J Mol Evol (1990) 30:463-476 Journal of Molecular Evolution (~ Springer-Verlag New York Inr 1990

Small Ribosomal Subunit RNA Sequences, Evolutionary Relationships among Different Life Forms, and Mitochondrial Origins

Yves Van de Peer, Jean-Marc Neefs, and Rupert De Wachter

Departement Biochemie, Universiteit Antwerpen (UIA), Universiteitsplein 1, B-2610 Antwerpen, Belgium

Summary. A tree was constructed from a struc- turally conserved area in an alignment of 83 small ribosomal subunit sequences of eukaryotic, archae- bacterial, eubacterial, plastidial, and mitochondrial origin. The algorithm involved computation and optimization of a dissimilarity matrix. According to the tree, only plant mitochondria belong to the eu- bacterial primary kingdom, whereas animal, fungal, algal, and ciliate mitochondria branch off from an internal node situated between the three primary kingdoms. This result is at variance with a parsi- mony tree of similar size published by Cedergren et al. (J Mol Evol 28:98-112, 1988), which postulates the mitochondria to be monophyletic and to belong to the eubacterial primary kingdom. The discrep- ancy does not follow from the use of conflicting sequence alignments, hence it must be due to the use of different treeing algorithms. We tested our algorithm on a set of sequences resulting from a simulated evolution and found it capable of faith- fully reconstructing a branching topology that in- volved very unequal evolutionary rates. The use of more limited or more extended areas of the com- plete sequence alignment, comprising only very con- served or also more variable portions of the small ribosomal subunit structure, does have some influ- ence on the tree topology. In all cases, however, the nonplant mitochondria seem to branch off before the emergence ofeubacteria, and the differences are limited to the branching pattern among different types of mitochondria.

Key words: Small ribosomal subunit RNA -- Eu- karyotes -- Archaebacteria -- Eubacteria -- Plastids -- Mitochondria -- Simulated evolution

Abbreviation: srRNA, small ribosomal subunit RNA Offprint requests to: R. De Wachter

Introduction

Approximately 150 complete nucleotide sequences of small ribosomal subunit RNA (srRNA) have been published. Because the genes for small and large ribosomal subunit R N A s are coded by the genomes of the cellular life forms (eubacteria, archaebacteria, eukaryotes) as well as the genomes of mitochondria and plastids, and because sequences representative of each of these genome types are available, it is now possible to measure the evolutionary relation- ships among these life forms by means of a univer- sal, although possibly distorted, molecular yard- stick.

A eubacterial origin has been proposed for both plastids and mitochondria. The case for plastids having a common ancestor with cyanobacteria is particularly strong. Their genes are strikingly similar in organization and mode of expression to those of eubacteria (Gray and Doolittle 1982; Gray 1988). The affiliation of plastids to cyanobacteria has been demonstrated by evolutionary trees constructed from ferredoxins and c type cytochromes (Schwartz and Dayhoff 1978; Hunt et al. 1985), from 5S rRNA sequences (Hori and Osawa 1987; Van den Eynde et al. 1988), from 16S rRNA sequences (Woese 1987; Cedergren et al. 1988; Giovannoni et al. 1988), and from 23S rRNA sequences (Cedergren et al. 1988).

In the case of mitochondria, too, a eubacterial origin is usually assumed. John and Whatley (1975) postulated a close evolutionary relationship be- tween mitochondria and Paracoccus denitrificans, a member of the a subdivision of the purple bacteria and relatives (sensu Woese 1987) or Proteobacteria (sensu Stackebrandt et al. 1988), on the basis of comparison of biochemical parameters. This rela- tionship between mitochondria and Proteobacteria a has also been supported by structural comparison

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o f c y t o c h r o m e c m o l e c u l e s ( D i c k e r s o n et al. 1976). H o w e v e r , b e c a u s e the m i t o c h o n d r i a l c y t o c h r o m e s are c o d e d by the n u c l e a r g e n o m e o f the hos t cell , a gene t r ans fe r m u s t be i n v o k e d in o r d e r for th i s ar- g u m e n t to s u p p o r t a b a c t e r i a l o r ig in for m i t o c h o n - d r ia .

In genera l , the large v a r i a b i l i t y in s ize o f m i t o - c h o n d r i a l (mt ) D N A ( G r a y 1982) a n d i ts gene or - g a n i z a t i o n a n d m o d e o f e x p r e s s i o n ( G r a y 1982; B e n n e a n d S l o o f 1987) c o m p l i c a t e the i n t e r p r e t a t i o n o f m i t o c h o n d r i a l e v o l u t i o n . M o r e speci f ica l ly , e v e n w h e n a u n i v e r s a l m o l e c u l a r c lock such as s r R N A is a v a i l a b l e , the o c c u r r e n c e o f d i f fe ren t e v o l u t i o n a r y ra tes in the m i t o c h o n d r i a l as c o m p a r e d to the cel- l u l a r genes m a y o b s c u r e the e v o l u t i o n a r y r e c o n - s t r uc t i on f r o m s e q u e n c e a l i g n m e n t s . N e v e r t h e l e s s , resu l t s p o i n t i n g to an e v o l u t i o n a r y af f i l ia t ion o f t he m i t o c h o n d r i a wi th the P r o t e o b a c t e r i a a g roup , b a s e d on a l i g n m e n t s o f s m a l l a n d large s u b u n i t r R N A s , h a v e r ecen t ly been r e p o r t e d . Y a n g et al. (19 85) con- s t r u c t e d an e v o l u t i o n a r y t ree b a s e d on c o m p a r i s o n o f c o n s e r v e d a reas o f s r R N A sequences f r o m six e u b a c t e r i a , one a r c h a e b a c t e r i u m , a n d fou r m i t o - c h o n d r i a . T h e y u sed a d i s t a n c e m a t r i x m e t h o d w i th the s t ruc tu ra l s i m i l a r i t y b e t w e e n a p a i r o f s equences be ing c a l c u l a t e d o n the bas i s o f t he n u m b e r o f p o - s i t i ons i d e n t i c a l in the s e q u e n c e p a i r a n d d i f fe ren t in the r e m a i n i n g sequences . In d o i n g so, t hey f o u n d m i t o c h o n d r i a to be m o s t c lose ly r e l a t ed to Agro- bacterium tumefaciens, a m e m b e r o f the P r o t e o - b a c t e r i a a g roup . A s i m i l a r r e l a t i o n s h i p was f o u n d b y C e d e r g r e n et al. (1988) , w h o used a p a r s i m o n y m e t h o d c o m b i n e d w i th a b o o t s t r a p t e c h n i q u e to b u i l d d e n d r o g r a m s b a s e d on m u c h m o r e c o m p r e - h e n s i v e s m a l l a n d large s u b u n i t r R N A s e q u e n c e a l i g n m e n t s . B o t h resul t s , h o w e v e r , c o n t r a d i c t p r e - v i o u s t ree t o p o l o g i e s a l so o b t a i n e d f r o m s r R N A a l i g n m e n t s . Kf in t ze l a n d K S c h e l (1981) f o u n d an i - m a l a n d fungal m i t o c h o n d r i a to be p a r a p h y l e t i c ( sensu W o l t e r s a n d E r d m a n 1989) a n d to n o t b e l o n g to the e u b a c t e r i a l r e a l m . M c C a r r o l l e t al. (1983) p e r c e i v e d m i t o c h o n d r i a as m o n o p h y l e t i c , b u t f o r m - ing a c lus te r s e p a r a t e f r o m the eubac t e r i a . G r a y et al. (1984) f o u n d the w h e a t m i t o c h o n d r i o n to b r a n c h o f f i n the e u b a c t e r i a l c lus te r a t a n o d e s e p a r a t e f r o m t h a t l e a d i n g to the a n i m a l a n d fungal m i t o c h o n d r i a .

