///us ac/doca/dar/us, by in Acholep/asma, ~C in Ureaplasma urealyticum~ The term Actinobacteria refers to actinomyretes and relatives, Firmibacteria to elostridia and relatives, and Rhodoba¢teria to purple beeteria and relatives. Position numbering is according to the general system of Erdmann and Wolters (1986).

212 Erythrobacter Iongus 01:95UC 34: 48

Helix

28C~A~ G2~4C 5eG~U[

Agrobecterlurn tumefaciens

I Helix 2 B He, f Loapout 56G--U x ~ 59A~G 11:109 81U~A --

3.3. Fitting plastids into a eukaryote phylogeny

"Achromobacter" cycloclastes

Acldiphiliurncryptum Thlobaclllus novellus

o(-2

Rhodopseudomonas palust ris Protomonas extorquens

A cladistic analysis of eukaryotic 5S rRNAs confirms particular monophyletic taxa listed in Table 2. Details of the analysis are published elsewhere (Wolters and Erdman, 1986). With respect to photosynthesis the following aspects are of special importance.

(1) Within the Heterokontophyta (also called Chromophyta or Chrysophyta sensu

Rhodospirlllum rubrum ~rn~ochondrla (4) J

Helix IE S-4-5

7 C A

o(.-4

Thiobaciilus acidophUus

c~o 7-UYAA-~

m

Rhodobacter denltriflcans

TABLE 2

Rhodobacter sphaeroldes Rhodobactercapsulatus N8254 Rhodobacter capsulatus 23782

o(-3

Thloba¢illus versutus Paracoccusdenitrificans

Fig. 3. Cladogram of the alpha subdivision of rhodobacteria as deduced from 5S rRNA secondary and primary structures. Conventions are as in Fig. 2.

and relatives), namely from the alpha subdivision (Villaneuva et al., 1985). In order to elucidate this question it has to be recognised that 5S rRNA does not offer any synapomorphic characters for defining rhodobacteria besides a Helix E/Loop e typical for the alpha subdivision: a 7 base-pair stem with a NNAA loop. There are, however, three positions of low variability, namely 27, 56 and 59, that appear to have back mutated within the alpha subdivision (Table 1) and together with other medium variable positions result in the cladogram shown in Fig. 3. The phenetic distinction between four subgroups (Woese et al., 1984) seems to be well reflected in the cladistic analysis too. Although an affiliation of angiosperm mitochondria with the alpha-1 and alpha-3 subgroups is suggested, Paracoccus, a member of the alpha-3 subgroup, cannot be the closest ancestor of these mitochondria. That the cytochrome c analysis confirms the latter, offers a third variant which suggests a relation to the alpha-2 subgroup (Dickerson, 1980). The possibility that mitochondria are polyphyletic has not been dismissed.

Synapomorphic characters in 5S rRNA which define major monophyletic taxa among eukaryotos. DEL, deletion; INS, insertion; colon denotes base-pairing, uncertain synapomorphies are in parentheses, position numbering as in Table 1. Monophyletic taxon

Synapomorphy

Rhodophyta (red algae) Chlorobionta (green algae and land plants) Metazoa (and Mesozoa) A group of Zygomycota (HarpeUales, Mucorales, and Entomophthorales, but excluding Kickxeilales) A group of Ascomycota (Endomycetidae and Ascomycetidae, but excluding Schizosaccharomyces and

20.1 INS (41 RE-INS) 43 C to A, 73:103 odd AC 81:95 odd UU 1:119 1 INS

52.1 INS

Protomyces) A group of Basidiomycota (Type A') A group of Basidiomycota (Type Bb) Euglenozoa (trypanosomatids, euglenids and Cyanophora) Oophyta (a group of Heterokontophyta comprising Phaeophyceae (brown algae), BaeiUariophyceae (diatoms) and Oomycetes (water moulds) but excluding the chrysophyte

104.3 INS (3:117 odd UU) 47 C to U 45 A to C

Hydrurus) "According to Gottschalk and Blanz (1985).

