Plasmodial ribosomal RNA as phylogenetic probe: a cautionary note

Letter to the Editor Plasmodial Ribosomal RNA as PhylogeneticProbe: A Cautionary Note’ Vladimir Corredor and Vincenzo Enea Department

of Medical

and Molecular

Parasitology,

New

York

University

Medical

Center

If one is to draw reliable taxonomical inferences from the analysis of molecular sequences, the phyletic relations between the genes used to build trees must be orthologous, i.e., the same as those of the corresponding organisms (Nei 1987, p. 288). Barring xenology, this condition is met when a gene either is single copy or evolves in so concerted a manner as to behave like one. The latter is generally the case for the ribosomal RNA (rRNA) genes, and it is in this sense that one refers to the rRNA sequence of an organism (Nei 1987, p. 129). A partial exception to this state of affairs is found in Plasmodium. These parasites are known to harbor distinct rRNA genes that are developmentally controlled. Type A genes are expressed in the asexual stages, and type B genes are expressed in the sexual stages (Gunderson et al. 1987; Waters et al. 1989a). The available sequences of the small ribosomal RNA subunit (SRS) indicate that the A and B genes do not evolve in a fully concerted manner (Enea and Corredor 199 1). This complicates their use as probes to ascertain phylogenetic relationships within the genus. For example, consider the cladograms that include the complete plasmodial SRS sequences available (seven type A and three type B) (fig. 1) . The B sequences lie close to their cognate A sequences, which might imply that the establishment of developmentally controlled SRS occurred three times, once each in P. berghei and P. falciparum and again in the P. cynomolgi/ P. fragile lineage. Although possible, this interpretation is unparsimonious because it requires separate duplications of the structural moieties of the gene, plus de novo establishment of the appropriate stage-specific control sequences. This entails that the trait of developmentally controlled rRNA isoforms, for all its rarity, is not intrinsic to the plasmodial ontogenetic plan. No such difficulties are met if one views the trait as ancestral and the similarity between the cognate isoforms as stemming from sequence homogenization (gene conversion, nonreciprocal recombination, and the like). If this is the case, it should not be assumed that the gene tree will be isomorphic with the corresponding species tree. Consider three taxa whose phyletic relationship is [ ( 1,2) 3 ] and whose A and B genes originally grouped as [((A 1,A2)A3) ( ( B 1,B2) B3)]. If, through the action of rare but relatively recent gene conversions, Bl and B3 came to resemble their A paralogues, whereas A2 came to resemble its B paralogue, the resulting gene tree would be [((A 1,B 1 )( A3,B3))( A2,B2)], which is not isomorphic with the species tree. In this light, consider the placement of berA on the tree (fig. 1). On the one hand, it is separated from the genes of the other species by the longest internal branch; on the other hand, it is much closer to its cognate (berB) than cynA and falA are to theirs. Thus berA may be the most divergent of the A genes, either because P. berghei actually is the most divergent of the taxa or because, through conversion, it has come to look more like the berB gene. There seems to be no reason to favor the first possibility: 1. Key words: Address Parasitology,

Plasmodium,

rRNA.

for correspondence and reprints: Dr. Vincenzo Enea, Department New York University Medical Center, 341 East 25th Street, New

1993. 0 1993 by The University of Chicago. All rights reserved. 0737-4038/93/1004-00 13$02.00 Mol. Biol. Evol. 10(4):924-926.

924

of Medical York, New

and Molecular York 10010.