W e m a i n t a i n a c o m p l e t e co l l e c t i on o f a l i gne d s r R N A sequences ( D a m s et al. 1988), w h i c h is reg- u l a r ly u p d a t e d wi th n e w l y p u b l i s h e d sequences , a n d h a v e u sed th i s d a t a set for the c o n s t r u c t i o n o f a n e v o l u t i o n a r y t ree c o m p r i s i n g spec ies f r o m all p r i - m a r y k i n g d o m s as wel l as p l a s t i d s a n d m i t o c h o n - d r ia . W e used a d i s t a n c e m a t r i x m e t h o d d e v e l o p e d b y D e Soe te (1983). Because o u r resu l t s a re a t v a r i - ance w i th t h o s e o f Y a n g et al. (1985) a n d C e d e r g r e n et al. (1988) r e g a r d i n g the e v o l u t i o n a r y o r ig in o f m i t o c h o n d r i a , we h a v e e x a m i n e d w h e t h e r th i s d i s -

c r e p a n c y is d u e to d i f fe rences in the a l i g n m e n t used as p r o g r a m inpu t , o r to the fact t ha t d i f fe ren t al- g o r i t h m s r e c o n s t r u c t d i f fe ren t e v o l u t i o n a r y p i c t u r e s f r o m the s a m e d a t a set. A s t h e r e is e v i d e n c e t ha t m i t o c h o n d r i a l g e n o m e s , i n c l u d i n g the r R N A genes, a c c u m u l a t e m u t a t i o n s a t a s u b s t a n t i a l l y h ighe r ra te t h a n ce l lu l a r g e n o m e s , we h a v e a lso t e s t ed the ca - p a c i t y o f o u r t r ee ing a l g o r i t h m to r e c o n s t r u c t a cor - rect e v o l u t i o n a r y p i c tu re f r o m p r e s e n t - d a y se- q u e n c e s . F o r t h i s p u r p o s e , w e s i m u l a t e d t h e d i v e r g e n c e o f an ances t r a l n u c l e o t i d e sequence , a l - l owing d i f fe ren t m u t a t i o n a l ra tes in the b r a n c h e s , a n d t h e n u sed the resu l t ing s equences as i n p u t for ou r t ree c o n s t r u c t i o n p r o g r a m .

Methods

Sequence Alignments. The srRNA sequence alignment published by Dams et al. (1988) has been gradually updated as new se- quences were published. The present data set contains sequences from 62 eukaryotes, 14 archaebacteria, 30 eubacteria, 7 plastids, and 26 mitochondria. The following published srRNA sequences, not referenced in Dams et al. (1988), were used for tree construc- tion: the archaebacteria Thermococcus celer (Achenbach-Richter et al. 1988) and Thermoplasma acidophilum (Ree et al. 1989), the eubacteriurn Brucella abortus (Dorsch et al. 1989), and the mitochondrion of Rana catesbeiana (Nagae et al. 1988). Minor shifts in the alignment have been introduced since the published version (Dams et al. 1988). The largest of these modifications is situated in the area corresponding to helix P21-1 on the schematic representation of the srRNA secondary structure model in Fig. 1. This area, which has quite a different structure in bacteria and eukaryotes and is missing altogether in many sequences, was not used for tree construction. The alignment as used in the present study is obtainable from the authors in computer-readable form.

Figure 1 reveals four fractions of increasing variability, in- dicated by different shading, on the srRNA secondary structure model. Three areas of increasing size were derived from these fractions, each area containing the preceding one(s) plus an ad- ditional more variable fraction of the structure. Area I comprises only the part that is most conserved in sequence and covers 19 helices of the secondary structure model, most of them in their entirety but some only in part. It comprises 485 positions in our alignment, most of which are occupied by nucleotides in all se- quences, but a few of which can be empty in certain species due to deletions or insertions assumed in the process of alignment. Area 2, corresponding with 961 alignment positions, comprises area 1 plus 13 additional helices of the secondary structure model, which are slightly more variable in structure. Area 3 covers areas 1 and 2 plus three additional helices and extends over 1123 alignment positions. Alignments covering areas 1, 2, and 3 were used for the construction of different trees. The remaining most variable fraction of the complete alignment, which contains nu- merous empty positions, was not used for tree construction. The exact boundaries of areas 1, 2, and 3 in terms of alignment po- sitions are defined in Table 1.

The alignment used by Cedergren et al. (1988) for construction of an srRNA tree was obtained from these authors. It comprises 477 positions in 76 nucleotide sequences. The borders of the sequence tracts comprised in our area 1 alignment were selected so as to coincide exactly with those of Cedergren's alignment. The slight difference in length--477 versus 485 positions--is due to eight extra deletion/insertion events that we assume in our alignment.