213 Chilomonas

(Cryptophyta)

Crypt hecodinium

(101

(Dinophyta)

Y~G ~Ciliophora (10)

I

~s :11@

YR~RY ~Thraustochytrlcea Hydrurus

(2)

J

CChrysophyceae)

r o

A45c

Diatoma

(Bacillariophyceae~

a

Fig. 4. Cladogram of Chromobionta as deduced from 5S rRNA secondary and primary structures. Conventions are as in Fig. 2. Uncertain synapomorphies are in parentheses.

lato) brown algae, diatoms and 0omycetes form a monophyletic group (Fig. 4). This is not surprising since Pringsheim had already suggested in 1858 (Pringsheim, 1858) that water moulds were derived from the Xanthophyceae, a group not represented by 5S rRNA sequence data but certainly expected to be included in this lineage which some authors call Oophyta. The phylogeny of the other groups within the Chromobionta remains tentative. (2) While the loss of plastids is obvious within the latter group, this question remains unresolved concerning the Euglenozoa. Euglenida and Kinetoplastida, which have been proposed to have a common ancestor, based on ultrastructural data (Taylor, 1976), are allied with the cyaneile-containing Cyanopkor~ Whether phototrophic euglenids evolved by the uptake of a cyanobacterium (Cyanopkorastage) or whether they evolved separately by the uptake of a eukaryotic volvophytean algae (Gibbs, 1978) remains speculative. (3) Within the Chlorobionta, the charophyte NiteUa represents the sister group of embryophytes (land plants), and the volvophytean algae have to be excluded from the chlorophytes to make the latter monophyletic (Fig. 5). For this subset the cladistic analysis

agrees with the phenetic analysis of Hori et al. (1985). Using 5S rRNA data, our restrictive approach does not elucidate the phylogeny of the higher eukaryotic taxa defined in Table 2. When this method is applied to 16S rRNA, the data are too scarce to always fulfil the criterion 5 (a higher taxon must be represented by at least two species) and only part of the eukaryotic tree can be resolved (Fig. 6). Again, like in 5S rRNA, a close relationship between Eugienida and Kinetoplastida is substantiated, here by three synapomorphies. This relationship was not detected by the additive tree method used by Sogin et al. (1986) until the inclusion of a microsporidian 16S rRNA sequence by Vossbrinck et al. (1987). Three synapomorphies (positions 122, 343, and 1397:1491, E. coli numbering) resolve the position of the Euglenozoa leading to a common origin with Ascomycota and Metazoa. This is the major difference to the above-mentioned analyses, which place them much closer to the root of eukaryotes. We would like to argue in favour of our analysis since it is supported by another synapomorphy: the use of the a-aminoadipic acid (AAA) instead of the diamin~ pimilic acid (DAP) pathway for lysine

84


Loopout

C3~

J

/Chlorophyta (5)

43 C ~ A 73 : 103 ~AC

81$:90

~ \ -

~ A U

~

Zygnematophyceae (1) Chlorocoec~les (3) - UloLrichales (i)

84~84.1

I 23G~UI 74 Y ~ A I 1101 Y--GI

NiteUa CCharophyta)

w2c-u I 84.1

Loopout[ C ( ~

~

/Embryophyta ~24~ - Bryophyta (4) - Pteridophyta (4) - SpermaLophyLa (16)

Fig. 5. Cladogram of Chlorobionta as deduced from 5S rRNA secondary and primary structures. Conventions are as in Fig. 2.

214 "~Cilioohora 0 ' )

]

Plasmodium ~Sporozoa~ 1397: 1491 CG~CoU

Vairimorpha CMicrosporidla) Dictyostellum Prorocentrum CDInophyta) Acanthamoeba CAmoeblna~

777 A~C

122

A~U

343

~ Spermat°phytaC3) I

1397:

343 ~ Kinetoplastida ('.2~) ] c~u

785 C~U 1388 C~A

Euglena (Euglenlda)

1491

1397 : 1491 CG~CA

~Ascomycota (2)

~Metazoa(8)

I

l

Fig. 6. Cladogram of Eukaryota as deduced from complete 16S rRNA sequences. Poeition numbering is according to the K coli sequence (IUB convention). Other conventions are as in Fig. 2.

s y n t h e s i s in E u g l e n i d a , E u m y c o t a (Chytridio-, Zygo-, Asco-, Basidiomycota) and M e t a z o a ( W h i t t a k e r , 1969, Corliss, 1984). H i g h e r r a t e s of evolution, typical of t h e t r y p a n o s o m a t i d i a n p a r a s i t e and also indicated a t l e a s t for t h e 5S r R N A of Euglena plastid, still cause s e v e r e p r o b l e m s e v e n for s o p h i s t i c a t e d c o m p u t e r analyses, so t h a t one has to be v e r y cautious w h e n dealing w i t h s y m b i o n t s or p a r a s i t e s . Acknowledgements W e t h a n k A n g e l a S c h r e i b e r for t h e elabor a t e d r a w i n g s , M a r t i n D i g w e e d for t h e c o r r e c t i o n of t h e E n g l i s h t e x t , and t h e D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t (Sfbg-B5) and t h e F o n d s d e r C h e m i s c h e n I n d u s t r i e for financial s u p p o r t .

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