Phylogeny of protozoa deduced from 5S rRNA sequences

Journal of MolecularEvolution

I Mol Evol (1983) 19:411-419

Q Springer-Verl ag 1983

Phylogeny of Protozoa Deduced from 5S rRNA Sequences Tsutomu Kumazaki 1 , Hiroshi Hori, and Syozo Osawa Laboratory of Molecular Genetics, Department of Biology, Faculty of Science, Nagoya University, Nagoya 464, Japan

Summary. The nucleotide sequences o f 5S rRNAs from three protozoa, Bresslaua vorax, Euplotes woodruffi and Chlarnydomonas sp. have been determined and aligned together with the sequences o f 12 p r o t o z o a species ineluding unicellular green algae already reported by the authors and others. Using this alignment, a phylogenic tree o f the 15 species o f protozoa has been constructed. The tree suggests that the ancestor for protozoa evolved at an early time o f eukaryotic evolution giving two major groups o f organisms. One group, which shares a COmmon ancestor with vascular plants, contains a unicellular green flagellate (Chlamydomonas) and unicellular green algae. The other group, which shares a common ancestor with the multicellular animals, includes various flagellated protozoa (including Euglena), ciliated Protozoa and slime molds. Most o f these protozoa appear to have separated from one another at a fairly early period o f eukaryotic evolution.

o f molecular evolution studies, it has become possible to construct phylogenic trees b y comparing sequences o f a certain informational macromolecule derived from various organisms. The usefulness o f the 5S rRNA sequence for this purpose has been shown first b y Kimura and Ohta (1973) and then by Hori and Osawa (1979) and others. However, the number of protozoa, whose 5S rRNA sequences have been determined, has still not been sufficient to understand the phylogenic structure o f protozoa. Under these circumstances, we have determined the sequences of 5S rRNAs from eight protozoa [see preliminary report b y Kumazaki et al. (1982a,b,d) and present paper] over the last three years. Thus we now have the 5S rRNA sequences from 15 representative protozoa species (Table 1). Using these sequences, we have constructed a phylogenic tree o f these protozoa.

Key words: 5S rRNA - RNA sequence - Protozoa Secondary structure - Phylogenetic tree

Materials and Methods

.Preparation of 5S rRNAs and Sequencing. Bresslaua vorax, Euplotes woodruffi and Chlamydomonas sp., kindly provided Introduction The positions in phylogenetic trees o f organisms o f variOus protozoa, including Chlamydomonas and unicellular green algae as well as their classification or interrelationship, have not been well established in classical biology due mainly to the lack o f definite and unified criteria for mutual comparisons. With the development

1present address." Department of Biochemistry and Biophysics, Research Institute for Nuclear Medicine and Biology, Hiroshima University, Hiroshima 734, Japan

Offprint requests to: T. Kumazaki at his present address

by Dr. M. Suhama and Dr. T. Kosaka of Hiroshima University, were cultured in bacterial wheat-infusion at 20~ - 30~C. The cells collected by centrifugation were lysed in a buffer containing SDS (1% SDS, 10 mM Tris-HCl (pH 7.7), 1 mM MgCl2), and shaken directly with 90% phenol to release rRNAs to the water phase without preparing ribosomes (Kumazaki etal. 1982c). The crude 5S rRNA was separated from other rRNAs by electrophoresis on a 15% polyacrylamide gel containing 7 M urea, 0.1 M Tris-borate (pH 8.3) and 1 mM EDTA. The 5S rRNA band on the gel was cut out and was eluted with 0.5 M ammonium acetate, 0.1 mM EDTA and 0.1% SDS at 37~ overnight and precipitated with 3 volumes of ethanol. This process was repeated once to purify the 5S rRNA. The sequences were determined by the chemical method of Peattie (1979) using y-32p-RNA and by the enzymatic method of Donis-Keller (1980) using 5' or y-32p-RNA. In some cases, electrophoresis was carried out on a hot plate at 70~ (Nazar

412 Table 1. Taxonomic positions* of protozoa and unicellular green algae whose 5S rRNA sequences have been known Phylum

Subphylum

Species

ReE for sequence

Bresslaua vorax Paramecium tetraurelia Tetrahymena thermophila Blepharisma/aponicum Euplotes woodruffi

This paper Kumazaki et al. 1982b Kumazaki et al. 1982b Kumazaki et al. 1982b This paper

Mastigophora

Chilomonas paramecium Crypthecodinium cohnii Euglena gracilis Chlamydomonas sp. Crithidia fasciculata

Kumazaki et al. 1982d Hinnebusch et al. 1981 Kumazaki et al. 1982a This paper MacKay et al. 1980

Sarcodina

Acanthamoeba castellanii Dictyostelium discoideum Physarum polycephalum

MacKay and Doolittle 1981 Hori et al. 1980 Komiya and Takemura 1981

Chlorella sp. Scenedesmus obliquus (Chlamydomonas sp.)