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Fig. 1. srRNA structural variability and alignment areas. The secondary structure model for prokaryotic srRNAs and the helix numbering system are as in Dams et al. (1988). Alignment areas of increasing length can be distinguished as follows: area l --structures indicated in solid black (e.g., helix 5); area 2--area 1 plus the structures shaded in gray (e.g., helix 15); area 3--areas l and 2 plus the structures drawn in thick lines (e.g., helix 14). The remaining part of the structure is drawn in thin lines (e.g., helix 6) or in broken lines in the case of sequences only found in certain mitochondrial srRNAs (e.g., helix P17-1). Eukaryotic srRNAs show structural differences with this model outside area 3 only.

Although our alignment comprises srRNA sequences from mitochondria of the flagellates Trypanosoma bruceL Leishmania tarentolae, and Crithidiafasciculata, these were not used in tree constructions. The main reason is that the monotony of these (A+U)-rich sequences as well as their short chain length results in uncertainty concerning the optimal alignment with other se-

quences and the placement of deletions. As a result, conflicting secondary structure models have been proposed (de la Cruz et al. 1985; Sloofet al. 1985; Dams et al. 1988).

Tree Construction. Dissimilarity values (D) between each pair of sequences A and B, were computed according to the

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Table 1. Definition of alignment areas used for tree construction

Alignment position a Nucleotide position b

Area 1 Area 2 Area 3 Area 1 Area 2 Area 3

36-73 34-90 31-90 11--47 9-62 80-90 52-62

252-282 252-282 113-142 613-711 588-807 240-314

750-778 737-778 351-379 339-379 788-807 788-807 384-403 384-403

1094-1152 1094-1188 1094-1188 500-556 500-587 1845-1952 1819-2061 666-732

1996-2051 1968-2061 766-817 741-827 2266-2324 2253-2384 2253-2384 880-933 873-990 2331-2384 938-990

2466-2510 2466-2548 3116-3139 3084-3139 3077-3139 1215-1238 3302-3309 3302-3416 3302-3416 1308-1314 3321-3353 1323-1357 3364--3416 1367-1418 3574--3622 3574-3630 3574-3635 1483-1521

1046-1086 1190-1238 1308-1418

1483-1529

6--62

113-142 220-403

500-587 656-827

873-990

1046-1117 1183-1238 1308-1418

1483-1534

a These numbers correspond with positions of the alignment as published by Dams et al. (1988) b Escherichia coli srRNA sequence (Brosius et al. 1981) positions, numbered without interruption from the 5'-terminus

equation (Huysmans and De Wachter 1986; Dams et al. 1987):

3[ 4(s)][ o D A ~ = - ~ l n 1 - 3 1 - + u (1)

where I is the number of identical nucleotides, S is the number of positions showing a substitution, and G is the number of gaps in one sequence with respect to the other. T is the sum of I, S, and G. The first term of the equation accounts for substitutions and comprises a correction factor for multiple mutations per site (Jukes and Cantor 1969; Kimura and Ohta 1972). The second term accounts for deletions and insertions. Adjacent gaps are treated as a single gap. The resulting dissimilarity matrix served as the input for an algorithm (De Soete 1983) that finds a matrix of distances corresponding to an additive tree and differing min- imally from the input matrix according to a least squares crite- rion.

The tree construction program, written in FORTRAN, was run on a microVAX II from Digital Equipment Corporation (Maynard, MA). The time required rises with the fourth power of the number of sequences, which puts a practical limit on the size of the trees that can be constructed. It took the computer about 30 h of CPU time to build a tree from 83 srRNA sequences.

Computer Simulation of Nucleotide Sequence Evolution. The capacity of the tree construction program to reproduce the correct tree topology for an evolutionary process involving unequal rates of mutation fixation in different branches was assessed as follows. A sequence of 477 nucleotides was produced by means of a random number generator (Press et al. 1985), giving each of the four nucleotides an equal chance of occurring at each site. Fix- ation of mutations was simulated by selecting random positions and giving the nucleotide present at that site an equal chance of being replaced by each of the three possible substituents. Tree branching and species divergence were simulated by duplicating a sequence and mutating each copy independently. In order to obtain trees with unequal branch lengths, sequences were mutated until a desired number of substitutions had accumulated with respect to the ancestral sequence at the last branching point. Trees

were reconstructed from the resulting set of sequences as de- scribed above for the actual srRNA sequence alignments or seg- ments thereof.

Results

Reconstructing Evolutionary Relationships among Cellular and Organelle Genomes

Figure 2 r ep resen t s the ou t l i ne o f an u n r o o t e d t ree

c o n s t r u c t e d f r o m area 2 (see M e t h o d s , Fig. 1, a n d

T a b l e 1) o f an a l i g n m e n t o f 83 s r R N A sequences .

T h i s a rea c o v e r s a r eg ion a p p r o x i m a t e l y twice as

large as t ha t used by C e d e r g r e n et al. (1988) in the i r

r ecen t s tudy o f e v o l u t i o n a r y r e l a t i onsh ips a m o n g

life f o r m s based on s r R N A sequences . T h e set o f 83

s r R N A sequences was se lec ted because it was no t

poss ib le to p rocess all 151 h i t h e r t o k n o w n s r R N A s

due to c o m p u t e r t i m e b e c o m i n g p r o h i b i t i v e . T h e

set inc ludes all 76 o r g a n i s m s a n d organe l les used in

the s tudy o f C e d e r g r e n et al. (1988), m i n u s Schizo- saccharomyces pombe m i t o c h o n d r i o n a n d Chlam- ydomonas eugametos ch lo rop las t , w h o s e s r R N A se-

quences are u n p u b l i s h e d a n d hence n o t a v a i l a b l e to

us o v e r the en t i re a rea 2. A d d i t i o n a l l y i n c l u d e d were

five a r chaebac t e r i a l s equences in o r d e r to o b t a i n a

be t t e r b a l a n c e d r e p r e s e n t a t i o n o f the th ree p r i m a r y

k i n g d o m s , plus th ree newly p u b l i s h e d m i t o c h o n -

dr ia l s r R N A sequences , p lus Brucella abortus, a m e m b e r o f the P r o t e o b a c t e r i a a subgroup . A c c o r d -

ingly, the t ree in Fig. 2 c o m p r i s e s 26 euka ryo t i c , 14

a rchaebac te r i a l , 16 eubac te r i a l , 4 p las t id ia l , a n d 23

m i t o c h o n d r i a l s r R N A sequences .