Letter

to the Editor

p--c berB 6.0 2.1

cynB

1.2

100

925

cynA

2.1 100

1.4

fraA

5.8 0.8

malA 3.2

90

IopA

2.5 100

0.6 68

2.5 3.4

67

galA falA

9.7

falB

FIG. 1.-Cladogram of 10 plasmodial SRSs. The tree was obtained, by neighbor joining (Saitou and Nei 1987)) from a matrix of pairwise differences obtained by using the Jukes-Cantor correction (Jukes and Cantor 1969) on an alignment produced by PILEUP (Devereux et al. 1984), with a gap-opening penalty of 2 and a gap-extension penalty of 0.2. The sequences are denoted with the first three letters of the corresponding species (i.e., berghei, cynomolgi, falciparum, fragile, gallinaceum, lophurae, and malariae), followed by A or B to denote the gene type. Lengths (in substitutions/ 100 nucleotides) are indicated above the branches. The numbers below the interior branches refer to the strict-consensus trees that resulted from 100 bootstrappings (Felsenstein 199 1). If the B sequences are removed from this tree, the remaining topology is that obtained when the A sequences alone are used to build a tree. Trees were also built from a number of other alignments, with different methods, and also under the assumption that a fraction of the unvaried sites were in fact invariable (Shoemaker and Fitch 1989). The only significant difference was that, in some cases, the falA sequence grouped weakly with galA and lopA. The sources for the sequences are as follows: Plasmodium berghei A and B genes, Gunderson et al. ( 1987); P. cynomologi A and B genes, Corredor and Enea (submitted); P. falciparum A and B genes, McCutchan et al. ( 1988); P. fragile gene A, Waters et al. ( 1991); P. gallinaceum gene A, Waters et al. ( 1991); P. lophurae gene A, Waters et al. ( 1989b); and P. malariae gene A, Goman et al. ( 199 I ). The alignment has been deposited with the EMBL data base (DS13648).

both are consistent with the observed long internal branch, and the second is also consistent with the observed short terminal branch. Similarly, the grouping of falA with galA and 1opA should be considered in conjunction with the relatively long terminal branch of falB. It may be the case that P.

926 Letter to the Editor P. gallinaceum, and P. lophurae are monophyletic, as proposed by Waters et al. ( 199 1 ), but it may also be that they have remained relatively untouched by conversion (which is consistent with the long terminal branch of falB) and that it just groups with whatever other A genes have also remained relatively untouched.

falciparum,

Acknowledgments

We thank John E. Hill for help with the mainframe computer. Computing was supported by NSF grant DIR8908095. This work was supported by the McArthur Foundation. LITERATURE CITED CORREDOR,V., and V. ENEA. The small-subunit ribosomal RNA in Plasmodium cynomolgi. (submitted). DEVEREUX,J., M. HAEBERLI,and 0. SMITHIES.1984. A comprehensive set of sequenceanalysis programs for the VAX. Nucleic Acids Res. 12:387-395. ENEA,V., and V. CORREDOR.199 1. The evolution of plasmodial stage-specificrRNA genes is dominated by gene conversion. J. Mol. Evol. 32: 183-186. FELSENSTEIN, J. 199 1. PHYLIP manual, version 3.4. University of Washington, Seattle. GOMAN, M., B. MONS,and J. SCAIFE.199 1. The complete sequence of a Plasmodium malariae SSUrRNA gene and its comparison to other plasmodial SSUrRNA genes. Mol. Biochem. Parasitol. 45:28 l-288. GUNDERSON, J. H., M. L. SOGIN,G. WOLLETT, M. HOLLINGDALE, V. F. DE LA CRUZ, A. P. WATERS, and T. F. MCCUTCHAN. 1987. Structurally distinct, stage specific ribosomes occur in Plasmodium. Science 238:933-937. JUKES,T. H., and C. R. CANTOR. 1969. Evolution of protein molecules. Pp. 2 l-l 32 in H. R. MUNRO, ed. Mammalian protein metabolism. Academic Press,New York. MCCUTCHAN, T. F., V. F. DE LA CRUZ, A. A. LAL, J. H. GUNDERSON, H. J. ELWOOD, and M. L. SOGIN. 1988. Primary sequence of two small subunit ribosomal RNA genes from Plasmodium falciparum. Mol. Biochem. Parasitol. 28:63-68. NEI, M. 1987. Molecular evolutionary genetics. Columbia University Press,New York. SAITOU, N., and M. NEI . 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425. SHOEMAKER, J. S., and W. M. FITCH. 1989. Evidence from nuclear sequencesthat invariable sites should be considered when sequence divergence is calculated. Mol. Biol. Evol. 6:270289. WATERS, A. P., D. G. HIGGINS,and T. F. MCCUTCHAN. 199 1. Plasmodium falciparum appears to have arisen as a result of lateral transfer between avian and human hosts. Proc. Natl. Acad. Sci. USA 88:3 140-3 144. WATERS,A. P., C. SYIN, and T. F. MCCUTCHAN. 1989a. Developmental regulation of stagespecific ribosome populations in Plasmodium. Nature 342:438-44 1. WATERS, A. P., T. R. UNNASCH, D. F. WIRTH, and T. F. MCCUTCHAN. 19898. Sequence of a small ribosomal RNA gene from Plasmodium lophurae. Nucleic Acids Res. 17: 1763. WALTER M. FITCH, reviewing editor

Received January 26, 1993; revision received March 2, 1993 Accepted March 4, 1993