Luehrsen and Fox Green et al. 1982

Ciliophora

Sarcomastigophora

Chlorophyta

1981

*According to Levine et al. (1980)

and Wildeman 1981) to resolve some ambiguous parts for reading.

Secondary Structures and Sequence Alignment. The secondary structures of 5S rRNAs were constructed according to the basic method of Tinoco et al. (1971), as adopted by Hori (1976) and Hori and Osawa (1979). For the alignment of the fifteen 5S rRNA sequences, all the 5S rRNA secondary structures were juxtaposed to obtain the alignment for helical regions. The bestmatch alignments of the non-base-paired regions were then obtained with minimum gap insertions.

Construction of Phylogenetic Tree. The rate of nucleotide substitution, Knuc, and the standard error of Knuc, Ok, between sequences i and ] were calculated by the following equations according to Kimura (1980).

Knuc=-171n[(1-2P-Q)~ 1 - 2 Q ]

(1)

o k-- 1

(2)

[,/(a2P + b2Q)_ (aP + Q)2' ]

where P and Q were the fractions of nucleotide sites showing transition- and tmnsversion-type differences, respectively, n was the number of nueleotide sites to be compared, and a and b were calculated by the following equations. a=

1

(3)

1-Ze-Q

1[

b= 2-

,

1-2e-a

,

1]

-i-:TO-

values obtained as above can be used for determination of the branching order and the relative evolutionary distance in the construction of phylogenetic trees. All pairs of organisms were rearranged in the order of increasing Knuc values, and the pair to be formed first was determined simply by choosing the pair with the smallest Knuc value. The value of 1/2 Knuc of the pair was then taken to fix the branching point between them. The branching points between two or more pairs were determined from the average number of 1/2 Knuc between the pairs. AssUna" ing that Knuc is proportional to the number of years that have elapsed since the evolutionary divergence of the two molecules from their common ancestor (Jukes and Cantor 1969), the value of 1/2 Knuc was taken as a relative time scale in the tree.

Results

Sequence T w o e x a m p l e s o f the sequencing gels are s h o w n in Fig. 1 and 2, where nucleotides o f position 4 - 1 1 8 are readable for the Chlamydomonas 5S r R N A (Fig. 1) and those of position 1 - 9 0 are readable for the Bresslaua 5S rRNA (Fig. 2.) The 5S r R N A n u c l e o t i d e sequences o f Bresslaua, Euplotes and Chlamydomonas d e t e r m i n e d here, together w i t h those o f o t h e r p r o t o z o a so far k n o w n , were able to be aligned easily with few gap insertions (Fig. 3). All these R N A s were I 1 8 - 1 2 2 nucleotides long.

(4)

One gap (represented by - in alignment in Fig. 3) or one blank (represented by blank space) vs one nucleotide was counted as equal to one transversion type substitution. A phylogenetic tree was constructed by the UPG method (e.g., Sheath and Sokal 1973; Nei 1975; Hori 1975). The Knuc

Secondary Structure All the e u k a r y o t i c 5S r R N A s e x a m i n e d revealed basical" ly the same secondary structure (Hori et al. 1980) having five base-paired regions ( A - A ' B-B', C-C', D-D'

413 G

A>G C>U

U

XC-

N N

11.

8P8-

N

=

I

B

A G

A

Fig. 1 A and B. Autoradiograms of [y.32p] 5S rRNA of Chlamydomonas sp. The partial chemical digests (Peattie 1979) were fractionated on 12% polyaerylamide gel in 7 M urea, 0.1 M Trisborate (pH 8.3) and 1 mM EDTA at 1500 volts for 1.5 h A or 6 h B. Abbreviations: XC and BPB; marker dyes: xylene cyanol and bromophenol blue, respectively