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Dissimilarity 0.1 animal mitochondria

A3 /

B I

A1 / B 2

algal mitochondrion

fungal ......... ~ mltoehondrla

Archaebacteria ~ / / ~Eubacteria ~ ciliate plastids mitochondria

plant mitochondria

Fig. 2. Tree derived from alignment area 2 of 83 srRNA sequences. Clusters formed by eukaryotes, archaebacteria, and eubacteria are represented as isosceles triangles with a base proportional to the number of terminal nodes, and a height equal to the mean dissimilarity, measured from the first branching point within the cluster to each terminal node. Mitochondrial clusters are similarly represented by shaded triangles. The plant mitochondrial cluster branches off within the eubacterial cluster. The dissimilarity scale is indicated on top. A~, A2, A3, B~, and B2 are possible root locations mentioned in the Discussion. The detailed structure of the archaebacterial, eubacterial, and mitochondrial clusters is given in Fig. 3.

The main clusters distinguishable in the tree are represented by triangles. The detailed structure of each cluster can be seen in Fig. 3, except for the eukaryotic cluster that had a structure similar to the previously published (Hendriks et al. 1988) tree con- structed exclusively from eukaryotic srRNA se- quences. One can distinguish three clusters formed by eukaryotes, archaebacteria, and eubacteria, which is in accordance with the division of cellular life forms into three primary kingdoms as suggested by Woese and Fox (1977). Consequently, our tree, like the one published by Cedergren et al. (1988), does not support the hypothesis of Lake (1987, 1988), which assumes a polyphyletic origin of the archae- bacteria.

In contrast with the results ofYang et al. (1985) and Cedergren et al. (1988), mitochondria appear as polyphyletic. Indeed, three distinct mitochon- drial clusters can be distinguished. One cluster, whose detailed structure is shown in Fig. 3a, comprises all animal mitochondria. A second cluster, shown in detail in Fig. 3b, is formed by the mitochondrion of the alga Chlamydomonas reinhardtiL a subcluster

consisting of two fungal mitochondria, and one con- taining three ciliate mitochondria. The third cluster comprises the land plant mitochondria and is embedded in the eubacterial cluster, shown in Fig. 3c, where it has the Proteobacteria ct subgroup as closest relatives. Wherever the root of the tree is assumed to be (see Discussion for possible loca- tions), in no case is it compatible with the mito- chondria as a whole being monophyletic. Only re- garding the origin of the land plant mitochondria is our tree in accordance with the results of Cedergren et al. (1988). On the other hand, the common an- cestry of cyanobacteria and plastids is again con- firmed by the topology of the eubacterial cluster (Fig. 3c).

Figure 3d is a detailed representation of the ar- chaebacterial cluster of Fig. 2. Usually three distinct phenotypes, viz. extreme thermophiles, methano- gens, and extreme halophiles, are distinguished among the archaebacteria, although their phyloge- netic relationships do not correspond with these di- visions. The root of the archaebacterial subtree di- vides the archaebacteria into two main groups, one

468

a Mus musculus mit.

Rattus norveKicus mit.

Bos taurus mit.

Pan troglodytes mif.

Pan paniscus mit.

Homo sapiens mit.

Gorilla Korilla mit.

Pongo piKmaeus mit.

Xenopus laevis mit.

Rand catcsbeiana mif.

- - Drosophila ~akuba mit.

~-- Drosophila wlrills mit.

b Paramecium primaurelia mit.

Paramecium tetraurelid mit.

Tefrahymend pyriformis mit.

AsperKillus nidulans mit.

Saccharomyces cerevisiae mit.

Chlamydomonas reinhardfli mit.

C _ _ • Glycine max mit.

Oenothera s p . mit.

Zea mays mit.

diploperennis m i t . Zea - Triticum aestivum mit.

Roehalimaea quintana

--Brucella abortus

Agrobacterium tumefaciens

Eseherichia cell

Proteus vulffaris

Pseudomonas testosferonl

Desulfovibrio desulfuricans

_~______Baeil]us subfilis

Hellobacterium chlorum

- - MyXOCOCCUS xanthus

Zea mays c h l , Nicotiana fabacum chl.

---------~E Chlamydomonas reinhardtil ch|.

uglena Kracilis chI.

Anacystis nidulans

_ ~ - - Bacferoides fraKilis

- - CytophaKa heparina

Mycoplasma caprieolum

--------~Mycoplasma PG50

Chlamydia psiftaci

d ~ A r c h a e o g l o b u s

- - T h e r m o c o c c u s c e l e r

M e t h a n o b a c t e r i u m f o r m i c i c u m

Mefhanobacterium thermoautotrophicum

Mefhanococeus vannielii

Halobacterium volcanii

H a l o c o c c u s m o r r h u a e H a l o b a c t e r i u m c u t i r u b r u m

L H a l o b a c t e r i u m h a t o b i u m

I t -- - -- Thermoplusma acidophilum

I r~ " - Sulfolobus soifataricus

[ J ' Desulfuroeoceus mobilis

t Thermoproteus tenax

Dissimilarity 0.I I i

Fig. 3. Structural details of the tree derived from alignment area 2. The complete structure of the following clusters, outlined in Fig. 2, is shown, a Animal mitochondria; b ciliate, fungal, and algal mitochondria; e eubacteria, plastids, and plant mitochondria; d archaebacteria. Dissimilarities are drawn to the scale indicated at the bottom. Dots at the root of a cluster correspond with the branching point to other clusters.

formed by the three sulfur-metabolizing extreme thermophi les Sulfolobus soIfataricus, Desulfurocoe- cus mobilis, and Thermoproteus tenax and the other formed by the halobacteria, the methanogens, the e x t r e m e t h e r m o p h i l e s Thermococcus celer and Thermoplasma acid@hilum, and the sulfate-reduc- ing extreme thermophi le Archaeoglobus. The divi- sion of the archaebacteria into two main branches is the same as found by Woese and Olsen (1986). The close evolut ionary relationship between Meth- anospirillum hungatei and the halobacteria (Woese and Olsen 1986; Achenbach-Richter et al. 1987, 1988) is also confirmed. Still, there are some dis- crepancies with the results of Woese and Olsen (1986) regarding the branching order o f the extreme ther- mophiles T. eeler and T. acidophilum relative to the halobacteria and methanogens.

