Infection, Genetics and Evolution 1 (2002) 297–301 Short communication Phylogenetic analysis of the genus Plasmodium based on the gene encoding adenylosuccinate lyase Lukasz Kedzierski a , Ananias A. Escalante b,c , Raul Isea d , Casilda G. Black a , John W. Barnwell c , Ross L. Coppel a,* a Department of Microbiology, P.O. Box 53, Monash University, 3800 Vic., Australia b Instituto Venezolano de Investigaciones Cientificas, Apartado 21827, Caracas 1020-A, Venezuela c Centers for Disease Control and Prevention, Mailstop F13, 4770 Buford Hwy NE, Atlanta, GA 30341, USA d Centro de Calculo Cient´ ıfico de la Universidad de Los Andes, Mérida, Venezuela Received 10 September 2001; received in revised form 17 December 2001; accepted 24 December 2001 Abstract Phylogenetic studies of the genus Plasmodium have been performed using sequences of the nuclear, mitochondrial and plastid genes. Here we have analyzed the adenylosuccinate lyase (ASL) gene, which encodes an enzyme involved in the salvage of host purines needed by malaria parasites for DNA synthesis. The ASL gene is present in several eukaryotic as well as prokaryotic organisms and does not have repeat regions, which facilitates the accuracy of the alignment. Furthermore, it has been shown that ASL is not subject to positive natural selection. We have sequenced the ASL gene of several different Plasmodium species infecting humans, rodents, monkeys and birds and used the obtained sequences along with the previously known P. falciparum ASL sequence, for structural and phylogenetic analysis of the genus Plasmodium. The genetic divergence of ASL is comparable with that observed in other nuclear genes such as cysteine proteinase, although ASL cannot be considered conserved when compared to aldolase or superoxide dismutase, which exhibit a slower rate of evolution. Nevertheless, a protein like ASL has a rate of evolution that provides enough information for elucidating evolutionary relationships. We modeled 3D structures of the ASL protein based on sequences used in the phylogenetic analysis and obtained a consistent structure for four different species despite the divergence observed. Such models would facilitate alignment in further studies with a greater number of plasmodial species or other Apicomplexa. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Malaria; Adenylosuccinate lyase; Phylogenetic analysis; Plasmodium Malaria is an infectious disease caused by parasites of the genus Plasmodium and remains one of the major health problems in tropical regions. Considerable interest has focussed on investigating the phylogenetic relationship between malaria species. Such relationship should be deri- ved from as large a number of genes as possible to max- imize certainty in the observed trees. The genes already used in analyses include the 18S small subunit (SSU) rRNA genes (Escalante and Ayala, 1994; Escalante et al., 1997; Qari et al., 1996; Waters et al., 1991), the circumsporozoite protein (CSP) gene (Escalante et al., 1995), the cytochrome b gene from the mitochondria (Escalante et al., 1998) and the gene encoding the caseinolytic protease CLpC from the plastid (Rathore et al., 2001). Early studies suggested that Plasmodium falciparum shares a common ancestor with Abbreviations: ASL, adenylosuccinate lyase; PCR, polymerase chain reaction; SSU rRNA, small subunit ribosomal RNA; NJ, neighbor-joining * Corresponding author. Tel.: +61-3-9905-4822; fax: +91-3-9905-4811. E-mail address: ross.copple@med.monash.edu.au (R.L. Coppel). avian parasites (Waters et al., 1991; Waters et al., 1993), but subsequent inclusion of P. reichenowi (chimpanzee malaria species) sequences into phylogenetic analyses, showed that P. falciparum shares a more recent ancestor with this species (Escalante and Ayala, 1994; Escalante et al., 1995; Escalante et al., 1998; Qari et al., 1996). Those studies were based on 18S SSU rRNA and CSP sequences. However, the pattern of expression of ribosomal genes in Plasmodium is complex (Corredor and Enea, 1993; McCutchan et al., 1995) and thorough analysis of SSU rRNA sequences incorporating secondary structure needs to be performed in order to obtain a reliable tree (Escalante et al., 1997). Similar problems are encountered when analysis is based on CSP data. The CSP is a surface protein and as such is under selection by the host immune system. Consequently, the CSP is polymor- phic (Hughes, 1991), accumulates mutations rapidly, which may lead to the distortion of the phylogenetic inferences. Genes that are relatively conserved across different species and not subject to immune selection are preferable for determining evolutionary relationship. One such gene is 1567-1348/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII:S1567-1348(02)00031-X