A+U C+U

X

G

A

A+U C+U

X

IC-

~emulD

m

~

8P6- ~

e-o

A

B

Fig. 2 A and B. Autoradiograms of [5'-32p| 5S rRNA of Bresslaua vorax. The partial digests (DonisKeller 1980) obtained by incubat: ing [5'-32P1 5S rRNA with RNase T1 (G), RNase U 2 (A), RNase phyM (A+U), Bacillus cereus RNase (C+U) and alkali (X) were subjected to eleetrophoresis on 20% polyacrylamide gels at 1500 volts for4 h A o r 15 hB

414

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Fig. 4a

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Fig. 4 A-G. Secondary structure models of 5S rRNAs from sea anemone (A, a representative of multicellular animals; Kumazaki

G.USO U--A A--U C--G A--UG C--G C--G A G 90G U

et al. 1982c), Bresslaua vorax B, Euplotes woodruffi C, ChlamY" domonas sp. D, Chlorella sp. (E, Luehrsen and Fox 1981),

G

Aspergig~s

n~duganS

Fig. 4g

spinach (F, a representative o f vascular plants; Delihas et al. 1981) and Aspergillus nidulans (G, a representative of fungi; Piechulla et al. 1981). Dotted lines indicate potential base pairs according to Peattie et al. (1981) ANIMALS

~ - : ~ ....

~--~'" -,

EUGLENA CRITHIDIA BRESSLAUA PARAMECIUM TETRAHYMENA BLEPHARISMA EUPLOTES CRYPTHECODINIUM ACANTHAMOEBA PHYSARUM DICTYOSTELIUM

! . . . .

CHILOMONAS

4

CHLAMYDOMONAS v

CHLORELLA

"L-SCENEDESMUS

VASCULAR PLANTS , ASCOMYCETES I 0.3

i

I 0.2

i

I 0,1

and E-E') and five loop regions (aLb, bLc-c'Lb', cLc', eLd-d'Le' and dLd') (for these symbols, see Fig. 3, 4; also Hori and Osawa 1979; Hori et al. 1980). It has been pointed out that the D-D' stem of the secondary structure has a bulge of G in fungi, a bulge of U in plants and an A/C mismatch in multicellular animals (Fig. 4; Kumazaki et al. 1982d; see also Delihas et al. 1981; MacKay and Doolittle 1981). Thus, the secondary structures of eukaryotic 5S rRNA appear to be more or less group-specific, although they are basically the same. The 5S rRNAs from Crypthecodinium, Acanthamoeba, Physarurn, Crithidia, Dictyostelium, Chilomonas and all the ciliates, as well as Chlamydomonas, had a bulge of G

al/2

KNUC

Fig. 5. Phylogenie tree of eukaryotes. ~- - - -I: range of standard error computed bY Eq. (2)

in the D-D' stem, while Chlorella and Scenedesmus had a bulge of U at the same position. The position of the bulge of G or U is the same as that of the bulge of G ir~ fungi, but shifts up by one-base as compared with that of the plants 5S rRNA (see Fig. 4). As an exception, no bulge existed in Euglena. The significance of these patterns will be discussed later. In the aLb region, two base-pairs can generally be made in all the eukaryotic 5S rRNAs including most of the protozoa 5S rRNAs (e.g., Fig. 4 for ChlamydornonaS 5S rRNA; see Peattie et al. 1981). However, the 5S rRNA from all the ciliates studied was able to show only

417 one base-pair with a bulge of one base as shown in Fig. 4 for the Bresslauaand Euplotes 5S rRNAs.

Phylogenetic Tree The Knuc values of every possible pair of 94 eukaryotic 5S rRNA species so far reported were calculated according to Eq. (1). From these values, a phylogenetic tree of eukaryotes was constructed according to the method described in Materials and Methods with special reference to the protozoa (Fig. 5).

Discussion

to one another. Especially notable is that Chilomonas, Crypthecodinium and Crithidia/Euglena bear one or more flagella, while the sequence difference between them is as big as the difference between these protozoa and ciliates or amoeba/slime molds. These protozoa appear to have separated from one another in the early period of evolution at the points between 0.23 and 0.25 + 0.05 of 1/2 Knuc value, i.e., 0.9 and 1.0 -+ 0.18 billion years ago. The five ciliates studied are closely related to one another (78%-88% identity; see Table 2). Also an amoeba (Acanthamoeba) and a true slime mold Physarum are relatively near (81% identity; see Table 2), but they are only remotely related to a cellular slime mold Dictyoste-