Possible Causes of Discrepancy with Other Results

Our results concerning the polyphyletic origin o f mi tochondr ia are clearly at variance with recently published trees also based on s rRNA structure, and which demonst ra te all mi tochondr ia to have a com- m on ancestor with Proteobacter ia subgroup o~ and to be monophylet ic . Yang et al. (1985) used a matr ix me thod and a least squares criterion for selecting the best fitting tree. However , dissimilarities were calculated only on the basis o f positions that are identical in a pair o f sequences while being dissim- ilar in all other sequences. Cedergren et al. (1988) used a pars imony me thod combined with local op- t imizat ion, as pars imony cannot tackle large num- bers o f sequences simultaneously. Two possible causes for the discrepancy between the latter results and those shown in Fig. 2 come to mind. (1) The input data for tree construct ion are different, and (2) the same data lead to different tree topologies when different tree construction algorithms are used.

The first cause o f discrepancy would occur i f the sequences are aligned differently by different in- vestigators or i f different areas o f the al ignment are used, one comprising only very conserved and another involving more variable areas. In order to address this question, we constructed trees f rom alignments of different origin and different length. As for the second possibility, al though different tree construct ion methods should ideally yield identical tree topologies, this is not so in practice because the sensitivity to various causes o f error differs among the algorithms. One impor tan t source o f error that may give a distorted picture in the case o f trees involving mi tochondr ia l sequences is that muta t ion fixation does not occur at the same rate in each branch. In order to examine the influence o f this source o f error on our algorithm, we applied it to

469

sequences resulting from a simulated evolut ion in- volving different branch lengths.

lnfluence of the Alignment on the Tree Topology

Figure 4a is a simplified representat ion of the tree topology reported by Cedergren et al. (1988), using their pars imony method on an al ignment covering 477 positions in 76 sequences. Only the main clus- ters are shown, so as to facilitate compar ison with our results (Fig. 2) and to emphasize the main dif- ference in topology, which is that all mi tochondr ia originate within the eubacterial cluster. When we applied our tree construct ion me thod to the data set ofCedergren et al. (1988), we obtained the tree shown in Fig. 4b, which has essentially the same topology as the tree of Fig. 2, constructed on area 2 (961 positions) of our alignment, and which also shows only the plant mi tochondr ia among the eubacteria. The differences between the trees o f Fig. 2 and Fig. 4b are restricted to branch lengths and to a few branching orders within the main clusters, but the topology o f the main clusters remains the same. Exactly the same topology as in Fig. 4b was obta ined when a tree was constructed f rom area 1 o f our alignment, which extends over 485 rather than 477 posit ions (see Methods).

These results demonst ra te that the conflicting tree topologies obtained by Cedergren et al. (1988) and by us are not due to al ignment problems, as our tree construct ion me thod yields exactly the same topol- ogy when we use their al ignment or a corresponding section of our alignment. The extent o f the align- ment used, however, does have some influence on the tree topology. Although we obta ined essentially the same topology when using area 1 (Fig. 4b) and area 2 (Fig. 2), a somewhat different topology re- sulted when area 3 was used. In this case, illustrated in Fig. 5, the animal mi tochondr ia l branch joins the one bearing the algal-, ciliate-, and fungal mi to- chondria, rather than originating f rom a separate node. In no case, however, did we observe a com- m o n ancestry specific for all mitochondria .

Accelerated Evolution o f Mitochondrial srRNAs

How much faster do mitochondria l genomes ac- cumulate mutat ions than the nuclear genomes o f their hosts? In the case o f s rRNA genes a quanti- tative measure o f this accelerated evolut ion is avail- able because in several species the mi tochondr ia l as well as the nuclear s rRNA sequences have been de- termined. The factor by which a mi tochondr ia l s rRNA gene evolves faster than the nuclear s rRNA gene o f its host can be est imated from the ratio

470

I00 mutations

Archaebacteria

animal mitochondria ~iiiiii~

a Jiii jiV ....

Eubacteria / mitochondrion

~ ~ p l a n t mitochondria ~ ciliate

mitochondria

Dissimilarity 0.1 b ~ n i m a l mitochondria

i / / 1 ~ ~ ciliate / I k Eubacteria mitochondria

plant mitochondria

Fig. 4. Trees derived from alignment area l of 76 srRNA sequences. These trees were constructed on the alignment obtained from Cedergren r al. (1988), which corresponds with our alignment area i. Clusters are symbolized as in Fig. 2. a Outline of the tree published by Cedergren et al. (1988). As this is a parsimony tree, the distance scale measures net substitutions rather than dissimilarity. b Outline of the tree constructed by our algorithm on the alignment of Cedergren et al. (1988).

Dissimilarity 0.1 ~ animal mitochondria

471

Eukaryota

algal mltochondrion

B 2

A 3

~ f u n g a l ~'~mitochondria

ciliate mitochondria

& p l a s t i d s

p I a n t mitochondria

Fig. 5. Tree derived from alignment area 3 of 83 srRNA sequences. Clusters are symbolized as in Fig. 2. A~, A=, A3, Bt, and B2 are possible root locations mentioned in the Discussion.

R - [DABL (2) [DA~].

Table 2. Rat ios o f d iss imi lar i ty between cor responding mi to - chondrial and nuclear sequence pairs

where [DAB]m is the dissimilarity between the mi- tochondrial srRNA sequences of two species A and B, and [DAB]n is the dissimilarity between the nu- clear srRNA sequences of the same pair of species. Values for R, calculated for the available pairs of species, are listed in Table 2. The top matrix gives the values calculated from the alignment of Ced- ergren et al. (1988) (477 positions), which corre- sponds to area 1 of our alignment. The second ma- trix lists the values obtained for area 2 of our alignment (961 positions). It can be seen that the ratios of the first matrix are of the same magnitude as, although not identical to, those of the second matrix, which is based on an alignment that is ap- proximately twice as long. This means that the extra positions included in area 2 are evolutionarily not much more variable than those of area 1, a fact consistent with the congruency of the trees in Figs. 2 and 4b.