lium. The tree shows that the protozoa can be divided phylogenically into two groups which, after the emergence of Ascomycetes (fungi), separated from each other in an early stage of evolution. The separation took place at about the point of 0.27 + 0.05 of 1/2 Knuc value. This COrresponds to about 1.1 -+ 0.18 x 109 (billion) years ago, if the yeast-animal divergence time is taken to be 1.2 billion years ago (Kimura and Ohta 1973). One group contains unicellular green flagellate Chlamydomonas and Unicellular green algae Chlorella/Scenedesmus (and also plants). Chlamydomonas, Chlorella/Scenedesmus and plants separated at about the same time. Thus, it is not improbable that the common ancestor for the plants and Chlorella/Scenedesmus is flagellated. This view is consistent with the notion that the plants originated from some type of green-flagellated protists. Although Chlamydomonas, green algae and plants belong to the same branch in the tree, their diversification seems to have occurred in a relatively early stage of evolution (about 0.8 +--0.14 billion years ago). Chlorella and Scenedesmus are closely related to each other as expected. (Lim et al. (1983) recently found that the 5S rRNA sequence of a multicellular green alga is close to those of unicellular green algae). The other group contains so called flagellated protozoa (including Euglena), ciliated protozoa, true and cellular slime molds, and multicellular animals. These organisms separated from the Chlamydomonas/green algae/plants stem about 1.1 -+ 0.18 billion years ago, and diverged as illustrated. Especially interesting is the fact that the flagellated protozoa (Crithidia/Euglena) and multicellular animals share a direct common ancestor in the tree. This is consistent with the view that the ancestor for the multicellular animals would be some kind of flagellated protozoon. The detailed branching order deduced here should, of course, be taken as tentative because relatively large standard errors from Eq. (2) are involved (see Fig. 5). Even so, the Knuc values from Eq. (1) and the phylogenic tree indicate that, with probable exceptions of rather closer relationships between Physarum and amoeba and between five ciliates, various protozoa studied here are only remotely related

As already mentioned under Results, the 5S rRNAs from most of the protozoa including slime molds and Chlarnydomonas have a bulge of G in the D-D' stem, while those from Chlorella and Scenedesmus have a bulge of U at the same position. Since the bulge at this position is G in fungi, these bulge profiles and the phylogenic tree in Fig. 5, when considered together, suggest that the G-,U transversion took place at this position in the green algae after their separation from Chlamydomonas/plants. The plants 5S rRNA has also a bulge of U in the D-D' stem, but its position shifts down by onebase. Thus more complicated substitutions would have occurred in the plants 5S rRNA after separation from Chlamydomonas/green algae. In the multicellular animals, the nucleotide substitution took place so as to have an A/C mismatch in the D-D' stem after their separation from the flagellated protozoa. Now, how is the phylogenetic relationships of the protozoa deduced from the 5S rRNA sequences consistent with the classification by taxonomic criteria? The taxonomic positions of the protozoa dealt with here may be summarized in Table 1 according to the scheme that has been generally accepted by protozoologists (Levine et al. 1980). Here, Chlorophyta, which are routinely treated as a phylum in the Plantae, are added as a phylum of the protozoa for convenience, because Chlamydomonas is treated either as a protozoon or as a member of the Chlorophyta. The first phylum Ciliophora includes all the ciliates studied here. The second phylum Sarcomastigophora is further divided into two subphyla, Mastigophora and Sarcodina. The former includes flagellated protozoa (including Chlarnydomonas) and the latter includes the amoeba and slime molds. The third phylum Chlorophyta contains the unicellular green algae Chlorella and Scenedesrnus (and sometimes

Chtarnydomonas). As has been discussed before, the ciliates of at least five species studied here are intimately related to one another. Thus the grouping of these ciliates seems to be reasonable. Corliss (1979) has suggested that in the ciliates the class I species are the "most primitive" and firstly separated from the ancestor common to "more

418 Table 2. Matrix of similarity (%) Ani Animals*

Bresslaua 67 Paramecium 64 Tetrahymena 66 Blepharisma 67 Euplotes 65 Chilomonas 64 Crypthecodinium 70 Euglena 72 Chlamydomonas 59 Crithidia 62 Acanthamoeba 66 Dictyostelium 62 Physarum 70 Chlorella 67 Scenedesmus 69 Plants* Ascomycetes*

63 60

Bre

Par

Tet Ble Eup

Chi

Cry

Eug

Chin Cri

Aea

Dic Phy

Chr

See Pin

AsC

67

64 88

66 87 88

64 66 65 65 63 63

70 74 73 72 78 76 66

72 70 66 70 66 72 62 67

59 63 64 60 66 60 59 59 63

66 70 68 68 72 68 62 70 68 63 67

62 62 59 61 59 63 66 59 68 57 65 66

67 63 61 64 64 63 61 68 66 70 63 65 56 66

69 66 63 66 66 66 62 68 68 70 65 67 57 68 97

60 60 57 57 59 62 60 62 57 55 57 63 56 67 54 55 56

88 87 85 88 66 74 70 63 62 70 62 77 63 66 65 60

88 86 80 65 73 66 64 60 68 59 73 61 63 71 57

78 80 65 72 70 60 67 68 61 73 64 66 64 57

67 85 86 78 79 63 78 66 66 61 72 59 75 64 66 68 59

65 88 80 80 79 63 76 72 60 61 68 63 73 63 66 63 62

66 62 59 67 62 66 67 61 62 62 60

67 59 58 70 59 67 68 68 64 62

63 72 68 68 73 66 68 60 57

59 63 57 68 70 70 71 55

62 62 60 67 61 61 67 58 72 59 67 65 71 63 65 54 57

66 81 65 67 61 63

70 56 57 51 56

70 77 73 73 75 73 67 67 73 68 71 81 70 66 68 66 67

97 70 54

71 55

63 65 71 64 68 63 62 64 60 71 54 61 51 66 70 71 56

*The mean similarity values were calculated from the sequences of 46 multiceUular animals, 10 vascular plants and 8 Ascomycetes species. For the sources of the sequences, see Table 1 and Kumazaki et al. (1983)

advanced" class II and the "most advanced" class III species, followed by the separation of these two classes. The 5S rRNA tree suggests, however, that the class III (Blepharisma and Euplotes) separated first from the ancestor common to the class I (Bresslaua) and the class II species (Paramecium and Tetrahymena), followed by the separation of the class I and class II more recently. The protozoa placed in the phylum Sarcomastigophora are very heterogeneous when deduced from the 5S rRNA sequences. In the subphylum Mastigophora, Euglena and Crithidia are, as already mentioned, somewhat related to each other (72% identity; see Table 2), b u t Chilomonas, Crypthecodinium, Chlamydomonas and Euglena/Crithidia are only distantly related (58%- 67% identity; see Table 2), and they appear to have diverged at an early time in eukaryotic evolution. Euglena has been classified either as related to plants (by botanists) or to animals (by zoologists). The sequence o f 5S rRNA suggests that it is much closer to animals than to plants. This has been also suggested by other biochemical data (Ragan and Chapman 1978; Chang et al. 1981), as well as by the classification scheme in Table 1. On the other hand, the 5S rRNA sequence of Chlamydomonas is closer to the sequences of green algae and plants than to other protozoa and animals. In the subphylum Sarcodina, Acanthamoeba and a true-slime mold Physantm are also close to each other from the 5S rRNA sequence data, b u t a cellular-slime mold Dictyostelium is very different either from amoeba or Physarum. The slime molds have been often classified in old textbooks as members of fungi, but the 5S rRNA data support the classification system in Table 1 where the slime molds, at least Physarum, and amoeba are placed in the same subphylum Sarcodina. In spite of such a partial consis-

tency, the phylum Sarcomastigophora appears to be, as a whole, composed of a number of mutually unrelated species and thus seems to be quite artificial. Chlorella and Scenedesmus are close to each other from the 5S rRNA data and, thus, placing these orga" nisms (plus Chlamydomonas) in the same phylum maY not be unreasonable. In conclusion, the 5S rRNAs from various protozoa and slime molds share the ancestor c o m m o n to those from the multicellular animals and in this sense they can be grouped as "protozoa", if desired, although the divergence times of many of them are very remote. On the other hand, Chlamydomonas, Chlorella and Scene" desmus are closer to plants than to the protozoa. Also, Chlorella and Scenedesmus are very close to a multic el~ lular green alga (Lim et al. 1983). Thus these three orga" nisms may be placed in the Chlorophyta or treated as members of the kingdom Plantae, if necessary.

Acknowledgements. We thank Dr. T. Nakano of Hiroshima U~i" versity for the identification of Chlamydomonas sp. This work was supported by grants 56480377, 56570178, and 57121003 (Special Project Research) from the Ministry of Education of Japan.

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Received February 21, 1983/Revised May 20, 1983