The ratios in Table 2 demonstrate that all mi- tochondrial srRNAs, except those of land plants, diverge considerably faster than the corresponding nuclear-coded srRNAs. The difference is especially remarkable for mammalian mitochondria, with the dissimilarity between mouse and human srRNAs being about 100 times that between the correspond-

H.s. M . m . X . 1 . C.r. T . t . P . t . S . c . Z . m . G . m .

H.s. - - 80 37 8.4 4 6.5 8.1 6.6 6.5 M.m. 100 -- 31 8.5 3.7 6.5 7.6 6.6 6.6 X.1. 21 22 -- 8.1 3.7 6.6 7.3 6.4 6.3 C.r. 8.2 8.6 8.5 - - 3.3 8.9 9.2 32 19 T.t . 4.3 4.0 4.6 4.5 - - 1.5 3.9 2.5 2.5 P.t. 5.8 6.2 6.4 8.5 3.9 - - 7.4 5.5 5.7 S.c. 7.4 7.3 7.6 9.6 4.5 8.2 - - 8.6 9.3 Z.m. 7.0 7.2 6.9 16 3.5 5.6 8.3 - - 0.8 G.m. 6.8 7 6.7 14 3.6 5.8 8.7 1.0 - -

Figures in the mat r ix are ratios [Eq. (2)] o f the diss imilar i ty [Eq. (1)] be tween two mi tochondr ia l sequences to the diss imilar i ty between the cor responding nuclear sequences. U p p e r f ight ha l f matr ix: ratios compu ted on the basis o f the a l ignment o f Ced- ergren et al. (1988), which cor responds with our a l ignment area 1. Lower left ha l f matr ix: rat ios c o m p u t e d on the basis o f align- m e n t area 2. H.s., Homo sapiens; M.m. , Mus musculus; X.I. Xenopus laevis; C.r., Chlamydomonas reinhardtii; T.t., Tetra- hymena thermophila; P.t. Paramecium tetraurelia; S.c., Saccha- romyces cerevisiae; Z.m. Zea mays; G.m. Glycine max

ing nuclear genes. These values seem to point not just to a faster evolution but to an acceleration of mitochondrial srRNA evolution in certain taxa, no- tably the vertebrates.

We have also attempted to calculate the corre- sponding ratios by measuring branch lengths in the srRNA tree of Cedergren et al. (1988). Although

472

! .00

g -~ .75 T,

.50

g ~ .25

4 -

Fig. 6.

| / ' ~ J

,2 .4 .6 .8 1.0 1.2

divergence time (years x 10 -9)

Fraction of substitutions in homologous positions of mitochondrial srRNA as a function of divergence time. The fol- lowing divergence times were assumed: Homo sapiens to Pan troglodytes and to Gorilla gorilla, and P. troglodytes to G, gorilla, 5 million years (Myr) (Sarich and Wilson 1967); Pongopygmaeus to other primates, 8 Myr (Sarich and Wilson 1967); primates to cow and to mouse, and cow to mouse, 80 Myr (Brown et al. 1982); monocotyledons to dicotyledons, 200 Myr (Emberger 1968); mammals to amphibians, 350 Myr (Rensch 1972); ver- tebrates to arthropods, 540 Myr (Rensch 1972); metazoa to fungi, 1000 Myr (Clemmey 1976; Brown et al. 1982). For each com- parison between two taxa, the fraction of substitutions was count- ed for all possible sequence pairs. As an example, 20 comparisons were made of vertebrate versus arthropod sequences, as the set contains 10 vertebrates and 2 arthropods (cE Fig. 3a). Black bars consist of overlapping squares for close measuring points. The continuous curve connects all points except those corresponding to divergences among mammals (upper broken curve) and the monocotyledon--dicotyledon divergence (lower broken curve).

several ratios could not be calculated precisely be- cause some very short branches could not be mea- sured accurately, the results are in line with those of Table 2, with two exceptions. Land plant mito- chondrial srRNAs seem to diverge about twice as fast as the nuclear equivalents, whereas mammalian mitochondrial srRNAs diverge only 15 times as fast as the nuclear equivalents.

Brown et al. (1982) compared a mtDNA sequence coding for proteins and tRNAs in five primates and two other mammals. They found that recently di- verged sequences differ by about 10 times as many transitions as transversions. Also, a plot of sequence differences against estimated divergence times in Fig. 4 of their paper might suggest that mitochondrial sequences become saturated with substitutions after about 20 million years of divergence. If such were the case, then it would be impossible to infer evo- lutionary distances among mitochondrial sequences separated by divergence times of the order of a bil- lion years or more, whether by a distance matrix method or by a parsimony method. However, in Fig. 6 we have plotted the number of substitutions

observed among mitochondrial srRNA sequences over a much larger scale of divergence times, in order to show that a different interpretation is pos- sible, at least for rRNA-coding sequences. The figure shows a steadily rising fraction of observed substi- tutions, when the divergences between mammals and amphibians, between vertebrates and arthro- pods, and between metazoa and fungi are taken into account. It also suggests that it is difficult to fit the same curve to divergences among mammals, e s - pecially primates, for which a much larger substi- tution rate seems to prevail, and to divergences among plants, to which corresponds a much lower substitution rate. This graphic representation of ob- served substitution fractions is in line with the data of Table 2, which also suggest an exceptionally high rate for mammals, an exceptionally low rate for plants, and a much more consistent rate among oth- er taxa. From these observations we conclude that mitochondrial srRNA sequences do not become sat- urated with substitutions after a few million years, and that a calculation of evolutionary distances can be meaningful within the time scale studied here.

Brown et al. (1982) assumed that the high ratio of transitions to transversions, observed upon com- parison of mtDNA sequences from the recently di- verged primates, reflects the actual ratio of transi- tions to transversions among fixed mutations. The ratio observed upon comparison of longer diverged sequences would fall because multiple transitions are obliterated by rare transversions. Comparison of mitochondrial srRNA sequences suggests a dif- ferent picture. The ratio of observed transitions to transversions is 10 or higher among primate se- quences, but is about 1 when sequences from other recently diverged species are compared, such as mouse and rat, or Drosophila yakuba and Drosoph- ila virilis. This suggests that the bias toward tran- sitions is much stronger in primate mitochondrial srRNAs than in other species. Nevertheless, because Eq. (1) assumes an equal probability for all substi- tutions, i.e., a transition to transversion ratio of 0.5, we computed an alternative dissimilarity matrix for alignment area 2. Correction for multiple mutations was according to the equation to Kimura (1980), which, contrary to the equation of Jukes and Cantor (1969), accounts for unequal probabilities of tran- sitions and transversions. Construction of a tree from the resulting matrix did not reveal any difference in topology with the tree shown in Fig. 2, but only minor changes in branch length.

Influence of Unequal Branch Lengths on Tree Topology

If evolution had actually proceeded according to the tree published by Cedergren et al. (1988), would our

473

treeing algorithm have been able to reconstruct the correct tree topology in spite of the unequal evo- lutionary rates in different branches of evolution? In order to address this question, the divergence of an ancestral sequence of 477 nucleotides into 76 present-day sequences was simulated as described in the Methods section.

The branching pattern and the branch lengths were chosen as in the srRNA (SSU rRNA) tree of Fig. 2 in Cedergren et al. (1988). The scale provided with this figure was used to determine the branch lengths, i.e., the net number of substitutions im- posed after each sequence duplication until the next duplication. The root of the simulation was chosen at the junction of the nuclear, archaebacterial, and eubacterial-mitochondrial subtrees of the afore- mentioned figure. A tree was reconstructed, by the algorithm described in the Methods section, from the 76 sequences resulting from the simulation. The outline of the resulting tree is drawn in Fig. 7, with the root coinciding with the ancestral sequence of the simulation, hence the first divergence is a triple branching point leading to a nuclear, archaebacteri- al, and eubacterial-mitoehondrial cluster. The tree has exactly the same topology as the unrooted srRNA tree published by Cedergren et al. (1988). This dem- onstrates that our treeing algorithm is capable of correctly reconstructing an evolutionary process, as- suming the branching topology and differences in evolutionary rates described in the latter paper.

Discussion

Reconstructions of evolutionary history on the basis ofsrRNA sequence alignments have been published by several research groups (Kiintzel and K/Schel 1981; McCarroll et al. 1983; Gray et al. 1984; Hunt et al. 1985; Yang et al. 1985; Lake 1987; Woese 1987; Achenbach-Richter et al. 1988; Cedergren et al. 1988; Field et al. 1988; Hendriks et al. 1988). The trees published by Cedergren et al. (1988) and those shown in Figs. 2, 4b, and 5 of this paper are the most comprehensive ones to our knowledge. The main discrepancy in the trees presented here with the results of Cedergren et al. (1988) is that we per- ceive the mitochondria to be polyphyletic. The clus- ters consisting of animal, algal, fungal, and ciliate mitochondria seem to have ancestors that do not belong to any of the three primary kingdoms. Only the ancestor of the land plant mitochondria is sit- uated among the eubacteria and seems related to the Proteobacteria o~ subgroup.

The discrepancy with the tree topology of Ced- ergren et al. (1988) is not due to the use of conflicting alignments, as we find essentially the same results (Fig. 4b) when using their alignment for area 1 of

the srRNA structure or our own alignment for the same area. This topology does not change when we use the alignment for area 2 (Fig. 2). When the align- ment for area 3 is used, however, a change in tree topology results (Fig. 5), with the animal mitochon- dria appearing to form a common cluster with the algal, fungal, and ciliate mitochondria. Yet the mi- tochondria remain polyphyletic, as those from land plants retain their eubacterial ancestry.

Tree construction on a set of sequences obtained by computer simulation of an evolutionary process, assuming branches of unequal length and of the size postulated by Cedergren et al. (1988), demonstrates that the treeing algorithm employed in this paper reconstructs the correct tree topology under such conditions. We are not aware of a similar test having been performed on the parsimony-bootstrap treeing algorithm.

If the tree topology of Fig. 2 is a correct repre- sentation of evolutionary relationships among life forms, what would be the implications for the evo- lutionary history of mitochondria with respect to cellular life forms? In order to facilitate this discus- sion, a number of possible places for the root have been indicated on the tree of Fig. 2. If the root were situated in A~, Az, or A3, this would imply a pri- meval divergence into three branches leading to the primary kingdoms, eukaryotes, archaebacteria, and a third kingdom comprising the mitochondria and the eubacteria. In the latter kingdom, however, the ancestor of the present eubacteria would arise later than the ancestor of the animal mitochondria and that of the algal, fungal, and ciliate mitochondria. Only for the land plant mitochondria would the emergence come after that of eubacteria. On the other hand, if the root were situated in B~ or B2, the primeval life form would have the animal mito- chondria or the algal, fungal, and ciliate mitochon- dria as the most direct present-day descendants. The topology of a tree rooted in B~ would bear some resemblance, although it would not be identical, to the tree postulated by Mikelsaar (1987). In his ar- chigenetic hypothesis, Mikelsaar assumes that the first branches of the evolutionary tree led to the present animal and fungal mitochondria, preceding the branching that gave rise to the three primary kingdoms as well as the plant mitochondria. A sep- arate endosymbiosis event giving rise to the plant mitochondria has also been envisioned by Gray et al. (1989), although in the framework of the hy- pothesis that all mitochondria are derived from the Proteobacteria a subgroup.

The tree'ofFig. 5, constructed on the area 3 align- ment, gives a picture slightly different from the tree of Fig. 2. The essential distinction is that animal mitochondria appear as monophyletic with algal, fungal, and ciliate mitochondria. The land plant mi-

474

Dissimilarity 0. I I I

animal mitochondria

-- algal mitochondrlon

~ fungal mitochondria

~ ciliate mitochondria

iiiiiiiiiiiiii i' -- DesuZfovibrio desuZfurieans

Mymoooeeus xanthus

Chlamvdia psittaei

Archaebacteria

Eukaryota

Fig. 7. Reconstruction of a simulated evolution. The divergence of an ancestral random nucleotide sequence of 477 nucleotides into 76 descendant sequences was simulated as described in Methods. Branch lengths and branching pattern were exactly as in the srRNA tree of Cedergren et al. (1988) (Fig. 4a). The root was chosen at the divergence between eukaryotes, archaebacteria, and eubacteria plus mitochondria. The tree reproduced from the resulting sequences by our algorithm is drawn with the root at the ancestral sequence in order to show clearly the differences in evolutionary rate. Although all resulting sequences are random, each was named after the species whose srRNA evolution it mimicks. Clusters are symbolized as in Fig. 2.

tochondria remain the only ones that arise among the eubacteria. I f the root were at location B~ (Fig. 5), the first divergence would be between the ances- tors of animal, fungal, algal, and ciliate mitochon- dria on the one hand, and the ancestors of cellular life forms plus plant mitochondria on the other hand. If the root were at B2, the most direct descendants

of the universal ancestor would be the animal mi- tochondria, and the picture would fit nearly perfectly with Mikelsaar's (1987) archigenetic hypothesis.

It may be slightly disappointing that the use of a more comprehensive alignment (area 3) gives a tree topology (Fig. 5) somewhat different from a more restricted alignment (area 2, Fig. 2). There are few

475

a r g u m e n t s for p r e f e r r i ng one t o p o l o g y o v e r the o th - er. O n e p o i n t in f a v o r o f the t o p o l o g y o f Fig. 2 is t h a t i t is f o u n d b o t h o n the bas i s o f a r e a 1 o f the a l i g n m e n t a n d o n the bas i s o f a r ea 2, w h i c h is twice as large. O n l y w h e n an e x t r a 162 p o s i t i o n s a re a d d e d to a r r i v e at a r ea 3 does the p i c t u r e change , w i th the a n i m a l m i t o c h o n d r i a l c lus t e r j o i n i n g the a l g a l - f u n - g a l - c i l i a t e m i t o c h o n d r i a l c lus ter . A n a r g u m e n t in f a v o r o f the l a t t e r t o p o l o g y (Fig. 5), w i th a r o o t in B~ o r BE, w o u l d be t ha t i t is c o n s i s t e n t w i t h a p r i m - i t i v e gene t ic c o d e r e s e m b l i n g the p r e s e n t - d a y c o d e s o f a n i m a l , fungal , a n d c i l i a te m i t o c h o n d r i a , w h i c h h a v e m a n y c o d o n a s s i g n m e n t s in c o m m o n (Benne a n d S l o o f 1987; F o x 1987). In the l ight o f t h i s a r - g u m e n t the t o p o l o g y o f Fig. 2 is less a t t r a c t i v e be - cause , i f the r o o t were s i t u a t e d in B1 o r B 2 a n d the p r i m i t i v e gene t i c c o d e were m i t o c h o n d r i a - l i k e , th i s w o u l d neces s i t a t e a n i n d e p e n d e n t change t o w a r d the p r e s e n t - d a y code in t w o b r a n c h e s , one l e a d i n g to a r c h a e b a c t e r i a a n d e u k a r y o t e s , t he o t h e r to e u b a c - t e r i a a n d p l a n t m i t o c h o n d r i a . I f t he r o o t were p l a c e d in one o f the A l o c a t i o n s a n d the p r i m i t i v e c o d e r e s e m b l e d the p r e s e n t one , t he p i c tu r e w o u l d be m o r e p l a u s i b l e b u t w o u l d sti l l neces s i t a t e pa ra l l e l c hanges in the b r a n c h e s l e a d i n g to the a n i m a l m i - t o c h o n d r i a on the one h a n d a n d the a l g a l - f u n g a l - c i l i a te m i t o c h o n d r i a o n t h e o the r .

T h e d i s c r e p a n c y o f o u r resu l t s w i th t h o s e o f Ced - e rg ren et al. (1988) r e m a i n s e m b a r r a s s i n g to exp la in . T h e s e a u t h o r s d o n o t m e n t i o n w h e t h e r t h e i r t r ee ing a l g o r i t h m has been t e s t ed for r e c o n s t r u c t i o n o f a s i m u l a t e d e v o l u t i o n w i th u n e q u a l b r a n c h lengths . A n a r g u m e n t t ha t t hey c i te in f a v o r o f the d e p e n - d e n d a b i l i t y o f t h e i r resu l t s is t h a t t rees c o n s t r u c t e d f r o m large a n d s m a l l s u b u n i t r R N A sequence a l ign- m e n t s a re cong ruen t . H o w e v e r , a l t h o u g h b o t h t rees s h o w the m i t o c h o n d r i a as m o n o p h y l e t i c , the o r d e r o f d i v e r g e n c e o f l a n d p lan t , fungal , c i l ia te , a lgal , a n d a n i m a l m i t o c h o n d r i a is n o t t he s ame . Also , b e c a u s e fas t e v o l u t i o n a r y ra tes in m i t o c h o n d r i a l b r a n c h e s a r e p r o b a b l y c o m m o n to the two mo lecu l e s , th i s c o u l d d i s t o r t t he two t rees s i m i l a r l y i f the a l g o r i t h m is s ens i t i ve to d i s t o r t i o n . P e n d i n g a d d i t i o n a l ev i - dence , we p r o p o s e t h a t the p i c tu re s re f lec ted b y the t rees o f Figs . 2 a n d 5 be t a k e n i n to a c c o u n t as pos - s ib ly co r r ec t o u t l i n e s o f t he e v o l u t i o n a r y r e l a t i o n s a m o n g l ife fo rms .

Acknowledgments. We thank Dr. R. Cedergren for supplying the small and large ribosomal subunit sequence alignments used by Cedergren et al. (1988) and Dr. G. De Soete for making avail- able the FORTRAN program for constructing trees from a dis- similarity matrix. Our research was supported in part by the Fund for Medical Scientific Research and by the Incentive Program on Fundamental Research in Life Sciences of the Otfice for Science Policy Programming. Y. Van de Peer and J. Neefs are recipients of a scholarship from the Institute for Scientific Research in Industry and Agriculture.

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Received September 5, 1989/Revised October 30, 1989